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.

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'.

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.

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.

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.

Structural insights into HDAC6 tubulin deacetylation and its selective inhibition.

We report crystal structures of zebrafish histone deacetylase 6 (HDAC6) catalytic domains in tandem or as single domains in complex with the (R) and (S) enantiomers of trichostatin A (TSA) or with the HDAC6-specific inhibitor nexturastat A. The tandem domains formed, together with the inter-domain linker, an ellipsoid-shaped complex with pseudo-twofold symmetry. We identified important active site differences between both catalytic domains and revealed the binding mode of HDAC6 selective inhibitors. HDAC inhibition assays with (R)- and (S)-TSA showed that (R)-TSA was a broad-range inhibitor, whereas (S)-TSA had moderate selectivity for HDAC6. We identified a uniquely positioned α-helix and a flexible tryptophan residue in the loop joining α-helices H20 to H21 as critical for deacetylation of the physiologic substrate tubulin. Using single-molecule measurements and biochemical assays we demonstrated that HDAC6 catalytic domain 2 deacetylated α-tubulin lysine 40 in the lumen of microtubules, but that its preferred substrate was unpolymerized tubulin.

Challenges in the development of an M4 PAM preclinical candidate: The discovery, SAR, and biological characterization of a series of azetidine-derived tertiary amides.

Herein we describe the continued optimization of M4 positive allosteric modulators (PAMs) within the 5-amino-thieno[2,3-c]pyridazine series of compounds. In this letter, we disclose our studies on tertiary amides derived from substituted azetidines. This series provided excellent CNS penetration, which had been challenging to consistently achieve in other amide series. Efforts to mitigate high clearance, aided by metabolic softspot analysis, were unsuccessful and precluded this series from further consideration as a preclinical candidate. In the course of this study, we found that potassium tetrafluoroborate salts could be engaged in a tosyl hydrazone reductive cross coupling reaction, a previously unreported transformation, which expands the synthetic utility of the methodology.

Challenges in the development of an M4 PAM in vivo tool compound: The discovery of VU0467154 and unexpected DMPK profiles of close analogs.

This letter describes the chemical optimization of a novel series of M4 positive allosteric modulators (PAMs) based on a 5-amino-thieno[2,3-c]pyridazine core, developed via iterative parallel synthesis, and culminating in the highly utilized rodent in vivo tool compound, VU0467154 (5). This is the first report of the optimization campaign (SAR and DMPK profiling) that led to the discovery of VU0467154, and details all of the challenges faced in allosteric modulator programs (steep SAR, species differences in PAM pharmacology and subtle structural changes affecting CNS penetration).

Optimization of M4 positive allosteric modulators (PAMs): The discovery of VU0476406, a non-human primate in vivo tool compound for translational pharmacology.

This letter describes the further chemical optimization of the 5-amino-thieno[2,3-c]pyridazine series (VU0467154/VU0467485) of M4 positive allosteric modulators (PAMs), developed via iterative parallel synthesis, culminating in the discovery of the non-human primate (NHP) in vivo tool compound, VU0476406 (8p). VU0476406 is an important in vivo tool compound to enable translation of pharmacodynamics from rodent to NHP, and while data related to a Parkinson's disease model has been reported with 8p, this is the first disclosure of the optimization and discovery of VU0476406, as well as detailed pharmacology and DMPK properties.

Discovery and optimization of a novel series of highly CNS penetrant M4 PAMs based on a 5,6-dimethyl-4-(piperidin-1-yl)thieno[2,3-d]pyrimidine core.

This Letter describes the chemical optimization of a novel series of M4 positive allosteric modulators (PAMs) based on a 5,6-dimethyl-4-(piperidin-1-yl)thieno[2,3-d]pyrimidine core, identified from an MLPCN functional high-throughput screen. The HTS hit was potent and selective, but not CNS penetrant. Potency was maintained, while CNS penetration was improved (rat brain:plasma Kp=0.74), within the original core after several rounds of optimization; however, the thieno[2,3-d]pyrimidine core was subject to extensive oxidative metabolism. Ultimately, we identified a 6-fluoroquinazoline core replacement that afforded good M4 PAM potency, muscarinic receptor subtype selectivity and CNS penetration (rat brain:plasma Kp>10). Moreover, this campaign provided fundamentally distinct M4 PAM chemotypes, greatly expanding the available structural diversity for this exciting CNS target.

A concise Diels-Alder strategy leading to congeners of the ABC ring system of the marine alkaloid 'upenamide.

A second-generation approach to the BC spirocycle of 'upenamide is reported. Central to the synthesis is an endo selective Diels-Alder reaction between 1-(t-butyldimethylsiloxy)-1,3-butadiene and bromomaleic anhydride followed by a radical mediated allylation of the ring fusion bromide. Functional group manipulation provides three (9-11) advanced synthetic intermediates ready for coupling with the remaining half (DE bicycle) of 'upenamide.

Microplate-based assay for identifying small molecules that bind a specific intersubunit interface within the assembled HIV-1 capsid.

Despite the availability of >30 effective drugs for managing HIV-1 infection, no current therapy is curative, and long-term management is challenging owing to the emergence and spread of drug-resistant mutants. Identification of drugs against novel HIV-1 targets would expand the current treatment options and help to control resistance. The highly conserved HIV-1 capsid protein represents an attractive target because of its multiple roles in replication of the virus. However, the low antiviral potencies of the reported HIV-1 capsid-targeting inhibitors render them unattractive for therapeutic development. To facilitate the identification of more-potent HIV-1 capsid inhibitors, we developed a scintillation proximity assay to screen for small molecules that target a biologically active and specific intersubunit interface in the HIV-1 capsid. The assay, which is based on competitive displacement of a known capsid-binding small-molecule inhibitor, exhibited a signal-to-noise ratio of >9 and a Z factor of >0.8. In a pilot screen of a chemical library containing 2,400 druglike compounds, we obtained a hit rate of 1.8%. This assay has properties that are suitable for screening large compound libraries to identify novel HIV-1 capsid ligands with antiviral activity.

Substituted indoles as selective protease activated receptor 4 (PAR-4) antagonists: Discovery and SAR of ML354.

Herein we report the discovery and SAR of an indole-based protease activated receptor-4 (PAR-4) antagonist scaffold derived from a similarity search of the Vanderbilt HTS collection, leading to MLPCN probe ML354 (VU0099704). Using a novel PAC-1 fluorescent αIIbß3 activation assay this probe molecule antagonist was found to have an IC50 of 140nM for PAR-4 with 71-fold selectivity versus PAR-1 (PAR-1IC50=10µM).

A structural model for binding of the serine-rich repeat adhesin GspB to host carbohydrate receptors.

GspB is a serine-rich repeat (SRR) adhesin of Streptococcus gordonii that mediates binding of this organism to human platelets via its interaction with sialyl-T antigen on the receptor GPIbα. This interaction appears to be a major virulence determinant in the pathogenesis of infective endocarditis. To address the mechanism by which GspB recognizes its carbohydrate ligand, we determined the high-resolution x-ray crystal structure of the GspB binding region (GspB(BR)), both alone and in complex with a disaccharide precursor to sialyl-T antigen. Analysis of the GspB(BR) structure revealed that it is comprised of three independently folded subdomains or modules: 1) an Ig-fold resembling a CnaA domain from prokaryotic pathogens; 2) a second Ig-fold resembling the binding region of mammalian Siglecs; 3) a subdomain of unique fold. The disaccharide was found to bind in a pocket within the Siglec subdomain, but at a site distinct from that observed in mammalian Siglecs. Confirming the biological relevance of this binding pocket, we produced three isogenic variants of S. gordonii, each containing a single point mutation of a residue lining this binding pocket. These variants have reduced binding to carbohydrates of GPIbα. Further examination of purified GspB(BR)-R484E showed reduced binding to sialyl-T antigen while S. gordonii harboring this mutation did not efficiently bind platelets and showed a significant reduction in virulence, as measured by an animal model of endocarditis. Analysis of other SRR proteins revealed that the predicted binding regions of these adhesins also had a modular organization, with those known to bind carbohydrate receptors having modules homologous to the Siglec and Unique subdomains of GspB(BR). This suggests that the binding specificity of the SRR family of adhesins is determined by the type and organization of discrete modules within the binding domains, which may affect the tropism of organisms for different tissues.

Spirocyclic replacements for the isatin in the highly selective, muscarinic M1 PAM ML137: the continued optimization of an MLPCN probe molecule.

This Letter describes the further optimization of an MLPCN probe molecule (ML137) through the introduction of 5- and 6-membered spirocycles in place of the isatin ketone. Interestingly divergent structure-activity relationships, when compared to earlier M1 PAMs, are presented. These novel spirocycles possess improved efficacy relative to ML137, while also maintaining high selectivity for the human and rat muscarinic M1 receptor subtype.

Isatin replacements applied to the highly selective, muscarinic M1 PAM ML137: continued optimization of an MLPCN probe molecule.

This Letter describes the continued optimization of an MLPCN probe molecule (ML137) with a focused effort on the replacement/modification of the isatin moiety present in this highly selective M(1) PAM. A diverse range of structures were validated as viable replacements for the isatin, many of which engendered sizeable improvements in their ability to enhance the potency and efficacy of acetylcholine when compared to ML137. Muscarinic receptor subtype selectivity for the M(1) receptor was also maintained.

Discovery of a selective M4 positive allosteric modulator based on the 3-amino-thieno[2,3-b]pyridine-2-carboxamide scaffold: development of ML253, a potent and brain penetrant compound that is active in a preclinical model of schizophrenia.

Herein we report a next generation muscarinic receptor 4 (M(4)) positive allosteric modulator (PAM), ML253 which exhibits nanomolar activity at both the human (EC(50)=56 nM) and rat (EC(50)=176 nM) receptors and excellent efficacy by the left-ward shift of the ACh concentration response curve (fold shift, human=106; rat=50). In addition, ML253 is selective against the four other muscarinic subtypes, displays excellent CNS exposure and is active in an amphetamine-induced hyperlocomotion assay.

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.

Development of a more highly selective M(1) antagonist from the continued optimization of the MLPCN Probe ML012.

This Letter describes the continued optimization of an MLPCN probe molecule (ML012) through an iterative parallel synthesis approach. After exploring extensive modifications throughout the parent structure, we arrived at a more highly M(1)-selective antagonist, compound 13l (VU0415248). Muscarinic subtype selectivity across all five human and rat receptors for 13l, along with rat selectivity for the lead compound (ML012), is presented.