N-Myristoyltransferase (NMT) represents
a promising
drug target for human African trypanosomiasis (HAT), which is caused
by the parasitic protozoa Trypanosoma brucei. We
report the optimization of a high throughput screening hit (1) to give a lead molecule DDD85646 (63), which
has potent activity against the enzyme (IC50 = 2 nM) and T. brucei (EC50 = 2 nM) in culture. The compound
has good oral pharmacokinetics and cures rodent models of peripheral
HAT infection. This compound provides an excellent tool for validation
of T. brucei NMT as a drug target for HAT as well
as a valuable lead for further optimization.
e We report here a series of five chemically diverse scaffolds that have in vitro activities on replicating and hypoxic nonreplicating bacilli by targeting the respiratory bc 1 complex in Mycobacterium tuberculosis in a strain-dependent manner. Deletion of the cytochrome bd oxidase generated a hypersusceptible mutant in which resistance was acquired by a mutation in qcrB. These results highlight the promiscuity of the bc 1 complex and the risk of targeting energy metabolism with new drugs.
A potent, non-cytotoxic indazole sulfonamide was identified by
high-throughput screening of >100,000 synthetic compounds for activity
against Mycobacterium tuberculosis (Mtb). This
non-cytotoxic compound did not directly inhibit cell wall biogenesis but
triggered a slow lysis of Mtb cells as measured by release of
intracellular green fluorescent protein (GFP). Isolation of resistant mutants
followed by whole-genome sequencing showed an unusual gene amplification of a 40
gene region spanning Rv3371 to Rv3411c and in
one case a potential promoter mutation upstream of guaB2
(Rv3411c) encoding inosine monophosphate dehydrogenase
(IMPDH). Subsequent biochemical validation confirmed direct inhibition of IMPDH
by an uncompetitive mode of inhibition and growth inhibition could be rescued by
supplementation with guanine, a bypass mechanism for the IMPDH pathway. Beads
containing immobilized indazole sulfonamides specifically interacted with IMPDH
in cell lysates. X-ray crystallography of the IMPDH-IMP-inhibitor complex
revealed that the primary interactions of these compounds with IMPDH were direct
pi-pi interactions with the IMP substrate. Advanced lead compounds in this
series with acceptable pharmacokinetic properties failed to show efficacy in
acute or chronic murine models of tuberculosis (TB). Time-kill experiments
in vitro suggest that sustained exposure to drug
concentrations above MIC for 24 hours were required for a cidal effect, levels
that have been difficult to achieve in vivo. Direct measurement
of guanine levels in resected lung tissue from tuberculosis infected animals and
patients revealed 0.5–2 mM concentrations in caseum and normal lung
tissue. The high lesional levels of guanine and the slow lytic, growth-rate
dependent, effect of IMPDH inhibition pose challenges to developing drugs
against this target for use in treating TB.
Mycobacterium tuberculosis (MTb) possesses
two nonproton pumping type II NADH dehydrogenase (NDH-2)
enzymes which are predicted to be jointly essential for respiratory
metabolism. Furthermore, the structure of a closely related bacterial
NDH-2 has been reported recently, allowing for the structure-based
design of small-molecule inhibitors. Herein, we disclose MTb whole-cell structure–activity relationships (SARs) for a series of 2-mercapto-quinazolinones which target the ndh encoded NDH-2 with nanomolar potencies. The compounds were inactivated by glutathione-dependent adduct formation as well as quinazolinone oxidation in microsomes. Pharmacokinetic studies demonstrated modest bioavailability and compound exposures. Resistance to the compounds in MTb was conferred by promoter mutations in the alternative nonessential NDH-2 encoded by ndhA in MTb. Bioenergetic analyses revealed a decrease in oxygen consumption rates in response to inhibitor in cells in which membrane potential was uncoupled from ATP production, while inverted membrane vesicles showed mercapto-quinazolinone-dependent inhibition of ATP production when NADH was the electron donor to the respiratory chain. Enzyme kinetic studies further demonstrated noncompetitive inhibition, suggesting binding of this scaffold to an allosteric site. In summary, while the initial MTb SAR showed limited improvement in potency, these results, combined with structural information on the bacterial protein, will aid in the future discovery of new and improved NDH-2 inhibitors.
Coenzyme A (CoA) is a fundamental co-factor for all life, involved in numerous metabolic pathways and cellular processes, and its biosynthetic pathway has raised substantial interest as a drug target against multiple pathogens including Mycobacterium tuberculosis. The biosynthesis of CoA is performed in five steps, with the second and third steps being catalysed in the vast majority of prokaryotes, including M. tuberculosis, by a single bifunctional protein, CoaBC. Depletion of CoaBC was found to be bactericidal in M. tuberculosis. Here we report the first structure of a full-length CoaBC, from the model organism Mycobacterium smegmatis, describe how it is organised as a dodecamer and regulated by CoA thioesters. A high-throughput biochemical screen focusing on CoaB identified two inhibitors with different chemical scaffolds. Hit expansion led to the discovery of potent and selective inhibitors of M. tuberculosis CoaB, which we show to bind to a cryptic allosteric site within CoaB.
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