RESUMEN
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.
Asunto(s)
Señalización del Calcio/efectos de los fármacos , Neuronas/metabolismo , Receptores de Glutamato Metabotrópico/administración & dosificación , Receptores de Glutamato Metabotrópico/agonistas , Receptores de Glutamato Metabotrópico/metabolismo , Animales , Línea Celular , Diseño de Fármacos , Humanos , Ratones , Estructura Molecular , Neuronas/efectos de los fármacos , Corteza Prefrontal/citología , Células Piramidales , Receptores de Glutamato Metabotrópico/genética , Relación Estructura-ActividadRESUMEN
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'.
Asunto(s)
Cumarinas/química , Furanos/química , Receptor del Glutamato Metabotropico 5/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Regulación Alostérica , Cumarinas/metabolismo , Furanos/metabolismo , Humanos , Receptor del Glutamato Metabotropico 5/química , Receptores de Glutamato Metabotrópico/química , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Amidas/farmacología , Receptor Muscarínico M4/antagonistas & inhibidores , Regulación Alostérica/efectos de los fármacos , Amidas/síntesis química , Amidas/química , Relación Dosis-Respuesta a Droga , Humanos , Enlace de Hidrógeno , Ligandos , Estructura Molecular , Receptor Muscarínico M4/metabolismo , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Regulación Alostérica/inmunología , Receptor Muscarínico M4/inmunología , Estructura Molecular , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Aldehído Oxidasa/metabolismo , Miotonía Congénita/metabolismo , Receptor Muscarínico M4/metabolismo , Animales , Descubrimiento de Drogas , Humanos , Ratas , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/metabolismo , Ácidos Hidroxámicos/farmacología , Tubulina (Proteína)/metabolismo , Proteínas de Pez Cebra/antagonistas & inhibidores , Proteínas de Pez Cebra/metabolismo , Acetilación/efectos de los fármacos , Animales , Biocatálisis , Histona Desacetilasa 6 , Inhibidores de Histona Desacetilasas/química , Histona Desacetilasas/química , Humanos , Ácidos Hidroxámicos/química , Modelos Moleculares , Relación Estructura-Actividad , Tubulina (Proteína)/química , Pez Cebra , Proteínas de Pez Cebra/químicaRESUMEN
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.
Asunto(s)
Amidas/química , Azetidinas/química , Receptor Muscarínico M4/metabolismo , Regulación Alostérica , Amidas/metabolismo , Evaluación Preclínica de Medicamentos , Humanos , Unión Proteica , Piridazinas/síntesis química , Piridazinas/química , Piridazinas/metabolismo , Receptor Muscarínico M4/antagonistas & inhibidores , Relación Estructura-ActividadRESUMEN
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).
Asunto(s)
Piridazinas/farmacología , Receptor Muscarínico M4/agonistas , Tiofenos/farmacología , Animales , Humanos , Ligandos , Proteínas de Transporte de Nucleósidos/metabolismo , Piridazinas/administración & dosificación , Piridazinas/síntesis química , Piridazinas/farmacocinética , Ratas Sprague-Dawley , Relación Estructura-Actividad , Tiofenos/administración & dosificación , Tiofenos/síntesis química , Tiofenos/farmacocinéticaRESUMEN
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.
Asunto(s)
Descubrimiento de Drogas , Piridazinas/farmacología , Tiofenos/farmacología , Investigación Biomédica Traslacional , Regulación Alostérica , Animales , Cristalografía por Rayos X , Enlace de Hidrógeno , Piridazinas/química , Ratas , Relación Estructura-Actividad , Tiofenos/químicaRESUMEN
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.
Asunto(s)
Piperidinas/farmacología , Pirimidinas/farmacología , Quinazolinas/farmacología , Receptor Muscarínico M4/metabolismo , Tiofenos/farmacología , Regulación Alostérica , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Humanos , Microsomas Hepáticos/metabolismo , Piperidinas/síntesis química , Piperidinas/metabolismo , Pirimidinas/síntesis química , Pirimidinas/metabolismo , Quinazolinas/síntesis química , Quinazolinas/metabolismo , Ratas , Receptor Muscarínico M4/agonistas , Receptor Muscarínico M4/antagonistas & inhibidores , Relación Estructura-Actividad , Tiofenos/síntesis química , Tiofenos/metabolismoRESUMEN
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.
RESUMEN
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.
Asunto(s)
Fármacos Anti-VIH/metabolismo , Proteínas de la Cápside/metabolismo , Cápside/metabolismo , Fármacos Anti-VIH/farmacología , Línea Celular , VIH-1/efectos de los fármacos , Humanos , Replicación Viral/efectos de los fármacosRESUMEN
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).
Asunto(s)
Proteínas Reguladoras de la Apoptosis/antagonistas & inhibidores , Descubrimiento de Drogas , Indoles/farmacología , Proteínas Reguladoras de la Apoptosis/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Indoles/síntesis química , Indoles/química , Estructura Molecular , Relación Estructura-ActividadRESUMEN
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 at the LBDs by glutamate and synthetic orthosteric compounds or at the TMDs by allosteric modulators. Despite the potential of allosteric compounds as therapeutics, an understanding of the functional and structural basis of their effects is limited. Here we use multiple approaches to dissect the 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 internalization of group II and III mGluRs. The effects of PAMs are pleiotropic, boosting the maximal response to orthosteric agonists and serving independently as internalization-biased agonists across mGluR subtypes. Motivated by this and intersubunit FRET analyses, 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.
Asunto(s)
Microscopía por Crioelectrón , Receptores de Glutamato Metabotrópico , Receptores de Glutamato Metabotrópico/metabolismo , Receptores de Glutamato Metabotrópico/química , Receptores de Glutamato Metabotrópico/agonistas , Regulación Alostérica , Humanos , Células HEK293 , Ligandos , Animales , Transferencia Resonante de Energía de Fluorescencia , Dominios ProteicosRESUMEN
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.
Asunto(s)
Adhesinas Bacterianas/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Serina/metabolismo , Streptococcus gordonii/genética , Adhesinas Bacterianas/metabolismo , Animales , Sitios de Unión , Plaquetas/metabolismo , Endocarditis Bacteriana/metabolismo , Endocarditis Bacteriana/microbiología , Femenino , Humanos , Lectinas/metabolismo , Microscopía Fluorescente , Mucinas/metabolismo , Mutagénesis Sitio-Dirigida , Complejo GPIb-IX de Glicoproteína Plaquetaria/metabolismo , Mutación Puntual , Unión Proteica , Estructura Secundaria de Proteína , Ratas , Ratas Sprague-Dawley , Análisis de Secuencia de ADN , Lectinas Similares a la Inmunoglobulina de Unión a Ácido SiálicoRESUMEN
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.
Asunto(s)
Isatina/análogos & derivados , Receptor Muscarínico M1/antagonistas & inhibidores , Compuestos de Espiro/química , Regulación Alostérica , Animales , Humanos , Isatina/química , Isatina/metabolismo , Unión Proteica , Pirrolidinas/síntesis química , Pirrolidinas/química , Pirrolidinas/metabolismo , Ratas , Receptor Muscarínico M1/metabolismo , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Amidas/química , Encéfalo/metabolismo , Piridinas/química , Receptor Muscarínico M4/metabolismo , Tiofenos/química , Regulación Alostérica , Amidas/farmacocinética , Amidas/uso terapéutico , Animales , Encéfalo/efectos de los fármacos , Colinérgicos/química , Colinérgicos/farmacocinética , Colinérgicos/uso terapéutico , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Semivida , Humanos , Unión Proteica , Piridinas/farmacocinética , Piridinas/uso terapéutico , Ratas , Receptor Muscarínico M4/química , Esquizofrenia/tratamiento farmacológico , Relación Estructura-Actividad , Tiofenos/farmacocinética , Tiofenos/uso terapéuticoRESUMEN
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.
Asunto(s)
Isatina/análogos & derivados , Isatina/síntesis química , Receptor Muscarínico M1/efectos de los fármacos , Concentración 50 Inhibidora , Isatina/química , Isatina/farmacología , Sondas Moleculares/química , Sondas Moleculares/farmacología , Estructura Molecular , Inhibidores de la Monoaminooxidasa/farmacologíaRESUMEN
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.