Contribution of direct InhA inhibitors to novel drug regimens in a mouse model of tuberculosis.

Isoniazid is an important first-line medicine to treat tuberculosis (TB). Isoniazid resistance increases the risk of poor treatment outcomes and development of multidrug resistance, and is driven primarily by mutations involving katG, encoding the prodrug-activating enzyme, rather than its validated target, InhA. The chemical tractability of InhA has fostered efforts to discover direct inhibitors of InhA (DIIs). In this study, we bridge the gap in understanding the potential contribution of DIIs to novel combination regimens and demonstrate a clear distinction of DIIs, like GSK693 and the newly described GSK138, from isoniazid, based on activity against clinical isolates and contribution to novel drug regimens. The results suggest that DIIs, specifically GSK138 and GSK693, could be promising partners in novel drug regimens, including those used against isoniazid-resistant TB, potentially enhancing their efficacy and/or preventing the selection of resistant mutants and supporting the continued exploration of InhA as a promising target for TB drug development.

Combinations of ß-Lactam Antibiotics Currently in Clinical Trials Are Efficacious in a DHP-I-Deficient Mouse Model of Tuberculosis Infection.

We report here a dehydropeptidase-deficient murine model of tuberculosis (TB) infection that is able to partially uncover the efficacy of marketed broad-spectrum ß-lactam antibiotics alone and in combination. Reductions of up to 2 log CFU in the lungs of TB-infected mice after 8 days of treatment compared to untreated controls were obtained at blood drug concentrations and time above the MIC (T>MIC) below clinically achievable levels in humans. These findings provide evidence supporting the potential of ß-lactams as safe and mycobactericidal components of new combination regimens against TB with or without resistance to currently used drugs.

Discovery of pyrazolopyrrolidinones as potent, broad-spectrum inhibitors of Leishmania infection.

Introduction: Leishmaniasis is a parasitic disease that affects more than 1 million people worldwide annually, predominantly in resource-limited settings. The challenge in compound development is to exhibit potent activity against the intracellular stage of the parasite (the stage present in the mammalian host) without harming the infected host cells. We have identified a compound series (pyrazolopyrrolidinones) active against the intracellular parasites of Leishmania donovani and L. major; the causative agents of visceral and cutaneous leishmaniasis in the Old World, respectively. Methods: In this study, we performed medicinal chemistry on a newly discovered antileishmanial chemotype, with over 100 analogs tested. Studies included assessments of antileishmanial potency, toxicity towards host cells, and in vitro ADME screening of key drug properties. Results and discussion: Members of the series showed high potency against the deadliest form, visceral leishmaniasis (approximate EC50 ≥ 0.01 µM without harming the host macrophage up to 10.0 µM). In comparison, the most efficient monotherapy treatment for visceral leishmaniasis is amphotericin B, which presents similar activity in the same assay (EC50 = 0.2 µM) while being cytotoxic to the host cell at 5.0 µM. Continued development of this compound series with the Discovery Partnership with Academia (DPAc) program at the GlaxoSmithKline Diseases of the Developing World (GSK DDW) laboratories found that the compounds passed all of GSK's criteria to be defined as a potential lead drug series for leishmaniasis. Conclusion: Here, we describe preliminary structure-activity relationships for antileishmanial pyrazolopyrrolidinones, and our progress towards the identification of candidates for future in vivo assays in models of visceral and cutaneous leishmaniasis.

The repurposing of Tebipenem pivoxil as alternative therapy for severe gastrointestinal infections caused by extensively drug-resistant Shigella spp.

Background: Diarrhoea remains one of the leading causes of childhood mortality globally. Recent epidemiological studies conducted in low-middle income countries (LMICs) identified Shigella spp. as the first and second most predominant agent of dysentery and moderate diarrhoea, respectively. Antimicrobial therapy is often necessary for Shigella infections; however, we are reaching a crisis point with efficacious antimicrobials. The rapid emergence of resistance against existing antimicrobials in Shigella spp. poses a serious global health problem. Methods: Aiming to identify alternative antimicrobial chemicals with activity against antimicrobial resistant Shigella, we initiated a collaborative academia-industry drug discovery project, applying high-throughput phenotypic screening across broad chemical diversity and followed a lead compound through in vitro and in vivo characterisation. Results: We identified several known antimicrobial compound classes with antibacterial activity against Shigella. These compounds included the oral carbapenem Tebipenem, which was found to be highly potent against broadly susceptible Shigella and contemporary MDR variants for which we perform detailed pre-clinical testing. Additional in vitro screening demonstrated that Tebipenem had activity against a wide range of other non-Shigella enteric bacteria. Cognisant of the risk for the development of resistance against monotherapy, we identified synergistic behaviour of two different drug combinations incorporating Tebipenem. We found the orally bioavailable prodrug (Tebipenem pivoxil) had ideal pharmacokinetic properties for treating enteric pathogens and was effective in clearing the gut of infecting organisms when administered to Shigella-infected mice and gnotobiotic piglets. Conclusions: Our data highlight the emerging antimicrobial resistance crisis and shows that Tebipenem pivoxil (licenced for paediatric respiratory tract infections in Japan) should be accelerated into human trials and could be repurposed as an effective treatment for severe diarrhoea caused by MDR Shigella and other enteric pathogens in LMICs. Funding: Tres Cantos Open Lab Foundation (projects TC239 and TC246), the Bill and Melinda Gates Foundation (grant OPP1172483) and Wellcome (215515/Z/19/Z).

Inhibition with spontaneous reactivation of carboxyl esterases by organophosphorus compounds: paraoxon as a model.

In this work kinetic data were obtained for different paraoxon concentrations incubated with chicken serum and the soluble fraction of chicken peripheral nerve. A kinetic model equation was deduced by assuming a multienzymatic system with three different simultaneously occurring molecular phenomena: (1) inhibition; (2) simultaneous spontaneous reactivation; (3) "ongoing" inhibition (inhibition during the substrate reaction). A three-dimensional fit of the model was applied to analyze the experimental data versus the concentration of the inhibitor and the preincubation time in an inhibition experiment. The best-fitting model in the soluble fraction of chicken peripheral nerve was compatible with a resistant component (22%) and with two sensitive enzymatic entities (37 and 41%). The corresponding second-order rate constants of inhibition (k(i) = 1.8 × 10(-3) and 5.1 × 10(-3) nM(-1) min(-1), respectively) and the spontaneous reactivation constants (k(r) = 0.428 and 0.011 min(-1), respectively) were estimated. The best-fitting model in chicken serum was compatible with a resistant component (5.6%) and with two sensitive enzymatic entities (22.1 and 72.3%). The corresponding second-order rate constants of inhibition (k(i) = 5.8 × 10(-2) and 2.0 × 10(-3) nM(-1) min(-1), respectively) and the spontaneous reactivation constants (k(r) = 0.0044 and 0.0091 min(-1), respectively) were estimated. These parameters were similar to those observed in spontaneous reactivation experiments with preinhibited paraoxon samples. The consistency of the results of all the experiments is considered an internal validation of the methodology. The results are also consistent with a significant ongoing inhibition. The proportion of enzymatic components shown in this work by the inhibition and reactivation of paraoxon is similar to that previously observed in inhibition experiments with mipafox in both tissues, demonstrating that this kinetic approach provides consistent results in complex enzymatic systems. The high sensitivity (at nanomolar concentrations) of these esterases suggests that they may either play a role in toxicity in low-level long-term exposure of organophosphate compounds or have a protective effect related with the spontaneous reactivation.

Synthesis and Structure-Activity Relationships of the Novel Antimalarials 5-Pyridinyl-4(1 H)-Pyridones.

Malaria is still one of the most prevalent parasitic infections in the world, with half of the world's population at risk for malaria. The effectiveness of current antimalarial therapies, even that of the most recent class of antimalarial drugs (artemisinin-combination therapies, ACTs), is under continuous threat by the spread of resistant Plasmodium strains. As a consequence, there is still an urgent requirement for new antimalarial drugs. We previously reported the identification of 4(1 H)-pyridones as a novel series with potent antimalarial activities. The low solubility was identified as an issue to address. In this paper, we describe the synthesis and biological evaluation of 4(1 H)-pyridones with potent antimalarial activities in vitro and in vivo and improved pharmacokinetic profiles. Their main structural novelties are the presence of polar moieties, such as hydroxyl groups, and the replacement of the lipophilic phenyl rings with pyridines on their lipophilic side chains.

Isoxazolopyrimidine-Based Inhibitors of Plasmodium falciparum Dihydroorotate Dehydrogenase with Antimalarial Activity.

Malaria kills nearly 0.5 million people yearly and impacts the lives of those living in over 90 countries where it is endemic. The current treatment programs are threatened by increasing drug resistance. Dihydroorotate dehydrogenase (DHODH) is now clinically validated as a target for antimalarial drug discovery as a triazolopyrimidine class inhibitor (DSM265) is currently undergoing clinical development. We discovered a related isoxazolopyrimidine series in a phenotypic screen, later determining that it targeted DHODH. To determine if the isoxazolopyrimidines could yield a drug candidate, we initiated hit-to-lead medicinal chemistry. Several potent analogues were identified, including a compound that showed in vivo antimalarial activity. The isoxazolopyrimidines were more rapidly metabolized than their triazolopyrimidine counterparts, and the pharmacokinetic data were not consistent with the goal of a single-dose treatment for malaria.

Antitubercular drugs for an old target: GSK693 as a promising InhA direct inhibitor.

Despite being one of the first antitubercular agents identified, isoniazid (INH) is still the most prescribed drug for prophylaxis and tuberculosis (TB) treatment and, together with rifampicin, the pillars of current chemotherapy. A high percentage of isoniazid resistance is linked to mutations in the pro-drug activating enzyme KatG, so the discovery of direct inhibitors (DI) of the enoyl-ACP reductase (InhA) has been pursued by many groups leading to the identification of different enzyme inhibitors, active against Mycobacterium tuberculosis (Mtb), but with poor physicochemical properties to be considered as preclinical candidates. Here, we present a series of InhA DI active against multidrug (MDR) and extensively (XDR) drug-resistant clinical isolates as well as in TB murine models when orally dosed that can be a promising foundation for a future treatment.

Properties of phenyl valerate esterase activities from chicken serum are comparable with soluble esterases of peripheral nerves in relation with organophosphorus compounds inhibition.

Chicken serum, the usual in vivo animal for testing organophosphorus delayed neuropathy, has long been reported not to contain a homologous activity of the neuronal neuropathy target esterase (NTE) activity when it is assayed according to standard methods as the phenyl valerate esterase (PVase) activity, which is resistant to paraoxon and sensitive to mipafox. However, a PVase activity (1000-1500 nmol/min/ml) can be measured in serum that is extremely sensitive to both paraoxon, a non-neuropathic organophosphorus compound and mipafox, a model neuropathy inducer. The inhibition was time progressive in both cases, suggesting a covalent phosphorilating reaction. The fixed time inhibition curves suggest at least two sensitive components. The IC50 for 30 min, at 37 degrees C are 6 and 51 nM for paraoxon and 4 and 110 nM for mipafox, for every sensitive component. When paraoxon was removed from a serum sample pretreated with the inhibitor, the paraoxon sensitive PVase activity was recovered, in spite of showing a time progressive inhibition suggesting that hydrolytic dephosphorylating reaction recovered at a significant rate. The reactivation of the phosphorylated enzyme could explain that the time progressive inhibitions curves for long time with paraoxon tend to reach a plateau depending on the inhibition concentration. However, with mipafox, the curve approached the same maximal inhibitions at all concentrations as expected for a permanent covalent irreversible phosphorylation, which is coherent with the observations that the activity remained inhibited after removing the inhibitor. Data of serum esterases described in this paper showed similar properties to those previously reported for peripheral nerve soluble phenylvalerate esterase: (1) extremely high sensitivity to paraoxon and mipafox; (2) time progressive kinetic with two sensitive components; (3) recovery of activity after removal of paraoxon; and (4) permanent inhibition with mipafox. These properties of serum esterases are very similar to those of soluble fraction of peripheral nerves. So, serum PVases could be considered as appropriate biomarkers, as a mirror for the neural soluble paraoxon and mipafox sensitive soluble esterases that could be used for biomonitoring purpose.

The inhibition of the high sensitive peripheral nerve soluble esterases by mipafox. A new mathematical processing for the kinetics of inhibition of esterases by organophosphorus compounds.

In the study of organophosphorus (OP) sensitive enzymes, careful discrimination of specific components within a complex multienzymatic mixture is needed. However, standard kinetic analysis gives inconsistent results (i.e., apparently different kinetic constants at different inhibitor concentration) with complex multienzymatic mixtures. A strategy is now presented to obtain consistent kinetic parameters. In the peripheral nerve, soluble carboxylesterases measured with the substrate phenylvalerate (PV) are found with extremely high sensitivity to some inhibitors. Tissue preparations were preincubated with mipafox at nanomolar concentrations (up to 100 nM) for different inhibition times (up to 180 min). Inhibition data were analyzed with model equations of one or two sensitive (exponential) components, with or without resistant components. The most complex model was %act=A1e-k1It+A2e-k2It+AR (step 1). From the curve with the highest mipafox concentration (100 nM), the amplitude for the resistant component was determined as AR=15.1% (step 2). The model equation with a fixed AR value was again applied (step 3) to deduce the second-order inhibition rate constants (k1=2.6 x 10(6) M-1 min-1 and k2=0.28 x 10(6) M-1 min-1), being conserved consistently throughout all mipafox concentrations. Finally, using fixed values of AR, k1, and k2, the amplitudes for the two exponential (sensitive) components (A1 and A2) were re-estimated (A1=50.2% and A2=34.2%). The operational process was internally validated by the close similarity with values obtained by directly fitting with a three-dimensional model equation (activity versus time and inhibitor concentration) to the same inhibition data. Carboxylesterase fractions separated by preparative chromatography showed kinetic properties consistent with the kinetically discriminated components. As practical conclusion, for routine analysis of esterases in toxicological studies, a simplified procedure using the inhibition with mipafox at 30 nM, 1 microM, and 1 mM for 30 min is suggested to discriminate the main esterase components in soluble fraction preparations.

Cyclopropyl Carboxamides: A New Oral Antimalarial Series Derived from the Tres Cantos Anti-Malarial Set (TCAMS).

Rapid triaging of three series of related hits selected from the Tres Cantos Anti-Malarial Set (TCAMS) are described. A triazolopyrimidine series was deprioritized due to delayed inhibition of parasite growth. A lactic acid series has derivatives with IC50 < 500 nM in a standard Plasmodium falciparum in vitro whole cell assay (Pf assay) but shows half-lives of < 30 min in both human and murine microsomes. Compound 19, from a series of cyclopropyl carboxamides, is a highly potent in vitro inhibitor of P. falciparum (IC50 = 3 nM) and has an oral bioavailability of 55% in CD-1 mice and an ED90 of 20 mg/kg after oral dosing in a nonmyelo-depleted P. falciparum murine model.

Increase of R-/S-methadone enantiomer concentration ratio in serum of patients treated with either nevirapine or efavirenz.

An increased methadone enantiomer ratio (R/S) was associated to both nevirapine (179%, n=5) and efavirenz (36%, n=9) treatments when compared with that of controls (n=52). Additionally, in four follow-up patients, both R- and S-methadone normalized concentrations decreased (19%-93%) while R/S increased (22%-314%) following nevirapine/efavirenz treatment. R/S decreased (42%) after non-compliance with efavirenz treatment. Therefore, the methadone-maintenance-treatment outcome should be evaluated when patients are treated with drugs which are supposed to induce CYP3A4 and CYP2B6 isoforms.