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Antimicrobial Agents and Chemotherapy, November 2007, p. 3915-3923, Vol. 51, No. 11
0066-4804/07/$08.00+0     doi:10.1128/AAC.00311-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Small-Molecule Scaffolds for CYP51 Inhibitors Identified by High-Throughput Screening and Defined by X-Ray Crystallography{triangledown}

Larissa M. Podust,1,{dagger}* Jens P. von Kries,2,{dagger}* Ali Nasser Eddine,3 Youngchang Kim,4 Liudmila V. Yermalitskaya,5 Ronald Kuehne,2 Hugues Ouellet,1 Thulasi Warrier,3 Markus Alteköster,3 Jong-Seok Lee,3 Jörg Rademann,2 Hartmut Oschkinat,2 Stefan H. E. Kaufmann,3 and Michael R. Waterman5

Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94158,1 Screening Unit and Department of Medicinal Chemistry, Leibniz Institute for Molecular Pharmacology (FMP), Berlin 13125,2 Max-Planck-Institute for Infection Biology, Berlin 10117, Germany,3 Structural Biology Center, Argonne National Laboratory, Argonne, Illinois 60439,4 Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 372325

Received 5 March 2007/ Returned for modification 30 April 2007/ Accepted 19 August 2007

Sterol 14{alpha}-demethylase (CYP51), a major checkpoint in membrane sterol biosynthesis, is a key target for fungal antibiotic therapy. We sought small organic molecules for lead candidate CYP51 inhibitors. The changes in CYP51 spectral properties following ligand binding make CYP51 a convenient target for high-throughput screening technologies. These changes are characteristic of either substrate binding (type I) or inhibitor binding (type II) in the active site. We screened a library of 20,000 organic molecules against Mycobacterium tuberculosis CYP51 (CYP51Mt), examined the top type I and type II binding hits for their inhibitory effects on M. tuberculosis in broth culture, and analyzed them spectrally for their ability to discriminate between CYP51Mt and two reference M. tuberculosis CYP proteins, CYP130 and CYP125. We determined the binding mode for one of the top type II hits, {alpha}-ethyl-N-4-pyridinyl-benzeneacetamide (EPBA), by solving the X-ray structure of the CYP51Mt-EPBA complex to a resolution of 1.53 Å. EPBA binds coordinately to the heme iron in the CYP51Mt active site through a lone pair of nitrogen electrons and also through hydrogen bonds with residues H259 and Y76, which are invariable in the CYP51 family, and hydrophobic interactions in a phylum- and/or substrate-specific cavity of CYP51. We also identified a second compound with structural and binding properties similar to those of EPBA, 2-(benzo[d]-2,1,3-thiadiazole-4-sulfonyl)-2-amino-2-phenyl-N-(pyridinyl-4)-acetamide (BSPPA). The congruence between the geometries of EPBA and BSPPA and the CYP51 binding site singles out EPBA and BSPPA as lead candidate CYP51 inhibitors with optimization potential for efficient discrimination between host and pathogen enzymes.

* Corresponding author. Mailing address for Larissa M. Podust: Department of Pharmaceutical Chemistry, University of California, 600 16th St., San Francisco, CA 94158. Phone: (415) 502-4728. Fax: (415) 502-4728. E-mail: larissa.podust{at}ucsf.edu. Mailing address for Jens P. von Kries: Leibniz-Institut fur Molekulare Pharmakologie FMP, Robert Roessle Str. 10, Berlin 13125, Germany. Phone: 49 (0)30 9406 2982. Fax: 49 (0)30 9406 2922. E-mail: kries{at}fmp-berlin.de

{triangledown} Published ahead of print on 10 September 2007.

{dagger} These authors contributed equally to the work.

Antimicrobial Agents and Chemotherapy, November 2007, p. 3915-3923, Vol. 51, No. 11
0066-4804/07/$08.00+0     doi:10.1128/AAC.00311-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Podust, L. M., Ouellet, H., von Kries, J. P., Ortiz de Montellano, P. R. (2009). Interaction of Mycobacterium tuberculosis CYP130 with Heterocyclic Arylamines. J. Biol. Chem. 284: 25211-25219 [Abstract] [Full Text]  
  • Sheng, C., Miao, Z., Ji, H., Yao, J., Wang, W., Che, X., Dong, G., Lu, J., Guo, W., Zhang, W. (2009). Three-Dimensional Model of Lanosterol 14{alpha}-Demethylase from Cryptococcus neoformans: Active-Site Characterization and Insights into Azole Binding. Antimicrob. Agents Chemother. 53: 3487-3495 [Abstract] [Full Text]  
  • Eddine, A. N., von Kries, J. P., Podust, M. V., Warrier, T., Kaufmann, S. H. E., Podust, L. M. (2008). X-ray Structure of 4,4'-Dihydroxybenzophenone Mimicking Sterol Substrate in the Active Site of Sterol 14{alpha}-Demethylase (CYP51). J. Biol. Chem. 283: 15152-15159 [Abstract] [Full Text]  


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