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Potential agonists and inhibitors of protein kinase C.
Thesis presented for the degree of Doctor of Philosophy
by Philip Charles Gordge.
Department of Pharmacognosy,
The School of Pharmacy,
Univeristy of London.
August, 1992. Abstract
Daphnane and tigliane diterpenoid hydrocarbon nuclei were isolated from the
latex of Euphorbia poissonii.Pax. Euphorbiaceae using the techniques of liquid-liquid
partition, column chromatography, centrifugal liquid chromatography (CLC) and
preparative partition and adsorption thin layer chromatography. Six naturally occurring
diterpene esters were isolated from the latex and a further eight were semi-synthesised
by selective esterification of the C 2 0 primary alcohol of these resiniferonol and 12-
deoxyphorbol nuclei. These compounds were characterised by their ^-NM R, mass
spectral, UV and IR properties.
Computer-assisted molecular modelling of these compounds in conjunction
with standard phorbol ester probes enabled the assessment of their minimum free
energy values; resiniferonol derivatives were found to have much lower minimum free
energy levels than corresponding 12-deoxyphorbol derivatives. The molecular co
ordinates of the potent irritant, resiniferatoxin, were also assigned.
An in vivo mouse ear erythema assay was used to evaluate the pro-
inflammatory response of these compounds and the ability of some resiniferonol derivatives to inhibit erythema induced by phorbol ester and neurogenic (capsaicin)
irritants. The pro-inflammatory response induced by resiniferatoxin was found to be
of mixed aetiology, comprising of neurogenic and phorbol ester components.The ability of the semi-synthetic compounds to induce epidermal hyperplasia of mouse skin was assessed as a potential correlation for tumour promotion activity.
To further understand the biochemical mechanisms of action of these synthetic compounds, their ability to activate or inhibit a mixed isozymic pool of the phorbol ester receptor protein, protein kinase C, isolated from rat brain was studied.
The ability of 12-deoxyphorbol- 13-O-phenylacetate-20-O-acetate (DOPPA) to selectively activate isozymic forms of protein kinase C isolated from human platelets was also assessed. 3
To
Joan and Harry, who’ve shown me love and support when I needed it,
and for the loan of the bung. 4
Table of Contents.
Page No.
Title. 1
Abstract 2
Table of Contents. 4
List of Figures. 11
List of Tables and Schemes. 14
Abbreviations. 16
Chapter 1: Introduction. 18 1.1. The plant families Euphorbiaceae and Thymelaeaceae. 19 1.1.1. The plant family Euphorbiaceae. 19 1.1.2. The plant family Thymelaeaceae. 20 1.1.3. Commercial uses of plants from the families Euphorbiaceae 20 and Thymelaeaceae. 1.1.4. Non-diterpenoid constituents of theEuphorbiaceae and 21 Thymelaeaceae.
1.2. Macrocyclic diteipenes of theEuphorbiaceae. 22 1.2.1. Lathyrane diteipenes. 22 1.2.2. Jatrophane diteipenes. 24 1.2.3. Jatropholane and Crotofolane diterpenes. 27 1.2.4. Rhamnofolane and Myrsinol diteipenes. 27 1.2.5. Casbane diteipenes. 27
1.3. Tigliane, Ingenane and Daphnane diteipenes. 29 1.3.1. Tigliane diterpenes. 29 1.3.2. Ingenane diterpenes. 37 1.3.3. Daphnane diterpenes. 39 5
1.4. Biological effects of the phorbol esters. 45 1.4.1. Tumour promotion. 45 1.4.2. Pro-inflammatory response. 53 1.4.3. Platelet aggregation. 56 1.4.4. Epstein-Barr virus Early Antigen expression. 58 1.4.5. Other effects of phorbol esters. 60
1.5. Protein kinase C - the phorbol ester receptor site. 62 1.5.1. The discovery of protein kinase C. 62 1.5.2. The phosphatidylinositol signal transduction cycle. 64 1.5.3. PKC - identity with the phorbol ester receptor. 72 1.5.4. PKC isozymes. 73 1.5.5. Translocation and down-regulation of PKC. 81 1.5.6. Inhibitors of PKC. 83 1.5.7. Substrates of PKC. 94 1.5.8. Phorbol ester interactions with other kinases. 105
1.6. Objective of this work. 106
Chapter 2: Materials and Methods. 107 2.1. Materials. 108 2.2. Apparatus. I l l 2.3. Methods. 113 2.4. Buffers. 115
Chapter 3: Isolation and synthetic modification of 12- 118 deoxvphorbol and resiniferonol diterpene nuclei. 3.1. Introduction. 119
3.2. Isolation of compounds from plant material. 120 3.2.1. Materials and methods. 120 3.2.1.1. Plant material. 120 3.2.1.2. Partitioning of extract 120 3.2.1.3. Purification of the ether extract 122 6
3.2.1.3.1. Charcoal column chromatography. 122 3.2.1.3.2. Centrifugal liquid chromatography (CLC) of 12- 125 deoxyphorbol mono- and di- ester fractions. 3.2.1.3.3. Preparative partition and adsorption TLC. 128 3.2.2. Results. 132 3.2.2.1. Resiniferonol derivatives. 132 % 3.2.2.1.1. 9,13,14-orthophenylacetylresiniferonol-20-O- 132 homovanillate (Resiniferatoxin, Rx). 3.2.2.1.2. 9,13,14-orthophenylacetylresiniferonol (Resiniferonol, 136 Ro). 3.2.2.2. 12-deoxyphorbol derivatives. 138 3.2.2.2.1. 12-deoxyphorbol- 13-O-phenylacetate-20-O-acetate 138 (DOPPA). 3.2.2.2.2. 12-deoxyphorbol-13-O-phenylacetate (DOPP). 139 3.2.2.2.3. 12-deoxyphorbol-13-O-angelate-20-O-acetate (DAA). 142 3.2.2.2.4. 12-deoxyphorbol-13-O-angelate (DA). 143
3.3. Selective esterification of the primary hydroxyl group of 145 resiniferonol (Ro) and 12-deoxyphorbol-13-O-angelate (DA). 3.3.1. Materials and methods. 145 3.3.1.1. Esterification with propionic acid derivatives and p- 145 bromobenzoic acid. 3.3.1.1.1. Preparation of acid chlorides. 145 3.3.1.1.2. Diterpene ester reaction with the acid chlorides. 146 3.3.1.2. Esterification with 3-methoxy-4-hydroxy-phenylacetic acid 146 (Homovanillic acid). 3.3.2. Results. 147 3.3.2.1. Ro and DA propionic acid derivative and p-bromobenzoic 147 acid diesters. 3.3.2.I.1.9,13,14-orthophenylacetylresiniferonol-20-O-[DL-2-(3- 147 benzylphenyl)-propionate] (RoK). 3.3.2.1.2. 9,13,14-orthophenylacetylresiniferonol-20-O-[DL-2-(4- 150 isobutylphenyl)-propionate] (Rol). 7
3.3.2.1.3. 9,13,14-orthophenylacetylresiniferonol-20-O-p- 151 bromobenzoate (RoB). 3.3.2.1.4. 12-deoxyphorbol-13-O-angelate-20-O-[DL-2-(3- 153 benzylphenyl)-propionate] (DAK). 3.3.2.1.5. 12-deoxyphorbol-13-O-angelate-20-O-[DL-2-(4- 155 isobutylphenyl)-propionate] (DAI). 3.3.2.1.6. 12-deoxyphorbol-13-O-angelate-20-O-p-bromobenzoate 157 (DAB). 3.3.2.2. Ro and DA homovanillic acid diesters. 158 3.3.2.2.1. 9,13,14-orthophenylacetylresiniferonol-20-O- 158 homovanillate (Resiniferatoxin, Rx). 3.3.2.2.2. 12-deoxyphorbol-13-O-angelate-20-O-homovanillate 159 (DAHV).
3.4. Discussion. 160
Chapter 4: Computer-assisted molecular modelling of diterpene 164 esters. 4.1. Introduction. 165 4.2. Materials and methods. 170 4.3. Results. 170 4.3.1. Minimum free energy values and molecular structures of 170 derivatives. 4.3.2. Molecular co-ordinates of resiniferatoxin. 172 4.4. Discussion. 176
Chapter 5: Pro-inflammatory, anti-inflammatory and epidermal 180 hyperplasiogenic activities of diterpene esters. 5.1. Introduction. 181
5.2. Materials and methods. 182 5.2.1.Test agonists and antagonists. 182 5.2.2. Mouse ear erythema assay. 183 5.2.2.1. Determination of ID^ value. 183 8
5.2.2.2. Inhibition and pre-treatment experiments. 184 5.2.3. Hyperplasia experiments. 184
5.3. Results. 185 5.3.1. Determination of ID^ values of compounds. 185 5.3.2. Effect of pre-treatment with RoK and Rol on phorbol ester- 188 induced erythema. 5.3.3. Studies on resiniferatoxin-induced erythema. 190 5.3.4. Induction of epidermal hyperplasia. 199
5.4. Discussion. 199
Chapter 6: The effects of synthetic derivatives of 12- 203 deoxyphorbol-13-O-angelate and resiniferonol on a mixture of isoforms of protein kinase C isolated from rat brain. 6.1. Introduction. 204
6.2. Materials and methods. 205 6.2.1. Purification of mixed isozymic forms of protein kinase C 205 from rat brain. 6.2.2. PKC activation assay. 205
6.3. Results. 206 6.3.1. Purification of mixed protein kinase C isotypes from rat 206 brain. 6.3.2. Activation of mixed rat brain PKC by 12-deoxyphorbol-13- 208 O-angelate (DA). 6.3.3. Activation of mixed rat brain PKC by synthetic 12- 208 deoxyphorbol- 13-O-angelate derivatives. 6.3.3.1. Activation of mixed rat brain PKC by C^ propionic acid 208 derivatives. 6.3.3.2. Activation of mixed rat brain PKC by 12-deoxyphorbol- 211 13-O-angelate-20-O-p-bromobenzoate. 6.3.4. Activation of mixed rat brain PKC by synthetic resiniferonol derivatives. 6.3.4.1. Activation of mixed rat brain PKC by 9,13,14-ortho- phenylacetylresiniferonol-20-O-p-bromobenzoate. 6.3.4.2. Activation of mixed rat brain PKC by Qo propionic acid derivatives.
6.4. Discussion.
Chapter 7: Studies on the activation of platelet protein kinase C bv the non-aggregatorv phorbol ester. 12-deoxyphorbol-13-0- phenvlacetate-20-Q-acetate (POPPA) - a specific fo PKC isotvpe activator. 7.1. Introduction.
7.2. Materials and methods. 7.2.1. Platelet isolation. 7.2.2. Purification of mixed isoforms of platelet PKC. 7.2.3. Purification of isoforms of platelet PKC.
7.3. Results. 7.3.1. Studies on mixed PKC isolated from human platelets. 7.3.1.1. Purification of platelet mixed PKC. 7.3.1.2. Activation of mixed platelet PKC isozymes by 12- deoxyphorbol- 13-O-phenylacetate-20-O-acetate (DOPPA). 7.3.2. Studies on PKC isoforms isolated from human platelets. 7.3.2.1. Purification of platelet PKC isoforms. 1 3 2 .2 . Activation of platelet PKC peaks "a" and "b" by DOPPA. 1 3 .2 3 . The effect of altering Ca2* concentration on the activation of platelet PKC peaks "a" and "b" induced by DOPPA.
7.4. Discussion.
Chapter 8: Final discussion and concluding remarks. 10
References. 253
Publications from this work. 297
Acknowledgements. 299
Appendix 1: Induction of epidermal hyperplasia bv diterpene 301 esters. 11
List of Figures.
Page No.
Fig.l. Structures of macrocyclic diterpene hydrocarbon skeletons. 23
Fig.2. Classes of lathyrane macrocyclic diterpenes. 25
Fig.3. Jatrophane, jatropholane and crotofolane macrocyclic 26 diterpenes.
Fig.4. Rhamnofolane, myrsinol and casbane macrocyclic 28 diterpenes.
Fig.5. Tigliane, ingenane and daphnane hydrocarbon skeletons. 30
Fig.6. 4p- and 4a- phorbol. 32
Fig.7. Some biologically important esters with different phorbol 36 nuclei.
Fig.8. Ingenane diterpenes. 38
Fig.9. Daphnane diterpene types, and the absolute configuration of 41 resiniferonol.
Fig. 10. Daphnane diterpene esters. 43
Fig.l 1. la-alkyldaphnane type esters. 44
Fig. 12a. The concept of tumour promotion. 49 Fig. 12b. First and second stage tumour promotion. 50
Fig. 13. The phosphatidylinositol signal transduction system. 69
Fig. 14. Phosphatidylinositol turnover. 71
Fig. 15. Transfer of 32P from y-32P-ATP onto acceptor serine or 74 threonine residues of protein substrate by PKC.
Fig. 16. Structure of PKC isotypes. 77
Fig. 17. Structures of commonly usedPKC inhibitors. 92 Fig. 18. Structures of compounds isolated from the latex of E.poissonii.Pax.
Fig. 19. 500 MHz *H-NMR of resiniferatoxin (Rx) in CDC13.
Fig.20. Thermospray mass spectrum of resiniferonol (Ro).
Fig.21. 250 MHx *H-NMR of 12-deoxyphorbol-13-0- phenylacetate-20-O-acetate (DOPPA) in CDQ3.
Fig.22. Structures of derivatives synthesised from 12- deoxyphorbol-13-O-angelate and resiniferonol nuclei.
Fig.23. Structures of tumour promoters previously analysed by computer-assisted molecular modelling.
Fig.24. Structures of minimum free energy conformer of tigliane and daphnane derivatives.
Fig.25. Effect of pre-treatment with 0.5 and 5 pg/5pl RoK and Rol on phorbol ester induced erythema at tp^.
Fig.26. Time taken to reach, and effect of dose on, tp^ for capsaicin, Rx and Sap D.
Fig.27. Induction of desensitisation of erythema response by repeated application of capsaicin.
Fig.28. Effect of increasing pre-treatment dose of capsaicin on total number of animals responding to a sub-threshold dose of sapintoxin D.
Fig.29. Effect of hydrocortisone or indomethacin pre-treatment before resiniferatoxin application.
Fig.30. Activation assay profile of mixed PKC isozymes eluted from DEAE-cellulose column.
Fig.31. Activation of mixed rat brain PKC by 12-deoxyphorbol- 13-O-angelate (DA) in comparison to 12-O-tetradecanoylphorbol- 13-O-acetate (TPA). 13
Fig.32. Activation of mixed rat brain PKC by 12-deoxyphorbol- 210 13-O-angelate propionic acid derivatives.
Fig.33. Activation of mixed rat brain PKC by 12-deoxyphorbol- 212 13-O-angelate-20-O-p-bromobenzoate (DAB) and 9,13,14-ortho- phenylacetylresiniferonol-20-O-p-bromobenzoate (RoB).
Fig.34. Effect of synthetic resiniferonol propionic acid derivatives 214 on activation of mixed isozyme PKC from rat brain, in the absence of PS.
Fig.35. Effect of synthetic resiniferonol propionic acid derivatives 215 on activation of mixed isozyme PKC from rat brain, in the presence of PS.
Fig.36. Inhibition of mixed rat brain PKC induced by 9,13,14- 216 orthophenylacetylresiniferonol-20-O-[DL-2-(3-benzoylphenyl)- propionate] (RoK).
Fig.37. Activation assay profile of mixed PKC isozymes eluted 226 from DEAE-cellulose column.
Fig.38. Activation of mixed platelet PKC isozymes by DOPPA in 228 the presence and absence of Ca2*.
Fig.39. Induction of p47 phosphorylation by DOPPA and TPA in 229 intact platelets in the presence and absence of Calcium ionophore, A23187.
Fig.40. Activation assay profile of PKC isozymes eluted by 231 hydroxylapatite chromatography.
Fig.41. Activation of platelet PKC peaks "a" and "b" by 500nM 232 DOPPA in the presence and absence of Ca2*.
Fig.42. Effect of altering Ca2* concentration on activation of peak 234 "a” and peak "bM platelet PKC by DOPPA. 14
List of Tables and Schemes.
Page No.
Table 1. Phorbol-related diterpene nuclei with different 33 oxygenation states.
Table 2. Comparison of the biological properties of initiating and 47 promoting agents.
Table 3. Structure-activity relationships for the induction of 55 inflammation.
Table 4. Agonists capable of stimulating phosphatidylinositol 65 turnover.
Table 5. Examples of tissue-specific protein kinase C isozyme 76 distribution.
Table 6. Activators of protein kinase C. 79
Table 7. Protein kinase C inhibitors. 85
Table 8. Protein substrates for protein kinase C. 96
Table 9. Analytical TLC of dried ether extract 123
Table 10. Composition of fractions eluted from the charcoal 124 column.
Table 11a. Composition of CLC fractions of mono- and di- ester 127 mixture. Table lib. Composition of CLC fractions of mono- and di- ester 127 mixture after acid- or base- catalysed hydrolysis.
Table 12. Preparative partition and adsorption TLC Rf values of 130 fractions previously semi-purified by CLC or charcoal column chromatography.
Table 13. Summary of compounds isolated and synthesised. 163 15
Table 14. Comparison of structural requirements of different 167 tumour promoters for activity.
Table 15. Structural requirements for activity for diacylglycerol 169 compared to 12-O-tetradecanoylphorbol-13-acetate.
Table 16. Computer-generated minimum free energy values of 171 derivatives of phorbol related nuclei.
Table 17. The molecular co-ordinates of resiniferatoxin. 177
Table 18. Irritant dose^ (ID^) values of isolated and synthesised 186 compounds.
Table 19. Comparison of the potency and duration of response of 191 resiniferatoxin, sapintoxin D and capsaicin.
Table 20. Effect of capsaicin pre-treatment on time to peak 197 irritancy of erythema response to resiniferatoxin.
Table 21. A summary of the effects of synthetic derivatives of 12- 218 deoxyphorbol-13-O-angelate and resiniferonol on mixed rat brain PKC.
Table 22. Summary of biological activities of compounds studied. 242
Scheme 1. Partitioning of latex. 121
Scheme 2. Summary of purification of ether extract of latex. 131 Abbreviations.
‘H-NMR Proton nuclear magnetic resonance. 3-TI 3-O-tetradecanoyl-ingenol. 3H-PDBu Tritiated phorbol-12,13-dibutyrate. 4-O-MeTPA 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate. A23187 Calcium ionophore. AA Arachidonic acid. ADP Adenosine diphosphate. ATP Adenosine triphosphate. Ca2+ Calcium ion. CHCI3 Chloroform. CIMS Chemical ionisation mass spectroscopy. CLC Centrifugal liquid chromatography. CytP450 Cytochrome P450. DA 12-deoxyphorbol-13-O-angelate. DAA 12-deoxyphorbol-13-O-angelate-20-O-acetate. DAB 12-deoxyphorbol-13-O-angelate-20-O-p-bromobenzoate. DAG Diacylglycerol DAHV 12-deoxyphorbol-13-O-angelate-20-O-(3-methoxy-4-hydroxy)- phenylacetate. [20-O-homovanillate] DAI 12-deoxyphorbol-13-0-angelate-20-O-[DL-2-(4-isobutylphenyl)- propionate]. DAK 12-deoxyphorbol-13-O-angelate-20-O-[DL-2-(3-benzoylphenyl)- propionate]. DEAE Diethylaminoethyl cellulose. DMBA Dimethylbenz[a] anthracene. DNA Deoxyribonucleic acid. DOPP 12-deoxyphorbol-13-O-phenylacetate. DOPPA 12-deoxyphorbol-13-O-phenylacetate-20-O-acetate. EBV Epstein-Barr virus. EBV-EA Epstein-Barr virus early antigen. EDTA Ethylenediaminetetra-acetic acid. EGTA Ethylene glycol bis (p-aminoethylether)-N,N,N\N’-tetra-acetic acid. EIMS Electron impact mass spectroscopy. EtOH Ethanol. FPLC Fast protein liquid chromatography. H-7 1 -(5-isoquinolinesulphonyl)-2-methylpiperazine (dihydrochloride). HA Hydroxylapatite. HETE Hydroxyeicosatetraenoic acid. HPETE Hydroxypereicosatetraenoic acid. HVA 3-methoxy-4-hydroxy-phenyacetic acid (homovanillic acid). ® 5 0 Irritant dose 50%. Ins-l-P Inositol-1 -monophosphate. Ins-1,3,4,5-P4 Inositol-1,3,4,5-tetrakisphosphate. Ins-1,4-P2 Inositol-1,4-biphosphate. Ins-1,4,5-P3 Inositol-1,4,5-trisphosphate. Ins-4-P Inositol-4-monophosphate. IR Infra-red. kDa kilodalton. M Molar. MAG Monoacylglycerol. MeOH Methanol. Mez Mezerein. Mr Relative molecular mass. MS Mass spectroscopy. Na2C03 Sodium carbonate. NaCl Sodium chloride (salt). PAH Poly-aromatic-hydrocarbon. PC Phosphatidylcholine. PDBu Phorbol-12,13-dibutyrate. PG Prostaglandin. Pi Inorganic phosphate. PKA Protein kinase A (cAMP-dependent protein kinase) PKC Protein kinase C PKM Protein kinase M (proteolytic catalytic domain fragment of PKC). PLA2 Phospholipase A2. PLC Phospholipase C. PLD Phospholipase D. ppm Parts per million. PRP Platelet rich plasma. PS Phosphatidylserine. Ptdlns Phosphatidylinositol PtdIns-4-P Phosphatidylinositol-4-monophosphate. PtdIns-4,5-P2 Phosphatidylinositol-4,5-biphosphate. RA Retinoic acid. RNA Ribonucleic acid. Ro Resiniferonol; 9,13,14-orthophenylacetylresiniferonol. RoB 9,13,14-orthophenylacetylresiniferonol-20-O-p-bromobenzoate. Rol 9,13,14-orthophenylacetylresiniferonol-20-O-[DL-2-(4-isobutylphenyl)- propionate]. RoK 9,13,14-orthophenylacetylresiniferonol-20-O-[DL-2-(3-benzoylphenyl)- propionate], RPA 12-O-retinoylphorbol- 13-acetate. Rx Resiniferatoxin; 9,13,14-orthophenylacetylresiniferonol-20-O-(3- methoxy-4-hydroxy)-phenylacetate. [-20-O-homovanillate] Sap D Sapintoxin D; 12-0-(2-methylaminobenzoyl)-phorbol-13-acetate. Sap A Sapintoxin A; 12-0-(2-methylaminobenzoyl)-4-deoxyphorbol-13-acetate. SDS-PAGE Sodium dodecylsulphate polyacrlyamide gel electrophoresis. TAS Transferable aggregating substance. Thy A Thymeleatoxin A; 9,13,14-orthobenzoyl-6,7-epoxy-5-hydroxy- resiniferonol-12-O-cinnamate. TLC Thin layer chromatography. TPA 12-O-tetradecanoylphorbol-13-acetate, t ^ Time to peak irritancy. TRE TPA responsive element UV Ultraviolet W-7 N-(6-aminohexyl)-5-chloro-1 -naphthalenesulphonamide. Chapter 1: Introduction. 1.1. The plant families Euphorbiaceae and Thymelaeaceae.
The plant families Euphorbiaceae and Thymelaeaceae have provided diterpene
compounds which, due to their potent and toxic effects, have been critical in
furthering our understanding of the states of cancer and inflammation. There has been
some disagreement on the botanical classification of these plant families (reviewed by
Webster, 1987); the family Thymelaeaceae being considered closer to the Malvales
order than the Euphorbiales order. These two families are, clearly, closely related and
they are the only plant families to exhibit these toxic diterpene compounds.
1.1.1. The plant family Euphorbiaceae.
The Euphorbiaceae, or Spurge family, consists of approximately 7000 species within approximately 300 genera, and is the fourth largest plant family (Punt, 1987).
The family Euphorbiaceae is widely distributed throughout the world (although is predominantly tropical) and species exhibit widely differing morphological characteristics, from large desert succulents to trees and herbaceous plants. The family was divided into four sub-families by Pax and Hoffman (1931), based on morphological differences: Phyllanthoideae; Crotonoideae; Peranteroideae;
Ricinocarpoideae. More recently, Webster (1975) has proposed five sub-families:
Phyllanthoideae; Oldfieldoideae; Acalyphoideae; Crotonoideae; Euphorboideae. A screening of 235 extracts, representing 75 genera (Beutler et al , 1989) has recently confirmed that biologically active diterpene esters are restricted to the Crotonoideae sub-family (Crotonoideae and Euphorboideae sub-families of Webster’s classification).
The Crotonoideae sub-family contains the largest genera of the Euphorbiaceae, the
Euphorbia (1600 species) and Croton (700 species); other genera producing diterpene toxins include Jatropha (150 species), Macaranga (240 species), as well as
Pedilanthus, Stillingia, Sapium and Elaeophorbia (Airy-Shaw, 1975).
1.1.2. The plant family Thymelaeaceae.
The Thymelaeaceae, or daphnane family, comprises of approximately 500
species within approximately 55 genera. These plants are mainly found in tropical and
temperate climates, and are usually erect shrubs (Nevling, 1962). There are four sub families within the Thymelaeaceae: Gonystyloideae; Aquilarioideae; Gilgiodaphoideae;
Thymelaeoideae (Domke, 1934). Genera which contain toxic diterpenes include
Daphne , Gnidia and Pimelea , and are present within the Thymelaeoideae sub-family.
1.1.3. Commercial uses of plants from the families Euphorbiaceae and Thymelaeaceae.
There are numerous and wide-ranging uses of plants from the families
Euphorbiaceae and Thymelaeaceae (reviewed by Schultes, 1987), including medicinal uses for treating a wide range of conditions. Croton repens is used to treat dysentery and stomach-ache (Central America); Croton lechleri (Dragon’s blood) is used to aid wound healing (Vaisberg et al, 1989); some Euphorbia species have been used as purgatives, and Jatropha gossypiifolia has been used to treat leprosy, venereal disease, ulcers and cancer (Schultes, 1987). Some of these medicinal uses can be attributed to the action of diterpene esters , although in these cases the possibility that their action is simply a generally toxic one cannot be excluded. For example, mezerein, isolated from Daphne mezereum, has been shown to be anti-leukaemic against P-388 lymphocytic leukaemia in mice (Kupchan and Baxter, 1974), yet is well documented as a second stage tumour promoter (Slaga et al , 1980; see section 1.4.1). Toxic diterpenes are also the anti-leukaemic constituents of Croton tiglium.L., Euphorbia esula.L. (Kupchan et al, 1976a) and Gnidia species (Kupchan et al, 1975,1976b). The uses of plants of the Euphorbiaceae and Thymelaeaceae as medicinal agents and ordeal poisons varies (Rizk, 1987) and originates from folk-lore and historical use by a particular people.
Euphorbiaceous plants also have uses of considerable importance and ecomonic impact in modem society. There are several sources of oils: Aleurites fordii.Hcmsl.
(tung oil); Amoluccana.WiM. (candlenut oil); Croton tiglium.L. (croton oil); and of great importance and wide use, Ricinus communis.L. (castor bean oil). Oils from other
Euphorbiaceous plants may also be of use as fuel oils (Calvin, 1987).
Manihot esculanta , better known as cassava, is one of the twelve or thirteen plants that feed the human race, particularly in tropical regions (the others include: rice, wheat, maize, soybean, groundnut, sugar cane and potato). Cassava does, however, contain cyanogenic glycosides and is potentially toxic (Cook, 1982).
Hevea brasiliensis is one of the most economically important discoveries of the twentieth century; this single species provides 98% of the world’s needs of natural rubber, providing 4 million tonnes of rubber from 7.5 million hectares per year
(Archer and Audley, 1987).
1.1.4. Non-diterpenoid constituents of the Euphorbiaceae and Thymelaeaceae.
A large number of non-diterpenoid constituents have been shown to be present in many of the Euphorbiaceae and Thymelaeaceae; unlike the diterpenes, their presence is not restricted to certain sub-families. Since many are biologically active compounds they undoubtably contribute to the physiological effect of these plants when used medicinally. Rizk (1987) and Rizk and El-Missiry (1986) have reviewed the non-diterpenoid constituents of these plant families, which include: -Triterpenes and sterols.
-Flavonoids (mainly flavanone, flavonol and flavone types, and some anthocyanins and one chalcone).
-Coumarins (mainly bicoumarins).
-Lignans.
-Tan riit\s
-Phenanthrenes and quinones.
-Other phenolics (including phenolic acids).
-Alkaloids. Classes include: isoquinoline (aporphines, proaporphines, dihydroproaporphines); morphinandienones; quinolines; pyrrolidines; pyrimidine and guanidines; purines; quinolizidines; pyridines; piperidines; indoles; imidazoles.
-Cyanogenic glycosides.
1.2. Macrocyclic diterpenes of the Euphorbiaceae.
There are seven classes of macrocyclic diterpenes that have been isolated from the Euphorbiaceae (see Fig.l.) (reviewed by Evans, 1986a), and in general they are neither pro-inflammatory nor tumour promoting (Adolf and Hecker, 1971), unlike the phorbol esters (see sections 1.3 and 1.4). Macrocyclics have, however, been shown to exhibit analgesic (Uemura and Hirata, 1975) and anti-ulcer (Kitazawa and Ogiso,
1981) activities, as well as in vivo anti-tumour (Kupchan et al , 1976c; Torrance et al ,
1976; Sahai et al , 1981) and in vitro cytotoxic (Abo and Evans, 1981; Schroeder et al , 1979) actions.
1.2.1. Lathvrane diterpenes. 20 20
16
17
Lathyrane Jatrophane
20 16 16 19
Jatropholane Crotofolane
12
19
20 20 Casbane Rhamnafolane
Myrsinol
Figure 1 Structures of macrocyclic diterpene hydrocarbon skeletons The lathyranes are the largest group of macrocyclic diterpenes, and 6,17- epoxylathyrol (see Fig.2.) was the first isolated, from the seed oil of Euphorbia lathyris (Zechmeister et al, 1970). 6,17-epoxylathyrol is a typical lathyrol derivative, with the cyclopropane ring cis fused to G, and Cu, and the cyclopentane and cycloundecane rings in the trans configuration.
The jollrinols (e.g. jolkinol A, see Fig.2.) are closely related lathyrane diterpenes, isolated from Euphorbia jolkini (Uemura et al , 1976). Other lathyrane derivatives include bertyadionols (e.g. bertyadionol, see Fig.2.) (Ghisalberti et al,
1973) from Bertya cupressoidea and the ingol diterpenes (e.g. ingol, see Fig.2.)
(Opferkuch and Hecker, 1973), isolated from Euphorbia ingens.
In common with the lathyrols, the jolkinols, bertyadionols, and ingols have a cyclopropane C ring cis fused to the cycloundecane B ring which is, in turn, trans fused with the cyclopentane A ring.
1.2.2. Jatrophane diterpenes.
Jatrophane macrocyclics are of a similar structure to the lathyrane diterpenes but are characterised by the opening of the cyclopropane ring, resulting in a bicyclic molecule. Jatrophane (see Fig.3.) was isolated from the roots of Jatropha gossypiifolia as a potent in vivo anti-leukaemic agent against P-388 leukaemia in mice, and it was proposed that its mechanism of action was by selective alkylation of growth regulatory macromolecules (Kupchan et al , 1976c).
Kansuinines A and B (see Fig.3.) were isolated from Euphorbia kansuiliou. by Uemura and Hirata (1975), and were the first jatrophane diterpenes to be shown to possess analgesic and anti-writhing properties in mice.
The jatrophane diterpenes are typically poly-oxygenated, often with epoxide HO
'"H
6,17 epoxylathyrol Jolkinol A Lathyrol derivative Jolkinol derivative
20
i OH OH OH
Bertyadionol Ingol Beryadionol derivative Ingol derivative
Figure 2 Classes of lathyrane macrocyclic diterpenes Jatrophone
J?-0\ #
io OAc
R=3xAcetate Kansuinine A Kansuinine lxbenzoate
Jatrophane diterpenes
OH 20 16 o
OH
Jatropholone A Crotofolin A
Jatropholane diterpene Crotofolane diterpene
Figure 3 Jatrophane, Jatropholane and Crotofolane macrocyclic diterpenes and/or ketone functions centred round C12, C13, C14, or C 15 atoms.
1.2.3. Jatropholane and crotofolane diterpenes.
The jatropholane and crotofolane diteipenes are related compounds and differ only in that the jatropholane cyclopropane ring has been opened to form an isopropylidene side chain in the crotofolane type. Jatropholanes (e.g. jatropholane A, see Fig.3.) were isolated from the roots of Jatropha gossypiifolia (Purushothaman and
Charasekharan, 1979) and crotofolanes (e.g. crotofolin A, see Fig.3.) were isolated from Croton corylifolius (Chan et al, 1975).
1.2.4. Rhamnofolane and mvrsinol diterpenes.
A rhamnofolane diterpene was isolated from Croton rhamnifolius and identified by its synthetic acetate as 13,15-seco-4,12-dideoxy-4a-phorbol-20-acetate (see Fig.4.)
(Stuart and Barrett, 1969). More recently (Rentzea et al, 1982), the tetracyclic myrsinol diteipenes were discovered in Euphorbia mysinites.L. The myrsinol diterpenes (see Fig.4) are non-irritant although pro-inflammatory ingenane diterpenes
(see section 1.3.3) are also present in this plant
1.2.5. Casbane diterpenes.
The casbane diterpenes are bicyclic and have been proposed as biosynthetic precursors to the tigliane, ingenane and daphnane diterpenes (Schmidt, 1986, 1987).
Soluble enzyme preparations of Ricinus communis converted mavelonic acid to the macrocyclic casbane (see Fig.4) (Robinson and West 1970).
Crotonitenone (see Fig.4.) was the first oxygenated casbane macrocyclic to be isolated from Croton nitens (Burke et al, 1979), in attempts to produce precursors of CHoOAc 20
13,15-seco-4 #12-dideoxy-4 c* -phorbol-20-acetate
Rhamnofolane Diterpene
20
r 1 #R^ typically simple aliphatic esters
C 2 -C5 length
R3 simple aliphatic ester or COC 5 H 4 N
Myrsinol diterpenes
20
Casbene Crotonitenone
Casbane diterpenes
Figure 4 Rhamnofolane, Myrsinol and Casbane macrocyclic diterpenes the crotofolane diteipenes.
The casbane macrocyclics seem to be implicated in the biosynthesis of other
diteipenes and it has been suggested that they are derived from i) a head-to-tail
condensation or ii) a "concertina" cyclisation of geranyl-geranyl pyrophosphate
(derived from the acetate-mevalonate pathway) (Schmidt, 1986,1987). However, the
biosynthetic pathways to diterpenes have not been greatly studied and therefore these
hypotheses remain unconfirmed.
1.3. Tigliane, Ingenane and Daphnane diterpenes.
Esters based upon the tigliane, ingenane and daphnane diterpene nuclei (see
Fig.5) possess potent and toxic biological properties (reviewed by Hecker and
Schmidt, 1974; Evans and Soper, 1978; Evans and Taylor, 1983). Tigliane derivatives were shown to be responsible for the tumour promoting effects of the seed oil of
Croton tiglium.L. (Hecker, 1968; see section 1.4.1.). The subsequent use of these diterpenes to study the mechanisms of disease states has resulted in some confusion of nomenclature; these three distinct classes of diterpene are now often termed the
"phorbol esters", although the term "phorbol" strictly refers only to tigliane derivatives.
1.3.1. Tigliane diterpenes.
Tigliane diteipenes are tetracyclic, consisting of a five-membered A ring, a seven-membered B ring, a six membered C ring and a cyclopropane D ring. The parent alcohol, phorbol, exhibits an a,p-unsaturated ketone at Q , tertiary hydroxyl groups at C4, C^and C13, a secondary hydroxyl group at C 12 and a primary hydroxyl at Qo (see Fig. 6 a.). The A/B and B/C ring junctions are both in the trans configuration whereas the C/D rings are cis fused. The isomer of phorbol characterised J \J
12
20
Tigliane
Ingenane
20
Daphnane
Figure 5 Tigliane, Ingenane and Daphnane hydrocarbon skeletons by cis fusion of the A/B rings and the C 4 hydroxyl being a-orientated (termed 4a- phorbol) has a different shape to phorbol (see Fig. 6 b.). This alteration of A/B ring stereochemistry has been proposed as the reason for esters of 4a-phorbol possessing no biological activity (Hecker, 1968; Hecker and Schmidt, 1974).
Phorbol esters occur naturally as 12,13-diesters. The ester functions can be either aliphatic or aromatic and have been classified as an "A" or "B" series (Hecker and Schmidt, 1974). The "A” series has a high molecular weight acyl residue at C 12 and a low molecular weight acyl residue at C13; in the "B" series, the high molecular weight residue is at C 13 and the low molecular weight residue at C12. C12, C 13 and tri-esters, termed "C" series or "cryptic esters", are also found naturally and are so called as removal of the acyl group is required before these esters can induce a pro-inflammatory response in the test system of Hecker and Schmidt (1974).
Various chemical reactions of both functional groups and the phorbol nucleus can be performed (reviewed by Evans, 1986b) and were important for the structure determination of phorbol (Crombie et al, 1968). Some of the most significant include those altering the ester functions of C12, C 13 or C^, which can be easily acetylated using acetic anhydride at room temperature (Boehm et al, 1935). Further acetylation of the G, and C 4 hydroxyls is possible, forming the penta-acetate, in acetic anhyride/p- toluene sulphonic acid (Boehm et al, 1935). Selective alkylation of the C4-hydroxy group (Kreibich and Hecker, 1968) has enabled the production of 4-O-methyl derivatives which have been of use in understanding the tumour promotion process
(see section 1.4.1.). Replacement of long chain esters at C 12 and C 13 to form the dibutyrate or dipropionate, followed by tritiation at C^, has enabled the study of binding of phorbol esters to their receptor sites (Dreidger and Blumberg, 1980;
Schmidt et al, 1983; see section 1.5.3.). A typical "A" series phorbol diester, which h 3c
CH,OH 20 L
4 /? - phorbol (biologically active)
CH3
4 oC -phorbol (biologically inactive)
Figure 6 4j3 and 4oc - phorbol Table 1. Phorbol-related diterpene nuclei with different oxygenation states. CO ON ON r- *o (A ON OO CO ON a* ON 00 T 3 C g OS £ pC/3 "O B p g CL. p o h O h X) O % P o P © wQ O n vo i n # 03 O n o r^» ON «G ON o o r - t" - P ONON CO T3 M < s M* £ c3 00 oo G c r - r - ON S3 3 I ON m c 2 > > E ON r** WW o ON J 3 ■a 00 • a 00 CO r- s ON 103 CO • o e E H—* H-* *> N o o T 3 s P JS .G V) £ T3 00 o o G «n »G P I £ G J 3 j 0 0 JS P P o U 2 CO CO 1 § o £ a £ 5 ,«<1 2 CLh c § s& g & £ 3 i a I £ s Oh p S - i <*3 8 o § A ? a £ *cs • 2 .£ .Q .§• •Si a Q 3 £ £ •£> • £ a , o 6 3 o , s .5 5 t*a u) i*a t*a t*3 hi t*5 Oh O • e O JS f t X 0 p T 3 1 VO cs CO M M 00 u< T 3 ON Ox00 ON 00 r^ •a ON ON M ON 00 ON *9 no oo ON CO §■% S te l - l .-0 COCH3 c h 2o h 1 2 -0 -tetradecanoyl 1 2 -0 -[2 -methylaminobenzoyl] phorbol-13-acetate -4-deoxyphorbol-13-acetate (TPA) (Sapintoxin A) \ 1 A -series phorbol diester 4-deoxyphorbol ester H H ' O C O C H j© 0 H O C H ,0 C 0 C H3 12-deoxyphorbol-13- 12-deoxyphorbol-13- phenylacetate phenylacetate- 2 0 -acetate (DOPP) (DOPPA) Figure 7 Some biologically important esters with different phorbol nuclei. was the original tumour promoting ester isolated from croton oil (Hecker, 1968), is 12-O-tetradecanoylphorbol-13-acetate, or TP A (also known as phorbol-12-myristate- 13-acetate, PMA) (see Fig.7.). TPA is a generally toxic substance and is the usual positive control phorbol ester used in biological tests, since it possesses a wide range of actions (see section 1.4.). The phorbol nucleus also exists naturally in different states of oxygenation. These states are listed in Table 1 and some compounds based upon these nuclei which have been of biological interest are shown in Fig.7. The different nuclei and their esters are identified by differences in their spectroscopic characteristics (e.g. MS; *H- NMR; 13C-NMR; UV; IR; CD). 1.3.2. Ingenane diterpenes. Ingenane diterpenes, like the tiglianes, are tetracyclic. The parent alcohol, ingenol (see Fig. 8 .), exhibits a five-membered A ring and seven-membered B ring in the trans configuration; the C ring is also seven-membered and is cis fused to the cyclopropane D ring. The C 1 2 and double bonds, the C 4 -p-tertiary hydroxyl group and the primary hydroxyl group are also present Ingenol exhibits secondary hydroxyl groups at C 3 and C5, and a p,y-unsaturated ketone at Cg. Naturally occurring aliphatic or aromatic esters of ingenol are linked to the nucleus via the C 3 and C 5 hydroxyls (as opposed to C 12 and C 13 hydroxyls for phorbol) as well as via the C 20 hydroxyl. Isolation of ingenane diterpenes is technically difficult and therefore screening of the genus Euphorbia for the presence of ingenanes has relied upon the preparation and subsequent detection of ingenol-3,5,20-triacetate by semi-synthetic methods (Evans and Kinghom, 1977; Upadhyay et al, 1980). This has led to the suggestion RO HO OH CI-UOH 2 0 Ingenol R=H 3-0-tetradecanoylingenol (3TI) R=CO(CH2 )i2CH3 Ingenol derivatives (includes configuration) HO OH OH CH3 5-deoxyingenol 2 0 -deoxyingenol HO —CH-OH HO OH HO OH OH CH3 C^OH 16-hydroxy-20-deoxyingenol 13-hydroxyingenol — c h 2 o h H O OH OH CH2 0H OH C ^ O H 16-hydroxyingenol 13,19-dihydroxyingenol Figure 8 Ingenane diterpenes (Evans, 1986c) that the ingenanes are probably the largest group of pro-inflammatory compounds found within the Euphorbiaceae and, interestingly, have not been reported outside the genus Euphorbia. In common with many tigliane derivatives, some ingenanes have been reported to be tumour promoting (e.g. Hergenhahn et al, 1974), although ingenol-3,20-dibenzoate has been reported to be anti-leukaemic (Kupchan et al, 1976a) and ingenol and 20-deoxyingenol esters from E.paralias latex are cytotoxic in vitro (Sayed et al, 1980). Apart from these activities, the biological effects of the ingenanes do not seem to have been extensively studied and they are, in general, considered to be of similar activity to the tiglianes; 3-O-tetradecanoylingenol (see Fig.8 ) is a full promoter and appears to have similar properties to TPA. Molecular modelling of ingenanes (Wender et al, 1986; see chapter 4) and recent studies on the binding of ingenol to protein kinase C (Hasler et al, 1992) support this supposition that ingenanes are biologically similar to tiglianes. Identification of ingenanes is heavily reliant upon MS and !H-NMR (Evans, 1986c). The ingenol nucleus occurs naturally in various states of oxygenation, including: 5-deoxyingenol (Evans and Kinghom, 1977); 20-deoxyingenol (Uemura et al, 1974a); 16-hydroxy-20-deoxyingenol (Lin etal, 1983); 13-hydroxyingenol (Uemura et al, 1974b); 16-hydroxyingenol (Opferkuch and Hecker, 1974); 13,19- dihydroxyingenol (Ott and Hecker, 1981) (see Fig. 8 .). All additional hydroxy functions can be esterified. 1.3.3. Daphnane diterpenes. The daphnane diteipenes are found in both the plant families Euphorbiaceae and Thymelaeaceae but their distribution is limited; esters based upon the 5- deoxydaphnetoxin and la-alkyldaphnane nuclei are present only in the Thymelaeaceae; resiniferonol esters are only present in the Euphorbiaceae, whilst daphnetoxin and 1 2 -hydroxydaphnetoxin esters are present in both plant families. This may provide some chemosystematic evidence for a relationship between these families (Schmidt, 1986b; see Fig.9.). The daphnane ring structure is identical to that of the tiglianes except that the cyclopropane D ring has been opened to form an isopropenyl side chain (see Fig.9.). The daphnanes also exhibit an ortho ester function bridging the a-orientated hydroxyl groups at Q, C 13 and C 14 of the parent alcohol, resiniferonol (see Fig.9.). Only one compound, pro-resiniferatoxin (Schmidt, 1977), is an exception to this and it is thought to be the immediate biosynthetic precursor to resiniferatoxin. The first daphnane diterpene was isolated in 1970 from a mixture of barks of Daphne mezereum, D.laureola and D.gnidium (Thymelaeaceae) (Stout et al, 1970) and was termed "daphnetoxin" (see Fig. 10.). Other esters occur naturally based upon this 5p-hydroxyresiniferonol-6a-7a-oxide (or daphnetoxin) nucleus and the benzoate ortho- ester can be replaced by a long chain (> 8 C) acyl residue, e.g. Simplexin (see Fig. 10.), a complete tumour promoter isolated from Pimelea simplex (Hafez et al , 1983). 5p,12p-dihydroxyresiniferonol-6a,7a-oxide ("12-hydroxydaphnetoxin") esters have been of use in understanding the process of tumour promotion (see section 1.4.1). Mezerein (see Fig. 10.), the first compound of this type, was isolated from Daphne mezereum (Ronlan and Wickberg, 1970) and shown to possess anti-leukaemic properties against P-388 leukaemia in mice (Kupchan and Baxter, 1974), although it is now recognised as a second stage tumour promoter (Slaga et al , 1980). X-ray crystallography enabled the absolute structure of mezerein to be determined (Nyborg and la Cour, 1975), confirming esterification at C12, which generates diagnostic signals in the !H-NMR spectra. For mezerein, the C 12 proton integrates for a 1H singlet at 85.12; in the daphnetoxin spectra, the signal is a 2H multiplet at 82.36. This illustrates 1 HO CH2 0R Absolute configuration Daphnetoxin type of Resiniferonol R OH CHoOR CHjOR 12-Hydroxydaphnetoxin type Resiniferonol type r o h o 0 HO c h 2 o h HO CH2 0R 5-Deoxydaphnetoxin type loc- alkyldaphnane type 5-deoxysimplexin R =-(CH2)8CH3 Figure 9 Daphnane diterpene types , and absolute configuration of Resiniferonal R = Ester function, (at C2Q#r is usually H) one difference in these nuclei and *H-NMR is the spectroscopic method of choice for analysis of daphnane diterpenes (Schmidt, 1986). Other anti-leukaemic principles, Gnidicin, Gniditrin and Gnididin, have been isolated from Gnidia lamprantha (Kupchan et al, 1975) and, more recently, gnidicin has been isolated from Thymelaea hirsuta (Rizk et a/,1984) along with a series of other compounds, termed Thymeleatoxins. Thymeleatoxin A (Gnidicin) has been shown, like mezerein, to be a second stage tumour promoter (Brooks et al, 1989) and this further suggests that the anti-cancer and co-carcinogenic actions of these diterpene esters may be difficult to distinguish. The resiniferonol daphnane esters (see Fig.9.) most closely resemble those of phorbol in that they retain the Q -Q double bond. The first to be characterised was resiniferatoxin, which was isolated from Euphorbia resinifera (Hergenhahn et al, 1975). This compound has a phenolic side chain, which was concluded to be (4- hydroxy-3-methoxy)-phenylacetic acid on the basis of *H-NMR spectra (Adolf et al , 1982) (see Fig. 10.) Resiniferatoxin is the most potent diterpene ester in its ability to induce erythema of the mouse ear (Schmidt and Evans, 1979) and the mechanism(s) by which resiniferatoxin exerts this effect has recently been of interest (see section 1.4.2. and chapter 5). One ester, 5-deoxysimplexin, has been isolated from the roots of Daphnopsis racemosa (Adolf and Hecker, 1982) and is based upon the resiniferonol-6a,7a-oxide (5-deoxydaphnetoxin) nucleus (see Fig.9.). la-alkyldaphnane esters (see Fig.9.) are found only in the plant family Thymelaeaceae. These esters possess a long chain aliphatic orthoester joined to Q of the A ring producing a fourth, macrocyclic, ring. The Q-Cj double bond is saturated, la-alkyldaphnane esters occur as: daphnetoxin esters (Hafez et al, 1983); macrinol Daphnetoxin R-CgH^ Simplexin R*(CH2)3CH3 Daphnetoxin type 0 HO HO c h 2 o h Gnidicin c o .c h =c h c 6h 5 (also termed Thymeleatoxin A) Gniditrin CO(CH=CH)3(CH2)2CH3 Gnididin CO(CH=CH)2(CH2)4CH3 Mezerein CO(CH=CH)2C6H5 12-Hydroxydaphnetoxin type HO 'o c o .c h 2 c6h 5 c h 2 o r CH20C0CH2-@ -0 H Resiniferonol 9,13, 14 orthophenylacetate R - H Proresiniferatoxin Resiniferatoxin o c h 3 R = CO •ch 2 - @ - 0 H Resiniferonol type Figure 10 Daphnane diterpene esters Pimelea factor Sg: R-(C-l)- C H (CH 3 )-(CH 2 )7 - Daphnetoxin type R^O HO >— < - 0 OH C ^O H Gnidimacrin R 1 *(C—1)-CH(CH 3 )(CH 2 )6 - CH (O H ) - Macrinol ester Linifolin A R=(C-l)-CH(CH3 )(CH 2 )7 - 12-Hydroxy-18 - deoxymacrinol ester R OH CH2 0H Linifolin B R=(C-l)-CH(CH3 )(CH 2 )7 18-deoxymacrinol ester Figure 1 1 lo< -alkyldaphnane type esters Daphnopsis factor R 6 R=(C-1)CH(CH3 ) (CH 2 )7 esters (Kupchan et al, 1976); 12-hydroxy- 18-deoxymacrinol esters (Tyler and Howden, 1981); 18-deoxymacrinol esters (Zayed et al, 1977b) and 5,18-dideoxymacrinol esters (Adolf and Hecker, 1982). Examples of these esters are shown in Fig. 11. The biological actions of la-alkyldaphnane esters has not been extensively investigated although they possess piscicidal (Tyler and Howden, 1981), anti-leukaemic (Kupchan et al, 1976) and pro-inflammatory (Zayed et al, 1977b) activities. 1.4. Biological effects of the phorbol esters. The effects of phorbol esters on the normal physiological function of mammalian systems are numerous and wide-ranging. Ultimately, phorbol esters can induce gross morphological changes in mammals but the discrete effects on biochemical mechanisms which presumably lead to this are still poorly understood. TPA is the most common phorbol ester used to study biological function but this agent is perhaps too toxic and, therefore, phorbol esters with different and selective actions may be the ones to provide critical breakthroughs in our understanding of systems studied. In discussing the biological effects of the phorbol esters, I shall attempt to emphasize the contribution that this class of compounds has made, and not just TPA. 1.4.1. Tumour promotion. In 1941, Berenblum first devised experiments, the protocols for which are essentially the same today, to study the mechanism of carcinogenesis. Polyaromatic hydrocarbons (PAHs), for example dimethylbenz[a]anthracene (DMBA) and benz[a]pyrene, were recognised carcinogens. That is, if the appropriate single dose was applied to the dorsal skin of an animal, tumours were observed (see Fig. 12a.). If a subthreshold dose was applied, no tumours developed. Application of a subthreshold dose of carcinogen, followed by weekly applications of Croton oil (the seed oil of Croton tiglium) resulted in the development of tumours (Berenblum, 1941). Berenblum later showed (Berenblum and Shubik, 1947) that application of the croton oil only produced tumours if it was applied after the carcinogen; application of croton oil followed by carcinogen produced no tumours. Furthermore, the administration of croton oil could be delayed for upto 43 weeks following carcinogen application and still promote tumour formation (Berenblum and Shubik, 1949). This and other work (Freidewald and Rous, 1944) resulted in the concept of a two stage mechanism for carcinogenesis: an "initiation" stage, and a "promotion" stage. Initiators and promoters have distinct biological properties (see Table 2.) but, essentially, the initiator must be carcinogenic, must be exposed to the host before the promoter and its action is irreversible. However, urethane and some of its derivatives are initiators but are not themselves carcinogenic (Salaman and Roe, 1953) and this has led to them being termed "incomplete carcinogens". These properties imply that the initiator acts via a different mechanism to the promoter; initiators are now thought to be metabolically activated, forming electrophiles that can covalently bind to cellular macromolecules (Weinstein et al, 1978; Kinsella, 1986). This is thought to explain the correlation of initiation with DNA alkylation (Bowden and Boutwell, 1974) and gene depression (Slaga et al, 1973). Interestingly, tumour promoters possess properties of reversible gene depressors (Hennings and Boutwell, 1970). In 1968, Hecker isolated the most potent tumour promoting substance from croton oil, 12-O-tetradecanoylphorbol-13-acetate (TPA). The isolation of TPA and other phorbol related diterpenes from natural sources (see section 1.3) enabled further studies on the mechanisms of carcinogenesis, in particular some of the cellular and Table 2 . Comparison of the biological properties of initiating and promoting agents. (Weinstein et al, 1978; Kinsella, 1986) Initiating agents. Promoting agents. Carcinogenic themselves Not carcinogenic alone Must be given before promoting agent Must be given after initiator Single exposure sufficient Repeated and prolonged exposure required Action is irreversible Action reversible upto a certain stage No apparent threshold Probable threshold Yield electrophiles that covalently No evidence of covalent binding bind to cellular macromolecules Do not alter gene expression at low Alter gene expression; induce initiating doses; no altered cellular hyperplasia; cause enzyme induction; morphology detectable induce viral genes; cause gene amplification Mutagenic themselves Not mutagenic DNA binding and resultant repair are Plethora of biological and biochemical detectable responses responses morphological changes that occur on treatment with TPA. These include: a sustained stimulation of RNA, DNA and protein synthesis in mouse epidermis (Baird et al, 1971; Raick, 1973); increased phospholipid synthesis (Rohrschneider et al, 1972; Balmain and Hecker, 1974); increased prostaglandin synthesis (Bresnik et al, 1979); elevated ornithine decarboxylase (ODC) activity (O’Brien et al, 1975; Yuspa et al , 1976) and induction of dark basal keratinocytes (Raick, 1973). A large number of dark cells are present in skin papillomas and carcinomas (Raick, 1974) and the ability to induce dark basal cells, together with ODC activity, were thought to be the best correlations with promoting ability (Raick, 1974; O’Brien et al, 1975; Baxter et al, 1989). The observation by Slaga’s group (Klein-Szanto et al, 1980) that mezerein was less able to induce dark cells than TPA implied a diversity of tumour promoting abilities within the phorbol ester series. At this time, an optimum dosage regimen for tumour promoting experiments had been proposed (Verma and Boutwell, 1980) as a twice weekly application of lOnM TPA, for 18 weeks following initiation. It was later confirmed that decreasing frequency of application of TPA caused a decrease in tumour yield (Chouroulinkov et al, 1989). Slaga then demonstrated (Slaga et al, 1980), by using different phorbol esters, that skin tumour promotion comprised of two stages (see Fig. 12b). Mezerein, itself only a weak (non) promoter, was shown to strongly promote tumour production using an 18 week protocol if TPA was used as a promoter for two weeks after initiation (the TPA doses were themselves not sufficient to induce tumour production). Mezerein was, therefore, termed a "second stage" tumour promoter. The C 4 hydroxy- methylated derivative of TPA, 4-O-methyl-TPA (40MeTPA), was shown to be a "first stage" promoter not capable on its own of tumour promotion, but capable of inducing the same effects as TPA to subsequently promote tumour production, if a second stage o 3 ’p o + + + M a + + i i + i < Pn M + + + H p o E Oi p H 0 u 0 -M >1 <3 <] B < < O < < < < A *H •rH U O 0 M u .c 0 0 H < < < < <3 O u 0 4 3 < 3 < o o o •H Oi O CD Eh •H P 1 P d) d) 0 < 1 o o o N 2 E d) O O 2 i P Oi < o o o < • & o i • P Eh • Ol 3 d) • O < o o o • d) E 01 p • 0) p - 2 i •rH < rH rH o o o 0 X £! 0) S s (0 M CO CD < < 3 < CD * o 0) CD u c phorbol ester tumour promotion was a two stage process, by semi-synthetic manufacture of 12-O-retinoylphorbol- 13-acetate (RPA), a compound which was also a potent second stage promoter in NMRI mice. RPA was later shown to be a complete promoter in Senear mice (Fischer et al, 1985). This is of interest as retinoids (Vitamin A analogues) had previously been shown to inhibit TPA induced tumour promotion (Verma et al, 1978; Weeks et al, 1979) as had leupeptin (a protease inhibitor) (Hozumi et al, 1972) and fluocinolone acetonide (a steroidal anti-inflammatory agent) (Weeks et al, 1979). Mouse strain-specific susceptibility to tumour promotion raises the question of the significance of this biological phenomenon to carcinogenesis in man. In the mouse, the Senear (Sensitive to carcinogenesis) strain is the most susceptible to carcinogenesis (DiGiovanni et al, 1980) and was bred for this purpose. CD-I and NMRI mice are also susceptible to carcinogenesis whilst B6C3F1 mice are resistant Shoyab et al (1982) proposed that a phorbol-12,13-diester-12-ester-hydrolase enzyme may be responsible for prevention of phorbol ester induced tumour promotion in skin. This enzyme converts 12,13-diesters to 13-monoesters and was shown to be present in rat, guinea pig, hamster and rabbit but not in mouse, cat, dog, monkey or human. They proposed that species possessing this enzyme were resistant to tumour promotion although no in vivo experiments demonstrated this hypothesis, and by implication of the species distribution of the enzyme, humans were susceptible. Hecker has provided evidence that phorbol esters could be risk factors of cancer in man (reviewed by Hecker, 1987). The Caribbean island of Curasao has a high incidence of oesophageal cancer. The population’s drinking water was found to have been contaminated with petrol from 1930-1964 and this may have caused an initiating dose by a PAH carcinogen. Furthermore, the population regularly take a tea made from the plant Croton flavens (termed "Welensali tea") and chew the roots of this bush. Hecker isolated the active constituents of this plant (Hecker et al, 1983), which were tumour promoting esters of 16-hydroxy- and 4-deoxy-16-hydroxy-phorbol. The use of this bush in local custom suggested that the promoting stage of the disease could be attributed to the bush (Hecker, 1983, 1984). This hypothesis was circumstantially supported by the fact that on neighbouring islands, which did not use the bush in local custom, the incidence of oesophageal cancer was lower. Recently, the use of another phorbol ester probe, Sapintoxin A (Sap A), by Evans’s group has enabled further understanding of the tumour promotion mechanism. This fluorescent phorbol ester was shown to activate protein kinase C but to be neither a complete nor second stage promoter in its own right (Brooks et al, 1987b). However, if co-administered with sub-hyperplasiogenic doses of the calcium ionophore A23187, Sap A was found to be a complete tumour promoter, inducing tumours in a dose-dependent manner (Brooks et al, 1989). This points to an involvement of Ca2* in the tumour promotion process and the tumour promoting sesquiterpene lactone, Thapsigargin, has been shown to act by discharging release of Ca2* from stores in the endoplasmic reticulum by specific inhibition of the endoplasmic reticulum Ca2+- ATPase (Thalstrup et al, 1990). The involvement of other biochemical systems (e.g. Ca2+ levels) in the tumour promotion process reflects the complex nature of this phenomenon and may help explain why different classes of compounds act as tumour promoters. Structure/activity relationships for phorbol ester induced tumour promotion beyond the basic requirements of: i) A/B rings in the trans configuration, ii) esterification at C 12 and C 13 (C3 for ingenols), of which at least one must be of high molecular weight, iii) C 20 primary hydroxyl group (although "cryptic 11 esters could be metabolically activated), are ill defined. C 12 deoxygenation leads to production of weak (non) promoters. Unsaturation of the acyl ester side chain is thought to impart second stage promoting ability (Sharkey et al, 1989). Methylation of C4-OH imparts first stage promoting ability (but consider Sap A, a C4-deoxyphorbol ester, in combination with A23187). Furthermore, are there inherent requirements of the nucleus structure for tumour promotion? For the phorbol ester series, other biological effects have been studied in an attempt to find a rapid assay that correlates for tumour promotion. Unfortunately, many non-correlations exist and mechanisms of tumour promotion, the grossest toxicological effect induced by phorbol esters, may remain poorly understood until the biochemical basis of this effect are better characterised. 1.4.2. Pro-inflammatory response. Although tumour promotion induced by phorbol esters implicates these compounds as potential co-carcinogenic agents, this biological effect is not valuable for the rapid analysis of a plant (or commercially available plant based material) since it is a chronic effect. Furthermore, since not all phorbol esters are tumour promoting, compounds with other biological effects could.be missed if tumour promotion was used to screen for activity. The acute activity of phorbol esters which has been utilised to screen for their presence is their ability to induce an inflammatory response. Phorbol ester induced inflammation is characterised by classical symptoms (Evans, 1978) of redness, swelling, pain and thickening of tissues. Skin burning by Euphorbia species can result in open and weeping pustules which may lead to dry flaking skin, scab formation and finally tissue necrosis (Schmidt and Evans, 1980). Ingestion results in burning of the lips, tongue and mucous membranes, and may lead to intestinal pain, vomiting and severe purging (Kinghom and Evans, 1975); contact with the eyes leads to conjunctivitis, swelling of the eye lids and eye closure due to oedema (Evans and Kinghom, 1975; Markby et al, 1989) which may, ultimately, result in blindness. Isolation of phorbol esters is therefore typically by bio-assay guided fractionation utilising the pro-inflammatory effect of these compounds on mouse ear. This has lead to the development of the "mouse ear erythema assay" (Hecker et al, 1966; Evans and Schmidt, 1979), an "all-or-nothing" assay, enabling the calculation of the dose of agonist that will induce erythema in 50% of the test animals (Irritant dose, or ID^ value). Hecker measures this as the dose that will induce erythema in 50% of animals 24 hours after application of agonist whereas Evans measures the time to peak irritancy (t^ ), the duration of the inflammatory response and reads the ID^ value at t,**. There is, however, general agreement on the structural requirements for pro-inflammatory activity (Hecker, 1978; Schmidt and Evans, 1979, 1980; Adolf et al , 1982; Evans and Edwards, 1987; Sorg et al, 1987). In common with the activity requirements for tumour promotion, the structure-activity relationships for induction of inflammation are generalised and not absolute; A/B rings in the trans configuration and an acyl residue at C 12/C13 or C 13 are the only requirements (Evans and Edwards, 1987; see Table 3). Features increasing potency include: a primary hydroxyl at C^; unsaturation in the C 12/C13 residue; an aromatic residue at C 12/C13. Pro-inflammatory activity has been used as a guide for tumour promoting activity although this is a non-correlation; whilst all tumour promoting phorbol esters are pro-inflammatory, not all pro-inflammatory phorbol esters are tumour promoting (Evans, 1986b). The sapintoxins and resiniferatoxin are examples of this situation. Sap A is a tumour promoter only in the presence of calcium ionophore (Brooks et al , Table 3. Structure-activity relationships for the induction of inflammation. (Evans and Edwards, 1987) Requirements for activity. A/B rings in the trans configuration. Acyl residue at C12/C13 or at C13. Features increasing potency. Primary hydroxyl at C^. Unsaturation in C 12/C13 acyl residue. Aromatic residue at C 12/C13. Features not necessary for activity. C 12 hydroxyl or acyl group. Free hydroxyl at C^. C4 tertiary hydroxyl. Cyclopropane D ring. Methyl group at C16. 1989) yet it is of similar inflammatory potency to TPA (Taylor et al, 1981a); resiniferatoxin (Rx) is not a tumour promoter (Adolf et al, 1982) yet it is one hundred fold more potent at inducing erythema than TPA (Schmidt and Evans, 1979). The mechanisms of resiniferatoxin induced erythema have recently been extensively studied; Blumberg has proposed that Rx acts as an ultrapotent analogue of Capsaicin, the neurogenic irritant (Szallasi and Blumberg, 1989a, 1989b), whilst Evans maintains that Rx induced inflammation is partially neurogenic but that its most potent action is as a typical phorbol ester (Gordge, Evans and Evans, 1990; Evans et al, 1992; see chapter 5). Gschwendt et al (1984) have proposed that measurement of oedema, as opposed to inflammation (per se), is a more accurate correlation with tumour promoting properties of phorbol esters. However, they have not tested their hypothesis on a range of promoting and non-promoting phorbol esters (for example, Sap A, Rx, 1 2 -deoxyphorbol esters) and this proposition must, therefore, be viewed with some scepticism. They measure oedema induction after six hours, by weight of an ear plug, and since the time taken to induce oedema (and inflammation) by different phorbol esters varies, this could further result in confusion when interpreting results. Essentially, induction of a pro-inflammatory response remains an accurate assay method for determining phorbol ester presence and guiding fractionation but there are too many non-correlations for its use to rapidly assess tumour-promoting activity. 1.4.3. Platelet aggregation. TPA was the first phorbol ester to be shown capable of inducing a dose- dependent aggregation of human platelets (Zucker, Troll and Balman, 1974). It was shown to cause vacuole formation within storage granules and dilation of the open canicular system of platelets (Estensen and White, 1974; White and Estensen, 1974). Studies on the aggregating ability of a range of phorbol esters indicated that daphnane ortho esters were unable to induce aggregation of platelets (Williamson, Westwick and Evans, 1980) yet 12-deoxyphorbol esters could induce platelet aggregation (Westwick, Williamson and Evans, 1980). Furthermore, in order to induce platelet aggregation, phorbol esters were required to possess: i) A/B rings in the trans configuration, ii) a free Qo primary hydroxyl group, and iii) a C 13 ester function. The C 4 tertiary hydroxyl and a C 12 ester function were not requirements for activity (Westwick, Williamson and Evans, 1980; Edwards et al , 1983a). Studies on the mechanism of aggregation induced by 12-deoxyphorbol-13- phenylacetate (DOPP) (Williamson et al , 1981) indicated that it was a true aggregation and not a non-specific agglutination induced by a surfactant mimicking action. EDTA was able to inhibit phorbol ester induced aggregation, again pointing to the importance of Ca2+ presence for phorbol ester induced biological responses. In inducing platelet aggregation, DOPP also stimulated the secretion of a biologically active factor termed a Transferable Aggregating Substance (TAS) into human plasma. This substance was so termed since if a lOOpl aliquot of plasma whose platelets had been aggregated by 0.86pM DOPP was transferred to fresh recipient platelets, then an aggregation was observed (Williamson et al, 1981). The concentration of DOPP transferred (0.14pM) was not itself sufficient to induce platelet aggregation. Although phorbol esters are known to be potent stimulators of aggregation in rabbit platelets (Edwards et al, 1983a), TAS was shown to not be induced in this system. On the basis of trypsin sensitivity and Sephadex G-25 elution, TAS was shown to be a protein (Edwards et al , 1987) and to be pharmacologically distinct from other known platelet aggregators, including phorbol esters. The half-life of TAS in plasma at 37°c is 20 minutes (Edwards et al, 1987) but recent use of a Sepharose 2B column in conjunction with Fast Protein Liquid Chromatography (FPLC) techniques has provided material that can be stored at -20°c for two weeks without loss of activity. This has enabled further characterisation of TAS, confirming it as a protein, with two bands in the 20kDa range when examined by SDS-PAGE electrophoresis (Evans,A.T., et al, 1991a). Biochemical studies using phorbol esters possessing different abilities to induce platelet aggregation have revealed that this activity may correlate with phosphorylation of a 47kDa protein substrate for protein kinase C (Brooks, 1989; Brooks et al, 1990). The aggregating phorbol esters, TPA and DOPP were able to stimulate phosphorylation in the presence and absence of Ca 2+-ionophore. Sap A, a weaker aggregatory agent, stimulated phosphorylation of the 47kDa protein, which was enhanced in the presence of Ca^-ionophore; the calcium dependency of this probe is in agreement with its tumour promoting properties, for which calcium is also required (Brooks et al, 1989). The non-aggregatory phorbol ester, 12-deoxyphorbol-13- phenylacetate-20-acetate (DOPPA), was able to induce phosphorylation only in the presence of ionophore; its k, value for PKC activation decreased from 530nM to 120nM in the presence of calcium and DOPPA seemed to preferentially stimulate one PKC isoform (Brooks et al, 1990; see chapter 7). These studies re-enforce the need to use different phorbol esters to dissect understanding of a biological response and confirm that Ca2+ presence may be critical for phorbol ester induced platelet aggregation, although the role of TAS in the aggregatory response requires further clarification. 1.4.4. Epstein-Barr virus Early Antigen expression. A relatively rapid (-5-7 days) in vitro assay technique for the detection of diterpene ester presence relies on the ability of these compounds to induce expression of Epstein-Barr virus Early Antigen (EBV-EA). In cell lines that normally spontaneously produce low levels of capsid antigen (e.g. P3HR-1, B95-8, termed "producer " cell lines), TPA induced expression of 20-40 times more viral DNA than untreated controls (Zur Hausen et al, 1979). In "non-producing" cell lines (those that normally produce no "Early", the equivalent of virus-capsid, antigen) induction lead exclusively to the synthesis of Early Antigen (EA) (Zur Hausen et al, 1979). Ito (Ito et al, 1981, 1983) subsequently showed that the ether extract of soil samples under diterpene ester containing plants of the Euphorbiaceae could induce EBV-EA expression in the Raji cell line. This has led to the possibility that areas of South China where there is a high incidence of nasopharyngeal cancer and Burkitt’s lymphoma associated with EBV expression could coincide with areas that use Euphorbiaceae derived materials in ethnomedicinal practices and as technological products (particularly tung oil, from Aleurites fordii) (reviewed Evans, 1986d; Hecker, 1987). Analysis of EBV-EA expression induced by different phorbol esters indicated that the non-promoting compounds DOPP, Sap A, and Rx could induce EBV-EA to a similar extent as TPA, suggesting a poor correlation with tumour promoting properties (Evans, Brooks and Evans, 1990). Recently, induction of EBV lytic cycle by tumour promoting and non-promoting phorbol esters has been shown to require active PKC (Davies et al, 1992). Substitution at C 12 or C 13 was thought to be the only structural requirement for EBV-EA expression, whilst A/B rings in the trans configuration, a free primary hydroxyl and a free C 4 p-tertiary hydroxyl were features thought to increase potency (Evans, Brooks and Evans, 1990). EBV-EA expression may be a useful in vitro technique to screen for diterpene 60 ester presence, although it is a slower method than in vivo induction of erythema in the mouse ear model. 1.4.5. Other effects of phorbol esters. As previously mentioned, the biological effects of phorbol esters are wide- ranging. The ability of phorbol esters to promote tumour formation and induce a pro- inflammatory response led to the use of these compounds to study the disease state mechanisms of cancer and inflammation. Phorbol esters can induce both proliferation and differentiation of cell types (Dicker and Rozengurt, 1980). These compounds induce mitogenic and co-mitogenic responses in human lymphocytes (Edwards et al, 1983b; Edwards, Evans and Evans, 1989) which correlates with their ability to stimulate PG Ej production in synovial cells (Edwards et al, 1985). In mouse 3T3 cells, proliferation induced by phorbol esters can be mimicked by diacylglycerol (Rozengurt et al, 1984) and the proliferative mechanism is different from that induced by the tumour promoter and phosphatase inhibitor, okadaic acid (Herschman et al, 1989). In mouse epidermal cultures, TPA is able to inhibit Epidermal Growth Factor (EGF) binding (Strickland et al , 1985) in addition to the well documented induction of ornithine decarboxylase and transglutaminase enzyme activities (Yuspa et al, 1983). Interestingly, some of these effects are partially blocked and some partially paralleled by Bryostatin 1 (Sako et al, 1987). Bryostatin can also antagonise terminal differentiation induced by TPA in a human colon cancer cell line (McBain et al, 1988) and HL-60 cells (Kraft et al, 1986); phorbol esters inhibit HL-60 cell proliferation before inducing differentiation, the relative differentiating potency correlating with their ability to activate protein kinase C (Evans et al, 1989). TPA can induce Interleukin-1 and Tumour Necrosis Factor (TNF) in mice sensitive and resistant to tumour promotion (Updyke et al, 1989); in Senear (promotion sensitive) but not C57BL/6J (promotion resistant) mice, phorbol ester induction of 8 -lipoxygenase correlated with hyperplasia, oedema and oxidant generation (Fischer et al , 1988). Arachidonic acid potentiates superoxide production induced by TPA in peritoneal macrophages (Czemiedd and Witz, 1989) and this has led to the suggestion that 8 -lipoxygenase activity and/or oxygen radical production may form the basis for sensitivity or resistance in mouse strains to phorbol ester induced tumour promotion (Fischer et al , 1988; Czemiecki and Witz, 1989); the link between superoxide production and tumour promotion has been reviewed (Cerutti, 1985). There would, therefore, appear to be a role for induction of inflammation in the tumour promoting effect of phorbol esters, although as earlier discussed, direct correlations are still difficult to reconcile. TPA enhances the adhesive and lung-colonising ability of Lewis lung carcinoma cells (Takenaga and Takahashi, 1986) and Walker 256 carcinosarcoma (Varani and Fantone, 1982) and may point to a contribution to metastatic (or malignant) potential of cancers by phorbol esters. TPA can also alter gene expression of many oncogenes, including c-myc, c-fos and c-jun by binding to TPA Responsive Elements (TRE’s, also termed AP-1 regions) (Angel et al, 1987, 1988). Interaction of c-jun and c-fos is thought to enhance their DNA binding (Allegretto et al, 1990) and the TRE is possibly a convergent point for PKA- and PKC-mediated signal transduction pathways (Hoeffler et al, 1989). Interestingly, TPA induced activation of PKC (see section 1.5.) is thought to decrease phosphorylation of c-jun at sites negatively regulating its DNA binding activity (Boyle et al , 1991), whilst the normally recognised effect of PKC activation is an increased phosphorylation (see section 1.5.), reflecting further the complexity of phorbol ester (and PKC) induced effects. Isolation of a cyclic nucleotide independent kinase and its pro-enzyme from bovine brain (Takai et al , 1977), the identification of this enzyme as the receptor site for tumour promoting phorbol esters (Castagna et al , 1982; Leach, James and Blumberg, 1983; Niedel, Kuhn and Vandenbark, 1983) and the involvement of this enzyme in phosphatidylinositol signal transduction mechanisms intensified research into the biochemical mechanisms of cancer. It was now possible to start to comprehend biochemical effects that phorbol esters induce when exerting biological actions. 1.5. Protein kinase C - the phorbol ester receptor site. 1.5.1. The discovery of protein kinase C. In 1977, Nishizuka reported the isolation of a novel protein kinase from bovine cerebellum (Takai et al, 1977). This protein kinase, which was termed protein kinase M (PKM), was active independent of the presence of cyclic nucleotides or Ca2* and capable of phosphorylating histone or protamine. It was suggested that PKM was produced by limited proteolysis of a pro-enzyme. An accompanying paper (Inoue et al, 1977) described the purification of the pro-enzyme from rat brain; it had an approximate molecular weight of 77kDa and the protease converting this enzyme to PKM required Ca2*. The pro-enzyme and accompanying protease were present in many tissues including: lung, liver, kidney, cerebellum, heart, skeletal muscle and adipose tissue. Nishizuka subsequently demonstrated (Takai et al , 1979) that the pro enzyme was able to phosphorylate five histone fractions and muscle phosphorylase kinase. The pro-enzyme required the presence of Ca2+ and a membrane associated factor for activity. Other divalent cations were unable to activate the pro-enzyme, but the membrane factor could be replaced by phosphatidylinositol, phosphatidylserine, phosphatidic acid or diphosphatidylglycerol. Other phospholipids (including phosphatidylcholine, phosphatidylethanolamine and sphingomyelin) were less capable of replacing the membrane factor. The pro-enzyme was additionally shown to be non- proteolytically activated in the presence of phospholipid and limited calcium ion; higher calcium ion concentrations were required to convert the pro-enzyme to PKM. Furthermore, activation of the pro-enzyme by Ca2* resulted in its conversion from a soluble form to a membrane-associated form. Nishizuka suggested that kinase activity induced by the non-proteolytic activation of the pro-enzyme, since it was reversible and required only low Ca2* concentrations, may be more physiologically significant than kinase activity induced by the irreversible proteolytic conversion to PKM. He therefore, considered the pro-enzyme a protein kinase in its own right, dependent for activity on the presence of Ca2* and phospholipid, and termed the pro-enzyme Protein Kinase C (PKC). Nishizuka subsequently demonstrated that PKC could be activated in vitro by diacylglycerol at concentrations of less than 5%w/w the concentration of phospholipid (Kishimoto et al, 1980). Diacylglycerol alone exerted little effect on the enzyme activity, over a wide Ca2+ range, indicating that phospholipid presence was required for activity. The enzyme stimulation by diacylglycerol was apparently due to an increased affinity of PKC for phospholipid and concomitant decrease in k, for Ca2* from lO' 4 to 10' 6 M. Mono- and tri-glycerols were inactive in stimulating the enzyme, and diglycerols with an unsaturated fatty acid at Cj (particularly arachidonic acid) possessed a greater stimulatory activity than those with a saturated Cj fatty acid. Since phosphatidylinositol was a potent phospholipid activator of PKC, and phosphatidylinositol turnover (first described by Hokin and Hokin, 1955) was expected to yield diacylglycerols with an unsaturated fatty acid side chain (Michell, 1979), Nishizuka proposed that PKC activation by diacylglycerol was due to the release of this substance from phosphatidylinositol metabolism (Kishimoto et al , 1980). 1.5.2. The phosphatidylinositol signal transduction cycle. The binding of a neurotransmitter or hormonal factor to its cell surface receptor initiates a sequence of events that ultimately results in a physiological response. The opening of a ligand-gated ion channel (e.g. Acetylcholine binding to the nicotinic receptor, resulting in the influx of Na+ ions and corresponding change in electrophysiological potential) is an effect induced directly by the binding of the ligand. Alternatively, the agonist may, on binding to its cell surface receptor,initiate a series of events that result in the physiological response, the primary signal (or "message") being passed through the cell membrane to "secondary messengers". Before the physiological event is observed, secondary messengers cause amplification of the signal by involvement of enzyme cascades. In this case, the ligand receptor is present in the cell membrane and can transiently couple a G-protein (Guanine- nucleotide-binding protein); these proteins link the receptor (and hence the extracellular signal) with one or more of the following signal transduction pathways: i) cAMP/adenylate cyclase system, ii) phospholipase C/phosphatidylinositol cycle, iii) ion channel regulation (reviewed by Drummond and Hughes, 1987). Agonists capable of stimulating phosphatidylinositol turnover are numerous (see Table 4) and include: neurotransmitters (e.g. adrenalin, GABA, 5HT, histamine); growth factors (e.g. epidermal growth factor, platelet derived growth factor); hormones (e.g. adrenocorticotropic hormone, thyroid stimulating hormone); insulin; inflammatory Table 4. Agonists capable of stimulating phosphatidylinositol turnover. VO ON oo O CO 00 n ON oo 73 0 0 ON oo ^ 73 O O 2 * ON 00 «n 73 CA CA J H U t3 P o o E § S £ ^ *—H ONON © ON 1 ^ a ^ ^ a ^ o o oo ^ « o 8* a, S cn S « § ON ON v V P h on k * ON 00 ON 00 00 VO ON oo u D« CA ON 00 oo 73 73 ON «n 00 o:> P «n x: ooON Q ON CA ONr— s p 0 VO s 0 00 § 05s 0 ^ 0 c ON <5 p . 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T3 *C »o h 0 0 ON m u i- i CA CO 0 0 CA •q c >* o c CA <4-1 V x> 9 £ •& CO mediators (e.g. leukotriene D4, prostaglandin F^, substance P, bradykinin). This is indicative of the importance of this signal transduction mechanism in cellular communication, proliferation and differentiation. Inositol phospholipid turnover has been reviewed (Hirasawa and Nishizuka, 1985; Berridge, 1987; Benidge and Irvine, 1989; Potter, 1990); the important inositol in terms of signal transduction is recognised as being phosphatidylinositol-4,5- biphosphate (PtdIns- 4 ,5 -P2) although this constitutes only a small percentage of total inositol phospholipids. PtdIns-4,5-P 2 is formed by sequential phosphorylation from phosphatidylinositol (Berridge and Irvine, 1984). Agonist binding to its cell surface receptor and transient coupling to a G- protein activates a membrane bound phosphodiesterase, phospholipase C (PLC). PLC hydrolyses PtdIns-4,5-P 2 to the secondary messengers, inositol-1,4,5-trisphosphate (Ins- 1,4,5-P3) and diacylglycerol (DAG) (see Fig. 13.), thereby forming a bifurcating signalling pathway. Ins-1,4,5-P 3 is water soluble and diffuses into the cytosol causing the release of calcium ions from intracellular stores, which are probably part of the endoplasmic reticulum (Ross et al, 1989). Elevation of intracellular Ca2* levels enables the activation of Ca2+-dependent kinases, including PKC, thereby inducing substrate protein phosphorylation and a subsequent physiological response. DAG remains within the cell membrane lipid bilayer and, on binding to PKC, enables PKC to become membrane associated, with a lower Ca2* requirement, and activated. PKC, in this membrane associated state, phosphorylates its protein substrates to induce a physiological response. Rozengurt et al (1983) showed that phosphorylation of an 80kDa substrate protein in intact quiescent 3T3 cells was a result of direct (using phorbol esters) or indirect (by addition of PLC or endogenous agonist, platelet derived growth factor) PKC activation and, therefore, demonstrated a direct relationship A g o n i s t O HU IT) •H » M m CO •H O -OC5 » I O O Pu 3 4 J 4-> M Q )a Pu (a a •J *H a CM •H - - CO t—I - 4-1 ) bo co tn a o) a 4-> m M •H B P sf M iH I CM M m •H 4-> G ss I -H y *p 43 tu 43 m h • - u 4-1 - *4U 4-> u tu tu E a O co 4-i a co co y y Q y y CO tu u co 10 3 j t y co CO «H 4-> CO tu o g tu y CO CO G co o between phosphatidylinositol turnover, PKC activation and protein substrate phosphorylation. Once Ins-1,4,5-P 3 or DAG have fulfilled their second messenger function, metabolic deactivation is necessary for the normal basal state of the cell to be retained. In the case of Ins-1,4,5-P3, which clearly has a critical role in the regulation of cellular calcium mobilisation and homeostasis (Mennitt et al> 1991), a complex metabolic pathway exists which is mediated by specific kinases (reviewed by Shears, 1989). Ins- 1,4,5-P3 can be metabolised to inositol-1,4-biphosphate and thence inositol-1- and inositol-4- monophosphates and finally inositol (Shears, 1989) (see Fig. 14.). Alternatively, inositol tetrakisphosphates (Batty et al, 1985; Shears et al, 1987) and thence pentakis- and hexakis-phosphates (Shears et al, 1987; Balia et al, 1987; Stephens et al, 1988) can be formed from Ins-1,4,5-P3. The physiological function of these inositol phosphates is poorly understood and although some of the higher polyphosphates may provide intracellular and extracellular signals to the brain (Vallejo et al, 1987), it is unlikely that they are involved in signal transduction (Potter, 1990). DAG can be metabolised to phosphatidic acid which, after subsequent phosphorylation and addition of cytidine monophosphate, can be coupled with D-myo inositol, thereby forming phosphatidylinositol (Ptdlns) which re-enters the phosphatidylinositol cycle. DAG can alternatively be hydrolysed to monoacylglycerol, releasing arachidonic acid (AA) (reviewed by Smith, 1989). AA can then be further metabolised to produce eicosanoid inflammatory mediators (see Fig. 14.). The regulation and turnover of the phosphatidylinositol cycle is thus complex; PLC exists as an isoenzymic family (Hirasawa and Nishizuka, 1985) and initial agonist - receptor complex interactions with G-protein subunits may cause preferential Complex metabolic pathway,involving CO the formation of tetrakis, -pentaki I CO m and hexakis - phosphates M HH HHH HH CM sr HH .CO CO ■H M m •H HH HH HH m O co co O c o < co co & <0 o * co (X -H < u T5 n i CO O u P ■H 2 co / \ y u P i—I T3 •H C In O co co 00 CO e CO co S activation of one or more isozyme and may partially explain cellular responses to a particular agonist. Interactions with other phospholipid pathways (e.g. phospholipase D metabolism of phosphatidylcholine, as an additional source of DAG) may also contribute to the response of the cell. I shall not discuss these complex feedback and regulatory mechanisms of phosphatidylinositol turnover further, but consider instead the implications of its turnover, focusing on one arm of this bifurcating signal transduction mechanism - activation of PKC. 1.5.3. PKC - identity with the phorbol ester receptor. At the same time that Nishizuka discovered PKC and, due to its activation by DAG, proposed its involvement in phosphatidylinositol turnover (Kishimoto et al, 1980), Blumberg was studying the in vitro binding of phorbol esters to potential receptor sites. The lipophilicity of TPA hindered early binding studies due to high levels of non-specific binding. Blumberg therefore proposed that phorbol-12,13- dibutyrate (PDBu) would possess an optimal ratio of binding activity to lipophilicity. The use of PDBu tritiated at C^ ( 3H-PDBu) enabled him (Dreidger and Blumberg, 1980) and others (Shoyab and Todaro, 1980) to demonstrate specific high affinity binding of phorbol esters to their receptor site(s). Similarities between PKC and the "phorbol ester receptor" were apparent: i) activity was highest in brain, high in lung and neutrophils and lower, but present, in other tissues; ii)phospholipid and low calcium ion concentration was required in both cases for activity. Nishizuka (Castagna et al, 1982) demonstrated that under conditions of limiting Ca2* and phospholipid, phorbol esters stimulated PKC activity. Several laboratories (including those of Nishizuka and Blumberg) then demonstrated that phorbol ester binding activity and PKC enzymatic activity co-purified, ultimately to homogeneity, implicating PKC as the phorbol ester receptor (Kikkawa et al, 1983; Leach, James and Blumberg, 1983; Niedel, Kuhn and Vandenbark, 1983; Sando and Young, 1983; Ashendel, Staller and Boutwell, 1983; Parker, Stabel and Waterfield, 1984). However, if PKC was the phorbol ester receptor, an endogenous PKC activator should competitively inhibit 3H- PDBu binding to PKC. Since DAG (in common with phorbol esters) was known to activate PKC under limiting conditions of Ca2+ and phospholipid, Blumberg tested this theory using DAG as the putative endogenous activator. He showed that DAG did competitively inhibit 3H-PDBu binding to PKC (Sharkey, Leach and Blumberg, 1984), thereby confirming PKC as the phorbol ester receptor and DAG as the endogenous activator of PKC. The stoichiometry of DAG binding to PKC was later shown to be 1:1 (Konig, DiNitto and Blumberg, 1985; Hannun, Loomis and Bell, 1985). The discovery of PKC as the receptor site for tumour promoting phorbol esters (reviewed by Hecker,1985; Blumberg, 1988) provided: i) a putative biochemical mechanism to mediate the biological actions of phorbol esters, ii) an enzyme implicated in events that lead ultimately to tumour formation, and probes to study its biochemical effects and iii) indicated signal transduction systems, particularly the phosphatidylinositol cycle, as critical participants in cellular communication, proliferation and differentiation. Not surprisingly, research in this area intensified. 1.5.4. PKC isozymes. The importance of PKC in signal transduction and the complex co-factor requirements for its action as a serine/threonine protein kinase (see Fig. 15.) led to intense investigations into the protein structure. Tryptic digestion of PKC purified from bovine brain enabled the determination of the primary amino acid sequence of PKC by Parker et al (1986). An accompanying paper (Coussens et al, 1986) indicated d) 0 I c + j 0 -H (0 X C M I CO • & o 4-» O SC 0 CO 0 (0 u u o u X3 dc a: 1 a: x x: P u - u - 0 ^ - 0 Oh -M C/3 g C0 || &4 Q TJ I o 0) < 0 c 4-> •H (0 P r—I I I d) >1 o 0 co cd u CM o c 1 o a: a: ' a ; x: *H X U - U - O CM . - o a C p4 a: C 0 || CO o CO z o d) o t O x: u x: a * x 04 + j u o d) c •H U d) CO M + O . O CM ■P «0 O M a u < (U 0) Q -t-> o CO o + CO Q) <0 u T3 u o • H i—I o u * -M ft O ■M * a. u • H X ) C O4 (0 H M 0 44 O O > 1 o x x: a< x> u f t f t H CO < 0 O 1 4-1 X! Oh (0 O4 CM M CO 4-» I CO * 3 CO oe c M -H CM 44 d) Oh O4-1 CM M co & <0 d) 4-4 44 0=04 - o * c CO c 0 0 I X 0 O X ■H 4-J u u G \ 14 0 04 CO X X X o 1 CO 0 0 Eh •H u - 0 - 0 0 0 44 0 < X a: u 4-> 0 TJ I z X •H C *H • i Eh 0 0 0 O X Oh — O CO O) U 0 X • M Eh U CM Q) p “ 0 CO 0 4-> I f t X) £, (Ono et al, 1987b) and eta, T|, (Osada et al, 1990; Bacher et al , 1991) PKC isoforms. The heterogeneity of PKC (reviewed by Nishizuka, 1988; Parker et al, 1992) and subtle enzymological properties of isotypes poses the question of individual isotype role in cellular function. Although PKC function is poorly understood, several properties of isotypes may contribute to particular roles: i) Distinct patterns of tissue expression and intracellular localisation (see Table 5.). ii) Specific substrates for a specific isotype (or a specific function) iii) Selective activation (or non-activation) of an isotype by different diacylglycerol agonists. (This could be the result of extracellular agonist stimulation of phosphatidylinositol turnover, or generation of diacylglycerols from metabolism of other phospholipids.) PKC is a monomeric protein, consisting of a protein kinase (or "catalytic") and a regulatory domain (see Fig. 16.). a, ft, ft,, and y isotypes possess four conserved (C,- C4) and five variable (Vr V5) regions. 8 , e, £, and T) isotypes lack the Q conserved region, which presumably contains the Ca2+ binding region, since these isotypes are not dependent on the presence of Ca2* for activation (Schaap et al, 1989; Ono et al , 1988, 1989). The conserved C, region contains a tandem repeat of a cysteine rich sequence, Cys-X 2-Cys-X13(14)-Cys-X2-Cys-X7 -Cys-X7 -Cys, where X represents any Table 5. Examples of tissue-specific protein kinase C isozyme distribution. Tissue. Isozymes Reference. present Adrenocortical a Pelosin, Vilgrain and Chambrez, 1987 Brain a, p, y, 8 , Bacher et al, 1991; Brandt et al, 1987; e, t 1 1 . Huang et al, 1986; Huang et al, 1987; Kosaka et al, 1988; Osada et al, 1990; Schaap et al, 1989. Epidermis 8 ,ii Bacher et al, 1991; Osada et al, 1990. Heart a, P, e, ii Kosaka et al, 1988; Osada et al, 1990; Schaap et al, 1989. Kidney a, p, £, ii Kosaka el al, 1988; Osada e/ al, 1990; Schaap ef u/, 1989. Liver a, P, e, ii Kosaka et al, 1988; Osada et al, 1990; Schaap et al, 1989. Lung a, P, e, ii Bacher et al, 1991; Kosaka et al, 1988; Osada et al, 1990; Schaap et al, 1989. Neutrophils O.P.C Petremoli et al, 1990; Sekiguchi et al, 1987; Stasin et al, 1990. Pancreatic acinar a Wooten and Wren, 1988. cells Platelets V and "b" Brooks et al, 1990. Spleen a, P, 11 Brandt et al, 1987; Kosaka et al, 1988; Osada et al, 1990. Testes Ot, P, Tl Kosaka et al, 1988; Osada et al, 1990. T-lymphocytes a, p Berry et al, 1989; Shearman et al, 1988. Regulatory Domain Protein Kinase (catalytic) Domain O co.cn, O l _ CM i M U > u > U > u > 0 > (N CM ro ro in CM + h a)•h •H ■H E *0 -H G - JG .P •H •H £3 +JU U 'O P TJ -P < E a* Pi P to 0 C 1 > c co a) to c 0) to c 01 G CD c h 1 a) to p p ) uu U) JO l_J o o CM > > U U > L> > CM ro ro •<* in HPi •H •H »p w •M ■P U vD (U * 4-> P Ol 0) P u P p p P 0) 0 CO O Pi d) CO >1 • •H •H iH > 0 > £3 •H 4-1 (p 'd Ot O to 'O p U co CP u u 01 0 c * P 0) 0 to P to d) > * R. in CO d) 0 O CO C /—i CO •H CO CO O O P O G p to P > d) 1=1 M* 1 H 1 1 ) - 1 fM + rH •H X U o t CP * U u K •H •H u) 'O •H •H X3 T3 u -P -M O tu P oG to d) u to c 0) d) Ol 0 G O) CM U G G Ol P d> C71 O P 0 G to d) CO to G c to C d) E t H • tp •H •H XS JS •p -P iH «P •H •H - ■p ■P x: h P d) d) (0 O Pi to to 0 1 > CO (U 0) P (0 G CO 0) O1 0 P G to CO 1 > O G d) — OCO CO d) 1 > O p 0 CO d) C 0) 0 CO O1 a) u p c 0 • ' •H O : s •H T3 •H CP JD -o < 0H ip (p ■H ■H T3 s iH 00 •H z £ X CO (0 G CO G O1 d) P d) 0 G o> ro G 0 (0 P 0 E N to • amino acid (£-PKC has only one set of this cysteine rich sequence; Ono et al, 1989). This is similar to the consensus sequence of a cysteine-zinc-DNA-binding finger found in mettalo-proteins and DNA-binding proteins although there is no evidence that PKC binds to DNA (Nishizuka, 1988). The conserved C 3 region has an ATP binding sequence, Gly-X-Gly-X-X-Gly....Lys. C4 contains a similar sequence, Gly-X-Gly-X-X- Gly....(X), although its significance is unknown (Nishizuka, 1988). Phorbol ester/diacylglycerol and phospholipid binding sites are presumably contained in the Q region. Arachidonic acid and other unsaturated fatty acids are capable of activating PKC (McPhail, Clayton and Snyderman, 1984; Sekiguchi et al, 1987), although the synergistic addition of diacylglycerol is required for activation of p- and a- subspecies (Shearman et al, 1991) but not for activation of the y-subspecies (Shearman et al, 1989). The potency of diacylglycerols and unsaturated fatty acids for activation of PKC is within the micromolar dose range; this renders these activators much less potent than phorbol esters, other tumour promoters (e.g. teleocidins, aplysiatoxin) and bryostatin, which are capable of activating PKC at nanomolar dose levels (see Table 6.). Activation studies on a mixture of PKC isozymes from rat brain by phorbol esters with different biological activities revealed that phorbol ester activators could be classified into four groups (Ellis et al, 1987; Evans, 1989): i) Compounds, which like TPA, could activate PKC at low (upto lOOnM) dose levels, e.g. Sap A, Sap D. ii) Derivatives only able to maximally stimulate PKC to -30% of the activity induced by TPA. e.g. DOPPA, Rx. iii) Compounds capable of fully activating PKC, but at greater dose levels than Table 6 . Activators of protein kinase C. "O *U ON oo in 0 0 CN ON 00 ON ON 00 «o CN CN CN 3 T ON 00 00 oo oo ON CO. ON 00 VO oo U ON 5-J O 00 CO CO S> >H O C CO O CA n ON , oo on k * ON OO OO *P CN 0 0 3 T ON OO VO OO on 00 CO o oo C w VO as 00 •g as oo CO CA CA ON O VO » c C 0 0 ON 00 ON 00 VO CO 7y 80 on 00 O n VO VO 00 oo OO ON o\ ON 00 ON 00 ON «o «n »n oo oo oo 00 ON ON ON ON CO CO Tf 00 ON oo ON 00 0 0 00 00 0 0 VO ON ON ON 0 0 ’■“< ON oo 00 •o ON 0 0 ► CA CO O CN VO 00 CN CN CNi I i 00 o o CO o CN •5 *9 T3 a cx c o *? g £ • £ CO VO JS 81 required by TPA (1,000 - 10,000nM). e.g. a-Sap A. iv) Compounds which failed to activate PKC at 10,000nM dose levels, e.g. a- sapine, phorbol. (These compounds have no pro-inflammatory or other biological effects.) However, the ability of phorbol esters to activate specific isotypes had not been studied until the groups of Evans and Parker collaborated to investigate the actions of TPA, Sap A, Rx, DOPP, DOPPA and Thymeleatoxin A (Thy A) on the purified a, ft, y, 8 and e PKC isoforms. These studies (Evans et al, 1991; Ryves et al, 1991) confirmed that a, p and y PKC isoforms were dependent on the presence of Ca2+ for activation, whereas 8 and e isoforms were Ca^-independent Furthermore, the range of phorbol esters used to study the isoforms of PKC gave differential responses in terms of activating ability: i) DOPPA was shown to be a specific ft-isotype activator (see also chapter 7); ii) Sap A was a specific 8 -isotype non-activator upto concentrations of lOOOng/ml. This data supports the possibility that PKC isotypes may have distinct roles in cellular function, since isotypes can be selectively activated/non-activated by a particular agonist, and was the first description of probes that could be utilised to selectively study PKC isotypes. 1.5.5. Translocation and down-reeulation of PKC. Binding of phorbol esters/DAG, phosphatidylserine and Ca2+ co-factors of PKC cause activation of the enzyme and a concomitant translocation of PKC from the cell cytoplasm, enabling PKC to become associated with the cell membrane (Kraft et al, 1982; Kraft and Anderson, 1983; Ashendel and Minor, 1986). Membrane association of PKC has been presumed to be a pre-requisite for substrate phosphorylation (Bell, 82 1986). Myristoylated (therefore membrane associated) and non-myristoylated (therefore non-membrane associated) forms of a PKC-specific substrate protein, with an approximate molecular weight of 80kDa (also termed the Myristoylated alanine-rich C kinase substrate, MARCKS, protein) are phosphorylated by PKC (Graff, Gordon and Blackshear, 1989), suggesting that whilst membrane association is required for phosphorylation, substrate membrane association is not Phosphorylation of the MARCKS protein by PKC has been proposed as a regulatory mechanism for MARCKS protein membrane association (Thelen et al , 1991). Activation and translocation of PKC and concomitant phosphorylation of substrates is therefore complex, and the situation is probably dynamic; activation of PKC (particularly by phorbol esters) is likely to increase the rate and quantity of PKC membrane association, thereby increasing protein substrate phosphorylation and (ultimately) raising cellular physiological responses above their normal basal level. A correlation has been shown between TPA induced PKC activation and membrane association above basal levels and the number of metatastic nodules produced by B16 melanoma cell sublines (Gopalakrishna and Barsky, 1988). Following activation of PKC and its translocation to the membrane, PKC is proteolytically degraded, or "down-regulated" (Rodriguez-Pena and Rozengurt, 1984; Ballester and Rosen, 1985), to its regulatory domain and catalytic domain (PKM) (Huang and Huang, 1986). Down-regulation can be mediated by the action of trypsin (Huang et al , 1989) or Calcium dependent Neutral Protease (Calpain) proteins I or II (Kishimoto et al, 1989), and the activated form of PKC is thought to be required for down-regulation, since the rate of down-regulation is enhanced by the presence of PKC co-factors (Huang et al, 1988). Initial predictions (Parker et al, 1986) that the V 3 region of PKC was the major site of proteolytic degradation were proved correct 83 (Huang and Huang, 1986; Lee and Bell, 1986). Investigations into the rate of down- regulation of a, (3, and y isozymes of PKC by trypsin and calpains I and II revealed that these isozymes were differentially down-regulated (Huang et al, 1989; Kishimoto et al, 1989). y-PKC was the most rapidly down-regulated isozyme by both trypsin and calpain, followed by p-PKC, whilst a-PKC was the most resistant to proteolysis. Prolonged TPA treatment (now a recognised method of inducing down-regulation) in intact rat basophilic leukaemia RBL-2H3 cells (which contain (3- and a-PKC isozymes) confirmed the in vitro results; TPA induced a faster down-regulation of p- PKC than a-PKC (Huang et al, 1989). These results suggested that susceptibilities of PKC isozymes to proteolysis at the V3 region may contribute to the mediation of responses induced by PKC isozymes under different physiological conditions, and further understanding of the cellular function of PKC isozymes. 1.5.6. Inhibition of PKC. The importance of PKC in signal transduction and its implicated role in cell proliferation and differentiation has led to considerable interest in potential PKC inhibitors. Compounds which inhibit PKC would enable further clarification and dissection of PKC function in normal and abnormal cellular states, and may ultimately provide novel anti-inflammatory and anti-cancer drugs. To fulfill their potential and to minimise side-effects and effects on other cellular enzyme cascades, PKC inhibitors (like any enzyme inhibitor) would be required to be both potent and specific (possibly specific for a particular PKC isoform?). The complex co-factor requirements for PKC mean that there are several potential sites for inhibitors to bind and act upon PKC. However, although the primary amino acid sequence for PKC is known (Parker et al, 1986), the three dimensional 84 structure of the protein is unresolved. Thus, apart from knowing which of the conserved regions PKC co-factors interact with, the absolute binding regions, in terms of amino acid/co-factor interactions, remain poorly understood. Therefore, the majority of reported PKC inhibitors (see Table 7) are of a relatively weak potency or are un specific (or both), and some have multiple mechanisms of PKC inhibition. These compounds remain of little use in studying PKC function. However, some inhibitors of PKC have been utilised which might provide genuine insight into PKC mediated effects. The structures of the most useful PKC inhibitors are shown in Fig. 17. N-(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide (W-7) (Hidaka et al, 1978) was originally shown to be a calmodulin inhibitor (Hidaka et al, 1980), and then a PKC inhibitor acting by competative inhibition of phospholipid binding (Tanaka et a/,1982; Schatzman et al, 1983). W-7 has also been shown to inhibit PKC through the ATP binding site (O’Brien and Ward, 1989a). Therefore, W-7 and related compounds, isoquinolinesulfonamides, e.g. l-(5-isoquinolinesulphonyl)-2-methylpiperazine (H-7) which also inhibit PKC competitively with respect to ATP (Hidaka et al, 1984), are problematical to use as PKC inhibitors since they inhibit other cyclic nucleotide dependent kinases. Staurosporine (Tamaoki et al, 1986) and K252a (Yamada et al, 1987) are polycyclic compounds isolated from microbial origin which are more potent (nM dose range required) PKC inhibitors by competitive inhibition of ATP binding. They have, therefore, recently superceded the use of W-7 and H-7 to study PKC, although the inherent problem of specificity for PKC over other cyclic nucleotide dependent kinases (Elliott et al, 1990) remains unresolved. Since the ATP binding region amino acid sequence is well conserved within the PKC family (Parker et al , 1986) and many other kinases utilise ATP, and hence need an ATP binding sequence similar to Gly-X-Gly-X-X-Gly....Lys (Nishizuka, 1988), searching for specific and Table 7. Protein kinase C inhibitors. £ 6 *o U 5 o oo 1 0 8 o 3 3 S *K 8 - S < S-*2 au *3 g 1 a> e 00 oo U-J *c T3 « ON ii 4> o o o -2 co ^ c?ffl § & * 5 _ ON 8 ON 3 T3© O O X) •a a S ■§S T3 ii o CN ffi O Q -O OO oo 2 >* .2 2 * 2 6 S 3 - a !x '< 1 ^r a o ON s - »n 3 X CAO c 53 ^ 00 c CA 2 2 § 00 00 8 3 , g M 0 oo _ #k • eO ^ 3 § s « 5 S S 00 z b O b 3 g 3 8 8 t>O CA ^ O eO . XT*13 ON ON b s ^ !s ■*** b S »# * • e O ON CO 1 0 3 3 I 4> N T3 O oo £ ■a ON ON o § o s o c a n O oo 00 2 ’S ,2 pO a N ■«p—I°N 00 “ b 00 a - a „ b ^ tii 3 ' S s ■S *“« ON Q X T3 CQ «0 ON b x s -x «2 i n o 3 30 J j - i i t: NO S - 53 2 « oo 0 -<**0 1 os -g 3 ^ 8 8 O 69 00 IS «*S * 6 ) ^ 4) o *> H * eo o 3"3 *o SC o O O S s CN 2 *n 3 3 8 a . N •o t oo m S5 •a 'S ON O O O *o ON ON 00 o •§ ’o S | a 3 *o l b l 2 g T3 s 1 •o b a s i i 2 a 1 3 #* • o a 00 OS 3 * • 4> 3 3 s 53os oo . 8 C CA •§ £ CA - 0 b 3 3 8 NJ .2 I . n 3 O «o o o ON ON ON O ON OO ON 3 00 00 00 CN CN o s w Q m o cn p^ 00 CN »n b ts * • 3 o £ o • 1 1 1 n ON oo 00 ffi • w4 a ON ON o T3 PC b J3 CO O O 4> e3 O 3 #» • CA £ H Sh u £ oo c3 8 > #» 85 86 4> 3 x : »n oo 00 .fl08 ON ON O *“ • CO *J *3 2 c g O« ai t: u T 3 <£* *C T 3 ^ pq a ? . 41a oo £ 00 s2 00 i i o n 00 oo O *"H o n m 00 ON S «g ooS H ONOO «3 u2? X) c3 G3 3 O Q 13 2 ON a G E J3 i i 3W 00 s PQ *o X3 o s g 00 •a £ •I ON 3 oo i OO oo On H ON C ^ oo ON §0 G ON2? o\ i g ^ .S *> £ ON N rf s _* 00 3 CA B Q <3 < .5 g 3 •a *0A .3 o «- -9 g •a * B *> 3 ■ s * •a 5 5 :* 5.h ~ *0G s « •g .s O 3 s *§ o ON ^ ... § • #» X) 2 O 00 o , - H CA ON o ro s ^ OO o - 3 ON g ON CA oo o ^ O ON « 2 sc *> #* _, G 93 3►» s o o *) CA JS ■V* 13 * g c *c o O o t> a « 3 | s X PQ O *4 o ' 5 s O3 . s s o 3. o o I 3 . 3 . in O 3. o o o O OO «o Sc o I O I o o »n o o CO B O VCA ••8 > 4> b9 *X33 ► > ii •c a a o 9 T3 u U ■s .2 o G CA D 2 « C a> G e3 >» §o c •e uG 'S u b S 2 .s 1*5o 33 9*! 0ex a 0 CA g. „ E j :-§ i x 0 a *3 1 jb a 0 o 1 S 3 1 x: *X3 __ a>B a .& c . 60 € SJ CO 1 s ^ CS 9 < CO B< 87 o n 00 O s O s 00 ON CO 00CO ON 00 Q OO o o 00 O s CO ft ON o o ON CO o o ON 00CN ON CN ON *g «n "g ON 00 00 o o g ON 00 o n 8 ON 00CO O n .« 00 CO CO C O o o CO 00 o o 00 00 00 ON ON ON ON ON ON i—i 00 ON fU 8 I f a. 3 ON NO o o CN i CO NO CN (0 ob P« •a 88 ON ON Tf00 ON00 ON ON 00VO ON 00OO 00 ON ON 00 CA ON 00 00 ON 00 ON 00 T3 VO "P ON ON 00 ON ON CO00 ON ON CO o mi o »n o vo *T3 ■© T3 8 -s 00 00VO Ov § 2? ’S : a CO 00 ON OO ov oo ON CA 00Ov OV OV OV 00 Ov Wh “> s .2 C CA oo CN 00 •» 8 » S i ii OS CA *3 X 00 S' nThis phorbol ester derivative is a non-activator of PKC, and at high doses prevents activation of PKC by TPA (presumably by binding site saturation). It is not an inhibitor of PKC in its own right. 14Sphingosine inhibition of PKC at the active site may be dependant on assay conditions employed; sphingosine also inhibits PKM (Nakadate, Jeng and Blumberg, 1988). 90 91 potent PKC inhibitors acting by competative inhibition of ATP binding could be problematical. It is surprising, therefore, that Roche have developed a series of inhibitors based upon K252a and staurosporine, which retain potency for PKC and are approximately 100-fold more specific for PKC than either PKA or phosphorylase kinase (Davis et al, 1989; Elliott et al, 1990). The initial lead compound, Ro 318220, has been developed to Ro 318425 and Ro 318830 to attempt to maximise potency and specificity and to enhance pharmacokinetic properties (Nixon et al, 1992). These compounds can inhibit some phorbol ester-induced effects both in vitro and in vivo (Davis et al, 1989; Nixon et al, 1992) and will undoubtably be of use in furthering our understanding of PKC mediated events. PKC inhibitors that are likely to aid the elucidation of PKC function and particularly that of individual isotypes, are those which will inhibit PKC by binding to the regulatory domain. Furthermore, since only the a, p, and y isoforms are Ca2+- dependent (Nishizuka, 1988; Schaap et al, 1989; Osada et al, 1990; Bacher et al, 1991; Ryves et al, 1991) and phorbol esters can selectively activate/non-activate PKC isotypes (Evans et al, 1991; Ryves et al, 1991), inhibitors whose mechanism of action is via the phorbol ester/DAG binding site would be critical to understand PKC mediated effects, and may provide templates for new drug design. Current inhibitors which affect phorbol ester binding to PKC (active site inhibitors) (see Table 7, and footnotes) are still problematical. Sphingosine (Hannun et al, 1986), gossypol (Nakadate, Jeng and Blumberg, 1988) and aminoacridines (Hannun and Bell, 1988) may additionally act at other co-factor binding sites and their ability to inhibit PKC may depend on the assay conditions employed (Nakadate, Jeng and Blumberg, 1988; Hannun and Bell, 1988). Synthetic pseudosubstrate peptides (House and Kemp, 1987) are still contentious, since they may not directly inhibit phorbol ester binding, and the 92 SO„NH(CHJ,NH H3 C, > - N W-7 H H-7 NHCH3 Staurosporine OH 0 N' 'N H~Y°y~cH3 OH 0 Calphostin C V o" C°2 CH3 H K252a SC-NHo II L R N R=NH2 : Ro-318425 H R=N(CH3 )2 • Ro-318830 Ro-318220 Figure 17 Structures of commonly used PKC inhibitors 93 pseudosubstrate site has not been unequivically designated the phorbol ester binding site. Additionally, in common with the diacylglycerol analogue, l-O-hexadecyl-2-O- methylglycerol (Van Blitterswijk et al, 1987), the potency of pseudosubstrates is somewhat low (20-100pM range). The A/B cis ring phorbol ester, a-sapine, is itself biologically inactive and a non-activator of PKC (Ellis et al, 1987; Evans, 1989). At high doses, a-sapine can inhibit TPA-induced activation of PKC (Ellis et al, 1987), although the biologically active Sap A can also induce this inhibition (Ellis et al, 1987); the mechanism of this inhibition is therefore likely to be via saturation of the binding site by high concentrations of a-sapinine. However, a-sapine cannot, per se, inhibit PKC activity. Calphostin C (see Fig. 17.), a microbial compound isolated from Cladosporium cladosporioides (Kobayashi et al, 1989), is a potent (50-500nM dose range) and selective (compared to PKA and calpain) PKC inhibitor acting at the phorbol ester binding site (Koyabashi et al, 1989; Tamaoki, 1991). Calphostin C has been shown to suppress tumour promoter (teleocidin) enhancement of Epstein-Barr virus induced growth transformation (Kinoshita et al, 1990). However, calphostin C inhibitory activity is light-dependent (Bruns et al, 1991) and this compound is cytotoxic at doses of 0.15-0.25pM (Tamaoki, 1991), suggesting that its use may be limited. Recently, (Coleman and Grant, 1991) synthesis of the naphthalene subunits of calphostin C has been undertaken, indicating that synthetic modification of this compound may be attempted, to dissect the active pharmacophore(s). There is, therefore, still a need for potent and selective inhibitors of PKC, preferably acting at the phorbol ester/DAG binding site, to probe the role of this family of isotypes in normal and abnormal cellular states. 94 1.5.7. Substrates of PKC. Extracellularly induced or direct (e.g.phorbol ester induced) activation of PKC results in events that ultimately lead to a change in cellular physiological function. The first step in this chain of events is substrate phosphorylation by PKC. However, whilst numerous proteins have been shown to be phosphorylated by PKC, the effects and relevance of this phosphorylation remain, in most cases, poorly characterised. Indeed, whether or not some of these proteins are physiological PKC substrates is contentious. In vitro and in vivo techniques have been used to examine the characteristics of potential PKC substrates. These normally utilise 32P radiolabelled phosphate to assess incorporation into the substrate as a measure of phosphorylation. In vivo experiments on intact cells involve loading the culture media with 32P-ortho-phosphate until the intracellular phosphate pool has been labelled to equilibrium. Cells can then be treated with activator (phorbol ester or diacylglycerol) and the 32P-phosphoproteins can be analysed by autoradiography following one or two dimensional electrophoresis on SDS-PAGE gels (Laemmeli, 1970). The ultimate aim of this analysis is to determine the amino acid sequence of the substrate protein, enabling assessment of the serine/threonine residues phosphorylated by PKC, in an attempt to clarify the physiological significance of substrate phosphorylation. There appears to be no absolute concensus sequence required for PKC-induced substrate phosphorylation, although PKC recognition shows independence from other serine/threonine kinases (Gallis et al , 1986). Experiments with synthetic substrates (O’Brian et al, 1984; Ferrari et al, 1985; Schaap et al, 1989) seem to suggest that basic amino acids (arginine or lysine) are required on both the carboxy- and amino- sides of the target residue for PKC phosphorylation. For a protein to be considered a potential physiological target for PKC 95 mediated phosphorylation, the following minimum conditions should be fulfilled: i) Correlation of in vitro and in vivo phosphorylation sites. ii) Functional difference of phosphorylated/dephosphorylated forms of the substrate. iii) Stoichiometric in vitro phosphorylation of substrate is dependent on the presence of PKC co-factors under conditions that will induce activation of PKC. iv) In vivo accessability of the substrate to PKC (i.e. co-localisation). Table 8 shows the numerous substrates that are phosphorylated by PKC; these include: receptor/signalling proteins; enzyme proteins; structural/contractile proteins; nuclear proteins; and some undesignated proteins. Many of these substrates can be phosphorylated by other kinases, either at the same serine/threonine residue e.g. C- protein (Lim, Sutherland and Walsh, 1985), at different residues e.g. GABA-modulin (Wise, Guidotti and Costa, 1983), or at a mixture of these e.g. MAP-2 (Akiyama et al, 1986). Therefore, the physiological significance of PKC induced phosphorylation is often unclear. Identification of PKC substrate proteins can be further hampered by several factors: i) Substrate species variation; the 87kDa protein from bovine sources is an 80kDa protein in rat (Albert et al, 1986); ii) Changes in isoelectric properties on phosphorylation (McFarlane, 1986); iii) Tissue-specific forms of a substrate exhibiting differences in phosphorylation (Wu et al, 1982; Rider and Hue, 1986); iv) Changes in substrate with maturation (Turner et al, 1984); v) Generation of PKC phosphoprotein fragments following autophosphorylation (Mochley-Rosen and Koshland, 1987; Huang et al, 1986); vi) Phosphatase action (Cantrell et al, 1989; Hannun, Loomis and Bell, 1985). The role of endogenous PKC inhibitory proteins (McDonald and Walsh, 1985a, 1985b, 1986; Swantke and Le Peuch, 1984; Toker et al, 1990; Aitken et al, 1990) in Table 8. Protein substrates for protein kinase C. 00 00 *o5 ON 00 ON 00 C/2 CO rHO mU ON OO * a CA 00 V5 I-* CA s § § .s I & s *2 g S3 * oo y ^ o oo y O « 00 00 a s COL ON 00 00 Tj- ON cs OS 00 o 3 7 ON 00 O ON 00 M ON oo «n T3 "O ON 00 O oo CO NO »n n 96 97 CA 00vo P «o co ON . oo 00 Tj- £ ON ON ON »o vn oo p »o 00 Q oo . 3 oo ► 00 ON on ON 2 ON Q ®h p 3 OO a *> o CA 4) OO m 2 . CO o J3 ON C \C •2 1 • PN o *8 . 3 •a -g oo § ON > U s 3 C 73 § 2 . U . 3 p § 2 o P 2 ON »0 2 p *H e3 ’S 2 O h P 0 P a p & CA tH 8 +■»2 * o 03 2 e O h £* S' oo P c M 0 r 3 P 8 • 3 •S tH £ c3 • a s 3 >* 8 • »»H 3 C • 0 00 ■8 a j P p o § CA c3 8 8 a -8 2 3 1 3 0 * t3 8 3 g 8 c3 S § 1 f P p 2 J3 ►5 Q 5 Q ps (3 ON p T 3 B p 3 8 2 5 A p o - - • « . p 9cA n dJ -I VO CQ Uh P ^2 < | | I P cP rv i 3 ,£> a - a ■ s o CQ •O —. — to a p X & ... 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Phosphorylation induced by phorbol ester activation of PKC is more prolonged than that induced by diacylglycerols (Wise, Guidotti and Costa, 1983; Gould et al , 1985; Wang et al , 1984), and phosphorylation patterns induced by phorbol esters may be different from those observed with an endogenous hormone agonist (Brooks, Evans and Aitken, 1987). These properties of phorbol esters may reflect their ability to push PKC (and hence PKC-mediated events) from a normal, regulated state to an abnormal, uncontrolled one. A further factor involved in the study of PKC substrate proteins is the heterogeneity of PKC isozymes. Different isotypes may be triggered by particular agonists (or phorbol esters) and may possess their own set of protein substrates, or may phosphorylate substrates at different rates e.g. in vitro EGF-receptor phosphorylation has slight variation a>p>y (Ido et a/,1987). These possibilities have implications in terms of co-localisation and tissue/cell-specific distribution of both PKC isotypes and the protein substrate, and the in vivo role of PKM following activation and down-regulation of PKC is still not understood. Reports of selective activation/non-activation of PKC isotypes (Evans et al, 1991; Ryves et al , 1991) by selected phorbol esters may help clarification and identification of PKC substrate protein function. However, the biologically inactive a-sapine acetate has been shown to stimulate phosphorylation to some extent in GH3 pituitary cells (Brooks, Evans and Aitken, 1987), and therefore interactions of other kinase systems with PKC (Yoshimasa et al, 1987) or even phorbol esters with other kinases, could contribute to regulation of PKC mediated substrate phosphorylation and subsequent physiological 105 responses. 1.5.8. Phorbol ester interactions with other kinases. In addition to the eight recognised isotypes of PKC (a, f t , ftj, y, 5, e, £ and T|; see section 1.5.4), the existance of other PKC or PKC-related kinases have been reported. These include two components of Type HI (a-) PKC (Buday et al , 1989) with different substrate specificities and Ca2+-dependencies and a calcium- unresponsive, phorbol ester/phospholipid activated protein kinase (Gschwendt, Leibersperger and Marks, 1989). The use of the PKC inhibitor K252a by Gschwendt, Leibersperger and Marks (1989) suggests that their kinase is not a PKC isotype, since this inhibitor was unable to inhibit the novel 76kDa kinase. However, since K252a has not been tested for differential activity on PKC isotypes (particularly the 8, e, £ and T| isotypes), the possibility that their kinase is one of these isotypes cannot be eliminated. Equally, the novel Type ID component reported by Buday et al (1989) could be a member of the nPKC sub-family. Beh, Schmidt and Hecker (1989) have reported differences in activation by TPA of PKC isozymes in HL-60 cells, which are likely to be a and p isozymes. More convincing data concerning phorbol ester stimulation of novel kinases has been provided by the group of Evans. Using TPA and Thymeleatoxin A (Thy A), they have shown that these probes can stimulate seven kinase peaks from rat brain (Evans et al, 1991). Peaks appear to contain conventional PKC isotypes, as Western blotting analysis using anti-PKC a, ft, y and e antisera blot under these peaks. This suggests that other phorbol ester stimulated kinases, other than the PKC isotypes at present defined, exist Evans has also identified a novel kinase, termed Rx- 106 kinase, which is stimulated by resiniferatoxin (Rx) to a greater extent than by TPA. This kinase has been isolated from human mononuclear cells (Ryves et al, 1989; Ryves, 1991), mouse peritoneal macrophages (Evans et al, 1990), and its tissue distribution has been investigated (Evans, A.T., et al, 1991b). The Rx-kinase elutes at a higher phosphate gradient concentration than conventional PKCs and it is, therefore, likely that this kinase is distinct from known isotypes of PKC. It is, therefore, a possibility that some of the biological effects of phorbol esters could be mediated by novel kinases, which may be part of the PKC family but which, at present, are distinct from known forms of PKC. 1.6. Objective of this work. Due to the present lack of potent and specific inhibitors of PKC, particularly those acting at the regulatory site of this enzyme, it was thought necessary to isolate phorbol ester nuclei from natural sources and to subsequently synthetically modify their structure, with a view to rationally designing novel inhibitors of PKC which were intended to act via the phorbol ester/DAG binding domain of PKC. In the first instance, these potential compounds should be non-inflammatory, and therefore pro-inflammatory assessment of these should be undertaken. Additionally, a detailed analysis of the pro-inflammatory response induced by resiniferatoxin should be undertaken, since this could provide information concerning the different mechanisms of actions of phorbol esters. These experiments would hopefully provide a means to clarify the structural requirements for phorbol ester induced PKC activation and inhibition. Chapter 2: Materials and Methods. 108 2.1. Materials. Throughout this project, the following materials were used, from the supplier listed. For biochemical work, all reagents were of "AnalaR" grade, or equivalent. 2.1.1. From Merck-BDH. Poole. UK. 20cm x 20cm Silica gel G F ^ 0.25mm thick pre-coated analytical TLC plates. Acetic acid. Acetic anhydride. All solvents, which were routinely re-distilled and stored over molecular sieve. Anhydrous sodium sulphate. Calcium chloride (CaCy. Celite (washed four times with acetone before use). Diethylene glycol (Digol). Dipotassium hydrogen orthophosphate (K2HP04). Disodium tetraborax. Drummond microcaps, 5pi. Ethylene-diamine-tetra-acetic acid (EDTA). Ethylene-glycol-bis-(P-aminoethylether)-N,N,N’,N’-tetra-acetic acid (EGTA). Glycine. Hydrochloric acid (HC1). Kieselguhr G (washed two times with acetone before use). Magnesium chloride (MgCy. N-2-hydroxyethylpiperazine-N-ethane-sulphonic acid (HEPES). Orthophosphoric acid. Perchloric acid. Potassium hydroxide (KOH). Potassium dihydrogen orthophosphate (KH2P04). Pyridine. Silica gel G F ^ (washed two times with acetone before use). Sodium citrate. Sodium hydroxide (NaOH). Sodium chloride (NaCl). Sodium carbonate (NajCOj). Sucrose. Sulphuric acid (H2S04). Thionyl chloride. Triethylamine. Water, AnalaR molecular biology grade. 2.1.2. From Sigma. Poole. UK. P-mercaptoethanol. 2-fluoro-1 -methylpyridinium-p-toluenesulphonate. 3-methoxy-4-hydroxyphenylacetic acid (homovaniUic acid). 4-bromobenzoic acid (p-bromobenzoic acid). 8-methyl-N-vanillyl-6-nonenamide (Capsaicin). 12-O-tetradecanoylphorbol-13-acetate (TPA). Adenosine triphosphate. Ammonium persulphate. Brilliant blue G (Coomassie blue). Dithiothreitol (DTT). Glycerol. Histone Type ni-S. Leupeptin hemisulphate. Phenylmethylsulphonylfluoride (PMSF). Sodium dodecyl sulphate (SDS). Trisma base (Tris). Triton X-100. 2.1.3. From Whatmann, Ashford. UK. 0.2pm filters. All filters used. Diethylaminoethyl cellulose (DEAE; DE52). Phosphocellulose ion exchange chromatographic paper. 1.4. From Bio-Rad. Hemel Hempstead. UK. Hydroxylapatite (HA). N,N,N’N’-tetramethylenediamine (TEMED). 2.1.5. From Rhone-Poulenc, Dagenham. UK. Charcoal (exhaustively soxhleted with chloroform before use). D,L-2-(3-benzoylphenyl)-propionic acid (KetoprofenR). 2.1.6. From Boots. Nottingham. UK. D,L-2-(3-isobutylphenyl)-propionic acid (IbuprofenR). 2.1.7. From Boehringer-Manheim. Lewis. UK. Acrylamide. N,N’ -methylenebisacrylamide. 110 2.1.8. From Pharmacia. Milton Kevnes. UK. Low molecular mass protein standard mixture (M, 94, 66, 43, 30, 20.1, 14.4 kDa). 2.1.9. From ICN biomedicals. High Wycombe. UK. Adenosine-5’-[y-32P]-triphosphate triethylammonium salt (32P-ATP), at 4500 Ci/mmol. 2.1.10. From Lipid Products. Knutford. UK. L-a-phosphatidylserine (PS). 2.1.11. From Vemon-Carus Ltd.. Preston. UK. Cotton wool (exhaustively soxhleted with acetone before use). 2.1.12. Phorbol esters. All phorbol esters were stored at -20°c under nitrogen; stock solutions and dilutions were made up in acetone and similarly stored. The following phorbol esters were isolated from natural sources in the laboratory of Professor Fred. J. Evans, Department of Pharmacognosy, The School of Pharmacy, London, UK: 9,13,14-ortobenzoyl-6,7-epoxy-5-hydroxy-resiniferonol- 12-cinnamate(Thymeleatoxin A, Thy A). 12-0-[2-methylaminobenzoyl]-4-deoxyphorbol-13-acetate (Sapintoxin A, Sap A). 12-0-[2-methylaminobenzoyl]phorbol-13-acetate (Sapintoxin D, Sap D). Other phorbol esters utilised throughout this project were isolated or synthesised as described in Chapter 3. 2.1.13. Experimental mice, from Charles River Ltd.. Margate. UK. Femake CD-I or BKTO mice, weighing <20g were housed three to a cage on I ll sawdust. Food and water were available ad libatum ; temperature and humidity were controlled to British legal specifications. 2.2. Apparatus. 2.2.1. *H-NMR spectroscopy. !H-NMR spectra were recorded on the following machines: i) 80MHz: Bruker WP 80 SY spectrometer, using TMS as internal standard (5 = O.OOOppm). ii) 250MHz: Bruker WM 250 spectrometer, using TMS as internal standard ( 8 = O.OOOppm). iii) 500MHz: Bruker AM 500 spectrometer, using TMS as internal standard ( 8 = O.OOOppm). 2.2.2. Mass spectrometry (MSI. All mass spectra were recorded on a VG Masslab 12-250 Quadrupole machine. i) Electron impact (EIMS): By direct insertion at 70eV at 170-220°c. ii) Chemical ionisation (CIMS): Using ammonia (NH*) as ionising gas at 170- 2 2 0 °c. iii) Thermospray MS: Using ammonium acetate buffer ionisation technique at 180-220°c. 2.2.3. Ultraviolet spectroscopy (UV). UV spectra were recorded on a Pye SP 8-100 spectrometer, using methanol as solvent. 112 2.2.4. Infrared spectroscopy (IR). ER spectra were recorded on a Perkin-Elmer 841 spectrometer, as a surface film on potassium bromide (KBr) discs. 2.2.5. Preparative thin layer chromatography (TLC1. Adsorption and partition preparative TLC plates were prepared using a Jobling moving spreader. 2.2.6. Centrifugal liquid chromatography (CLC). Kieselgel 60 P F ^ discs were used in conjunction with a Harrison Research model 7924 T chromatotron. 2.2.7. Scintillation counting. 32P samples were counted on a Beckmen LS 6000 TA liquid scintillation counter by Cherenkov counting. 2.2.8. Fast liquid protein chromatography (FPLC). Protein chromatography was conducted on a Pharmacia FPLC LCC 500plus system, with a Frac-100 fraction collector. 2.2.9. Centrifuges. The following centrifuges were used: i) MSE High Speed 18 centrifuge. ii) MSE bench top Micro-fuge. 113 iii) MSE bench top Chilspin 2 centrifuge. 2.2.10. pH meter. A Pye PW 9418 pH meter was used. 2.3. Methods. 2.3.1. Preparative thin layer chromatography (TLC). 2.3.1.1. Preparative adsorption TLC. 0.5mm thick silica gel G F ^ chromatographic plates buffered at pH 7.0 (see section 2.4, "buffers") were prepared using a Jobling moving spreader and activated at 1 1 0 °c for 1 - 2 hours, and allowed to cool before application of sample (normally not more than 10-15mg sample per plate). 2.3.1.2. Preparative partition TLC. 0.5mm thick kieselguhr G chromatographic plates buffered at pH 7.0 (see section 2.4, "buffers") were prepared using a Jobling moving spreader and activated at 110°c for 1-2 hours. After cooling, the plates were fully developed in 20% diethyleneglycol in acetone. They were then allowed to air dry for at least 30 minutes before application of sample (normally not more than 10-15mg sample per plate). 2.3.2. Centrifugal liquid chromatography (CLC). 2mm thick kieselgel 60 P F ^ discs were prepared by mixing 65g of silica with 130ml pH 7.0 buffers (see section 2.4, "buffers") (both cooled to 0-5°c before use), and the slurry poured onto a rotating glass disc. The silica was allowed to air dry for 48 hours at room temperature, before scraping excess silica off with the Harrison 114 Research scraping tools. The disc was placed into the chromatotron with a constant stream of nitrogen (15ml/min) and samples eluted at 6 -8 ml/min. Gradient systems employed are detailed in chapter 3, section 3. 2.3.3. Acetvlation of diterpene esters. Acetylation of mono-esters to di-esters was performed in a slight excess of pyridine/acetic anhydride (2:1) for 1 hour at 100°c (oil bath) under N2, or for 4 hours at room temperature. Reagents were evaporated in a stream of N 2 and the C^ acetates purified by preparative adsorption or preparative partition TLC. 2.3.4. Mild-base catalysed hydrolysis of diterpene esters. Di-esters were hydrolysed to mono-esters in a slight excess of 0.1 M KOH in dry MeOH under N 2 for 30-60 minutes at room temperature. The reaction was monitered by analytical TLC, and on completion the reaction products were tipped into ice cold distilled water, before being partitioned against CH 2C12, washed with water and then dried over anhydrous sodium sulphate. The products were purified by preparative adsorption or preparative partition TLC. 2.3.5. Mild-acid catalysed hydrolysis of diterpene esters. Di-esters were hydrolysed to mono-esters in a slight excess of 1% perchloric acid in dry MeOH under N 2 for 15 hours at room temperature. The reaction products were taken up in excess CH 2C12 and washed with: i) 5% NaCl and ii) water, before drying over anhydrous sodium sulphate. The products were purified by preparative adsorption or preparative partition TLC. 115 2.3.6. Protein kinase C activation assays. Protein kinase C activity was detected, and then utilised for further studies using the following assay, which contained: lOpl 3.5mg/ml Histone Type UI-S. lOpl enzyme fraction. lOpl 0.5mM CaCl 2 (or assay buffer). 5pi 2mg/ml phosphatidylserine (PS) (or assay buffer). lOul 32P-ATP mixture. 45pi Total volume. The assay was started by addition of lOpl 32P-ATP mixture, and terminated after 10 minutes incubation at 25°c by spotting 30pl of reaction mixture onto Whatman phosphocellulose ion exchange paper. The papers were washed four times in 5% acetic acid, or 70mM orthophosphoric acid, and the 32P incorporation into Histone counted by Cherenkov counting. For studies using phorbol ester probes, 5pl of the required dilution was added to the assay (giving a total volume of 50pl), and 35pl spotted onto the phosphocellulose paper on termination of the assay. Ca2+ and/or PS can be omitted from the assay reaction mixture to enable calcium/phospholipid dependencies of the enzyme or probes to be studied. 2.4. Buffers. 2.4.1. Kolthofrs Borax phosphate buffer (for preparative TLC and CLC1. The pH 7.0 buffer solution contained: 16.6g KH2P0 4 14.9g Borax Distilled water to 11. 116 This buffer was diluted 1:1 with distilled water immediately prior to use. 2.4.2. Anti-coagulant buffer. 72mM citric acid 90mM trisodium citrate lOOmM glucose. 2.4.3. Protein isolation/purification buffers. 2.4.3.1. Homogenisation buffer. 20mM Tris/HCl pH 7.5 (Stock kept at 4°c) 0.25M sucrose lOmM EGTA 2mM EDTA ImM DTT (added fresh) lOOpg/ml leupeptin (added fresh). Stored at 4°c. 2.4.3.2. Chromatography buffer "A". 20mM Tris/HCl pH 7.5, 4°c 5mM EGTA 2mM EDTA 60mM p-mercaptoethanol. 2.4.3.3. Chromatography buffer "B". As for chromatography buffer A, with 0.4M NaCl. 2.4.3.4. Potassium phosphate buffer. KH2P04; 1 part K2HP04; 4 parts : adjust to pH 7.5 2.4.3.5. Chromatography buffer "C". 20mM potassium phosphate buffer (above), pH 7.5, 4°c 5mM EGTA 117 2mM EDTA 60mM p-mercaptoethanol 2.4.3.6. Chromatography buffer "D". As chromatography buffer C, but 250mM potassium phosphate. 2.4.3.7. Chromatography buffer ME". As chromatography buffer C, but 500mM potassium phosphate. All chromatographic buffers were filtered through a 0.2pm filter and degassed by vacuum. 2.4.4. Protein kinase C assay buffers. 2.4.4.1. Assay buffer. 20mM Tris/HCl pH 7.5, stored at 4°c. 2.4.4.2. Substrate. 3.5mg/ml Histone Type ni-S made up in assay buffer (prepared fresh). 2.4.4.3. Phosphatidvlserine. For 1ml of 2mg/ml PS mixture: 70pl PS 20mg/ml (stock). Dry down under N2, then add a further 30pl 20mg/ml PS, and 970pl assay buffer. Vortex, and then sonicate, until a clear, translucent suspension is formed. 2.4.4.4.32P-ATP mixture. 0.145 mg ATP (cold) lOOmM MgCl2 0.04mCi 32P-ATP (adjusted to ensure specific activity of -300,000 cpm/assay) Water to 2ml. Water used for protein isolation/purification and enzyme assays was AnalaR molecular biology grade, or equivalent 118 Chapter 3: Isolation and synthetic modification of 12-deoxyphorbol and resiniferonol diterpene nuclei. 119 3.1. Introduction. Esters of 12-deoxyphorbol were originally isolated from the succulent Euphorbia species, E.triangularis (Gschwendt and Hecker, 1969,1974). Subsequently, esters of this phorbol nucleus were shown to occur naturally in eight other Euphorbia species (Hergenhahn, Kusomoto and Hecker, 1974; Evans, Kinghom and Schmidt, 1975; Kinghom and Evans, 1975a; Schmidt and Evans, 1977a, 1980a; Evans, 1978; Evans and Schmidt, 1979) (see Chapter 1, section 1.3.1 and Table 1), and two further genera of the Euphorbiaceae, Croton (Chavez et al, 1982) and Baliospermum (Ogura et al, 1978). One 12-deoxyphorbol ester, Prostratin (12-deoxyphorbol-13-acetate), has been isolated from Pimelea prostrata, of the plant family Thymelaeaceae (Casmore et al , 1976; Zayed et al, 1977a). The resiniferonol derivative, Resiniferatoxin (Rx), has been isolated from three species of the Euphorbia genus, E.resinifera, E.poissonii and E.unispina (Hergenhahn, Adolf and Hecker, 1975; Schmidt and Evans, 1976) (these species also represent three of those from which 12-deoxyphorbol esters have been isolated). Resiniferonol derivatives exhibit a A 6,7 double bond and are deoxygenated at C5. These features distinguish them from the classical daphnetoxin derivatives which possess a C 6 7 a- epoxide and a C 5 hydroxyl groups (see Chapter 1, section 1.3.3). Both 12-deoxyphorbol and resiniferonol derivatives differ in the spectrum of their biological activities when compared to TPA (Evans and Edwards, 1987). For example, 12-deoxyphorbol-13-O-phenylacetate (DOPP) is capable of stimulating production of a Transferable Aggregating Substance (TAS) from human platelets (Westwick, Williamson and Evans, 1980; Williamson et al, 1981). Rx is a more potent 120 pro-inflammatory agent than TPA on the mouse ear erythema model (Schmidt and Evans, 1979), yet is not a tumour promoter, and may exert its action via a novel kinase (Ryves et al, 1989). Thus, differences in these two nuclei from that of phorbol would seem to impart some diversity of biological action on these derivatives. The 12- deoxyphorbol and resiniferonol nuclei were, therefore, considered good candidates for synthetic modification, prior to pharmacological and biochemical studies, which would attempt to clarify their mechanism(s) of action. 3.2. Isolation of compounds from plant material. 3.2.1. Materials and methods. 3.2.1.1. Plant material. Euphorbia poissonii. Pax. (Euphorbiaceae) was identified by Professor David T Okpako, University of Ibadan, Nigeria, near Ibadan, Nigeria and the latex was collected into methanol. On arrival at the Department of Pharmacognosy, The School of Pharmacy, the methanol was reduced to a small volume under reduced pressure at <40°c. The latex was then re-suspended in acetone, stored at 4°c for two weeks and used for further chemical isolation. This acetone extract gave a positive "phorbol colour reaction" (Hecker, 1968), that is a deep red colour on brief boiling with a few drops of concentrated HC1. 3.2.1.2. Partitioning of extract. The partitioning process is summarised in Scheme 1. The acetone extract (500mls) was filtered and its volume decreased under reduced pressure at <40°c, giving a viscous residue. The residue was re-dissolved in methanol (MeOH) using a water bath if necessary (not greater than 60°c). The methanolic solution (~200ml) was then diluted to 90% MeOH by the addition of distilled water. 121 Remove MeOH 500ml latex in MeOH 500ml latex in Acetone Resuspend in Stand at 4°C for 2 weeks acetone +ve phorbol reaction Redissolve in Filtered, acetone removed MeOH under reduced pressure at 90% MeOH fraction < 40°C adjust to 90% Partition v s . Hexane until colourless Hexane layer -ve phorbol reaction. Discard 90% MeOH layer Dilute to 40% 40% MeOH fraction Partition vs. ether until colourless ______40% MeOH layer -ve phorbol reaction. Discard Ether layer +ve phorbol reaction i) Combine ether layers and reduce volume to 150ml (<40°C, reduced pressure ) ii) Back wash with a) 0.25% Na2C03 b) 5% NaCl 0.25% Na2C03 layer -ve phorbol reaction. Discard 5% NaCl layer -ve phorbol reaction. Discard Ether layer Remove ether 1.81g dry material Filter over anhydrous used for further sodium sulphate purification under reduced pressure at < 4 0 ° C Scheme 1 Partitioning of Latex 122 The 90% MeOH extract was then partitioned against hexane (3 x 100ml), until the hexane fraction was colourless and gave a negative phorbol colour reaction, to remove any steroids and other lipid material. The MeOH layer was further diluted to 40% MeOH, before partitioning against ether (3 x 100ml, until the ether layer was colourless). The ether layers, which gave a positive phorbol colour reaction, were combined and the volume reduced to ~ 150ml (at <40°c, under reduced pressure), before being back-washed with: i) 3 x 40ml 0.25% NajCOj solution, and ii) 3 x 40ml 5% NaCl solution. These back-washings were confirmed to contain no phorbol esters (negative phorbol colour reaction). The combined ether layers were then filtered over anhydrous sodium sulphate and the solvent removed under reduced pressure at <40°c. This gave a yield of 1.8lg of dry off white/yellow coloured material. Analytical TLC of this material (solvent system: CHCl/Acetone 8:2) gave the results shown in Table 9. This suggested that the extract contained 12-deoxyphorbol mono- and di-esters, resiniferatoxin and 9,13,14-orthophenylacetylresiniferonol (Ro), which would require further purification. 3.2.1.3. Purification of the ether extract. 3.2.1.3.1. Charcoal column chromatography. A charcoal column (15cm x 1cm) over cotton wool and celite (3cm x 1cm) was poured and equilibrated with 100% ethanol (EtOH). 300mg of the ether extract, dissolved in a minimum volume of EtOH, was then applied to the column. 25ml fractions were eluted from the column and monitored by analytical TLC in CHCyacetone (8:2); identical fractions were combined. The solvent gradient applied and the yields of compounds are shown in Table 10. The use of this column thus enabled further separation of the components of 123 Table 9. Analytical TLC of dried ether extract Rf value. Colour of spot Inference. After spraying with Under 356nm UV 60% HjSO*. light 0 . 2 1 Orange/brown/black Bright orange Resiniferonol 0.26 Orange/brown Bright orange 1 2 -deoxyphorbol monoesters 0.32 Orange/brown Bright orange 0.46 Brown No colour Unknown - Contaminant? 0.63 Black Bright orange Resiniferatoxin 0.70 Orange/brown Bright orange 1 2 -deoxyphorbol diesters 0.78 Orange/brown Bright orange The analytical TLC was run on 0.25mm thick pre-coated aluminium-backed TLC plates. The solvent system used was CHCyacetone (8:2). 124 Table 10. Composition of fractions eluted from the charcoal column. Fraction number. Solvent gradient Compounds Weight of present*. compound. 1 90% EtOH 1 2 -deoxyphorbol 194mg mono- and di esters elute as a 2 100% EtOH 3 mixture 4 5 6 EtOH/CHClj (9:1) Resiniferatoxin 55mg starts to elute 7 EtOH/CHClj (8:2) Resiniferatoxin 8 elutes 9 1 0 1 1 EtOH/CHCl3 (7:3) Resiniferonol 26mg 1 2 elutes 13 100% CHC13 Cleans column, 20-25mg 14 ensures all 15 material has 16 eluted. Non- phorbol ester material elutes. ‘Deduced from analytical TLC run in CHCyacetone (8:2), by comparison of Rf value and spot colour with original ether extract Some cross-contamination at adjacent fractions (e.g. 5/6; 10/11) was observed. 125 the ether extract. However, whilst Rx and Ro were eluted in a semi-purified form, separation of the di-ester and mono-ester components of the ether fraction was not as satisfactory as had been anticipated. These components could each be separated into two spots, of Rf value 0.21 and 0.26, and 0.70 and 0.78, for the mono-esters and di- esters respectively, by analytical TLC. Since large quantities of these components were eluted from the column, I decided to use the technique of Centrifugal Liquid Chromatography (CLC) for further purification. 3.2.1.3.2. Centrifugal liquid chromatography (CLQ of 12-deoxyphorbol mono- and di- ester fractions. CLC operates on essentially the same principle as TLC. The circular plate rotates at a constant rate. The plate is saturated with solvent The sample is then applied at the centre of the plate, eluted with solvent (gradient systems can be utilised) and fractions are collected. CLC has several advantages over TLC in that: i) a larger quantity of sample can be applied, ii) CLC is a rapid technique, iii) no silica dust is generated (this can be a particular problem of TLC separation of toxic diterpene esters) and, iv) separation by CLC with a particular solvent system should be almost identical to that observed by TLC separation using the same solvent system. In this instance, adsorption CLC with a 2mm thick plate was the method of choice, although partition methods can be developed. The plate was made as detailed in Chapter 2, section 2.3.2. The plate was saturated with the first solvent and 250mg sample, dissolved in a minimum volume of CHClj/acetone (1:1), was applied. 5ml fractions were collected; identical fractions by analytical TLC were combined and finally purified by preparative TLC (see section 3.2.1.3.3.). 3.2.1.3.2.1. CLC of mono- and di- ester mixture. The following gradient system was used to elute the mono- and di- ester 126 mixture from the charcoal column: 1. 30ml CHC13 2. 100ml CHCyacetone (8:2) 3. 30ml CHCyacetone (1:1) 4. 50ml acetone (wash). The elution profile of the mixture of components is shown in Table 11a. 3.2.1.3.2.2. CLC of mono- and di- ester mixture after hydrolysis. An alternative purification method by CLC was to hydrolyse the mixture of mono- and di- esters using either acid or base to catalyse the reaction (see Chapter 2, sections 2.3.4. and 2.3.5.). This yields a mixture of mono-esters which are then further purified by CLC. In this instance, the following gradient system was used: 1. 100ml CHCyacetone (8:2) 2. 50ml CHCyacetone (1:1) 3.50ml acetone (wash). The elution profile of components after hydrolysis is shown in Table lib. CLC purification of the mixture of components eluted from the charcoal column was a rapid process enabling the collection of relatively large quantities of well resolved compounds. Hydrolysis of diesters to monoesters was particularly useful for further synthetic work (see section 3.3). However, there was still some cross- contamination of compounds and initial 80MHz !H-NMR analysis indicated that all compounds contained some degree of lipid contamination, which was not observed on analytical TLC. This contaminant was probably either i) a remnant from initial partitioning and extraction of the E.poissonii latex, or ii) had arisen from the charcoal column purification stage (despite the charcoal being exhaustively soxhleted with CHCI3). Alternatively, the lipid contamination could represent a combination of these factors. Therefore, final purification of these compounds was by preparative TLC. 127 Tables 11a and lib . Composition of fractions after CLC purification. Fraction number. Rf value*. Compounds present*. Weight of compound. 1-4 - - - 5-7 0.78 Diester component 1 ~85mg 8 - 1 0 0.70 Diester component 2 ~50mg 1 1 - 1 2 0.63 Resiniferatoxin (Rx) ~8 mg 13-19 --- 2 0 - 2 2 0.32 Monoester component 1 ~50mg 23-25 0.26 Monoester component 2 ~2 0 mg 26-27 0 . 2 1 Resiniferonol (Ro) ~2 mg 28-32 + wash - - - Table 11a. Composition of CLC fractions of mono- and di- ester mixture. Fraction number. Rf value*. Compounds present*. Weight of compound. 1-16 --- 17-20 0.32 Monoester component 1 ~135mg 21-24 0.26 Monoester component 2 ~70mg 25-26 0 . 2 1 Resiniferonol (Ro) ~ 1 2 mg 27-30 + wash - - - Table lib. Composition of CLC fractions of mono- and di- ester mixture after acid- or base- catalysed hydrolysis. *Run by analytical TLC in CHCyacetone (8:2). +By comparison of Rf value and spot colour with original TLC extract Some cross-contamination of adjacent fractions was observed. 128 3.2.1.3.3. Preparative partition and adsorption TLC. 3.2.1.3.3.1. Preparative partition TLC. 0.5mm thick preparative partition TLC plates impregnated with digol were prepared as described in Chapter 2, section 2.3.1.2. 10-12mg of compound was streaked onto each plate, which was then run in cyclohexane/ethylacetate (7:3), using the technique of triple development to ensure resolution and narrowing of bands. One side of the plate was sprayed with 60% H 2S0 4 to visualise the bands (the rest of the plate being covered by a glass plate). Bands were then scraped off the plate and eluted over cotton wool and celite (2cm x 1cm) with CHCyacetone (2:1). The solvent was evaporated off at <40°c under reduced pressure, resulting in a small volume of glycol solution. The glycol solution was taken up with 1 vol. (15mls) CH 2C12 and partitioned against 5% NaCl solution (3x2 vol.) The CH 2C12 layer was filtered over anhydrous sodium sulphate and the filter washed with a further 10ml CH 2C12. The CH 2C12 solution was evaporated to dryness at <40°c under reduced pressure, the compound taken up in 2-3mls acetone and filtered through an HPLC grade filter. An analytical TLC was run in CHCyacetone (8:2) to ensure purity before drying under N 2 and storing at -20°c. Compounds thus purified were then sent for *H-NMR and mass spectral analysis. 3.2.1.3.3.2. Preparative adsorption TLC. 0.5mm thick preparative adsorption TLC plates were prepared as described in Chapter 2, section 2.3.1. 10-12mg of compound was streaked onto each plate, which was then run in CHCyacetone (8:2), using the technique of triple development to ensure resolution and narrowing of bands. The side of one plate was sprayed with 60% H2S0 4 to visualise the bands and, assuming a clear separation was observed, the other plates were then visualised by 254nm UV light thereby ensuring a maximum 129 yield of each compound. The bands were then scraped off the plate and eluted over cotton wool and celite (2cm x 1cm) with CHCyacetone (1:1). The solvent was evaporated off at <40°c under reduced pressure. The clear, colourless resin produced was taken up in 5-10mls acetone before filtering over anhydrous sodium sulphate and the filter washed with a further 5mls acetone. This was then evaporated off at <40°c under reduced pressure, and the compound taken up in 2-3mls acetone and filtered through an HPLC grade filter. Purity was confirmed by analytical TLC in CHCyacetone (8:2), before drying under N 2 and storing at -20°c. Compounds thus purified were then sent for *H-NMR and mass spectral analysis. Table 12 shows the partition and adsoiption TLC Rf values obtained for the components purified by CLC and charcoal column chromatography. Although all components were separable by both preparative partition and adsoiption TLC systems, the band width of components was greater in the partition system than the adsorption system. Additionally, digol coating of plates for partition TLC and subsequent extraction of compounds from the digol solution was time consuming. These factors suggest that the preparative TLC method of choice is the adsorption technique. Preparative adsorption TLC does, however, generate silica dust and therefore care must be taken when isolating these toxic phorbol esters by this method. If preparative adsoiption TLC plates were left open to the atmosphere for 2-3 days, a bright yellow band would appear (Rf=0.46). This was presumably the lipid contaminant, which had become visible on oxygenation (however, no confirmatory spectra were run). This band constituted -65-70% of the mass of compounds applied to each preparative TLC plate. Spectra of isolated compounds (see results section, 3.2.2.) indicated that these compounds were pure, and suitable for pharmacological and biochemical analysis. 130 Table 12. Preparative partition and adsorption TLC Rf values of fractions previously semi-purified by CLC or charcoal column chromatography. Compound. Source. Rf value*. Partition Adsorption Resiniferatoxin Charcoal column 0.43 0.63 Resiniferonol 0.26 0 . 2 1 Diester component 1 CLC 0.58 0.78 Diester component 2 0.53 0.70 Resiniferatoxin 0.43 0.63 Monoester component 1 0.36 0.32 Monoester component 2 0.32 0.26 Resiniferonol 0.26 0 . 2 1 Monoester component 1 CLC after acid- 0.36 0.32 or base- Monoester component 2catalysed 0.32 0.26 Resiniferonol hydrolysis 0.26 0 . 2 1 ’Solvent systems: Adsorption TLC: CHCyacetone (8:2), triple developed. Partition TLC: Cyclohexane/ethylacetate (7:3), triple developed. Rf values shown are those of the major component Minor components that had cross-contaminated were separable and, subsequent to *H-NMR and MS confirmation, were pooled with the respective major component 131 300mg ether extract Gradient charcoal Column chromatography Pure Rx i) Rx fraction (fract.no. 6-10) • Purification by __ Pure Ro Preparative \k ii) Ro fraction (fract.no. 11-11) Partition/. Some di and Adsorption TLC monoesters i i iii) Mixed di and monoester fraction ------1 (fract.no. 1-5) Acid/base hydrolysis Gradient CLC Mixed monoesters Diester component 1 Gradient CLC Diester component 2 Resiniferatoxin Monoester component 1 Monoester component 1 Monoester component 2 Monoester component 2 Resiniferonol Resiniferonol Preparative Partition/Absorption TLC Pure compounds for Spectral Analysis Pure Rx Pure Diester 1 Pure Monoester 1 Pure Ro Pure Diester 2 Pure Monoester 2 (used for synthetic work) Scheme 2 Summary of Purification of Ether Extract of Latex Compounds isolated from the latex of E.poissonii. were also used as starting material for synthetic work (see section 3.3.). Scheme 2 summarises the purification steps performed on the original ether extract of the latex of E.poissonii. 3.2.2. Results. The following compounds were isolated from the latex of E.poissonii. by the above methods. Their structures (see Fig. 18.) were assigned on the basis of their 1H- NMR, MS, UV and IR spectroscopic characteristics, by comparison with authentic spectra. 3.2.2.1. Resiniferonol derivatives. 3.2.2.1.1.9.13.14-orthophenvlacetvlresiniferonol-20-Q-homovanillate (Resiniferatoxin. Rx). This compound was the major component of the resiniferatoxin fraction eluted from the charcoal column and the minor component of the resiniferonol and mixed mono- and di- ester fractions. Hence, additional material was isolated by the CLC fractionation of the mono- and di- ester mixture. Rx was recovered as a clear, colourless resin, forming a brittle white foam when final traces of solvent were removed under high vacuum, in final yield of 92mg/500ml latex. Rx had an Rf value of 0.63 in CHCyacetone (8:2) and was a single black spot after spraying with 60% H 2S04, which flouresced bright orange under 356nm UV light. Rx exhibited the following spectral characteristics: EIMS: Peaks at m/z: 628 (NT, 27%, C j^ O ,) 428 (<5%) 310 (36%, QoHzA)610 (<5%) 310 (36%, QoHzA)610 492 (<5%) 292 (5%) 474 (<5%) 137 (100%) 446 (<5%) 133 CHoOR 20 L Resiniferonol Derivatives R OMe 9,13,14,orthophenylacetylresiniferonOl- 20-0-homovanillate (Rx) frCH2-^@-°H 0 9,13,14,orthophenylacetylresiniferonol (Ro) H 12-Deoxyphorbol Derivatives J?2— 12-deoxyphorbol-13-0-phenylacetate- — C— CHo” \0/ ”C“ CHq 20-0-acetate II Z N' II ^ 0 0 12-deoxyphorbol-13-0-phenylacetate M H CH. CH. 1 I 12-deoxyphorbol-13-0-angelate- C“ C = C “C“ CH^ 20-0-acetate \ n n H 0 12-deoxyphorbol-13-0-angelate n H Figure 18 Structures of compounds isolated from the latex of E.poissonii. Pax. 134 UV (MeOH): at: 276, 228 nm IR (KBr disc, surface film): at: 3460, 2950, 1720, 1520, 1280, 1020, 910, 700 cm 1 *H-NMR (500MHz, CDC13) (see Fig. 19.): 8 , ppm Integration. Multiplicity. Inference. 7.43 1H s H-l 7.27-7.37 5H m Aromatics 6.79 3H m Qo-aromatics 5.87 1H s H-7 4.71 2H s H-16 4.52, 4.54} 2H dd 2H-20 4.58, 4.60} (J=17.12Hz) 4.19, 4.20 1H d H-14 (J=2.6Hz) 3.87 3H s C20-Ar-O-Ch 3 3.60 2H s Cjo-^-CHj 3.20 2H s 4>-ch 2 3.08 1H bs H-10 3.04 1H bs H- 8 2.63 1H m H -ll 2.12, 2.17 2H ABq 2H-5 (J=l 7.19Hz) 2.06 2H m 2H-12 1.82 3H m 3H-19 1.53 3H s 3H-17 0.95, 0.96 3H d 3H-18 (J=7.08Hz) A one proton signal at 6=5.52ppm was not evident after addition of D 20. Acid/base catalysed hydrolysis (see Chapter 2, sections 2.3.4. and 2.3.5.) of Rx produced 9,13,14-orthophenylacetylresiniferonol (Resiniferonol, Ro) in approximately 80% yield (confirmed by JH-NMR and mass spectra, as for Ro, below). CDC13 ro J L CSI CO ao \o IO co o m ‘O O O v CM o ' ,5 ro o o O CM CM 00 ro CM -M CM CM CM o CM O CM . o O in vo VO o in in in in in O ro (O in CO O o CM in CM rH lo in Ok a* S • • • • • F igu re 19 500 MHZ. 'h-NMR of r e s i n i f e r a t o x i n (Rx) in CDCI3 136 3.2.2.1.2. 9.13,14-orthophenvlacetvlresiniferonol (Resiniferonol. Ro). This compound was the major component of the resiniferonol fraction eluted from the charcoal column and a minor component of the resiniferatoxin fraction; additional material was isolated by the CLC fractionation of the mono- and di- ester mixture and after hydrolysis of this fraction (Rx converted to Ro). Ro was recovered as a clear, colourless resin in final yield of 46mg/500ml latex. Ro had an Rf value of 0.21 in CHCyacetone (8:2), and was a single black spot after spraying with 60% H2S04, which flouresced bright orange under 356nm UV light Ro exhibited the following spectral characteristics: 'H-NMR (250MHz, CDC13): 5, ppm. Integration. Multiplicity. Inference. 7.52 1H s H-l 7.35 5H m Aromatics 5.87 1H s H-7 4.71 2H s H-16 4.27 2H s 2H-20 4.09 1H d H-14 (J=10.0Hz) 3.64 1H s H-10 3.57 1H s H- 8 3.21 2H s <|>-ch 2 2.35 2H s H-5 2.04-2.17 3H m 2H-12, H -ll 1.82 3H m 3H-19 1.55 3H s 3H-17 0.97 3H d 3H-18 (J=7.1Hz) A one proton signal at 5=2.95 was not evident after addition of D 20. MS (Thermospray) (see Fig.20.): m/z peaks at: 465 (M++l, 100%, C^HjA ) 311 (55%) 447 (5%) 293 (37%) 429 (<5%) 138 (22%) 329 (12%) 465 oeuq aTes % aATqexsH aouepunqe 137 138 UV (MeOH): at: 232nm IR (KBr disc, surface film): Vmtx at: 3440, 2920, 1700, 1460, 1380, 1240, 1020, 910, 760, 700 cm 1. 3.2.2.2. 12-deoxvphorbol derivatives. 3.2.2.2.I. 12-deoxvphorbol-13-O-phenvlacetate-20-Q-acetate (POPPA). This compound was the major constituent of diester component 2 and a minor constituent of diester component 1 of the CLC fractionation of the mono- and di- ester mixture eluted from the charcoal column; additional material was also isolated from the resiniferatoxin fraction eluted from the charcoal column. DOPPA was recovered as as a clear, colourless resin in final yield of 66mg/500ml latex. DOPPA had an Rf value of 0.70 in CHCyacetone (8:2), and was a single brown/orange spot after spraying with 60% H 2S04, which flouresced bright orange under 356nm UV light DOPPA exhibited the following spectral characteristics: MS (Ammonia Cl): peaks at m/z: 526 (M++NH/, 8 %) 400 (15%) 508 (M+, <5%, C 30H3A) 355 (90%) 449 (<5%) 340 (5%) 431 (8 %) 313 (30%) 418 (<5%) 295 (100%, Q oHjA) UV (MeOH): at: 251, 225nm IR (KBr disc, surface film): Vm„ at: 3460, 2920, 2840, 2320, 1740 cm 1 139 *H-NMR (250MHz, CDC13) (see Fig.21): 5, ppm Integration. Multiplicity. Inference. 7.59 1H bs H-l 7.29 5H m Aromatics 5.69 1H d H-7 (J=4.1Hz) 4.44 2H s 2H-20 3.61 2H s 0 . 8 6 3H d 3H-18 (J=6.3HZ) 0.75 1H d H-14 (J=5.4Hz) A one proton signal at 5=5.30 was not evident on addition of D 20. Acid/base catalysed hydrolysis (see Chapter 2, sections 2.3.4. and 2.3.5.) of DOPPA produced 12-deoxyphorbol-13-0-phenylacetate (DOPP) in approximately 80% yield (confirmed by *H-NMR and mass spectra as for Dopp, below). 3.2.2.2.2. 12-deoxvphorbol-13-0-phenvlacetate (DOPP). This compound was the major constituent of monoester component 2, a minor constituent of monoester component 1 and the resiniferonol component of the CLC fractionation of the mono- and di- ester mixture eluted from the charcoal column. DOPP was also isolated from the hydrolysis of diesters and their subsequent CLC fractionation (DOPPA converted to DOPP). DOPP was recovered as a clear, colourless resin in final yield of 27mg/500ml latex. DOPP had an Rf value of 0.26 in CH^O £ VO X 1 L rH X 1 CM o J L O ro oo in ON ON CO m u X (M i O 0 0 ro iH ,LJ ,LJ ° 5 o J L X o J L ro on 1 CNl m CM CM o CM X I rH X 1 141 CHCyacetone (8:2) and was a single brown/orange spot after spraying with 60% H2S04, which flouresced bright orange under 356nm UV light DOPP exhibited the following spectral characteristics: ‘H-NMR (250MHz, CDC13): ______ 8 , ppm. Integration. Multiplicity. Inference. 7.57 1H bs H-l 7.27 5H m Aromatics 5.64 1H d H-7 (J=4.8Hz) 4.00 2H s 2H-20 3.60 2H s 4>-ch 2 3.25 1H m H-10 2.96 1H m H- 8 2.46 2H bs H-5 2.06-2.17 3H m 2H-12, H -ll 1.77 3H m 3H-19 1.03 6 H s 3H-16, 3H-17 0.87 3H d 3H-18 (J=6.3Hz) 0.75 1H d H-14 (J=5.2Hz) A one proton signal at 8=5.30 was not evident on addition of D 20. MS (Ammonia Cl): peaks at m/z: 484 (M++NH4+, 8 %) 357 (30%) 466 (M \ <5%, Q gH jA ) 339 (7%) 449 (<5%) 312 (25%) 430 (<5%) 294 (100%, QoH^O^ 375 (5%) UV (MeOH): ^ at: 253, 226nm IR (KBr disc, surface film): Vmtx at: 3420, 2920, 2840, 2300, 1740 cm 1 Acetylation of DOPP (see Chapter 2, section 2.3.3.) produced 12- deoxyphorbol-13-O-phenylacetate-20-O-acetate (DOPPA) in approximately 80% yield 142 (confirmed by *H-NMR and mass spectra as for DOPPA, above). 3.2.2.2.3. 12-deoxvphorbol-13-0-angelate-2Q-Q-acetate (DAA). This compound was the major one isolated from diester component 1 and the minor one isolated from diester component 2 of the CLC fractionation of the mono- and di- ester mixture eluted from the charcoal column; additional material was also isolated from the resiniferatoxin fraction eluted from the charcoal column. DAA was recovered as a clear, colourless resin in final yield of 11 lmg/500ml of latex. DAA had an Rf value of 0.78 in CHCyacetone (8:2) and was a single brown/orange spot when sprayed with 60% H 2S04, which flouresced bright orange under 356nm UV light. DAA exhibited the following spectral characteristics: MS (Thermospray): peaks at m/z: 490 (M++18, 12%) 330 (<5%) 473 (M++l, 5%, C ^ C V f l) 313 (95%) 391 (<5%) 295 (100%, Q o H ^ + l) 348 (9%) UV (MeOH): at: 249nm IR (KBr disc, surface film): Vm„ at: 3400, 2840, 1760, 1360, 1320, 1240, 1160, 760 cm 1 143 'H-NMR (250MHz, CDC13): 5, ppm. Integration. Multiplicity. Inference. 7.61 1H bs H-l 6.18, 6.16 1H dd =CH(CH3) (J=2.4Hz, 5.0Hz) 5.71 1H d H-7 (J=5.1Hz) 4.44 2H s 2H-20 3.28 1H m H-10 3.04 1H m H- 8 2.49 2H bs 2H-5 2.04-2.13 3H m 2H-12, H -ll 2 . 0 2 3H s CHa-CO 1.87 3H d OC-C(CH3)=CH- (J=2.4Hz) 1.76 3H m 3H-19 1.24 3H d =CH-CH3 (J=6.1Hz) 1.04 6 H s 3H-16, 3H-17 0.87 3H d 3H-18 (J=6.0Hz) 0.77 1H d H-14 (J=5.4Hz) A one proton signal at 5=5.8 lppm was not evident on addition of D 20. Acid/base catalysed hydrolysis (see Chapter 2, section 2.3.4 and 2.3.5.) of DAA produced 12-deoxylphorbol- 13-O-angelate (DA) in approximately 80% yield (confirmed by ]H-NMR and mass spectra as for DA, below). 3.2.2.2.4. 12-deoxvphorbol-13-O-angelate (DA). DA was the major compound isolated from monoester component 1 and a minor constituent of monoester component 2, from the CLC fractionation of the mono- and di- ester mixture eluted from the charcoal column. Additional material was also isolated from the resiniferatoxin fraction eluted from the charcoal column and from the hydrolysis of diesters and their subsequent fractionation by CLC (DAA converted to DA). DA was recovered as a clear, colourless resin in final yield of 57mg/500ml of latex. DA had an Rf value of 0.32 in CHCyacetone (8:2) and was a single brown/orange spot when sprayed with 60% H 2S04, which flouresced bright orange under 356nm UV light DA exhibited the following spectral characteristics: 'H-NMR (250MHz, CDC13): 5, ppm. Integration. Multiplicity. Inference. 7.60 1H bs H-l 6.18, 6.16 1H dd CH(CH3) (J=2.5HZ, 5.2Hz) 5.70 1H d H-7 (J=5.2Hz) 4.02 2H s 2H-20 3.27 1H m H-10 3.03 1H m H- 8 2.50 2H bs 2H-5 2.01-2.17 3H m 2H-12, H -ll 1.87 3H s OC-C(CH3)=CH- 1.76 3H m 3H-19 1.25 3H d =CH-CH3 (J=6.3Hz) 1.03 6 H s 3H-16, 3H-17 0 . 8 8 3H d 3H-18 (J=6.0Hz) 0.76 1H d H-14 (J=5.4Hz) A one proton signal at 8=5.13ppm was not evident on addition of D 20. 145 MS (Thermospray): peaks at m/z: 448 (M++18, 14%) 348 (7%) 431 (NT+1, 5%, C ^ H ^ + l) 330 (6 %) 413 (22%) 313 (97%) 395 (<5%) 295 (100%, QoH^Oj+l) UV (MeOH): at: 247nm IR (KBr disc, surface film): Vm#x at: 3400, 2920, 1700, 1380, 1320, 1240, 1160, 760 cm 1 Acetylation (see Chapter 2, section 2.3.3.) of DA produced 12-deoxyphorbol- 13-O-angelate-20-O-acetate (DAA) in approximately 80% yield (confirmed by ]H- NMR and mass spectra as for DAA, above). 3.3. Selective esterification of the C™ primary hydroxyl group of Resiniferonol (Ro) and 12-deoxvphorbol-13-O-angelate (DA). 3.3.1. Methods and materials. 3.3.1.1. Esterification with propionic acid derivatives and p-bromobenzoic acid. Two anti-inflammatory propionic acid derivatives, DL-2-(3-benzoylphenyl)- propionic acid (Ketoprofen, Rhone-Poulenc) and DL-2-(3-isobutylphenyl)-propionic acid (Ibuprofen, Boots), and p-bromobenzoic acid were used for selective esterification of the Cjq primary hydroxyl of Ro and DA. 3.3.1.1.1. Preparation of acid chlorides. Acid chlorides of the three acids were prepared as follows: Approximately 500mg of the acid was dissolved in excess thionyl chloride and heated (oil bath) under reflux for 30 minutes. Excess thionyl chloride was removed in a stream of N2, and the residue taken up in dry toluene. This mixture was allowed to boil freely to expel traces of thionyl chloride, hydrogen chloride and sulphur 146 dioxide, and then boiled down to a small volume before the remainder of the toluene was removed in a stream of N2. This left the acid chloride residue which was used without further purification. 3.3.1.1.2. Diterpene ester reaction with the acid chlorides. Qo esterification of Ro and DA with the respective acid chlorides was performed as follows: A slight excess of the acid chloride was added to solution of the diterpene monoester (5mg) in dry pyridine (1ml). The mixture was allowed to stand at room temperature for 2 hours (15 hours for the reaction with p-bromobenzoic acid chloride). The reaction mixture was then taken up with CH 2C12 (20mls) and washed with 0.25% N a^O j solution (2 x 20ml) and 5% NaCl solution (2 x 20ml). Subsequent evaporation of the CH 2C12 at <40°c under reduced pressure gave a crude diester residue. The diester residue was purified by preparative adsorption TLC, using CHCyacetone (8:2) as a solvent system (plates were triple developed), as described in section 3.2.I.3.3.2. These reactions produced six compounds (see section 3.3.2.1) for pharmacological (see Chapter 5) and biochemical (see Chapter 6 ) analysis. 3.3.1.2. Esterification with 3-methoxv-4-hvdroxv-phenvlacetic acid (Homovanillic acid). C^o esterification of Ro and DA with 3-methoxy-4-hydroxy-phenylacetic acid (homovanillic acid) was performed as follows: 10-12mg of the diterpene monoester was dissolved in 1.3ml CH 2C12, to which 27mg (37.5pl) triethylamine was added. 80mg 2-flouro-l-methylpyridinium-p-toluene sulphate was added to the reaction mixture, which was then allowed to stand at room temperature for 30 minutes. The solvent was evaporated off under a stream of N 2 and 147 1.3ml of toluene/acetone (1:1) added. 45mg (62.5pl) triethylamine and 80mg homovanillic acid were added to the reaction mixture, which was stirred for 1.5 hours at 60°c (oil bath). Subsequently, 5mls Kolthoff s buffer (see Chapter 2, sectioin 2.4) was added and the reaction stirred. The reaction mixture was then extracted with 3 x 10ml ethylacetate and these fractions combined. The ethylacetate was dried over anhydrous sodium sulphate and then evaporated to dryness at <40°c under reduced pressure giving a crude diester residue. The diester residue was purified by preparative adsorption TLC, using CHCyacetone (8:2) as a solvent system (plates were triple developed), as described in section 3.2.1.3.3.2. These reactions produced two compounds (see section 3.3.2.2.) for pharmacological analysis (see Chapter 5). 3.3.2. Results. 3.3.2.1. Ro and DA propionic acid derivative and p-bromobenzoic acid diesters. The following compounds were synthesised by the above methods (section 3.3.1.1); their structures (see Fig.22.) were assigned on the basis of their ^-NM R, MS, UV and IR spectroscopic properties: 3.3.2.1.1. 9.13.14-orthophenvlacetvlresiniferonol-20-Q-rDL-2-(3-benzovlphenvl)- propionatel (RoK). This compound was synthesised by the reaction of Ro with DL-2-(3- benzoylphenyl)-propionic acid chloride, giving a yield of approximately 70%. RoK had an Rf value of 0.71 in CHCyacetone (8:2), and was a single black spot after spraying with 60% H 2S04, which fluoresced bright orange under 356nm UV light. RoK exhibited the following spectral characteristics: WH 3 XH ch 2 or c h 2o r Resiniferonol 12-deoxyphorbol-13-0-angelate Derivatives Derivatives R = - C - C H - CHo n DL-2-(3benzoylphenyl) propionate 0 (Ketoprofen Rhone-Poulenc) R = - C - C H - C H 3 DL-2-(4-isobutylphenyl) propionate (Ibuprofen f Boots) R p-bromobenzoate OMe 0 3-Methoxy-4-hydroxyphenyl R - c - c h 2 OH II u acetate 0 (Homovanillate) Figure 22 Structures of C2 0 derivatives synthesised from 12-deoxyphorbol-13-0-angelate and resiniferonol nuclei. 149 'H-NMR (250MHz, CDQj): 8 , ppm. Integration. Multiplicity. Inference. 7.62-7.61 5H m J^Aromatics (O ) 7.59 1H s H-l 7.44-7.50 4H m Aromatics 7.33-7.41 5H m Ro aromatics 5.87 1H bs H-7 4.70 2H s 2H-16 4.53 2H s 2H-20 4.22 1H dd OC-CH-CH3 (J=11.5Hz) 4.18 1H d H-14 (J=7.4Hz) 3.18 2H s (|)-CH2 3.13 1H s H-10 3.00 1H s H- 8 2.63 2H s 2H-5 2.17 6 H m CH3-CH, 2H-12, H -ll 1.78 3H m 3H-19 1.51 3H s 3H-17 0.96 3H d 3H-18 (J=7.2Hz) MS (Theimospray): peaks at m/z: 718 (M*+18, <5%) 311 (5%) 701 (M*+l, 15%, Q.H .A ) 293 ( 8 %) 595 (<5%) 283 (12%) 465 (<5%) 255 (100%, C16H1302+18) 428 (<5%) 238 ( 6 %, C16H130 2+1) 343 (60%) 329(6%) 209 (17%) 150 UV (MeOH): at: 249nm IR (KBr disc, surface film): V ,^ at: 3420, 2920, 2240, 1730, 1640, 1380, 1180, 1020, 820, 700 cm *1 3.3.2.1.2. 9,13.14-orthophenvlacetvlresiniferonol-20-Q-rDL-2-(4-isobutvlphenvl)- propionatel (Roll. This compound was synthesised by the reaction of Ro with the DL-2-(4- isobutylphenyl)-propionic acid chloride, giving a yield of approximately 70%. Rol had an Rf value of 0.74 in CHClj/acetone (8:2) and was a single black spot after spraying with 60% H 2 S04, which flouresced bright orange under 356nm UV light. Rol exhibited the following spectral characteristics: MS (Thermospray): peaks at m/z: 653 (M++l, 14%, Q jHA) 295 (56%) 500 (80%) 239 (100%) 491 (<5%) 224 (10%, C13H1802+18) 331 (<5%) 206 (15%, Cu HjjOj) 313 (7%) 161 (50%) UV (MeOH): at: 267, 259(shoulder), 252(shoulder), 247(shoulder) IR (KBr disc, surface film): Vmjlx at: 3420, 2860, 2360, 1740, 1380, 1160, 1020, 780, 700 cm 1 151 *H-NMR (250MHz, CDC13): 8 ,ppm. Integration. Multiplicity. Inference. 7.43 1H s H-l 7.17-7.37 5H m Ro aromatics lAromatics 7.05-7.11 4H m 5.80 1H bs H-7 4.69 2H s 2H-16 4.52 2H s 2H-20 4.21 1H d H-14 (J=4.8Hz) 4.13 1H d d o c -c h - c h 3 (J=3.4Hz) 3.69 2H m -CHj-CH 3.51 1H m CH2-CH-(CH3) 2 3.20 2H s 4>-ch 2 3.04 2H m H-8 ,H-10 2.45 2H s 2H-5 2.42 3H d CH3-CH-CO (J=3.1Hz) 2.02-2.33 3H m 2H-12, H -ll 1.81 3H m 3H-19 1.51 3H s 3H-17 0.90 6 H d (CH3)?-CH-CH2 (J=2.1Hz) 0.87 3H d 3H-18 (J=2.0Hz) 3.3.2.1.3. 9.13.14-orthophenvlacetvlresiniferonol-20-O-rp-bromobenzoatel (RoB). This compound was synthesised by the reaction of Ro with the p-bromobenzoic acid chloride, giving a yield of approximately 70%. RoB had an Rf value of 0.72 in CHClj/acetone (8:2), and was a single black spot after spraying with 60% H 2S04, 152 which flouresced bright orange under 356nm UV light RoB exhibited the following spectral characteristics: ‘H-NMR (250MHz, CDQ,): ____ 8 , ppm Integration. Multiplicity. Inference. 7.91, 7.87} 4H d d 7.61, 7.57} (J=8 .8 , 8.5Hz) ? Br 7.55 1H s H-l 7.35 5H m Ro aromatics 6 . 0 1 1H bs H-7 4.78 2H s 2H-16 4.71 2H s 2H-20 4.27 1H d H-14 (J=7.4Hz) 3.21 2H s <|)-CH2 3.16 2H m H-10, H- 8 2.35 1H m H -ll 2.16 2H s 2H-5 2.09 2H m 2H-12 1.83 3H m 3H-19 1.55 3H s 3H-17 0.96 3H d 3H-18 (J=7.0Hz) MS (Theimospray): peaks at m/z: 649 (8 IBrM++l, 35%, C,5H3J0 78 lBr) 446 (<5%) 647 ( 79 BrM++l, 28%, CjjHjA^Br) 428 (<5%) 494 (<5%) 328 (5%) 492 (<5%) 311 (15%) 482 (<5%) 291 (100%, C y ijA ) 473 (15%) 229 (60%) 471 (7%) 185 (37%, C fl,0,"B r) 464 (5%) 183 (31%, C 7 H40 , 79 Br) 149 (45%) UV (MeOH): 7 at: 277 (shoulder), 272(shoulder), 266(shoulder), 241nm IR (KBr disc, surface film): V ^ at: 3440, 2860, 2360, 1720, 1370, 1010, 760 cm 1 153 3.3.2.1.4.12-deoxvphorbol-13-0-angelate-2Q-0-rDL-2-(3-benzovlphenvl)-propionate1 (DAK). This compound was synthesised by the reaction of DA with the DL-2-(3- benzoylphenyl)-propionic acid chloride, giving a yield of approximately 70%. DAK had an RF value of 0.73 in CHClj/acetone (8:2) and was a single orange/brown spot after spraying with 60% H 2S04, which flouresced bright orange under 356nm UV light. DAK exhibited the following spectral characteristics: MS (Thermospray): peaks at m/z: 684 (M++18, 27%) 345 (12%) 667 (M++l, 29%, Q jH A + l) 330 (10%) 649 (<5%) 312 (15%) 531 (5%) 290 (22%) 433 (5%) 255 (100%, C16H1302+18) 392 (7%) 238 (5%, C 16H130 2+1) 362 (10%) 209 (8 %) UV (MeOH): at: 246 (shoulder), 235nm IR (KBr disc, surface film): Vm4X at: 3420, 2910, 2360, 1700, 1380, 1280, 1020, 800, 700 cm 1 154 'H-NMR (250MHz, CDQj): 8 , ppm Integration. Multiplicity. Inference. 7.69-7.81 5H m (Q) Aromatics 7.54 1H bs H-l JLAromatics 7.48-7.54 4H m ' e y y 6.17 1H m = c h -c h 3 5.74 1H d H-7 (J=7.1Hz) 4.30 2H s 2H-20 4.28 1H dd < x -c h -c h 3 (J=7.0Hz) 3.37 1H m H-10 3.23 1H m H- 8 2.38 2H bs 2H-5 2.32 3H d CIt-CH-CO (J=7.6Hz) 2.10-2.17 3H m 2H-12, H -ll 1 . 8 8 3H s OC-C(CH3)=CH- 1.74 3H m 3H-19 1.19 3H d CH-QL (J=7.4Hz) 1.03 6 H s 3H-16, 3H-17 0 . 8 8 3H d 3H-18 (J=7.0Hz) 0.82 1H d H-14 (J=6 .8 Hz) 155 3.3.2.1.5. 12-deoxvphorbol-13-O-angelate-20-O-rDL-2-(4-isobutvlphenvl)-propionatel (DAI). This compound was synthesised by the reaction of DA with the DL-2-(4- isobutylphenyl) propionic acid chloride, giving a yield of approximately 70%. DAI had an RF value of 0.76 in CHClj/acetone (8:2) and was a single orange/brown spot after spraying with 60% H 2S04, which flouresced bright orange under 356nm UV light. DAI exhibited the following spectral characteristics: MS (Thermospray): peaks at mjz: 636 (M++18, 7%) 313 (5%) 619 (M++l, <5%, QjH^Og+l) 295 (8 %) 601 (<5%) 290 (17%) 518 (5%) 267 (40%) 460 (<5%) 235 (100%) 413 (<5%) 190 (<5%, C13H17 0,+1) 391 (10%) 161 (37%) 330 (<5%) UV (MeOH): \ max at: 292, 230 (shoulder), 222 (shoulder) nm IR (KBr disc, surface film): Vmix at: 3440, 2920, 1700, 1340, 1280, 1040, 720 cm *1 156 ‘H-NMR (250MHz, CDC1,): 5, ppm. Integration. Multiplicity. Inference. 7.53 1H s H-l 4H m 7.07-7.19 & ^Aromatics 6.19, 6.21 1H dd =CH(CH3) (J=2.7, 5.4Hz) 5.70 1H d H-7 (J=5.4Hz) 4.35 2H s 2H-20 4.21 1H dd OC-CH-CH3 (J=5.6Hz) 3.65 2H m -CHj-CH- 3.57 1H m CH2-CH-(CH3) 2 3.27 1H m H-10 3.06 1H m H- 8 2.43 2H bs 2H-5 2.36 3H d CHa-CH-CO (J=5.2Hz) 2.05-2.17 3H m 2H-12, H -ll 1.85 3H s OC-C(CH3)=CH- 1.75 3H m 3H-19 1 . 2 2 3H d CH-QL (J=5.4Hz) 1 . 0 2 6 H s 3H-16, 3H-17 0.90 6 H d (CH,),-CH-CH,- (J=5.3Hz) 0 . 8 6 3H d 3H-18 (J=5.2Hz) 0.78 1H d H-14 (J=5.6Hz) 157 3.3.2.1.6. 12-deoxvphorbol-13-O-angelate-20-O-rp-bromobenzoatel (DAB). This compound was synthesised by the reaction of DA with the p- bromobenzoic acid chloride, giving a yield of approximately 70%. DAB had an Rf value of 0.75 in CHGj/acetone (8:2), and was a single brown/orange spot after spraying with 60% H 2S04, which fluoresced bright orange under 356nm UV light DAB exhibited the following spectral characteristics: *H-NMR (250MHz, CDC13): 6 , ppm. Integration. Multiplicity. Inference. 7.88, 7.87} 7.58, 7.55} 4H dd (J=8 .6 , 8 .6 Hz) Aromatics Br 7.64 1H s H-l 6.17 1H dd = c h -c h 3 (J=1.6, 7.3Hz) 5.82 1H d H-7 (J=5.5Hz) 4.72 2H s 2H-20 3.35 1H m H-10 3.05 1H m H- 8 2.51 2H bs 2H-5 2.02-2.18 3H m 2H-12, H -ll 1.87 3H s OC-C(CH,)=CH- 1.74 3H m 3H-19 1.23 3H d CH-CH 3 (J=6.2Hz) 1.03 6 H s 3H-16, 3H-17 0 . 8 8 3H d 3H-18 (J=3.0Hz) 0.80 1H d H-14 (J=5.9Hz) 158 MS (Thermospray): peaks at mjz: 667 (81BrM++18, <5%) 497 (7%) 665 (79BrM++18, <5%) 495 (10%) 649 (81 BrM+, 5%, 395 (<5%) 647 ( 79 BrM+, <5%, C35H360 779 Br) 313 (15%) 631 (<5%) 295 (100%, Q o H ^ + l) 629 (<5%) 267 (25%) 614 (<5%) 213 (<5%, Q U O ^ r+ lS ) 612 (5%) 211 (5%, C7H4079Br+18) 597 (<5%) 185 (<5%, 595 (<5%) 183 (<5%, CjH40 79Bt) 149 (17%) UV (MeOH): at: 277, 240nm IR (KBr disc, surface film): Vmtx at: 3440, 2920, 2340, 1740, 1700, 1400, 1370, 1320, 1010, 780 cm 1 3.3.2.2. Ro and DA homovanillic acid diesters. The following compounds were synthesised by the above methods (see section 3.3.1.2.); their structures (see Fig.22.) were assigned on the basis of their ^-NMR, MS, UV and IR spectroscopic properties. 3.3.2.2.1.9.13,14-orthophenvlacetviresiniferonol-20-Q-homovanillate (Resiniferatoxin. Rx). This compound was synthesised by the reaction of Ro with homovanillic acid, giving a yield of approximately 60%. Rx had an Rf value of 0.63 in CHCl^acetone (8:2), and was a single black spot after spraying with 60% H 2S04, which flouresced bright orange under 356nm UV light The synthesised Rx exhibited identical spectral characteristics to Rx isolated from the latex of E. poissonii. (see section 3.2.2.1.1.), hence confirming that the reaction method was correct and providing further evidence to support the authenticity of the isolated Ro. 159 3.3.2.2.2. 12-deoxvphorbol-13-0-angelate-2Q-0-homovanillate (DAHV). This compound was synthesised by the reaction of DA with homovanillic acid, giving a yield of approximately 60%. DAHV had an Rf value of 0.65 in CHClj/acetone (8:2), and was a single brown/orange spot after spraying with 60% H2S04, which flouresced bright orange under 356nm UV light DAHV exhibited the following spectral characteristics: ‘H-NMR (250MHz, CDC13): 5, ppm. Integration. Multiplicity. Inference. 7.60 1H bs H-l 6.78 3H m Aromatics 6.20, 6.17 1H dd =CH(CH3) (J=1.6, 5.6Hz) 5.67 1H d H-7 (J=5.5Hz) 4.49 2H s 2H-20 3.87 3H s -o c h 3 3.53 2H s <|)-CH2 3.23 1H m H-10 2.98 1H m H- 8 2.41 2H bs 2H-5 2.03-2.17 3H m 2H12, H ll 1 . 8 8 3H s OC-C(CH,)=CH- 1.78 3H m 3H-19 1.25 3H d CH-CH 3 (J=6.7Hz) 1.04 6 H s 3H-16,3H-17 0.90 3H d 3H-18 (J=2.0Hz) 0.78 1H d H-14 (J=4.2Hz) 160 MS (Thermospray): peaks at m/z: 612 (M++18, 5%) 331 (5%) 594 (M \ <5%, C ^ A ) 313 (12%) 577 (<5%) 295 (26%) 531 (<5%) 267 (10%) 477 (<5%) 137 (7%) 413 (7%) 110 (100%) 391 (5%) UV (MeOH): ^ at: 285 (shoulder), 276, 220 (shoulder)nm IR (KBr disc, surface film): Vm„ at: 3400, 2920,2360, 1700, 1600, 1520, 1430, 1380, 1270, 1160, 1040,760 cm ' 1 3.4. Discussion. The objective of this work was to isolate different diterpene nuclei and synthetically modify them before studying the pharmacological and biochemical profiles of the compounds isolated and synthesised. The latex of Euphorbia poissonii.Pax. was selected to provide compounds with the 1 2 -deoxyphorbol and resiniferonol nuclei. Compounds with these nuclei appear to possess a different spectrum of biological responses when compared to the classical 12,13-dioxygenated phorbol nucleus (Evans and Edwards, 1987), and I therefore thought that compounds with these nuclei may be useful in dissecting the mechanisms of biological responses induced by the phorbol ester diterpenes. Charcoal column, centrifugal liquid and finally preparative partition/adsorption thin layer chromatography have provided relatively rapid isolation techniques for diterpene mono- and di- esters in the pure form necessary for biological analysis. The problem of lipid contamination (most likely arising from the charcoal column step) was overcome by subsequent purification steps (confirmed by spectral analysis). In particular, the centrifugal liquid chromatographic stage enabled the semi-separation of 161 a large quantity of extracted material, in a relatively short time (~l- 2 hrs.), with the additional advantage of a lower likelyhood of decomposition of compounds. Final purification was found to be more rapid and with better resolution of individual compounds when the preparative adsorption TLC method was utilised rather than the preparative partition TLC method. The compounds isolated by these methods and their yields are in agreement with those previously reported for E.poissonii. latex (Schmidt and Evans, 1977a; Evans and Schmidt, 1979). Selctive esterification of the C 20 primary hydroxyl group of the 12- deoxyphorbol and resiniferonol nuclei additionally provided further compounds for structure/activity analysis. The rationale for C 20 esterification with the non-steroidal anti-inflammatory drugs (NSAIDs), Ketoprofen and Ibuprofen, was to provide compounds which, when delivered to the phorbol ester receptor site, might: i) Inhibit/erase the biological (pro-inflammatory) action of phorbol esters by inhibition of the enzyme receptor (PKC). ii) Esterase-mediated cleavage at Qo could cause the release of a known anti inflammatory agent at the required site of action. p-bromobenzoic acid was also used for Qo esterification to provide additional control compounds and enable further investigation of the structural requirements for activity. For example, would an electronegative group linked to an aromatic ring system cause similar effects to Qq- homovanillic acid substituted compounds or to C20- aromatic propionic acid derivative substituted compounds? Structural elucidation of compounds utilised !H-NMR, MS, UV and IR spectral techniques. 250MHz JH-NMR and MS were the most diagnostic techniques. Retention of the biologically active A/B trans ring isomers was confirmed in all cases by the presence of an H-l peak at ~5=7.65ppm in the NMR spectra. NMR data additionally 162 provided distinction between 12-deoxyphorbol and resiniferonol nuclei. The downfield shift from -5=0.88 to -5=4.70 ppm of H-16 protons is characteristic of the cyclopropane ring D or the isopropenyl side chain of the the 12-deoxyphorbol or resiniferonol nucleus respectively (integration confirming 2 protons in the resiniferonol case). A downfield shift from -5=4.00 to -5=4.40 ppm for 2H-20 protons is observed for Qq esterified compounds and confirms that the desired esterification has occurred, along with the presence of signals for protons of the esterifying group. In the mass spectra, El analysis was somewhat variable and this technique was therefore not usually sufficient for structural elucidation, due mainly to low abundance levels (<5%) of the molecular ion peak (with the notable exception of resiniferatoxin). Variability in El fragmentation of phorbol esters has been previously observed (Evans, 1986b). Chemical ionisation was a more reliable technique but there was a degree of variability. The thermospray soft ionisation technique was therefore used and a higher abundance M++l or M++18 peak was observed. M++18 peaks are due to the use of ammonium acetate buffer ionisation techniques. XH-NMR and thermospray MS were thus the main tools for structural elucidation, with UV and IR serving as confirmatory methods (the similarities in the spectra of compounds using these techniques rendered them somewhat non-diagnostic for individual compounds). ^-NM R and MS also enabled confirmation that compounds were of a desired purity for phamacological (see Chapter 5) and biochemical (see Chapter 6 ) studies. Table 13 summarises the compounds isolated/synthesised during the course of this work. 163 Table 13. Summary of compounds isolated and synthesised. Compound. Abbreviation. Yield. (mg/500ml latex.) 1. Isolated compounds. 9,13,14-orthophenylacetylresiniferonol- Rx 92 20-O-homovanillate (Resiniferatoxin). 9,13,14-orthophenylacetylresiniferonol Ro 46 (Resiniferonol). 12-deoxyphorbol-13-O-phenylacetate- DOPPA 6 6 20-O-acetate. 12-deoxyphorbol-13-O-phenylacetate. DOPP 27 12-deoxyphorbol-13-O-angelate-20-O- DAA 1 1 1 acetate. 12-deoxyphorbol-13-O-angelate. DA 57 2 . Synthesised compounds. 9,13,14-orthophenylacetylresiniferonol- RoK 20-O-[DL-2-(3-benzoylphenyl)- propionate]. 9,13,14-orthophenylacetylresiniferonol- Rol 20-O-[DL-2-(4-isobutylphenyl)- propionate]. 9,13,14-orthophenylacetylresiniferonol- RoB - 2 0 -0 - [p-bromobenzoate]. 9,13,14-orthophenylacetylresiniferonol- Rx - 20-O-homovanillate (Resiniferatoxin). 12-deoxyphorbol-13-O-angelate-20-O- DAK - [DL-2-(3-benzoylphenyl)-propionate]. 12-deoxyphorbol-13-O-angelate-20-O- DAI - [DL-2-(4-isobutylphenyl)-propionate]. 12-deoxyphorbol-13-O-angelate-20-O- DAB - [p-bromobenzoate]. 12-deoxyphorbol-13-O-angelate-20-O- DAHV - homovanillate. 164 Chapter 4: Computer-assisted molecular modelling of diterpene esters. 165 4.1. Introduction. Computer-assisted molecular modelling has been used to attempt to rationalise the structural requirements necessary for different classes of tumour promoters to induce tumour promotion and to analyse their requirements for protein kinase C activation (Jeffrey and Liskamp, 1986; Wender et al, 1986, 1988; Itai et al, 1988; Nakamura et al, 1989). This has led to the proposition of a common pharmacophore for compounds from the phorbol ester, ingenol, teleocidin, bryostatin and aplysiatoxin families of tumour promoters. Their structures are shown in Fig.23. and proposed structural equivalents required for activity in Table 14. Additionally, requirements for activation of PKC by its endogenous activator, DAG, compared to phorbol diesters have been proposed (Nishizuka, 1984; Leli, Hauser, and Froimowitz, 1990) (see Table 15). However, these propositions do not provide additional information regarding structure/activity requirements above those already suggested for tumour promotion activity by classical in vivo techniques (Evans and Edwards, 1987). Minor chemical alterations in the phorbol ester structure (for example, methylation of the C 4 hydroxyl group, or removal of the C 12 or C 13 long chain acyl group) can cause a decrease/loss of tumour promoting capability (see Chapter 1, section 1.4.1.) whilst still retaining the nucleus and atoms required by these models. This suggests that there are other, more subtle, structural requirements for phorbol ester-induced tumour promotion. The modelling of these different classes of tumour promoter has concentrated on superimposing compounds onto the phorbol ester class of tumour promoters, particularly TPA, and to correlate this with PKC activation. These hypotheses do not take account of the possibility that these compounds could be acting via additional/different mechanisms to PKC activation, or for differences in PKC isotype 166 C0(CH2)12 ch 3 N n 1 0 0 3 , 0 C0 Ch 3 Q CHoOH 20 L Phorbol ester,TPA Teleocidin B OMe B r o 2 1 « CH^CH^CD OH CHnOH 2090 L Ingenol-3TI OH Aplysiatoxin O^CHjCHg 5 Bryostatin I A cO H Figure 23 Structures of tumour promoters previously analysed by computer-assisted molecular modelling. Table 14. Comparison of structural requirements of different classes of tumour promoters for activity. co l-H "O O CO cs -a ON 00 VO 3 T co is CO CO c 00 >» XS EC U On > 00 o C )D 3 T EC U ex 167 oo a ON28 <- ■s' «d 3 3 S ON •a 0 0 ►5 on >—1 Z ^ o J2 xs .5 c cd cuq 0?S ai O 09 ex"I 2 . P oS * >*^ c « DO >* 5° Oo O *S 73 CG c — -XJ M s “ O cd CA Oh * O c c 0 fe O h . O C/5 •“ <4H D O ■§!U si u C •Sis 00 .hc .O 233 ow « £ 3 « 8 < *>e « 8 o =3 a. V 9 9 O 9eo 9_ 2 cd o u d5 d SB SB M (H 01 o I o 9 * «n ^ e o’ z" U U o T 3 EC o 33 SB EC X o 0 9 9 1 £ d d d e a • a o T 3 •H-* • *■4 cd u o o l—H *E h p £ £ < CO 169 Table 15. Structural requirements for activity for diacyglycerol compared to 12-0-tetradecanoylphorbol-13-acetate. Compound. Correlating atoms. Reference. TPA1 C12, C13 diester unit Nishizuka, 1984. DAG2 0 atoms TPA C9-0, C12-0, C13-0 Wender et al, 1986. DAG 0 atoms TPA C9-0, C12-0, C13-0, and Leli, Hauser and additional binding site at Froimowitz, 1990. A/B ring junction. o o O * N DAG 9* #* 112-O-tetradecanoylphorbol-13-acetate. 2Diacylglycerol. 170 activation/function in the tumour promotion process. It is known that phorbol esters can selectively activate particular PKC isotypes (Evans et al, 1991; Ryves et al, 1991; see Chapter 1, section 1.5.4.). Thus, assessment of the ability of different classes of tumour promoters to activate different PKC isotypes may be a more rational method to understanding the mechanisms of tumour promotion across tumour promoter classes rather than (or in conjunction with) molecular super-positioning. I therefore decided to investigate the molecular modelling characteristics of a range of naturally occurring and synthetic phorbol ester derivatives with different biological activities. 4.2. Materials and methods. A Desk Top Molecular Modelling programme (from Oxford University graphics) run on an IBM compatible personal computer with maths co-processor was used for molecular modelling analysis. The cartesian co-ordinates of phorbol (Pack, 1981) were used to create the initial nucleus and other compounds for study were built from this using the computer programme. These molecules then had their free energy minimised (using 1000 iterations unless otherwise stated), as the minimum free energy conformer was reasoned to be the conformer most likely to bind to a receptor site and subsequently exert a biological effect. All molecular structures created were the biologically active A/B ring trans isomers. 4.3. Results. 4.3.1. Minimum free energy values and molecular structures of derivatives. In total, the minimum free energy of seventeen naturally occurring or synthetic phorbol related diterpenes was assessed; this reflected five classes of nucleus, the 171 Table 16. Computer-generated minimum free energy values of derivatives of phorbol related nuclei. Nucleus. Compound* Minimum % relative Number of free energy to TPA. iterations.* (kcalmol*1) Phorbol TPA 164.9 100 Phorbol 114.8 69.6 4-deoxyphorbol Sap A 154.4 93.6 500 12-deoxyphorbol DOPP 109.3 66.3 DOPPA 109.0 66.1 DA 110.3 66.8 DAK 122.3 74.1 737 DAI 127.0 77.0 187 DAB 98.5 59.7 DAHV 114.4 69.3 195 Resiniferonol Ro 19.7 11.9 Rx 15.5 9.4 RoK 27.5 16.6 700 Rol 21.2 12.9 754 RoB 23.9 14.4 200 Daphnetoxin Mezerein 154.4 93.6 721 Thy A 151.7 92.1 187 - Capsaicin 111.9 67.8 287 +Abbreviations: DA 12-deoxyphorbol-13-O-angelate. DAB 12-deoxyphorbol-13-O-angelate-20-O-p-bromobenzoate. DAHV 12-deoxyphorbol-13-O-angelate-20-O-homovanillate. DAI 12-deoxyphorbol-13-O-angelate-20-O-[DL-2-(4-isobutylphenyl)- propionate]. DAK 12-deoxyphorbol-13-O-angelate-20-O-[DL-2-(3-benzylphenyl)- propionate]. DOPP 12-deoxyphorbol-13-O-phenylacetate. DOPPA 12-deoxyphorbol-13-O-phenylacetate-20-O-acetate. Ro 9,13,14-orthophenylacetylresiniferonol. RoB 9,13,14-orthophenylacetylresiniferonol-20-O-p-bromobenzoate. Rol 9,13,14-orthophenylacetylresiniferonol-20-O-[DL-2-(4-isobutylphenyl)- propionate]. RoK 9,13,14-orthophenylacetylresiniferonol-20-O-[DL-2-(3-benzylphenyl)- propionate]. Rx 9,13,14-orthophenylacetylresiniferonol-20-O-homovanillate. Sap A Sapintoxin A. Thy A Thymeleatoxin A. TPA 12-O-tetradecanoylphorbol-13-acetate. *A11 compounds were energy minimised for 1000 iterations unless otherwise stated. 172 tigliane phorbol, 4-deoxyphorbol and 12-deoxyphorbol nuclei, and the daphnane resiniferonol and daphnetoxin nuclei. Additionally, the structure of Capsaicin was generated and its free energy minimised. Table 16 shows the computer-generated minimum free energy values for the various derivatives and Fig.24 the corresponding minimum free energy conformer of compounds from each class of derivative. Compounds based upon the phorbol nucleus with C12 and C13 ester functions (TPA and Sap A) appear to possess the highest, and similar, minimum free energy values. Since 12-deoxyphorbol analogues generally have a lower minimum free energy value ( -60-75% of that of TPA), this suggests that some interaction between the ester groups of TPA is causing an increased free energy value. Additionally, interactions of the C13 ester group with the rigid cyclopropane D ring could cause an increase in this free energy value. By contrast, the minimum free energy of resiniferonol derivatives is much lower than that of TPA. It would seem that the 9,13,14-ortho- ester group acts to stabilise the molecule, and the opening of the D ring to form an isopropenyl side chain may place less constraint on the molecule. For the daphnetoxin group, the presence of the 6,7-a-epoxide group and the C12 ester group appear to increase the minimum free energy possessed by these compounds compared to resiniferonol derivatives. 4.3.2. Molecular co-ordinates of resiniferatoxin. The cartesian co-ordinates of the lowest energy conformer of resiniferatoxin (Rx) are shown in Table 17; in this conformation, the six membered C ring exists in the chair configuration (see Fig.24.) as previously observed for biologically active phorbol diesters (Leli, Hauser and Froimowitz, 1990). Rx is of interest since it is a non-tumour promoter yet is an extremely potent irritant (see Chapter 1, section 1.4). 173 Fig.24. Structures of minimum free energy conformer of tigliane and daphnane derivatives. T #:gv 12-O-tetradecanoylphorbol-13-O-acetate (TPA) Sapintoxin A (Sap A) 174 12-deoxyphorbol-13-O-phenylacetate-20-O-acetate (DOPPA) 'W:W' Resiniferatoxin (Rx) 9,13,14-orthophenylacetylresiniferonol-20-O-[DL-2-(3- benzylphenyl)-propionatel (RoK) Thymeleatoxin A (Thy A) 176 It has been suggested (DeVries and Blumberg, 1989; Szallasi and Blumberg, 1989b) that Rx acts as an ultrapotent analogue of the neurogenic irritant, capsaicin, although a novel receptor kinase has been isolated (Ryves et al, 1989; Evans et al , 1990). The pro-inflammatory response induced by Rx has also been shown to be partially neurogenic and partially non-neurogenic (Gordge, Evans and Evans, 1990; Evans et al, 1992; for more detail, see Chapter 5). Since the minimum free energy of Rx is much lower than that of TPA or capsaicin, this may indicate that the lowest energy conformer may bind to alternative/additional site(s). The cartesian co-ordinates of this conformer may therefore be useful in further understanding the biochemical mechanism(s) of action of Rx, since it is probable that Rx-induced effects are not solely PKC-mediated. 4.4. Discussion. Previous molecular modelling analysis of tumour promoting compounds has concentrated on the super-positioning of these molecules onto TPA (Jeffrey and Liskamp, 1986; Wender et al, 1986, 1988; Itai et al, 1988; Nakamura et al, 1989). In the analysis done here, I have concentrated solely on phorbol related diterpene esters (both naturally occurring and semi-synthetic) in an attempt to explain the action of these compounds since both promoters and non-promoters can activate PKC isotypes to a similar extent to TPA (Evans et al, 1991; Ryves et al, 1991). Subtle differences in activation/non-activation of one or more isotype and corresponding protein substrate phosphorylation may confer tumour promoting properties. In this case, these studies on the lowest energy conformers of these compounds may help to understand the tumour promotion process, and the role of PKC in this process. However, it is acknowledged that before structure/activity relationships can be fully understood, the Ill Table 17. The molecular co-ordinates of Resiniferatoxin (Rx). Atom. Cartesian co-ordinates Connectivity. XY Z 1 C -2.87718 0.23664 -0.61286 2 10 27 2 C -4.02756 -0.42949 -0.20922 1 3 19 3 C -3.57795 -1.68791 0.47866 2 4 21 4 C -2.08536 -1.74514 0.63586 3 5 10 22 5 C -1.48122 -3.10357 0.28561 4 6 28 29 6 C -0.12515 -3.03566 -0.43032 5 7 20 7 C 0.80468 -2.00266 -0.27713 6 8 30 8 C 0.62082 -0.82517 0.68132 7 9 14 31 9 C -0.42668 0.17662 0.16586 8 10 11 23 IOC -1.64099 -0.61641 -0.31903 1 4 9 32 11 C -0.76741 1.17540 1.28702 9 12 18 33 12 C 0.42225 1.37366 2.22931 11 13 34 24 13 C 1.73958 1.28418 1.46599 12 14 15 25 14 C 1.94654 -0.08788 0.81959 8 13 35 50 15 C 2.93743 1.61198 2.35511 13 16 17 16 C 4.04038 2.28671 1.82978 15 36 37 17 C 2.95950 1.14122 3.80638 15 38 39 40 18 C -1.23925 2.52984 0.76131 11 41 42 43 19 C -5.47901 -0.00667 -0.40294 2 44 45 46 20 C 0.28562 -4.18972 -1.34094 6 26 47 48 21 0 -4.34433 -2.49599 0.97690 3 22 0 -1.83442 -1.45642 2.05606 4 49 23 0 0.10885 0.88718 -1.02397 9 51 24 H 0.39840 0.58848 2.99899 12 25 0 1.65779 2.18888 0.30701 13 51 26 0 -0.12612 -5.50898 -0.82403 20 66 27 H -2.86772 1.15885 -1.18212 1 28 H -2.18528 -3.65446 -0.35518 5 29 H -1.34599 -3.67247 1.21678 5 30 H 1.73052 -2.06640 -0.83755 7 31 H 0.33859 -1.21190 1.66942 8 32 H -1.36495 -1.07918 -1.27802 10 33 H -1.59227 0.76932 1.88714 11 34 H 0.35179 2.35500 2.72080 12 35 H 2.72154 -0.67484 1.33220 14 36 H 4.89753 2.49542 2.45930 16 37 H 4.04596 2.59469 0.79063 16 38 H 2.42039 0.18598 3.89093 17 39 H 3.99972 1.00680 4.13778 17 40 H 2.47002 1.89562 4.43954 17 41 H -0.56318 2.90631 -0.01922 18 178 42 H -1.26594 3.25207 1.59036 18 43 H -2.25270 2.43640 0.34876 18 44 H -6.11627 -0.90003 -0.47779 19 45 H -5.79691 0.59824 0.45836 19 46 H -5.56841 0.59152 -1.32170 19 47 H -0.18640 -4.03872 -2.32237 20 48 H 1.37638 -4.18030 -1.48168 20 49 H -2.34217 -2.05178 2.59488 22 50 0 2.34156 0.28493 -0.53732 14 51 51 C 1.49044 1.40273 -0.91890 23 25 50 63 52 C 0.81871 5.75029 -2.27908 53 60 62 53 C 1.61859 4.62394 -2.07209 52 54 55 54 C 1.13164 3.35398 -2.39191 53 56 63 55 H 2.61649 4.73601 -1.66380 53 56 C -0.15908 3.20912 -2.90717 54 58 57 57 H -0.54100 2.22341 -3.14582 56 58 C -0.96048 4.33481 -3.10953 56 60 59 59 H -1.96177 4.22259 -3.50814 58 60 C -0.47024 5.60537 -2.79872 52 58 61 61 H -1.09167 6.47899 -2.95716 60 62 H 1.19702 6.73608 -2.03389 52 63 C 1.99763 2.12143 -2.15466 51 54 64 65 64 H 3.04407 2.42218 -1.99772 63 65 H 1.93291 1.44751 -3.02142 63 66 C 0.36784 -5.93822 0.32178 26 67 68 67 0 0.03347 -7.05236 0.68974 66 68 C 1.23517 -5.13292 1.25780 66 71 80 81 69 C 4.32748 -3.08551 0.36093 70 77 79 70 C 3.10399 -3.47414 0.91178 69 71 72 71 C 2.58250 -4.74434 0.65509 68 70 73 72 H 2.54809 -2.78205 1.53299 70 73 C 3.30699 -5.64688 -0.12709 71 75 74 74 H 2.91438 -6.63873 -0.31910 73 75 C 4.54400 -5.27186 -0.65679 73 77 76 76 H 5.10917 -5.97660 -1.25527 75 77 C 5.05451 -3.99332 -0.41716 69 75 78 78 0 6.21397 -3.66111 -0.90361 77 86 79 0 4.79438 -1.89380 0.61055 69 82 80 H 0.69904 -4.22381 1.56406 68 81 H 1.42936 -5.73095 2.16045 68 82 C 5.19312 -1.02088 -0.50843 79 83 84 85 83 H 4.73685 -0.02984 -0.36705 82 84 H 4.84255 -1.43901 -1.46370 82 85 H 6.28790 -0.91696 -0.52196 82 86 H 6.58048 -4.39732 -1.37960 78 179 tertiary structure of individual PKC isotypes is required. In the meantime, although critical interactions of the phorbol esters with their amino acid receptor sites within the protein cannot be determined, minimum free energy evaluations may be of contributory importance and constitute an advance in understanding over purely qualitative structure/activity correlations. The minimum free energies throughout the five diterpene nuclei studied suggest that the cyclopropane D ring and C12/C13 ester units increase the minimum free energy. De-oxygenation at C12 appears to lower this minimum free energy. Comparison of the synthetic 12-deoxyphorbol and resiniferonol derivatives suggests that the C^ ester function has little effect on the minimum free energy, which is determined to the greatest extent by the nucleus. 9,13,14-ortho- ester linkage appears to greatly reduce the minimum free energy, but the presence of a C12 ester group and a C6.7-a-epoxide (as observed for daphnetoxin derivatives) increases the minimum free energy. The minimum free energy of resiniferatoxin, and its cartesian co-ordinates may help further understanding of its mechanism of action. Interestingly, both capsaicin and the 12-deoxyphorbol analogue of Rx, 12-deoxyphorbol- 13-O-angelate-20-O- homovanillate (DAHV), possess higher minimum free energy values than Rx. This may indicate that they act, in part, by a different mechanism to Rx, although molecular modelling analysis will clearly need to be correlated with any future pharmacological and biochemical studies. 180 Chapter 5: Pro-inflammatory, anti-inflammatory and epidermal hyperplasiogenic activity of diterpene esters. 181 5.1. Introduction. Classical bio-assay guided fractionation of extracts from the plant families Euphorbiaceae and Thymelaeaceae utilise the pro-inflammatory response induced by the tigliane, daphnane and ingenane diterpenes (Evans and Edwards, 1987; see Chapter 1, section 1.4.2.). This acute pro-inflammatory effect of diterpene esters has led to the development of the "mouse ear erythema assay" (Hecker et al, 1966; Evans and Schmidt, 1979), an "all-or-nothing" assay, enabling calculation of an Irritant Dose (ID50) value, which is that dose of agonist that will induce erythema in 50% of the test animals. Following the isolation and synthetic modification of diterpene esters from the latex of E.poissonii. (see Chapter 3), I decided to assess their pro-inflammatory activity. Although pro-inflammatory response does not directly correlate with either tumour promoting or PKC activating ability (Evans, 1986b), all phorbol esters studied to date that activate one or more isoform of PKC are also capable of inducing erythema of the mouse ear to some extent (Ellis et al, 1987; Evans and Edwards, 1987; Brooks et al, 1989b; Evans et al, 1991). Therefore, this assay would enable: i) identification of pro-inflammatory agonists, which would probably activate one or more isoform of PKC, and ii) identification of non-inflammatory agents, which would be considered potential PKC non-activators/inhibitors. The experimental methodology would therefore: i) Initially characterise ID^ values of isolated and synthetic compounds. ii) Compounds which were non-inflammatory would be utilised to attempt to inhibit agonist-induced erythema. iii) Hyperplasia induced by compounds would be studied since this may be a guide to tumour promoting activity. 182 Additionally, I decided to study the erythema response induced by resiniferatoxin (Rx) in some detail. The Qo ester side chain of Rx (the homovanillic acid moiety) resembles the polar head group of the neurogenic irritant isolated from the Capsicum (or red pepper) family, Capsaicin. Blumberg has proposed that Rx acts as ultrapotent analogue of capsaicin (De Vries and Blumberg, 1989; Szallasi and Blumberg, 1989a, 1989b, 1990a). Furthermore, Blumberg has demonstrated that 3H-Rx binding to vanilloid receptors in dorsal root ganglion can be inhibited by capsaicin (Szallasi and Blumberg, 1990b, 1991a, 1991b; Szallasi, Szallasi and Blumberg, 1990). At the same time, Evans has shown that Rx activates a calcium-independant (Ca2+- inhibited?) kinase, isolated from human mononuclear cells (Ryves et al, 1989) and mouse peritoneal macrophages (Evans et al> 1990), that is distinct from known isoforms of PKC on the basis of its elution by hydroxylapatite column-FPLC techniques and its tissue distribution (Evans, A.T., et al, 1991; Ryves, 1991). This kinase, termed the "Rx-kinase", is activated by Rx to a greater extent than by TPA, and is not activated by capsaicin (Ryves et al, 1989; Ryves, 1991). Thus, the mechan/Sr} of action of Rx would appear to be confusing. I therefore hoped that the study of Rx-induced erythema would help to clarify this somewhat anomalous situation. 5.2. Materials and methods. 5.2.1. Test agonists and antagonists. Diterpene esters were isolated from E.poissonii. latex and synthetically modified as described in Chapter 3; Sapintoxins were isolated in the laboratory of Professor Fred. J. Evans from the fruits of Sapiwn indicum (Evans, 1986b). TPA, capsaicin, hydrocortisone and indomethacin were purchased from Sigma (Poole, UK). 183 Compounds were diluted in acetone (all diterpenes and capsaicin) or ethanol (indomethacin and hydrocortisone) immediately before use. 5.2.2. Mouse ear erythema assay. 5.2.2.1. Determination of IDgn value. IDsq values were assessed essentially by the method of Schmidt and Evans (1979), as follows: 1. 1 mg/ml (=5pg/5pl) solution of compound was made, and two fold dilutions of this stock solution were used. Pilot assays were performed on two mice for each sequential dilution until no erythema was observed. The highest dose used was 4 x 5pg/5pl applications; a compound not capable of inducing erythema at this dose was considered non-inflammatory. Pilot assays can be used to determine the time to peak irritancy ( t^ ) (see below). 2. Main assays were performed on female BKTO mice (20-25g body weight), randomly assigned to groups of six animals each. Six dose levels, including the lowest capable of inducing erythema, were utilised. 3. 5pi of test agonist in acetone was applied to the inner surface of the right ear, 5pl of vehicle was applied to the inner surface of the left ear as a control. 4. Erythema was assessed subjectively at at appropriate time intervals by comparison of the treated ear with the control ear. 5. Erythema was assigned a score at each reading as follows: 0 = No reddening ± = Reddening just visible + = Distinct reddening. 6. This assay protocol enabled the following parameters to be measured: i) The time to peak irritancy ( t^ ) - the time at which the maximum 184 number respond. If the same maximal number respond throughout several time points, then tp^ is subjectively assessed as the time when the irritancy is at its most intense. ii) Total number responding - for the different dose levels, a different number of mice show a positive response at tp^. The number responding at each dose level at tp^ is then analysed by log-probit computer analysis, enabling determination of the ID^ value. iii) Duration of response - this is expressed as a cumulative erythema score for each mouse. The assay is performed at least three times for assessment of ID50 value and the coded cages are randomised for reading after application of agonist by an independant helper. 5.2.2.2. Inhibition and pre-treatment experiments. Antagonist or pre-treatment agonist compounds were applied 50 minutes prior to the agonist compound being studied ensuring adequate time for delivery to the site of action. In these experiments, mice were randomly assigned to groups of 10 animals each. 5.2.3. Hyperplasia experiments. Hyperplasia was assessed by application of test compound in 200pl acetone to the dorsal surface of female CD-I mice (20-25g body weight), following shaving and removal of any residual hair using a diluted solution of depilatory agent (Imrnac*; 1 part immac: 5 parts water). The mice were sacrificed after 48 hours and the skin removed for sectioning. Skin (~lcm x 1cm) was fixed in 10% buffered formalin, dehydrated and embedded in 56°c melting point paraffin wax. 6pm sections were cut on a Reichert 2030 rotary microtome and stained with Haematoxylin and Eosin. The sections were then viewed and photographed under a Nikon Microphot 185 photomicroscope using Kodak T.Max 100 film. 5.3. Results. 5.3.1. Determination of IDW values of compounds. The ID^ values for the isolated and synthesised compounds are shown in Table 18. The values for isolated compounds are in agreement with previously published values (Schmidt, 1977; Schmidt and Evans, 1979, 1980) as are those for other standard phorbol ester agonists. acetylation of the 12-deoxyphorbol nucleus appears to have litde effect on the ID50 value; this may reflect conversion of C13, Qo diesters to C13 monoesters by endogenous esterase activity. However, the time to peak irritancy is not prolonged in these cases, and C 2 0 acetylation prevents conferment of platelet aggregation activity (Williamson et al, 1981), suggesting a lack of esterase- mediated metabolism. Esterification with more complex compounds (Ketoprofen, Ibuprofen and p-bromobenzoic acid) reduces the IDjq value, although not to a great extent; the time to peak irritancy is unaltered. Qo esterification with homovanillic acid (DAHV) greatly reduces the ID^ value and shortens tp^. In this instance, the ID50 value and are reduced to similiar levels to those observed for Rx., the recognised potent irritant (Schmidt and Evans, 1979). However, the de-esterified parent alcohol, Ro, has a higher ID50 value compared to 12-deoxyphorbol monoesters. This suggests that the irritancy of Rx and DAHV is not due to a metabolic product The parent acid, homovanillic acid, is itself non-inflammatory and capsaicin is of lower potency than DAHV or Rx, implying that the potent pro-inflammatory nature of DAHV and Rx is a combination of endogenous phorbol ester effects which are enhanced by the C 2 0 homovanillate substitution. Szallasi, Sharkey and Blumberg (1989) have synthesised Cjo-homovanillate analogues of DOPP and mezerein, and have found their potency to be similar to that of Rx, and have proposed that this mechanism is purely neurogenic. Table 18. Irritant Doseso (ID50) values of isolated and synthesised compounds. ■o a. CM -o o o VO o © o CO I M 8 CM CM ■S o 00 CO o O 2 B CM CM 00 s cm o CO © o CM r- o OV CO CM ex 00 CM o o o 00 00 VO VO r- o cno o o 0.74 0.076 0.055 0.042 o A >28.5 o o >109.9 00 o\00 910*0 00 o 100 VO Tj- CM o >20 o A >20 o 0.047 0.052 0.022 o 1 I I 1.0 1.0 1.5 1.5 4.0 0.15 s a 12-deoxyphorbol-13-0-angelate-20-0-[DL-2-(3- 12-deoxyphorbol-13-0-angelate-20-0-[DL-2-(4- 12-O-tetradecanoylphorbol-13-0-acetate+ 12-O-tetradecanoylphorbol-13-0-acetate+ (TPA) 12-0-[2-methylaminobenzoyl]-4-deoxyphorbol- 13-0-acetate+ [2-methylaminobenzoyl]-phorbol-13-0-12-0- (Sap A) benzylphenyl)-propionate] benzylphenyl)-propionate] (DAK) [DL-2-(4-isobutylphenyl)-propionate] (Rol) [DL-2-(3-benzylphenyl)-propionate] (RoK) 8-methyl-N-vanillyl-6-nonenamide 8-methyl-N-vanillyl-6-nonenamide (Capsaicin) 3-methoxy-4-hydroxyphenylacetic acid (Homovanillic acid) isobutylphenyl)-propionate] isobutylphenyl)-propionate] (DAI) 9,13,14-orthophenylacetylresiniferonol-20-O-[p- bromobenzoate] (RoB) 9,13,14-orthophenylacetylresiniferonol-20-O- acetate+ (Sap (Sap acetate+ D) 9,13,14-orthophenylacetylresiniferonol-20-O- 3. 3. Other compounds. 188 However, they have pharmacologically tested solely for neurogenic activity and have therefore selected out possible phorbol ester-like effects (see also section 5.3.3.). The corresponding complex Qo synthetic resiniferonol compounds (RoB, RoK and Rol) show an interesting spectrum of activity. RoB exhibits similar potency to Ro, whilst RoK and Rol (that is those with an anti-inflammatory ester function) are, themselves, non-inflammatory. This points to an inherent difference between the 12- deoxyphorbol and resiniferonol nuclei in confering pro-inflammatory activity since DAK and DAI are active. RoK and Rol were therefore considered compounds which, due to their possesion of a daphnane nuclei with an anti-inflammatory side chain, could be potential inhibitors of phorbol ester induced biological effects which, it was hoped, would interact at the same site of action as phorbol esters. I therefore decided to use these compounds as pre-treatment agents to attempt to inhibit the erythema response induced by standard phorbol ester agonists. 5.3.2. Effect of pre-treatment with RoK and Rol on phorbol ester induced erythema. RoK and Rol were applied as a 50 minute pre-treatment at doses of 5pg/5pl and 0.5pg/5pl before application of the standard phorbol ester agonists TPA, Sap A, DOPPA and Rx. The dose of agonist applied was the minimum dose which would normally cause 100% of the test animals to respond at t ^ . Therefore, inhibition would be observed if less than 100% of the animals responded to agonist treatment. The results of RoK and Rol pre-treatment experiments are shown in Fig.25. This demonstrates that for each of the agonists, TPA, Sap A, DOPPA and Rx, at the minimum dose causing 100% of animals to respond, pre-treatment with 0.5pg/5pl or 5pg/5pl RoK or Rol was unable to inhibit the agonist induced erythema. Due to the complex nature of the inflammatory response, I decided to test RoK and Rol for in +j C -M a) -M C £ C Q) +J a) E (0 £ ■M a) ■M to u (0 299993 (1) C o t—\ (0 +J in to CO •H cn u C o in 6 uTpuodsaj jsquinu xe^ox 190 vitro PKC inhibition (see Chapter 6), since it is possible that these compounds are potential PKC inhibitors, but that the phorbol esters are such potent agonists that inhibition of their biological effects is not possible unless high dose levels are utilised. Furthermore, there could be problems in the delivery of the potential inhibitors to the required site of action in the in vivo situation. 5.3.3. Studies on resiniferatoxin-induced erythema. Due to the potency of resiniferatoxin-induced erythema compared to other phorbol esters, the similar potency of other C^-homovanillate substituted compounds (see above and Szallasi, Sharkey and Blumberg, 1989), and the conflicting evidence that the mechanism of action of Rx is by kinase stimulation (Ryves et al, 1989; Evans et al , 1990) or as an ultrapotent neurogenic irritant (DeVries and Blumberg, 1989; Szallasi and Blumberg, 1989a, 1989b), I thought that the mechanism of Rx-induced erythema warranted closer investigation. Table 19 shows a comparison of the potency and duration of erythema response for Rx, Capsaicin and Sapintoxin D (Sap D), a control phorbol ester agonist of similar potency to TPA in terms of erythema response and in vitro PKC activation (Ellis et al, 1987b). At the minimum dose causing 100% of animals to respond, the duration of Rx induced erythema resembles that of Sap D (>3 hours), whereas capsaicin induced erythema lasts for 11 minutes, which at a high dose can be extended to 80 minutes. At the minimum dose causing 100% of animals to respond, the response was a generalised vasodilation of ear blood vessels, whereas for Rx and Sap D, a generalised ear reddening and erythema was observed. This suggests that phorbol ester induced irritancy is more potent and prolonged than that of capsaicin, and that the irritancy induced by Rx resembles that of Sap D rather than capsaicin. Fig.26. shows the time taken to reach peak irritancy, t ^ , for these three 191 Table 19. Comparison of the potency and duration of response of Resiniferatoxin, Sapintoxin D and Capsaicin. Compound. Minimum dose causing Maximum dose tested. 100% of animals to respond. pg/5pl Duration. pg/5pl Duration. Sapintoxin D (Sap D) 0.031 >3 hours 0.375 >12 hours Resiniferatoxin (Rx) 0.0008 >3 hours 5 >8 hours Capsaicin 0.0625 11 (±2)* min 35 80 (±13)' min 'Values are ±SEM, with data from four experiments. All doses were tested in parallel groups of 10 animals each. 192 60 - i c •h 40 - c U> 1 c (0 ■M •H U U •H «0 0) 20 “ a o +j 0.055 0.16 0.0008 0.013 5 0.031 0.375 Capsaicin Resiniferatoxin Sapintoxin D Agonist and dose applied (ug/5jul) Points are mean ± SEM from four experiments. Each of lower selected doses (/jg/5jil) are minimum d o se s c a u sin g 100% of animals to respond. Higher Rx doses are those producing response >3Hf and>8Hf and for Sap D >12H respectively. Figure 26 Time taken to reach, and effect of dose on Irpeak for Capsaicin, Rx, and Sap D. 193 compounds. For each agonist, increasing the dose applied causes a reduction in tp^. At the minimum dose causing 100% of animals to respond, tp^ for Rx (47 minutes) resembles that of Sap D (59 minutes) rather than capsaicin (13 minutes). Application of higher doses of Rx results in a larger decrease in tp^ than that observed for Sap D, and the Rx induced erythema is visible almost immediately after Rx application. Repeated capsaicin treatments have been reported to cause desensitisation (Bevan et al, 1987), which results in loss of pharmacological response (in this case, erythema of the mouse ear). This phenomenon was confirmed for this animal model of irritancy (see Fig.27.) using higher doses than that required to cause 100% of animals to respond. At these doses (0.08pg/5pl and 0.16pg/5pl, the initial inflammatory response was of approximately 40 minutes duration (see Fig.27.), and the second application of capsaicin elicited a response from only a small number (-5%) of animals. 50 minutes was therefore considered an adequate pre-treatment time after the first capsaicin application to ensure desensitisation to neurogenic inflammatory responses. Capsaicin pre-treatment should have no effect on typical phorbol ester induced erythema, since the phorbol esters are non-neurogenic irritants. Fig.28. shows the effect of increasing doses of capsaicin pre-treatment, 50 minutes before application of a sub-threshold (0.002pg/5pl) dose of Sap D (ID^ = 0.0076pg/5pl). This demonstrates that capsaicin pre-treatment does, in fact, dramatically augment phorbol ester induced inflammatory effects, and that phorbol ester induced irritancy cannot be desensitised by neurogenic irritants. The mechanism of phorbol ester induced irritancy is thus different from that of neurogenic inflammation. Pre-treatment of mice with 0.25pg/5pl capsaicin 50 minutes prior to application of 0.0008pg/5pl (minimum dose causing 100% of animals to respond) or 0.013pg/5pl o c o CO •H 4-> ■M c 10 Q) U TJ £ o •rH Q) H 10 £ a> X3 4 4 > i a) C o •H (4-1 ro E o \ Q) c E o •H •H H 4-» (0 CO •H 4 J *H o CO CM G a ) co a; (44 G o •H O h i c •H o (0 •H CO 4 J ft U (0 TJG u C (44 O h i CM in a) u c 013 •H •H O <0 CO ft o o o (0 o vo CN u ( % ) Buxpuodsaj jaquinu iBqoj, 195 100 o> 60 - 20 - 0 0.04 0.08 0.16 0.32 Capsaicin pre-treatment dose (yjg/5pl) Capsaicin pre-treatment dose applied 50 minutes before subthreshhold (0 .0 0 2 ^1 9 /5^ 1 ) dose of Sap D. Points are mean ± SEM from three experiments, each using 10 mice for each data point. Figure 28 Effect of increasing pre-treatment dose of Capsaicin on total number of animals responding to a subthreshold dose of Sapintoxin D. 196 Rx revealed that capsaicin pre-treatment had little effect on Rx potency; that is, at both dose levels, all mice exhibited erythema and there was no observed desensitisation. However, capsaicin pre-treatment did affect tp^ (see Table 20). Without pre-treatment, t ^ for the 0.0008pg/5pl Rx dose level is 47 minutes; tp^ is 12 minutes when 0.013pg/5pl Rx is applied. 0.25pg/5pl capsaicin pre-treatment results in the respective reduction and enlargement of these tp^ values; for both doses, the tp^ following capsaicin pre-treatment is 28 minutes. This may be due to the desensitisation of a more prominant neurogenic component for the higher dose of Rx application, thereby causing an increase in tp^, the latency of which is more typical of phorbol esters. In the case of the lower dose Rx, which may exhibit a smaller (or ‘ non-existent) neurogenic component, tp^ may be reduced by the synergistic action of capsaicin with the phorbol ester component of the Rx response. This possibility is supported by the inhibitory effects of non-steroidal (indomethacin) and steroidal (hydrocortisone) anti-inflammatory agents on the erythema induced by 0.013pg/5pl Rx (see Fig.29.). Indomethacin causes an abolition of the early phase resiniferatoxin response, whereas hydrocortisone causes a slight reduction in the total number responding. However, at later time points, hydrocortisone caused a 50% inhibition of the Rx-induced erythema, and indomethacin did not. This would suggest that the early phase of Rx-induced erythema is of a neurogenic nature and the later phase is typical of phorbol ester induced erythema, since Williamson and Evans (1981) have shown that hydrocortisone is capable of causing 50-60% inhibition of erythema induced by 12-deoxyphorbol esters, althlough indomethacin could only cause 10% inhibition. 197 Table 20. Effect of capsaicin pre-treatment on time to peak irritancy of erythema response to resiniferatoxin. Dose of resiniferatoxin Time to peak irritancy, t ^ (mean ±SEM) applied (pg/5pl) No pre-treatment Capsaicin pre-treatment* 0.0008 47 (±7) 28 (±11) 0.013 12 (±1) 28 (±3) *0.25pg/5pl capsaicin applied 50 minutes before resiniferatoxin treatment Data from five experiments, each dose consisting of 10 mice tested in parallel groups per data point a) Hydrocortisone H rH vo rH O o i h 6 Tuda jraqumu ieq.o uTpuodsaa o CM o CO CM CM O O o in CM CM in JZ •H -P M 0 E a> ID c u C VO O .1 o % CM o CM (N CM o CM o in co o CM co o o o in in •H ■H •H •H •H ov 0 rH rH ■— rH JZ z s in' 'O TJ in rH in TJ in »—1 ■P -P X -P M 0 o> 0 CO (0 G E a) G CO (0 04 0 a^ CD G O a) 0 a) E c O CO 04 O) P 0 O 0 P a. 0 (0 (0 c a. •0 U £ O. 0) 0 2 a. >1 # r -p •rH •H •rH MH •rH CO N. IT) 0 0 rH rH -P •P ■P X -P ■P ■p OS p 0 OCD CO G CD (0 P CD CO E (0 . a O) G CO 0 G CD £ . a X * • •rH (p •rH T3 •H •H •H JZ cp sz T3 JZ £ JZ -P cn -p ■P CL. ■ W 2 ■P CO P 0) G a> 1 + CO co p 0 E p a> CO < 0 G CO CO 0 p (DCD «0 CO G E a) X 04 a) P E a) G 0 0 0 £ £ d - Figure 29 Effect of Hydrocortisone or Indomethacin pre-treatment before Resiniferatoxin application. 199 5.3.4. Induction of epidermal hyperplasia. Compounds were assessed for their ability to induce epidermal hyperplasia by a single application of 50nM of test compound. This dose level was selected, since compounds which cause tumour promotion do so at doses ranging from 17-100nM (Brooks et al, 1989). Therefore, if any of the isolated or synthesised compounds were hyplastic at 50nM, they would be considered potential tumour promoters, since epidermal hyperplasia is necessary (but in itself insufficient) for tumour promotion (Marks and Furstenberger, 1984). Appendix 1 shows the ability of these compounds to induce epidermal hyperplasia. TPA (Appendix 1, a.) induced epidermal hyperplasia when compared to a control application of acetone (Appendix 1, b.). All other compounds tested, that is: DOPPA (Appendix 1, c.), Rx (Appendix 1, d.) DAB (Appendix 1, e.), DAI (Appendix 1, f.), DAK (Appendix 1, g.), DAHV (Appendix 1, h.), RoB (Appendix 1, i.), Rol (Appendix 1, j.) and RoK (Appendix 1, k.) were unable to induce epidermal hyperplasia and are, therefore, considered non-tumour promoting agents. These findings support the supposition (Evans and Edwards, 1987) that pro-inflammatory activity does not directly correlate with tumour promoting activity, and point to the existence of more subtle and complex mechanisms by which compounds act as tumour promoters compared to pro-inflammatory agents. 5.4. Discussion. Pharmacological testing has revealed that all isolated and synthesised compounds (except Rol and RoK) were pro-inflammatory agents in the mouse ear erythema model. Where applicable, the ID50 values (see Table 18) obtained were in agreement with those previously reported for isolated compounds (Schmidt, 1977; 200 Taylor, 1982; Brooks, 1988). C^-esterification appears to decrease the inflammatory potency of these compounds, except in the case of C^-esterification with homovanillic acid (Rx, DAHV). DAHV and Rx are the most potent pro-inflammatory agents (typically 100 fold more potent than parent compounds). Whilst homovanillic acid itself is non-inflammatory, C^-homovanillate substitution appears to confer this potent activity on compounds, since other C^-homovanillate esters, for example 12- deoxyphorbol-13-O-phenylacetate-20-O-homovanillate and mezerein-20-O- homovanillate (Szallasi, Sharkey and Blumberg, 1989) are of a similar potency to Rx and DAHV. The striking similarity of the C^-homovanillate side chain with the polar head group of the neurogenic irritant, capsaicin, has led to the proposition that Rx is an ultrapotent capsaicin analogue (DeVries and Blumberg, 1989; Szallasi and Blumberg, 1989a, 1989b). Studies on the mechanism of Rx-induced erythema do point to a neurogenic component in the inflammatory response to Rx. However, there also appears to be a phorbol ester-like component The erythema response induced by Rx resembles that of Sap D rather than capsaicin, both in terms of latency and duration of response (Table 19). Increasing the dose of application of Sap D, Rx and capsaicin caused a decrease in tp^ (Fig.26.). Repeated applications of capsaicin cause a desensitisation of the mice to capsaicin-induced erythema (Fig.27.), but capsaicin pre- treatment before application of a sub-threshold dose of Sap D causes augmentation of the phorbol ester response (Fig.28.). Capsaicin pre-treatment before Rx application has little effect on Rx potency although tp^ is affected (increased or decreased, depending on the dose of Rx applied). This data suggests that the erythema response to Rx consists of an early neurogenic phase and a later phorbol ester phase. The neurogenic phase may be more evident at higher dose levels. The early neurogenic response can 201 be inhibited by indomethacin but not by hydrocortisone, whereas the later (phorbol ester) response can be 50% inhibited by hydrocortisone and only 10% inhibited by indomethacin (see Fig.29). This evidence supports the later phase of Rx induced erythema being of a phorbol ester nature, as hydrocortisone is capable of inhibiting erythema induced by 12-deoxyphorbol esters, and indomethacin is not (Williamson and Evans, 1981). In summary, these features of dual-inhibitor sensivity, dose- dependent latency and interaction with neurogenic irritants point to a mixed aetiology in the erythema response to Rx, which consists of an early neurogenic phase and a later phorbol ester phase. It is likely, therefore, that Rx activity is not solely attributable to a single target enzyme (or family of enzymes), or a single mechanism. This may explain the conflicting reports that Rx acts as an ultrapotent neurogenic irritant (Szallasi and Blumberg, 1989a, 1989b), for which binding sites have been demonstrated (Szallasi and Blumberg, 1990b, 1991a, 1991b), or as a novel kinase activator (Ryves et al, 1989; Evans et al, 1990). However, how these mechanisms interact during the response of a host to external or internal Rx (or Rx-like) inflammatory stimuli warrants further investigation. The non-inflammatory semi-synthetic compounds, RoK and Rol, were tested for their ability to inhibit erythema induced by TP A, Sap A, DOPPA and Rx. RoK and Rol were unable to inhibit the erythema induced by this range of phorbol ester agonists (see Fig.25.) This could be due partially to problems with their delivery to their site of action (PKC), as well as possible lower affinity for the receptor site than the standard agonists. I decided that biochemical investigation of their activity should be pursued (see Chapter 6), to attempt to clarify this position. C^-esterification would appear to be an important factor when considering pro-inflammatory effects; C^- homovanillate substitution caused a large increase in potency, yet Qo-esterification 202 with anti-inflammatory agents caused a complete loss of pro-inflammatory activity. The importance of the ester group in pro-inflammatory structure/activity relationships may be more important than originally thought Experiments to test the ability of these compounds to induce epidermal hyperplasia would suggest that none of the compounds isolated or synthesised are tumour promoting agents, since none were able to induce epidermal hyperplasia. Assessment of epidermal hyperplasia activity is a rapid technique, and is useful to assess tumour promoting activity, since epidermal hyperplasia is a requisite (although not sufficient alone) for tumour promotion (Marks and Furstenberger, 1984). These experiments suggest that correlations between pro-inflammatory and tumour promoting activities are tenuous, since all compounds tested (except Rol and RoK) are pro- inflammatory (DAHV and Rx being 100 fold more potent than TPA), yet only the complete tumour promoter TPA was hyperplasiogenic. These anomolies between pharmacological activities of phorbol esters do beg the question concerning correlation of activation of PKC, as the phorbol ester receptor site, and tumour promotion (and even other biological effects). It wouldtherefore seem that in order to understand the mechanisms of action of the phorbol esters, the best approach may be to consider the pharmacological and biochemical profiles of compounds on an individual basis, before correlations can be made. 203 Chapter 6: The effects of synthetic derivatives of 12-deoxyphorboI-13-0- angelate and resiniferonol on a mixture of isoforms of protein kinase C isolated from rat brain. 204 6.1. Introduction. Protein kinase C has been defined as the receptor site for tumour promoting phorbol esters (Castagna et al, 1982; Leach, James and Blumberg, 1983; Niedel, Kuhn and Vandenbark, 1983; see Chapter 1, section 1.5). Ellis et al (1987b) have demonstrated that tumour promoting and non-promoting phorbol esters can activate a mixture of isoforms of PKC from rat brain. They have proposed that four groups of phorbol esters exist, based on their ability to activate PKC (Ellis et al, 1987b; Evans, 1989): i) Compounds which fully activate PKC at low dose levels, in a similar manner to TPA, for example, Sap A. Sap A is a non-tumour promoter (Brooks et al, 1989). ii) Compounds capable of stimulating PKC to -30% of the activity of TPA, for example, DOPPA, Rx. iii) Compounds capable of fully activating PKC, but at 10-100 times higher dose levels than TPA, for example a-Sap A. iv) Substances that are unable to activate PKC, for example, a-Sapine, phorbol. Clearly, some of these effects could be due to the selective binding to and activation of different PKC isoforms, and/or different co-factor requirements for PKC activation exhibited by different phorbol esters (or different PKC isoforms). Recently, purified PKC isoforms have been utilised to study the actions of different phorbol esters (Evans et al, 1991; Ryves et al, 1991); DOPPA has been shown to be a selective pr isoform activator and Sap A a selective 8-isoform non-activator. Following pharmacological evaluation of the isolated and synthetic phorbol esters (see Chapter 5, section 5.3), it is important to assess the abilities of these compounds to exert effects at a biochemical level, viz activation of a mixture of rat brain PKC isoforms. 205 6.2. Materials and methods. 6.2.1. Purification of mixed isozvmic forms of protein kinase C from rat brain. Two female rats (200-250g) were killed by cervical dislocation and their brains removed. These were immediately homogenised in 10ml Homogenisation buffer (see Chapter 2, section 2.4.3.1.). This and all subsequent procedures were performed at 4°c. The homogenate was centrifuged at 15,000g for 15 minutes and the supernatant (containing cytosolic PKC) applied at lml/min. to a 2.5ml DEAE cellulose column (10cm x 0.5cm), equilibrated with buffer A (see Chapter 2, section 2.4.3.2.), connected to an FPLC machine (Pharmacia). PKC was eluted at lml/min. by application of a linear gradient of 0-0.4M NaCl in buffer A, using a total volume of 20ml and collecting 1ml fractions. 6.2.2. PKC activation assay. Fractions were immediately assayed for PKC activity (see Chapter 2, section 2.3.6.). The peak of activity was found to elute between 0.18-0.22M NaCl, and the peak fractions were pooled and stored at -70°c in 50%v/v glycerol. This purification method gave a mixed isozymic pool of rat brain PKC which was used for further studies. Initial PKC activation was assessed in the presence of phospholipid and Ca2+ compared to basal enzyme activity (without either phospholipid or Ca2+ presence) and in the absence of added phorbol ester. Fig.30 shows a typical activation assay profile and protein trace of the fractions obtained from the purification of rat brain PKC. In order to study the dependency of each phorbol ester probe on phospholipid and Ca2+ co-factor presence for activity, PKC activity was assayed in the following conditions: i) In the presence of phosphatidylserine (PS), and 0.5mM Ca2+ added ("+PS, +Ca2+"). 206 ii) In the presence of PS, and without added Ca2* ("+PS, -Ca2+"). iii) In the absence of PS, with 0.5mM Ca2* added ("-PS,-t‘Ca2+"). iv) In the absence of PS, and without added Ca2* ("-PS, -Ca2+"; basal activity). In conditions where Ca2+ was not added, ImM EGTA was added to the assay, thus giving the without Ca2+ (-Ca2+) assay. In conditions where PS was absent, 5pl 20mM Tris/HCl pH 7.5 was added in its place. Assays were only considered valid if the following control basal activities of the enzyme were satisfied, due to normal co- factor dependencies of the enzyme: i) +PS, +Ca2+ activity> -PS, +Ca2+ activity. ii) +PS, H-Ca2* activity> +PS, -Ca2+ activity. iii) -PS, +Ca2+ activity> -PS, -Ca2+ activity. iv) +PS, -Ca2* activity> -PS, -Ca2* activity. All activations shown are expressed as a percentage greater (or less) than the basal activity(=100% activity level) for that particular condition. 6.3. Results. 6.3.1. Purification of mixed protein kinase C isotvpes from rat brain. Fig.30. shows a typical activation assay profile of mixed PKC isotypes purified from rat brain by the above methods. The graph is shown as a percentage of activity in the presence of both phosphatidylserine and Ca2* (+PS, +Ca2+) greater than activity in the absence of PS and Ca2+ (-PS, -Ca2+). This enables purification of a mixture of PKC isotypes (measured by activity in the presence of both PS and Ca2+ co-factors) from nominal phosphorylation activity (measured in the absence of co-factors) which could also have been eluted by the DEAE cellulose purification of the rat brain. A typical UV protein trace obtained from the FPLC during column elution is superimposed over the activation profile. This indicates that protein presence and 2U/ >1 0 0 U c 0 0 B 0 -P iH P 0 c -P iH 0 0 c •H c O 0 •H ■H 0 0 0 -P 0 U ■P (0 0 t—I D> 0 o > -H iH •H < a 2 D 0 A 1 i u 1 • I • w • i i < I w I 1 Q B 0 M &p dp dp ip o O sq-Tun/souBqjcosqv O VO T3 0 -P D WAuaxpejfi tdbn 0 0 cm E o o N VO o 0 •H U fU* TJ 0 X •H E 4h 0 CM U (0 0 .Q t—I E •H P «P c 0 o iH cuo ft ■H >1 c0 ■P 0 o O 0 •H (0 0 ■P U 0 O C 10 0 u 00 •H fc. ■P 0 > •H ■P O < O m 0 M o o o 01D o o o •H in m fct (-[Bseq SAoqe uoTq.exnuiT^s %) D)Iti 208 enzyme activity co-purify, suggesting that centrifugation and initial washing of the column remove other kinase activities, leaving the mixed isotypes of PKC for elution by application of the NaCl gradient The peak fractions of PKC activity thus purified are then pooled, giving "pooled” or "mixed" PKC. 6.3.2. Activation of mixed rat brain PKC bv 12-deoxyphorbol-13-0-angelate. The ability of 12-deoxyphorbol-13-O-angelate (DA) to activate mixed rat brain PKC was compared to that of TPA in each of the four assay conditions (+PS, +Ca2+; +PS, -Ca2+; -PS, +Ca2+; -PS, -Ca2+; see above) (see Fig.31.). DA was capable of activating mixed PKC in all four assay conditions, and its activation of PKC was not dependent on the presence of other PKC co-factors (PS and Ca2+). This data suggests that in all four assay conditions, DA is able to activate mixed rat brain PKC isoforms to a similar extent as, and in a similar manner to, TPA. DA was therefore considered to be a member of the "group i)" phorbol ester compounds, that is, those compounds which fully activate PKC at low dose levels (see Introduction, above). DA was therefore used as a positive control in all subsequent experiments (as well as normal co-factor controls). 6.3.3. Activation of mixed rat brain PKC bv synthetic 12-deoxyphorbol-l3-O-angelate derivatives. 6.3.3.1. Activation of mixed rat brain PKC bv C™ propionic acid derivatives. The activation of mixed rat brain PKC isotypes by 12-deoxyphorbol-13-0- angelate-20-O-[DL-2-(3-benzoylphenyl)-propionate] (DAK) and 12-deoxyphorbol-13- O-angelate-20-O-[DL-2-(4-isobutylphenyl)-propionate] (DAI) (for synthesis, see Chapter 3, section 3.3.2.1.) is shown in Fig.32. DAI and DAK activate PKC to a similar extent, except for the -PS, +Ca2+ condition, where DAI maximally activates PKC to a greater extent than DAK. The activations of mixed PKC induced by these 209 +PS -Ca 2 + 200 n + PS + Ca2 + h 180 -| CO (0 X> a) > 160 - o X(0 i o 140 •H <0 •H> +J U 120 - < CAP 100 -PS -Ca 2 + 200 -PS +Ca2+ h(0 180 . CO (0 J2 > 160 o (0 ! 140 ■M <0 •H> u 120 < 100 5nM DA 50nM DA 5nM TPA 5nm DA 50nM DA 5nM TPA Figure 31 Activation of mixed rat brain PKC by 12-deoxy- phorbol-13-0-angelate ( DA ) in comparison to 12-0- tetradecanoylphorbol-13-0-acetate (TPA) Points are % activation above basal for each condition, mean ± SEM (n=3), assayed in duplicate 210 2 + PS -C a2 + 180 _ +PS +Ca <0 m 10 X3 160- -PS -Ca2+ -PS +Ca 2 + 1801 (0 CO (0 160“ dP 100 2.5nM 25nM 2.5nM 25nM 2.5nM 25nM 2.5nM 25nM DAK DAI DAK DAI Figure 32 Activation of mixed rat brain PKC by 12-deoxyphorbol- 13-0-angelate propionic acid derivatives. Points are % activation above basal for each condition, mean ± SEM (n=3) assayed in duplicate 211 two probes appear to be dose dependent; this is more noticeable in conditions where PS is absent In the conditions -PS, -Ca2* and +PS, +Ca2*, the activation is of a similar magnitude to that induced by DA. In the presence of both PS and Ca2*, the activation induced by DAI and DAK is 120% compared to basal; this is also the case for DA and TPA (see Fig.31.), and is probably indicative that PKC is almost completely activated in the presence of these two co-factors - the addition of a phorbol ester causes only a slight further elevation in PKC activity. Conversely, in the -PS, - Ca2+ condition, the largest activation above basal is observed, reflecting the ability of these phorbol ester derivatives to activate PKC in the absence of other co-factors. 6.3.3.2. Activation of mixed rat brain PKC bv 12-deoxyphorbol- 13-O-angelate-20-Q-p- bromobenzoate. The activation of mixed rat brain PKC by 12-deoxyphorbol- 13-O-angelate-20- O-p-bromobenzoate (DAB) (for synthesis, see Chapter 3, section 3.3.2.l.)is shown in Fig.33. DAB is able to activate PKC in all four assay conditions; this activation is dose-dependent (except for the -PS, -f-Ca2* condition where a decreased activation is observed at 50nM DAB). However, the activation induced by DAB is not as great as that induced by DAK or DAI in the -PS, -Ca2* or +PS, -Ca2* condition. This may suggest that DAB activation of PKC is partly dependent on Ca2* presence; the ability of this probe to activate PKC is enhanced in the +Ca2* conditions, particularly the -PS, +Ca2* condition. 6.3.4. Activation of mixed rat brain PKC bv synthetic resiniferonol derivatives. 6.3.4.1. Activation of mixed rat brain PKC bv 9.13.14-orthophenvlacetvlresiniferonol- 20-Q-p-bromobenzoate. The activation of mixed rat brain PKC by 9,13,14- orthophenylacetylresiniferonol-20-O-p-bromobenzoate (RoB) (for synthesis, see 212 +PS -Ca 2 + 1 6 0 —1 PS +Ca (0 caCO £ 2 140 - 0) > o £ 2 to G 120 “ o •H -P (0 •H> 100 ■P U < c*P -PS - Ca 2 + 160 -PS +Ca 2 + to co to £2 140 - CD > o £«0 2 120 ~ C o •H ■P (0 100 •H> ■PU < <#> 5nM 50nM 5nM 50nM 5nM 50nM 5nM 50nM DAB RoB DAB RoB Figure 33 Activation of mixed rat brain PKC by 12-deoxyphorbol -13-0-angelate-20-0-p-bromobenzoate (DAB) and 9,13,14-orthophenylacetylresiniferonol-20-0-p- bromobenzoate (RoB). Points are % activation above basal for each condition, mean ± SEM (n=3), assayed in duplicate 213 Chapter 3, section 3.3.2.1.) is shown in Fig.33. RoB is able to activate PKC above basal levels at the 50nM dose level in the presence of PS, and in the -PS, -Ca2+ condition, although in this condition the observed activation is lower than that observed for DAB, DA and the DA-propionic acid derivatives, DAK and DAI. PS presence enhances the activation induced by RoB, whereas the effect of Ca2+ presence appears to be minimal. Since RoB is a weak activator of PKC, and then only under certain assay conditions, there may be an inherent difference in the PKC activating ability between resiniferonol and 12-deoxyphorbol-13-O-angelate nuclei. 6.3.4.2. Activation of mixed rat brain PKC bv C™ propionic acid derivatives. The activation of mixed rat brain PKC induced by 9,13,14- orthophenylacetylresiniferonol-20-O-[DL-2-(3-benzoylphenyl)-propionate (RoK) and 9,13,14-orthophenylacetylresiniferonol-20-O-[DL-2-(4-isobutylphenyl)-propionate (Rol) (for synthesis, see Chapter 3, section 3.3.2.1.) are shown in Figs.34. and 35. In all assay conditions, both Rol and RoK cause a decrease in PKC activity below basal levels. In the +PS, +Ca2+ condition, the 5nM dose of RoK and Rol cause a slight activation.The activation is, however, less than that induced by the positive control (50nM DA), and may reflect a difficulty in reducing the activity of PKC in the presence of both PS and Ca2+ co-factors, when the enzyme would be phosphorylating its protein substrate (histone) at its greatest rate (due to co-factor activation). The inhibition induced by Rol appears to be of a similar level, whether the final assay concentration of Rol is 5nM or 50nM. This suggests that inhibition by Rol may be dosage-independent. The inhibition induced by RoK is more dose-dependent, and this probe may therefore be a more useful potential inhibitor. However, if the dose level of RoK in the assay is further increased (see Fig.36.), the inhibition of PKC induced by RoK is decreased, and in conditions where Ca2+ is not present, RoK causes a slight 214 +PS -Ca 2 + n c i 5nM 50nM 5nM 50nM 50nM 40 5nM 50nM 5nM 50nM 50nM RoK Rol DA RoK Rol DA Figure 34 Effect of synthetic resiniferonol C-20 propionic acid derivatives on activation of mixed isozyme PKC from rat brain, in absence of PS. Points are % Activation above basal (=100%), in presence and absence of Ca^+, mean ± SEM (n=3), assayed in duplicate using 50nM DA as positive control. ZJ.D (0 co 2 + PS -Ca2+ (0 200 +PS +Ca X3 T* 120 - 100 & 8 0- 60 5nM 50nM 5nM 50nM 50nM 5nM 50nM 5nM 50nM 50nM RoK Rol DA Rok Rol DA 40 -1 Points are % activation above basal (100%), in presence and absence of Ca2+; mean ± SEM (n=3), assayed in duplicate using 50nM DA as positive control. Figure 35 Effect of synthetic resiniferonol C-20 propionic acid derivatives on activation of mixed isozyme PKC from rat brain in presence of PS. Activation above basal iue3 Ihbto o ie rt ri PC nue by induced PKC brain rat mixed of Inhibition 36 Figure 100 120 80 - 80 60 - mean of three separate assays, each with duplicate with each assays, separate three of mean ue i ec condition. each in tubes ons r % ciain oprd o aa (=100%), basal to compared activation % are Points 5 - L23bnolhnl point] (ROK) propionate] [0- DL-2(3-benzoylphenyl) 9,13,14-orthophenylacetyl-resiniferonol-20- D cnetain (nM) concentration RDK 50 — ps ~Ca2+o—o - +Ca S P - # « —A A— 4 +S +Ca ^+PS p "Ca2+ +ps 500 2 2 + + Basal 216 217 activation above basal enzyme levels. PKC inhibition induced by Rol (see Figs.34. and 35.) appears greatest when one of the PS or Ca2* co-factors is present, although there is no dependency on either PS or Ca2+ presence for inhibitory activity. RoK inhibitory activity is greatest in the absence of both co-factors and the inhibition is more noticeable in the absence of Ca2+, although higher dose levels may lead to the loss of inhibitory activity, (see Fig.36.). 6.4. Discussion. The ability of the synthetic esters of 12-deoxyphorbol and resiniferonol to activate/inhibit mixed PKC isotypes from rat brain are summarised in Table 21. 12- deoxyphorbol-13-O-angelate appears to be a group i) PKC activator of similar potency to the standard phorbol ester, TPA (see Fig.31.). This is because DA is capable of activating mixed PKC at low nanomolar dose levels, in each of the four assay conditions studied. DA-induced activation of PKC in vitro is not dependent on the presence of either of the PKC co-factors, phosphatidylserine and Ca2*, in the assay mixture. In the presence of both PS and Ca2* in the assay, the percentage activation of PKC above basal levels induced by DA is lower than in conditions where only one (or none) of the PKC co-factors are present; the reason for this is probably that the enzyme is phosphorylating the protein substrate at its highest rate in the presence of both co-factors and thus the addition of a phorbol ester, whilst causing an increase in phosphorylation, does not cause such a dramatic enhancement of enzyme activity as that observed when only one (or none) of the co-factors are present The esterified C^ derivatives of DA were also capable of activating mixed PKC. However, their mechanism of inducing activation was more complex and it is not therefore possible to classify them in the phorbol ester groups previously defined 218 Table 21. A summary of the effects of synthetic derivatives of 12- deoxyphorboI-13-O-angelate and resiniferonol on mixed rat brain PKC. Probe. Effect on PKC. 12-deoxyphorbol-13-O-angelate Activator, of comparable potency to TPA in all assay conditions. 12-deoxyphorbol-13-O-angelate-20-O- Activator, of comparable potency to [DL-2-(3-benzylphenyl)-propionate] DA in +PS, +Ca** and -Ca2* (DAK) conditions. 12-deoxyphorbol-13-O-angelate-20-O- Activator, similar activity levels to [DL-2-(4-isobutylphenyl)-propionate] DAK, but more potent in the -PS, (DAI) +Ca2+ condition (but not as potent as DA). 12-deoxyphorbol-13-O-angelate-20-O- Weaker activator, Ca2* presence p-bromobenzoate (DAB) required (?). PS presence causes decreased activity. 9,13,14-orthophenylacetylresiniferonol- Weak activator, PS presence required 20-O-p-bromobenzoate (RoB) (?); activity spectrum varied. 9,13,14-orthophenylacetylresiniferonol- Potential inhibitor; inhibition is dose- 20-0- [DL-2-(3-benzylphenyl)- dependent, although less inhibition at propionate] (RoK) 500nM dose level. 9,13,14-orthophenylacetylresiniferonol- Potential inhibitor; effect not as dose- 20-0- [DL-2-(4-isobutylphenyl)- dependent as that of RoK. Less potent propionate] (Rol) than RoK. 219 by Ellis et al (1987b) and Evans (1989). DAB appears to require the presence of Ca2+ for PKC activating ability (see Fig.33.), although some activation of PKC above basal levels is observed in all four assay conditions. The C 2 0 propionic acid derivatives of DA, DAK and DAI, exhibit a similar spectrum of activity; both are capable of inducing PKC activation in all assay conditions, and their activity is enhanced in the absence of Ca2*. Indeed, in the -PS, +Ca2* condition, at the 2.5nM dose level, DAK slightly inhibits PKC activity and DAI induces only a slight activation of PKC above basal levels. The most potent effects of DA and its C 20 derivatives occur in the absence of both PS and Ca2*. This is unsurprising, as the addition of a phorbol ester in the -PS, -Ca2* assay condition would be expected to cause a dramatic increase in ability of the enzyme to phosphorylate its substrate. In the presence of one or other of the co- factors, addition of these derivatives causes an increase in phosphorylation, although the increase is generally less than that observed for the -PS, -Ca2* condition. In the presence of both co-factors, the enzyme is already phosphorylating at an increased rate and the presence of one of these phorbol esters does not cause such a large increase in substrate phosphorylation above basal levels as that observed in conditions of limited co-factor presence. One possible explanation for the difference in activity between DA and its Qo derivatives could be an ability of DA to increase the affinity of PKC for Ca2*, particularly in the absence of PS, since DA is able to induce higher levels of activation than the C ^ derivatives in the -PS, +Ca2* condition. DOPPA is recognised as a weaker activator of mixed rat brain PKC when compared to TPA (Ellis et al, 1987b) and is now known to selectively activate the fr-PKC isotype (Evans et al, 1991; Ryves et al, 1991). There is therefore also a possibility that these C^-derivatives 220 of DA could be selectively activating one (or more) of the PKC isotypes present in the mixture of isotypes purified by DEAE cellulose chromatography from rat brain. If this were so, some of the differences in activation (increases or decreases) induced by a particular phorbol ester could be accounted for by differences in the activation of selected isotypes; clearly different co-factor requirements of the selected isotype(s) thus activated would then make an important contribution to the overall activation profile of the mixed PKC in terms of phospholipid and/or Ca2+ dependencies. It would appear that esterification of the C^ hydroxyl group could be important in conferring isotype-specific activating properties on a phorbol ester probe. The Cjq resiniferonol p-bromobenzoate derivative, RoB, is a weak (or non-) activator of mixed PKC from rat brain and the presence of PS may be required for this activity; in conditions where PS is absent, the activation induced by RoB is lower than that induced by the 12-deoxyphorbol analogue, DAB. Some inhibition is observed in varying conditions when RoB is utilised as a potential agonist, and the results are not clear. This could point to this probe possessing a PKC isotype-specific activity, probably for a Ca2+-dependent isotype, since the presence of Ca2* would seem to enhance activity. The Cjq resiniferonol propionic acid derivatives, Rol and RoK, both inhibit PKC-induced substrate phosphorylation. RoK appears to inhibit PKC in a more dose- dependent manner than Rol, although both these compounds inhibit PKC activity in all four assay conditions. At higher doses of RoK, the decreased inhibition (resulting in a slight activation of PKC above basal levels) may be due to an inherent PKC activating ability of the resiniferonol nucleus at higher doses. Another resiniferonol derivative, Rx, can activate mixed PKC (Ellis et al, 1987b) and some PKC isotypes (Ryves et al , 1991), although higher dose levels, which induce a lower maximal 221 activation, are required for activity when compared to TPA. Rx has, however, been shown to be capable of inducing activation of a separate kinase, the Rx-kinase, to a greater extent than TPA (Ryves et al, 1989). These factors suggest that one possibility concerning the mechanism of RoK and Rol inhibition of mixed PKC isotypes could be due to differences in PKC activating capability of the resiniferonol nucleus compared to the phorbol (and 12-deoxyphorbol) nucleus, in combination with an inhibitory activity of the esterifying group at Qo- At the present, the only potent and specific PKC inhibitor whose mechanism of action is to interact with the phorbol ester binding domain of PKC is Calphostin C (Kobayashi et al, 1989; see Chapter 1, section 1.5.6.). However, the use of calphostin C may be limited, due to apparent non-specific cytotoxicity (Tamaoki, 1991). The synthesis of these 12-deoxyphorbol and resiniferonol derivatives has provided compounds which, due to their differences in activity spectrum, have enabled further understanding of the interactions of phorbol esters with their receptor site, PKC. In addition to providing compounds which are potential PKC activators (and possibly PKC isotype specific activators), compounds have been synthesised which may be phorbol ester inhibitors of PKC. However, further understanding of the mechanism of PKC inhibition by RoK and Rol, both in terms of isotype-specific action and binding activities, are required before they can be designated as such. 222 Chapter 7: Studies on the activation of platelet protein kinase C by the non- aggregatory phorbol ester, 12-deoxyphorbol-13-0-phenylacetate-20-0-acetate (DOPPA) - a specific ft-PKC isotype activator. 223 7.1. Introduction. Platelets are anucleate cells which circulate the blood in a resting, inactive form. Activation by a number of transmitters (for example, Platelet Activating Factor, PAF) can induce functional changes, for example, shape change (to a spheroidal shape), secretion of granular contents (which include ATP, ADP, Ca2+, Mg2+, serotonin, and proteins e.g. fibrinogen, acid hydrolase and clotting factors), increase adherence and ultimately result in platelet aggregation. Aggregation consists of two phases; a primary, reversible phase and a secondary, irreversible phase. A potent biological activity of the phorbol esters is their ability to induce platelet aggregation (Zucker, Troll and Balman, 1974; see also Chapter 1, section 1.4.3.). This, together with the fact that numerous hormone and neurotransmitter agonists acting on platelets (for example, thrombin, collagen, thromboxanes and PAF) stimulate phosphatidylinositol turnover and subsequent production of both DAG and Ins-1,4,5-P3 second messengers, implicates a role for PKC in platelet function. However, this role is unclear, since PKC exhibits both inhibitory, e.g. inhibition of PtdIns-4,5-P2 hydrolysis (Watson and Lapetina, 1985); inhibition of Ca2* mobilisation (Krishnamurthi, Joseph and Kakkar, 1986), and stimulatory, e.g. aggregation (Nishizuka, 1984); secretion (Kaibuchi et al, 1983), effects. Various protein substrates for PKC have been identified in platelets. These include a specific PKC protein substrate of apparent molecular weight 40-47kDa, often termed "p47" (but see also Chapter 1, section 1.5.7.). p47 is phosphorylated in response to a number of stimuli, including the calcium ionophore, A23187, (Kaibuchi et a ly 1983), dioctanoylglycerol (Lapetina et aly 1985), thrombin (Kaibuchi et al, 1983) PAF (Lapetina and Seigel, 1983) and TPA (Castagna et al, 1985). More recently, (Brooks et al , 1990), other phorbol esters have been shown to be capable of inducing 224 PKC-mediated p47 phosphorylation. However, the physiological consequence(s) of this phosphorylation and the necessity of this phosphorylation for platelet aggregation have not been resolved. p47 phosphorylation does provide a useful marker for PKC activity, and Brooks et al, (1990) have shown a requirement for calcium ionophore presence for less potent (or non-) aggregatory phorbol esters to induce p47 phosphorylation. The objective of this work was to measure the activation (if any) of PKC by the non- aggregatory phorbol ester, 12-deoxyphorbol-13-O-phenylacetate-20-O-acetate (DOPPA), and to investigate any effect the presence of Ca2+ might have on DOPPA- induced activation of platelet PKC(s). 7.2. Materials and methods. 7.2.1. Platelet isolation. 40ml freshly drawn venous blood was collected from healthy male donors, who denied taking medication for the previous 10 days, into 1/10 volume of anticoagulant buffer (see Chapter 2, section 2.4.2.). The blood was incubated at room temperature for 15 minutes, and then centrifuged at 200g for 10 minutes. The platelet rich plasma (PRP) was re-centrifuged at 280g for 10 minutes to pellet any residual red and white blood cells. The PRP was then centrifuged at 1400g for 5 minutes to pellet the platelets. All centrifugations were performed at room temperature; all following procedures were performed at 4°c. 7.2.2. Purification of mixed isoforms of platelet PKC. After removal of the supernatant, the pelleted platelets were re-suspended in 2ml homogenisation buffer (see Chapter 2, section 2.4.3.1.), and rapidly lysed by sonication for 30 seconds. The lysate was then centrifuged on an MSE bench top microfuge for 30 minutes. The resultant supernatant, containing cytosolic PKC, was 225 applied to a 2.5ml DEAE cellulose column (10cm x 0.5cm), equilibrated with buffer A (Chapter 2, section 2.4.3.2.), connected to an FPLC machine (Pharmacia). PKC was eluted at lml/min by application of a linear gradient of 0-0.4M NaCl in buffer A, using a total volume of 20ml and collecting 1ml fractions. Fractions were then assayed for PKC activity (see Chapter 2, section 2.3.6.). The peak of activity was found to elute between 0.18-0.22M NaCl (see results, below), and the peak fractions were pooled. This purification step gave a mixed isozymic pool of platelet PKC, and is termed "mixed" or "pooled" PKC. 7.2.3. Purification of isoforms of platelet PKC. The mixed PKC was applied to a pre-packed Sephadex G-25 gel filtration column (PD-10, Pharmacia), equilibrated with buffer C (see Chapter 2, section 2.4.3.5.). PKC was eluted under gravity and activity was recovered between 3-5mls buffer C. Active fractions were loaded onto a 0.5cm x 2.5cm hydroxylapatite column, equilibrated with buffer C, connected to an FPLC machine. PKC was eluted at 0.5ml/min by application of a linear gradient of 20-250mM potassium phosphate buffer (buffers C and D, see Chapter 2, sections 2.4.3.5. and 2.4.3.6.) using a total volume of 84ml and collecting 1ml fractions. These fractions were assayed for PKC activity (see Chapter 2, section 2.3.6) and the resultant PKC isozyme fractions were used for further studies. Assays were only considered valid if normal co-factor control activities of the enzyme were satisfied (see Chapter 6, section 6.2). 7.3. Results. 7.3.1. Studies on mixed PKC isolated from human platelets. 7.3.1.1. Purification of platelet mixed PKC. As stated above, the peak of activity of mixed isozymic forms of PKC isolated o lsq OAoqe uoTq.eTnun:}S(leseq %) 600 n Fractions Pooled VO O 0>P o o •H •H •H 4-1 1— 1 < 4-> 4-> O > 10 o C (0 0o f0 CO CO (0 M 0 a CD 4U3Tpej:6 iDew e D i 6 : j e p T 3 U /4 W •H sq-Tun/soucqjosqv i—1 T3 z U 4-> (0 00)O) U (0 0) G • 1 1 1 O CM *H D > 4-> 4J M 0 G u d) a i • i i i i A} tat O o CM } oe o)id (N O p d O P d VO VO 00 ,Q En V4 (0 u c c E o p 226 •H CO CJ TJ •H O. •H •H •H •H < ■M <+4 i—I i—I 4-> vw CO T3 iH H o 4-» N w < w 0) Q X E cn P M 0 O < 0 ( E > (0 o c U CD O CD (0 CO CO (0 >i a O (0 <1) O 0 o P E c P 7.3.1.2. Activation of mixed platelet PKC isozymes bv 12-deoxvphorbol-13-0- phenvlacetate-20-Q-acetate (POPPA). The ability of DOPPA to activate a mixed isozymic pool of platelet PKC was assessed in the presence of phosphatidylserine (PS) and 0.5mM added Ca2+ or ImM EGTA (that is, +PS, +Ca2+ and +PS, -Ca2* conditions; see Fig.38.). The results are expressed as a percent of the maximal activation induced by the presence of all three co-factors (PS, Ca2+ and DOPPA), ±SEM, from four separate experiments (at least two different purification preparations), each data point being performed in duplicate for each experiment This revealed that the k, value for DOPPA activation of mixed platelet PKC was 120 ±30nM in the presence of Ca2* and 530 ±30nM in the absence of Ca2+ (values are mean ±SEM, n=4). This suggests that DOPPA requires the presence of Ca2+ for activation of PKC. This correlates with parallel studies on DOPPA induced phosphorylation of the p47 PKC substrate protein in platelets (Brooks, 1989; Brooks et al, 1990), which indicate that concurrent addition of the calcium ionophore, A23187, is required for DOPPA- induced p47 phosphorylation (see Fig.39). Since more than one PKC isozyme are known to be present in human platelets (Tsukuda et al, 1988), the activation of mixed PKC isozymes induced by DOPPA could represent preferential activation of one (or 228 <4-1 <4-4 0 + 0 CM c •H < 00 0* PM CM o a > i 43 (0 00 c <4-1 hC H 0 0 o •H + J a> (0 o > c •H 0) -M CO O £> < to o o o o o oo vo CM 00 CO MQ) 3 Aq.TATq.oe xBUITXBUI ^ u eo x aj D> •H En Points are mean ± SEM of four separate experiments, assayed in duplicate. 03 0 rH 0 4-» 0 u to to X3 rH c P P 0 0 p to O rH 0 0 a a x rH 0 0) a 0 p (0 *4H P a 03 0 x P 0 0) -P 0 0 2 p 4-> ■H a X C 0 C £ 4-J •H £ 0 0 4-> 4 J • c C CO X 1 c •H o o c 0 1 0 o o •rH • 0 H 0 0) O) -P s CO 4-> x • * c 0 c to 0 Eh 0 •H rH -_ £ p C * 0 04 • 0 P ■P < c 03 rH 0 0 04 0 • (U 0 £ rH Eh •H p X a -M 0) a P 0 C 10 -P to 0 0 ■p 0 X CO p -P £ o •H 3 a) o i CO 0 o -P O 0 0 0 -P H to c rH •H X c P M o 03 rH 5 *H -P X to 0 C • P p X3 * C 0 PO 0 P 0 *H o 3 X 4-> O * c 0 a 3 x 0 < 0 £ to a o a u 4H p Eh rH 0 CO x (!) •H 4-> • 03 P 0) Q) •H a C H 0 o 01 £ • 0 m c C/3 0) Q) o < u CO X 1 P 04 • I------1------1------1------r o 0) 4-> 1 Q 04 t"- e ' p o o 00 in o in o m en a 03 > i Q rH • • • • • C p X CO 7.3.2. Studies on PKC isoforms isolated from human platelets. 7.3.2.1. Purification of platelet PKC isoforms. Platelet PKC isoforms were purified by hydroxylapatite column chromatography as described in section 7.2.3. Fig.40. shows a typical activation assay profile of the fractions obtained from hydroxylapatite column purification of mixed platelet PKC, and the accompanying UV protein trace. Peaks of kinase activity eluted at 89mM and 120mM phosphate gradient which correspond to the elution positions of Types II and III PKC from rat brain (Tsukuda et al, 1988). Type HI platelet PKC has been shown to be identical to Type HI (or a-) PKC isolated from rat brain (Tsukuda et al, 1988; Watanabe et al, 1988), yet immunoblot and kinetic analysis has suggested that Type II platelet PKC is distinct from Type II (or P-) PKC isolated from rat brain (Tsukuda et al, 1988; Watanabe et al, 1988) (although could this difference reflect species variation of the kinase or species antibody recognition problems?). Therefore, the isolated peaks are labelled "peak a" and "peak b", conforming to the previously published nomenclature of platelet PKC isotypes (Tsukuda et al, 1988). 7.3.2.2. Activation of platelet PKC peaks "a" and "b" bv DOPPA. Activation of platelet PKC peaks "a" and "b" by 500nM DOPPA in the presence of PS and the presence and absence of Ca2+ is shown in Fig.41. This indicates that in the absence of Ca2+, DOPPA is capable of maximally activating peak "a" PKC to a level comparable to PKC activation induced by Ca2+ in the absence of DOPPA. In the +PS, +Ca2+ condition, DOPPA is capable of fully activating peak "a" PKC. This suggests that activation of peak "a" PKC by DOPPA is dependent on the Peak o vo o ee Qoe oqxuxs / Aq.TATq.OY %/ DMd uoTqexnuixqs QAoqe teseq CM o in U/uxeB qqsq uinxsseqod: aqeqdsoqd WUi/quaxpejB o o CM O O O in sq.Tun/boueqjosqv o o o CM o © (X> in o oo o 00 oo vo CM (N CO vo in vo CM •H 4-J P e >1 H C •H i s a a* to 4-> •P E 0 C0-H i P to 0 CO Q* (0 (0 03Pi P (0T3 i o 0 H • P H • H • H • (4-1 H • (4-1 I—I Pm < 4-> cu u * •H 4-> TJ J 4 01 3 P 03 U > (0 o c 0) 0) o to CO CO (0 (0 o i > Pi P 0 N E 0) (0 03 (0 3 •H > 4-J D 4-J p 0) o 0 p by Hydroxylapatite Chromatography. 0) iue 41 Figure Peak maximal activation 0 - 100 80 - 20 - sas are ot n rsne f .m Ca2+ mean 0.5mM of presence in out carried Assays 'a' peaks by PKC and'b platelet of Activation +Ca 0n DPA n h peec ad bec o Ca2+ of absence and presence the in DOPPA 500nM DPA DPA DPA +DOPPA -DOPPA +DOPPA -DOPPA or eaae rprtos sae i duplicate. in assayed preparations separate four +a o IM GA -a . eut ae the are Results ). + (-Ca2 EGTA ImM ) or + (+Ca2 2 + C2 +a+ +Ca +Ca2+ -Ca2+ + ± E f eemntos are ot on out carried determinations of SEM ek'* Peak'b* Peak 'a* 2 + -Ca + 2 + +Ca + 2 + 233 presence of Ca2+. In these saturating conditions of DOPPA and Ca2+ presence, peak "b" platelet PKC is relatively unresponsive to co-factor induced activation; the observed activation is only slightly higher than that induced by the presence of Ca2+ alone. In the presence of DOPPA, but absence of Ca2+, peak "b" is slightly more responsive, although activation levels are low when compared to the maximal activation levels of peak "a". This data suggests that DOPPA is less able to activate peak "b" PKC than peak "a" PKC; in the presence of Ca2+, DOPPA would appear to selectively activate peak "a". Peak "b" PKC would also appear to be less responsive to Ca2+ presence than peak "a". 7.3.2.3. The effect of altering Ca2+ concentration on the activation of platelet PKC peaks "a" and "b" induced bv DOPPA. To further investigate the requirement of Ca2* presence for activation of peaks "a” and "bM PKC by DOPPA, the Ca2* concentration was adjusted whilst maintaining PS and DOPPA presence. The range of Ca2+ added to the assay was lO.OmM-O.lmM, as well as 1.0 and 5.0mM EGTA; the final concentration of DOPPA in the assay was 250nM. Fig.42. indicates that maximal activation of peak "a" PKC by DOPPA occurs only in the presence of Ca2+; activation is lower in the presence of EGTA. Increasing the Ca2* concentration does not seem to affect the extent of maximal activation, although there is possibly some inhibitory effects at a high (lOmM) Ca2+ concentration. Conversely, peak "b" PKC is maximally activated in the presence of EGTA and increasing Ca2+ concentration appears to result in a decreased activity of the enzyme. It is noticeable that the activation of peak "a” PKC induced by DOPPA is greater than the activation of peak "b" PKC, independent of the Ca2+ concentration employed; this supports earlier evidence that DOPPA selectively activates human platelet peak "a” PKC over peak "b" PKC. O CO Peak X d CD <0 0T/uido c O © O VO CM CO CM CM + . N •H u U < -P ■P W H U P3 u a> o c a U (0 o c to 0 O CM + •H •H X tp •H •H CM d tp tp tp U H ■P -P •H u ■P ■P d H H -P ■P d u c o (U c 0) <0 o U to c c c c o (1)to to C D) to o o c 10 U to o -P O * to rH X3 o d < d +J d Q <0 234 CM + o •H tp CM in •H •H •H o •o 2 Q d d P +J < d C/3 u O H < u < c (0 o o o o a) c U (0 o c co O c o to to C to u c O) to c co co to >i •0 M P a) o p c Q* u Q) co 0) > Results are mean ± SEM on three separate experiments assayed in duplicate. 235 7.4.Discussion. 12-deoxyphorbol-13-O-phenylacetate-20-O-acetate (DOPPA) was capable of inducing activation of mixed isoforms of PKC isolated from human platelets. The k, values for this activation were 120nM (±30nM) in the +PS, +Ca2+ condition and 530nM (±30nM) in the +PS, -Ca2+ condition. The maximal activation of mixed platelet PKC was similar in both these conditions. The k, value for DOPPA in the +PS, +Ca2+ condition was similar to its k, value for the activation of mixed PKC isolated from rat brain (Ellis et al, 1987b). Unlike the C^-deacetylated derivative, DOPP, DOPPA is a non-aggregatory phorbol ester, and is unable to induce platelet aggregation in either a direct manner or indirectly, by causing the release of the Transferable Aggregating Substance (TAS) (Williamson, Westwick and Evans, 1980; Westwick, Williamson and Evans, 1980; Edwards et al, 1983). This is in agreement with structure/activity analysis of platelet aggregation activity for phorbol esters (Evans and Edwards, 1987). Thus, the activation of platelet PKC by DOPPA at normal phorbol ester concentration levels (nM) could be considered somewhat anomalous. However, the platelet PKC activating ability of DOPPA, which appears to require the presence of Ca2+ (since the k, value is reduced in the presence of Ca2+), does correlate with the ability of DOPPA to induce phosphorylation of the p47 PKC marker substrate protein present in platelets (Brooks, 1989; Brooks et al, 1990). There are therefore several possibilities that could explain the PKC-activating but non-aggregatory actions of DOPPA: i) phorbol ester induced platelet aggregation may occur partly via distinct biochemical pathways from PKC activation. ii) DOPPA could preferentially activate one isoform of platelet PKC, which may not be involved in events that result in platelet aggregation. iii) DOPPA may preferentially activate one platelet PKC isoform, and 236 activation of all isoforms may be required for induction of aggregation. iv) DOPPA-induced activation of PKC would appear to require the presence of Ca2+. Thus, the Ca2* concentration may be critical in events leading to platelet aggregation, and DOPPA may be unable to induce an adequate level of PKC activation required for an aggregatory response at resting Ca2+ levels. v) DOPPA induced phosphorylation of the p47 platelet PKC substrate protein is also synergised by Ca2* presence. This also points to a required increase in the levels of Ca2+ before DOPPA can induce any cellular response, although it should be remembered that the physiological function of p47 has yet to be determined. Thus, whilst DOPPA can induce phosphorylation of p47, it may not be capable of inducing phosphorylation of a protein substrate whose phosphorylation is critical for platelet aggregation. In order to attempt to further understand the actions of DOPPA on platelet PKC, I decided to separate the PKC isoforms present in platelets by hydroxylapatite column chromatography. This yielded two isoforms, peaks "a" and Mb", which is in agreement with previous purifications of platelet PKC (Kaibuchi et al, 1983; Tsukuda et al, 1988). However, immunoblotting analysis of rat brain PKC (Evans et al, 1991; Ryves et al, 1991) has revealed that novel PKC isoforms (5, e) may elute underneath a, (3, or y PKC activity peaks (this will presumably be the case for £ and T] PKC as well). DOPPA was shown to fully activate peak "a" platelet PKC in the presence of Ca2+. Peak "b" was less responsive to DOPPA, even in the presence of Ca2+. This suggests that DOPPA may preferentially activate peak "a" platelet PKC, which is likely to be a p or p-related PKC, since peak "a" reacts with specific (3-PKC monoclonal antibodies, although its kinetic properties are different, to rat brain (3-PKC (Watanabe et al, 1988; Tsukuda et al, 1988). Subsequently, the group of Evans have 237 demonstrated that DOPPA specifically activates ft-PKC in a Ca2+-dependent manner at nanomolar concentrations by the use of pure PKC isoforms (Evans et al, 1991; Ryves et al, 1991). This evidence supports peak "a" platelet PKC being a p or p- related PKC. The slight activation of peak "b" platelet PKC induced by DOPPA may be due to cross-contamination of peak "a" PKC into peak "b" PKC since it is known that PKC isoforms isolated from rat brain and separated by hydroxylapatite chromatography cross-contaminate when immunologically analyzed by specific antisera (Evans et al, 1991). The requirement of Ca2+ for DOPPA-induced activation of peak "a" platelet PKC may provide one explanation for DOPPA being a non-aggregatory phorbol ester; Ca2+ presence (introduced via A23187) is also required for DOPPA induced phosphorylation of p47. The situation is similar for the weak aggregatory agent, Sap A, in that ionophore presence is required for Sap A to induce phosphorylation of p47 (Brooks, 1989; Brooks et al, 1990), and whilst Sap A can activate all PKC isoforms (except 5-PKC) (Evans et al, 1991; Ryves et al, 1991), activation of P- (and a- and y-) PKC is dependent on the presence of Ca2+. Conversely, the potent platelet aggregatory agents, TPA and DOPP, are capable of maximal phosphorylation of p47 in the absence of A23187 (Brooks, 1989; Brooks et al, 1990) and are capable of maximal activation of purified a, p, y, 8 and e PKC to the same extent in the presence or absence of Ca2+ (Evans et al, 1991; Ryves et al, 1991). Thus Ca2* concentration could be critical in events that lead to phorbol ester induced platelet aggregation. However, selective (or non-selective) activation of individual PKC isoforms (which may have their own substrate specificities and functions within the cell) may also be critical in understanding the aggregatory response induced by phorbol esters. Chapter 8: Final discussion and concluding remarks. 239 The development of the concept of tumour promotion by the use of repeated applications of croton oil (Berenblum, 1941) resulted in intensive research to identify the biologically active constituents of the plant families Euphorbiaceae and Thymelaeaceae. Approximately twenty-five years later, Hecker (1968) and Crombie, Games and Pointer (1968) elucidated the structure of TPA and its parent alcohol, phorbol. Subsequent to this, many laboratories isolated biologically active diterpene esters based upon the tigliane, daphnane and ingenane nuclei from the plant families Euphorbiaceae and Thymelaeaceae (Hecker and Schmidt, 1971; Evans and Taylor, 1983). The phorbol esters had been shown to possess a large spectrum of biological activities, but it was the discovery of high affinity receptor sites (Dreidger and Blumberg, 1980), which were shown by several laboratories to co-purify with protein kinase C (PKC) (Castagna et al, 1982; Leach, James and Blumberg, 1983; Niedel, Kuhn and Vandenbark, 1983; Sando and Young, 1983; Ashendel, Staller and Boutwell, 1983; Parker, Stabel and Waterfield, 1984) that enabled the identification of the biochemical receptor site of the phorbol esters. This established a unique position for the phorbol esters as probes of cellular communication signals and disease states (particularly inflammatory and cancerous conditions). The existence of PKC as a multigene family, consisting of the calcium-dependent a, ft, pn and y isoforms (Coussens et al, 1986; Parker et al, 1986) and the calcium-independent 5, e, £ and ti isoforms (Ono et al, 1987b; Osada et al, 1990; Bacher et al, 1991), as well as the complexity of regulation of the phosphatidylinositol cycle (Berridge and Irvine, 1989; Potter, 1990), has further hindered the understanding of cellular signal transduction and how PKC and the phorbol esters exert their ultimate biological effects. These problems have generated considerable interest in the structure, localisation and function of PKC substrates (e.g. Rozengurt et al, 1983; see Chapter 1, section 1.5.7. 240 for more detail) and mechanisms of regulating (particularly inhibiting) PKC activity. As described in Chapter 1, section 1.5.6., PKC inhibitors catalogued to date are generally non-specific and of low potency. However, the ATP-binding site inhibitors (Ro 318425 and Ro 318830) developed by Roche based upon staurosporine (Davis et al, 1989; Elliott et al, 1990; Nixon et al, 1992) and Calphostin C , which acts by interaction with the phorbol ester binding domain of PKC (Koyabashi et al , 1989; Tamaoki, 1991), could prove to be critical in the dissection of PKC-mediated events. However, since the inhibitory action of calphostin C is light-dependent (Bruns et al , 1991) and it exhibits cytotoxic properties (Tamaoki, 1991), the use of this compound may be limited. Therefore, there is still a requirement for the development of PKC inhibitors (which could have potential as therapeutic agents), particularly those acting at the endogenous (DAG/phorbol ester) binding site. Inhibitors acting at the DAG/phorbol ester binding site would have the advantage of acting solely upon PKC, and after PKC down-regulation would have no effect on PKM (unlike ATP-binding site inhibitors). The objective of this work was to design compounds which would be potential inhibitors of PKC activity by interaction with the phorbol ester/DAG binding domain. In the first instance, I decided that it was necessary to isolate diterpene nuclei from natural sources which could be used as starting material for subsequent semi synthetic modification. The latex of Euphorbia poissonii was selected to provide 12- deoxyphorbol and resiniferonol nuclei since compounds previously isolated from this species of the Euphorbiaceae have been shown to possess a more limited spectrum of biological activities when compared to the classical 12,13-phorbol diesters (e.g. TP A) (Evans and Edwards, 1987). Esters of these 12-deoxyphorbol and resiniferonol nuclei would therefore enable further dissection of the requirements for activity of the 241 phorbol esters which, if not immediately providing PKC inhibitory compounds, could provide a rationale for the design of such compounds. Separation of biologically active diterpenes by solvent partition methods, followed by the use of charcoal column, centrifugal liquid and preparative partition and adsorption thin layer chromatographic techniques enabled the isolation of six compounds from the latex of E.poissonii. Centrifugal liquid chromatography was particularly useful for the initial separation of larger quantities of extracts, due to the quantity of material applied to the plate and the rapidity of the technique, with the added advantage of not generating silica dust After the final separation of compounds by preparative TLC, the following compounds were identified on the basis of their MS, ^-NM R, UV and IR spectroscopic characteristics: 12-deoxyphorbol-13-0- phenylacetate-20-O-acetate (DOPPA); 12-deoxyphorbol-13-O-phenylacetate (DOPP); 12-deoxyphorbol-13-O-angelate-20-O-acetate (DAA); 12-deoxyphorbol- 13-O-angelate (DA); 9,13,14-orthophenylacetylresiniferonol (Ro); 9,13,14-orthophenylacetyl- resiniferonol-20-O-homovanillate (Rx), in yields that agree with those obtained by other workers (Schmidt and Evans, 1977a; Evans and Schmidt, 1979). DA and Ro were then utilised as starting material for selective esterification of the Qo primary alcohol. Esterification with DL-2-(4-isobutlyphenyl)-propionic acid (IbuprofenR), DL- 2-(3-benzoylphenyl)-propionic acid (KetoprofenR), p-bromobenzoic acid and 3- methoxy-4-hydroxy-phenylacetic acid (homovanillic acid) provided two sets of four compounds each based upon the resiniferonol and 12-deoxyphorbol nuclei (see Table 13) for pharmacological and biochemical analysis. Pharmacological analysis (see Table 22, which summarises the biological activities of compounds) utilised the acute pro-inflammatory action of diterpene esters and was quantified by the mouse ear erythema assay (Schmidt and Evans, 1979). This Table 22. Summary of biological activities of compounds studied. m vo o o o OV cn 2 ^ V & o n Op n c o o 00 m CN ^ O CN n c o o 00 m o CN o ^ CO u ^ CN VO s a 00 «n 00 o o vo & 3 C CN o o n c Pu& 6 .2 a n c «o o o oo 0 . Op J. 5 cn 00 4> o a 1 c c 1 £ 242 243 I-* * o c n oo * «o CN VO «n »o oo o c n CN 8 00 o CN OO o rH CN o o •a 00 73 00 *se a « T3 <-» CN O a CN vs c& ji 0> 13 CN Vh .a oo fHo vs o X) A u ■» O Oh «3 % > , a GO a o O h e o *o cn y cn p c n O 'B h CN O o o ^ 6 2 OV CN OV CN O v CN O h 244 enabled the assessment of the IDjo and tp^ values for each of the isolated and semi- synthetic compounds (see Table 18). 12-deoxyphorbol compounds were approximately three to five fold less potent in their ability to induce erythema than the standard phorbol ester TPA (except for DAHV, see discussion of Rx, below). esterification, whether with acetate or more complex molecules, caused a slight reduction in inflammatory potency; the largest decrease in inflammatory potency was observed when the 12-deoxyphorbol nucleus was esterified with p-bromobenzoic acid. Ro has an IDso value of 0.72pg/5pl; this value is -70 fold lower than that of TPA and -20 fold lower than the ID^ value of DOPP, whose C13-phenylacetate ester is not dissimilar in structure from the 9,13,14-orthophenylacetyl ester of Ro. These factors point to an inherent difference in the pro-inflammatory activity of the resiniferonol and 12-deoxyphorbol nuclei (although p-bromobenzoic acid esterification of Ro increases the ID50 value of RoB to 0.48pg/5pl). Cjo esterification of Ro with propionic acid derivatives renders these compounds non-inflammatory in their own right Rol and RoK were therefore considered potential inhibitors of phorbol ester-induced erythema and used as a pre-treatment to attempt to inhibit the erythema response induced by the phorbol ester agonists, TPA, DOPPA, Sap A and Rx. However, Rol and RoK were unable to inhibit the erythema induced by these agonists, and therefore cannot be classified as in vivo inhibitors of phorbol ester induced erythema under the assay conditions employed here. Additional experiments to re-investigate the anti- erythema activity of RoK and Rol would enable clarification of whether these compounds are potential in vivo antagonists of phorbol ester induced effects, or whether there are pharmacokinetic problems of delivery of these compounds to their site of action within the cell. Such experiments could include increasing the time of antagonist application before agonist application and increasing the dose of antagonist 245 applied. Furthermore, other inflammatory models could be utilised to study the effects of Rol and RoK on different components of the inflammatory response; for example, studies on the antagonism of induction of oedema in the mouse ear or paw (assessed by weight measurement) and in vitro inhibition of superoxide ion production via NADPH-oxidase by a range of biologically active phorbol esters could provide clues concerning the potential use of these inhibitors. The erythema response induced by the Qo-homovanillate derivatives Rx and DAHV are respectively 100 and 50 fold more potent than that induced by TPA, although homovanillic acid itself is non-inflammatory. The extreme inflammatory potency of Rx and DAHV correlates with that of other homovanillate phorbol esters (Szallasi, Sharkey and Blumberg, 1989). However, the irritancy of Rx (and presumably of other Qo homovanillate esters) would not seem to be entirely of a neurogenic nature, as has been proposed (DeVries and Blumberg, 1989; Szallasi and Blumberg, 1989a, 1989b, 1990a, 1990b). Detailed study of the pro-inflammatory response induced by Rx including the use of desensitisation by capsaicin pre- treatment, comparison of latency and duration of response with capsaicin and standard phorbol esters, and differential inhibition of erythema by non-steroidal and steroidal anti-inflammatory agents, revealed that Rx induced erythema was of a mixed aetiology. The inflammatory response to Rx appears to consist of an early, neurogenic phase and a classical phorbol ester late phase (Gordge, Evans and Evans, 1990; Evans et aly 1992). This suggests that the biochemical mechanism of action of Rx may involve activation of Rx-kinase (Ryves et aly 1989; Evans et aly 1990), although how this enzyme activation interacts with biochemical mechanisms of nociceptive stimuli is unclear. One possibility is an interaction with ion channel regulation; it has been proposed that both PKC and PKA can phosphorylate purified subunits of the GABAa receptor (Browning et aly 1990; Whiting, McKeman and Iversen, 1990), and that TPA induced PKC activation can cause down-modulation of a series of different recombinant GABAa receptor subunits, when compared to a-TPA treated subunits (Sigel, Baur and Malherbe, 1991). The significance of these observations is unclear (partly since in vivo subunit composition of the GABAa receptor is not known), however, there is an indication that ion channels may be partially regulated by kinase mediated phosphorylations. Thus, the observed neurogenic activity of Rx could partially involve Rx activation of Rx-kinase and subsequent ion channel regulation (either complete or partial) by Rx-kinase induced phosphorylations. This regulation of neurogenic ion channels by Rx-kinase may be in addition to any direct effect of Rx on the ion channel; clearly, kinase regulation of ion channels (if any) is reliant upon complex feedback mechanisms involving the endogenous extracellular agonist as well as normal regulatory mechanisms of the kinase pathways. Further experiments to attempt to clarify the properties of Rx-kinase could use 3H-Rx to study Rx binding and inhibition of binding by phorbol esters (and other homovanillate phorbol esters) to Rx-kinase, and could follow the translocation (if any) of Rx-kinase from cytosol to membrane on phorbol ester activation. The Rx-kinase may itself exist as a series of isoforms, all of which could be different from recognised forms of PKC; studies could also be undertaken to assess whether Rx-kinase was a "daphnane receptor", that is whether other daphnanes would bind to Rx-kinase in preference to PKC. Chemical synthesis could produce compounds with different residues attached to homovanillic acid to investigate further the process of neurogenic inflammation. Additionally, homovanillate esterification at different positions of the phorbol nucleus (e.g. C12, C13, C4, C16) could be attempted, to assess whether C^ homovanillate substitution is required to impart neurogenic activity. Parallel synthesis of this work could be 247 performed using ingenane diterpenes as starting material, which would enable further structure/activity conclusions (at both the pharmacological and biochemical level), concerning the phorbol esters as a class of compounds, to be drawn. Computer-assisted molecular modelling analysis of the isolated and synthesised compounds points to a difference in the resiniferonol nucleus from the phorbol and 12-deoxyphorbol nuclei, in that the minimum free energy of Ro and its derivatives (including complex C^ esters) is much lower than naturally occurring phorbol esters or parallel synthetic 12-deoxyphorbol esters. The significance of this is unclear, although further studies on the effects of these compounds in their ability to activate PKC isoforms (and other kinases) may enable clarification of the situation. The importance of the opening of the phorbol D ring to form an isopropenyl side chain is also unclear. This has previously been thought to be unimportant in terms of structure/activity analysis, although other subtle changes in the phorbol nucleus (involving hydrocarbon groups) are known to cause alterations of biological action (see below). The three dimensional structure of PKC (and that of individual isoforms) will be required before the interaction(s) of phorbol esters with their binding domain can be fully understood. This molecular modelling analysis may contribute to the understanding of how phorbol esters, which possess different biological effects, interact with their receptor site. It is, however, clear that molecular modelling analysis, which involves the super-positioning of different classes of tumour promoter (Jeffrey and Liskamp, 1986; Wender et al, 1986,1988; Itai et al , 1988; Nakamura et al, 1989) in an attempt to rationalise the pharmacophore(s) necessary for tumour promotion activity, is unsatisfactory because all phorbol esters possess these requirements, yet not all are tumour promoters. Indeed, subtle modification of phorbol ester structure can result in a significant change in tumour promoting ability, for example, unsaturation 248 of a C12 or C13 acyl ester is thought to impart second stage promoter activity (from complete promoter activity) (Sharkey et al, 1989), and C4-hydroxy methylation is recognised to impart first stage promoter activity (from complete promoter activity) (Hecker, 1987). Bearing this in mind, clearly the opening of the cyclopropane D ring of the phorbol nucleus, to form the isopropenyl side chain of the resiniferonol nucleus, is also of importance in terms of structure/activity analysis. Experiments to assess the ability of the synthetic compounds to induce epidermal hyperplasia revealed that none of the compounds were hyperplasiogenic when compared to the acetone control, whereas the complete tumour promoter, TPA, was capable of inducing epidermal hyperplasia. On this basis, none of the synthetic compounds would appear to be tumour promoters, should induction of epidermal hyperplasia prove to be a rapid correlation for ability to induce tumour promotion. Biochemical analysis of the synthetic derivatives of 12-deoxyphorbol-13-0- angelate and resiniferonol revealed that there were differences between parallel compounds in their ability to activate mixed PKC isolated from rat brain. DA was a potent activator of PKC in a manner similar to that of TPA. DAK and DAI were also capable of activating PKC. DAB was a weaker activator, possibly requiring the presence of Ca2+ for activation; RoB was also a weak activator, possibly requiring PS presence for activity. These activations could reflect isoform specific activations and, therefore, further experiments would involve the use of hydroxylapatite-separated (or purified) PKC kinases. Since all these synthetic compounds were pro-inflammatory, it would seem that if a compound exhibits pro-inflammatory activity, it will be capable of inducing PKC activation to some extent This is a significant observation because PKC effects are currently linked to tumour promoting actions (Castagna et al, 1982; Hecker, 1985; Blumberg, 1988), whilst induction of erythema are still the most widely reported effects of the phorbol esters. Hence, the role of PKC in events leading to tumour promotion remains unresolved. The TRE’s discovered in oncogenes (Angel et aly 1987, 1988) could provide alternative receptor sites for tumour promoting phorbol esters. In the normal experimental models of tumour promotion (the dorsal skin of the mouse), application of an initiator could cause some alteration of gene expression, enabling the gene to become responsive to tumour promoting phorbol esters (that is, to possess a TRE). Subsequent applications of a tumour promoter then cause the production of tumours, the biochemical mechanism of this being driven by tumour promoter binding to a TRE. Experiments to study the binding and effects of phorbol esters with different tumour promoting profiles (such as Sap A, DOPP, DOPPA, Thy A, Rx) to TRE’s, would enable clarification of this situation. Further studies of phorbol ester-PKC mediated events may provide clues as to possible PKC substrates and roles in the inflammatory (and tumour promoting) response. These could include further experiments on the stimulation and phosphorylation of the NADPH-oxidase complex by PKC isotypes (Sharma et al, 1991) and the effects on cellular Ca2+ influx (Menitt et aly 1992) by phorbol esters. The Ro propionic acid derivatives, RoK and Rol, were inhibitory of PKC phosphorylation activity. RoK was a more potent inhibitor than Rol and RoK inhibition of PKC appeared to be dose-dependent, except at higher concentrations. This could point to a difference in the PKC activating ability of the resiniferonol nucleus compared to the 12-deoxyphorbol nucleus. Further experiments would require an assessment of inhibition of 3H-PDBu binding to PKC (preferably for each isoform), an assessment of activation/inhibition potential against Rx-kinase and inhibition of 3H- Rx binding to Rx-kinase by these compounds. This would enable clarification of the PKC (and/or other kinase) inhibitory potential of these compounds. If these 250 compounds had a higher binding affinity for Rx-kinase than PKC, it would provide further evidence for the existence of novel class of enzyme receptor sites for the daphnane diterpenes rather than PKC. These experiments studying the effect of derivatives of 12-deoxyphorbol and resiniferonol on mixed rat brain PKC indicate that C 2 0 substitution can greatly affect the ability of a compound to activate PKC. It is possible that the inhibitory activity of RoK and Rol is a combination of changes in activity as a result of the resiniferonol nucleus and the C^ anti-inflammatory substituent. These compounds are the first reported inhibitors of PKC based upon the phorbol structure, and may be useful in the future to help dissection of PKC mediated responses. RoK and Rol could also provide templates for the design of other phorbol ester inhibitors of PKC (e.g. by Hansch analysis). Further work with Rol and RoK in a variey of in vitro and in vivo test systems (e.g. platelet aggregation, generation of superoxide ion, effect on Ca2+ flux, effect on levels of substrate phosphorylation in vivo) is now required to assess the usefulness of these compounds as biochemical/pharmacological probes of PKC function. The importance of the C^ substituent is emphasised by the actions of DOPPA on platelet PKC. Platelet PKC was separated into two isoforms by hydroxylapatite chromatography, peaks "a" (probably P-) and "b" (a-) PKC. DOPPA preferentially activated peak "a” platelet PKC in a calcium-dependent manner, and the presence of Ca2+ caused a reduction in the k* value for DOPPA-induced activation of mixed platelet PKC from 530nM in the absence of Ca2+ to 120nM in the presence of Ca2f. Activation studies using pure PKC isotypes (Evans et aly 1991; Ryves et al, 1991) have recently confirmed that DOPPA is a selective ft-PKC activator. Further studies using this compound in intact cells may enable researchers to follow the physiological 251 effects of phorbol ester induced PKC activation and substrate phosphorylation. Rol and RoK could also be used in this environment to attempt to inhibit PKC-mediated events and delivery of these compounds to their site of action may be easier in intact cells than in an animal model (of, for example, inflammation). However, moving from the in vitro biochemical situation to the in vivo one (whether cellular or animal) is complex in that Ca2+ concentrations would appear to be one of the most important factors which could influence phorbol ester effects, due to the dependency of some phorbol esters (e.g. Sap A, DOPPA) on the presence of Ca2+ for activity, and the dependency/independency (/inhibition?) of PKC (and other kinase, e.g. Rx-kinase) isoforms on the presence of Ca2+ for activity. The implications of Ca2+ concentration effects mean that regulation of inositol phosphate concentrations (particularly Ins- 1,4,5-P3) are also critical. Additionally, generation of Ca2+ (from non-ins-1,4,5-P3- dependent stores) and DAG (for example, from PLD mediated phosphatidylcholine metabolism) are likely to influence the final response of the cell endogenous agonist stimuli (particularly in terms of long-term effects?). Many other factors will affect phorbol ester/PKC mediated cellular responses, including: feedback mechanisms with the Ptdlns cycle, as well as with other signal transduction pathways; PKC-isotype distribution; substrate distribution, function and co-localisation with the relevant phosphorylating PKC isotype; PKC down-regulation and PKM-induced phosphorylations. Thus, the complexity of cellular signalling systems would indicate that our understanding of the biochemical basis of how the phorbol esters induce such gross pharmacological effects at nanomolar concentrations (for example, tumour promotion, inflammation) is still relatively poor. This work has described, for the first time, potential in vitro phorbol ester inhibitors of PKC. 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Evans,F.J., Parker,P.J., Olivier,A.R., Thomas,S., Ryves,W.J., Evans,A.T., Gordge,P., and SharmaJP., (1991) Phorbol ester activation of the isotypes of protein kinase C from bovine and rat brain. Biochem.Soc.Trans. 19 397-402. Evans,A.T., Gordge,P.C., Sahni,V., and Evans,F.J., (1992) The potent irritancy of the daphnane orthoester, resiniferatoxin, exhibits features of a mixed aetiology. J.PharmBharmacol. 44 361-363. Ryves,W.J., Sharma,P., Evans,A.T., Gordge,P.G, Hassan,N.M., Thomas,N.S.B., and Evans,F.J., (1992) Stimulation of pre- and post- protein kinase C isotype protein kinase activities from rat brain by different phorbol esters. FEBSLetts. (Submitted). Gordge,P.G, Evans,A.T., Aitken,A., and Evans,F,J., (1992) Comparison of the phorbol and daphnane diterpene nuclei by molecular modelling and the molecular co-ordinates of the potent irritant, resiniferatoxin. Cancer Letts. (Submitted) 2. Conference communications. Gordge,P.C., Evans,A.T., and Evans,F.J., (1990) Evidence of a mixed aetiology in features of the erythema response to the potent irritant, Resiniferatoxin. J.Pharm.Pharmacol. 42 32P. Autore,G., Gordge,P.C., and Evans,F.J., (1991) Leukotriene D4 is a potent activator of protein kinase C and inhibits 3H-phorbol-dibutyrate binding. JPharm.Pharmac. 43 5P. Evans,A.T., Duggan,C., Gordge,P.C., Sharma,P., and Evans,F.J., (1991) A 20kDa protein comprises the major portion of the phorbol ester stimulated platelet Transferable Aggregating Substance (TAS) activity. JBharmBharmacol. 43 19P. Evans,A.T., Gordge,P.C., Sharma,P., Ryves,W.J., and Evans,F.J., (1991) Investigation of the tissue distribution of the calcium-independent kinase, Rx-kinase, which is distinct from known isozymes of protein kinase C. BiochemSoc.Trans. 19 157S. 3. Posters. Gordge,P.C., Sharma,P., Ryves,W.J., Evans,A.T., and Evans,F.J., (1990) The substitution in the phorbol vs. the daphnane nucleus induce opposing effects on irritant potency which are linked to relative activation of PKC or Rx-kinase. 6th International 298 Symposium on Cellular Endocrinology, August 1990, Lake Placid, USA. p47. Evans,A.T., Sharma,P., West,R., Ryves,W.J., Gordge,P.C., and Evans,FJ., (1992) Induction of calcium-inhibited, phorbol ester-stimulated kinase activity in macrophages and neutrophils by soluble stimuli. Strasburg, France. Sharma,P., Ryves,WJ., Evans,A.T., Gordge,P.C., and Evans,F.J., (1992) Stimulation of a protein kinase in human neutrophils by a daphnane orthoester, TxA, in a Ca2+- independent manner. Strasburg, France. 299 Acknowledgements. I would like to give special thanks to my supervisor, Professor Fred J Evans, for his guidance and direction of the project, and for the discussions which have arisen as a result of this. I am also grateful to my external supervisor, Dr Alastair Aitken, for his support of this project and for the provision of laboratory space at NIMR. The other members of the group, to whom I am particularly grateful for being good friends as well as excellent colleagues, and who have provided the opportunity for many discussions are Dr A Tudor Evans, Dr W Jonathon Ryves and Pawan Sharma. Thanks are also due to the following people of the Department of Pharmacognosy: Professor J David Phillipson for providing the facilities of his department; Dr Margaret F Roberts for advice; Maureen Pickett and Gus Rongren for technical advice and support; Annie Cavanagh for the excellent photographs and drawings. I am grateful to Curt Homeyer and Graham Florence of the Computer Centre for their help and patience. I would like to thank the following for running spectra for me: Wilf Baldeo (80 MHz !H-NMR); Dr Graham Anderson (500 MHz XH-NMR); Jane Hawkes and staff of Kings College (250 MHz X-H-NMR); Drs. Dave Carter, Mark Harrison and Kevin Welham for mass spectra. Thanks are also due to Dave McCarthy for preparing, staining and sectioning mouse epidermal sections. The p47 phosphorylation data was provided by Dr Sue Brooks, for which I am 300 grateful. Thanks to my many Mends at the School, who have made my time here enjoyable, particularly Ades, Sab, Marie, Si, Dave, Mel, Clare, Krys, Claire, Mich, John, Dan, and Abs, for everything. Finally, I wish to thank the SERC and NIMR, Mill Hill, for financial support 301 Appendix 1: Induction of epidermal hyperplasia by diterpene esters. 302 a. 50nM TPA. b. 200pl acetone control. 303 c. 50nM DOPPA. d. 50nM Rx. e. 50nM DAB. f. 50nM DAI. 305 g. 50nM DAK. h. 50nM DAHV. 306 i. 50nM RoB. j. 50nM Rol. 307 k. 50nM RoK. All compounds were topically applied in 200pl acetone. Stain: Haematoxylin and Eosin. Magnification: x 100 Scale: 1mm = 90pm. U -4-4CM 33 3 £ 0) co U 3 o — ~ m _e* ^ 3 « 04 u >,r4 •S O > O 4-* 3 X & *1 . S « 2 B 3 <4-1 3 3 * s • § 1 i CM 3 ■s 3 cd II 2 X P* U 3 c* £ 1 3 1 3 si & PQ M <