Volume 294. number 3. 183-186 FEBS 10458 Dc~:cmlx-r IO91 ~ 1991 Federation of European Biochemical Societies 00145793/91/$3._~t) Structure/function relationships in the inhibition of thimet oligopcpti,tase by carboxyphenyl propyl-peptides C. Graham Knight and Alan J. Barrett Der~rcs~ncnl of Bi¢~chemistrt'. Strangeway$ Research Laboratory. EVorts Causeway. Cambridge CBI 4RN. UN Received 26 September I(~I; revised version received 17 October 1991 Some novel N-[l(RS)-carboxy-3-phenylpropyl]trilaeptide p-aminobcnzoates hawe bexm synthesised as inhibitors of lhimet oligopeptidas¢ (EC 3.4.24.151. The~e compounds are considered to bind as substrate analogues with the Cpp group in SI and the l~ptide portion in the S" ~ite~. The most Dotc,t inhibitor is Cpp-Ala-Pro-Phe-pAb. which has a K. - 7 nM. Substitution of (~ly for Ala at Pl" leads to weaker binding which can be ascribed to increa.~d rotational tr~x'dom. Good substrates often have Pro at P2" and Pro is favoured over Ala at this position in the inhihitors, too. When P2" is Pro. Phe is preferr~ over Tyr and Tip in P3 +. The p-amlnobenToate group makes an ;ml~,rtant contribution to the binding, probably by forming a .salt bridge, and removal of the ('-terminal negative 4zhargc results in much less potcnl inhibitors. EC 3.4.24. I S; Fm, ym© inhibition; Subsite specificity i. INTRODUCTION tide-pAb are competitive inhibitors, and since the most potent have peptide structures analogous to those of Thimet oligopeptidase (EC 3.4.24.15) is ~" thiol-de- good substrates [4], the amino acid sidechains pre- pendent metallo-endopeptidase widely distributed in sumably occupy the same specificity subsites on the dells and tissues, and previously known by the names enzyme. By convention [7], these subsites are labelled S Pz-peptidase, endo-oligopeptidase A and soluble endo- to the N-terminal side of the scissile bond and S" to the peptidase 24.15 [I]. Characteristic features of the en- C-terminal side, numbering being away from the bond zyme are the inhibition by both chelators and thiol- cleaved, the scissile bond. The amino acid residues reactive reagents and activation by low concentrations which occupy these subsites are labelled P and P', re- of thiols. The biological function of thimct oligo- spectively. Although it is not yet possible to define the peptidase is unknown, but is capable in vitro of generat- characteristics of the subsites precisely [I], good sub- ing or destroying a number of pharmacologically active strates and inhibitors of thimet oligopeptidase com- peptides and is found in the highest concentrations in monly have hydrophobic residues at Pi and PY [2,4]. the soluble protein fraction of testis, brain and pituitary In addition to their use in biological experiments, [2]. lnhibitors may provide the best approach to inves- inhibitors more potent than those currently available tigating the physiological role of the enzyme [3]. are needed for active site titration of the enzyme, so that Thimet oligopeptidase is selectively and reversibly in- kinetic studies can be placed on a quantitative basis. hibited by N-[l(RS)-carboxy-3-phenylpropyi]tripeptide Also needed are inhibitors more resistant to degrada- p-aminobenzoates (Cpp-peptide-pAb) [4,5]. The mecha- tion, which currently complicates their use in studies of nism of inhibition may be similar to that proposed for the role of thimet oligopeptidase in vivo [3]. We now thermolysin and Cpp-Leu-Trp [6], where X-ray crystal- report our initial studies to investigate the relationships lography has revealed the coordination of the carboxyl between inhibitor structure and function. ol the Cpp group to the Zn atom in the active site of the enzyme. In thimet oligopeptidase, interaction of the 3- 2. EXPERIMENTAL phenyl ring with the hydrophobic S I subsite also makes 2.]. Maaet4ais an important contribution to the binding [4] Cpp-pep- Boc-amino acid N-hydroxysuc~nimide esters, p-aminobenzoic acid and L-phenylalanine derivatives ~ from Sigma. Uoc-amino acid Abbrevlatlons: Ahx, L-2-aminohexanoic acid; Boc, t-butyloxycarbo- p.aminobenzoates were made as de~ribed [8]. Ethyl; 2-keto-4-phenyl- ttyl; CI~, N-[It(RSk-¢arboxy-3-phenylpropyli; Drip, dinitrophenyl; butanoat© from Scbeizerhall (South Plainfield, NJ 07080) was saponi- Moc. 7-melhoxyooumarin-3<arboxylyl; ONSu, N-hydroxystt~ni- fled with NaOH to yield the sodium salt. Cpl~Ala-Ala-Tyr-pAb was mide ester; pAb. p-aminobenzoat¢; Pz. phenylaz.obertzyloxycarbonyl. a gift from Dr. M. Orlowski. Mount Sinai School of Medicine, New York, USA. Moc-Pro-I..eu-Oly-Pro-D-Lys(Dnp) was made by solid Corresptmdence address: C.O. Knight, Departmem of Bk~hcmistry, phase pepcide ~tnthe~ 19L Rat t,n~t~ mim~ ~ w.as puri- Strangeways Research Laboratory, Worts Causeway, Cambridge fied by a method similar to that used for the chicken enzyn~ 15|. and CBI 4RN, UK. ga~e a single band of "/6 kDa in SDS?l~amli~ ~ doctralphorcsh. Pub/is&ed by E./sev~r Sck,nce Pub/t~'rs B. g,'. 183 Votume .~4. number 3 UEBS LETTERS December 1991 2.2. 5;Ynth,'s:.~ ¢,I Cpp-.4la-Pro-Ph*'-p.4~ was controlled, and data colh_.cted and analys~d, hy use of the This was made step-wire by standard ~ltstion-pha~ mctht~cls [4. I0]. VI.USYS software Dackag~ [I I ]. Whe en,wm¢ was pro-activated with Reactions ~crt" ft~llowt~J by HP[.C on a C1~4 L'olumn, clulcd with tt S mM dithiothreitol in assay buffer (50 mM Tris-HCI, DH 7.g, contain- gradient of 5 100~4;, aL~tomtrilc containing 0 I¢IF trIfluorowuctlc acid Ing tlOSqF Itr U iS) for 5 rain at 21"( and then diluted IO*fold and kept (Iml,min) over 25 rain. Tht, colulnn ¢Itiant ~Jas monitored at ".10 rim. at 0"('. A~ay~, were made with OR nM em, yme. based on protein I~--Phe-p,Ab 1384 rag. I retool) gas treatL,~J with trifluolat~llccYic ;lt:iti/ concentration. For the determination of K,. the rate of substrat¢ hy- water I 19 I. ~'~'v. :q) ml) for 20 n~it~ at 21 ~'C to remove the B¢~.- group droly~i~ in the absem.'¢ of inhibitor was first recorded, and then the The acid was removed at 44)°(" in vacuo and the t~sidue was dissolved inhibitor was add~ in a negligible volume and the new steady stale in dimcthylfomlamidc (Sail, To this ,~tuiion was addc~l B~'-Pro- was monitored Rapid equilibration was observed in all cases. Ap- ONSu (330 rag, I I mmolL l-hydroxyl~n~otria~c~le (I 35 g, I ntm~i) parent inhibition constants A'.=r~., were calculated by fitting Ihe frac- and di-isopropylethylamine (O.7 ml. 4 mmolL After stirring overnight, tional activity darn to II1¢ Mlorrisort L~quation [I 2| by nonlin~r relgl~o analysis by HPLC sho~k~t."d the reaction to the complete. The dimethyl- sion, using the p¢ogt am En~litter ! I-Ascvier-Biosofl, Cambridge, UK). formam/d~ was evaporated at 40°(" in vacuo and chloroform (~) ml) Values wct'¢ corrected lor the effoet ofsubstrate by using equation (t). was adld~. After eglra~liom with ~ tw/v} citric acid (2 ~< ~ mlL water 120 ml) and saturaled Na('l (20 roll the chloroform solution was K, = K.~.~/(I ÷ [SFK.) (l) dried (M8SO4) and evaporated to leave crude Boc-Pro-Phe-pAh_ This was Ptx-rystalli~L'd from ethi*nol/water (yielding 0.46 g. 95~[-I and A value of 16 .u M was dctcrmin*~! for Km. The different:c~ in K, va|tt~ treated with tri~luoroa,cctic acid/water as described above, The residue I'~twL~n hthibitors were converted into differences in the fr~ energy after evaporation was dissolved in dimethylformamkle and reacted of binding t~(~. d hy equation (2), with Eloc.Ala.~)NS¢ (2~ mr. I mmoh. I-hydroxybenzotriazote and di-isop~xspylethylamine. As before, the solvent was evaporated after --. -2.303RT- Iogl reac*ion overnight and the residue dissolved in chloroform, extracted 66G~,.a K,,tK',:) (2) and dried. The crude Ek~-Ala-Pro-Phe-pAb was 97q~, pure by HPL(" analysis and was treated with trifluoroa~i~tic acid without further purification. The residue after evaporation was dissolved in methanol/ 3. RESULTS AND DISCUSSION water ( 1:1 v/v, ~ ml) and brought to pH 7 with 5 M NaOH. Sodium 3. I. A lanine is fitvoured in Pl" 3-phcnyl-2-ketobutyrate ( I g, 5 retool) and sodium cyanoborohydride (0.32 g. 5 retool) were added and She mixture stirr~ at 2 I~'C. HPLC Good peptide substrates of thimct oligopeptidase analysis showed that reaction was ~.~.~mplete after 24 h. The solveats commonly have hydrophobic residuesin PI and P3". and were evaporated, water (50 ml) was added and the ,solution acidified arginine is often found in Pl" [2], suggesting that the SI" with 6 M HCI ( 1.5 ml) to precipitate Cpp-Ala-Pro-Phe-pAb.HCI. The site of the enzyme is accessible to large side chains. This crude prodtu:t wa,~ washed with ethyl acetate, and reerystallised from ethanol and water, yielding 0.33g ¢49~-), m,p. IS? 188°C. Analysis: conclusion is supported by studies with inhibitors, since calculated for C,,H~N~O~.H(?I (M, 679.2) C. 62.72; H, 6.O4; N. 8.60; the K, of Cpp-Ph¢-Ala-Phe-pAb is only 3-fold that of t~)und (', 62.98: H, 6.1 I; N. 8.60. Cpp-Ala-Phe-pAb [4] (Table 1). Leucine. however, is The other inhibitors w~re mad~ by similar methods.