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

Abstract

The development of novel antimycobacterial agents against Mycobacterium tuberculosis (Mtb) is urgently required due to the appearance of multidrug resistance (MDR) combined with complicated long‐term treatment. CYP121 was shown to be a promising novel target for inhibition of mycobacterial growth. In this study, we describe the rational discovery of new CYP121 inhibitors by a systematic screening based on biophysical and microbiological methods. The best hits originating from only one structural class gave initial information about molecular motifs required for binding and activity. The initial screening procedure was followed by mode‐of‐action studies and further biological characterizations. The results demonstrate superior antimycobacterial efficacy and a decreased toxicity profile of our frontrunner compound relative to the reference compound econazole. Due to its low molecular weight, promising biological profile, and physicochemical properties, this compound is an excellent starting point for further rational optimization.

Structure‐Activity Relationship and Mode‐Of‐Action Studies Highlight 1‐(4‐Biphenylylmethyl)‐1H‐imidazole‐Derived Small Molecules as Potent CYP121 Inhibitors

“…For 79 compounds, which showed a visible shift of the absorption maximum at concentrations of 20 μM, the binding constant K D was determined by concentration dependent titration of the enzyme (respective K D values are listed in SI, table S1). All of these binders showed a type-II shift identical to the previously determined I:47, [20] as expected because of the structural similarity (discussion on actual binding mode in "complex crystallization" paragraph). 38 of the approved binders (hit rate 40 %) exhibited a binding constant below or equal to I:47 (K D = 5 μM).…”

“…The screening library was examined in a UV/Vis heme binding assay regarding affinity toward CYP121 as previously described. [20] The compounds were screened at concentrations of 100 μM and 20 μM. For 79 compounds, which showed a visible shift of the absorption maximum at concentrations of 20 μM, the binding constant K D was determined by concentration dependent titration of the enzyme (respective K D values are listed in SI, table S1).…”

“…UV/Vis heme P450 binding assay: Optical titration experiments were performed and analyzed as described previously. [20,40] Determination of BCG MIC 50 by OD 600 assay: A pre-culture of M. bovis BCG was grown in 7H9 medium supplemented with ADC Enrichment until OD 600 reached a value of 0.7 and was then used for susceptibility testing in the following 72 h. The assay was performed in 48 well plates (Greiner, Kremsmünster, AT). Prior to culture addition, compounds were serially diluted in DMSO to fit a final DMSO concentration of 1 %.…”

“…However, none of these compounds was shown to exhibit any in vitro activity toward Mtb (or M. bovis BCG). Recently, we described a screening approach based on a CYP‐inhibitor library which resulted in the identification of the first potent inhibitors of CYP121 with activity against M. bovis BCG and Mtb [20] …”

Design and Synthesis of Imidazole and Triazole Pyrazoles as Mycobacterium Tuberculosis CYP121A1 Inhibitors

“…The role of mycocylosin in Mtb remains unclear, although cyclodipeptides have important biological effects, such as inhibition of production of bacterial virulence factors and biofilm formation . Drug design for CYP121A1 inhibitors has followed several approaches: (1) a fragment‐based approach has been very effective in developing compounds with low nM enzyme binding affinity, however this has not translated to antimycobacterial activity; (2) a recent biophysical screening of a library of described P450 inhibitors has led to a promising lead compound with both good binding affinity and antimycobacterial activity; (3) our own approach has focused on developing a series of different compound scaffolds as mimics of cYY, resulting in both good binding affinity and promising antimycobacterial activity . Our previous research identified derivatives of (1,3‐diphenyl)‐4‐((1,3‐imidazol‐1‐yl)methyl)‐1 H ‐pyrazole and 1‐((1,3‐diphenyl‐1 H ‐pyrazol‐4‐yl)methyl)‐1 H ‐triazole as inhibitors of CYP121A1 ( e. g .…”