Fragment-Based Approaches to the Development of Mycobacterium tuberculosis CYP121 Inhibitors

Abstract

The essential enzyme CYP121 is a target for drug development against antibiotic resistant strains of Mycobacterium tuberculosis. A triazol-1-yl phenol fragment 1 was identified to bind to CYP121 using a cascade of biophysical assays. Synthetic merging and optimization of 1 produced a 100-fold improvement in binding affinity, yielding lead compound 2 (KD = 15 μM). Deconstruction of 2 into its component retrofragments allowed the group efficiency of structural motifs to be assessed, the identification of more LE scaffolds for optimization and highlighted binding affinity hotspots. Structure-guided addition of a metal-binding pharmacophore onto LE retrofragment scaffolds produced low nanomolar (KD = 15 nM) CYP121 ligands. Elaboration of these compounds to target binding hotspots in the distal active site afforded compounds with excellent selectivity against human drug-metabolizing P450s. Analysis of the factors governing ligand potency and selectivity using X-ray crystallography, UV–vis spectroscopy, and native mass spectrometry provides insight for subsequent drug development.

Biophysical Screening of a Focused Library for the Discovery of CYP121 Inhibitors as Novel Antimycobacterials

“…Due to the fact that CYP121i sap otential target for Mtb treatment, some efforts have been undertaken to identify potent inhibitors. Hudson et al [13] and Kavanagh et al [14] published several compounds designed for selectiveC YP121 binding and inhibition. However,n one of them weres hown to be effective against Mtb.…”

Substrate Fragmentation for the Design of M. tuberculosis CYP121 Inhibitors

“…Publishedb yWiley-VCH Verlag GmbH &Co. KGaA, Weinheim 2.50-2.65), as is observed for CYP121 bound to econazole( g z = 2.48, g y = 2.24, g x = 1.90). [36] Theo pposite trend in the g values of EPR spectra collected for CYP121 in complex with the current series of tryptophan( aliphatic amine) ligands, and the previous series of aniline ligands, in addition to their characteristic type II opticals pectra and crystallographic evidence for direct aniline-Fe 3 + binding interactions, [25] providesn ovel insight into the spectroscopice ffect of weak donor ligandso nt he P450 heme environment.…”

“…[33] We have previously reportedt he developmento fp otent ligands against CYP121, however,t hese compounds had suboptimal cellular activity,o wing to either poor permeability or efflux. [25] In the current study,i tw as reasoned that the deconstruction of the CYP121s ubstrates into their component fragments might provide access to novel scaffolds and insight into the interactions governing enzyme-ligand recognition. Substrate fragments were preferentially identified to bind to CYP121w hen they were screened as part of al ibraryo fc hemically similar compounds by thermal shift and UV/vis spectroscopy assays.…”

“…One of these fragments, compound 1fcontained an aniline functionalgroup, which we have previously reported as ap referred CYP121h eme bindingm otif. [25,35] The second, was the d-tryptophan methyl ester 1a,w hich produced ar ed shift in the Soret l max of + 4.5 nm. The remaining fragments, including the methyl ester of d-tyrosine 1b,d id not perturb the opticals pectrum of CYP121.…”

“…[44] However,arecently characteriseds eries of ligands that directly coordinate to the heme iron of CYP121 using ap rimary aniline as the nitrogen ligand (PDB ID:5 IBE) also produce relatively small shifts in the Soret (l max = 422 nm at saturating ligand concentrations), despite their having low nanomolar affinity. [25] Electron paramagnetic resonance (EPR) spectra of CYP121i n complex with fragment 1a and fourteen of the elaborated compounds, were collected to gain furtheri nsight into their bindingi nteractions( Ta ble 3, Supporting Information). All compounds generated ar hombic triplet of g values that are consistent with al ow spin, cysteinate-coordinated ferric P450 (Figure 4a).…”