Enoyl-acyl carrier protein reductase (InhA) is a key enzyme in the type II fatty acid biosynthesis pathway (FASII) in Mtb. InhA was first identified as a clinical target based on the therapeutic potential of isoniazid in TB treatment [235,236]. InhA catalyzes the NADH-dependent reduction in long-chain trans-2-enoyl-acyl carrier proteins (ACPs) [235]. Isoniazid, an essential component of the TB drug regimens, targets the InhA of Mtb, and it is a prodrug activated by KatG—a mycobacterial catalase-peroxidase enzyme [237,238]. The dependency of isoniazid on KatG activation is the main clinical weakness (particularly drug resistance) associated with its application due to mutation occurrences in katG [239]. Thus, identifying inhibitors that directly bind to InhA without the requirement for activation by KatG (direct InhA inhibitors) may represent a valid strategy to overcome isoniazid resistance in TB treatment [240]. GSK-693 and NITD-916 (Figure 11) are novel direct InhA inhibitors of Mtb currently studied as potential substitutes for isoniazid in current TB treatment regimens.

Lead optimization studies by GlaxoSmithKline (GSK) led to the identification of the thiadiazole chiral compound GSK-693 as a novel, selective, and promising lead compound with attractive antitubercular properties that overcome isoniazid resistance. GSK and other research units have carried out high-throughput screening against InhA using a collection of GSK compounds and identified the thiadiazole series to be the most promising compound class [241]. Despite the interesting in vitro antitubercular profile obtained from the initial SAR studies, hits were affected by a number of compound development requirements such as physicochemical properties, drug metabolism, and pharmacokinetics (DMPK) profiles [241].

Further tests confirmed that GSK-693 does not require KatG activation and retain the antitubercular activity against DS, MDR, and XDR clinical isolates [241]. Like other InhA direct inhibitors, thiadiazoles bind to the enzyme–NADH complex, but they do not establish any direct interaction with active site residue Tyr158 [242]. Unlike the isoniazid-NADH adduct, GSK-693 does not cause the flipping of the Phe149 side chain, and there is no interaction with the isonicotinic acid-binding pocket [241]. In addition, compounds inhibiting InhA without requiring activation by KatG could be active under anaerobic conditions where catalase-mediated activation is suppressed by the lack of oxygen.

GSK-693 displayed equal potency (MICs of 0.2 µg/mL) against the Mtb H37Rv inside and outside of macrophages, along with a good CYP 3A4 inhibition profile [241]. The compound demonstrates decreased lipophilicity, enhanced solubility, and reduced metabolic liabilities coupled with simultaneous good oral bioavailability at different doses in acute and chronic mouse models [165]. Lastly, early safety assessments done across different enzymatic assays indicated that GSK-693 did not show any sign of cytotoxicity and no hERG potassium channel inhibition [243], suggesting a low risk for general toxicity and cardiotoxicity observed in isoniazid.

More efforts to overcome isoniazid resistance in Mtb led to the discovery of a new class of direct InhA inhibitors called 4-hydroxy-2-pyridones, identified using phenotypic high-throughput screening [241,244]. The lead compound of the 4-hydroxy-2-pyridones series known as NITD-916 exhibits good activity against isoniazid-resistant MDR-TB clinical isolates and displays good efficacy in vivo in acute and established mouse TB infection models [95]. To inhibit InhA, NITD-916 forms a ternary complex with InhA-NADH, which blocks access to the enoyl substrate-binding site with a consequential reduction in the biosynthesis of mycolic acids and eventually cell death [245,246]. Based on the co-crystal structure, the 4-hydroxy-2-pyridones (including NITD-916) interact with the InhA–NADH complex through hydrogen bonding, pi-stacking, and hydrophobic interactions [247]. However, this mechanism of InhA inhibition was recently challenged by Flint et al. (2020) using killing kinetic studies to suggest that inhibition of InhA is bactericidal against nutrient-starved non-replicating Mtb, regardless of the binding mechanism [248]. This corroborates earlier findings that isoniazid reduces CFU in starved bacteria [249,250].

NITD-916 displays superior MIC against Mtb H37Rv over isoniazid (0.08 µg/mL vs. 0.8 µg/mL respectively) [251]; and also proves to have a lower frequency of resistance (1 × 10⁻⁸) than isoniazid (1 × 10⁻⁵) in vitro [95]. The difference in frequency of resistance is because isoniazid is a prodrug, and mutations in the KatG enzyme occur at a high frequency [251]. To this effect, NITD-916 overcomes a key liability associated with the application of isoniazid in current TB treatment regimens [251]. Thus, portraying potential as a novel TB therapeutic agent that is useful in guiding the development of improved hydroxy-pyridines and other direct InhA inhibitors.

The β-lactams are the most populous class of antibiotics and are the mainstay of most antibacterial drug treatment regimens; however, their efficacy against Mtb has always been limited [72]. Nonetheless, β-lactams generally offer a well-defined safety profile than other second-line alternatives [252]. Therefore, β-lactams: faropenem and ertapenem (Figure 12) briefly discussed below are being studied in phase II clinical trials [37].

Faropenem is an orally bioavailable (72–84%) penem antibiotic that is more resistant to hydrolysis by β-lactamases than cephalosporins and carbapenems [253]. It is structurally similar to the carbapenems; however, this penem ring system is slightly less strained and consequently has improved chemical stability [254]. Additionally, it has been modified to a prodrug ester (faropenem medoxomil), which permits oral administration, both of which are desirable advantages for treating MDR-TB [72]. The drug is already approved for the treatment of respiratory infections in humans, but it also displayed promising bactericidal activity in both active and non-replicating Mtb cells comparable to meropenem [72,253].

Faropenem demonstrates potent biochemical activity independent of clavulanate (β-lactamase inhibitor) and inactivate Mtb L,D-transpeptidase enzymes more efficiently than meropenem [253]. Thus, the drug inhibits the L,D-transpeptidases, which perform the last cross-linking step of peptidoglycan synthesis. The MIC of faropenem against Mtb is 1.3 µg/mL with or without clavulanate, as opposed to an 8-fold increase in MIC for meropenem and many folds for other β-lactams tested in the absence of clavulanate [253]. Whilst using the dehydropeptidase inhibitor, probenecid; Dhar and colleagues demonstrated that Mtb-infected mice had a small but significant reduction in lung burden (7.7 and 7.5 log₁₀ CFU/mouse at the beginning and end of treatment, respectively) after 9 days of treatment with a combination of faropenem/clavulanate/probenecid [253,255].

In summary, faropenem’s stability, oral bioavailability, superior biochemical potency against its molecular target, and proven efficacy in the treatment of infections caused by nontuberculous mycobacteria all justify its potential in the chemotherapy of MDR-TB and XDR-TB [256].

The second β-lactam agent to have shown promising clinical results and favorable pharmacokinetic properties in the treatment of Mtb is ertapenem, a member of the carbapenem class of antibiotics [151]. Amongst all β-lactams, the carbapenems are the most suitable for treating Mtb as they potently target the high molecular weight penicillin-binding proteins and also inactivate the unusual L,D-transpeptidases that form the 3→3 crosslinks found in the unique Mtb cell wall [253,257]. Carbapenems inhibit the peptidase domain of penicillin-binding proteins, leading to autolysis and peptidoglycan weakening of the cell wall [258]. The development of carbapenems for TB treatment has raised considerable interest recently because the presence of clavulanic acid establishes uniform activity against XDR-Mtb and kills both growing and dormant forms of the bacilli [258]. The carbapenems are less favorable substrates for the Mtb β-lactamase BlaC due to rapid acylation and slow deacylation [72]. As such, unlike other β-lactams, they are not rapidly hydrolyzed by BLaC and thus maintain their activity against Mtb [259]. Although encouraging results have recently been obtained using meropenem for the treatment of XDR-TB in humans, the compound is unstable and has poor bioavailability [253]. As a result, frequent intravenous administrations of the drug are required, making its general clinical application limited [165].

Following reconstitution and dilution, Ertapenem degradation is temperature-dependent. Thus, the proposed in-use shelf life for ertapenem is 6 h at room temperature, 24 h at 2–8 °C [151]. Since Mtb has a doubling time of at least 24 h under the best of circumstances [260], microbial killing and inhibition of growth by the most effective of antibiotics, especially by β-lactams, are slow (requiring several days) because the drugs depend on cell wall turnover [151]. Consequently, drugs such as ertapenem, already appearing unstable at 37 °C [259], are likely to be degraded before killing or inhibiting slow-growing bacteria, especially semi-dormant Mtb. However, drug susceptibility tests show that ertapenem is likely to have a good sterilizing effect in TB [151]. This might be attributed to its rapid inactivation of L,D-transpeptidases (which is more rapid than imipenem and meropenem), as well as due to its reversible protein binding nature [257]. To reach maximal bactericidal activity for Gram-positive, Gram-negative, and anaerobic bacterial infections, ertapenem is given intravenously at a dose of 1000 mg once daily [261]. Ertapenem has a long plasma half-life of 4 h, compared to other carbapenems, enabling once-daily dosing, which is an advantage for MDR-TB treatment [262,263]. The AUC_0–24 of ertapenem is 544.9 h·mg/L with a Cmax of 127.5 mg/L in MDR-TB patients [259].

Generally, ertapenem treatment is well tolerated during MDR-TB treatment [259,264]. There are no drug-drug interactions, and the drug is not metabolized by cytochrome P450, nor is it a substrate for p-glycoprotein [265,266]. Renal excretion is the main route of elimination for ertapenem. Therefore, the pharmacokinetics of the drug are altered to a significant extent clinically in patients with severe renal impairment [267]. In such cases, a dose reduction to 500 mg once a day is recommended [266].

Recently, boron-containing compounds known as oxaboroles have been shown to inhibit LeuRS (Leucyl-tRNA synthetase) by the oxaborole tRNA-trapping mechanism [268]. The aminoacyl-tRNA synthetases are a family of essential enzymes that are required for protein synthesis in all cells [269]. The boron atom is an integral part of Mtb LeuRS inhibitors, as it forms a bidentate covalent adduct with the terminal adenosine nucleotide Ade76 of tRNALeu [269]. Consequently, this covalent adduct traps the 3′ end of the tRNALeu in the editing site forming a nonproductive complex, blocking leucylation and bacterial protein synthesis [268].

Another product of potential interest discovered by the GSK group is GSK-3036656, alternatively known as GSK-070 (Figure 13) is currently in a phase II clinical trial for the treatment of TB [270]. GSK-070 is a novel 3-aminomethyl-4-chloro-benzoxaborole with inhibitory activity against the Mtb enzyme LeuRS [271]. Thus, GSK-070 binds to the catalytic site of LeuRS that is responsible for the hydrolysis of incorrectly ligated aminoacylated tRNAs [179]. Notably, the binding is necessitated by the presence of the amino group of the (S)-aminomethyl side chain at C-3 through hydrogen-bonding interactions [271]. Whereas the halogen substituents (Cl or Br) improves the Mtb’s LeuRS and antitubercular activity against Mtb H37Rv as well as selectivity against other bacteria [272]. GSK-070 exhibited promising activity against laboratory strains of Mtb as well as against selected DS-TB, MDR-TB, and XDR-TB clinical isolates [271].

The efficacy of GSK-070 was assessed against replicating and nonreplicating/slow-replicating mycobacteria in mice models of acute and chronic Mtb H37Rv lung infections [273,274]. In the acute model, a maximum difference of 3.6 log₁₀ CFU was obtained in the lungs compared to untreated mice for 8 days; whereas the chronic cohort produced a drop of 2.1 log₁₀ CFU after 2 months of daily oral treatment at maximum doses of 1.1 and 1.3 mg/kg, respectively [273]. A recent phase 1 clinical study evaluating the safety, tolerability, and pharmacokinetics of single and repeat oral doses of GSK-070 met all primary endpoints and showed no serious adverse events [179,275]. The study determined potent inhibition of Mtb LeuRS (IC₅₀ = 0.20 µM) and in vitro antitubercular activity (Mtb H37Rv MIC = 0.08 µg/mL) [272]. GSK-070 showed a dose-proportional increase following single-dose administration after dosing for 14 days. Additionally, the drug showed 2–3 fold increase in Cmax and AUC accumulations with repeated administration after the dosing period that was unaltered in the presence of food [271]. Furthermore, GSK-070 is a low-hepatic-clearance compound with high solubility and moderate passive permeability and is not a p-glycoprotein substrate; hence it undergoes rapid absorption and exhibited very high oral bioavailability in the nonclinical species (mouse, rat, and dog) [271]. Finally, in vitro data suggest that GSK-070 has a low plasma protein binding in the nonclinical species (mice, rats, and dogs, and humans) [271]. The main route of elimination for GSK-070 is in the urine.

Table 1 below summarizes the mutation event(s) associated with resistance against novel anti-TB drugs and drug candidates.