RESUMEN
Glioblastomas (GBMs) are heterogeneous, treatment-resistant tumors driven by populations of cancer stem cells (CSCs). However, few molecular mechanisms critical for CSC population maintenance have been exploited for therapeutic development. We developed a spatially resolved loss-of-function screen in GBM patient-derived organoids to identify essential epigenetic regulators in the SOX2-enriched, therapy-resistant niche and identified WDR5 as indispensable for this population. WDR5 is a component of the WRAD complex, which promotes SET1 family-mediated Lys4 methylation of histone H3 (H3K4me), associated with positive regulation of transcription. In GBM CSCs, WDR5 inhibitors blocked WRAD complex assembly and reduced H3K4 trimethylation and expression of genes involved in CSC-relevant oncogenic pathways. H3K4me3 peaks lost with WDR5 inhibitor treatment occurred disproportionally on POU transcription factor motifs, including the POU5F1(OCT4)::SOX2 motif. Use of a SOX2/OCT4 reporter demonstrated that WDR5 inhibitor treatment diminished cells with high reporter activity. Furthermore, WDR5 inhibitor treatment and WDR5 knockdown altered the stem cell state, disrupting CSC in vitro growth and self-renewal, as well as in vivo tumor growth. These findings highlight the role of WDR5 and the WRAD complex in maintaining the CSC state and provide a rationale for therapeutic development of WDR5 inhibitors for GBM and other advanced cancers.
Asunto(s)
Glioblastoma , Humanos , Glioblastoma/tratamiento farmacológico , Glioblastoma/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Factores de Transcripción , Células Madre Neoplásicas/patología , Péptidos y Proteínas de Señalización Intracelular/genéticaRESUMEN
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
Cancer stem cells drive tumor growth and survival via self-renewal and therapeutic resistance, but the upstream mechanisms are not well defined. In this issue of PLOS Biology, a study in colon cancer reveals a new signalling network that links epigenetic regulation to these phenotypes.
Asunto(s)
Neoplasias del Colon , Resistencia a Antineoplásicos , Humanos , Resistencia a Antineoplásicos/genética , Epigénesis Genética , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/genética , Células Madre Neoplásicas , Fenotipo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Proteínas de Neoplasias , Péptidos y Proteínas de Señalización IntracelularRESUMEN
Interferon (IFN)-stimulated gene 15 (ISG15), a ubiquitin-like protein, is covalently conjugated to host immune proteins such as MDA5 and IRF3 in a process called ISGylation, thereby promoting type I IFN induction to limit the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, whether SARS-CoV-2 proteins can be directly targeted for ISGylation remains elusive. In this study, we identified the nucleocapsid (N) protein of SARS-CoV-2 as a major substrate of ISGylation catalyzed by the host E3 ligase HERC5; however, N ISGylation is readily removed through deISGylation by the papain-like protease (PLpro) activity of NSP3. Mass spectrometry analysis identified that the N protein undergoes ISGylation at four lysine residues (K266, K355, K387, and K388), and mutational analysis of these sites in the context of a SARS-CoV-2 replicon (N-4KR) abolished N ISGylation and alleviated ISGylation-mediated inhibition of viral RNA synthesis. Furthermore, our results indicated that HERC5 targets preferentially phosphorylated N protein for ISGylation to regulate its oligomeric assembly. These findings reveal a novel mechanism by which the host ISGylation machinery directly targets SARS-CoV-2 proteins to restrict viral replication and illuminate how an intricate interplay of host (HERC5) and viral (PLpro) enzymes coordinates viral protein ISGylation and thereby regulates virus replication.IMPORTANCEThe role of protein ISGylation in regulating host cellular processes has been studied extensively; however, how ISG15 conjugation influences the activity of viral proteins, particularly coronaviral proteins, is largely unknown. Our study uncovered that the nucleocapsid (N) protein of SARS-CoV-2 is ISGylated by the HERC5 ISGylation machinery and that this modification impedes the functional assembly of N into oligomers ultimately inhibiting viral RNA synthesis. This antiviral restriction mechanism is antagonized by the PLpro deISGylation activity of SARS-CoV-2 NSP3. This study deepens our understanding of SARS-CoV-2 protein regulation by posttranslational modifications and may open new avenues for designing antiviral strategies for COVID-19.
RESUMEN
Rett syndrome (RS) is a neurodevelopmental disorder that shares many symptomatic and pathological commonalities with idiopathic autism. Alterations in protein synthesis-dependent synaptic plasticity (PSDSP) are a hallmark of a number of syndromic forms of autism; in the present work, we explore the consequences of disruption and rescue of PSDSP in a mouse model of RS. We report that expression of a key regulator of synaptic protein synthesis, the metabotropic glutamate receptor 5 (mGlu5) protein, is significantly reduced in both the brains of RS model mice and in the motor cortex of human RS autopsy samples. Furthermore, we demonstrate that reduced mGlu5 expression correlates with attenuated DHPG-induced long-term depression in the hippocampus of RS model mice, and that administration of a novel mGlu5 positive allosteric modulator (PAM), termed VU0462807, can rescue synaptic plasticity defects. Additionally, treatment of Mecp2-deficient mice with VU0462807 improves motor performance (open-field behavior and gait dynamics), corrects repetitive clasping behavior, as well as normalizes cued fear-conditioning defects. Importantly, due to the rationale drug discovery approach used in its development, our novel mGlu5 PAM improves RS phenotypes and synaptic plasticity defects without evoking the overt adverse effects commonly associated with potentiation of mGlu5 signaling (i.e. seizures), or affecting cardiorespiratory defects in RS model mice. These findings provide strong support for the continued development of mGlu5 PAMs as potential therapeutic agents for use in RS, and, more broadly, for utility in idiopathic autism.
Asunto(s)
Trastorno Autístico/genética , Receptor del Glutamato Metabotropico 5/genética , Síndrome de Rett/genética , Convulsiones/genética , Adulto , Regulación Alostérica/genética , Animales , Trastorno Autístico/tratamiento farmacológico , Trastorno Autístico/patología , Autopsia , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Hipocampo/efectos de los fármacos , Hipocampo/patología , Humanos , Masculino , Proteína 2 de Unión a Metil-CpG/genética , Ratones , Ratones Noqueados , Corteza Motora/efectos de los fármacos , Corteza Motora/patología , Plasticidad Neuronal/efectos de los fármacos , Pirazoles/administración & dosificación , Pirimidinonas/administración & dosificación , Receptor del Glutamato Metabotropico 5/biosíntesis , Síndrome de Rett/tratamiento farmacológico , Síndrome de Rett/patología , Convulsiones/tratamiento farmacológico , Convulsiones/patología , Transducción de Señal/efectos de los fármacos , Adulto JovenRESUMEN
This letter describes the chemical optimization of a new series of M1 positive allosteric modulators (PAMs) based on a novel benzomorpholine core, developed via iterative parallel synthesis, and culminating in the highly utilized rodent in vivo tool compound, VU0486846 (7), devoid of adverse effect liability. This is the first report of the optimization campaign (SAR and DMPK profiling) that led to the discovery of VU0486846 and details all of the challenges faced in allosteric modulator programs (both steep and flat SAR, as well as subtle structural changes affecting CNS penetration and overall physiochemical and DMPK properties).
Asunto(s)
Descubrimiento de Drogas , Morfolinas/farmacología , Pirazoles/farmacología , Receptor Muscarínico M1/agonistas , Animales , Células CHO , Cricetulus , Humanos , Estructura Molecular , Morfolinas/síntesis química , Morfolinas/química , Pirazoles/síntesis química , Pirazoles/química , Ratas , Relación Estructura-ActividadRESUMEN
A second-generation small molecule P2X3 receptor antagonist has been developed. The lead optimization strategy to address shortcomings of the first-generation preclinical lead compound is described herein. These studies were directed towards the identification and amelioration of preclinical hepatobiliary findings, reducing potential for drug-drug interactions, and decreasing the projected human dose of the first-generation lead.
Asunto(s)
Analgésicos/uso terapéutico , Benzamidas/uso terapéutico , Dolor/tratamiento farmacológico , Antagonistas del Receptor Purinérgico P2X/uso terapéutico , Piridinas/uso terapéutico , Receptores Purinérgicos P2X3/metabolismo , Analgésicos/síntesis química , Analgésicos/química , Analgésicos/farmacocinética , Animales , Benzamidas/síntesis química , Benzamidas/química , Benzamidas/farmacocinética , Perros , Diseño de Fármacos , Interacciones Farmacológicas , Glucuronosiltransferasa/antagonistas & inhibidores , Semivida , Hiperbilirrubinemia/prevención & control , Estructura Molecular , Antagonistas del Receptor Purinérgico P2X/síntesis química , Antagonistas del Receptor Purinérgico P2X/química , Antagonistas del Receptor Purinérgico P2X/farmacocinética , Piridinas/síntesis química , Piridinas/química , Piridinas/farmacocinética , Ratas , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Human platelets display a unique dual receptor system for responding to its primary endogenous activator, α-thrombin. Because of the lack of efficacious antagonists, the field has relied on synthetic peptides and pepducins to describe protease-activated receptor PAR1 and PAR4 signaling. The precise contributions of each receptor have not been established in the context of thrombin. We took advantage of newly discovered PAR antagonists to contrast the contribution of PAR1 and PAR4 to thrombin-mediated activation of the platelet fibrin receptor (GPIIbIIIa). PAR1 is required for platelet activation at low but not high concentrations of thrombin, and maximal platelet activation at high concentrations of thrombin requires PAR4. As the concentration of thrombin is increased, PAR1 signaling is quickly overcome by PAR4 signaling, leaving a narrow window of low thrombin concentrations that exclusively engage PAR1. PAR4 antagonism reduces the maximum thrombin response by over 50%. Thus, although the PAR1 response still active at higher concentrations of thrombin, this response is superseded by PAR4. Truncation of a known PAR4 antagonist and identification of the minimum pharmacophore converted the mechanism of inhibition from noncompetitive to competitive, such that the antagonist could be outcompeted by increasing doses of the ligand. Fragments retained efficacy against both soluble and tethered ligands with lower cLogP values and an increased free fraction in plasma. These reversible, competitive compounds represent a route toward potentially safer PAR4 antagonists for clinical utility and the development of tools such as radioligands and positron emission tomography tracers that are not currently available to the field for this target.
Asunto(s)
Plaquetas/metabolismo , Integrina beta3/metabolismo , Complejo GPIb-IX de Glicoproteína Plaquetaria/metabolismo , Receptor PAR-1/metabolismo , Receptores de Trombina/metabolismo , Trombina/farmacología , Plaquetas/efectos de los fármacos , Humanos , Ligandos , Receptor PAR-1/antagonistas & inhibidores , Receptores de Trombina/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacosRESUMEN
Negative allosteric modulators (NAMs) of metabotropic glutamate receptor subtype 5 (mGlu5) have potential applications in the treatment of fragile X syndrome, levodopa-induced dyskinesia in Parkinson disease, Alzheimer disease, addiction, and anxiety; however, clinical and preclinical studies raise concerns that complete blockade of mGlu5 and inverse agonist activity of current mGlu5 NAMs contribute to adverse effects that limit the therapeutic use of these compounds. We report the discovery and characterization of a novel mGlu5 NAM, N,N-diethyl-5-((3-fluorophenyl)ethynyl)picolinamide (VU0477573) that binds to the same allosteric site as the prototypical mGlu5 NAM MPEP but displays weak negative cooperativity. Because of this weak cooperativity, VU0477573 acts as a "partial NAM" so that full occupancy of the MPEP site does not completely inhibit maximal effects of mGlu5 agonists on intracellular calcium mobilization, inositol phosphate (IP) accumulation, or inhibition of synaptic transmission at the hippocampal Schaffer collateral-CA1 synapse. Unlike previous mGlu5 NAMs, VU0477573 displays no inverse agonist activity assessed using measures of effects on basal [(3)H]inositol phosphate (IP) accumulation. VU0477573 acts as a full NAM when measuring effects on mGlu5-mediated extracellular signal-related kinases 1/2 phosphorylation, which may indicate functional bias. VU0477573 exhibits an excellent pharmacokinetic profile and good brain penetration in rodents and provides dose-dependent full mGlu5 occupancy in the central nervous system (CNS) with systemic administration. Interestingly, VU0477573 shows robust efficacy, comparable to the mGlu5 NAM MTEP, in models of anxiolytic activity at doses that provide full CNS occupancy of mGlu5 and demonstrate an excellent CNS occupancy-efficacy relationship. VU0477573 provides an exciting new tool to investigate the efficacy of partial NAMs in animal models.
Asunto(s)
Agonistas del GABA/farmacología , Ácidos Picolínicos/farmacología , Receptor del Glutamato Metabotropico 5/efectos de los fármacos , Regulación Alostérica/efectos de los fármacos , Animales , Ansiolíticos/farmacología , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Conducta Animal/efectos de los fármacos , Encéfalo/metabolismo , Relación Dosis-Respuesta a Droga , Descubrimiento de Drogas , Agonistas del GABA/farmacocinética , Células HEK293 , Humanos , Fosfatos de Inositol/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ácidos Picolínicos/farmacocinética , Piridinas/metabolismo , Ensayo de Unión Radioligante , Ratas , Receptor del Glutamato Metabotropico 5/metabolismo , Transmisión Sináptica/efectos de los fármacosRESUMEN
This letter describes the further deconstruction of the known PAR4 inhibitor chemotypes (MWs 490-525 and with high plasma protein binding) to identify a minimum PAR4 pharmacophore devoid of metabolic liabilities and improved properties. This exercise identified a greatly simplified 2-methoxy-6-arylimidazo[2,1-b][1,3,4]thiadiazole scaffold that afforded nanomolar inhibition of both activating peptide and γ-thrombin mediated PAR4 stimulation, while reducing both molecular weight and the number of hydrogen bond donors/acceptors by â¼50%. This minimum PAR4 pharmacophore, with competitive inhibition, versus non-competitive of the larger chemotypes, allows an ideal starting point to incorporate desired functional groups to engender optimal DMPK properties towards a preclinical candidate.
Asunto(s)
Agregación Plaquetaria/efectos de los fármacos , Receptores de Trombina/antagonistas & inhibidores , Tiadiazoles/química , Tiadiazoles/farmacología , Plaquetas/citología , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Humanos , Inhibidores de Agregación Plaquetaria/química , Inhibidores de Agregación Plaquetaria/farmacología , Receptores de Trombina/metabolismo , Trombina/metabolismoRESUMEN
This Letter describes the continued chemical optimization of the VU0453595 series of M1 positive allosteric modulators (PAMs). By surveying alternative 5,6- and 6,6-heterobicylic cores for the 6,7-dihydro-5H-pyrrolo[3,4-b]pyridine-5-one core of VU453595, we found new cores that engendered not only comparable or improved M1 PAM potency, but significantly improved CNS distribution (Kps 0.3-3.1). Moreover, this campaign provided fundamentally distinct M1 PAM chemotypes, greatly expanding the available structural diversity for this valuable CNS target, devoid of hydrogen-bond donors.
Asunto(s)
Fármacos del Sistema Nervioso Central/farmacología , Sistema Nervioso Central/efectos de los fármacos , Descubrimiento de Drogas , Compuestos Heterocíclicos/farmacología , Piridinas/farmacología , Pirroles/farmacología , Receptor Muscarínico M1/metabolismo , Regulación Alostérica/efectos de los fármacos , Animales , Fármacos del Sistema Nervioso Central/síntesis química , Fármacos del Sistema Nervioso Central/química , Relación Dosis-Respuesta a Droga , Compuestos Heterocíclicos/síntesis química , Compuestos Heterocíclicos/química , Estructura Molecular , Piridinas/síntesis química , Piridinas/química , Pirroles/síntesis química , Pirroles/química , Ratas , Relación Estructura-ActividadRESUMEN
As part of our efforts to identify a suitable back-up compound to our recently disclosed mGlu5 positive allosteric modulator (PAM) clinical candidate VU0490551/JNJ-46778212, this letter details the investigation and challenges of a novel series of 6,7-dihydropyrazolo[1,5-a]pyrazin-4-one derivatives. From these efforts, compound 4k emerged as a potent and selective mGlu5 PAM displaying overall attractive in vitro (pharmacological and ADMET) and PK profiles combined with in vivo efficacy in preclinical models of schizophrenia. However, further advancement of the compound was precluded due to severely limiting CNS-related side-effects confirming the previously reported association between excessive mGlu5 activation and target-related toxicities.
Asunto(s)
Regulación Alostérica/efectos de los fármacos , Antipsicóticos/uso terapéutico , Pirazinas/uso terapéutico , Pirazoles/uso terapéutico , Receptor del Glutamato Metabotropico 5/metabolismo , Esquizofrenia/tratamiento farmacológico , Animales , Antipsicóticos/química , Antipsicóticos/farmacocinética , Células HEK293 , Humanos , Masculino , Pirazinas/química , Pirazinas/farmacocinética , Pirazoles/química , Pirazoles/farmacocinética , Ratas Sprague-Dawley , Esquizofrenia/metabolismoRESUMEN
A series of substituted hydroxymethyl piperidine small molecule inhibitors of the protein-protein interaction between menin and mixed lineage leukemia 1 (MLL1) are described. Initial members of the series showed good inhibitory disruption of the menin-MLL1 interaction but demonstrated poor physicochemical and DMPK properties. Utilizing a structure-guided and iterative optimization approach key substituents were optimized leading to inhibitors with cell-based activity, improved in vitro DMPK parameters, and improved half-lives in rodent PK studies leading to MLPCN probe ML399. Ancillary off-target activity remains a parameter for further optimization.
Asunto(s)
Proteína de la Leucemia Mieloide-Linfoide/antagonistas & inhibidores , Piperidinas/química , Piperidinas/farmacología , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Animales , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Cristalografía por Rayos X , Diseño de Fármacos , Humanos , Técnicas In Vitro , Ratones , Modelos Moleculares , Proteína de la Leucemia Mieloide-Linfoide/química , Piperidinas/farmacocinética , Dominios y Motivos de Interacción de Proteínas/efectos de los fármacos , Proteínas Proto-Oncogénicas/química , Ratas , Relación Estructura-ActividadRESUMEN
Results from a 2012 high-throughput screen of the NIH Molecular Libraries Small Molecule Repository (MLSMR) against the human muscarinic receptor subtype 1 (M1) for positive allosteric modulators is reported. A content-rich screen utilizing an intracellular calcium mobilization triple-addition protocol allowed for assessment of all three modes of pharmacology at M1, including agonist, positive allosteric modulator, and antagonist activities in a single screening platform. We disclose a dibenzyl-2H-pyrazolo[4,3-c]quinolin-3(5H)-one hit (DBPQ, CID 915409) and examine N-benzyl pharmacophore/SAR relationships versus previously reported quinolin-3(5H)-ones and isatins, including ML137. SAR and consideration of recently reported crystal structures, homology modeling, and structure-function relationships using point mutations suggests a shared binding mode orientation at the putative common allosteric binding site directed by the pendant N-benzyl substructure.
Asunto(s)
Descubrimiento de Drogas , Pirazoles/farmacología , Quinolinas/farmacología , Quinolonas/química , Receptor Muscarínico M1/química , Receptor Muscarínico M1/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Regulación Alostérica , Sitio Alostérico , Humanos , Modelos Moleculares , Simulación del Acoplamiento Molecular , Estructura Molecular , Pirazoles/química , Quinolinas/química , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-ActividadRESUMEN
This Letter describes the progress and challenges in the continued optimization of the mGlu5 positive allosteric modulator (PAM) clinical candidate VU0490551/JNJ-46778212. While many analogs addressed key areas for improvement, no one compound possessed the amalgamation of improvements needed within the (2(phenoxymethyl)-6,7-dihydrooxazolo[5,4-c]pyridine-5(4H)-yl(aryl)methanone scaffold to advance as a back-up clinical candidate. However, many analogs displayed excellent solubility and physiochemical properties, and were active in the amphetamine-induced hyperlocomotion (AHL) model. Moreover, the SAR was robust for this series of PAMs, and both polar and hydrogen-bond donors were found to be tolerated, leading to analogs with overall attractive profiles and good ligand efficiencies.
Asunto(s)
Receptor del Glutamato Metabotropico 5/uso terapéutico , Esquizofrenia/genética , Regulación Alostérica , Descubrimiento de Drogas , Humanos , Estructura Molecular , Receptor del Glutamato Metabotropico 5/química , Relación Estructura-ActividadRESUMEN
We report the discovery and SAR of two novel series of imidazopyrimidinones and dihydroimidazopyrimidinones as metabotropic glutamate receptor 5 (mGlu5) positive allosteric modulators (PAMs). Exploration of several structural features in the western and eastern part of the imidazopyrimidinone core and combinations thereof, revealed compound 4a as a mGlu5 PAM with good in vitro potency and efficacy, acceptable drug metabolism and pharmacokinetic (DMPK) properties and in vivo efficacy in an amphetamine-based model of psychosis. However, the presence of CNS-mediated adverse effects in preclinical species precluded any further in vivo evaluation.
Asunto(s)
Antipsicóticos/química , Compuestos Heterocíclicos con 2 Anillos/química , Imidazoles/química , Pirimidinonas/química , Receptor del Glutamato Metabotropico 5/química , Regulación Alostérica , Animales , Antipsicóticos/síntesis química , Antipsicóticos/farmacocinética , Encéfalo/metabolismo , Evaluación Preclínica de Medicamentos , Semivida , Compuestos Heterocíclicos con 2 Anillos/síntesis química , Compuestos Heterocíclicos con 2 Anillos/farmacocinética , Humanos , Imidazoles/síntesis química , Imidazoles/farmacocinética , Locomoción/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Unión Proteica , Pirimidinonas/síntesis química , Pirimidinonas/farmacocinética , Ratas , Receptor del Glutamato Metabotropico 5/metabolismo , Relación Estructura-ActividadRESUMEN
We report the optimization of a series of metabotropic glutamate receptor 5 (mGlu5) positive allosteric modulators (PAMs) from an acyl dihydropyrazolo[1,5-a]pyrimidinone class. Investigation of exocyclic amide transpositions with this unique 5,6-bicyclic core were conducted in attempt to modulate physicochemical properties and identify a suitable backup candidate with a reduced half-life. A potent and selective PAM, 1-(2-(phenoxymethyl)-6,7-dihydropyrazolo[1,5-a]pyrimidin-4(5H)-yl)ethanone (9a, VU0462807), was identified with superior solubility and efficacy in the acute amphetamine-induced hyperlocomotion (AHL) rat model with a minimum effective dose of 3mg/kg. Attempts to mitigate oxidative metabolism of the western phenoxy of 9a through extensive modification and profiling are described.
Asunto(s)
Encéfalo/metabolismo , Pirazoles/farmacocinética , Pirimidinas/farmacocinética , Pirimidinonas/farmacocinética , Receptor del Glutamato Metabotropico 5/agonistas , Regulación Alostérica , Animales , Perros , Humanos , Ligandos , Masculino , Actividad Motora/efectos de los fármacos , Pirazoles/sangre , Pirazoles/síntesis química , Pirazoles/aislamiento & purificación , Pirazoles/farmacología , Pirimidinas/sangre , Pirimidinas/síntesis química , Pirimidinas/farmacología , Pirimidinonas/sangre , Pirimidinonas/síntesis química , Pirimidinonas/aislamiento & purificación , Pirimidinonas/farmacología , Ratas , Ratas Sprague-Dawley , Relación Estructura-ActividadRESUMEN
The IC50 of a beta-secretase (BACE-1) lead compound was improved â¼200-fold from 11 µM to 55 nM through the addition of a single methyl group. Computational chemistry, small molecule NMR, and protein crystallography capabilities were used to compare the solution conformation of the ligand under varying pH conditions to its conformation when bound in the active site. Chemical modification then explored available binding pockets adjacent to the ligand. A strategically placed methyl group not only maintained the required pKa of the piperidine nitrogen and filled a small hydrophobic pocket, but more importantly, stabilized the conformation best suited for optimized binding to the receptor.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Hidantoínas/química , Hidantoínas/farmacología , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Ácido Aspártico Endopeptidasas/metabolismo , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Humanos , Hidantoínas/síntesis química , Metilación , Modelos Moleculares , Estructura Molecular , Relación Estructura-ActividadRESUMEN
The bat coronavirus HKU4 belongs to the same 2c lineage as that of the deadly Middle East Respiratory Syndrome coronavirus (MERS-CoV) and shows high sequence similarity, therefore potentiating a threat to the human population through a zoonotic shift or 'spill over' event. To date, there are no effective vaccines or antiviral treatments available that are capable of limiting the pathogenesis of any human coronaviral infection. An attractive target for the development of anti-coronaviral therapeutics is the 3C-like protease (3CL(pro)), which is essential for the progression of the coronaviral life cycle. Herein, we report the screening results of a small, 230-member peptidomimetic library against HKU4-CoV 3CL(pro) and the identification of 43 peptidomimetic compounds showing good to excellent inhibitory potency of HKU4-CoV 3CL(pro) with IC50 values ranging from low micromolar to sub-micromolar. We established structure-activity relationships (SARs) describing the important ligand-based features required for potent HKU4-CoV 3CL(pro) inhibition and identified a seemingly favored peptidic backbone for HKU4-CoV 3CL(pro) inhibition. To investigate this, a molecular sub-structural analysis of the most potent HKU4-CoV 3CL(pro) inhibitor was accomplished by the synthesis and testing of the lead peptidomimetic inhibitor's sub-structural components, confirming the activity of the favored backbone (22A) identified via SAR analysis. In order to elucidate the structural reasons for such potent HKU4-CoV 3CL(pro) inhibition by the peptidomimetics having the 22A backbone, we determined the X-ray structures of HKU4-CoV 3CL(pro) in complex with three peptidomimetic inhibitors. Sequence alignment of HKU4-CoV 3CL(pro), and two other lineage C Betacoronaviruses 3CL(pro)'s, HKU5-CoV and MERS-CoV 3CL(pro), show that the active site residues of HKU4-CoV 3CL(pro) that participate in inhibitor binding are conserved in HKU5-CoV and MERS-CoV 3CL(pro). Furthermore, we assayed our most potent HKU4-CoV 3CL(pro) inhibitor for inhibition of HKU5-CoV 3CL(pro) and found it to have sub-micromolar inhibitory activity (IC50=0.54±0.03µM). The X-ray structures and SAR analysis reveal critical insights into the structure and inhibition of HKU4-CoV 3CL(pro), providing fundamental knowledge that may be exploited in the development of anti-coronaviral therapeutics for coronaviruses emerging from zoonotic reservoirs.
Asunto(s)
Infecciones por Coronavirus/enzimología , Coronavirus del Síndrome Respiratorio de Oriente Medio/genética , Inhibidores de Proteasas/uso terapéutico , Animales , Quirópteros , Infecciones por Coronavirus/virología , Modelos Animales de Enfermedad , Humanos , 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).