Identification of Small‐Molecule Scaffolds for P450 Inhibitors

Jens P. von Kries1, Thulasi Warrier2, Larissa M. Podust3

1 Screening Unit, Leibniz Institute for Molecular Pharmacology (FMP), Berlin, Germany, 2 Max‐Planck‐Institute for Infection Biology, Berlin, Germany, 3 Department of Pharmaceutical Chemistry, University of California, San Francisco, California
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 17.4
DOI:  10.1002/9780471729259.mc1704s16
Online Posting Date:  February, 2010
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Mycobacterium tuberculosis cytochrome P450 enzymes (CYP) attract ongoing interest for their pharmacological development potential, driving direct screening efforts against potential CYP targets with the ultimate goal of developing potent CYP‐specific inhibitors and/or molecular probes to address M. tuberculosis biology. The property of CYP enzymes to shift the ferric heme Fe Soret band in response to ligand binding provides the basis for an experimental platform for high‐throughput screening (HTS) of compound libraries to select chemotypes with high binding affinities to the target. Promising compounds can be evaluated in in vitro assays or in vivo disease models and further characterized by x‐ray crystallography, leading to optimization strategies to assist drug design. Protocols are provided for compound library screening, analysis of inhibitory potential, and co‐crystallization with the target CYP, as well as expression and purification of soluble CYP enzymes. Curr. Protoc. Microbiol. 16:17.4.1‐17.4.25. © 2010 by John Wiley & Sons, Inc.

Keywords: Mycobacterium tuberculosis; P450; CYP; high‐throughput screening; X‐ray crystallography; small‐molecule inhibitors

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Table of Contents

  • Introduction
  • Basic Protocol 1: High‐Throughput Binding Assay
  • Basic Protocol 2: Inhibition of M. tuberculosis Growth in Liquid Medium
  • Basic Protocol 3: Inhibition of M. tuberculosis Growth in Macrophage Cells
  • Basic Protocol 4: Co‐Crystallization of CYP Target with Screen Hits
  • Support Protocol 1: Expression of Bacterial CYP for Screening and Crystallization
  • Support Protocol 2: Purification of Bacterial CYP for Screening and Crystallization
  • Support Protocol 3: Preparation of Selenomethionine Protein Derivative
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: High‐Throughput Binding Assay

  • HTS assay buffer (see recipe)
  • Reference compounds known to bind CYP of interest via type I and/or type II mechanism
  • 10 mM library compound stock solutions in DMSO
  • 2 µM CYP of interest solution in HTS assay buffer (see recipe for HTS assay buffer)
  • 384‐well polypropylene deep‐well plates (Corning, cat. no. 3347)
  • 16‐channel automated dispenser (µFlo, BioTek)
  • Sciclone ALH‐3000 pipetting robot equipped with 384‐tip low‐volume head (CaliperLS)
  • 384‐well polystyrol plates (Corning, cat. no. 3702)
  • Aluminium foil
  • Ultrasonic water bath (Transsonic 460, Elma)
  • Centrifuge (Eppendorf 5810R, rotor A‐4‐81)
  • 384‐well microtiter plate reader (SafireII, Tecan AG)

Basic Protocol 2: Inhibition of M. tuberculosis Growth in Liquid Medium

  • 100 mM compounds of interest stock solutions in DMSO
  • Middlebrook 7H9 liquid medium with and without Tween‐80 (see recipe for complete medium)
  • 50 mg/ml hygromycin (Roche)
  • DMSO
  • Log phase culture of M.tuberculosis
  • Alamar blue reagent (AbD Serotec, cat. no. BUF012A)
  • 20% Tween‐80
  • 96‐well flat‐bottom plates, sterile (TC Microwell 96F, NUNC, cat. no. 167008)
  • Spectrometer
  • 37°C incubator
  • Parafilm
  • 0.22‐µm pore membrane filter
  • Microplate reader or digital camera

Basic Protocol 3: Inhibition of M. tuberculosis Growth in Macrophage Cells

  • 8‐ to 12‐week‐old female C57/Bl6 mice
  • Bone marrow macrophage differentiation medium (see recipe)
  • Phosphate buffered saline (PBS; appendix 2A), cold
  • Macrophage infection medium (see recipe)
  • Log phase culture of M. tuberculosis
  • 100 mM compounds of interest (test compounds) stock solutions in DMSO
  • 100 mM isoniazid stock solution in water
  • Cell lysis buffer: 0.5% Triton X‐100 ( appendix 2A) in PBS
  • Dilution buffer: 0.05% Tween‐80 in PBS
  • Middlebrook 7H11 agar plates (unit 10.1)
  • Plastic petri dishes (92‐mm diameter; Sarstedt, cat. no. 821473)
  • 37°C, 5% CO 2 incubator
  • Neubauer counting chamber
  • 96‐well flat‐bottom plates, sterile (TC Microwell 96F, Nunc, cat. no. 167008)
  • 1.5‐ml microcentrifuge tubes
  • 0.4‐µm needle
  • Spectrophotometer
  • Additional reagents and equipment for harvesting bone marrow cells (Zhang et al., )

Basic Protocol 4: Co‐Crystallization of CYP Target with Screen Hits

  • ∼1 mM purified CYP of interest stock solution (see protocol 5)
  • 100 mM compound of interest stock solutions in DMSO
  • 10 mM Tris⋅Cl, pH 7.5 ( appendix 2A)
  • Crystallization screening kits in 96‐well deep‐well block format:
    • Crystal Screen (Hampton Research, cat. no. HR2‐130)
    • Index (Hampton Research, cat. no. HR2‐134)
    • PEG/Ion (Hampton Research, cat. no. HR2‐139)
    • Grid Screen Salt (Hampton Research, cat. no. HR2‐248)
  • 100% glycerol
  • Liquid nitrogen
  • 96‐well plates, non‐sterile, no lid (compatible with the drop‐setting robot)
  • Nanoliter drop setting robot Mosquito (TTP LabTech)
  • Transparent 96‐well plate seal (TTP LabTech, cat. no. 4150‐05100)
  • Aluminium foil with DMSO‐resistant cement for re‐sealing screen deep‐well plates (Corning, cat. no. 9596)
  • Temperature‐controlled environment (20° to 25°C) for incubation of crystallization plates
  • 24‐well VDX plates with sealant (Hampton Research, cat. no. HR3‐170)
  • Orbital shaker
  • Siliconized circle cover slides (Hampton Research, cat. no. HR3‐233)
  • CryoTools (Hampton Research): long CryoTong, long CrystalWand, curved vial clamp, CrystalCap holder (cat. no. HR4‐705), CrystalCap plastic vials, socket driver (cat. no. HR4‐706) for height adjustment, 1‐liter stainless‐steel dewar (cat. no. HR4‐699), aluminum CryoCane, tall 1‐liter dewar
  • CrystalCap mounted CryoLoops of various sizes (Hampton Research)
  • Dry‐shipper

Support Protocol 1: Expression of Bacterial CYP for Screening and Crystallization

  • Expression vector encoding CYP of interest (see recipe)
  • HMS174(DE3) competent cells
  • Luria‐Bertani (LB) medium ( appendix 4A)
  • 40 to 50 mg/ml antibiotics
  • 1 M thiamin
  • 4000× microelement solution (see recipe)
  • Expression medium (see recipe)
  • 0.5 mM isopropyl β‐D‐thiogalactopyraniside (IPTG)
  • 1 M δ‐aminolevulinic acid
  • Suspension buffer (see recipe)
  • 1.5‐ml microcentrifuge tubes
  • 42°C hot plate
  • 15‐ml tubes (BD Falcon), sterile
  • 37°C incubator with platform shaker
  • 250‐ml culture flask
  • 2.8‐liter standard Fernbach flasks (do not use baffled flasks)
  • 500‐ml or 1‐liter centrifuge bottles, sterilized
  • 250‐ml plastic container

Support Protocol 2: Purification of Bacterial CYP for Screening and Crystallization

  • Crude protein extract cells in suspension buffer at −80°C (see protocol 5)
  • 20‐ml Ni‐NTA column
  • Ni‐NTA equilibration buffer (see recipe)
  • Ni‐NTA high‐salt wash buffer (see recipe)
  • Ni‐NTA no‐salt wash buffer (see recipe)
  • Ni‐NTA elution buffer (see recipe)
  • 40‐ml S‐Sepharose column
  • 40‐ml Q‐Sepharose column
  • S‐Sepharose/Q‐Sepharose equilibration buffer (see recipe)
  • S‐Sepharose/Q‐Sepharose wash buffer (see recipe)
  • 0 to 0.5 M NaCl gradient in Q‐Sepharose elution buffer (see recipe)
  • 5 M NaCl ( appendix 2A)
  • 250‐ml metal sonicator cup
  • Sonicator
  • 60‐ml ultracentrifuge tubes, sterile
  • Electronic balance
  • Refrigerated ultracentrifuge
  • 250‐ml Erlenmeyer flasks
  • 10‐ml tubes
  • Fraction collector
  • Centriprep YM‐50 centrifugal filter device (Millipore)

Support Protocol 3: Preparation of Selenomethionine Protein Derivative

  • Luria‐Bertani (LB) medium ( appendix 4A)
  • Antibiotic
  • Transformed cells (see protocol 5)
  • SelenoMet medium base (see recipe; Athena Enzyme Systems, cat. no. 0501)
  • 10 mg/ml methionine
  • SelenoMet nutrient mix (see recipe; Athena Enzyme Systems, cat. no. 0502)
  • Microelement solution (see recipe)
  • Selenomethionine (Sigma‐Aldrich, cat. no. S3132)
  • 0.5 mM isopropyl β‐D‐thiogalactopyraniside (IPTG)
  • 1 M δ‐aminolevulinic acid
  • Suspension buffer (see recipe), ice cold
  • 50‐ml centrifuge tubes, sterile
  • 37°C incubator with platform shaker
  • 250‐ml culture flasks
  • Spectrophotometer
  • 2.8‐liter standard Fernbach flasks
  • 500‐ml or 1‐liter centrifuge bottles
  • 250‐ml plastic container
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Literature Cited

Literature Cited
   Ahmad, Z., Sharma, S., and Khuller, G.K. 2005. In vitro and ex vivo antimycobacterial potential of azole drugs against Mycobacterium tuberculosis H37Rv. FEMS Microbiol. Lett. 251:19‐22.
   Ahmad, Z., Sharma, S., and Khuller, G.K. 2006a. Azole antifungals as novel chemotherapeutic agents against murine tuberculosis. FEMS Microbiol. Lett. 261:181‐186.
   Ahmad, Z., Sharma, S., and Khuller, G.K. 2006b. The potential of azole antifungals against latent/persistent tuberculosis. FEMS Microbiol. Lett. 258:200‐203.
   Ahmad, Z., Sharma, S., Khuller, G.K., Singh, P., Faujdar, J., and Katoch, V.M. 2006c. Antimycobacterial activity of econazole against multidrug‐resistant strains of Mycobacterium tuberculosis. Int. J. Antimicrob. Agents 28:543‐544.
   Aoyama, Y. 2005. Recent progress in the CYP51 research focusing on its unique evolutionary and functional characteristics as a diversozyme P450. Front. Biosci. 10:1546‐1557.
   Banfi, E., Scialino, G., Zampieri, D., Mamolo, M.G., Vio, L., Ferrone, M., Fermeglia, M., Paneni, M.S., and Pricl, S. 2006. Antifungal and antimycobacterial activity of new imidazole and triazole derivatives. A combined experimental and computational approach. J. Antimicrob. Chemother. 58:76‐84.
   Belin, P., Le Du, M.H., Fielding, A., Lequin, O., Jacquet, M., Charbonnier, J.‐B., Lecoq, A., Thai, R., Courcon, M., Masson, C., Dugave, C., Genet, R., Pernodet, J.‐L., and Gondry, M. 2009. Identification and structural basis of the reaction catalyzed by CYP121, an essential cytochrome P450 in Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. U.S.A. 106:7426‐7431.
   Byrne, S.T., Denkin, S.M., Gu, P., Nuermberger, E., and Zhang, Y. 2007. Activity of ketoconazole against Mycobacterium tuberculosis in vitro and in the mouse model. J. Med. Microbiol. 56:1047‐1051.
   Cali, J.J., Ma, D., Sobol, M., Simpson, D.J., Frackman, S., Good, T.D., Daily, W.J., and Liu, D. 2006. Luminogenic cytochrome P450 assays. Expert. Opin. Drug. Metab. Toxicol. 2:629‐645.
   Capyk, J.K., Kalscheuer, R., Stewart, G.R., Liu, J., Kwon, H., Zhao, R., Okamoto, S., Jacobs, W.R. Jr., Eltis, L.D., and Mohn, W.W. 2009. Mycobacterial cytochrome P450 125 (CYP125) catalyzes the terminal hydroxylation of C27 steroids. J. Biol. Chem. 284:35534‐35542.
   Chang, J.C., Harik, N.S., Liao, R.P., and Sherman, D.R. 2007. Identification of mycobacterial genes that alter growth and pathology in macrophages and in mice. J. Infect. Dis. 196:788‐795.
   Chen, C.‐K., Doyle, P.S., Yermalitskaya, L.V., Mackey, Z.B., Ang, K.K.H., McKerrow, J.H., and Podust, L.M. 2009. Trypanosoma cruzi CYP51 inhibitor derived from a Mycobacterium tuberculosis screen hit. PLoS Negl. Trop. Dis. 3:e372.
   Cole, S.T., Brosch, R., Parkhill, J., Garnier, T., Churcher, C., Harris, D., Gordon, S.V., Eiglmeier, K., Gas, S., Barry, C.E. 3rd, Tekaia, F., Badcock, K., Basham, D., Brown, D., Chillingworth, T., Connor, R., Davies, R., Devlin, K., Feltwell, T., Gentles, S., Hamlin, N., Holroyd, S., Hornsby, T., Jagels, K., Krogh, A., McLean, J., Moule, S., Murphy, L., Oliver, K., Osborne, J., Quail, M.A., Rajandream, M.A., Rogers, J., Rutter, S., Seeger, K., Skelton, J., Squares, R., Squares, S., Sulston, J.E., Taylor, K., Whitehead, S., and Barrell, B.G. 1998. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393:537‐544.
   Dunford, A.J., McLean, K.J., Sabri, M., Seward, H.E., Heyes, D.J., Scrutton, N.S., and Munro, A.W. 2007. Rapid P450 heme iron reduction by laser photoexcitation of Mycobacterium tuberculosis CYP121 and CYP51B1. Analysis of CO complexation reactions and reversibility of the P450/P420 equilibrium. J. Biol. Chem. 282:24816‐24824.
   Fai, P.B. and Grant, A. 2009. A rapid resazurin bioassay for assessing the toxicity of fungicides. Chemosphere 74:1165‐1170.
   Flynn, J.L. 2004. Immunology of tuberculosis and implications in vaccine development. Tuberculosis 84:93‐101.
   Hendrickson, W.A., Horton, J.R., Murthy, H.M., Pahler, A., and Smith, J.L. 1989. Multiwavelength anomalous diffraction as a direct phasing vehicle in macromolecular crystallography. Basic Life Sci. 51:317‐324.
   Holton, J.M. 2009. A beginner's guide to radiation damage. J. Synchrotron Radiat. 16:133‐142.
   Johnston, J., Kells, P.M., Podust, L.M., and Ortiz de Montellano, P.R. 2009. Biochemical and structural characterization of CYP124, a methyl‐branched lipid w‐hydroxylase from Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. U.S.A. 106:20687‐20692.
   Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680‐685.
   McLean, K.J., Cheesman, M.R., Rivers, S.L., Richmond, A., Leys, D., Chapman, S.K., Reid, G.A., Price, N.C., Kelly, S.M., Clarkson, J., Smith, W.E., and Munro, A.W. 2002. Expression, purification and spectroscopic characterization of the cytochrome P450 CYP121 from Mycobacterium tuberculosis. J. Inorg. Biochem. 91:527‐541.
   McLean, K.J., Carroll, P., Lewis, D.G., Dunford, A.J., Seward, H.E., Neeli, R., Cheesman, M.R., Marsollier, L., Douglas, P., Smith, W.E., Rosenkrands, I., Cole, S.T., Leys, D., Parish, T., and Munro, A.W. 2008. Characterization of active site structure in CYP121: A cytochrome P450 essential for viability of Mycobacterium tuberculosis H37Rv. J. Biol. Chem. 283:33406‐33416.
   Nasser Eddine, A., von Kries, J.P., Podust, M.V., Warrier, T., Kaufmann, S.H., and Podust, L.M. 2008. X‐ray structure of 4,4′‐dihydroxybenzophenone mimicking sterol substrate in the active site of sterol 14alpha‐demethylase (CYP51). J. Biol. Chem. 283:15152‐15159.
   Nwaka, S. and Hudson, A. 2006. Innovative lead discovery strategies for tropical diseases. Nat. Rev. Drug Discov. 5:941‐955.
   O'Brien, J., Wilson, I., Orton, T., and Pognan, F. 2000. Investigation of the Alamar blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur. J. Biochem. 267:5421‐5426.
   Ogura, H., Nishida, C.R., Hoch, U.R., Perera, R., Dawson, J.H., and Ortiz de Montellano, P.R. 2004. EpoK, a cytochrome P450 involved in biosynthesis of the anticancer agents epothilones A and B. Substrate‐mediated rescue of a P450 enzyme. Biochemistry 43:14712‐14721.
   Orme, I.M. 2003. The mouse as a useful model of tuberculosis. Tuberculosis 83:112‐115.
   Ortiz de Montellano, P.R. 2005. Cytochrome P450: Structure, Mechanism, and Biochemistry. Kluwer Academic/Plenum, New York.
   Ouellet, H., Podust, L.M., and de Montellano, P.R. 2008. Mycobacterium tuberculosis CYP130: Crystal structure, biophysical characterization, and interactions with antifungal azole drugs. J. Biol. Chem. 283:5069‐5080.
   Pieters, J. 2008. Mycobacterium tuberculosis and the macrophage: Maintaining a balance. Cell Host Microbe 3:399‐407.
   Podust, L.M., Yermalitskaya, L.V., Lepesheva, G.I., Podust, V.N., Dalmasso, E.A., and Waterman, M.R. 2004. Estriol bound and ligand‐free structures of sterol 14α‐demethylase. Structure 12:1937‐1945.
   Podust, L.M., von Kries, J.P., Nasser Eddine, A., Kim, Y., Yermalitskaya, L.V., Kuehne, R., Ouellet, H., Warrier, T., Altekoster, M., Lee, J.‐S., Rademann, J., Oschkinat, H., Kaufmann, S.H.E., and Waterman, M.R. 2007. Small molecule scaffolds for CYP51 inhibitors identified by high‐throughput screening and defined by x‐ray crystallography. Antimicrob. Agents Chemother. 51:3915‐3923.
   Podust, L.M., Ouellet, H., von Kries, J.P., and Ortiz de Montellano, P.R. 2009. Interaction of Mycobacterium tuberculosis CYP130 with heterocyclic arylamines. J. Biol. Chem. 284:25211‐25219.
   Rasmussen, E.S. 1999. Use of fluorescent redox indicators to evaluate cell prolifiration and viability. In Vitro Mol. Toxicol. 12:47‐58.
   Recchi, C., Sclavi, B., Rauzier, J., Gicquel, B., and Reyrat, J.M. 2003. Mycobacterium tuberculosis Rv1395 is a class III transcriptional regulator of the AraC family involved in cytochrome P450 regulation. J. Biol. Chem. 278:33763‐33773.
   Rosloniec, K.Z., Wilbrink, M.H., Capyk, J.K., Mohn, W.W., Ostendorf, M., van der Geize, R., Dijkhuizen, L., and Eltis, L.D. 2009. Cytochrome P450 125 (CYP125) catalyses C26‐hydroxylation to initiate sterol side‐chain degradation in Rhodococcus jostii RHA1. Mol. Microbiol. 74:1031‐1043.
   Sassetti, C.M. and Rubin, E.J. 2003. Genetic requirements for mycobacterial survival during infection. Proc. Natl. Acad. Sci. U.S. A. 100:12989‐12994.
   Saunders, B.M. and Britton, W.J. 2007. Life and death in the granuloma: Immunopathology of tuberculosis. Immunol. Cell Biol. 85:103‐111.
   Schenkman, J.B., Remmer, H., and Estabrook, R.W. 1967. Spectral studies of drug interaction with hepatic microsomal cytochrome. Mol. Pharmacol. 3:113‐123.
   Sheehan, D.J., Hitchcoch, C.A., and Sibley, C.M. 1999. Current and emerging azole antifungal agents. Clin. Microbiol. Rev. 12:40‐79.
   Sherman, D.H., Li, S., Yermalitskaya, L.V., Kim, Y., Smith, J.A., Waterman, M.R., and Podust, L.M. 2006. The structural basis for substrate anchoring, active site selectivity, and product formation by P450 PikC from Streptomyces venezuelae. J. Biol. Chem. 281:26289‐26297.
   Yajko, D.M., Madej, J.J., Lancaster, M.V., Sanders, C.A., Cawthon, V.L., Gee, B., Babst, A., and Hadley, W.K. 1995. Colorimetric method for determining MICs of antimicrobial agents for Mycobacterium tuberculosis. J. Clin. Microbiol. 33:2324‐2327.
   Zhang, X., Goncalves, R., and Mosser, D.M. 2008. The isolation and characterization of muringe macrophages. Curr. Protoc. Immunol. 83:14.1.1‐14.1.14.
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