Lipoamide Channel-Binding Sulfonamides Selectively Inhibit Mycobacterial Lipoamide Dehydrogenase

Tuberculosis remains a global health emergency that calls for treatment regimens directed at new targets. Here we explored lipoamide dehydrogenase (Lpd), a metabolic and detoxifying enzyme in <i>Mycobacterium tuberculosis</i> (Mtb) whose deletion drastically impairs Mtb’s ability to establish infection in the mouse. Upon screening more than 1.6 million compounds, we identified <i>N</i>-methylpyridine 3-sulfonamides as potent and species-selective inhibitors of Mtb Lpd affording >1000-fold selectivity versus the human homologue. The sulfonamides demonstrated low nanomolar affinity and bound at the lipoamide channel in an Lpd–inhibitor cocrystal. Their selectivity could be attributed, at least partially, to hydrogen bonding of the sulfonamide amide oxygen with the species variant Arg93 in the lipoamide channel. Although potent and selective, the sulfonamides did not enter mycobacteria, as determined by their inability to accumulate in Mtb to effective levels or to produce changes in intracellular metabolites. This work demonstrates that high potency and selectivity can be achieved at the lipoamide-binding site of Mtb Lpd, a site different from the NAD⁺/NADH pocket targeted by previously reported species-selective triazaspirodimethoxybenzoyl inhibitors

Design and implementation of HTS campaign.

<p><b>A</b>) Plate layout for 320-array with four sets of controls (n = 16 for each) in columns 1, 2, 23, and 24. <b>B</b>) Results from Blank Plate Validation using DMSO alone in all 320 assay wells.</p

Screening results.

<p><b>A</b>) Activity of compounds from LISSP4 (grey) and Diversity (black) libraries shown as percent inhibition against PanC_MTB. <b>B</b>) Concentration response curves (CRCs) for two representative hits and nafronyl oxalate. <b>C</b>) Structures of compound <b>1</b> and <b>2</b>.</p

Single-time point fluorescence assay for PanC.

<p><b>A</b>) Reaction schematic. Upper panel shows the reaction catalyzed by PanC and the enzyme cascade that is initiated by the reaction product AMP resulting in βNADH oxidation. Lower panel shows the final βNADH dependent fluorescence generating reaction that is coupled to the PanC-initiated enzyme cascade. <b>B</b>) Low-throughput assay: kinetic reaction monitoring the rate of βNADH oxidation. <b>C</b>) High-throughput assay: fluorescent resorufin signal generated by residual βNADH following the PanC initiated enzyme cascade. <b>D</b>) Fluorescence generated with varied βNADH in solution using the same conditions as in (B).</p

Biochemical characterization of Class 1 compounds.

<p>Michaelis-Menten plots with varied concentrations of pantoate and <b>A</b>) compound <b>1</b> and <b>B</b>) compound <b>2</b>. Graphpad Prism was used to fit the data to nonlinear regressions.</p

The representative aminothiazole 20 possesses bactericidal activity.

<p><i>M</i>. <i>tuberculosis</i> was inoculated to a starting of OD₅₉₀ of 0.1 in medium containing compound 20. CFU/mL was enumerated at indicated time points by serial dilution onto solid medium. The limit of detection was 20. Note that the lines for 0.625, 1.25 and 2.5 overlap.</p

Chemical synthesis of analogs comprised of thiazole core replacement.

<p>Reagents:: (i) 4-(t-butyl)PhCOCl, EtOH, 1 h; (ii) EtOH, reflux, 16 h; (iii) H₂SO₄, r.t., 16 h; (iv) EDC, CH₂Cl₂, r.t., 16 h.</p

SAR at C-4 position of thiazole core.

<p>^aMIC (μM) is the minimum concentration required to inhibit the growth of <i>M</i>. <i>tuberculosis</i> in liquid culture. MICs of active compounds are the average of two independent experiments. ^bToxic concentration (TC₅₀, in μM) is the concentration required to inhibit growth of Vero cells by 50%. Selectivity index (SI) is the ratio of TC₅₀ to MIC. Cpd = compound; Rif = rifampicin; ND = not determined.</p

Chemical synthesis of C-4 ketone and carboxamide analogs.

<p>Reagents: (i) EDC.HCl, HOBt, NCH₃(OCH₃), DIPEA, CH₂Cl₂, 16 h; (ii) CH₃MgBr, THF, -78°C–r.t., 2 h; (iii) 1-adamantanoyl chloride, Et₃N, THF, r.t., 1 h.</p

Chemical synthesis of pyrazole-based analogs.

<p>Reagents: (i) KO^<i>t</i>Bu, BuOH, reflux, 3 h; (ii) RCOCl, THF, r.t., 1 h; (iii) RNCO, THF, 1 h.</p