RESUMO
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.
Assuntos
Glioblastoma , Humanos , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Histona-Lisina N-Metiltransferase/metabolismo , Fatores de Transcrição , Células-Tronco Neoplásicas/patologia , Peptídeos e Proteínas de Sinalização Intracelular/genéticaRESUMO
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.
Assuntos
Descoberta de Drogas , Bibliotecas de Moléculas Pequenas , Animais , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala , Humanos , National Institutes of Health (U.S.) , Estados UnidosRESUMO
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.
Assuntos
Neoplasias do Colo , Resistencia a Medicamentos Antineoplásicos , Humanos , Resistencia a Medicamentos Antineoplásicos/genética , Epigênese Genética , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Células-Tronco Neoplásicas , Fenótipo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Proteínas de Neoplasias , Peptídeos e Proteínas de Sinalização IntracelularRESUMO
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.
Assuntos
Transtorno Autístico/genética , Receptor de Glutamato Metabotrópico 5/genética , Síndrome de Rett/genética , Convulsões/genética , Adulto , Regulação Alostérica/genética , Animais , Transtorno Autístico/tratamento farmacológico , Transtorno Autístico/patologia , Autopsia , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Humanos , Masculino , Proteína 2 de Ligação a Metil-CpG/genética , Camundongos , Camundongos Knockout , Córtex Motor/efeitos dos fármacos , Córtex Motor/patologia , Plasticidade Neuronal/efeitos dos fármacos , Pirazóis/administração & dosagem , Pirimidinonas/administração & dosagem , Receptor de Glutamato Metabotrópico 5/biossíntese , Síndrome de Rett/tratamento farmacológico , Síndrome de Rett/patologia , Convulsões/tratamento farmacológico , Convulsões/patologia , Transdução de Sinais/efeitos dos fármacos , Adulto JovemRESUMO
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).
Assuntos
Descoberta de Drogas , Morfolinas/farmacologia , Pirazóis/farmacologia , Receptor Muscarínico M1/agonistas , Animais , Células CHO , Cricetulus , Humanos , Estrutura Molecular , Morfolinas/síntese química , Morfolinas/química , Pirazóis/síntese química , Pirazóis/química , Ratos , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Analgésicos/uso terapêutico , Benzamidas/uso terapêutico , Dor/tratamento farmacológico , Antagonistas do Receptor Purinérgico P2X/uso terapêutico , Piridinas/uso terapêutico , Receptores Purinérgicos P2X3/metabolismo , Analgésicos/síntese química , Analgésicos/química , Analgésicos/farmacocinética , Animais , Benzamidas/síntese química , Benzamidas/química , Benzamidas/farmacocinética , Cães , Desenho de Fármacos , Interações Medicamentosas , Glucuronosiltransferase/antagonistas & inibidores , Meia-Vida , Hiperbilirrubinemia/prevenção & controle , Estrutura Molecular , Antagonistas do Receptor Purinérgico P2X/síntese química , Antagonistas do Receptor Purinérgico P2X/química , Antagonistas do Receptor Purinérgico P2X/farmacocinética , Piridinas/síntese química , Piridinas/química , Piridinas/farmacocinética , Ratos , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Plaquetas/metabolismo , Integrina beta3/metabolismo , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Receptor PAR-1/metabolismo , Receptores de Trombina/metabolismo , Trombina/farmacologia , Plaquetas/efeitos dos fármacos , Humanos , Ligantes , Receptor PAR-1/antagonistas & inibidores , Receptores de Trombina/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacosRESUMO
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.
Assuntos
Agonistas GABAérgicos/farmacologia , Ácidos Picolínicos/farmacologia , Receptor de Glutamato Metabotrópico 5/efeitos dos fármacos , Regulação Alostérica/efeitos dos fármacos , Animais , Ansiolíticos/farmacologia , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Comportamento Animal/efeitos dos fármacos , Encéfalo/metabolismo , Relação Dose-Resposta a Droga , Descoberta de Drogas , Agonistas GABAérgicos/farmacocinética , Células HEK293 , Humanos , Fosfatos de Inositol/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Ácidos Picolínicos/farmacocinética , Piridinas/metabolismo , Ensaio Radioligante , Ratos , Receptor de Glutamato Metabotrópico 5/metabolismo , Transmissão Sináptica/efeitos dos fármacosRESUMO
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.
Assuntos
Agregação Plaquetária/efeitos dos fármacos , Receptores de Trombina/antagonistas & inibidores , Tiadiazóis/química , Tiadiazóis/farmacologia , Plaquetas/citologia , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Humanos , Inibidores da Agregação Plaquetária/química , Inibidores da Agregação Plaquetária/farmacologia , Receptores de Trombina/metabolismo , Trombina/metabolismoRESUMO
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.
Assuntos
Fármacos do Sistema Nervoso Central/farmacologia , Sistema Nervoso Central/efeitos dos fármacos , Descoberta de Drogas , Compostos Heterocíclicos/farmacologia , Piridinas/farmacologia , Pirróis/farmacologia , Receptor Muscarínico M1/metabolismo , Regulação Alostérica/efeitos dos fármacos , Animais , Fármacos do Sistema Nervoso Central/síntese química , Fármacos do Sistema Nervoso Central/química , Relação Dose-Resposta a Droga , Compostos Heterocíclicos/síntese química , Compostos Heterocíclicos/química , Estrutura Molecular , Piridinas/síntese química , Piridinas/química , Pirróis/síntese química , Pirróis/química , Ratos , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Regulação Alostérica/efeitos dos fármacos , Antipsicóticos/uso terapêutico , Pirazinas/uso terapêutico , Pirazóis/uso terapêutico , Receptor de Glutamato Metabotrópico 5/metabolismo , Esquizofrenia/tratamento farmacológico , Animais , Antipsicóticos/química , Antipsicóticos/farmacocinética , Células HEK293 , Humanos , Masculino , Pirazinas/química , Pirazinas/farmacocinética , Pirazóis/química , Pirazóis/farmacocinética , Ratos Sprague-Dawley , Esquizofrenia/metabolismoRESUMO
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.
Assuntos
Proteína de Leucina Linfoide-Mieloide/antagonistas & inibidores , Piperidinas/química , Piperidinas/farmacologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Técnicas In Vitro , Camundongos , Modelos Moleculares , Proteína de Leucina Linfoide-Mieloide/química , Piperidinas/farmacocinética , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Proteínas Proto-Oncogênicas/química , Ratos , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Descoberta de Drogas , Pirazóis/farmacologia , Quinolinas/farmacologia , Quinolonas/química , Receptor Muscarínico M1/química , Receptor Muscarínico M1/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Regulação Alostérica , Sítio Alostérico , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Estrutura Molecular , Pirazóis/química , Quinolinas/química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Receptor de Glutamato Metabotrópico 5/uso terapêutico , Esquizofrenia/genética , Regulação Alostérica , Descoberta de Drogas , Humanos , Estrutura Molecular , Receptor de Glutamato Metabotrópico 5/química , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Antipsicóticos/química , Compostos Heterocíclicos com 2 Anéis/química , Imidazóis/química , Pirimidinonas/química , Receptor de Glutamato Metabotrópico 5/química , Regulação Alostérica , Animais , Antipsicóticos/síntese química , Antipsicóticos/farmacocinética , Encéfalo/metabolismo , Avaliação Pré-Clínica de Medicamentos , Meia-Vida , Compostos Heterocíclicos com 2 Anéis/síntese química , Compostos Heterocíclicos com 2 Anéis/farmacocinética , Humanos , Imidazóis/síntese química , Imidazóis/farmacocinética , Locomoção/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Ligação Proteica , Pirimidinonas/síntese química , Pirimidinonas/farmacocinética , Ratos , Receptor de Glutamato Metabotrópico 5/metabolismo , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Encéfalo/metabolismo , Pirazóis/farmacocinética , Pirimidinas/farmacocinética , Pirimidinonas/farmacocinética , Receptor de Glutamato Metabotrópico 5/agonistas , Regulação Alostérica , Animais , Cães , Humanos , Ligantes , Masculino , Atividade Motora/efeitos dos fármacos , Pirazóis/sangue , Pirazóis/síntese química , Pirazóis/isolamento & purificação , Pirazóis/farmacologia , Pirimidinas/sangue , Pirimidinas/síntese química , Pirimidinas/farmacologia , Pirimidinonas/sangue , Pirimidinonas/síntese química , Pirimidinonas/isolamento & purificação , Pirimidinonas/farmacologia , Ratos , Ratos Sprague-Dawley , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Hidantoínas/química , Hidantoínas/farmacologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Humanos , Hidantoínas/síntese química , Metilação , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Infecções por Coronavirus/enzimologia , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Inibidores de Proteases/uso terapêutico , Animais , Quirópteros , Infecções por Coronavirus/virologia , Modelos Animais de Doenças , Humanos , Relação Estrutura-AtividadeRESUMO
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).
Assuntos
Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Descoberta de Drogas , Indóis/farmacologia , Proteínas Reguladoras de Apoptose/metabolismo , Relação Dose-Resposta a Droga , Humanos , Indóis/síntese química , Indóis/química , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
We report the optimization of a series of novel metabotropic glutamate receptor 5 (mGlu5) positive allosteric modulators (PAMs) from a 5,6-bicyclic class of dihydropyrazolo[1,5-a]pyridin-4(5H)-ones containing a phenoxymethyl linker. Studies focused on a survey of non-amide containing hydrogen bond accepting (HBA) pharmacophore replacements. A highly potent and selective PAM, 2-(phenoxymethyl)-6,7-dihydropyrazolo[1,5-a]pyridin-4(5H)-one (11, VU0462054), bearing a simple ketone moiety, was identified (LE=0.52, LELP=3.2). In addition, hydroxyl, difluoro, ether, and amino variations were examined. Despite promising lead properties and exploration of alternative core heterocycles, linkers, and ketone replacements, oxidative metabolism and in vivo clearance remained problematic for the series.