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.

Adjunctive Host-Directed Therapy With Statins Improves Tuberculosis-Related Outcomes in Mice.

BACKGROUND: Tuberculosis (TB) treatment is lengthy and complicated and patients often develop chronic lung disease. Recent attention has focused on host-directed therapies aimed at optimizing immune responses to Mycobacterium tuberculosis (Mtb), as adjunctive treatment given with antitubercular drugs. In addition to their cholesterol-lowering properties, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) have broad anti-inflammatory and immunomodulatory activities. METHODS: In the current study, we screened 8 commercially available statins for cytotoxic effect, anti-TB activity, synergy with first-line drugs in macrophages, pharmacokinetics and adjunctive bactericidal activity, and, in 2 different mouse models, as adjunctive therapy to first-line TB drugs. RESULTS: Pravastatin showed the least toxicity in THP-1 and Vero cells. At nontoxic doses, atorvastatin and mevastatin were unable to inhibit Mtb growth in THP-1 cells. Simvastatin, fluvastatin, and pravastatin showed the most favorable therapeutic index and enhanced the antitubercular activity of the first-line drugs isoniazid, rifampin, and pyrazinamide in THP-1 cells. Pravastatin modulated phagosomal maturation characteristics in macrophages, phenocopying macrophage activation, and exhibited potent adjunctive activity in the standard mouse model of TB chemotherapy and in a mouse model of human-like necrotic TB lung granulomas. CONCLUSIONS: These data provide compelling evidence for clinical evaluation of pravastatin as adjunctive, host-directed therapy for TB.

The anti-tubercular activity of simvastatin is mediated by cholesterol-driven autophagy via the AMPK-mTORC1-TFEB axis.

The rise of drug-resistant tuberculosis poses a major risk to public health. Statins, which inhibit both cholesterol biosynthesis and protein prenylation branches of the mevalonate pathway, increase anti-tubercular antibiotic efficacy in animal models. However, the underlying molecular mechanisms are unknown. In this study, we used an in vitro macrophage infection model to investigate simvastatin's anti-tubercular activity by systematically inhibiting each branch of the mevalonate pathway and evaluating the effects of the branch-specific inhibitors on mycobacterial growth. The anti-tubercular activity of simvastatin used at clinically relevant doses specifically targeted the cholesterol biosynthetic branch rather than the prenylation branches of the mevalonate pathway. Using Western blot analysis and AMP/ATP measurements, we found that simvastatin treatment blocked activation of mechanistic target of rapamycin complex 1 (mTORC1), activated AMP-activated protein kinase (AMPK) through increased intracellular AMP:ATP ratios, and favored nuclear translocation of transcription factor EB (TFEB). These mechanisms all induce autophagy, which is anti-mycobacterial. The biological effects of simvastatin on the AMPK-mTORC1-TFEB-autophagy axis were reversed by adding exogenous cholesterol to the cells. Our data demonstrate that the anti-tubercular activity of simvastatin requires inhibiting cholesterol biosynthesis, reveal novel links between cholesterol homeostasis, the AMPK-mTORC1-TFEB axis, and Mycobacterium tuberculosis infection control, and uncover new anti-tubercular therapy targets.

Remembering the Host in Tuberculosis Drug Development.

New therapeutics to augment current approaches and shorten treatment duration are of critical importance for combating tuberculosis (TB), especially those with novel mechanisms of action to counter the emergence of drug-resistant TB. Host-directed therapy (HDT) offers a novel strategy with mechanisms that include activating immune defense mechanisms or ameliorating tissue damage. These and related concepts will be discussed along with issues that emerged from the workshop organized by the Stop TB Working Group on New Drugs, held at the Gordon Research Conference for Tuberculosis Drug Development in Lucca, Italy in June 2017, titled "Strategic Discussion on Repurposing Drugs & Host Directed Therapies for TB." In this review, we will highlight recent data regarding drugs, pathways, and concepts that are important for successful development of HDTs for TB.

Metformin Adjunctive Therapy Does Not Improve the Sterilizing Activity of the First-Line Antitubercular Regimen in Mice.

Preliminary preclinical and observational studies suggest the potential utility of metformin as an adjunctive, host-directed agent for treatment of tuberculosis (TB). In this study, we sought to investigate the bactericidal and sterilizing activities of human-like exposures of metformin when given in combination with the first-line regimen against chronic tuberculosis in BALB/c mice. Mice receiving metformin adjunctive therapy had similar lung bacillary burdens with control mice during treatment, and the proportion of mice with microbiological relapse was similar between the two groups.

Stringent Response Factors PPX1 and PPK2 Play an Important Role in Mycobacterium tuberculosis Metabolism, Biofilm Formation, and Sensitivity to Isoniazid In Vivo.

Mycobacterium tuberculosis remains a global health threat largely due to the lengthy duration of curative antibiotic treatment, contributing to medical nonadherence and the emergence of drug resistance. This prolonged therapy is likely due to the presence of M. tuberculosis persisters, which exhibit antibiotic tolerance. Inorganic polyphosphate [poly(P)] is a key regulatory molecule in the M. tuberculosis stringent response mediating antibiotic tolerance. The polyphosphate kinase PPK1 is responsible for poly(P) synthesis in M. tuberculosis, while the exopolyphosphatases PPX1 and PPX2 and the GTP synthase PPK2 are responsible for poly(P) hydrolysis. In the present study, we show by liquid chromatography-tandem mass spectrometry that poly(P)-accumulating M. tuberculosis mutant strains deficient in ppx1 or ppk2 had significantly lower intracellular levels of glycerol-3-phosphate (G3P) and 1-deoxy-xylulose-5-phosphate. Real-time PCR revealed decreased expression of genes in the G3P synthesis pathway in each mutant. The ppx1-deficient mutant also showed a significant accumulation of metabolites in the tricarboxylic acid cycle, as well as altered arginine and NADH metabolism. Each poly(P)-accumulating strain showed defective biofilm formation, while deficiency of ppk2 was associated with increased sensitivity to plumbagin and meropenem and deficiency of ppx1 led to enhanced susceptibility to clofazimine. A DNA vaccine expressing ppx1 and ppk2, together with two other members of the M. tuberculosis stringent response, M. tuberculosis rel and sigE, did not show protective activity against aerosol challenge with M. tuberculosis, but vaccine-induced immunity enhanced the killing activity of isoniazid in a murine model of chronic tuberculosis. In summary, poly(P)-regulating factors of the M. tuberculosis stringent response play an important role in M. tuberculosis metabolism, biofilm formation, and antibiotic sensitivity in vivo.

Statin adjunctive therapy shortens the duration of TB treatment in mice.

BACKGROUND: The repurposing of existing agents may accelerate TB drug development. Recently, we reported that the lipid-lowering drug simvastatin, when added to the first-line antitubercular regimen, reduces the lung bacillary burden in chronically infected mice. OBJECTIVES: We investigated whether the addition of simvastatin to the first-line regimen (isoniazid/rifampicin/pyrazinamide) shortens the duration of curative TB treatment in mice. METHODS: Mycobacterium tuberculosis-infected THP-1 cells were exposed to simvastatin to determine the effect of statins on the activity of first-line anti-TB drug activity and intracellular rifampicin concentration. Single-dose and steady-state pharmacokinetic studies guided optimized simvastatin dosing in vivo. BALB/c mice were aerosol-infected with M. tuberculosis H37Rv and drug treatment was initiated 6 weeks post-infection. Separate groups of mice received standard TB treatment with or without simvastatin. Relapse rates were assessed 3 months after discontinuation of each treatment regimen. MALDI-MS imaging was used to image the cholesterol content of mouse lung lesions. RESULTS: Simvastatin significantly enhanced the bactericidal activity of first-line drugs against intracellular M. tuberculosis without altering intracellular rifampicin concentrations. Adjunctive treatment with 60 mg/kg simvastatin shortened the time required to achieve culture-negative lungs from 4.5 to 3.5 months. Following 2.5, 3.5 and 4.5 months of treatment, relapse rates were 100%, 50% and 0%, respectively, in the control group and 50% (P = 0.03), 20% and 0%, respectively, in the statin group. Simvastatin did not alter plasma or lung lesion cholesterol levels. CONCLUSIONS: Statins are attractive candidates for host-directed, adjunctive TB therapy. Further preclinical studies are needed to define the optimal statin and dosing.

Characterization of a novel necrotic granuloma model of latent tuberculosis infection and reactivation in mice.

We sought to develop and characterize a novel paucibacillary model in mice, which develops necrotic lung granulomas after infection with Mycobacterium tuberculosis. Six weeks after aerosol immunization with recombinant Mycobacterium bovis bacillus Calmette-Guerin overexpressing the 30-kDa antigen, C3HeB/FeJ mice were aerosol infected with M. tuberculosis H37Rv. Six weeks later, mice were treated with one of three standard regimens for latent tuberculosis infection or tumor necrosis factor (TNF)-neutralizing antibody. Mouse lungs were analyzed by histological features, positron emission tomography/computed tomography, whole-genome microarrays, and RT-PCR. Lungs and sera were studied by multiplex enzyme-linked immunosorbent assays. Paucibacillary infection was established, recapitulating the sterilizing activities of human latent tuberculosis infection regimens. TNF neutralization led to increased lung bacillary load, disrupted granuloma architecture with expanded necrotic foci and reduced tissue hypoxia, and accelerated animal mortality. TNF-neutralized mouse lungs and sera showed significant up-regulation of interferon γ, IL-1ß, IL-6, IL-10, chemokine ligands 2 and 3, and matrix metalloproteinase genes. Clinical and microbiological reactivation of paucibacillary infection by TNF neutralization was associated with reduced hypoxia in lung granulomas and induction of matrix metalloproteinases and proinflammatory cytokines. This model may be useful for screening the sterilizing activity of novel anti-tuberculosis drugs, and identifying mycobacterial regulatory and metabolic pathways required for bacillary growth restriction and reactivation.

Sterilizing activity of thioridazine in combination with the first-line regimen against acute murine tuberculosis.

We recently reported that in lung tissue, thioridazine accumulates at high concentrations relative to serum levels, displaying modest synergy with isoniazid and reducing the emergence of isoniazid-resistant mutants in mouse lungs. In this study, we sought to investigate the sterilizing activity of human-equivalent doses of thioridazine when given in combination with the "Denver regimen" against acute murine tuberculosis. We found a trend toward a positive impact of thioridazine on the bacterial clearance and lowering relapse rates of the combined standard TB chemotherapy.

Reduced emergence of isoniazid resistance with concurrent use of thioridazine against acute murine tuberculosis.

The repurposing of existing drugs is being pursued as a means by which to accelerate the development of novel regimens for the treatment of drug-susceptible and drug-resistant tuberculosis (TB). In the current study, we assessed the activity of the antipsychotic drug thioridazine (TRZ) in combination with the standard regimen in a well-validated murine TB model. Single-dose and steady-state pharmacokinetic studies were performed in BALB/c mice to establish human-equivalent doses of TRZ. To determine the bactericidal activity of TRZ against TB in BALB/c mice, three separate studies were performed, including a dose-ranging study of TRZ monotherapy and efficacy studies of human-equivalent doses of TRZ with and without isoniazid (INH) or rifampin (RIF). Therapeutic efficacy was assessed by the change in mycobacterial load in the lung. The human-equivalent dose of thioridazine was determined to be 25 mg/kg of body weight, which was well tolerated in mice. TRZ was found to accumulate at high concentrations in lung tissue relative to serum levels. We observed modest synergy during coadministration of TRZ with INH, and the addition of TRZ reduced the emergence of INH-resistant mutants in mouse lungs. In conclusion, this study further illustrates the opportunity to reevaluate the contribution of TRZ to the sterilizing activity of combination regimens to prevent the emergence of drug-resistant M. tuberculosis.

Preliminary pharmacokinetic study of repeated doses of rifampin and rifapentine in guinea pigs.

Substitution of rifapentine (RFP) for rifampin (RIF) in the standard antituberculous regimen reduces the time required to cure chronic tuberculosis (TB) infection in mice, but not in guinea pigs. In order to gain insight into these discrepant findings, we conducted a steady-state pharmacokinetic (PK) study in healthy guinea pigs to study the metabolism and autoinduction of RIF and RFP. Both RFP and RIF 25-desacetyl metabolites (desRFP and desRIF, respectively), were detected at low concentrations in the serum of guinea pigs. The metabolite concentrations in guinea pigs are much lower than those seen in humans at steady state.

Potent rifamycin-sparing regimen cures guinea pig tuberculosis as rapidly as the standard regimen.

Strategies involving new drug combinations, as well as new uses of existing drugs, are urgently needed to reduce the time required to cure patients with drug-sensitive or multidrug-resistant (MDR) tuberculosis (TB). We compared the sterilizing activity of the standard first-line antitubercular regimen, rifampin-isoniazid-pyrazinamide (RHZ), with that of the novel regimen PA-824-moxifloxacin-pyrazinamide (PaMZ), which is currently being studied in clinical trials (NCT01498419), in the guinea pig model of chronic TB infection, in which animals develop necrotic granulomas histologically resembling their human counterparts. Guinea pigs were aerosol infected with ~2 log10 bacilli of wild-type Mycobacterium tuberculosis H37Rv, and antibiotic treatment was initiated 6 weeks after infection. Separate groups of animals received RHZ, PaMZ, or single or two-drug components of the latter regimen administered at human-equivalent doses 5 days/week for a total of 8 weeks. Relapse rates were assessed 3 months after discontinuation of treatment to determine the sterilizing activity of each combination regimen. PaMZ given at human-equivalent doses was safe and well tolerated for the entire treatment period and rendered guinea pig lungs culture negative more rapidly than RHZ did. After 1 month of treatment, 80% and 50% of animals in the RHZ and PaMZ groups, respectively, had lung culture-positive relapse. Both combination regimens prevented microbiological relapse when administered for a total of 2 months. Our data support the use of PaMZ as a novel isoniazid- and rifamycin-sparing regimen suitable for treatment of both drug-sensitive TB and MDR-TB.

Thioridazine lacks bactericidal activity in an animal model of extracellular tuberculosis.

OBJECTIVES: The antipsychotic drug thioridazine is active in the murine model of tuberculosis infection, which is predominantly intracellular in nature. Recent clinical reports suggest that thioridazine may play a role in the treatment of drug-resistant tuberculosis. We studied the tuberculocidal activity of thioridazine in guinea pigs, which develop necrotic lung granulomas histologically resembling their human counterparts. METHODS: Pharmacokinetic studies were performed in guinea pigs to establish human-equivalent doses of thioridazine. Guinea pigs were aerosol-infected with ∼100 bacilli of Mycobacterium tuberculosis and single-drug treatment was started 4 weeks later with a range of thioridazine doses daily (5 days/week) for up to 4 weeks. Control animals received no treatment or 60 mg/kg isoniazid. RESULTS: The human-equivalent dose of thioridazine was determined to be 5 mg/kg with saturable absorption noted above 50 mg/kg. At the start of treatment, the lung bacterial burden was ∼6.2 log10 cfu. Although isoniazid reduced bacillary counts more than 10-fold, thioridazine monotherapy showed limited killing over the range of doses tested, reducing lung bacillary counts by 0.3-0.5 log10 following 1 month of treatment. Thioridazine was tolerated up to 40 mg/kg. CONCLUSIONS: Thioridazine has limited bactericidal activity against extracellular bacilli within necrotic granulomas. Its contribution to the sterilizing activity of combination regimens against drug-susceptible and drug-resistant tuberculosis remains to be determined.

Aerosolized gentamicin reduces the burden of tuberculosis in a murine model.

Tuberculosis (TB) is a major infectious disease problem: 1.7 million people annually die due to TB. Emergence of drug-resistant Mycobacterium tuberculosis and the lack of new antibiotics have exacerbated the situation. There is an urgent need to develop or repurpose drugs against TB. We evaluated inhaled gentamicin as direct respiratory system-targeted therapy in a murine model of TB. Aerosolized-gentamicin-treated mice showed significantly reduced lung M. tuberculosis loads and fewer granulomas relative to untreated controls. These results suggest that direct delivery of antibiotics to the respiratory system may provide therapeutic benefit to conventional treatment regimes for treatment of pulmonary TB.

Rifapentine is not more active than rifampin against chronic tuberculosis in guinea pigs.

Rifamycins are key sterilizing drugs in the current treatment of active tuberculosis (TB). Daily dosing of rifapentine (P), a potent rifamycin with high intracellular accumulation, in place of rifampin (R) in the standard antitubercular regimen significantly shortens the duration of treatment needed to prevent relapse in a murine model of active TB. We undertook the current study to compare directly the activities of human-equivalent doses of P and R in a guinea pig model of chronic TB, in which bacilli are predominantly extracellular within human-like necrotic granulomas. Hartley strain guinea pigs were aerosol infected with ~200 bacilli of Mycobacterium tuberculosis H37Rv, and treatment given 5 days/week was initiated 6 weeks later. R at 100 mg/kg of body weight and P at 100 mg/kg were given orally alone or in combination with isoniazid (H) at 60 mg/kg and pyrazinamide (Z) at 300 mg/kg. Culture-positive relapse was assessed in subgroups of guinea pigs after completion of 1 and 2 months of treatment. Human-equivalent doses of R and P showed equivalent bactericidal activity when used alone and in combination therapy. In guinea pigs treated with rifampin, isoniazid, and pyrazinamide (RHZ) or PHZ, microbiological relapse occurred in the lungs of 8/10 animals treated for 1 month and in 0/10 animals treated for 2 months. Substitution of P for R in the standard antitubercular regimen did not shorten the time to cure in this guinea pig model of chronic TB. Data from ongoing clinical trials comparing the activity of these two drugs are awaited to determine the relevance of the guinea pig TB model in preclinical drug screening.

Reactivation of latent tuberculosis in rhesus macaques by coinfection with simian immunodeficiency virus.

BACKGROUND: Tuberculosis (TB) and AIDS together present a devastating public health challenge. Over 3 million deaths every year are attributed to these twin epidemics. Annually, ∼11 million people are coinfected with HIV and Mycobacterium tuberculosis (Mtb). AIDS is thought to alter the spontaneous rate of latent TB reactivation. METHODOLOGY: Macaques are excellent models of both TB and AIDS. Therefore, it is conceivable that they can also be used to model coinfection. Using clinical, pathological, and microbiological data, we addressed whether latent TB infection in rhesus macaques can be reactivated by infection with simian immunodeficiency virus (SIV). RESULTS: A low-dose aerosol infection of rhesus macaques with Mtb caused latent, asymptomatic TB infection. Infection of macaques exhibiting latent TB with a rhesus-specific strain of SIV significantly reactivated TB. CONCLUSIONS: Rhesus macaques are excellent model of TB/AIDS coinfection and can be used to study the phenomena of TB latency and reactivation.