Discovery of Imidazo[1,2-a]pyridine Ethers and Squaramides as Selective and Potent Inhibitors of Mycobacterial Adenosine Triphosphate (ATP) Synthesis.

The approval of bedaquiline to treat tuberculosis has validated adenosine triphosphate (ATP) synthase as an attractive target to kill Mycobacterium tuberculosis (Mtb). Herein, we report the discovery of two diverse lead series imidazo[1,2-a]pyridine ethers (IPE) and squaramides (SQA) as inhibitors of mycobacterial ATP synthesis. Through medicinal chemistry exploration, we established a robust structure-activity relationship of these two scaffolds, resulting in nanomolar potencies in an ATP synthesis inhibition assay. A biochemical deconvolution cascade suggested cytochrome c oxidase as the potential target of IPE class of molecules, whereas characterization of spontaneous resistant mutants of SQAs unambiguously identified ATP synthase as its molecular target. Absence of cross resistance against bedaquiline resistant mutants suggested a different binding site for SQAs on ATP synthase. Furthermore, SQAs were found to be noncytotoxic and demonstrated efficacy in a mouse model of tuberculosis infection.

In silico-based high-throughput screen for discovery of novel combinations for tuberculosis treatment.

There are currently 18 drug classes for the treatment of tuberculosis, including those in the development pipeline. An in silico simulation enabled combing the innumerably large search space to derive multidrug combinations. Through the use of ordinary differential equations (ODE), we constructed an in silico kinetic platform in which the major metabolic pathways in Mycobacterium tuberculosis and the mechanisms of the antituberculosis drugs were integrated into a virtual proteome. The optimized model was used to evaluate 816 triplets from the set of 18 drugs. The experimentally derived cumulative fractional inhibitory concentration (∑FIC) value was within twofold of the model prediction. Bacterial enumeration revealed that a significant number of combinations that were synergistic for growth inhibition were also synergistic for bactericidal effect. The in silico-based screen provided new starting points for testing in a mouse model of tuberculosis, in which two novel triplets and five novel quartets were significantly superior to the reference drug triplet of isoniazid, rifampin, and ethambutol (HRE) or the quartet of HRE plus pyrazinamide (HREZ).

Triaminopyrimidine is a fast-killing and long-acting antimalarial clinical candidate.

The widespread emergence of Plasmodium falciparum (Pf) strains resistant to frontline agents has fuelled the search for fast-acting agents with novel mechanism of action. Here, we report the discovery and optimization of novel antimalarial compounds, the triaminopyrimidines (TAPs), which emerged from a phenotypic screen against the blood stages of Pf. The clinical candidate (compound 12) is efficacious in a mouse model of Pf malaria with an ED99 <30 mg kg(-1) and displays good in vivo safety margins in guinea pigs and rats. With a predicted half-life of 36 h in humans, a single dose of 260 mg might be sufficient to maintain therapeutic blood concentration for 4-5 days. Whole-genome sequencing of resistant mutants implicates the vacuolar ATP synthase as a genetic determinant of resistance to TAPs. Our studies highlight the potential of TAPs for single-dose treatment of Pf malaria in combination with other agents in clinical development.

UDP-N-acetylmuramic acid l-alanine ligase (MurC) inhibition in a tolC mutant Escherichia coli strain leads to cell death.

The Mur ligases play an essential role in the biosynthesis of bacterial peptidoglycan and hence are attractive antibacterial targets. A screen of the AstraZeneca compound library led to the identification of compound A, a pyrazolopyrimidine, as a potent inhibitor of Escherichia coli and Pseudomonas aeruginosa MurC. However, cellular activity against E. coli or P. aeruginosa was not observed. Compound A was active against efflux pump mutants of both strains. Experiments using an E. coli tolC mutant revealed accumulation of the MurC substrate and a decrease in the level of product upon treatment with compound A ,: indicating inhibition of MurC enzyme in these cells. Such a modulation was not observed in the E. coli wild-type cells. Further, overexpression of MurC in the E. coli tolC mutant led to an increase in the compound A MIC by ≥16-fold, establishing a correlation between MurC inhibition and cellular activity. In addition, estimation of the intracellular compound A level showed an accumulation of the compound over time in the tolC mutant strain. A significant compound A level was not detected in the wild-type E. coli strain even upon treatment with high concentrations of the compound. Therefore, the lack of MIC and absence of MurC inhibition in wild-type E. coli were possibly due to suboptimal compound concentration as a consequence of a high efflux level and/or poor permeativity of compound A.

N-aryl-2-aminobenzimidazoles: novel, efficacious, antimalarial lead compounds.

From the phenotypic screening of the AstraZeneca corporate compound collection, N-aryl-2-aminobenzimidazoles have emerged as novel hits against the asexual blood stage of Plasmodium falciparum (Pf). Medicinal chemistry optimization of the potency against Pf and ADME properties resulted in the identification of 12 as a lead molecule. Compound 12 was efficacious in the P. berghei (Pb) model of malaria. This compound displayed an excellent pharmacokinetic profile with a long half-life (19 h) in rat blood. This profile led to an extended survival of animals for over 30 days following a dose of 50 mg/kg in the Pb malaria model. Compound 12 retains its potency against a panel of Pf isolates with known mechanisms of resistance. The fast killing observed in the in vitro parasite reduction ratio (PRR) assay coupled with the extended survival highlights the promise of this novel chemical class for the treatment of malaria.

1,4-azaindole, a potential drug candidate for treatment of tuberculosis.

New therapeutic strategies against multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis are urgently required to combat the global tuberculosis (TB) threat. Toward this end, we previously reported the identification of 1,4-azaindoles, a promising class of compounds with potent antitubercular activity through noncovalent inhibition of decaprenylphosphoryl-ß-D-ribose 2'-epimerase (DprE1). Further, this series was optimized to improve its physicochemical properties and pharmacokinetics in mice. Here, we describe the short-listing of a potential clinical candidate, compound 2, that has potent cellular activity, drug-like properties, efficacy in mouse and rat chronic TB infection models, and minimal in vitro safety risks. We also demonstrate that the compounds, including compound 2, have no antagonistic activity with other anti-TB drugs. Moreover, compound 2 shows synergy with PA824 and TMC207 in vitro, and the synergy effect is translated in vivo with TMC207. The series is predicted to have a low clearance in humans, and the predicted human dose for compound 2 is ≤1 g/day. Altogether, our data suggest that a 1,4-azaindole (compound 2) is a promising candidate for the development of a novel anti-TB drug.

Aminoazabenzimidazoles, a novel class of orally active antimalarial agents.

Whole-cell high-throughput screening of the AstraZeneca compound library against the asexual blood stage of Plasmodium falciparum (Pf) led to the identification of amino imidazoles, a robust starting point for initiating a hit-to-lead medicinal chemistry effort. Structure-activity relationship studies followed by pharmacokinetics optimization resulted in the identification of 23 as an attractive lead with good oral bioavailability. Compound 23 was found to be efficacious (ED90 of 28.6 mg·kg(-1)) in the humanized P. falciparum mouse model of malaria (Pf/SCID model). Representative compounds displayed a moderate to fast killing profile that is comparable to that of chloroquine. This series demonstrates no cross-resistance against a panel of Pf strains with mutations to known antimalarial drugs, thereby suggesting a novel mechanism of action for this chemical class.

Lead optimization of 1,4-azaindoles as antimycobacterial agents.

In a previous report, we described the discovery of 1,4-azaindoles, a chemical series with excellent in vitro and in vivo antimycobacterial potency through noncovalent inhibition of decaprenylphosphoryl-ß-d-ribose-2'-epimerase (DprE1). Nevertheless, high mouse metabolic turnover and phosphodiesterase 6 (PDE6) off-target activity limited its advancement. Herein, we report lead optimization of this series, culminating in potent, metabolically stable compounds that have a robust pharmacokinetic profile without any PDE6 liability. Furthermore, we demonstrate efficacy for 1,4-azaindoles in a rat chronic TB infection model. We believe that compounds from the 1,4-azaindole series are suitable for in vivo combination and safety studies.

Azaindoles: noncovalent DprE1 inhibitors from scaffold morphing efforts, kill Mycobacterium tuberculosis and are efficacious in vivo.

We report 1,4-azaindoles as a new inhibitor class that kills Mycobacterium tuberculosis in vitro and demonstrates efficacy in mouse tuberculosis models. The series emerged from scaffold morphing efforts and was demonstrated to noncovalently inhibit decaprenylphosphoryl-ß-D-ribose2'-epimerase (DprE1). With "drug-like" properties and no expectation of pre-existing resistance in the clinic, this chemical class has the potential to be developed as a therapy for drug-sensitive and drug-resistant tuberculosis.

Antidiabetic effect of Ficus racemosa Linn. stem bark in high-fat diet and low-dose streptozotocin-induced type 2 diabetic rats: a mechanistic study.

The present study was designed to investigate the effects of the ethanol extract of Ficus racemosa (FRE) on biochemical parameters in type 2-like diabetes, induced by a combination of standardised high-fat diet and low-dose streptozotocin (25mgkg(-1), i.p.) in rats. To elucidate the mode of action of FRE, its effects on a battery of targets involved in glucose homeostasis was evaluated. FRE (200 and 400mgkg(-1), p.o.), in a dose-dependent manner, altered the biochemical parameters and significantly improved glucose tolerance and HDL-c levels. In different bioassays, FRE showed inhibition of PTP-1B (IC50 12.1µg/mL) and DPP-IV (42.5%). FRE exhibited 82.6% binding to PPAR-γ. Furthermore FRE exhibited stimulation of glucose uptake by skeletal muscles (hemi-diaphragm). Bergenin was quantified in bioactive-FRE by high-performance liquid chromatography (0.15%w/w). This is the first report demonstrating the effectiveness of F. racemosa stem bark in type 2 diabetes and targets involved in it.

Antidiabetic effect of Dodonaea viscosa aerial parts in high fat diet and low dose streptozotocin-induced type 2 diabetic rats: a mechanistic approach.

CONTEXT: High fat diet (HFD) and low-dose streptozotocin (STZ) is an ideal model for type 2 diabetes mellitus (T2DM) that would closely reflect the natural history and metabolic characteristics of human T2DM and is also suitable for pharmacological screening. OBJECTIVE: The present study was designed to investigate the effect of the water extract (DVW) and the polar fraction of ethanol extract (DVE-4) of Dodonaea viscosa (L). Jacq. (Sapindaceae) on biochemical parameters in type 2 diabetes induced by a standardized HFD and low dose streptozotocin (25 mg/kg) in rats. Further, to elucidate the mode of action we evaluated its effects on a battery of targets involved in glucose homeostasis (in vitro studies). MATERIALS AND METHODS: Different doses of DVW and DVE-4 were administered once daily for two weeks to HFD + STZ diabetic rats. Quantification of biomarker quercetin was done using HPLC. RESULTS AND DISCUSSION: Both DVW and DVE-4 dose-dependently reduced blood glucose, serum insulin, homeostatic model assessment (HOMA), lipid profiles, and significantly improved glucose tolerance and HDL-c levels. In addition, the extract and fraction also decreased oxidative stress by improving endogenous antioxidants. In different, bioassays, DVW and DVE-4 showed inhibition of PTP-1B and at a concentration of 10 µg/mL showed 60 and 54.2% binding to PPARγ, respectively. Both extract/fraction exhibited stimulation of glucose uptake by skeletal muscles. CONCLUSION: Taken together, these results suggest that DVW and DVE-4 inhibits HFD + STZ-induced insulin resistance, lipid abnormalities and oxidative stress indicating that these effects may be mediated by interacting with multiple targets operating in diabetes mellitus.

Antidiabetic effect of Dodonaea viscosa (L). Lacq. aerial parts in high fructose-fed insulin resistant rats: a mechanism based study.

To study the effect and mode of action of water extract (DVW) and polar fraction of ethanol extract (DVE-4) of D. viscosa in high-fructose diet induced insulin resistance in male Wistar rats. D. viscosa's effects were evaluated on a battery of targets involved in glucose homeostasis (in vitro studies). Rats were rendered insulin resistant by feeding 66% (w/w) fructose and 1.1% (v/w) coconut oil mixed with normal pellet diet (NPD) for six weeks. DVW and DVE4 at different doses were administered simultaneously. At the end of the study, blood glucose, oral glucose tolerance test, lipid profile and insulin were estimated and homeostatic model assessment (HOMA) levels were calculated. In addition, enzymatic and nonenzymatic liver antioxidant levels were also estimated. Quantification of biomarker quercetin was done using HPLC. Fructose diet with DVW, DVE-4 significantly reduced blood glucose, serum insulin, HOMA, lipid profiles and significantly improved glucose tolerance and HDL-c levels. In addition, these extract and fraction also decreased oxidative stress by improving endogenous antioxidants. In different bioassays, DVW and DVE-4 inhibited protein tyrosine phosphatase-1B with IC50 65.8 and 54.9 microg/ml respectively and showed partial inhibition of dipeptidyl peptidase-IV. Moreover, DVW and DVE-4, at 10 microg/ml showed 60 and 54.2% binding to peroxisome proliferator-activated receptor-gamma. Further, 2.1% (w/w) of quercetin was quantified in bioactive-DVE-4 using HPLC method. The results provide pharmacological evidence of D. viscosa in treatment of prediabetic conditions and these effects may be mediated by interacting with multiple targets operating in diabetes mellitus.

Ficus racemosa Stem Bark Extract: A Potent Antioxidant and a Probable Natural Radioprotector.

Ethanol extract (FRE) and water extract (FRW) of Ficus racemosa (family: Moraceae) were subjected to free radical scavenging both by steady state and time resolved methods such as nanosecond pulse radiolysis and stopped-flow spectrophotometric analyses. FRE exhibited significantly higher steady state antioxidant activity than FRW. FRE exhibited concentration dependent DPPH, ABTS(*-), hydroxyl radical and superoxide radical scavenging and inhibition of lipid peroxidation with IC(50) comparable with tested standard compounds. In vitro radioprotective potential of FRE was studied using micronucleus assay in irradiated Chinese hamster lung fibroblast cells (V79). Pretreatment with different doses of FRE 1h prior to 2 Gy gamma-radiation resulted in a significant (P < 0.001) decrease in the percentage of micronucleated binuclear V79 cells. Maximum radioprotection was observed at 20 mug/ml of FRE. The radioprotection was found to be significant (P < 0.01) when cells were treated with optimum dose of FRE (20 mug/ml) 1 h prior to 0.5, 1, 2, 3 and 4 Gy gamma-irradiation compared to the respective radiation controls. The cytokinesis-block proliferative index indicated that FRE does not alter radiation induced cell cycle delay. Based on all these results we conclude that the ethanol extract of F. racemosa acts as a potent antioxidant and a probable radioprotector.

Antioxidant and radioprotective effect of the active fraction of Pilea microphylla (L.) ethanolic extract.

The ethanolic extract of Pilea microphylla (L.) was defatted, successively fractionated with acetone and the residue so obtained was found to be most potent when subjected to detailed free radical scavenging and in vivo radioprotection studies. The most active fraction reacts with free radicals, such as DPPH (50 microM), ABTS(.)(-) (100 microM) and (.)OH (generated by Fenton reaction) with IC(50) value of 23.15 microg/ml, 3.0 microg/ml and 310 microg/ml, respectively. The most active fraction inhibited iron-induced lipid peroxidation in phosphatidyl choline liposomes with an IC(50) of 13.74 microg/ml. The kinetics of scavenging of DPPH and ABTS(.)(-) radicals were followed at different concentrations of the fraction by employing stopped-flow studies. The observed first order decay rate constants at 200 microg/ml and 50 microg/ml of fraction with DPPH (50 microM) and ABTS(.)(-) (50 microM) were found to be 0.4s(-1) and 2.1s(-1), respectively. The fraction when screened for in vivo radioprotection in Swiss albino mice showed 80% protection at a dose of 900 mg/kg and with a DRF of about 1.12. The fraction was also found to protect livers of irradiated mice from depletion of endogenous antioxidant enzymes like glutathione, GST, SOD, catalase and thiols. The fraction also protected the villi height, increased the number of crypt cells while offering general protection to the intestine from acute radiation effects. The fraction also protected the hematopoietic system as assessed by endogenous spleen colony assay, contributing to the overall radioprotective ability.

Effect of sesamol on radiation-induced cytotoxicity in Swiss albino mice.

The radio-protective ability of sesamol (SM) at various doses viz., 0, 10, 25, 40, 50, 70 and 100mg/kg bw, administered intraperitoneally 30min prior to 9.5Gy whole-body gamma-irradiation was studied in Swiss albino mice. Radiation toxicity and mortality were observed during a period of 30 days and the percentage mortality was calculated. SM pretreatment with 50mg/kg bw was found to be the most effective dose in maintaining body weight and in reducing the percentage mortality, while 100mg/kg bw was found to be more effective in maintaining the spleen index and in stimulation of endogenous spleen colony-forming units. Pretreatment with SM (50mg/kg bw) in mice irradiated with 15Gy significantly reduced dead, inflammatory, mitotic and goblet cells in irradiated jejunum. SM at 50mg/kg bw also increased crypt cells, maintained villus height, and prevented mucosal erosion. Nuclear enlargement in epithelial cells was found less in SM-treated mice compared with the irradiated control. The radiation-induced decrease in endogenous antioxidant enzymes (GSH, GST, catalase) and the increase in lipid peroxidation were also reduced by pretreatment with SM [50 and 100mg/kg bw] at all monitored post-irradiation intervals. There was no protection at a dose less than 25mg/kg bw.

Evaluation and optimization of radioprotective activity of Coronopus didymus Linn. in gamma-irradiated mice.

PURPOSE: To evaluate and optimize the radioprotective ability of the most potent fraction of an aqueous extract of Coronopus didymus in whole body gamma-irradiated Swiss albino mice and to evaluate the antioxidant status and lipid peroxidation of the livers of the surviving mice. To correlate the free radical scavenging studies with in vivo radioprotection ability. MATERIALS AND METHODS: Swiss albino mice were treated with either vehicle or the different doses of extract/fraction suspension by an i.p. route, 30 min before exposure to 10 Gy gamma-irradiation and the animals were monitored twice daily for any signs of radiation toxicity and mortality. Radiation dose response (7-11 Gy), optimization of route, time of drug administration and evaluation of dose response factor (DRF) at the best dose of the fraction was studied. Endogenous antioxidant status and lipid peroxidation of the livers of the mice surviving on the 31st day was evaluated by using spectrophotometric methods. RESULTS: The most active free radical scavenging fraction (CDF1) as assessed by competition kinetic studies using pulse radiolysis showed maximum in vivo radioprotection of 70% at a dose of 400 mg/kg body weight (bw) compared to corresponding 10 Gy irradiated control. Optimum radioprotection was observed upon i.p. administration, 30 min prior to 10 Gy irradiation and DRF at a dose of 400 mg/kg bw for 30 day survival was found to be 1.07. The levels of endogenous antioxidant enzymes and lipid peroxidation in the CDF1 treated surviving mice were found to reverse back to their normal levels. CONCLUSIONS: The optimum dose, time and route of drug administration for maximum radioprotection by CDF1 were determined. The reversal of the levels of endogenous antioxidant enzymes and lipid peroxidation indicates reduced oxidative stress in CDF1 treated surviving mice.

Identification and evaluation of antioxidant, analgesic/anti-inflammatory activity of the most active ninhydrin-phenol adducts synthesized.

Treatment of phenols with ninhydrin in acidic medium afforded 2-hydroxy-2-(ortho-hydroxy-phenyl/naphthyl)-1,3-dioxoindanes, which being unstable were isolated in their hemiketal forms. These synthesized compounds were subjected to TLC screening for radical scavenging and in vitro lipoxgenase and cycloxygenase enzyme inhibition assays. The best compound was identified and studied in detail for steady-state and time-resolved free radical kinetics, viz., DPPH, ABTS(-), *OH and rate constants for these reactions were evaluated. The best compound was also subjected to in vivo anti-inflammatory and analgesic activities in which the compound showed good promise for further structural optimization.

Bioactivity-guided fractionation of Coronopus didymus: A free radical scavenging perspective.

The whole plant aqueous extract of Coronopus didymus Linn. was fractionated on the basis of polarity and resulting fractions were evaluated for free radical scavenging ability. The most non-polar fraction (CDF1) was found to be more active than other fractions in scavenging DPPH, ABTS(-), nitric oxide and hydroxyl radicals in steady-state conditions. Stop-flow spectrometric studies showed 58.13% inhibition of 100 microM DPPH at a concentration of 150 microg/ml of CDF1 in 1000 s and 32.31% scavenging of 960 microM ABTS(-) at a concentration of 300 microg/ml of CDF1 in 100 s. The reaction of CDF1 with hydroxyl radicals produced by pulse radiolysis showed a transient spectrum with absorption peaks at 320, 390 and 400 nm, indicating the presence of flavonoids/related components. Competition kinetics with potassium thiocyanate against scavenging of hydroxyl radicals showed a reactivity of 0.1326 against thiocyanate. CDF1 also protected against Fenton reagent-induced calf thymus DNA damage at a concentration of 400 mg/ml indicating it to be the most potent fraction.