AAC Accepts, published online ahead of print on 10 September 2007

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Antimicrob. Agents Chemother. doi:10.1128/AAC.00311-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

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

Larissa M. Podust^*, Jens P. von Kries^*, Ali Nasser Eddine, Youngchang Kim, Liudmila V. Yermalitskaya, Ronald Kuehne, Hugues Ouellet, Thulasi Warrier, Markus Alteköster, Jong-Seok Lee, Jörg Rademann, Hartmut Oschkinat, Stefan H.E. Kaufmann, and Michael R. Waterman

Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94158; Screening Unit and Department of Medicinal Chemistry, Leibniz Institute for Molecular Pharmacology (FMP), Berlin, 13125, Germany; Max-Planck-Institute for Infection Biology, Berlin, 10117, Germany; Argonne National Laboratory, Structure Biology Center, Argonne, IL 60439; Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232

^* To whom correspondence should be addressed. Email: larissa.podust{at}ucsf.edu. kries{at}fmp-berlin.de.

Sterol 14-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 it a convenient target for high throughput screening (HTS) 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 (MtCYP51) and examined the top type I and type II binding hits for their inhibitory effects against M. tuberculosis in broth culture and spectrally for their ability to discriminate between MtCYP51 and two reference M. tuberculosis CYP proteins, CYP130 and CYP125. We determined the binding mode for one of the top type II hits, -ethyl-N-4-pyridinyl-benzeneacetamide (EPBA), by solving the x-ray structure of the MtCYP51/EPBA complex to a resolution of 1.53 Å. EPBA binds coordinately to the heme iron in the MtCYP51 active site through a lone pair of nitrogen electrons and also through hydrogen bonds with residues H259 and Y76, which are invariant 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 EPBA, (2-[(2,1,3-benzothiadiazol-4-sulfonamide]-2-phenyl-N-pyridin-4-acetamide (BSPPA). The congruence between the geometries of EPBA and BSPPA and the CYP51 binding site singles them out as lead candidate CYP51 inhibitors, with optimization potential for efficient discrimination between host and pathogen enzymes.

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