The effect of the EP3 antagonist DG-041 on male mice with diet-induced obesity.

BACKGROUND/AIMS: The prostaglandin E2 (PGE2) EP3 receptor has a multifaceted role in metabolism. Drugs targeting EP3 have been proposed as therapeutics for diabetes; however, studies utilizing global EP3 knockout mice suggest that EP3 blockade increases obesity and insulin resistance. The present studies attempt to determine the effect of acute EP3 antagonist treatment on the diabetic phenotype. METHODS: DG-041 was confirmed to be a high affinity antagonist at the mouse EP3 receptor by competition radioligand binding and by blockade of EP3-mediated responses. DG-041 pharmacokinetic studies were performed to determine the most efficacious route of administration. Male C57BL/6 × BALB/c (CB6F1) mice were fed diets containing 10%, 45%, or 60% calories from fat to induce obesity. Changes to the metabolic phenotype in these mice were evaluated after one week treatment with DG-041. RESULTS: Subcutaneous injections of DG-041 at 20 mg/kg blocked the sulprostone-evoked rise in mean arterial pressure confirming the efficacy of this administration regime. Seven day treatment with DG-041 had minimal effect on body composition or glycemic control. DG-041 administration caused a reduction in skeletal muscle triglyceride content while showing a trend toward increased hepatic triglycerides. CONCLUSION: Short term EP3 administration of DG-041 produced effective blockade of the EP3 receptor and decreased skeletal muscle triglyceride content but had no significant effects on the diabetic phenotype.

SAR inspired by aldehyde oxidase (AO) metabolism: Discovery of novel, CNS penetrant tricyclic M4 PAMs.

This letter describes progress towards an M4 PAM preclinical candidate inspired by an unexpected aldehyde oxidase (AO) metabolite of a novel, CNS penetrant thieno[2,3-c]pyridine core to an equipotent, non-CNS penetrant thieno[2,3-c]pyrdin-7(6H)-one core. Medicinal chemistry design efforts yielded two novel tricyclic cores that enhanced M4 PAM potency, regained CNS penetration, displayed favorable DMPK properties and afforded robust in vivo efficacy in reversing amphetamine-induced hyperlocomotion in rats.

mGlu5 positive allosteric modulation normalizes synaptic plasticity defects and motor phenotypes in a mouse model of Rett syndrome.

Rett syndrome (RS) is a neurodevelopmental disorder that shares many symptomatic and pathological commonalities with idiopathic autism. Alterations in protein synthesis-dependent synaptic plasticity (PSDSP) are a hallmark of a number of syndromic forms of autism; in the present work, we explore the consequences of disruption and rescue of PSDSP in a mouse model of RS. We report that expression of a key regulator of synaptic protein synthesis, the metabotropic glutamate receptor 5 (mGlu5) protein, is significantly reduced in both the brains of RS model mice and in the motor cortex of human RS autopsy samples. Furthermore, we demonstrate that reduced mGlu5 expression correlates with attenuated DHPG-induced long-term depression in the hippocampus of RS model mice, and that administration of a novel mGlu5 positive allosteric modulator (PAM), termed VU0462807, can rescue synaptic plasticity defects. Additionally, treatment of Mecp2-deficient mice with VU0462807 improves motor performance (open-field behavior and gait dynamics), corrects repetitive clasping behavior, as well as normalizes cued fear-conditioning defects. Importantly, due to the rationale drug discovery approach used in its development, our novel mGlu5 PAM improves RS phenotypes and synaptic plasticity defects without evoking the overt adverse effects commonly associated with potentiation of mGlu5 signaling (i.e. seizures), or affecting cardiorespiratory defects in RS model mice. These findings provide strong support for the continued development of mGlu5 PAMs as potential therapeutic agents for use in RS, and, more broadly, for utility in idiopathic autism.

Discovery of 6-(pyrimidin-5-ylmethyl)quinoline-8-carboxamide negative allosteric modulators of metabotropic glutamate receptor subtype 5.

Based on previous work that established fused heterocycles as viable alternatives for the picolinamide core of our lead series of mGlu5 negative allosteric modulators (NAMs), we designed a novel series of 6-(pyrimidin-5-ylmethyl)quinoline-8-carboxamide mGlu5 NAMs. These new quinoline derivatives also contained carbon linkers as replacements for the diaryl ether oxygen atom common to our previously published chemotypes. Compounds were evaluated in a cell-based functional mGlu5 assay, and an exemplar analog 27 was >60-fold selective versus the other seven mGlu receptors. Selected compounds were also studied in metabolic stability assays in rat and human S9 hepatic fractions and exhibited a mixture of P450- and non-P450-mediated metabolism.

A novel in vitro allometric scaling methodology for aldehyde oxidase substrates to enable selection of appropriate species for traditional allometry.

1. Failure to predict human pharmacokinetics of aldehyde oxidase (AO) substrates using traditional allometry has been attributed to species differences in AO metabolism. 2. To identify appropriate species for predicting human in vivo clearance by single-species scaling (SSS) or multispecies allometry (MA), we scaled in vitro intrinsic clearance (CLint) of five AO substrates obtained from hepatic S9 of mouse, rat, guinea pig, monkey and minipig to human in vitro CLint. 3. When predicting human in vitro CLint, average absolute fold-error was ≤2.0 by SSS with monkey, minipig and guinea pig (rat/mouse >3.0) and was <3.0 by most MA species combinations (including rat/mouse combinations). 4. Interspecies variables, including fraction metabolized by AO (Fm,AO) and hepatic extraction ratios (E) were estimated in vitro. SSS prediction fold-errors correlated with the animal:human ratio of E (r2 = 0.6488), but not Fm,AO (r2 = 0.0051). 5. Using plasma clearance (CLp) from the literature, SSS with monkey was superior to rat or mouse at predicting human CLp of BIBX1382 and zoniporide, consistent with in vitro SSS assessments. 6. Evaluation of in vitro allometry, Fm,AO and E may prove useful to guide selection of suitable species for traditional allometry and prediction of human pharmacokinetics of AO substrates.

Allosteric activation of M4 muscarinic receptors improve behavioral and physiological alterations in early symptomatic YAC128 mice.

Mutations that lead to Huntington's disease (HD) result in increased transmission at glutamatergic corticostriatal synapses at early presymptomatic stages that have been postulated to set the stage for pathological changes and symptoms that are observed at later ages. Based on this, pharmacological interventions that reverse excessive corticostriatal transmission may provide a novel approach for reducing early physiological changes and motor symptoms observed in HD. We report that activation of the M4 subtype of muscarinic acetylcholine receptor reduces transmission at corticostriatal synapses and that this effect is dramatically enhanced in presymptomatic YAC128 HD and BACHD relative to wild-type mice. Furthermore, chronic administration of a novel highly selective M4 positive allosteric modulator (PAM) beginning at presymptomatic ages improves motor and synaptic deficits in 5-mo-old YAC128 mice. These data raise the exciting possibility that selective M4 PAMs could provide a therapeutic strategy for the treatment of HD.

Discovery of imidazo[1,2-a]-, [1,2,4]triazolo[4,3-a]-, and [1,2,4]triazolo[1,5-a]pyridine-8-carboxamide negative allosteric modulators of metabotropic glutamate receptor subtype 5.

Based on a hypothesis that an intramolecular hydrogen bond was present in our lead series of picolinamide mGlu5 NAMs, we reasoned that an inactive nicotinamide series could be modified through introduction of a fused heterocyclic core to generate potent mGlu5 NAMs. In this Letter, we describe the synthesis and evaluation of compounds that demonstrate the viability of that approach. Selected analogs were profiled in a variety of in vitro assays, and two compounds were evaluated in rat pharmacokinetic studies and a mouse model of obsessive-compulsive disorder. Ancillary pharmacology screening revealed that members of this series exhibited moderate inhibition of the dopamine transporter (DAT), and SAR was developed that expanded the selectivity for mGlu5 versus DAT.

VU0477573: Partial Negative Allosteric Modulator of the Subtype 5 Metabotropic Glutamate Receptor with In Vivo Efficacy.

Negative allosteric modulators (NAMs) of metabotropic glutamate receptor subtype 5 (mGlu5) have potential applications in the treatment of fragile X syndrome, levodopa-induced dyskinesia in Parkinson disease, Alzheimer disease, addiction, and anxiety; however, clinical and preclinical studies raise concerns that complete blockade of mGlu5 and inverse agonist activity of current mGlu5 NAMs contribute to adverse effects that limit the therapeutic use of these compounds. We report the discovery and characterization of a novel mGlu5 NAM, N,N-diethyl-5-((3-fluorophenyl)ethynyl)picolinamide (VU0477573) that binds to the same allosteric site as the prototypical mGlu5 NAM MPEP but displays weak negative cooperativity. Because of this weak cooperativity, VU0477573 acts as a "partial NAM" so that full occupancy of the MPEP site does not completely inhibit maximal effects of mGlu5 agonists on intracellular calcium mobilization, inositol phosphate (IP) accumulation, or inhibition of synaptic transmission at the hippocampal Schaffer collateral-CA1 synapse. Unlike previous mGlu5 NAMs, VU0477573 displays no inverse agonist activity assessed using measures of effects on basal [(3)H]inositol phosphate (IP) accumulation. VU0477573 acts as a full NAM when measuring effects on mGlu5-mediated extracellular signal-related kinases 1/2 phosphorylation, which may indicate functional bias. VU0477573 exhibits an excellent pharmacokinetic profile and good brain penetration in rodents and provides dose-dependent full mGlu5 occupancy in the central nervous system (CNS) with systemic administration. Interestingly, VU0477573 shows robust efficacy, comparable to the mGlu5 NAM MTEP, in models of anxiolytic activity at doses that provide full CNS occupancy of mGlu5 and demonstrate an excellent CNS occupancy-efficacy relationship. VU0477573 provides an exciting new tool to investigate the efficacy of partial NAMs in animal models.

Evaluating the Disposition of a Mixed Aldehyde Oxidase/Cytochrome P450 Substrate in Rats with Attenuated P450 Activity.

Marketed drugs cleared by aldehyde oxidase (AO) are few, with no known clinically relevant pharmacokinetic drug interactions associated with AO inhibition, whereas cytochrome P450 (P450) inhibition or induction mediates a number of clinical drug interactions. Little attention has been given to the consequences of coadministering a P450 inhibitor with a compound metabolized by both AO and P450. Upon discovering that VU0409106 (1) was metabolized by AO (to M1) and P450 enzymes (to M4-M6), we sought to evaluate the in vivo disposition of 1 and its metabolites in rats with attenuated P450 activity. Male rats were orally pretreated with the pan-P450 inactivator, 1-aminobenzotriazole (ABT), before an i.p. dose of 1. Interestingly, the plasma area under the curve (AUC) of M1 was increased 15-fold in ABT-treated rats, indicating a metabolic shunt toward AO resulted from the drug interaction condition. The AUC of 1 also increased 7.8-fold. Accordingly, plasma clearance of 1 decreased from 53.5 to 15.3 ml/min per kilogram in ABT-pretreated rats receiving an i.v. dose of 1. Consistent with these data, M1 formation in hepatic S9 increased with NADPH-exclusion to eliminate P450 activity (50% over reactions containing NADPH). These studies reflect possible consequences of a drug interaction between P450 inhibitors and compounds cleared by both AO and P450 enzymes. Notably, increased exposure to an AO metabolite may hold clinical relevance for active metabolites or those mediating toxicity at elevated concentrations. The recent rise in clinical drug candidates metabolized by AO underscores the importance of these findings and the need for clinical studies to fully understand these risks.

N-Alkylpyrido[1',2':1,5]pyrazolo-[4,3-d]pyrimidin-4-amines: A new series of negative allosteric modulators of mGlu1/5 with CNS exposure in rodents.

Selective negative allosteric modulators (NAMs) of each of the group I metabotropic glutamate receptors (mGlu1 and mGlu5) have been well characterized in the literature and offer potential as therapeutics in several disorders of the central nervous system (CNS). Still, compounds that are potent mGlu1/5 NAMs with selectivity versus the other six members of the mGlu family as well as the balance of properties required for use in vivo are lacking. A medicinal chemistry effort centered on the identification of a lead series with the potential of delivering such compounds is described in this Letter. Specifically, a new class of pyrido[1',2':1,5]pyrazolo[4,3-d]pyrimidin-4-amines was designed as a novel isosteric replacement for 4-aminoquinazolines, and compounds from within this chemotype exhibited dual NAM activity at both group I mGlus. One compound, VU0467558 (29), demonstrated near equipotent activity at both receptors, selectivity versus other mGlus, a favorable ancillary pharmacology profile, and CNS exposure in rodents.

Discovery and characterization of a novel series of N-phenylsulfonyl-1H-pyrrole picolinamides as positive allosteric modulators of the metabotropic glutamate receptor 4 (mGlu4).

Herein we report the synthesis and characterization of a novel series of N-phenylsulfonyl-1H-pyrrole picolinamides as novel positive allosteric modulators of mGlu4. We detail our work towards finding phenyl replacements for the core scaffold of previously reported phenyl sulfonamides and phenyl sulfone compounds. Our efforts culminated in the identification of N-(1-((3,4-dimethylphenyl)sulfonyl)-1H-pyrrol-3-yl)picolinamide as a potent PAM of mGlu4.

Discovery of 3-aminopicolinamides as metabotropic glutamate receptor subtype 4 (mGlu4) positive allosteric modulator warheads engendering CNS exposure and in vivo efficacy.

This letter describes the further chemical optimization of the picolinamide-derived family of mGlu4 PAMs wherein we identified a 3-amino substituent to the picolinamide warhead that engendered potency, CNS penetration and in vivo efficacy. From this optimization campaign, VU0477886 emerged as a potent (EC50=95nM, 89% Glu Max) mGlu4 PAM with an attractive DMPK profile (brain:plasma Kp=1.3), rat CLp=4.0mL/min/kg, t1/2=3.7h) and robust efficacy in our standard preclinical Parkinson's disease model, haloperidol-induced catalepsy (HIC).

Preliminary investigation of 6,7-dihydropyrazolo[1,5-a]pyrazin-4-one derivatives as a novel series of mGlu5 receptor positive allosteric modulators with efficacy in preclinical models of schizophrenia.

As part of our efforts to identify a suitable back-up compound to our recently disclosed mGlu5 positive allosteric modulator (PAM) clinical candidate VU0490551/JNJ-46778212, this letter details the investigation and challenges of a novel series of 6,7-dihydropyrazolo[1,5-a]pyrazin-4-one derivatives. From these efforts, compound 4k emerged as a potent and selective mGlu5 PAM displaying overall attractive in vitro (pharmacological and ADMET) and PK profiles combined with in vivo efficacy in preclinical models of schizophrenia. However, further advancement of the compound was precluded due to severely limiting CNS-related side-effects confirming the previously reported association between excessive mGlu5 activation and target-related toxicities.

VFV as a New Effective CYP51 Structure-Derived Drug Candidate for Chagas Disease and Visceral Leishmaniasis.

Sterol 14α-demethylases (CYP51) are the enzymes essential for sterol biosynthesis. They serve as clinical targets for antifungal azoles and are considered as targets for treatment of human Trypanosomatidae infections. Recently, we have shown that VNI, a potent and selective inhibitor of trypanosomal CYP51 that we identified and structurally characterized in complex with the enzyme, can cure the acute and chronic forms of Chagas disease. The purpose of this work was to apply the CYP51 structure/function for further development of the VNI scaffold. As anticipated, VFV (R)-N-(1-(3,4'-difluorobiphenyl-4-yl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-phenyl-1,3,4-oxadiazol-2-yl)benzamide, the derivative designed to fill the deepest portion of the CYP51 substrate-binding cavity, reveals a broader antiprotozoan spectrum of action. It has stronger antiparasitic activity in cellular experiments, cures the experimental Chagas disease with 100% efficacy, and suppresses visceral leishmaniasis by 89% (vs 60% for VNI). Oral bioavailability, low off-target activity, favorable pharmacokinetics and tissue distribution characterize VFV as a promising new drug candidate.

Phospholipase D facilitates efficient entry of influenza virus, allowing escape from innate immune inhibition.

Lipid metabolism plays a fundamental role during influenza virus replication, although key regulators of lipid-dependent trafficking and virus production remain inadequately defined. This report demonstrates that infection by influenza virus stimulates phospholipase D (PLD) activity and that PLD co-localizes with influenza during infection. Both chemical inhibition and RNA interference of PLD delayed viral entry and reduced viral titers in vitro. Although there may be contributions by both major isoenzymes, the effects on viral infectivity appear to be more dependent on the PLD2 isoenzyme. In vivo, PLD2 inhibition reduced virus titer and correlated with significant increases in transcription of innate antiviral effectors. The reduction in viral titer downstream of PLD2 inhibition was dependent on Rig-I (retinoic acid-inducible gene-1), IRF3, and MxA (myxovirus resistance gene A) but not IRF7. Inhibition of PLD2 accelerated the accumulation of MxA in foci as early as 30 min postinfection. Together these data suggest that PLD facilitates the rapid endocytosis of influenza virus, permitting viral escape from innate immune detection and effectors that are capable of limiting lethal infection.

A Screen of Approved Drugs Identifies the Androgen Receptor Antagonist Flutamide and Its Pharmacologically Active Metabolite 2-Hydroxy-Flutamide as Heterotropic Activators of Cytochrome P450 3A In Vitro and In Vivo.

Once thought to be an artifact of microsomal systems, atypical kinetics with cytochrome P450 (CYP) enzymes have been extensively investigated in vitro and found to be substrate and species dependent. Building upon increasing reports of heterotropic CYP activation and inhibition in clinical settings, we screened a compound library of clinically approved drugs and various probe compounds to identify the frequency of heterotropism observed with different drug classes and the associated CYP enzymes thereof (1A2, 2C9, 2D6, and 3A4/5). Results of this screen revealed that the prescribed androgen receptor antagonist flutamide activated the intrinsic midazolam hydroxylase activity of CYP3A in human hepatic microsomes (66%), rat and human hepatocytes (36 and 160%, respectively), and in vivo in male Sprague-Dawley rats (>2-fold, combined area under the curve of primary rat in vivo midazolam metabolites). In addition, a screen of the pharmacologically active metabolite 2-hydroxy-flutamide revealed that this principle metabolite increased CYP3A metabolism of midazolam in human microsomes (30%) and hepatocytes (110%). Importantly, both flutamide and 2-hydroxy-flutamide demonstrated a pronounced increase in the CYP3A-mediated metabolism of commonly paired medications, nifedipine (antihypertensive) and amiodarone (antiarrhythmic), in multispecies hepatocytes (100% over baseline). These data serve to highlight the importance of an appropriate substrate and in vitro system selection in the pharmacokinetic modeling of atypical enzyme kinetics. In addition, the results of our investigation have illuminated a previously undiscovered class of heterotropic CYP3A activators and have demonstrated the importance of selecting commonly paired therapeutics in the in vitro and in vivo modeling of projected clinical outcomes.

Optimization of a small molecule probe that restores e-cadherin expression.

E-cadherin is a ubiquitous trans-membrane protein that has important functions in cellular contacts and has been shown to play a role in the epithelial mesenchymal transition. We have previously reported the use of an HTS screen to identify compounds that are capable of restoring e-cadherin in cancer cells. Here, we report the additional medicinal chemistry optimization of these molecules, resulting in new molecules that restore e-cadherin expression at low micromolar concentrations. Further, we report preliminary pharmacokinetic data on a compound, ML327, that can be used as a probe of e-cadherin restoration.

Progress towards small molecule menin-mixed lineage leukemia (MLL) interaction inhibitors with in vivo utility.

A series of substituted hydroxymethyl piperidine small molecule inhibitors of the protein-protein interaction between menin and mixed lineage leukemia 1 (MLL1) are described. Initial members of the series showed good inhibitory disruption of the menin-MLL1 interaction but demonstrated poor physicochemical and DMPK properties. Utilizing a structure-guided and iterative optimization approach key substituents were optimized leading to inhibitors with cell-based activity, improved in vitro DMPK parameters, and improved half-lives in rodent PK studies leading to MLPCN probe ML399. Ancillary off-target activity remains a parameter for further optimization.

Further optimization of the M5 NAM MLPCN probe ML375: tactics and challenges.

This Letter describes the continued optimization of the MLPCN probe ML375, a highly selective M5 negative allosteric modulator (NAM), through a combination of matrix libraries and iterative parallel synthesis. True to certain allosteric ligands, SAR was shallow, and the matrix library approach highlighted the challenges with M5 NAM SAR within in this chemotype. Once again, enantiospecific activity was noted, and potency at rat and human M5 were improved over ML375, along with slight enhancement in physiochemical properties, certain in vitro DMPK parameters and CNS distribution. Attempts to further enhance pharmacokinetics with deuterium incorporation afforded mixed results, but pretreatment with a pan-P450 inhibitor (1-aminobenzotriazole; ABT) provided increased plasma exposure.

Further optimization of the mGlu5 PAM clinical candidate VU0409551/JNJ-46778212: Progress and challenges towards a back-up compound.

This Letter describes the progress and challenges in the continued optimization of the mGlu5 positive allosteric modulator (PAM) clinical candidate VU0490551/JNJ-46778212. While many analogs addressed key areas for improvement, no one compound possessed the amalgamation of improvements needed within the (2(phenoxymethyl)-6,7-dihydrooxazolo[5,4-c]pyridine-5(4H)-yl(aryl)methanone scaffold to advance as a back-up clinical candidate. However, many analogs displayed excellent solubility and physiochemical properties, and were active in the amphetamine-induced hyperlocomotion (AHL) model. Moreover, the SAR was robust for this series of PAMs, and both polar and hydrogen-bond donors were found to be tolerated, leading to analogs with overall attractive profiles and good ligand efficiencies.