RESUMO
Achieving ligand subtype selectivity within highly homologous subtypes of G-protein-coupled receptor (GPCR) is critical yet challenging for GPCR drug discovery, primarily due to the unclear mechanism underlying ligand subtype selectivity, which hampers the rational design of subtype-selective ligands. Herein, we disclose an unusual molecular mechanism of entropy-driven ligand recognition in cannabinoid (CB) receptor subtypes, revealed through atomic-level molecular dynamics simulations, cryoelectron microscopy structure, and mutagenesis experiments. This mechanism is attributed to the distinct conformational dynamics of the receptor's orthosteric pocket, leading to variations in ligand binding entropy and consequently, differential binding affinities, which culminate in specific ligand recognition. We experimentally validated this mechanism and leveraged it to design ligands with enhanced or ablated subtype selectivity. One such ligand demonstrated favorable pharmacokinetic properties and significant efficacy in rodent inflammatory analgesic models. More importantly, it is precisely due to the high subtype selectivity obtained based on this mechanism that this ligand does not show addictive properties in animal models. Our findings elucidate the unconventional role of entropy in CB receptor subtype selectivity and suggest a strategy for rational design of ligands to achieve entropy-driven subtype selectivity for many pharmaceutically important GPCRs.
Assuntos
Entropia , Simulação de Dinâmica Molecular , Receptores Acoplados a Proteínas G , Ligantes , Animais , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/química , Humanos , Ligação Proteica , Camundongos , Microscopia Crioeletrônica , Receptores de Canabinoides/metabolismo , Receptores de Canabinoides/química , Sítios de LigaçãoRESUMO
Chemokine receptors are integral to the immune system and prime targets in drug discovery that have undergone extensive structural elucidation in recent years. We outline a timeline of these structural achievements, discuss the intracellular negative allosteric modulation of chemokine receptors, analyze the mechanisms of orthosteric receptor activation, and report on the emerging concept of biased signaling. Additionally, we highlight differences of G-protein binding among chemokine receptors. Intracellular allosteric modulators in chemokine receptors interact with a conserved motif within transmembrane helix 7 and helix 8 and exhibit a two-fold inactivation mechanism that can be harnessed for drug-discovery efforts. Chemokine recognition is a multi-step process traditionally explained by a two-site model within chemokine recognition site 1 (CRS1) and CRS2. Recent structural studies have extended our understanding of this complex mechanism with the identification of CRS1.5 and CRS3. CRS3 is implicated in determining ligand specificity and surrounds the chemokine by almost 180°. Within CRS3 we identified the extracellular loop 2 residue 45.51 as a key interaction mediator for chemokine binding. Y2917.43 on the other hand was shown in CCR1 to be a key determinant of signaling bias which, along with specific chemokine-dependent phosphorylation ensembles at the G-protein coupled receptors (GPCR's) C-terminus, seems to play a pivotal role in determining the direction of signal bias in GPCRs.
Assuntos
Receptores de Quimiocinas , Transdução de Sinais , Receptores de Quimiocinas/metabolismo , Receptores de Quimiocinas/química , Humanos , Quimiocinas/metabolismo , Quimiocinas/química , Ligação Proteica , Regulação Alostérica , Modelos Moleculares , Animais , Sítios de Ligação , Conformação Proteica , LigantesRESUMO
Cannabis sativa is known for its therapeutic benefit in various diseases including pain relief by targeting cannabinoid receptors. The primary component of cannabis, Δ9-tetrahydrocannabinol (THC), and other agonists engage the orthosteric site of CB1, activating both Gi and ß-arrestin signaling pathways. The activation of diverse pathways could result in on-target side effects and cannabis addiction, which may hinder therapeutic potential. A significant challenge in pharmacology is the design of a ligand that can modulate specific signaling of CB1. By leveraging insights from the structure-function selectivity relationship (SFSR), we have identified Gi signaling-biased agonist-allosteric modulators (ago-BAMs). Further, two cryoelectron microscopy (cryo-EM) structures reveal the binding mode of ago-BAM at the extrahelical allosteric site of CB1. Combining mutagenesis and pharmacological studies, we elucidated the detailed mechanism of ago-BAM-mediated biased signaling. Notably, ago-BAM CB-05 demonstrated analgesic efficacy with fewer side effects, minimal drug toxicity and no cannabis addiction in mouse pain models. In summary, our finding not only suggests that ago-BAMs of CB1 provide a potential nonopioid strategy for pain management but also sheds light on BAM identification for GPCRs.
Assuntos
Microscopia Crioeletrônica , Receptor CB1 de Canabinoide , Transdução de Sinais , Receptor CB1 de Canabinoide/metabolismo , Receptor CB1 de Canabinoide/genética , Receptor CB1 de Canabinoide/química , Animais , Regulação Alostérica/efeitos dos fármacos , Camundongos , Humanos , Transdução de Sinais/efeitos dos fármacos , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/genética , Células HEK293 , Relação Estrutura-Atividade , Dronabinol/farmacologia , Dronabinol/química , Dronabinol/análogos & derivados , Cannabis/química , Cannabis/metabolismoRESUMO
Selectively targeting the cannabinoid receptor CB2 is an attractive therapeutic strategy for the treatment of inflammatory pain without psychiatric side effects mediated by the cannabinoid receptor CB1. Herein, we report the discovery of 4-(1,2,4-oxadiazol-5-yl)azepan-2-one derivatives as a new class of CB2 agonists. Systematic structure-activity relationship investigations resulted in the identification of the most potent compound 25r. This compound displayed high selectivity for CB2 against CB1 (CB2 EC50 = 21.0 nM, Emax = 87%, CB1 EC50 > 30 µM, ratio CB1/CB2 > 1428) with favorable pharmacokinetic properties. Especially, 25r demonstrated significant efficacy in the analgesic model of rodent inflammatory pain. All the results suggest that compound 25r could serve as a lead compound for treating inflammatory pain and deserves further in-depth studies.
Assuntos
Agonistas de Receptores de Canabinoides , Canabinoides , Humanos , Dor/tratamento farmacológico , Analgésicos/farmacologia , Analgésicos/uso terapêutico , Relação Estrutura-Atividade , Receptor CB2 de Canabinoide , Receptor CB1 de CanabinoideRESUMO
The gonadotrophin-releasing hormone (GnRH) is a central regulator of the human reproductive system and exerts physiological effects by binding to GnRH1R. The GnRH-GnRH1R system is a promising therapeutic target for the maintenance of reproductive function. There are several GnRH1R agonists on the market, but like GnRH, they are all peptide compounds and are limited by their way of administration (subcutaneous or intramuscular injection). To date, no published GnRH1R small molecule agonists have been reported. In this paper, the HTRF-based screening method has been used to screen our in-house chemical library, and we found and confirmed CD304 as a hit compound. Subsequently, structure optimization led to the discovery of compound 6d, exhibited with a certain GnRH1R activation activity (EC50: 1.59 ± 0.38 µM). Further molecular dynamics simulation experiments showed that 6d can well bind to the orthosteric site of GnRH1R through forming a hydrogen-bonding interaction with Y2836.51. Binding of 6d further induces conformational changes in TM6 and TM7, promoting the formation of a continuous water channel in GnRH1R, thereby promoting GnRH1R activation. This well-characterized hit compound will facilitate the further development of novel small molecule agonists of GnRH1R.
Assuntos
Hormônio Liberador de Gonadotropina , Receptores LHRH , Humanos , Hormônio Liberador de Gonadotropina/farmacologia , Receptores LHRH/agonistas , Receptores LHRH/química , Bibliotecas de Moléculas Pequenas/farmacologia , Ligação de HidrogênioRESUMO
Given the promising clinical value of allosteric modulators of G protein-coupled-receptors (GPCRs), mechanistic understanding of how these modulators alter GPCR function is of significance. Here, we report the crystallographic and cryo-electron microscopy structures of the cannabinoid receptor CB1 bound to the positive allosteric modulator (PAM) ZCZ011. These structures show that ZCZ011 binds to an extrahelical site in the transmembrane 2 (TM2)-TM3-TM4 surface. Through (un)biased molecular dynamics simulations and mutagenesis experiments, we show that TM2 rearrangement is critical for the propagation of allosteric signals. ZCZ011 exerts a PAM effect by promoting TM2 rearrangement in favor of receptor activation and increasing the population of receptors that adopt an active conformation. In contrast, ORG27569, a negative allosteric modulator (NAM) of CB1, also binds to the TM2-TM3-TM4 surface and exerts a NAM effect by impeding the TM2 rearrangement. Our findings fill a gap in the understanding of CB1 allosteric regulation and could guide the rational design of CB1 allosteric modulators.
Assuntos
Simulação de Dinâmica Molecular , Receptor CB1 de Canabinoide , Regulação Alostérica , Sítio Alostérico , Microscopia Crioeletrônica , Receptor CB1 de Canabinoide/genéticaRESUMO
Ataxia telangiectasia and Rad3-related (ATR) kinase is a key regulating protein within the DNA damage response (DDR), responsible for sensing replication stress (RS), and has been considered as a potential target for cancer therapy. Herein, we report the discovery of a series of 6,7-dihydro-5H-pyrrolo[3,4-d]-pyrimidine derivatives as a new class of ATR inhibitors. Among them, compound 5g exhibits an IC50 value of 0.007 µM against ATR kinase. In vitro, 5g displays good anti-tumor activity and could significantly reduce the phosphorylation level of ATR and its downstream signaling protein. Overall, this study provides a promising lead compound for subsequent drug discovery targeting ATR kinase.
Assuntos
Neoplasias , Inibidores de Proteínas Quinases , Proteínas Mutadas de Ataxia Telangiectasia , Dano ao DNA , Humanos , Neoplasias/tratamento farmacológico , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêuticoRESUMO
Bromodomain and PHD finger containing transcription factor (BPTF) is a multidomain protein that regulates the transcription of chromatin and is related to many cancers. Herein, we report the screening-based discovery of Cpd1, a compound with micromolar affinity to the BPTF bromodomain. Through structure-guided optimization, we synthesized a variety of new inhibitors. Among these compounds, Cpd8 and Cpd10 were highly potent and selective inhibitors, with KD values of 428 nM and 655 nM in ITC assays, respectively. The high activity was explained by the cocrystal structure of Cpd8 in complex with the BPTF bromodomain protein. Cpd8 and Cpd10 were able to stabilize the BPTF bromodomain protein in cells in a cellular thermal shift assay (CETSA). Cpd8 downregulated c-MYC expression in A549 cells. All experiments prove that these two compounds are potential BPTF inhibitors.
Assuntos
Proteínas do Tecido Nervoso/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Células A549 , Antígenos Nucleares/química , Antígenos Nucleares/genética , Calorimetria , Cristalografia por Raios X , Desenho de Fármacos , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Fluorometria , Regulação da Expressão Gênica/efeitos dos fármacos , Genes myc , Células HEK293 , Humanos , Modelos Moleculares , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Domínios Proteicos , Estabilidade Proteica/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/genética , Relação Estrutura-Atividade , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continually poses serious threats to global public health. The main protease (Mpro) of SARS-CoV-2 plays a central role in viral replication. We designed and synthesized 32 new bicycloproline-containing Mpro inhibitors derived from either boceprevir or telaprevir, both of which are approved antivirals. All compounds inhibited SARS-CoV-2 Mpro activity in vitro, with 50% inhibitory concentration values ranging from 7.6 to 748.5 nM. The cocrystal structure of Mpro in complex with MI-23, one of the most potent compounds, revealed its interaction mode. Two compounds (MI-09 and MI-30) showed excellent antiviral activity in cell-based assays. In a transgenic mouse model of SARS-CoV-2 infection, oral or intraperitoneal treatment with MI-09 or MI-30 significantly reduced lung viral loads and lung lesions. Both also displayed good pharmacokinetic properties and safety in rats.
Assuntos
Antivirais/farmacologia , Tratamento Farmacológico da COVID-19 , Proteases 3C de Coronavírus/antagonistas & inibidores , Inibidores de Proteases/farmacologia , Animais , Antivirais/química , Antivirais/uso terapêutico , COVID-19/patologia , COVID-19/virologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Quimiocina CXCL10/metabolismo , Modelos Animais de Doenças , Desenho de Fármacos , Humanos , Interferon beta/metabolismo , Pulmão/imunologia , Pulmão/patologia , Pulmão/virologia , Camundongos , Camundongos Transgênicos , Oligopeptídeos , Prolina/análogos & derivados , Inibidores de Proteases/química , Inibidores de Proteases/uso terapêutico , Inibidores de Proteases/toxicidade , Ratos , Ratos Sprague-Dawley , Carga Viral/efeitos dos fármacos , Replicação ViralRESUMO
BAZ1A is a bromodomain-containing protein, and has been recognized as a potential target for multiple diseases, particularly cancer. However, there is no BAZ1A inhibitor reported so far. In this study, we used a consensus docking/scoring strategy to screen for BAZ1A bromodomain inhibitors from commercial chemical libraries and an in-house chemical database. The retrieved hit compounds were evaluated experimentally and four compounds were found to be active against BAZ1A bromodomain. To the most active compounds, similarity and substructure searches were used to find more BAZ1A bromodomain inhibitors. Among all the obtained active compounds, Cpd-2 is the most potent one, which showed a KD value of 0.52 µM. The interaction model of Cpd-2 with BAZ1A bromodomain was revealed by molecular docking. In a cellular assay, Cpd-2 displayed good anti-viability activity against cancer cell lines expressing a high level of BAZ1A. Overall, we discovered a number of BAZ1A bromodomain inhibitors for the first time, which can be a good starting point for subsequent drug discovery targeting BAZ1A bromodomain.
Assuntos
Antineoplásicos/farmacologia , Proteínas Cromossômicas não Histona/antagonistas & inibidores , Descoberta de Drogas , Compostos Orgânicos/farmacologia , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Compostos Orgânicos/química , Relação Estrutura-AtividadeRESUMO
Indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO), which mediate kynurenine pathway of tryptophan degradation, have emerged as potential new targets in immunotherapy for treatment of cancer because of their critical role in immunosuppression in the tumor microenvironment. In this investigation, we report the structural optimization and structure-activity relationship studies of 1-phenyl-1H-naphtho[2,3-d][1,2,3]triazole-4,9-dione derivatives as a new class of IDO1/TDO dual inhibitors. Among all the obtained dual inhibitors, 1-(3-chloro-4-fluorophenyl)-6-fluoro-1H-naphtho[2,3-d][1,2,3]triazole-4,9-dione (38) displayed the most potent IDO1 and TDO inhibitory activities with IC50 (half-maximal inhibitory concentration) values of 5 nM for IDO1 and 4 nM for TDO. It turned out that compound 38 was not a PAINS compound. Compound 38 could efficiently inhibit the biofunction of IDO1 and TDO in intact cells. In LL2 (Lewis lung cancer) and Hepa1-6 (hepatic carcinoma) allograft mouse models, this compound also showed considerable in vivo anti-tumor activity and no obvious toxicity was observed. Therefore, 38 could be a good lead compound for cancer immunotherapy and deserving further investigation.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Triazóis/química , Triazóis/farmacologia , Triptofano Oxigenase/antagonistas & inibidores , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Desenho de Fármacos , Células HeLa , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Camundongos Endogâmicos C57BL , Relação Estrutura-Atividade , Triptofano Oxigenase/metabolismoRESUMO
We propose a frequency-scanning interferometry using the Kalman filtering technique for dynamic absolute distance measurement. Frequency-scanning interferometry only uses a single tunable laser driven by a triangle waveform signal for forward and backward optical frequency scanning. The absolute distance and moving speed of a target can be estimated by the present input measurement of frequency-scanning interferometry and the previously calculated state based on the Kalman filter algorithm. This method not only compensates for movement errors in conventional frequency-scanning interferometry, but also achieves high-precision and low-complexity dynamic measurements. Experimental results of dynamic measurements under static state, vibration and one-dimensional movement are presented.
RESUMO
Mode hopping is a critical performance limitation of external cavity diode lasers (ECDLs). We developed a method to suppress the mode hops by shifting the internal cavity longitudinal mode spectrum to match the other mode spectra. A model of longitudinal mode structure based on Littman-Metcalf configuration has been deduced for analyzing the characteristics of external cavity mode hop and internal cavity mode hop. The best mode matching point appears at the peak of output power when tuning the ECDL. Both the short-term and long-term wavelength stability were investigated in different laser diode current values while the tuning element maintains a fixed position. The experimental results demonstrated the effectiveness of the proposed mode matching methods for improving the mode stability and reducing the sensitivity to environment change.
RESUMO
We present an effective method to extend the mode-hop-free (MHF) tuning range of an external-cavity diode laser (ECDL) by synchronous tuning of the longitudinal modes of the external cavity and the internal cavity, with the mode also matched in the initial state. Both the principle of synchronous tuning and the condition of mode matching in a Littman-configuration ECDL are introduced. The necessary tuning parameters could simply be estimated by the output power curve of the tuning with a single photodiode. By using this tuning method, we increased the MHF tuning range of an ECDL with a nonoptimized reflector pivot position from several gigahertzes to over 78 GHz around 774.5 nm. The tuning performance of the ECDL could meet the requirement of frequency scanned interferometry.