Single-Dose Drug Development Candidate for Schistosomiasis.

Aryl hydantoins were identified in the early 1980s as a promising antischistosomal chemotype. However, as exemplified by Ro 13-3978, this compound series produced antiandrogenic side effects on the host, a not unexpected outcome given their structural similarity to the antiandrogenic drug nilutamide. The two key advances in our optimization of Ro 13-3978 were swapping the aryl trifluoromethyl substituent with a difluoroethyl to abolish antiandrogenic effects and replacing the hydrogen atoms of the gem-dimethyl substructure with deuterium atoms to increase metabolic stability. Combining these two structural changes led to the discovery of single-dose drug candidate AR102, a compound with potent, selective, and broad-spectrum activity against schistosomes, a long pharmacokinetic half-life in preclinical species, and an acceptable safety profile.

The effects of single and multiple resistance mechanisms on bacterial response to meropenem.

OBJECTIVES: Meropenem is commonly used against Pseudomonas aeruginosa. Traditionally, the time unbound antibiotic concentration exceeds the MIC (fT>MIC) is used to select carbapenem regimens. We aimed to characterize the effects of different baseline resistance mechanisms on bacterial killing and resistance emergence; evaluate whether fT>MIC can predict these effects; and, develop a novel Quantitative and Systems Pharmacology (QSP) model to describe the effects of baseline resistance mechanisms on the time-course of bacterial response. METHODS: Seven isogenic P. aeruginosa strains with a range of resistance mechanisms and MICs were used in 10-day hollow-fiber infection model studies. Meropenem pharmacokinetic profiles were simulated for various regimens (t1/2,meropenem = 1.5 h). All viable counts on drug-free, 3 × MIC, and 5 × MIC meropenem-containing agar across all strains, five regimens, and control (n = 90 profiles) were simultaneously subjected to QSP modeling. Whole genome sequencing was completed for total population samples and emergent resistant colonies at 239 h. RESULTS: Regimens achieving ≥98%fT>1×MIC suppressed resistance emergence of the mexR knockout strain. Even 100%fT>5 × MIC failed to achieve this against the strain with OprD loss and the ampD and mexR double-knockout strain. Baseline resistance mechanisms affected bacterial outcomes, even for strains with the same MIC. Genomic analysis revealed that pre-existing resistant subpopulations drove resistance emergence. During meropenem exposure, mutations in mexR were selected in strains with baseline oprD mutations, and vice versa, confirming these as major mechanisms of resistance emergence. Secondary mutations occurred in lysS or argS, coding for lysyl and arginyl tRNA synthetases, respectively. DISCUSSION: The QSP model well-characterized all bacterial outcomes of the seven strains simultaneously, which fT>MIC could not.

Identification of potent and reversible piperidine carboxamides that are species-selective orally active proteasome inhibitors to treat malaria.

Malaria remains a global health concern as drug resistance threatens treatment programs. We identified a piperidine carboxamide (SW042) with anti-malarial activity by phenotypic screening. Selection of SW042-resistant Plasmodium falciparum (Pf) parasites revealed point mutations in the Pf_proteasome ß5 active-site (Pfß5). A potent analog (SW584) showed efficacy in a mouse model of human malaria after oral dosing. SW584 had a low propensity to generate resistance (minimum inoculum for resistance [MIR] >109) and was synergistic with dihydroartemisinin. Pf_proteasome purification was facilitated by His8-tag introduction onto ß7. Inhibition of Pfß5 correlated with parasite killing, without inhibiting human proteasome isoforms or showing cytotoxicity. The Pf_proteasome_SW584 cryoelectron microscopy (cryo-EM) structure showed that SW584 bound non-covalently distal from the catalytic threonine, in an unexplored pocket at the ß5/ß6/ß3 subunit interface that has species differences between Pf and human proteasomes. Identification of a reversible, species selective, orally active series with low resistance propensity provides a path for drugging this essential target.

DNDI-6174 is a preclinical candidate for visceral leishmaniasis that targets the cytochrome bc1.

New drugs for visceral leishmaniasis that are safe, low cost, and adapted to the field are urgently required. Despite concerted efforts over the last several years, the number of new chemical entities that are suitable for clinical development for the treatment of Leishmania remains low. Here, we describe the discovery and preclinical development of DNDI-6174, an inhibitor of Leishmania cytochrome bc1 complex activity that originated from a phenotypically identified pyrrolopyrimidine series. This compound fulfills all target candidate profile criteria required for progression into preclinical development. In addition to good metabolic stability and pharmacokinetic properties, DNDI-6174 demonstrates potent in vitro activity against a variety of Leishmania species and can reduce parasite burden in animal models of infection, with the potential to approach sterile cure. No major flags were identified in preliminary safety studies, including an exploratory 14-day toxicology study in the rat. DNDI-6174 is a cytochrome bc1 complex inhibitor with acceptable development properties to enter preclinical development for visceral leishmaniasis.

Design, synthesis and evaluation of novel 2-phenyl-3-(1H-pyrazol-4-yl)pyridine positive allosteric modulators for the M4 mAChR.

Translation of muscarinic acetylcholine receptor (mAChR) agonists into clinically used therapeutic agents has been difficult due to their poor subtype selectivity. M4 mAChR subtype-selective positive allosteric modulators (PAMs) may provide better therapeutic outcomes, hence investigating their detailed pharmacological properties is crucial to advancing them into the clinic. Herein, we report the synthesis and comprehensive pharmacological evaluation of M4 mAChR PAMs structurally related to 1e, Me-C-c, [11C]MK-6884 and [18F]12. Our results show that small structural changes to the PAMs can result in pronounced differences to baseline, potency (pEC50) and maximum effect (Emax) measures in cAMP assays when compared to the endogenous ligand acetylcholine (ACh) without the addition of the PAMs. Eight selected PAMs were further assessed to determine their binding affinity and potential signalling bias profile between cAMP and ß-arrestin 2 recruitment. These rigorous analyses resulted in the discovery of the novel PAMs, 6k and 6l, which exhibit improved allosteric properties compared to the lead compound, and probative in vivo exposure studies in mice confirmed that they maintain the ability to cross the blood-brain barrier, making them more suitable for future preclinical assessment.

Increased In Vivo Exposure of N-(4-Hydroxyphenyl) Retinamide (4-HPR) to Achieve Plasma Concentrations Effective against Dengue Virus.

N-(4-hydroxyphenyl) retinamide (4-HPR, or fenretinide) has promising in vitro and in vivo antiviral activity against a range of flaviviruses and an established safety record, but there are challenges to its clinical use. This study evaluated the in vivo exposure profile of a 4-HPR dosage regime previously shown to be effective in a mouse model of severe dengue virus (DENV) infection, comparing it to an existing formulation for human clinical use for other indications and developed/characterised self-emulsifying lipid-based formulations of 4-HPR to enhance 4-HPR in vivo exposure. Pharmacokinetic (PK) analysis comprising single-dose oral and IV plasma concentration-time profiles was performed in mice; equilibrium solubility testing of 4-HPR in a range of lipids, surfactants and cosolvents was used to inform formulation approaches, with lead formulation candidates digested in vitro to analyse solubilisation/precipitation prior to in vivo testing. PK analysis suggested that effective plasma concentrations could be achieved with the clinical formulation, while novel lipid-based formulations achieved > 3-fold improvement. Additionally, 4-HPR exposure was found to be limited by both solubility and first-pass intestinal elimination but could be improved through inhibition of cytochrome P450 (CYP) metabolism. Simulated exposure profiles suggest that a b.i.d dosage regime is likely to maintain 4-HPR above the minimum effective plasma concentration for anti-DENV activity using the clinical formulation, with new formulations/CYP inhibition viable options to increase exposure in the future.

PPARα and PPARγ activation is associated with pleural mesothelioma invasion but therapeutic inhibition is ineffective.

Mesothelioma is a cancer that typically originates in the pleura of the lungs. It rapidly invades the surrounding tissues, causing pain and shortness of breath. We compared cell lines injected either subcutaneously or intrapleurally and found that only the latter resulted in invasive and rapid growth. Pleural tumors displayed a transcriptional signature consistent with increased activity of nuclear receptors PPARα and PPARγ and with an increased abundance of endogenous PPAR-activating ligands. We found that chemical probe GW6471 is a potent, dual PPARα/γ antagonist with anti-invasive and anti-proliferative activity in vitro. However, administration of GW6471 at doses that provided sustained plasma exposure levels sufficient for inhibition of PPARα/γ transcriptional activity did not result in significant anti-mesothelioma activity in mice. Lastly, we demonstrate that the in vitro anti-tumor effect of GW6471 is off-target. We conclude that dual PPARα/γ antagonism alone is not a viable treatment modality for mesothelioma.

Development of [18F]MIPS15692, a radiotracer with in vitro proof-of-concept for the imaging of MER tyrosine kinase (MERTK) in neuroinflammatory disease.

MER tyrosine kinase (MERTK) upregulation is associated with M2 polarization of microglia, which plays a vital role in neuroregeneration following damage induced by neuroinflammatory diseases such as multiple sclerosis (MS). Therefore, a radiotracer specific for MERTK could be of great utility in the clinical management of MS, for the detection and differentiation of neuroregenerative and neurodegenerative processes. This study aimed to develop an [18F] ligand with high affinity and selectivity for MERTK as a potential positron emission tomography (PET) radiotracer. MIPS15691 and MIPS15692 were synthesized and kinase assays were utilized to determine potency and selectivity for MERTK. Both compounds were shown to be potent against MERTK, with respective IC50 values of 4.6 nM and 4.0 nM, and were also MERTK-selective. Plasma and brain pharmacokinetics were measured in mice and led to selection of MIPS15692 over MIPS15691. X-ray crystallography was used to visualize how MIPS15692 is recognized by the enzyme. [18F]MIPS15692 was synthesized using an automated iPHASE FlexLab module, with a molar activity (Am) of 49 ± 26 GBq/µmol. The radiochemical purity of [18F]MIPS15692 was >99% and the decay-corrected radiochemical yields (RCYs) were determined as 2.45 ± 0.85%. Brain MERTK protein density was measured by a saturation binding assay in the brain slices of a cuprizone mouse model of MS. High levels of specific binding of [18F]MIPS15692 to MERTK were found, especially in the corpus callosum/hippocampus (CC/HC). The in vivo PET imaging study of [18F]MIPS15692 suggested that its neuroPK is sub-optimal for clinical use. Current efforts are underway to optimize the neuroPK of our next generation PET radiotracers for maximal in vivo utility.

Discovery and Structure-Activity Relationships of Quinazolinone-2-carboxamide Derivatives as Novel Orally Efficacious Antimalarials.

A phenotypic high-throughput screen allowed discovery of quinazolinone-2-carboxamide derivatives as a novel antimalarial scaffold. Structure-activity relationship studies led to identification of a potent inhibitor 19f, 95-fold more potent than the original hit compound, active against laboratory-resistant strains of malaria. Profiling of 19f suggested a fast in vitro killing profile. In vivo activity in a murine model of human malaria in a dose-dependent manner constitutes a concomitant benefit.

Cytochrome P450-Mediated Metabolism and CYP Inhibition for the Synthetic Peroxide Antimalarial OZ439.

OZ439 is a potent synthetic ozonide evaluated for the treatment of uncomplicated malaria. The metabolite profile of OZ439 was characterized in vitro using human liver microsomes combined with LC/MS-MS, chemical derivatization, and metabolite synthesis. The primary biotransformations were monohydroxylation at the three distal carbon atoms of the spiroadamantane substructure, with minor contributions from N-oxidation of the morpholine nitrogen and deethylation cleavage of the morpholine ring. Secondary transformations resulted in the formation of dihydroxylation metabolites and metabolites containing both monohydroxylation and morpholine N-oxidation. With the exception of two minor metabolites, none of the other metabolites had appreciable antimalarial activity. Reaction phenotyping indicated that CYP3A4 is the enzyme responsible for the metabolism of OZ439, and it was found to inhibit CYP3A via both direct and mechanism-based inhibition. Elucidation of the metabolic pathways and kinetics will assist with efforts to predict potential metabolic drug-drug interactions and support physiologically based pharmacokinetic (PBPK) modeling.

Potent Antimalarials with Development Potential Identified by Structure-Guided Computational Optimization of a Pyrrole-Based Dihydroorotate Dehydrogenase Inhibitor Series.

Dihydroorotate dehydrogenase (DHODH) has been clinically validated as a target for the development of new antimalarials. Experience with clinical candidate triazolopyrimidine DSM265 (1) suggested that DHODH inhibitors have great potential for use in prophylaxis, which represents an unmet need in the malaria drug discovery portfolio for endemic countries, particularly in areas of high transmission in Africa. We describe a structure-based computationally driven lead optimization program of a pyrrole-based series of DHODH inhibitors, leading to the discovery of two candidates for potential advancement to preclinical development. These compounds have improved physicochemical properties over prior series frontrunners and they show no time-dependent CYP inhibition, characteristic of earlier compounds. Frontrunners have potent antimalarial activity in vitro against blood and liver schizont stages and show good efficacy in Plasmodium falciparum SCID mouse models. They are equally active against P. falciparum and Plasmodium vivax field isolates and are selective for Plasmodium DHODHs versus mammalian enzymes.

Systematic evaluation of structure-property relationships and pharmacokinetics in 6-(hetero)aryl-substituted matched pair analogs of amiloride and 5-(N,N-hexamethylene)amiloride.

The K+-sparing diuretic amiloride elicits anticancer activities in multiple animal models. During our recent medicinal chemistry campaign aiming to identify amiloride analogs with improved properties for potential use in cancer, we discovered novel 6-(hetero)aryl-substituted amiloride and 5-(N,N-hexamethylene)amiloride (HMA) analogs with up to 100-fold higher potencies than the parent compounds against urokinase plasminogen activator (uPA), one of amiloride's putative anticancer targets, and no diuretic or antikaliuretic effects. Here, we report the systematic evaluation of structure-property relationships (lipophilicity, aqueous solubility and in vitro metabolic stability in human and mouse liver microsomes) in twelve matched pair analogs selected from our 6-substituted amiloride and HMA libraries. Mouse plasma stability, plasma protein binding, Caco-2 cell permeability, cardiac ion channel activity and pharmacokinetics in mice (PO and IV) and rats (IV) are described alongside amiloride and HMA comparators for a subset of the four most promising matched-pair analogs. The findings combined with earlier uPA activity/selectivity and other data ultimately drove selection of two analogs (AA1-39 and AA1-41) that showed efficacy in separate mouse cancer metastasis studies.

Repositioning and Characterization of 1-(Pyridin-4-yl)pyrrolidin-2-one Derivatives as Plasmodium Cytoplasmic Prolyl-tRNA Synthetase Inhibitors.

Prolyl-tRNA synthetase (PRS) is a clinically validated antimalarial target. Screening of a set of PRS ATP-site binders, initially designed for human indications, led to identification of 1-(pyridin-4-yl)pyrrolidin-2-one derivatives representing a novel antimalarial scaffold. Evidence designates cytoplasmic PRS as the drug target. The frontrunner 1 and its active enantiomer 1-S exhibited low-double-digit nanomolar activity against resistant Plasmodium falciparum (Pf) laboratory strains and development of liver schizonts. No cross-resistance with strains resistant to other known antimalarials was noted. In addition, a similar level of growth inhibition was observed against clinical field isolates of Pf and P. vivax. The slow killing profile and the relative high propensity to develop resistance in vitro (minimum inoculum resistance of 8 × 105 parasites at a selection pressure of 3 × IC50) constitute unfavorable features for treatment of malaria. However, potent blood stage and antischizontal activity are compelling for causal prophylaxis which does not require fast onset of action. Achieving sufficient on-target selectivity appears to be particularly challenging and should be the primary focus during the next steps of optimization of this chemical series. Encouraging preliminary off-target profile and oral efficacy in a humanized murine model of Pf malaria allowed us to conclude that 1-(pyridin-4-yl)pyrrolidin-2-one derivatives represent a promising starting point for the identification of novel antimalarial prophylactic agents that selectively target Plasmodium PRS.

Discovery of Potent and Fast-Acting Antimalarial Bis-1,2,4-triazines.

Novel 3,3'-disubstituted-5,5'-bi(1,2,4-triazine) compounds with potent in vitro activity against Plasmodium falciparum parasites were recently discovered. To improve the pharmacokinetic properties of the triazine derivatives, a new structure-activity relationship (SAR) investigation was initiated with a focus on enhancing the metabolic stability of lead compounds. These efforts led to the identification of second-generation highly potent antimalarial bis-triazines, exemplified by triazine 23, which exhibited significantly improved in vitro metabolic stability (8 and 42 µL/min/mg protein in human and mouse liver microsomes). The disubstituted triazine dimer 23 was also observed to suppress parasitemia in the Peters 4-day test with a mean ED50 value of 1.85 mg/kg/day and exhibited a fast-killing profile, revealing a new class of orally available antimalarial compounds of considerable interest.

Carvedilol blocks neural regulation of breast cancer progression in vivo and is associated with reduced breast cancer mortality in patients.

PURPOSE: The sympathetic nervous system drives breast cancer progression through ß-adrenergic receptor signalling. This discovery has led to the consideration of cardiac ß-blocker drugs as novel strategies for anticancer therapies. Carvedilol is a ß-blocker used in the management of cardiovascular disorders, anxiety, migraine and chemotherapy-induced cardiotoxicity. However, little is known about how carvedilol affects cancer-related outcomes. METHODS: To address this, we investigated the effects of carvedilol on breast cancer cell lines, in mouse models of breast cancer and in a large cohort of patients with breast cancer (n = 4014). RESULTS: Treatment with carvedilol blocked the effects of sympathetic nervous system activation, reducing primary tumour growth and metastasis in a mouse model of breast cancer and preventing invasion by breast cancer cell lines. A retrospective analysis found that women using carvedilol at breast cancer diagnosis (n = 136) had reduced breast cancer-specific mortality compared with women who did not (n = 3878) (5-year cumulative incidence of breast cancer deaths: 3.1% versus 5.7%; p = 0.024 and 0.076 from univariate and multivariable analyses, respectively) after a median follow-up of 5.5 years. CONCLUSIONS: These findings provide a rationale to further explore the use of the ß-blocker carvedilol as a novel strategy to slow cancer progression.

Selecting an anti-malarial clinical candidate from two potent dihydroisoquinolones.

BACKGROUND: The ongoing global malaria eradication campaign requires development of potent, safe, and cost-effective drugs lacking cross-resistance with existing chemotherapies. One critical step in drug development is selecting a suitable clinical candidate from late leads. The process used to select the clinical candidate SJ733 from two potent dihydroisoquinolone (DHIQ) late leads, SJ733 and SJ311, based on their physicochemical, pharmacokinetic (PK), and toxicity profiles is described. METHODS: The compounds were tested to define their physicochemical properties including kinetic and thermodynamic solubility, partition coefficient, permeability, ionization constant, and binding to plasma proteins. Metabolic stability was assessed in both microsomes and hepatocytes derived from mice, rats, dogs, and humans. Cytochrome P450 inhibition was assessed using recombinant human cytochrome enzymes. The pharmacokinetic profiles of single intravenous or oral doses were investigated in mice, rats, and dogs. RESULTS: Although both compounds displayed similar physicochemical properties, SJ733 was more permeable but metabolically less stable than SJ311 in vitro. Single dose PK studies of SJ733 in mice, rats, and dogs demonstrated appreciable oral bioavailability (60-100%), whereas SJ311 had lower oral bioavailability (mice 23%, rats 40%) and higher renal clearance (10-30 fold higher than SJ733 in rats and dogs), suggesting less favorable exposure in humans. SJ311 also displayed a narrower range of dose-proportional exposure, with plasma exposure flattening at doses above 200 mg/kg. CONCLUSION: SJ733 was chosen as the candidate based on a more favorable dose proportionality of exposure and stronger expectation of the ability to justify a strong therapeutic index to regulators.

Development of Novel 4-Arylpyridin-2-one and 6-Arylpyrimidin-4-one Positive Allosteric Modulators of the M1 Muscarinic Acetylcholine Receptor.

This study investigated the structure-activity relationships of 4-phenylpyridin-2-one and 6-phenylpyrimidin-4-one M1 muscarinic acetylcholine receptor (M1 mAChRs) positive allosteric modulators (PAMs). The presented series focuses on modifications to the core and top motif of the reported leads, MIPS1650 (1) and MIPS1780 (2). Profiling of our novel analogues showed that these modifications result in more nuanced effects on the allosteric properties compared to our previous compounds with alterations to the biaryl pendant. Further pharmacological characterisation of the selected compounds in radioligand binding, IP1 accumulation and ß-arrestin 2 recruitment assays demonstrated that, despite primarily acting as affinity modulators, the PAMs displayed different pharmacological properties across the two cellular assays. The novel PAM 7 f is a potential lead candidate for further development of peripherally restricted M1 PAMs, due to its lower blood-brain-barrier (BBB) permeability and improved exposure in the periphery compared to lead 2.

The Anti-Inflammatory Drug Aspirin Does Not Protect Against Chemotherapy-Induced Memory Impairment by Paclitaxel in Mice.

Inflammation has been proposed to play a causal role in chemobrain which-if true-would represent an opportunity to repurpose existing anti-inflammatory drugs for the prevention and treatment of chemobrain. Here, we show that the chemoagent paclitaxel induces memory impairment and anhedonia in mice within 24 h of treatment cessation, but inflammation is not present until 2 weeks after treatment. We find no evidence of brain inflammation as measured by cytokine analysis at any time point. Furthermore, treating with aspirin to block inflammation did not affect paclitaxel-induced memory impairment. These findings suggest that inflammation may not be responsible for memory impairment induced by paclitaxel. These results contrast with recent findings of a causal role for inflammation in cancer-induced memory deficits in mice that were prevented by treatment with oral aspirin, suggesting that cognitive impairment in cancer patients undergoing treatment may arise from multiple convergent mechanisms.

In the Loop: Extrastriatal Regulation of Spiny Projection Neurons by GPR52.

GPR52 is a Gαs-coupled orphan receptor identified as a putative target for the treatment of schizophrenia. The unique expression and signaling profile of GPR52 in key areas of dopamine and glutamate dysregulation suggests its activation may resolve both cortical and striatal dysfunction in the disorder. GPR52 mRNA is enriched in the striatum, almost exclusively on dopamine D2-expressing medium spiny neurons (MSNs), and to a lesser extent in the cortex, predominantly on D1-expressing pyramidal neurons. Synthetic, small molecule GPR52 agonists are effective in preclinical models of psychosis; however, the relative contribution of cortical and striatal GPR52 is unknown. Here we show that the GPR52 agonist, 3-BTBZ, inhibits phencyclidine-induced hyperlocomotor activity to a greater degree than amphetamine-induced motor effects, suggesting a mechanism beyond functional antagonism of striatal dopamine D2 receptor signaling. Using DARPP-32 phosphorylation and electrophysiological recordings in either striatopallidal or striatonigral MSNs, we were surprised to find no significant effect of 3-BTBZ in striatopallidal MSNs, but GPR52-mediated effects in striatonigral MSNs, where its mRNA is absent. 3-BTBZ increases phosphorylation of T75 on DARPP-32 in striatonigral MSNs, an effect that was dependent on cortical inputs. A similar role for GPR52 in regulating extrastriatal glutamatergic drive onto striatonigral MSNs was also evident in recordings of spontaneous excitatory postsynaptic currents and was shown to be dependent on the metabotropic glutamate (mGlu) receptor subtype 1. Our results demonstrate that GPR52-mediated regulation of striatal function depends heavily on extrastriatal inputs, which may further support its utility as a novel target for the treatment of schizophrenia.