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.

Anti-Inflammatory Effects of Auranamide and Patriscabratine-Mechanisms and In Silico Studies.

Auranamide and patriscabratine are amides from Melastoma malabathricum (L.) Smith. Their anti-inflammatory activity and nuclear factor erythroid 2-related factor 2 (NRF2) activation ability were evaluated using Escherichia coli lipopolysaccharide (LPSEc)-stimulated murine macrophages (RAW264.7) and murine hepatoma (Hepa-1c1c7) cells, respectively. The cytotoxicity of the compounds was assessed using a 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. The anti-inflammatory activity was determined by measuring the nitric oxide (NO) production and pro-inflammatory cytokines (Interleukin (IL)-1ß, Interferon (IFN)-γ, tumour necrosis factor (TNF)-α, and IL-6) and mediators (NF-κB and COX-2). NRF2 activation was determined by measuring the nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) quinone oxidoreductase 1 (NQO1), nuclear NRF2 and hemeoxygenase (HO)-1. In vitro metabolic stability was assessed using the mouse, rat, and human liver microsomes. The compounds were non-toxic to the cells at 10 µM. Both compounds showed dose-dependent effects in downregulating NO production and pro-inflammatory cytokines and mediators. The compounds also showed upregulation of NQO1 activity and nuclear NRF2 and HO-1 levels. The compounds were metabolically stable in mouse, rat and human liver microsomes. The possible molecular targets of NRF2 activation by these two compounds were predicted using molecular docking studies and it was found that the compounds might inhibit the Kelch domain of KEAP1 and GSK-3ß activity. The physicochemical and drug-like properties of the test compounds were predicted using Schrodinger small molecule drug discovery suite (v.2022-2).

A Systematic Study of the In Vitro Pharmacokinetics and Estimated Human In Vivo Clearance of Indole and Indazole-3-Carboxamide Synthetic Cannabinoid Receptor Agonists Detected on the Illicit Drug Market.

In vitro pharmacokinetic studies were conducted on enantiomer pairs of twelve valinate or tert-leucinate indole and indazole-3-carboxamide synthetic cannabinoid receptor agonists (SCRAs) detected on the illicit drug market to investigate their physicochemical parameters and structure-metabolism relationships (SMRs). Experimentally derived Log D7.4 ranged from 2.81 (AB-FUBINACA) to 4.95 (MDMB-4en-PINACA) and all SCRAs tested were highly protein bound, ranging from 88.9 ± 0.49% ((R)-4F-MDMB-BINACA) to 99.5 ± 0.08% ((S)-MDMB-FUBINACA). Most tested SCRAs were cleared rapidly in vitro in pooled human liver microsomes (pHLM) and pooled cryopreserved human hepatocytes (pHHeps). Intrinsic clearance (CLint) ranged from 13.7 ± 4.06 ((R)-AB-FUBINACA) to 2944 ± 95.9 mL min-1 kg-1 ((S)-AMB-FUBINACA) in pHLM, and from 110 ± 34.5 ((S)-AB-FUBINACA) to 3216 ± 607 mL min-1 kg-1 ((S)-AMB-FUBINACA) in pHHeps. Predicted Human in vivo hepatic clearance (CLH) ranged from 0.34 ± 0.09 ((S)-AB-FUBINACA) to 17.79 ± 0.20 mL min-1 kg-1 ((S)-5F-AMB-PINACA) in pHLM and 1.39 ± 0.27 ((S)-MDMB-FUBINACA) to 18.25 ± 0.12 mL min-1 kg-1 ((S)-5F-AMB-PINACA) in pHHeps. Valinate and tert-leucinate indole and indazole-3-carboxamide SCRAs are often rapidly metabolised in vitro but are highly protein bound in vivo and therefore predicted in vivo CLH is much slower than CLint. This is likely to give rise to longer detection windows of these substances and their metabolites in urine, possibly as a result of accumulation of parent drug in lipid-rich tissues, with redistribution into the circulatory system and subsequent metabolism.

Discovery of super soft-drug modulators of sphingosine-1-phosphate receptor 1.

The oral S1PR1 agonist ponesimod demonstrated substantial efficacy in a phase II clinical trial of psoriasis. Unfortunately, systemic side effects were observed, which included lymphopenia and transient bradycardia. We sought to develop a topical soft-drug S1PR1 agonist with an improved therapeutic index. By modifying ponesimod, we discovered an ester series of S1PR agonists. To increase metabolic instability in plasma we synthesised esters described as specific substrates for paraoxonase and butyrylcholinesterases, esterases present in human plasma.

Synthesis of New Shogaol Analogues as NRF2 Activators and Evaluation of Their Anti-Inflammatory Activity, Modes of Action and Metabolic Stability.

6-shogaol is a natural and the most potent bioactive vanilloid in dried Zingiber officinale rhizomes. Many scientific studies have reported the diverse biological activities of 6-shogaol. However, the major drawback of 6-shogaol is its instability at room temperature. We synthesised new shogaol thiophene compounds (STCs) by replacing the pentyl group in the sidechain with thiophene derivatives. The STCs were tested for their nuclear factor erythroid 2-related factor 2 (NRF2) activation ability in murine hepatoma cells (Hepa1c1c-7) by determining their NAD(P)H quinone oxidoreductase 1 (NQO1) inducing ability and expression of NRF2-associated antioxidant genes. The anti-inflammatory activity of STCs was determined in Escherichia coli lipopolysaccharide (LPSEc)-stimulated NR2-proficient and -silenced mouse microglial cells (BV-2) by measuring the inflammatory markers, cytokines, and mediators. The modes of action (interacting with the Kelch domain of KEAP1, covalent bonding with cysteines of KEAP1, and inhibition of GSK-3ß enzyme activity) of NRF2 activation by STCs were determined using commercially available kits. The in vitro metabolic stability of the STCs in liver microsomes (humans, rats, and mice) was also investigated. The molecular docking and molecular dynamics studies were conducted to identify the binding poses, stability, and molecular interactions of the STCs in the binding pockets of Kelch and BTB domains of KEAP1 and GSK-3ß enzyme. The new STCs were synthesised in good yields of > 85%, with a purity of about 95%, using a novel synthesis method by employing a reusable proline-proline dipeptide catalyst. The STCs are more potent than 6-shogaol in activating NRF2 and reducing inflammation. The nature of substituents on thiophene has a profound influence on the bioactivity of the STCs. Phenylthiophene STC (STC5) is the most potent, while thiophenes containing electron-withdrawing groups showed weaker bioactivity. The bioactivity of 6-shogaol is in the micromolar range, whereas STC5 showed bioactivity in the sub micromolar range. The STCs showed anti-inflammatory effects via NRF2-dependent and NRF2-independent mechanisms. The STCs improved NRF2 activity through multiple (KEAP1-independent and -dependent) mechanisms. The STCs showed decreased reactivity with thiols than 6-shogaol and thus may possess fewer side-effects than 6-shogaol. The STCs were more metabolically stable than 6-shogaol.

Swietenine Alleviates Nonalcoholic Fatty Liver Disease in Diabetic Mice via Lipogenesis Inhibition and Antioxidant Mechanisms.

Our previous studies have reported the effect of swietenine (a major bioactive component of Swietenia macrophylla seeds) in reversing and potentiating the effect of metformin in hyperglycemia and hyperlipidaemia in diabetic rats. Moreover, we reported that the anti-inflammatory effect of swietenine is mediated via the activation of nuclear factor erythroid 2-related factor 2 (Nrf2). This study evaluated the effect of swietenine and its mechanisms in nonalcoholic fatty liver disease (NAFLD) in high-fat diet/streptozotocin-induced diabetic mice. The effect was assessed by determining blood biochemical parameters (glucose, cholesterol, triglycerides, alanine transaminase (ALT), asparate transaminase (AST), alkaline phosphatase (ALP), glutathione (GSH), total antioxidant capacity (TAC), and malondialdehyde (MDA)) and liver biochemical parameters (liver index, cholesterol, and triglycerides). Hepatic lipid accumulation (initial causative factor in NAFLD) was determined by oil-O-red staining. Gene expression (qPCR) and immunohistochemical studies were performed to elucidate the mechanism of swietenine's effect in NAFLD. The critical regulators (genes and proteins) involved in lipogenesis (ACLY, ACC1, FASN, SREBP1c, and ChREBPß) and oxidative stress (Nrf2, NQO-1 and HO-1) pathways were determined. In mice fed with a high-fat diet followed by streptozotocin injection, the liver cholesterol, triglycerides, and lipids were elevated. These increases were reversed by the oral administration of swietenine, 80 mg/kg body weight, on alternate days for eight weeks. Gene expression and immunohistochemical studies showed that swietenine reversed the elevated levels of crucial enzymes of lipogenesis (ACLY, ACC1 and FASN) and their master transcription factors (SREBP1c and ChREBPß). Furthermore, swietenine activated the Nrf2 antioxidant defense mechanism, as evidenced by the upregulated levels of Nrf2, NQO-1, and HO-1. It is concluded that swietenine shows beneficial effects in diabetes-induced NAFLD via inhibiting lipogenesis and activating the Nrf2 pathway.

Identification and Optimization of Novel Inhibitors of the Polyketide Synthase 13 Thioesterase Domain with Antitubercular Activity.

There is an urgent need for new tuberculosis (TB) treatments, with novel modes of action, to reduce the incidence/mortality of TB and to combat resistance to current treatments. Through both chemical and genetic methodologies, polyketide synthase 13 (Pks13) has been validated as essential for mycobacterial survival and as an attractive target for Mycobacterium tuberculosis growth inhibitors. A benzofuran series of inhibitors that targeted the Pks13 thioesterase domain, failed to progress to preclinical development due to concerns over cardiotoxicity. Herein, we report the identification of a novel oxadiazole series of Pks13 inhibitors, derived from a high-throughput screening hit and structure-guided optimization. This new series binds in the Pks13 thioesterase domain, with a distinct binding mode compared to the benzofuran series. Through iterative rounds of design, assisted by structural information, lead compounds were identified with improved antitubercular potencies (MIC < 1 µM) and in vitro ADMET profiles.

The role of DMPK science in improving pharmaceutical research and development efficiency.

The successful regulatory authority approval rate of drug candidates in the drug development pipeline is crucial for determining pharmaceutical research and development (R&D) efficiency. Regulatory authorities include the US Food and Drug Administration (FDA), European Medicines Agency (EMA), and Pharmaceutical and Food Safety Bureau Japan (PFSB), among others. Optimal drug metabolism and pharmacokinetics (DMPK) properties influence the progression of a drug candidate from the preclinical to the clinical phase. In this review, we provide a comprehensive assessment of essential concepts, methods, improvements, and challenges in DMPK science and its significance in drug development. This information provides insights into the association of DMPK science with pharmaceutical R&D efficiency.

Synthesis and Anti-Inflammatory Activity of 2-Amino-4,5,6,7-tetrahydrobenzo[b]thiophene-Derived NRF2 Activators.

This is the first study investigating the nuclear factor (erythroid-derived 2)-like 2 (NRF2) activity of compounds containing a new scaffold, tetrahydrobenzo[b]thiophene. Eighteen compounds were synthesised and confirmed their NRF2 activation through NQO1 enzymatic activity and mRNA expression of NQO1 and HO-1 in Hepa-1c1c7 cells. The compounds disrupted the interaction between Kelch-like ECH-associated protein 1 (KEAP1) and NRF2 via interfering with the KEAP1's Kelch domain. The compounds exhibited anti-inflammatory activity in Escherichia coli Lipopolysaccharide (LPSEc )-stimulated RAW 264.7 cells. The anti-inflammatory activity of the compounds was associated with their ability to activate NRF2. The compounds reversed the elevated levels of pro-inflammatory cytokines (IL-1ß, IL-6, TNF-α, and IFN-γ) and inflammatory mediators (PGE2, COX-2, and NF-κB). The compounds were metabolically stable in human, rat, and mouse liver microsomes and showed optimum half-life (T1/2 ) and intrinsic clearance (Clint ). The binding mode of the compounds and physicochemical properties were predicted via in silico studies.

Optimization of TAM16, a Benzofuran That Inhibits the Thioesterase Activity of Pks13; Evaluation toward a Preclinical Candidate for a Novel Antituberculosis Clinical Target.

With increasing drug resistance in tuberculosis (TB) patient populations, there is an urgent need for new drugs. Ideally, new agents should work through novel targets so that they are unencumbered by preexisting clinical resistance to current treatments. Benzofuran 1 was identified as a potential lead for TB inhibiting a novel target, the thioesterase domain of Pks13. Although, having promising activity against Mycobacterium tuberculosis, its main liability was inhibition of the hERG cardiac ion channel. This article describes the optimization of the series toward a preclinical candidate. Despite improvements in the hERG liability in vitro, when new compounds were assessed in ex vivo cardiotoxicity models, they still induced cardiac irregularities. Further series development was stopped because of concerns around an insufficient safety window. However, the demonstration of in vivo activity for multiple series members further validates Pks13 as an attractive novel target for antitubercular drugs and supports development of alternative chemotypes.

Lysyl-tRNA synthetase, a target for urgently needed M. tuberculosis drugs.

Tuberculosis is a major global cause of both mortality and financial burden mainly in low and middle-income countries. Given the significant and ongoing rise of drug-resistant strains of Mycobacterium tuberculosis within the clinical setting, there is an urgent need for the development of new, safe and effective treatments. Here the development of a drug-like series based on a fused dihydropyrrolidino-pyrimidine scaffold is described. The series has been developed against M. tuberculosis lysyl-tRNA synthetase (LysRS) and cellular studies support this mechanism of action. DDD02049209, the lead compound, is efficacious in mouse models of acute and chronic tuberculosis and has suitable physicochemical, pharmacokinetic properties and an in vitro safety profile that supports further development. Importantly, preliminary analysis using clinical resistant strains shows no pre-existing clinical resistance towards this scaffold.

Optimisation of 6-substituted isoquinolin-1-amine based ROCK-I inhibitors.

Rho kinase is an important target implicated in a variety of cardiovascular diseases. Herein, we report the optimisation of the fragment derived ATP-competitive ROCK inhibitors 1 and 2 into lead compound 14A. The initial goal of improving ROCK-I potency relative to 1, whilst maintaining a good PK profile, was achieved through removal of the aminoisoquinoline basic centre. Lead 14A was equipotent against both ROCK-I and ROCK-II, showed good in vivo efficacy in the spontaneous hypertensive rat model, and was further optimised to demonstrate the scope for improving selectivity over PKA versus hydroxy Fasudil 3.

Discovery of potent and orally bioavailable heterocycle-based cannabinoid CB1 receptor agonists.

Novel 3-(1H-indol-3-yl)-1,2,4-oxadiazoles and -thiadiazoles were synthesized and found to be potent CB1 cannabinoid receptor agonists. The oral bioavailability of these compounds could be dramatically improved by optimization studies of the side chains attached to the indole and oxadiazole cores, leading to identification of a CB1 receptor agonist with good oral activity in a range of preclinical models of antinociception and antihyperalgesia.

Fragment-based discovery of 6-substituted isoquinolin-1-amine based ROCK-I inhibitors.

Fragment-based NMR screening of a small literature focused library led to identification of a historical thrombin/FactorXa building block, 17A, that was found to be a ROCK-I inhibitor. In the absence of an X-ray structure, fragment growth afforded 6-substituted isoquinolin-1-amine derivatives which were profiled in the primary ROCK-I IMAP assay. Compounds 23A and 23E were selected as fragment optimized hits for further profiling. Compound 23A has similar ROCK-1 affinity, potency and cell based efficacy to the first generation ROCK inhibitors, however, it has a superior PK profile in C57 mouse. Compound 23E demonstrates the feasibility of improving ROCK-1 affinity, potency and cell based efficacy for the series, however, it has a poor PK profile relative to 23A.

Design, synthesis and structure-activity relationships of (indo-3-yl) heterocyclic derivatives as agonists of the CB1 receptor. Discovery of a clinical candidate.

We report an expansion of the structure-activity relationship (SAR) of a novel series of indole-3-heterocyclic CB1 receptor agonists. Starting from the potent but poorly soluble lead, 1, a rational approach was taken in order to balance solubility, hERG activity and potency while retaining the desired long duration of action within the mouse tail flick test. This led to the discovery of compound 38 which successfully progressed into clinical development.

Spirocycle MmpL3 Inhibitors with Improved hERG and Cytotoxicity Profiles as Inhibitors of Mycobacterium tuberculosis Growth.

With the emergence of multi-drug-resistant strains of Mycobacterium tuberculosis, there is a pressing need for new oral drugs with novel mechanisms of action. A number of scaffolds with potent anti-tubercular in vitro activity have been identified from phenotypic screening that appear to target MmpL3. However, the scaffolds are typically lipophilic, which facilitates partitioning into hydrophobic membranes, and several contain basic amine groups. Highly lipophilic basic amines are typically cytotoxic against mammalian cell lines and have associated off-target risks, such as inhibition of human ether-à-go-go related gene (hERG) and IKr potassium current modulation. The spirocycle compound 3 was reported to target MmpL3 and displayed promising efficacy in a murine model of acute tuberculosis (TB) infection. However, this highly lipophilic monobasic amine was cytotoxic and inhibited the hERG ion channel. Herein, the related spirocycles (1-2) are described, which were identified following phenotypic screening of the Eli Lilly corporate library against M. tuberculosis. The novel N-alkylated pyrazole portion offered improved physicochemical properties, and optimization led to identification of a zwitterion series, exemplified by lead 29, with decreased HepG2 cytotoxicity as well as limited hERG ion channel inhibition. Strains with mutations in MmpL3 were resistant to 29, and under replicating conditions, 29 demonstrated bactericidal activity against M. tuberculosis. Unfortunately, compound 29 had no efficacy in an acute model of TB infection; this was most likely due to the in vivo exposure remaining above the minimal inhibitory concentration for only a limited time.

The discovery and SAR of indoline-3-carboxamides--a new series of 5-HT6 antagonists.

Antagonists of the 5-HT(6) receptor have been shown to improve cognitive function in a wide range of animal models and as such may prove to be attractive agents for the symptomatic treatment of cognitive disorders such as Alzheimer's disease (AD) and schizophrenia. We report herein the identification and SAR around N-(2-aminoalkyl)-1-(arylsulfonyl)indoline-3-carboxamides-a novel chemotype of 5-HT(6) antagonists.

Identification and Optimization of a Series of 8-Hydroxy Naphthyridines with Potent In Vitro Antileishmanial Activity: Initial SAR and Assessment of In Vivo Activity.

Visceral leishmaniasis (VL) is a parasitic infection that results in approximately 26 000-65 000 deaths annually. The available treatments are hampered by issues such as toxicity, variable efficacy, and unsuitable dosing options. The need for new treatments is urgent and led to a collaboration between the Drugs for Neglected Diseases initiative (DNDi), GlaxoSmithKline (GSK), and the University of Dundee. An 8-hydroxynaphthyridine was identified as a start point, and an early compound demonstrated weak efficacy in a mouse model of VL but was hampered by glucuronidation. Efforts to address this led to the development of compounds with improved in vitro profiles, but these were poorly tolerated in vivo. Investigation of the mode of action (MoA) demonstrated that activity was driven by sequestration of divalent metal cations, a mechanism which was likely to drive the poor tolerability. This highlights the importance of investigating MoA and pharmacokinetics at an early stage for phenotypically active series.

Discovery of Soft-Drug Topical Tool Modulators of Sphingosine-1-phosphate Receptor 1 (S1PR1).

In order to study the role of S1PRs in inflammatory skin disease, S1PR modulators are dosed orally and topically in animal models of disease. The topical application of S1PR modulators in these models may, however, lead to systemic drug concentrations, which can complicate interpretation of the observed effects. We set out to design soft drug S1PR modulators as topical tool compounds to overcome this limitation. A fast follower approach starting from the drug ponesimod allowed the rapid development of an active phenolic series of soft drugs. The phenols were, however, chemically unstable. Protecting the phenol as an ester removed the instability and provided a compound that is converted by enzymatic hydrolysis in the skin to the phenolic soft drug species. In simple formulations, topical dosing of these S1PR modulators to mice led to micromolar skin concentrations but no detectable blood concentrations. These topical tools will allow researchers to investigate the role of S1PR in skin, without involvement of systemic S1PR biology.

Selective relaxant binding agents for reversal of neuromuscular blockade.

Traditionally, reversal of neuromuscular blockade during anaesthesia was achieved by increasing the acetylcholine concentration in the neuromuscular junction using acetylcholinesterase inhibitors. However, this is ineffective against profound blockade. Furthermore, the increase in acetylcholine level is not limited to the neuromuscular junction, resulting in unwanted side effects requiring co-treatment with muscarinic antagonists. Selective relaxant binding agents offer a new approach for the reversal of neuromuscular blockade: encapsulation of the neuromuscular blocking agent, resulting in inactivation. As part of this new approach, cyclodextrin molecules have been designed that selectively encapsulate steroidal neuromuscular blocking agents. Both animal and human experiments have demonstrated that fast, effective and complete recovery from both normal and profound neuromuscular blockade is now possible. Furthermore, these cyclodextrin derivatives do not have the unwanted side effects of acetylcholinesterase inhibitors.