Structural basis of allosteric modulation of metabotropic glutamate receptor activation and desensitization.

The metabotropic glutamate receptors (mGluRs) are neuromodulatory family C G protein coupled receptors which assemble as dimers and allosterically couple extracellular ligand binding domains (LBDs) to transmembrane domains (TMDs) to drive intracellular signaling. Pharmacologically, mGluRs can be targeted either at the LBDs by glutamate and synthetic orthosteric compounds or at the TMDs by allosteric modulators. Despite the potential of allosteric TMD-targeting compounds as therapeutics, an understanding of the functional and structural basis of their effects on mGluRs is limited. Here we use a battery of approaches to dissect the distinct functional and structural effects of orthosteric versus allosteric ligands. We find using electrophysiological and live cell imaging assays that both agonists and positive allosteric modulators (PAMs) can drive activation and desensitization of mGluRs. The effects of PAMs are pleiotropic, including both the ability to boost the maximal response to orthosteric agonists and to serve independently as desensitization-biased agonists across mGluR subtypes. Conformational sensors reveal PAM-driven inter-subunit re-arrangements at both the LBD and TMD. Motivated by this, we determine cryo-electron microscopy structures of mGluR3 in the presence of either an agonist or antagonist alone or in combination with a PAM. These structures reveal PAM-driven re-shaping of intra- and inter-subunit conformations and provide evidence for a rolling TMD dimer interface activation pathway that controls G protein and beta-arrestin coupling. Highlights: -Agonists and PAMs drive mGluR activation, desensitization, and endocytosis-PAMs are desensitization-biased and synergistic with agonists-Four combinatorial ligand conditions reveal an ensemble of full-length mGluR structures with novel interfaces-Activation and desensitization involve rolling TMD interfaces which are re-shaped by PAM.

mGlu1-mediated restoration of prefrontal cortex inhibitory signaling reverses social and cognitive deficits in an NMDA hypofunction model in mice.

Extensive evidence supports the hypothesis that deficits in inhibitory GABA transmission in the prefrontal cortex (PFC) may drive pathophysiological changes underlying symptoms of schizophrenia that are not currently treated by available medications, including cognitive and social impairments. Recently, the mGlu1 subtype of metabotropic glutamate (mGlu) receptor has been implicated as a novel target to restore GABAergic transmission in the PFC. A recent study reported that activation of mGlu1 increases inhibitory transmission in the PFC through excitation of somatostatin-expressing GABAergic interneurons, implicating mGlu1 PAMs as a potential treatment strategy for schizophrenia. Here, we leveraged positive allosteric modulators (PAMs) of mGlu1 to examine whether mGlu1 activation might reverse physiological effects and behavioral deficits induced by MK-801, an NMDA receptor antagonist commonly used to model cortical deficits observed in schizophrenia patients. Using ex vivo whole-cell patch-clamp electrophysiology, we found that MK-801 decreased the frequency of spontaneous inhibitory postsynaptic currents onto layer V pyramidal cells of the PFC and this cortical disinhibition was reversed by mGlu1 activation. Furthermore, acute MK-801 treatment selectively induced inhibitory deficits onto layer V pyramidal cells that project to the basolateral amygdala, but not to the nucleus accumbens, and these deficits were restored by selective mGlu1 activation. Importantly, the mGlu1 PAM VU6004909 effectively reversed deficits in sociability and social novelty preference in a three-chamber assay and improved novel objection recognition following MK-801 treatment. Together, these findings provide compelling evidence that mGlu1 PAMs could serve as a novel approach to reduce social and cognitive deficits associated with schizophrenia by enhancing inhibitory transmission in the PFC, thus providing an exciting improvement over current antipsychotic medication.

Positive allosteric modulators (PAMs) of the group II metabotropic glutamate receptors: Design, synthesis, and evaluation as ex-vivo tool compounds.

This letter describes synthesis and evaluation of two series of dual mGlu2/mGlu3 positive allosteric modulators with moderate mGlu3 potency and robust mGlu2 potency in thallium flux assays. These compounds were profiled their ability to modulate mGlu3-mediated signaling in central neurons by co-application of a selective mGlu2 NAM to isolate mGlu3-selective effects. Using acute mouse brain slices from the prefrontal cortex, potentiation of group II mGlu receptor agonist Ca2+ signaling in PFC pyramidal cells with either the dual mGlu2/mGlu3 PAM 16e or 23d demonstrated effects mediated selectively via mGlu3.

Inhibition of Histone Deacetylases 1, 2, and 3 Enhances Clearance of Cholesterol Accumulation in Niemann-Pick C1 Fibroblasts.

Niemann-Pick disease type C1 (NPC1) is a rare genetic cholesterol storage disorder caused by mutations in the NPC1 gene. Mutations in this transmembrane late endosome protein lead to loss of normal cholesterol efflux from late endosomes and lysosomes. It has been shown that broad spectrum histone deacetylase inhibitors (HDACi's) such as Vorinostat correct the cholesterol accumulation phenotype in the majority of NPC1 mutants tested in cultured cells. In order to determine the optimal specificity for HDACi correction of the mutant NPC1s, we screened 76 HDACi's of varying specificity. We tested the ability of these HDACi's to correct the excess accumulation of cholesterol in patient fibroblast cells that homozygously express NPC1 I1061T , the most common mutation. We determined that inhibition of HDACs 1, 2, and 3 is important for correcting the defect, and combined inhibition of all three is needed to achieve the greatest effect, suggesting a need for multiple effects of the HDACi treatments. Identifying the specific HDACs involved in the process of regulating cholesterol trafficking in NPC1 will help to focus the search for more specific druggable targets.

Lead optimization of the VU0486321 series of mGlu1 PAMs. Part 4: SAR reveals positive cooperativity across multiple mGlu receptor subtypes leading to subtype unselective PAMs.

Further optimization of the VU0486321 series of highly selective and CNS-penetrant mGlu1 PAMs identified unique 'molecular switches' on the central aromatic ring that engendered positive cooperativity with multiple mGlu subtypes across the receptor family, resulting in compounds with comparable activity at Group I (mGlu1/5) and Group III (mGlu4/6/7/8) mGlu receptors, receptors. These exciting data suggests this PAM chemotype appears to bind to multiple mGlu receptors, and that subtype selectivity is dictated by the degree of cooperativity, not a subtype selective, unique allosteric binding site. Moreover, there is interesting therapeutic potential for mGlu1/4/7/8 PAMs, as well as the first report of a GPCR allosteric 'privileged structure'.

Development of a target-free high-throughput screening platform for the discovery of antileishmanial compounds.

Leishmania parasites are the causative agents of a wide spectrum of human diseases. The clinical manifestations of leishmaniasis range from self-healing skin lesions to fatality. The World Health Organization has classed leishmaniasis as a category 1 neglected tropical disease. Leishmaniasis represents a major international health challenge, affecting 12 million people per year and with nearly 310 million people at risk. The first-line chemotherapies used to treat leishmaniasis are intravenous pentavalent antimonials; however, these drugs are highly toxic. As the use of oral treatment options such as paromomycin and miltefosine has increased, the incidence of disease relapse has increased and drug resistance to antimonials has developed, emphasizing the importance of identifying new chemotherapies. A novel, target-free fluorometric high-throughput screen with an average Z-score of 0.73 +/- 0.13 has been developed to identify small molecules with antileishmanial activity. Screening of 10,000 small molecules from the ChemBridge DIVER-set™ library cassette #5 yielded 210 compounds that killed 80% of parasites, resulting in a hit rate of 2.1%. One hundred and nine molecular scaffolds were represented within the hit compounds, and one scaffold that exhibited potent antileishmanial activity was 2,4-diaminoquinazoline. Host cell toxicity was determined prior to in-vitro infection of human THP-1 macrophages with Leishmania donovani mCherry expressing promastigotes; successful drug treatment was considered when the half maximal inhibitory concentration was <10 µM. BALB/c mice were infected with Leishmania major mCherry promastigotes and treated with small molecules that were successful during in-vitro infections. Several small molecules tested were as efficacious at resolving cutaneous leishmaniasis lesions in mice as known antimonial treatments.

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.

VU6005806/AZN-00016130, an advanced M4 positive allosteric modulator (PAM) profiled as a potential preclinical development candidate.

This letter describes progress towards an M4 PAM preclinical candidate that resulted in the discovery of VU6005806/AZN-00016130. While the thieno[2,3-c]pyridazine core has been a consistent feature of key M4 PAMs, no work had previously been reported with respect to alternate functionality at the C3 position of the pyridazine ring. Here, we detail new chemistry and analogs that explored this region, and quickly led to VU6005806/AZN-00016130, which was profiled as a putative candidate. While, the ß-amino carboxamide moiety engendered solubility limited absorption in higher species precluding advancement (or requiring extensive pharmaceutical sciences formulation), VU6005806/AZN-00016130 represents a new, high quality preclinical in vivo probe.

2-Hydroxypropyl-ß-cyclodextrin is the active component in a triple combination formulation for treatment of Niemann-Pick C1 disease.

Niemann-Pick type C1 (NPC1) disease is a fatal neurovisceral disease for which there are no FDA approved treatments, though cyclodextrin (HPßCD) slows disease progression in preclinical models and in an early phase clinical trial. Our goal was to evaluate the mechanism of action of a previously described combination-therapy, Triple Combination Formulation (TCF) - comprised of the histone deacetylase inhibitor (HDACi) vorinostat/HPßCD/PEG - shown to prolong survival in Npc1 mice. In these studies, TCF's benefit was attributed to enhanced vorinostat pharmacokinetics (PK). Here, we show that TCF reduced lipid storage, extended lifespan, and preserved neurological function in Npc1 mice. Unexpectedly, substitution of an inactive analog for vorinostat in TCF revealed similar efficacy. We demonstrate that the efficacy of TCF was attributable to enhanced HPßCD PK and independent of NPC1 protein expression. We conclude that although HDACi effectively reduce cholesterol storage in NPC1-deficient cells, HDACi are ineffective in vivo in Npc1 mice.

The succinct synthesis of AT13387, a clinically relevant Hsp90 inhibitor.

AT13387 is an orally bioavailable clinical candidate developed to inhibit theheat shock protein 90 (Hsp90). This article describes a modified synthetic route for the multi-gram production of AT13387 in 46% overall yield. The modified synthetic route is short, avoids stringent reaction conditions and difficult purifications, which led to increase in an overall yield.

Discovery of Tricyclic Triazolo- and Imidazopyridine Lactams as M1 Positive Allosteric Modulators.

This Letter describes the chemical optimization of a new series of muscarinic acetylcholine receptor subtype 1 (M1) positive allosteric modulators (PAMs) based on novel tricyclic triazolo- and imidazopyridine lactam cores, devoid of M1 agonism, e.g., no M1 ago-PAM activity, in high expressing recombinant cell lines. While all the new tricyclic congeners afforded excellent rat pharmacokinetic (PK) properties (CLp < 8 mL/min/kg and t1/2 > 5 h), regioisomeric triazolopyridine analogues were uniformly not CNS penetrant ( Kp < 0.05), despite a lack of hydrogen bond donors. However, removal of a single nitrogen atom to afford imidazopyridine derivatives proved to retain the excellent rat PK and provide high CNS penetration ( Kp > 2), despite inclusion of a basic nitrogen. Moreover, 24c was devoid of M1 agonism in high expressing recombinant cell lines and did not induce cholinergic seizures in vivo in mice. Interestingly, all of the new M1 PAMs across the diverse tricyclic heterocyclic cores possessed equivalent CNS MPO scores (>4.5), highlighting the value of both "medicinal chemist's eye" and experimental data, e.g., not sole reliance (or decision bias) on in silico calculated properties, for parameters as complex as CNS penetration.

Novel M4 positive allosteric modulators derived from questioning the role and impact of a presumed intramolecular hydrogen-bonding motif in ß-amino carboxamide-harboring ligands.

This letter describes a focused exercise to explore the role of the ß-amino carboxamide moiety found in all of the first generation M4 PAMs and question if the NH2 group served solely to stabilize an intramolecular hydrogen bond (IMHB) and enforce planarity. To address this issue (and to potentially find a substitute for the ß-amino carboxamide that engendered P-gp and contributed to solubility liabilities), we removed the NH2, generating des-amino congeners and surveyed other functional groups in the ß-position. These modifications led to weak M4 PAMs with poor DMPK properties. Cyclization of the ß-amino carboxamide moiety by virtue of a pyrazole ring re-enforced the IMHB, led to potent (and patented) M4 PAMs, many as potent as the classical bicyclic ß-amino carboxamide analogs, but with significant CYP1A2 inhibition. Overall, this exercise indicated that the ß-amino carboxamide moiety most likely facilitates an IMHB, and is essential for M4 PAM activity within classical bicyclic M4 PAM scaffolds.