Development and profiling of mGlu7 NAMs with a range of saturable inhibition of agonist responses in vitro.

We describe here a series of metabotropic glutamate receptor 7 (mGlu7) negative allosteric modulators (NAMs) with a saturable range of activity in inhibiting responses to an orthosteric agonist in two distinct in vitro pharmacological assays. The range of inhibition among compounds in this scaffold provides highly structurally related ligands with differential degrees of receptor blockade that can be used to understand inhibitory efficacy profiles in native tissue or in vivo.

Synthesis and SAR of a series of mGlu7 NAMs based on an ethyl-8-methoxy-4-(4-phenylpiperazin-1-yl)quinoline carboxylate core.

A High-Throughput Screening (HTS) campaign identified a fundamentally new mGlu7 NAM chemotype, based on an ethyl-8-methoxy-4-(4-phenylpiperazin-1-yl)quinolone carboxylate core. The initial hit, VU0226390, was a potent mGlu7 NAM (IC50 = 647 nM, 6% L-AP4 min) with selectivity versus the other group III mGlu receptors (>30 µM vs. mGlu4 and mGlu8). A multi-dimensional optimization effort surveyed all regions of this new chemotype, and found very steep SAR, reminiscent of allosteric modulators, and unexpected piperazine mimetics (whereas classical bioisosteres failed). While mGlu7 NAM potency could be improved (IC50s ~ 350 nM), the necessity of the ethyl ester moiety and poor physiochemical and DMPK properties precluded optimization towards in vivo tool compounds or clinical candidates. Still, this hit-to-lead campaign afforded key medicinal chemistry insights and new opportunities.

Discovery of a novel 2,3-dimethylimidazo[1,2-a]pyrazine-6-carboxamide M4 positive allosteric modulator (PAM) chemotype.

This Letter details our efforts to discover structurally unique M4 PAMs containing 5,6-heteroaryl ring systems. In an attempt to improve the DMPK profiles of the 2,3-dimethyl-2H-indazole-5-carboxamide and 1-methyl-1H-benzo[d][1,2,3]triazole-6-carboxamide cores, we investigated a plethora of core replacements. This exercise identified a novel 2,3-dimethylimidazo[1,2-a]pyrazine-6-carboxamide core that provided improved M4 PAM activity and CNS penetration.

Surveying heterocycles as amide bioisosteres within a series of mGlu7 NAMs: Discovery of VU6019278.

This letter describes a diversity-oriented library approach to rapidly assess diverse heterocycles as bioisosteric replacements for a metabolically labile amide moiety within a series of mGlu7 negative allosteric modulators (NAMs). SAR rapidly honed in on either a 1,2,4- or 1,3,4-oxadizaole ring system as an effective bioisostere for the amide. Further optimization of the southern region of the mGlu7 NAM chemotype led to the discovery of VU6019278, a potent mGlu7 NAM (IC50 = 501 nM, 6.3% L-AP4 Min) with favorable plasma protein binding (rat fu = 0.10), low predicted hepatic clearance (rat CLhep = 27.7 mL/min/kg) and high CNS penetration (rat Kp = 4.9, Kp,uu = 0.65).

Discovery of a novel 3,4-dimethylcinnoline carboxamide M4 positive allosteric modulator (PAM) chemotype via scaffold hopping.

This Letter details our efforts to replace the 2,4-dimethylquinoline carboxamide core of our previous M4 PAM series, which suffered from high predicted hepatic clearance and protein binding. A scaffold hopping exercise identified a novel 3,4-dimethylcinnoline carboxamide core that provided good M4 PAM activity and improved clearance and protein binding profiles.

Multiomic Profiling of Human Clonal Hematopoiesis Reveals Genotype and Cell-Specific Inflammatory Pathway Activation.

Clonal hematopoiesis (CH) is an age-associated phenomenon that increases risk for hematologic malignancy and cardiovascular disease. CH is thought to enhance disease risk through inflammation in the peripheral blood1. Here, we profile peripheral blood gene expression in 66,968 single cells from a cohort of 17 CH patients and 7 controls. Using a novel mitochondrial DNA barcoding approach, we were able to identify and separately compare mutant TET2 and DNMT3A cells to non-mutant counterparts. We discovered the vast majority of mutated cells were in the myeloid compartment. Additionally, patients harboring DNMT3A and TET2 CH mutations possessed a pro-inflammatory profile in CD14+ monocytes through previously unrecognized pathways such as galectin and macrophage Inhibitory Factor (MIF). We also found that T cells from CH patients, though mostly un-mutated, had decreased expression of GTPase of the immunity associated protein (GIMAP) genes, which are critical to T cell development, suggesting that CH impairs T cell function.

Isocitrate dehydrogenase mutations are associated with altered IL-1ß responses in acute myeloid leukemia.

Mutations in isocitrate dehydrogenase 2 (IDH2) have been noted to impact cellular differentiation in addition to DNA and histone methylation. However, little is known about the impact of IDH2 mutations on intracellular signaling. Using an isogenic cell line model, we investigated both differentiation and signaling responses in IDH2 mutant cells and show augmented responses to inflammatory immune ligands. Using phospho-specific flow and mass cytometry, we demonstrate IDH2 mutant cells were significantly more sensitive to IL-1ß at multiple downstream readouts. Further, bulk RNA sequencing confirmed increases in cytokine-related signaling pathways and NF-κB target genes. Single-cell RNA sequencing of unstimulated and stimulated cells confirmed altered IL-1ß transcriptional responses in the IDH2 mutant cells. Targeted inhibition of the IKK complex reduced IL-1ß responses and induced cell death in primary IDH-mutated leukemia samples. Together, these results confirm altered IL-1ß signaling in IDH2 mutant cells and identify this pathway as a potential therapeutic target.

The delta isoform of phosphatidylinositol-3-kinase predominates in chronic myelomonocytic leukemia and can be targeted effectively with umbralisib and ruxolitinib.

Chronic myelomonocytic leukemia (CMML) is a myelodysplastic syndrome/myeloproliferative neoplasm overlap syndrome characterized by monocytic proliferation in the presence of dysplastic bone marrow changes, inflammatory symptoms, and propensity for transformation to acute myeloid leukemia (AML), with a poor prognosis and limited treatment options. Unlike the α and ß isoforms, the phosphatidylinositol-3-kinase (PI3K)-δ signaling protein is predominantly expressed by hematopoietic cells and therefore has garnered interest as a potential target for the treatment of lymphomas and leukemias. We revealed a pattern of increased PIK3CD:PIK3CA ratio in monocytic M5 AML patients and cell lines, and this ratio correlated with responsiveness to pharmacological PI3K-δ inhibition in vitro. Because CMML is a disease defined by monocytic clonal proliferation, we tested the PI3K-δ inhibitor umbralisib as a single agent and in combination with the JAK1/2 inhibitor ruxolitinib, in CMML. Our ex vivo experiments with primary CMML patient samples revealed synergistic inhibition of viability and clonogenicity with this combination. Phospho-specific flow cytometry revealed that dual inhibition had the unique ability to decrease STAT5, ERK, AKT, and S6 phosphorylation simultaneously, which offers a mechanistic hypothesis for the enhanced efficacy of the combination treatment. These preclinical data indicate promising activity by co-inhibition of PI3K-δ and JAK1/2 and support the use of ruxolitinib + umbralisib combination therapy in CMML under active clinical investigation.

Phenotypic profiling of mGlu7 knockout mice reveals new implications for neurodevelopmental disorders.

Neurodevelopmental disorders are characterized by deficits in communication, cognition, attention, social behavior and/or motor control. Previous studies have pointed to the involvement of genes that regulate synaptic structure and function in the pathogenesis of these disorders. One such gene, GRM7, encodes the metabotropic glutamate receptor 7 (mGlu7 ), a G protein-coupled receptor that regulates presynaptic neurotransmitter release. Mutations and polymorphisms in GRM7 have been associated with neurodevelopmental disorders in clinical populations; however, limited preclinical studies have evaluated mGlu7 in the context of this specific disease class. Here, we show that the absence of mGlu7 in mice is sufficient to alter phenotypes within the domains of social behavior, associative learning, motor function, epilepsy and sleep. Moreover, Grm7 knockout mice exhibit an attenuated response to amphetamine. These findings provide rationale for further investigation of mGlu7 as a potential therapeutic target for neurodevelopmental disorders such as idiopathic autism, attention deficit hyperactivity disorder and Rett syndrome.

Discovery of an Orally Bioavailable and Central Nervous System (CNS) Penetrant mGlu7 Negative Allosteric Modulator (NAM) in Vivo Tool Compound: N-(2-(1 H-1,2,4-triazol-1-yl)-5-(trifluoromethoxy)phenyl)-4-(cyclopropylmethoxy)-3-methoxybenzamide (VU6012962).

Herein, we report the discovery of a new, orally bioavailable and CNS-penetrant metabotropic glutamate receptor 7 (mGlu7) negative allosteric modulator (NAM) that achieves exposure in cerebral spinal fluid (CSF) 2.5× above the in vitro IC50 at minimum effective doses (MEDs) of 3 mg/kg in preclinical anxiety models.

Correlation between the Functional Movement Screen and Hip Mobility in NCAA Division II Athletes.

The Functional Movement Screen (FMS) is a series of seven exercises designed to assess athletes prior to competitive training to determine dysfunctional movement patterns. The exercises include the deep squat (DS), hurdle step (HS), inline lunge (IL), shoulder mobility (SM), active straight leg raise (ASL), pushup (PU), and rotary stability (RS), all of which comprise the composite score (FMS CS). The purpose of this study was to determine correlations between the FMS screens and hip mobility. The specific hip range-of-motion exercises used were bilateral internal/external rotation (IRR, IRL ERL, ERR), and flexion/extension (FL, FR, ER, EL). Participants of both genders (32 males, 13 females) were solicited from four sports (baseball, softball, and men's and women's cross-country). The most significant/intriguing correlations were between FMS HS/FL, FMS ILL/R, FMS PU/FL, FMS ALL and IRR/L and ERR/L and FMS SM/ER. All correlations produced were weak to moderate at the .05 level of significance. These results demonstrate that hip range-of-motion plays a minor role in FMS score. Future studies should utilize a larger sample size, including more females, as well as analyze range-of-motion across multiple joints.