Structure Based Discovery of Inhibitors of CYP125 and CYP142 from Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) was responsible for approximately 1.6 million deaths in 2021. With the emergence of extensive drug resistance, novel therapeutic agents are urgently needed, and continued drug discovery efforts required. Host-derived lipids such as cholesterol not only support Mtb growth, but are also suspected to function in immunomodulation, with links to persistence and immune evasion. Mtb cytochrome P450 (CYP) enzymes facilitate key steps in lipid catabolism and thus present potential targets for inhibition. Here we present a series of compounds based on an ethyl 5-(pyridin-4-yl)-1H-indole-2-carboxylate pharmacophore which bind strongly to both Mtb cholesterol oxidases CYP125 and CYP142. Using a structure-guided approach, combined with biophysical characterization, compounds with micromolar range in-cell activity against clinically relevant drug-resistant isolates were obtained. These will incite further development of much-needed additional treatment options and provide routes to probe the role of CYP125 and CYP142 in Mtb pathogenesis.

Non-classical Helices with cis Carbon-Carbon Double Bonds in the Backbone: Structural Features of α,γ-Hybrid Peptide Foldamers.

The impact of geometrically constrained cis α,ß-unsaturated γ-amino acids on the folding of α,γ-hybrid peptides was investigated. Structure analysis in single crystals and in solution revealed that the cis carbon-carbon double bonds can be accommodated into the 12-helix without deviation from the overall helical conformation. The helical structures are stabilized by 4→1 hydrogen bonding in a similar manner to the 12-helices of ß-peptides and the 310 helices of α-peptides. These results show that functional cis carbon-carbon double bonds can be accommodated into the backbone of helical peptides.

HBTU mediated 1-hydroxybenzotriazole (HOBt) conjugate addition: synthesis and stereochemical analysis of ß-benzotriazole N-oxide substituted γ-amino acids and hybrid peptides.

HBTU is a standard coupling agent commonly used for the activation of free carboxylic acids during the solution and solid phase peptide synthesis. 1-Hydroxybenzotriazole (HOBt) plays a significant role in reducing the racemization during peptide synthesis; hence it is regularly used as a coupling additive. Here, we are reporting the mild and facile conjugate addition of HOBt to E-vinylogous γ-amino acids mediated by the HBTU. The reaction is moderately diastereoselective and novel ß-benzotriazole N-oxide (ß-BtO) substituted γ-amino acids were isolated in moderate to good yields. The single crystal analysis of methyl esters of major (anti) and minor (syn) conjugate addition products infers the formation of exclusively N-alkylated benzotriazole N-oxides instead of O-alkylation of HOBt. In addition, we showed the utilization of ß-BtO substituted γ-amino acids in peptide synthesis and studied their conformations in single crystals.

A new strategy for hit generation: Novel in cellulo active inhibitors of CYP121A1 from Mycobacterium tuberculosis via a combined X-ray crystallographic and phenotypic screening approach (XP screen).

There is a pressing need for new drugs against tuberculosis (TB) to combat the growing resistance to current antituberculars. Herein a novel strategy is described for hit generation against promising TB targets involving X-ray crystallographic screening in combination with phenotypic screening. This combined approach (XP Screen) affords both a validation of target engagement as well as determination of in cellulo activity. The utility of this method is illustrated by way of an XP Screen against CYP121A1, a cytochrome P450 enzyme from Mycobacterium tuberculosis (Mtb) championed as a validated drug discovery target. A focused screening set was synthesized and tested by such means, with several members of the set showing promising activity against Mtb strain H37Rv. One compound was observed as an X-ray hit against CYP121A1 and showed improved activity against Mtb strain H37Rv under multiple assay conditions (pan-assay activity). Data obtained during X-ray crystallographic screening were utilized in a structure-based campaign to design a limited number of analogues (less than twenty), many of which also showed pan-assay activity against Mtb strain H37Rv. These included the benzo[b][1,4]oxazine derivative (MIC90 6.25 µM), a novel hit compound suitable as a starting point for a more involved hit to lead candidate medicinal chemistry campaign.