A Preclinical Candidate Targeting Mycobacterium tuberculosis KasA.

Published Mycobacterium tuberculosis ß-ketoacyl-ACP synthase KasA inhibitors lack sufficient potency and/or pharmacokinetic properties. A structure-based approach was used to optimize existing KasA inhibitor DG167. This afforded indazole JSF-3285 with a 30-fold increase in mouse plasma exposure. Biochemical, genetic, and X-ray studies confirmed JSF-3285 targets KasA. JSF-3285 offers substantial activity in an acute mouse model of infection and in the corresponding chronic infection model, with efficacious reductions in colony-forming units at doses as low as 5 mg/kg once daily orally and improvement of the efficacy of front-line drugs isoniazid or rifampicin. JSF-3285 is a promising preclinical candidate for tuberculosis.

Phase variation in Mycobacterium tuberculosis glpK produces transiently heritable drug tolerance.

The length and complexity of tuberculosis (TB) therapy, as well as the propensity of Mycobacterium tuberculosis to develop drug resistance, are major barriers to global TB control efforts. M. tuberculosis is known to have the ability to enter into a drug-tolerant state, which may explain many of these impediments to TB treatment. We have identified a mechanism of genetically encoded but rapidly reversible drug tolerance in M. tuberculosis caused by transient frameshift mutations in a homopolymeric tract (HT) of 7 cytosines (7C) in the glpK gene. Inactivating frameshift mutations associated with the 7C HT in glpK produce small colonies that exhibit heritable multidrug increases in minimal inhibitory concentrations and decreases in drug-dependent killing; however, reversion back to a fully drug-susceptible large-colony phenotype occurs rapidly through the introduction of additional insertions or deletions in the same glpK HT region. These reversible frameshift mutations in the 7C HT of M. tuberculosis glpK occur in clinical isolates, accumulate in M. tuberculosis-infected mice with further accumulation during drug treatment, and exhibit a reversible transcriptional profile including induction of dosR and sigH and repression of kstR regulons, similar to that observed in other in vitro models of M. tuberculosis tolerance. These results suggest that GlpK phase variation may contribute to drug tolerance, treatment failure, and relapse in human TB. Drugs effective against phase-variant M. tuberculosis may hasten TB treatment and improve cure rates.

Effect of C-2 substitution on the stability of non-traditional cephalosporins in mouse plasma.

A systematic study of the stability of a set of cephalosporins in mouse plasma reveals that cephalosporins lacking an acidic moiety at C-2 may be vulnerable to ß-lactam cleavage in mouse plasma.

Fluoroquinolone Efficacy against Tuberculosis Is Driven by Penetration into Lesions and Activity against Resident Bacterial Populations.

Fluoroquinolones represent the pillar of multidrug-resistant tuberculosis (MDR-TB) treatment, with moxifloxacin, levofloxacin, or gatifloxacin being prescribed to MDR-TB patients. Recently, several clinical trials of "universal" drug regimens, aiming to treat drug-susceptible and drug-resistant TB, have included a fluoroquinolone. In the absence of clinical data comparing their side-by-side efficacies in controlled MDR-TB trials, a pharmacological rationale is needed to guide the selection of the most efficacious fluoroquinolone. The present studies were designed to test the hypothesis that fluoroquinolone concentrations (pharmacokinetics) and activity (pharmacodynamics) at the site of infection are better predictors of efficacy than the plasma concentrations and potency measured in standard growth inhibition assays and are better suited to determinations of whether one of the fluoroquinolones outperforms the others in rabbits with active TB. We first measured the penetration of these fluoroquinolones in lung lesion compartments, and their potency against bacterial populations that reside in each compartment, to compute lesion-centric pharmacokinetic-pharmacodynamic (PK/PD) parameters. PK modeling methods were used to quantify drug penetration from plasma to tissues at human-equivalent doses. On the basis of these metrics, moxifloxacin emerged with a clear advantage, whereas plasma-based PK/PD favored levofloxacin (the ranges of the plasma AUC/MIC ratio [i.e., the area under the concentration-time curve over 24 h in the steady state divided by the MIC] are 46 to 86 for moxifloxacin and 74 to 258 for levofloxacin). A comparative efficacy trial in the rabbit model of active TB demonstrated the superiority of moxifloxacin in reducing bacterial burden at the lesion level and in sterilizing cellular and necrotic lesions. Collectively, these results show that PK/PD data obtained at the site of infection represent an adequate predictor of drug efficacy against TB and constitute the baseline required to explore synergies, antagonism, and drug-drug interactions in fluoroquinolone-containing regimens.

Synergistic Lethality of a Binary Inhibitor of Mycobacterium tuberculosis KasA.

We report GSK3011724A (DG167) as a binary inhibitor of ß-ketoacyl-ACP synthase (KasA) in Mycobacterium tuberculosis Genetic and biochemical studies established KasA as the primary target. The X-ray crystal structure of the KasA-DG167 complex refined to 2.0-Å resolution revealed two interacting DG167 molecules occupying nonidentical sites in the substrate-binding channel of KasA. The binding affinities of KasA to DG167 and its analog, 5g, which binds only once in the substrate-binding channel, were determined, along with the KasA-5g X-ray crystal structure. DG167 strongly augmented the in vitro activity of isoniazid (INH), leading to synergistic lethality, and also synergized in an acute mouse model of M. tuberculosis infection. Synergistic lethality correlated with a unique transcriptional signature, including upregulation of oxidoreductases and downregulation of molecular chaperones. The lead structure-activity relationships (SAR), pharmacokinetic profile, and detailed interactions with the KasA protein that we describe may be applied to evolve a next-generation therapeutic strategy for tuberculosis (TB).IMPORTANCE Cell wall biosynthesis inhibitors have proven highly effective for treating tuberculosis (TB). We discovered and validated members of the indazole sulfonamide class of small molecules as inhibitors of Mycobacterium tuberculosis KasA-a key component for biosynthesis of the mycolic acid layer of the bacterium's cell wall and the same pathway as that inhibited by the first-line antitubercular drug isoniazid (INH). One lead compound, DG167, demonstrated synergistic lethality in combination with INH and a transcriptional pattern consistent with bactericidality and loss of persisters. Our results also detail a novel dual-binding mechanism for this compound as well as substantial structure-activity relationships (SAR) that may help in lead optimization activities. Together, these results suggest that KasA inhibition, specifically, that shown by the DG167 series, may be developed into a potent therapy that can synergize with existing antituberculars.

Storage lipid studies in tuberculosis reveal that foam cell biogenesis is disease-specific.

Foam cells are lipid-laden macrophages that contribute to the inflammation and tissue damage associated with many chronic inflammatory disorders. Although foam cell biogenesis has been extensively studied in atherosclerosis, how these cells form during a chronic infectious disease such as tuberculosis is unknown. Here we report that, unlike the cholesterol-laden cells of atherosclerosis, foam cells in tuberculous lung lesions accumulate triglycerides. Consequently, the biogenesis of foam cells varies with the underlying disease. In vitro mechanistic studies showed that triglyceride accumulation in human macrophages infected with Mycobacterium tuberculosis is mediated by TNF receptor signaling through downstream activation of the caspase cascade and the mammalian target of rapamycin complex 1 (mTORC1). These features are distinct from the known biogenesis of atherogenic foam cells and establish a new paradigm for non-atherogenic foam cell formation. Moreover, they reveal novel targets for disease-specific pharmacological interventions against maladaptive macrophage responses.