RESUMO
State-of-the-art methods in photoproximity labeling center on the targeted generation and capture of short-lived reactive intermediates to provide a snapshot of local protein environments. Diazirines are the current gold standard for high-resolution proximity labeling, generating short-lived aryl(trifluoromethyl) carbenes. Here, we present a method to access aryl(trifluoromethyl) carbenes from a stable diazo source via tissue-penetrable, deep red to near-infrared light (600-800 nm). The operative mechanism of this activation involves Dexter energy transfer from photoexcited osmium(II) photocatalysts to the diazo, thus revealing an aryl(trifluoromethyl) carbene. The labeling preferences of the diazo probe with amino acids are studied, showing high reactivity toward heteroatom-H bonds. Upon the synthesis of a biotinylated diazo probe, labeling studies are conducted on native proteins as well as proteins conjugated to the Os photocatalyst. Finally, we demonstrate that the conjugation of a protein inhibitor to the photocatalyst also enables selective protein labeling in the presence of spectator proteins and achieves specific labeling of a membrane protein on the surface of mammalian cells via a two-antibody photocatalytic system.
Assuntos
Proteínas , Luz Vermelha , Animais , Proteínas/química , Metano/química , Diazometano/química , MamíferosRESUMO
The characterization of ligand binding modes is a crucial step in the drug discovery process and is especially important in campaigns arising from phenotypic screening, where the protein target and binding mode are unknown at the outset. Elucidation of target binding regions is typically achieved by X-ray crystallography or photoaffinity labeling (PAL) approaches; yet, these methods present significant challenges. X-ray crystallography is a mainstay technique that has revolutionized drug discovery, but in many cases structural characterization is challenging or impossible. PAL has also enabled binding site mapping with peptide- and amino-acid-level resolution; however, the stoichiometric activation mode can lead to poor signal and coverage of the resident binding pocket. Additionally, each PAL probe can have its own fragmentation pattern, complicating the analysis by mass spectrometry. Here, we establish a robust and general photocatalytic approach toward the mapping of protein binding sites, which we define as identification of residues proximal to the ligand binding pocket. By utilizing a catalytic mode of activation, we obtain sets of labeled amino acids in the proximity of the target protein binding site. We use this methodology to map, in vitro, the binding sites of six protein targets, including several kinases and molecular glue targets, and furthermore to investigate the binding site of the STAT3 inhibitor MM-206, a ligand with no known crystal structure. Finally, we demonstrate the successful mapping of drug binding sites in live cells. These results establish µMap as a powerful method for the generation of amino-acid- and peptide-level target engagement data.
Assuntos
Peptídeos , Proteínas , Ligantes , Proteínas/química , Sítios de Ligação , Peptídeos/química , Ligação ProteicaRESUMO
Over half of new therapeutic approaches fail in clinical trials due to a lack of target validation. As such, the development of new methods to improve and accelerate the identification of cellular targets, broadly known as target ID, remains a fundamental goal in drug discovery. While advances in sequencing and mass spectrometry technologies have revolutionized drug target ID in recent decades, the corresponding chemical-based approaches have not changed in over 50 y. Consigned to outdated stoichiometric activation modes, modern target ID campaigns are regularly confounded by poor signal-to-noise resulting from limited receptor occupancy and low crosslinking yields, especially when targeting low abundance membrane proteins or multiple protein target engagement. Here, we describe a broadly general platform for photocatalytic small molecule target ID, which is founded upon the catalytic amplification of target-tag crosslinking through the continuous generation of high-energy carbene intermediates via visible light-mediated Dexter energy transfer. By decoupling the reactive warhead tag from the small molecule ligand, catalytic signal amplification results in unprecedented levels of target enrichment, enabling the quantitative target and off target ID of several drugs including (+)-JQ1, paclitaxel (Taxol), dasatinib (Sprycel), as well as two G-protein-coupled receptors-ADORA2A and GPR40.
Assuntos
Sistemas de Liberação de Medicamentos , Transferência de Energia , Proteômica , Descoberta de Drogas , Espectrometria de MassasRESUMO
Modern proximity labeling techniques have enabled significant advances in understanding biomolecular interactions. However, current tools primarily utilize activation modes that are incompatible with complex biological environments, limiting our ability to interrogate cell- and tissue-level microenvironments in animal models. Here, we report µMap-Red, a proximity labeling platform that uses a red-light-excited SnIV chlorin e6 catalyst to activate a phenyl azide biotin probe. We validate µMap-Red by demonstrating photonically controlled protein labeling in vitro through several layers of tissue, and we then apply our platform in cellulo to label EGFR microenvironments and validate performance with STED microscopy and quantitative proteomics. Finally, to demonstrate labeling in a complex biological sample, we deploy µMap-Red in whole mouse blood to profile erythrocyte cell-surface proteins. This work represents a significant methodological advance toward light-based proximity labeling in complex tissue environments and animal models.
Assuntos
Biotina , Proteômica , Animais , Biotina/metabolismo , Luz , Proteínas de Membrana , Camundongos , Proteômica/métodos , Coloração e RotulagemRESUMO
We previously reported medicinal chemistry efforts that identified MK-5204, an orally efficacious ß-1,3-glucan synthesis inhibitor derived from the natural product enfumafungin. Further extensive optimization of the C2 triazole substituent identified 4-pyridyl as the preferred replacement for the carboxamide of MK-5204, leading to improvements in antifungal activity in the presence of serum, and increased oral exposure. Reoptimizing the aminoether at C3 in the presence of this newly discovered C2 substituent, confirmed that the (R) t-butyl, methyl aminoether of MK-5204 provided the best balance of these two key parameters, culminating in the discovery of ibrexafungerp, which is currently in phase III clinical trials. Ibrexafungerp displayed significantly improved oral efficacy in murine infection models, making it a superior candidate for clinical development as an oral treatment for Candida and Aspergillus infections.
Assuntos
Antifúngicos/farmacologia , Aspergillus/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Glicosídeos/química , Triterpenos/química , beta-Glucanas/metabolismo , Administração Oral , Animais , Antifúngicos/síntese química , Antifúngicos/farmacocinética , Antifúngicos/uso terapêutico , Aspergilose/tratamento farmacológico , Candidíase/tratamento farmacológico , Modelos Animais de Doenças , Glicosídeos/farmacocinética , Glicosídeos/farmacologia , Glicosídeos/uso terapêutico , Meia-Vida , Camundongos , Relação Estrutura-Atividade , Triterpenos/farmacocinética , Triterpenos/farmacologia , Triterpenos/uso terapêuticoRESUMO
Our previously reported efforts to produce an orally active ß-1,3-glucan synthesis inhibitor through the semi-synthetic modification of enfumafungin focused on replacing the C2 acetoxy moiety with an aminotetrazole and the C3 glycoside with a N,N-dimethylaminoether moiety. This work details further optimization of the C2 heterocyclic substituent, which identified 3-carboxamide-1,2,4-triazole as a replacement for the aminotetrazole with comparable antifungal activity. Alkylation of either the carboxamidetriazole at C2 or the aminoether at C3 failed to significantly improve oral efficacy. However, replacement of the isopropyl alpha amino substituent with a t-butyl, improved oral exposure while maintaining antifungal activity. These two structural modifications produced MK-5204, which demonstrated broad spectrum activity against Candida species and robust oral efficacy in a murine model of disseminated Candidiasis without the N-dealkylation liability observed for the previous lead.
Assuntos
Antifúngicos/química , Triazóis/química , beta-Glucanas/metabolismo , Administração Oral , Animais , Antifúngicos/metabolismo , Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Candida/efeitos dos fármacos , Candidíase/tratamento farmacológico , Modelos Animais de Doenças , Glucosiltransferases/antagonistas & inibidores , Glucosiltransferases/metabolismo , Glicosídeos/química , Meia-Vida , Camundongos , Testes de Sensibilidade Microbiana , Estereoisomerismo , Relação Estrutura-Atividade , Triazóis/metabolismo , Triazóis/farmacologia , Triazóis/uso terapêutico , Triterpenos/química , beta-Glucanas/químicaRESUMO
Many disease pathologies can be understood through the elucidation of localized biomolecular networks, or microenvironments. To this end, enzymatic proximity labeling platforms are broadly applied for mapping the wider spatial relationships in subcellular architectures. However, technologies that can map microenvironments with higher precision have long been sought. Here, we describe a microenvironment-mapping platform that exploits photocatalytic carbene generation to selectively identify protein-protein interactions on cell membranes, an approach we term MicroMap (µMap). By using a photocatalyst-antibody conjugate to spatially localize carbene generation, we demonstrate selective labeling of antibody binding targets and their microenvironment protein neighbors. This technique identified the constituent proteins of the programmed-death ligand 1 (PD-L1) microenvironment in live lymphocytes and selectively labeled within an immunosynaptic junction.
Assuntos
Antígeno B7-H1/metabolismo , Membrana Celular/metabolismo , Microambiente Celular , Linfócitos/metabolismo , Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas , Catálise , Membrana Celular/efeitos da radiação , Transferência de Energia , Humanos , Células Jurkat , Linfócitos/efeitos da radiação , Metano/análogos & derivados , Metano/química , Metano/efeitos da radiação , Processos Fotoquímicos , Raios UltravioletaRESUMO
Doxorubicin is a highly effective chemotherapy agent used to treat many common malignancies. However, its use is limited by cardiotoxicity, and cumulative doses exponentially increase the risk of heart failure. To identify novel heart failure treatment targets, a zebrafish model of doxorubicin-induced cardiomyopathy was previously established for small-molecule screening. Using this model, several small molecules that prevent doxorubicin-induced cardiotoxicity both in zebrafish and in mouse models have previously been identified. In this study, exploration of doxorubicin cardiotoxicity is expanded by screening 2271 small molecules from a proprietary, target-annotated tool compound collection. It is found that 120 small molecules can prevent doxorubicin-induced cardiotoxicity, including 7 highly effective compounds. Of these, all seven exhibited inhibitory activity towards cytochrome P450 familyâ 1 (CYP1). These results are consistent with previous findings, in which visnagin, a CYP1 inhibitor, also prevents doxorubicin-induced cardiotoxicity. Importantly, genetic mutation of cyp1a protected zebrafish against doxorubicin-induced cardiotoxicity phenotypes. Together, these results provide strong evidence that CYP1 is an important contributor to doxorubicin-induced cardiotoxicity and highlight the CYP1 pathway as a candidate therapeutic target for clinical cardioprotection.
Assuntos
Cardiomiopatias/prevenção & controle , Família 1 do Citocromo P450/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Cardiomiopatias/induzido quimicamente , Cardiomiopatias/patologia , Família 1 do Citocromo P450/antagonistas & inibidores , Família 1 do Citocromo P450/genética , Modelos Animais de Doenças , Doxorrubicina/toxicidade , Insuficiência Cardíaca , Mutagênese , Fenótipo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/uso terapêutico , Relação Estrutura-Atividade , Peixe-Zebra , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/genéticaRESUMO
GPR142 has been identified as a potential glucose-stimulated insulin secretion (GSIS) target for the treatment of type 2 diabetes mellitus (T2DM). A class of triazole GPR142 agonists was discovered through a high throughput screen. The lead compound 4 suffered from poor metabolic stability and poor solubility. Lead optimization strategies to improve potency, efficacy, metabolic stability, and solubility are described. This optimization led to compound 20e, which showed significant reduction of glucose excursion in wild-type but not in GPR142 deficient mice in an oral glucose tolerance test (oGTT) study. These studies provide strong evidence that reduction of glucose excursion through treatment with 20e is GPR142-mediated, and GPR142 agonists could be used as a potential treatment for type 2 diabetes.
RESUMO
Using structure based drug design, a novel class of potent coagulation factor IXa (FIXa) inhibitors was designed and synthesized. High selectivity over FXa inhibition was achieved. Selected compounds were evaluated in rat IV/PO pharmacokinetic (PK) studies and demonstrated desirable oral PK profiles. Finally, the pharmacodynamics (PD) of this class of molecules were evaluated in thrombin generation assay (TGA) in Corn Trypsin Inhibitor (CTI) citrated human plasma and demonstrated characteristics of a FIXa inhibitor.
Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Fator IXa/antagonistas & inibidores , Compostos Heterocíclicos com 3 Anéis/química , Compostos Heterocíclicos com 3 Anéis/farmacologia , Administração Oral , Animais , Cristalografia por Raios X , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/química , Compostos Heterocíclicos com 3 Anéis/síntese química , Humanos , Estrutura Molecular , RatosRESUMO
Using structure based drug design (SBDD), a novel class of potent coagulation Factor IXa (FIXa) inhibitors was designed and synthesized. High selectivity over FXa inhibition was achieved. Selected compounds demonstrated oral bioavailability in rat IV/PO pharmacokinetic (PK) studies. Finally, the pharmacodynamics (PD) of this class of molecules was evaluated in Thrombin Generation Assay (TGA) in Corn Trypsin Inhibitor (CTI) citrated human plasma and demonstrated characteristics of a FIXa inhibitor.
Assuntos
Aminas/farmacologia , Inibidores Enzimáticos/farmacologia , Fator IXa/antagonistas & inibidores , Administração Oral , Aminas/síntese química , Aminas/química , Animais , Disponibilidade Biológica , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Fator IXa/metabolismo , Humanos , Modelos Moleculares , Estrutura Molecular , Ratos , Relação Estrutura-AtividadeRESUMO
Two high-throughput screening hits were investigated for SAR against human factor IXa. Both hits feature a benzamide linked to a [6-5]-heteroaryl via an alkyl amine. In the case where this system is a benzimidazolyl-ethyl amine the binding potency for the hit was improved >500-fold, from 9 µM to 0.016 µM. For the other hit, which contains a tetrahydropyrido-indazole amine, potency was improved 20-fold, from 2 µM to 0.09 µM. X-ray crystal structures were obtained for an example of each class which improved understanding of the binding, and will enable further drug discovery efforts.
Assuntos
Anticoagulantes/química , Anticoagulantes/farmacologia , Fator IXa/antagonistas & inibidores , Sítios de Ligação , Descoberta de Drogas , Humanos , Modelos Moleculares , Estrutura Molecular , Conformação ProteicaRESUMO
Orally bioavailable inhibitors of ß-(1,3)-D-glucan synthase have been pursued as new, broad-spectrum fungicidal therapies suitable for treatment in immunocompromised patients. Toward this end, a collaborative medicinal chemistry program was established based on semisynthetic derivatization of the triterpenoid glycoside natural product enfumafungin in order to optimize in vivo antifungal activity and oral absorption properties. In the course of these studies, it was hypothesized that the pharmacokinetic properties of the semisynthetic enfumafungin analog 3 could be improved by tethering the alkyl groups proximal to the basic nitrogen of the C3-aminoether side chain into an azacyclic system, so as to preclude oxidative N-demethylation. The results of this research effort are disclosed herein.
Assuntos
Antifúngicos/síntese química , Inibidores Enzimáticos/síntese química , Glucosiltransferases/antagonistas & inibidores , Glicosídeos/química , Triterpenos/química , Administração Oral , Animais , Antifúngicos/química , Antifúngicos/farmacocinética , Candida albicans/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Glucosiltransferases/metabolismo , Glicosídeos/síntese química , Glicosídeos/farmacocinética , Meia-Vida , Camundongos , Relação Estrutura-Atividade , Triterpenos/síntese química , Triterpenos/farmacocinéticaRESUMO
Tetrahydrofluorenones which possess a C9a-fluoroalkyl substituent were efficiently converted to tetrahydrofluorenones which contain a ring bridging C9a-C2. Conditions include a stepwise sequence of conversion to an alkyl bromide followed by treatment with base, and a direct cyclization by treatment with lithium chloride in DMF heated to 150 degrees C.
Assuntos
Fluorenos/síntese química , Hidrocarbonetos Fluorados/química , Alquilação , Hidrocarbonetos Aromáticos com Pontes/síntese química , Catálise , Ciclização , Receptor beta de Estrogênio/agonistas , Estrutura MolecularRESUMO
Several tetrahydrofluorenones with a triazole fused across C7-C8 showed high levels of ERbeta-selectivity and were found to be potent ERbeta-agonists. As a class they demonstrate improved oral bioavailability in the rat over a parent class of 7-hydroxy-tetrahydrofluorenones. The most selective agonist displayed 5.7 nM affinity and 333-fold selectivity for ERbeta.