RESUMEN
Small-molecule probes can illuminate biological processes and aid in the assessment of emerging therapeutic targets by perturbing biological systems in a manner distinct from other experimental approaches. Despite the tremendous promise of chemical tools for investigating biology and disease, small-molecule probes were unavailable for most targets and pathways as recently as a decade ago. In 2005, the NIH launched the decade-long Molecular Libraries Program with the intent of innovating in and broadening access to small-molecule science. This Perspective describes how novel small-molecule probes identified through the program are enabling the exploration of biological pathways and therapeutic hypotheses not otherwise testable. These experiences illustrate how small-molecule probes can help bridge the chasm between biological research and the development of medicines but also highlight the need to innovate the science of therapeutic discovery.
Asunto(s)
Descubrimiento de Drogas , Bibliotecas de Moléculas Pequeñas , Animales , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Ensayos Analíticos de Alto Rendimiento , Humanos , National Institutes of Health (U.S.) , Estados UnidosRESUMEN
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
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.
Asunto(s)
Regulación Alostérica/fisiología , Ácido Glutámico/metabolismo , Enfermedad de Huntington/tratamiento farmacológico , Receptor Muscarínico M4/fisiología , Transmisión Sináptica/fisiología , Animales , Encéfalo/metabolismo , Fluorescencia , Enfermedad de Huntington/fisiopatología , Inmunohistoquímica , Ratones , Ratones Mutantes , Piridazinas/farmacología , Piridazinas/uso terapéutico , Prueba de Desempeño de Rotación con Aceleración Constante , Transmisión Sináptica/efectos de los fármacos , Tiofenos/farmacología , Tiofenos/uso terapéuticoRESUMEN
This letter details the continued chemical optimization of a novel series of M4 positive allosteric modulators (PAMs) based on a 5-amino-thieno[2,3-c]pyridazine core by incorporating a 3-amino azetidine amide moiety. The analogs described within this work represent the most potent M4 PAMs reported for this series to date. The SAR to address potency, clearance, subtype selectivity, CNS exposure, and P-gp efflux are described. This work culminated in the discovery of VU6000918, which demonstrated robust efficacy in a rat amphetamine-induced hyperlocomotion reversal model at a minimum efficacious dose of 0.3mg/kg.
Asunto(s)
Amidas/farmacología , Azetidinas/farmacología , Receptor Muscarínico M4/antagonistas & inhibidores , Regulación Alostérica/efectos de los fármacos , Amidas/síntesis química , Amidas/química , Animales , Azetidinas/síntesis química , Azetidinas/química , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Ratas , Relación Estructura-ActividadRESUMEN
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 PAMs based on a non-enolizable ketone core, identified from an MLPCN functional high-throughput screen. The HTS hit was potent, selective and CNS penetrant; however, the compound was highly cleared in vitro and in vivo. SAR provided analogs for which M4 PAM potency and CNS exposure were maintained; yet, clearance remained high. Metabolite identification studies demonstrated that this series was subject to rapid, and near quantitative, reductive metabolism to the corresponding secondary alcohol metabolite that was devoid of M4 PAM activity.
Asunto(s)
Descubrimiento de Drogas , Cetonas/farmacocinética , Receptor Muscarínico M1/agonistas , Regulación Alostérica , Animales , Sistema Nervioso Central/metabolismo , Humanos , Cetonas/síntesis química , Cetonas/química , Estructura Molecular , Relación Estructura-ActividadRESUMEN
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
Of the five muscarinic receptor subtypes, the M5 receptor is the only one detectable in midbrain dopaminergic neurons, making it an attractive potential therapeutic target for treating disorders in which dopaminergic signaling is disrupted. However, developing an understanding of the role of M5 in regulating midbrain dopamine neuron function has been hampered by a lack of subtype-selective compounds. Here, we extensively characterize the novel compound VU0238429 and demonstrate that it acts as a positive allosteric modulator with unprecedented selectivity for the M5 receptor. We then used VU0238429, along with M5 knock-out mice, to elucidate the role of this receptor in regulating substantia nigra pars compacta (SNc) neuron physiology in both mice and rats. In sagittal brain slices that isolate the SNc soma from their striatal terminals, activation of muscarinic receptors induced Ca2+ mobilization and inward currents in SNc dopamine neurons, both of which were potentiated by VU0238429 and absent in M5 knock-out mice. Activation of M5 also increased the spontaneous firing rate of SNc neurons, suggesting that activation of somatodendritic M5 increases the intrinsic excitability of SNc neurons. However, in coronal slices of the striatum, potentiation of M5 with VU0238429 resulted in an inhibition in dopamine release as monitored with fast scan cyclic voltammetry. Accordingly, activation of M5 can lead to opposing physiological outcomes depending on the location of the receptor. Although activation of somatodendritic M5 receptors on SNc neurons leads to increased neuronal firing, activation of M5 receptors in the striatum induces an inhibition in dopamine release.
Asunto(s)
Dopamina/metabolismo , Neuronas Dopaminérgicas/fisiología , Receptor Muscarínico M5/metabolismo , Animales , Animales Recién Nacidos , Encéfalo/citología , Células CHO , Calcio/metabolismo , Cricetulus , Neuronas Dopaminérgicas/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Técnicas In Vitro , Indoles/farmacología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Ratas , Ratas Sprague-Dawley , Receptor Muscarínico M5/genética , TransfecciónRESUMEN
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.
Asunto(s)
Imidazoles/química , Indoles/química , Receptor Muscarínico M5/química , Regulación Alostérica , Animales , Encéfalo/metabolismo , Semivida , Humanos , Imidazoles/metabolismo , Imidazoles/farmacocinética , Indoles/metabolismo , Indoles/farmacocinética , Microsomas Hepáticos/metabolismo , Unión Proteica , Ratas , Receptor Muscarínico M5/genética , Receptor Muscarínico M5/metabolismo , Relación Estructura-ActividadRESUMEN
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
A new class of CGRP receptor antagonists was identified by replacing the central amide of a previously identified anilide lead structure with ethylene, ethane, or ethyne linkers. (E)-Alkenes as well as alkynes were found to preserve the proper bioactive conformation of the amides, necessary for efficient receptor binding. Further exploration resulted in several potent compounds against CGRP-R with low susceptibility to P-gp mediated efflux.
Asunto(s)
Alquenos/farmacología , Antagonistas del Receptor Peptídico Relacionado con el Gen de la Calcitonina , Alquenos/síntesis química , Alquenos/química , Amidas/química , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Estructura Molecular , Relación Estructura-ActividadRESUMEN
This Letter describes the further chemical optimization of the M5 PAM MLPCN probes ML129 and ML172. A multi-dimensional iterative parallel synthesis effort quickly explored isatin replacements and a number of southern heterobiaryl variations with no improvement over ML129 and ML172. An HTS campaign identified several weak M5 PAMs (M5 EC50 >10µM) with a structurally related isatin core that possessed a southern phenethyl ether linkage. While SAR within the HTS series was very shallow and unable to be optimized, grafting the phenethyl ether linkage onto the ML129/ML172 cores led to the first sub-micromolar M5 PAM, ML326 (VU0467903), (human and rat M5 EC50s of 409nM and 500nM, respectively) with excellent mAChR selectivity (M1-M4 EC50s >30µM) and a robust 20-fold leftward shift of the ACh CRC.
Asunto(s)
Descubrimiento de Drogas , Indoles/farmacología , Receptores Muscarínicos/metabolismo , Animales , Relación Dosis-Respuesta a Droga , Humanos , Indoles/síntesis química , Indoles/química , Estructura Molecular , Ratas , Relación Estructura-ActividadRESUMEN
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 further exploration of two series of M(1) allosteric agonists, TBPB and VU0357017, previously reported from our lab. Within the TPBP scaffold, either electronic or steric perturbations to the central piperidine ring led to a loss of selective M(1) allosteric agonism and afforded pan-mAChR antagonism, which was demonstrated to be mediated via the orthosteric site. Additional SAR around a related M(1) allosteric agonist family (VU0357017) identified similar, subtle 'molecular switches' that modulated modes of pharmacology from allosteric agonism to pan-mAChR orthosteric antagonism. Therefore, all of these ligands are best classified as bi-topic ligands that possess high affinity binding at an allosteric site to engender selective M(1) activation, but all bind, at higher concentrations, to the orthosteric ACh site, leading to non-selective orthosteric site binding and mAChR antagonism.
Asunto(s)
Receptor Muscarínico M1/agonistas , Acetilcolina/metabolismo , Regulación Alostérica/efectos de los fármacos , Sitio Alostérico , Animales , Benzamidas/química , Benzamidas/farmacología , Bencimidazoles/química , Bencimidazoles/farmacología , Células CHO , Calcio/metabolismo , Cricetinae , Cricetulus , Humanos , Piperidinas/química , Piperidinas/farmacología , Ratas , Receptor Muscarínico M1/genética , Receptor Muscarínico M1/metabolismo , Receptores Muscarínicos/química , Receptores Muscarínicos/metabolismo , Relación Estructura-Actividad , TransfecciónRESUMEN
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.