Small-molecule scaffolds for CYP51 inhibitors identified by high-throughput screening and defined by X-ray crystallography

Abstract

Sterol 14alpha-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 (CYP51(Mt)), 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 CYP51(Mt) 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 CYP51(Mt)-EPBA complex to a resolution of 1.53 A. EPBA binds coordinately to the heme iron in the CYP51(Mt) 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.

FIG. 1.

Type I and type II spectral responses upon ligand binding in CYP51_Mt. (A) Type I spectral changes caused by binding of the nonsubstrate sterol estriol. (B) Type II spectral changes as a result of binding of an azole inhibitor, 4-phenylimidazole. The concentration dependence of the spectral changes allows the binding affinities of the ligands to be estimated.

FIG. 2.

Top HTS hits. (A) Type I HTS hits (K_D, ∼25 to 50 μM). (B) Type II HTS hits (K_D, ∼5 to 10 μM). Dissociation constants confirmed for selected compounds in manual binding assays are provided below their chemical structures. The N-(4-pyridyl)-formamide moiety is highlighted in gray. Chiral carbon centers are marked by an asterisk. (C) BSPPA, selected based on structural similarity to EPBA. Compounds are identified by the numbers in the ChemDiv, Inc., product library catalog. (D) Fluconazole, a clinical antifungal drug.

FIG. 3.

Inhibition of M. tuberculosis growth by DHBP and EPBA. The inhibition of M. tuberculosis (Mtb) growth in broth culture was assessed by the Alamar blue assay utilizing a resazurin color change from blue to pink upon reduction to resorufin in wells containing live mycobacteria. The experiment was performed in duplicate.

FIG. 4.

EPBA and BSPPA binding specificity. Linearization in the form of the S₀/ΔA-versus-S₀ plot of the UV-visible spectral titration data obtained by adding EPBA (A) or BSPPA (B) to CYP51_Mt or its F78L mutant form in 5 μM increments or by adding EPBA (C) to CYP125 or CYP130 in 100 μM increments.

FIG. 5.

CYP51 active-site residues across the different phyla. Fragments of the multiple sequence alignment of 69 sequences of CYP51 isoforms from different organisms show the residues of the substrate binding site in CYP51 as deduced from the estriol-bound form (26). Accession numbers for CYP51 in the Swiss-Prot/TrEMBL (http://us.expasy.org/sprot) or NCBI (http://www.ncbi.nlm.nih.gov) database are given next to the names of the host organisms. Alignment was performed using the maximum a posteriori algorithm as implemented in the BCM Search Launcher (http://searchlauncher.bcm.tmc.edu/multi-align/multi-align.html) (31). The figure was generated with ESPript (12). M. bovis, Mycobacterium bovis; M. avium, Mycobacterium avium; M. ulcerans, Mycobacterium ulcerans; M. smegmatis, Mycobacterium smegmatis; N. farcinica, Nocardia farcinica; M. capsulatus, Methylococcus capsulatus; T. brucei, Trypanosoma brucei; T.b. gambiense, Trypanosoma brucei gambiense; L. amazonensis, Leishmania amazonensis; L. major, Leishmania major; A. fumigatus, Aspergillus fumigatus; V. inaequalis, Venturia inaequalis, V. nashicola, Venturia nashicola; L. maculans, Leptosphaeria maculans; T. acuformis, Tapesia acuformis; O. yallundae, Oculimacula yallundae; B. graminis, Blumeria graminis; B. fuckeliana, Botryotinia fuckeliana; M. fructicola, Monilinia fructicola; U. necator, Uncinula necator; M. grisea, Magnaporthe grisea; N. crassa, Neurospora crassa; M. graminicola, Meloidogyne graminicola; P. digitatum, Penicillium digitatum; P. italicum, Penicillium italicum; E. nidulans, Emericella nidulans; S. cerevisiae, Saccharomyces cerevisiae; C. glabrata, Candida glabrata; A. gossypii, Ashbya gossypii; C. albicans, Candida albicans; C. tropicalis, Candida tropicalis; P. carinii, Pneumocystis carinii; S. pombe, Schizosaccharomyces pombe; C. neoformans, Cryptococcus neoformans; F. neoformans, Filobasidiella neoformans; U. maydis, Ustilago maydis; C. elegans, Cunninghamella elegans; and C. krusei, Candida krusei.

FIG. 6.

Stereo view of EPBA bound in the active site of CYP51_Mt. The electron densities of EPBA (green) and the active-site residues located within 4 Å of the ligand are represented by a fragment of a 2Fo-Fc map (cyan), where Fo is the observed structure factor and Fc is the calculated structure factor, contoured at 2.0 σ. Two water molecules mediating ligand-hydrogen bonding contacts between the amide nitrogen of EPBA and H259 and the carbonyl oxygen of EPBA and Y76 are shown as red spheres. Images were generated using the program SETOR (9).

FIG. 7.

Binding of EBPA and BSPPA in the CYP51_Mt active site. EPBA (A) and BSPPA (B) bound in the active site of CYP51_Mt, which is represented by a space-filled model. Both compounds (cyan) and the heme edge (green) are clearly seen through the active-site opening created by the bent I helix, an open conformation of the BC loop, and missing electron density for the C helix. The ethyl (A) and benzothiadiazole sulfonamide (B) groups protrude into the bulk solvent through an open space of the active-site entrance. In panel A, the heme propionate side chain is shown in two alternative conformations. In panel B, the benzothiadiazole ring of BSPPA flips over to make stacking contacts with the pyridine ring of the same molecule and the Y76 side chain. Some contacts are also made with the heme propionate chain apparently stabilizing it in a single conformation. Images were generated using VMD software (13).