DOCSLIB.ORG

Site-Directed Psoralen Crosslinking of DNA (Mercurated Pyrimidines/Restriction Nucleases/DNA Damage/Photochemistry) WILMA A

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Site-Directed Psoralen Crosslinking of DNA (Mercurated Pyrimidines/Restriction Nucleases/DNA Damage/Photochemistry) WILMA A Proc. Nati Acad. Sci. USA Vol. 79, pp. 4594-4598, August 1982 Biochemistry Site-directed psoralen crosslinking of DNA (mercurated pyrimidines/restriction nucleases/DNA damage/photochemistry) WILMA A. SAFFRAN., MERRLL GOLDENBERG, AND CHARLES R. CANTOR Department of-Human Genetics and Development, College of Physicians and Surgeons, Columbia University, New York, New York 10032 Communicated by Gilbert Stork, May 3, 1982 ABSTRACT A technique has-been developed for placing site- -,CH2-N), 2.4 (6H, s, CH3-N-), 2.5-2.2 (2H, s, H-N); mass spec- specific crosslinks into DNA by using a psoralen derivative con- trum (chemical ionization in methane) 177 (M + 1), 205 (M taining a sulfhydryl group. Plasmid pBR322 was nicked at the + 29), 217 (M + 41). unique BamHI restriction-site, and mercurated pyrimidines were Chloromethyltrimethylpsoralen (II). This compound was introduced by nick-translation. Then the psoralen was specifically made (with minor modifications) according to the literature (5): directed to this site in the dark through a Hg-S linkage prior to yield (not including mother liquor) 32%; mp 212-215°C (lit. irradiation to form a crosslink. The location of interstrand cross- 215-2170C); NMR (Varian T60) 8 7.5 (1H, s, phenyl), 6.17 (1H, links was examined by electron microscopy ofPst I-cut molecules s, lactone), 4.7 (2H, s, Cl-CH2-Ar), 2.5.(9H, m, methyls). *and found to be at or-near the BamHI site. Methylaminodiethoxyethanemethylaminomethyltrimethyl- psoralen (Ill). A mixture of 176 mg of II and 1.69 g of I was Interstrand crosslinks in nucleic acids are readily formed by heated with stirring in 20 ml ofdry toluene overnight. The re- reaction with psoralen or its derivatives (1, 2). Psoralens bind action was monitored by TLC on silica gel with enzene/meth- to-double-stranded DNA or RNA noncovalently in-the dark, by anol, or 1:1 (vol/vol) 95% ethanol/concentrated aqueous am- intercalation. Irradiation of this complex with 365-nm light re- monia, 4: 1, as eluants. The product (140 mg) was eluted from sults in the formation of a cycloadduct between a pyrimidine a silicagel (Baker, 60-200mesh)flashcolumnwith95% ethanol/ residue on one strand and the psoralen. This may then react concentrated aqueous ammonia, 4: 1, as a pale yellow oil: NMR photochemically with a second pyrimidine on the opposite (80 MHz) 8 7.68 (lH, s, phenyl), 6.2 (LH, s, lactone), 3.9-3.4 strand to form a crosslink. Although the relative rates ofreaction (1OH, m, -CH2-O-, -N-CH2-Ar), 3-2.2 (19H, m, -CH3 and -CH2- with various pyrimidines differ, there is little indication of any N-); mass spectrum (chemical ionization in methane) 417 (M more general sequence specificity. + 1), 445 (M + 29), 457 (M + 41). .In addition to its medical application in the treatment ofpso- 2-Pyridyldithioethylmethylamidodiethoxyethanerrthylami- riasis, the psoralen photoreaction has been used in studies of nomethyltrimethylpsoralen (IV) [site-specifwc psoralen (SSP)]. nucleic acid secondary structure, synthesis, mutagenesis, and N-Succinimidyl 3-(2-pyridyldithio)propionate (6) (SPDP; 29mg; DNA repair and recombination (3, 4). Although some of these Pharmacia) was dissolved in 200 ,ul ofdry methylene chloride. investigations have taken advantage ofthe double-strand spec- Then 35 mg ofIII dissolved in 100 ,ul ofdry methylene chloride ificity of the crosslinking photoreaction to map secondary struc- was added and the reaction mixture was stirred in the dark at ture in nucleic acids or to probe the organization ofchromatin, room temperature. The disappearance ofIll and SPDP and the the molecules under study reacted randomly with psoralen, and appearance of the product and N-hydroxysuccinimide were a heterogeneous mixture of products was produced. monitored by TLC (silica gel with methanol eluant). The re- *We describe here a method ofdirectingpsoralen crosslinking action was complete within 15 min. The productwas eluted with to a specific location in a DNA molecule by using a derivative benzene/methanol, 1:1, on a reversed-phase (silica gel 60, ofpsoralen containing a thiol group. We have prepared plasmid Merck) flash column. The fractions were analyzed on a linear pBR322 DNA molecules with interstrand crosslinks placed near KC18 plate. (On this TLC system, the RFS of the product, the unique BamHI restriction site by enzymatically incorpo- SPDP, N-hydroxysuccinimide, and Ill, were 0.24, 0.7, 0.46, rating mercurated nucleotides at this site and then directing and 0.04, respectively.) On all TLC plates the product spot ab- psoralen to the modified bases through -a Hg-S linkage. sorbs 260 nm light, fluoresces pale blue on irradiation with 370 nm light and is stained by iodine. Yield was 41 mg. The product MATERIALS AND METHODS was further characterized by UV spectroscopy. With a molar Synthesis of Site-Specific PsorAlen. The sequence. is shown extinction coefficient of6,884 (at 280 nm) for psoralen (5), 5,100 in Fig. 1. (at 280 nm) for 2-pyridyl disulfide, and (after reduction of the 1,2-Bis(2-nethylaminoethoxy)ethane (I). Twenty grams of disulfide with dithiothreitol) 8,080 (at 345 nm) for 2-thiopyri- 1,2-bis(2-chloroethoxy)ethane (Eastman) and 165 g of methyl- done (7), it was determined that the molar ratio of2-pyridyldi- amine (40% in water) (Matheson, Coleman, and Bell) were sulfide to psoralen was 1:1: mass spectrum (chemical ionization placed in a pressure bottle and, heated at 85°C for 3 days. The in methane) 614 (M + 1), 628 (M + 15), 642 (M + 29). solution was made basic with sodium hydroxide and the product Preparation of SSP Crosslinks in Plasmid DNA. Nick-trans- was extracted into chloroform, and dried over anhydrous so- lation with mercurated nucleotides. Plasmid pBR322 was iso- dium sulfate. After rotoevaporation ofthe solvent, the product lated by the method of Bolivar and Boekman (8). BamHI was (76% yield) was purified by vacuum distillation: bp at 12 mm prepared by the method ofWilson and Young-(9). Plasmid was .Hg 112-114'C; NMR(C2HC13, Varian T60).8 3.6 (8H, s, su- nicked with BamHI in the presence ofethidium bromide under perimposed on t, J = 5 Hz, -CH2-O-), 2.75 (4H, t, J = 8 Hz, the following conditions: 100 ,ug ofDNA per ml, 150 mM NaCl, 6 mM Tris-HCl (pH 8.0), 6 mM MgCl2, 0.5 mg ofbovine serum The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertise- Abbreviations: SSP, site-specific psoralen; SPDP, N-succinimidyl 3-(2- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. pyridyldithio)propionate. 4594 Downloaded by guest on September 25, 2021 Biochemistry: Saffran et al. Proc. Natl. Acad. Sci. USA 79 (1982) 4595 CH3 CH3 (5). Irradiation time was generally 10 min. H-N-(CH2)2-0-(CH2)-O0-CH2)Z-N-H R-Cl Crosslinks were detected by electrophoresis of native and I 1 denatured DNA on 1.2% agarose in Tris-acetate/EDTA pH 7.6 buffer (10). Equal aliquots ofsample, one native and one dena- tured in 0.2 M NaOH, were run in adjacent lanes. Denatured, 1000, toluene noncrosslinked DNA ran as single strands, with higher mobility than the double-stranded species; the rapidly renaturing cross- CH3 CH3 linked molecules had the same mobility as the native DNA. The H-N-(CH2)2-0-(CH2)2-O-(CH2)27N-R amount of crosslinked DNA in 32P-labeled samples was mea- m sured by cutting out the ethidium bromide-stained gel bands, SPDP, CH2CI2 dissolving them in 1 ml of formamide for 1 hr at 500C, and then assaying them in 10 ml of scintillation fluid. DNA was dena- O CH3 CH3 tured and spread by the method of Cech and Pardue (11). (S-S-(CH ),7C-N-'C`H2)2-O-(CH2)270-(CH )i-N-R N DTT RESULTS Synthesis and Properties of SSP. This psoralen derivative 0 CH3 CH3 was constructed to optimize the-chances ofsite-specific reaction HS-(CH2)2-C-N-(CH2)2-0-(CH2)2°--(CH2)2-N-R near mercurated bases in DNA. It has the following properties: (i) a disulfide group that is readily reduced to a thiol [this moiety CH3 binds specifically and tightly (Kd = 10-16 M) to mercury in the CH2 dark (12)]; (ii) is soluble in water; (iii) is positively charged, which R= increases the affinity for DNA; and (iv) has a long and flexible CH 0 0 linker between the furocoumarin and the thiol, which enables CH3 the psoralen moiety to intercalate into DNA while attached to a mercurated base through the sulfur at its other end. This FIG. 1. Synthesis of site-specific psoralen (SSP). linker is 20 A long, allowing intercalation three to four bases from the site ofattachment. Cystaminylmethyltrimethylpsoralen, albumin per ml, 25 ,g of ethidium bromide per ml, and 100 another thiol-containing psoralen derivative with a shorter (5 units of BamHI per ml. After incubation for 1 hr at 37°C, the A) linker (13), failed to produce any specific interstrand reaction mixture was extracted with 1 vol of phenol and then crosslinks. ethanol precipitated. Up to this step, all procedures were car- Preparation of pBR322 Crosslinked near the BamHI Site. ried out under red light to minimize random nicking by ethi- To prepare pBR322 DNA containing mercury near or at a spe- dium bromide. The pellet was washed once with 80% ethanol cific site, intact plasmid was first nicked with BamHI in the and resuspended in 10 mM Tris (pH 8.0) at a concentration of presence of ethidium bromide (Fig. 2A). Ethidium bromide 1 mg/ml. inhibits restriction endonucleases, and this treatment results Nick-translation was carried out in 50-mM Tris HCl, pH 7.6/ in the production of single-strand cuts rather than the normal 6.7 mM MgCl2/6 mM mercaptoethanol/10 ,M dATP/100 ,M staggered double-strand cuts (14).
Load more

Recommended publications
  • Phototoxicity of 7-Oxycoumarins with Keratinocytes in Culture T ⁎ Christophe Guillona, , Yi-Hua Janb, Diane E
    Bioorganic Chemistry 89 (2019) 103014 Contents lists available at ScienceDirect Bioorganic Chemistry journal homepage: www.elsevier.com/locate/bioorg Phototoxicity of 7-oxycoumarins with keratinocytes in culture T ⁎ Christophe Guillona, , Yi-Hua Janb, Diane E. Heckc, Thomas M. Marianob, Robert D. Rappa, Michele Jettera, Keith Kardosa, Marilyn Whittemored, Eric Akyeaa, Ivan Jabine, Jeffrey D. Laskinb, Ned D. Heindela a Department of Chemistry, Lehigh University, Bethlehem, PA 18015, USA b Department of Environmental and Occupational Health, Rutgers University School of Public Health, Piscataway, NJ 08854, USA c Department of Environmental Science, New York Medical College, Valhalla, NY 10595, USA d Buckman Laboratories, 1256 N. McLean Blvd, Memphis, TN 38108, USA e Laboratoire de Chimie Organique, Université Libre de Bruxelles, B-1050 Brussels, Belgium ARTICLE INFO ABSTRACT Keywords: Seventy-one 7-oxycoumarins, 66 synthesized and 5 commercially sourced, were tested for their ability to inhibit 7-hydroxycoumarins growth in murine PAM212 keratinocytes. Forty-nine compounds from the library demonstrated light-induced 7-oxycoumarins lethality. None was toxic in the absence of UVA light. Structure-activity correlations indicate that the ability of Furocoumarins the compounds to inhibit cell growth was dependent not only on their physiochemical characteristics, but also Psoralens on their ability to absorb UVA light. Relative lipophilicity was an important factor as was electron density in the Methoxsalen pyrone ring. Coumarins with electron withdrawing moieties – cyano and fluoro at C – were considerably less 8-MOP 3 Phototoxicity active while those with bromines or iodine at that location displayed enhanced activity. Coumarins that were PAM212 keratinocytes found to inhibit keratinocyte growth were also tested for photo-induced DNA plasmid nicking.
    [Show full text]
  • Phototoxicity of 7-Oxycoumarins with Keratinocytes in Culture T ⁎ Christophe Guillona, , Yi-Hua Janb, Diane E
    Bioorganic Chemistry 89 (2019) 103014 Contents lists available at ScienceDirect Bioorganic Chemistry journal homepage: www.elsevier.com/locate/bioorg Phototoxicity of 7-oxycoumarins with keratinocytes in culture T ⁎ Christophe Guillona, , Yi-Hua Janb, Diane E. Heckc, Thomas M. Marianob, Robert D. Rappa, Michele Jettera, Keith Kardosa, Marilyn Whittemored, Eric Akyeaa, Ivan Jabine, Jeffrey D. Laskinb, Ned D. Heindela a Department of Chemistry, Lehigh University, Bethlehem, PA 18015, USA b Department of Environmental and Occupational Health, Rutgers University School of Public Health, Piscataway, NJ 08854, USA c Department of Environmental Science, New York Medical College, Valhalla, NY 10595, USA d Buckman Laboratories, 1256 N. McLean Blvd, Memphis, TN 38108, USA e Laboratoire de Chimie Organique, Université Libre de Bruxelles, B-1050 Brussels, Belgium ARTICLE INFO ABSTRACT Keywords: Seventy-one 7-oxycoumarins, 66 synthesized and 5 commercially sourced, were tested for their ability to inhibit 7-hydroxycoumarins growth in murine PAM212 keratinocytes. Forty-nine compounds from the library demonstrated light-induced 7-oxycoumarins lethality. None was toxic in the absence of UVA light. Structure-activity correlations indicate that the ability of Furocoumarins the compounds to inhibit cell growth was dependent not only on their physiochemical characteristics, but also Psoralens on their ability to absorb UVA light. Relative lipophilicity was an important factor as was electron density in the Methoxsalen pyrone ring. Coumarins with electron withdrawing moieties – cyano and fluoro at C – were considerably less 8-MOP 3 Phototoxicity active while those with bromines or iodine at that location displayed enhanced activity. Coumarins that were PAM212 keratinocytes found to inhibit keratinocyte growth were also tested for photo-induced DNA plasmid nicking.
    [Show full text]
  • The Synergistic Anti-Neoplastic Activity of Combinations of Mitomycins with Either 6-Thioguanine Or @.-F1uorouracip
    CANCER RESEARCH VOLUME 25 OCTOBER 1965 NUMBER 9 The Synergistic Anti-neoplastic Activity of Combinations of Mitomycins with Either 6-Thioguanine or @.-F1uorouraciP ALAN C. SARTORELLI AND BARBARA A. BOOTH Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut SUMI@1ARY Mitomycin C or porfiromycin (methyl mitomycin) in combination with either 6- thioguanine or 5-fluorouracil produced synergistic inhibition of the growth of both Sar coma 180 and Lymphoma L1210 ascites cell neoplasms. i\Iitomycin C and porfiromy cm possessed the same therapeutic index in the L1210 lymphoma system, but mitomycin C was about 5 times more potent. Doses of mitomycin C that induced pro nounced inhibition of thymidine-3H incorporation into deoxyribonucleic acid (DNA) did not decrease the average DNA content of treated cells. The degree of inhibition of ribonucleic acid (RNA) synthesis by mitomycin C was less pronounced than that of DNA, and lysine-1-'4C fixation into residual protein was insensitive to the antibiotic. 6-Thioguanine induced a marked depression in the rate of incorporation of thymidine 3H and orotic acid-6-'4C into DNA and RNA, respectively. The combination of these agents produced subadditive to additive inhibition of the formation of nucleic acids and also caused inhibition of protein synthesis; incorporation of thymidine into DNA ap peared to be the metabolic path most sensitive to the combination of these agents. Measurement of thymidine kinase and thymidylate kinase activity in cells treated with these agents indicated that these enzymes were relatively resistant to the action of mi tomycin C ; however, some inhibition of thymidylate kinase activity was induced by the thioguanine treatment.
    [Show full text]
  • Part IV: Basic Considerations of the Psoralens
    CORE Metadata, citation and similar papers at core.ac.uk Provided by Elsevier - Publisher Connector THE CHEMISTRY OF THE PSORALENS* W. L. FOWLKS, Ph.D. The psoralens belong to a group of compoundsfled since biological activity of the psoralens and which have been considered as derivatives ofangelicins has been demonstrated. They appear coumarin, the furocoumarins. There are twelveto have specific biochemical properties which different ways a furan ring can be condensed withmay contribute to the survival of certain plant the coumarin molecule and each of the resultingspecies. Specifically these compounds belong to compounds could be the parent for a family ofthat group of substances which can inhibit certain derivatives. Examples of most of these possibleplant growth without otherwise harming the furocoumarins have been synthesized; but natureplant (2, 3, 5). is more conservative so that all of the naturally It is interesting that it was this property which occurring furocoumarins so far described turnled to the isolation of the only new naturally out to be derivativesof psoralen I or angelicinoccuring furocoumarin discovered in the United II (1). States. Bennett and Bonner (2) isolated tharn- nosmin from leaves of the Desert Rue (Tham- n.osma montana) because a crude extract of this 0 0 plant was the best growth inhibitor found among /O\/8/O\/ the extracts of a number of desert plants sur- i' Ii veyed for this property, although all the extracts showed seedling growth inhibition. One could I II speculate as to the role such growth inhibition plays in the economy of those desert plants These natural derivatives of psoralen and angeli-when survival may depend upon a successful cm have one or more of the following substituentsfight for the little available water.
    [Show full text]
  • Interactions of Antitumor Triazoloacridinones with DNA
    Vol. 54 No. 2/2007, 297–306 on-line at: www.actabp.pl Regular paper Interactions of antitumor triazoloacridinones with DNA * Marcin Koba and Jerzy Konopa Department of Pharmaceutical Technology and Biochemistry, Gdańsk University of Technology, Gdańsk, Poland Received: 12 February, 2007; revised: 20 March, 2007; accepted: 21 March, 2007 available on-line: 19 April, 2007 Triazoloacridinones (TA) are a new group of potent antitumor compounds, from which the most active derivative, C-1305, has been selected for extended preclinical trials. This study investi- gated the mechanism of TA binding to DNA. Initially, for selected six TA derivatives differing in chemical structures as well as cytotoxicity and antitumor activity, the capability of noncova- lent DNA binding was analyzed. We showed that all triazoloacridinones studied stabilized the DNA duplex at a low-concentration buffer but not at a salt concentration corresponding to that in cells. DNA viscometric studies suggested that intercalation to DNA did not play a major role in the mechanism of the cytotoxic action of TA. Studies involving cultured cells revealed that triazoloacridinone C-1305 after previous metabolic activation induced the formation of interstrand crosslinks in DNA of some tumor and fibroblast cells in a dose dependent manner. However, the detection of crosslink formation was possible only when the activity of topoisomerase II in cells was lowered. Furthermore, it was impossible to validate the relevance of the ability to crosslink DNA to biological activity of TA derivatives. Keywords: triazoloacridinones, DNA intercalation, DNA interstrand crosslinks, structure–activity relationship, biological activity, metabolic activation INTRODUCTION Previous investigations demonstrated that triazoloacridinones inhibited cleavable complexes of Triazoloacridinones (TA) (see Table 1, for topoisomerase II–DNA and also the catalytic activity structures) are a class of antitumor agents synthe- of this enzyme (Skladanowski et al., 1999; Lemke et sized in our laboratory (Cholody et al., 1990).
    [Show full text]
  • Characterization of SPRTN, the First Mammalian Metalloprotease That Repairs DNA-Protein-Crosslinks
    Aus dem Fachbereich Medizin der Johann Wolfgang Goethe-Universität Frankfurt am Main betreut am Gustav Embden-Zentrum der Biochemie Institut für Biochemie II (Kardiovaskuläre Biochemie) Direktor: Prof. Dr. Ivan Dikic Characterization of SPRTN, the first mammalian metalloprotease that repairs DNA-protein-crosslinks Dissertation zur Erlangung des Doktorgrades der Medizin des Fachbereichs Medizin der Johann Wolfgang Goethe-Universität Frankfurt am Main vorgelegt von Stefan Prgomet, dr. med. aus Düsseldorf Frankfurt am Main, 2019 1 Dekan: Prof. Dr. Stefan Zeuzem Referent/in: Prof. Dr. Ivan Dikic Korreferent/in: Prof. Dr. Jörg Trojan Tag der mündlichen Prüfung: 12.05.2020 2 Contents 1 Summary / Zusammenfassung .................................................................... 6 / 8 2 Introduction ...................................................................................................... 10 2.1 SPRTN regulates a progeria and tumorigenesis axis ............................ 10 2.1.1 RJALS-syndrome as the clinical manifestation of SPRTN malfunction 10 2.1.2 SPRTN acts in DNA damage repair ..................................................... 11 2.1.3 SPRTN’s essential function remains unknown ..................................... 13 2.2 DNA damage response (DDR) .................................................................. 16 2.2.1 Types of DNA-lesions ........................................................................... 16 2.2.2 DNA damage repair mechanisms ......................................................... 18 2.2.3
    [Show full text]
  • Chemotherapy and Drug Resistance
    Chemotherapy and Drug Resistance Prof. Ramesh Chandra Department of Chemistry University of Delhi The thought of having chemotherapy frightens many people. Almost everyone has heard stories about someone who was "on chemo." But we believe that knowing what chemotherapy is, how it works, and what to expect can often help calm your fears and give you more of a sense of control. Chemotherapy and Drug Resistance • History • Principles • Side effects • Categories of chemotherapeutics • Drug resistance What is chemotherapy? History: Started after World War II (mustard gas) 1950's-1970's e.g., lymphoma/ALL, germ cell tumors >>> effective solid tumors (>90%) >>> resistant 1970's- research on drug resistance palliative >>> aggressive (control, cure) e.g., pre- and post-treatment of breast cancer surgery combination with radiotherapy of osteosarcoma • Why chemotherapy is different from other treatments? (systematic) • Chemotherapy in clinical trials (depending on drugs) Cancer response to anticancer drugs High responsiveness: HLL, lymphoma Partial responsiveness: breast and ovarian cancer Poor responsiveness: melanoma, small cell lung cancer “Heterogeneous drug sensitivity“ in same type of cancers Chemotherapy and Drug Resistance • History • Principles • Side effects • Categories of chemotherapeutics • Drug resistance How does chemotherapy work? Proliferating cells M G1 Growth arrest G 2 Differentiation Apoptosis S Cell Cycle Phases G1 phase. Metabolic changes prepare the 18_02_four_phases.jpg Premitotic synth of cell for division. At a certain point - the structures, mol’s restriction point - the cell is committed to division and moves into the S phase. thesisS phase replicates. DNA the synthesisgenetic material.replicates Each thechromosome now consists of two sister chromatids.genetic material. Each chromosome now G2consists phase.ofMetabolictwo sister changeschromatids assemble.
    [Show full text]
  • Evaluation of Methoxsalen in the Treatment of Vitiligo Irving D
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector EVALUATION OF METHOXSALEN IN THE TREATMENT OF VITILIGO IRVING D. LONDON, M.D. Montgomery, Atabamo The value of the psoralen compounds in theviolet light from a General Electric R.S. Sunlamp treatment of vitiligo has been documented inbulb. The amount of exposure was gradually in- this country since 1952 (1—6). Vitiligo is a dis-creased, based on the patient's tolerance. Another ease of unknown etiology in which the pigmentgroup was treated by topical application of one rarely if ever has been known to return spon-per cent methoxsalen paint over small patches of vitiligo followed by exposure to sunlight or sun- taneously to the involved areas (see p. 281).lamp. At first, both 8-methoxypsoralen (methox- No statistical analysis will be attempted in this salen) and 8-isoamylenoxypsoralen were usedpaper for several reasons. First, several patients together in capsules for oral administration andwere lost from view after several months of treat- mixed in a cream for topical use. Since 1955,ment. Some were seen at distant clinics and these only methoxsalen has been used, after Kanofrecords were not available during the preparation (5) found that this compound was as effectiveof this paper. A few patients had to stop taking when used alone as when combined with 8-the drug because of toxic reactions. Lastly, the isoamylenoxypsoralen. When methoxsalen, eithertechnics of administration of the psoralens were not always comparable. I shall, therefore, discuss applied topically or taken orally, is followed bynot statistics but the results in general of the exposure to gradually increasing doses of sun-various technies especially as they pertain to light or artificial ultraviolet light, gradual re-individual patients.
    [Show full text]
  • Multiplexed Small Molecule Ligand Binding Assays by Affinity Labeling and DNA Sequence Analysis
    Multiplexed Small Molecule Ligand Binding Assays by Affinity Labeling and DNA Sequence Analysis Bo Cai and Casey J. Krusemark* Department of Medicinal Chemistry and Molecular Pharmacology, Purdue Center for Cancer Research, Purdue University, West Lafayette, Indiana 47907, United States Abstract: Small molecule binding assays to target proteins are a core component of drug discovery and development. While a number of assay formats are available, significant drawbacks still remain in cost, sensitivity, and throughput. To improve assays by capitalizing on the power of DNA sequence analysis, we have developed an assay method that combines DNA encoding with split-and-pool sample handling. The approach involves affinity labeling of DNA-linked ligands to a protein target. Critically, the labeling event assesses ligand binding and enables subsequent pooling of several samples. Application of a purifying selection on the pool for protein-labeled DNAs allows detection of ligand binding by quantification of DNA barcodes. We demonstrate the approach in both ligand displacement and direct binding formats and demonstrate its utility in determination of relative ligand affinity, profiling ligand specificity, and high-throughput small molecule screening. The identification and characterization of small molecule ligands to proteins are central to the development of drugs and chemical probes.1-4 While a variety of assay approaches are available for detecting protein-ligand interactions,5-7 significant limitations exist, particularly in the areas of throughput,
    [Show full text]
  • Psoralen Photochemistry and Nucleic Acid Structure
    0022-202X/ 8 1/ 770 1-00J9$02.00/ 0 THE JOURNAL OF I NV r, STIGATIVE DEIlMATOLOGY, 77:39-44, IH8 1 Vol. 77, No. I Copy righ t © 198 1 by The Williams & Wilkins Co. Printed ill U.S.A. Psoralen Photochemistry and Nucleic Acid Structure JOHN E. H EARST, PH.D. Departm.ent of Chemistry, University of California, B erkeley, California, U.S.A . Many new psorale n derivatives have b een synthesized nated in the production of many new psoralens, several of which in an effort to enhance their water solubility and their have superior photoreactivity with both DNA and RNA as binding to nucleic acids. Availability of the very soluble compared to TMP and 8-MOP. The 5 new derivatives shown strongly binding compounds has improved our abilities here are 4' adducts of TMP and their structW"es ru-e shown (3 - to follow the optical changes associated with the photo­ 7). T he complete characterization of methoxymethyltrioxsalen chemistry of psoralens with DNA. Changes in both ab­ (MMT, 4) , hydroxymethyltrioxsalen (HMT, 5) and aminome­ sorbance and fluoresce nce are prese nted in this r eview. thyltrioxsalen (AMT, 7) wi th respect to their reactivity with A kinetic model for the photochemistry concludes that nucleic acids, including a theoretical treatment, is described in the detailed kinetics is dominated by the equilibrium a recent paper by Isaacs et al [6]. A related set of soluble 8- constant for intercalation of the psoralen in the DNA, methoxypsoralen derivatives have been synthesized by Isaacs, the quantum yield for photoaddition to DNA once inter­ Chun, a nd Hearst [7] a nd ru'e listed as compounds 8-11.
    [Show full text]
  • Cross-Linking of DNA by Alkylating Agents and Effects on DNA Function in the Chick Embryo
    (CANCER RESEARCH 31, 1573-1579, November 1971] Cross-linking of DNA by Alkylating Agents and Effects on DNA Function in the Chick Embryo Jerome J. McCann, Timothy M. Lo, and D. A. Webster Department of Biology, Illinois Institute of Technology, Chicago, Illinois 60616 SUMMARY single-stranded DNA partitions into the dextran-rich lower phase (3). This assay has been used to demonstrate that many Drug-induced cross-links are found in the DNA of chick preparations of DNA normally contain some cross-linked embryos within 6 hr after injection of mitomycin C and DNA, which was postulated to arise when DNA is sheared methyl-di-(2-chloroethyl)amine into the egg and 24 hr after during purification (2, 3). In Escherichia coli, an enzyme injection of triethylene thiophosphoramide. Effects on the system, consisting of an exonuclease and a ligase, has been rates of synthesis of DNA, RNA, and protein were studied shown to cross-link DNA terminally (25). with chemical assays for total content of these When difunctional alkylating agents were injected onto the macromolecules as well as radioactive precursor incorporation. area vasculosa of 4-day chick embryos in ovo, within 24 hr a All three drugs inhibited DNA synthesis before RNA and specific type of macrophage was formed over the entire protein synthesis were affected, but there were discrepancies embryo (24). These macrophages were indistinguishable by between the two methods of measuring macromolecular light and electron microscopy from macrophages which are synthesis; thus, radioactive precursor incorporation is not formed during the process of "programmed cell death," a always a reliable measurement of macromolecular synthesis in phenomenon believed important for sculpturing the normal the chick embryo.
    [Show full text]
  • 3939.Full.Pdf
    (CANCER RESEARCH 46, 3939-3944. August 1986] Cytotoxicity and DNA Lesions Produced by Mitomycin C and Porfiromycin in Hypoxie and Aerobic EMT6 and Chinese Hamster Ovary Cells1 Paula M. Fracasso2 and Alan C. Sartorelli3 Department of Pharmacology and Developmental Therapeutics Program, Comprehensive Cancer Center, Yale University School of Medicine, Ne»'Haven, Connecticut 06510 ABSTRACT cally resistant group within solid tumors; thus, hypoxic neo- plastic cells may be capable of proliferating and causing tumor Solid neoplasms may contain deficient or poorly functional vascular beds, a property that leads to the formation of hypoxic tumor cells, which regrowth after treatment that produces tumor regression. Our form a therapeutically resistant cell population within the tumor that is laboratory has suggested that hypoxic cells in solid tumors may difficult to eradicate by ionizing irradiation and most existing chemother- exist in an environment conducive to reductive processes, which apeutic agents. As an approach to the therapeutic attack of hypoxic cells, might be exploited by the use of chemotherapeutic agents that we have measured the cytotoxicity and DNA lesions produced by the become cytotoxic after reductive activation (5, 6). This class of bioreductive alkylating agents mitomycin C and porfiromycin, two struc drugs, which we have called bioreductive alkylating agents, turally similar antibiotics, in oxygen-deficient and aerobic cells. Mito consists of compounds which require metabolic reduction to mycin C and porfiromycin were preferentially cytotoxic to hypoxic EMT6 form species capable of alkylating critical cellular macromole- cells in culture, with porfiromycin producing a greater differential kill of cules. The expectation is that hypoxic tumor cells would readily hypoxic EMT6 cells relative to their oxygenated counterparts than did activate and be preferentially susceptible to drugs of this class.
    [Show full text]