Comparative Analysis of Pharmacodynamics in the C3HeB/FeJ Mouse Tuberculosis Model for DprE1 Inhibitors TBA-7371, PBTZ169, and OPC-167832.

Multiple drug discovery initiatives for tuberculosis are currently ongoing to identify and develop new potent drugs with novel targets in order to shorten treatment duration. One of the drug classes with a new mode of action is DprE1 inhibitors targeting an essential process in cell wall synthesis of Mycobacterium tuberculosis. In this investigation, three DprE1 inhibitors currently in clinical trials, TBA-7371, PBTZ169, and OPC-167832, were evaluated side-by-side as single agents in the C3HeB/FeJ mouse model presenting with caseous necrotic pulmonary lesions upon tuberculosis infection. The goal was to confirm the efficacy of the DprE1 inhibitors in a mouse tuberculosis model with advanced pulmonary pathology and perform comprehensive analysis of plasma, lung, and lesion-centric drug levels to establish pharmacokinetic-pharmacodynamic (PK-PD) parameters predicting efficacy at the site of infection. Results showed significant efficacy for all three DprE1 inhibitors in the C3HeB/FeJ mouse model after 2 months of treatment. Superior efficacy was observed for OPC-167832 even at low-dose levels, which can be attributed to its low MIC, favorable distribution, and sustained retention above the MIC throughout the dosing interval in caseous necrotic lesions, where the majority of bacteria reside in C3HeB/FeJ mice. These results support further progression of the three drug candidates through clinical development for tuberculosis treatment.

Intracellular and in vivo evaluation of imidazo[2,1-b]thiazole-5-carboxamide anti-tuberculosis compounds.

The imidazo[2,1-b]thiazole-5-carboxamides (ITAs) are a promising class of anti-tuberculosis agents shown to have potent activity in vitro and to target QcrB, a key component of the mycobacterial cytochrome bcc-aa3 super complex critical for the electron transport chain. Herein we report the intracellular macrophage potency of nine diverse ITA analogs with MIC values ranging from 0.0625-2.5 µM and mono-drug resistant potency ranging from 0.0017 to 7 µM. The in vitro ADME properties (protein binding, CaCo-2, human microsomal stability and CYP450 inhibition) were determined for an outstanding compound of the series, ND-11543. ND-11543 was tolerable at >500 mg/kg in mice and at a dose of 200 mg/kg displayed good drug exposure in mice with an AUC(0-24h) >11,700 ng·hr/mL and a >24 hr half-life. Consistent with the phenotype observed with other QcrB inhibitors, compound ND-11543 showed efficacy in a chronic murine TB infection model when dosed at 200 mg/kg for 4 weeks. The efficacy was not dependent upon exposure, as pre-treatment with a known CYP450-inhibitor did not substantially improve efficacy. The ITAs are an interesting scaffold for the development of new anti-TB drugs especially in combination therapy based on their favorable properties and novel mechanism of action.

Pyrazolo[1,5- a]pyridine Inhibitor of the Respiratory Cytochrome bcc Complex for the Treatment of Drug-Resistant Tuberculosis.

Respiration is a promising target for the development of new antimycobacterial agents, with a growing number of compounds in clinical development entering this target space. However, more candidate inhibitors are needed to expand the therapeutic options available for drug-resistant Mycobacterium tuberculosis infection. Here, we characterize a putative respiratory complex III (QcrB) inhibitor, TB47: a pyrazolo[1,5- a]pyridine-3-carboxamide. TB47 is active (MIC between 0.016 and 0.500 µg/mL) against a panel of 56 M. tuberculosis clinical isolates, including 37 multi-drug-resistant and two extensively drug-resistant strains. Pharmacokinetic and toxicity studies showed promising profiles, including negligible CYP450 interactions, cytotoxicity, and hERG channel inhibition. Consistent with other reported QcrB inhibitors, TB47 inhibits oxygen consumption only when the alternative oxidase, cytochrome bd, is deleted. A point mutation in the qcrB cd2-loop (H190Y, M. smegmatis numbering) rescues the inhibitory effects of TB47. Metabolomic profiling of TB47-treated M. tuberculosis H37Rv cultures revealed accumulation of steps in the TCA cycle and pentose phosphate pathway that are linked to reducing equivalents, suggesting that TB47 causes metabolic redox stress. In mouse infection models, a TB47 monotherapy was not bactericidal. However, TB47 was strongly synergistic with pyrazinamide and rifampicin, suggesting a promising role in combination therapies. We propose that TB47 is an effective lead compound for the development of novel tuberculosis chemotherapies.

Discovery of a cofactor-independent inhibitor of Mycobacterium tuberculosis InhA.

New antitubercular agents are needed to combat the spread of multidrug- and extensively drug-resistant strains of Mycobacterium tuberculosis. The frontline antitubercular drug isoniazid (INH) targets the mycobacterial enoyl-ACP reductase, InhA. Resistance to INH is predominantly through mutations affecting the prodrug-activating enzyme KatG. Here, we report the identification of the diazaborines as a new class of direct InhA inhibitors. The lead compound, AN12855, exhibited in vitro bactericidal activity against replicating bacteria and was active against several drug-resistant clinical isolates. Biophysical and structural investigations revealed that AN12855 binds to and inhibits the substrate-binding site of InhA in a cofactor-independent manner. AN12855 showed good drug exposure after i.v. and oral delivery, with 53% oral bioavailability. Delivered orally, AN12855 exhibited dose-dependent efficacy in both an acute and chronic murine model of tuberculosis infection that was comparable with INH. Combined, AN12855 is a promising candidate for the development of new antitubercular agents.

Development of a Novel Lead that Targets M. tuberculosis Polyketide Synthase 13.

Widespread resistance to first-line TB drugs is a major problem that will likely only be resolved through the development of new drugs with novel mechanisms of action. We have used structure-guided methods to develop a lead molecule that targets the thioesterase activity of polyketide synthase Pks13, an essential enzyme that forms mycolic acids, required for the cell wall of Mycobacterium tuberculosis. Our lead, TAM16, is a benzofuran class inhibitor of Pks13 with highly potent in vitro bactericidal activity against drug-susceptible and drug-resistant clinical isolates of M. tuberculosis. In multiple mouse models of TB infection, TAM16 showed in vivo efficacy equal to the first-line TB drug isoniazid, both as a monotherapy and in combination therapy with rifampicin. TAM16 has excellent pharmacological and safety profiles, and the frequency of resistance for TAM16 is ∼100-fold lower than INH, suggesting that it can be developed as a new antitubercular aimed at the acute infection. PAPERCLIP.

Spectinamides are effective partner agents for the treatment of tuberculosis in multiple mouse infection models.

Objectives: New drug regimens employing combinations of existing and experimental antimicrobial agents are needed to shorten treatment of tuberculosis (TB) in humans. The spectinamides are narrow-spectrum semisynthetic analogues of spectinomycin, modified to avoid intrinsic efflux by Mycobacterium tuberculosis . Spectinamides, including lead 1599, have been previously shown to exhibit a promising therapeutic profile in mice as single agents. Here we explore the in vivo activity of lead spectinamides when combined with other agents. Methods: The efficacy of 1599 or 1810 was tested in combination in three increasingly advanced TB mouse models. Mice were infected by aerosol and allowed to establish acute or chronic infection, followed by treatment (≤4 weeks) with the spectinamides alone or in two- and three-drug combination regimens with existing and novel therapeutic agents. Bacteria were enumerated from lungs by plating for cfu. Results: Herein we show the following: (i) 1599 exhibits additive or synergistic activity with most of the first-line agents; (ii) 1599 in combination with rifampicin and pyrazinamide or with bedaquiline and pyrazinamide promotes significantly improved efficacy in the high-dose aerosol model; (iii) 1599 enhances efficacy of rifampicin or pyrazinamide in chronically infected BALB/c mice; and (iv) 1599 is synergistic when administered in combination with rifampicin and pyrazinamide in the C3HeB/FeJ mouse model showing caseous necrotic pulmonary lesions. Conclusions: Spectinamides were effective partner agents for multiple anti-TB agents including bedaquiline, rifampicin and pyrazinamide. None of these in vivo synergistic interactions was predicted from in vitro MIC chequerboard assays. These data support further development of the spectinamides as combination partners with existing and experimental anti-TB agents.

Origins of large rate enhancements in the Nazarov cyclization catalyzed by supramolecular encapsulation.

The self-assembled supramolecular host [Ga4L6](12-) (1; L=N,N-bis(2,3-dihydroxybenzoyl)-1,5-diaminonaphthalene) catalyzes the Nazarov cyclization of 1,3-pentadienols with extremely high levels of efficiency. The catalyzed reaction proceeds at a rate over a million times faster than that of the background reaction, an increase comparable to those observed in some enzymatic systems. A detailed study was conducted to elucidate the reaction mechanism of both the catalyzed and uncatalyzed Nazarov cyclization of pentadienols. Kinetic analysis and (18)O-exchange experiments implicate a mechanism, in which encapsulation, protonation, and water loss from substrate are reversible, followed by irreversible electrocyclization. Although electrocyclization is rate determining in the uncatalyzed reaction, the barrier for water loss and for electrocyclization are nearly equal in the assembly-catalyzed reaction. Analysis of the energetics of the catalyzed and uncatalyzed reaction revealed that transition-state stabilization contributes significantly to the dramatically enhanced rate of the catalyzed reaction.

Enzymelike catalysis of the Nazarov cyclization by supramolecular encapsulation.

The water-soluble, self-assembled, tetrahedral assembly K(12)Ga(4)L(6) (L = 1,5-biscatecholamidenaphthalene) catalyzes the Nazarov cyclization of 1,3-pentadienols with extremely high levels of efficiency. The catalyzed reaction proceeds over a million times faster than the background reaction, an increase comparable to those observed in some enzymatic systems. This catalysis operates under aqueous conditions at mild temperatures and pH, and the reaction is halted by the addition of an appropriate inhibitor. This unprecedented rate enhancement is attributed to both the stabilization of protonated reaction intermediates and the effect of constrictive binding on the bound guest.

Aza Cope rearrangement of propargyl enammonium cations catalyzed by a self-assembled "nanozyme".

The tetrahedral assembly [Ga4L6](12-) [L = N,N-bis(2,3-dihydroxybenzoyl)-1,5-diaminonaphthalene) encapsulates a variety of cations, including propargyl enammonium cations capable of undergoing the aza Cope rearrangement. For propargyl enammonium substrates that are encapsulated in the [Ga4L6](12-) assembly, rate accelerations by factors of up to 184 compared with the background reaction rate were observed. After rearrangement, the product iminium ion is released into solution and hydrolyzed, allowing for catalytic turnover. The activation parameters for the catalyzed and uncatalyzed reaction were determined, revealing that a decrease in the entropy of activation is responsible for the observed rate enhancements. The catalyzed reaction exhibits saturation kinetics: the rate data obeyed the Michaelis-Menten model of enzyme kinetics, and competitive inhibition using a nonreactive guest was demonstrated.