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1.
Cell Chem Biol ; 28(11): 1602-1615.e9, 2021 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-34111400

RESUMO

Genetic screening technologies to identify and validate macromolecular interactions (MMIs) essential for complex pathways remain an important unmet need for systems biology and therapeutics development. Here, we use a library of peptides from diverse prokaryal genomes to screen MMIs promoting the nuclear relocalization of Forkhead Box O3 (FOXO3a), a tumor suppressor more frequently inactivated by post-translational modification than mutation. A hit peptide engages the 14-3-3 family of signal regulators through a phosphorylation-dependent interaction, modulates FOXO3a-mediated transcription, and suppresses cancer cell growth. In a crystal structure, the hit peptide occupies the phosphopeptide-binding groove of 14-3-3ε in a conformation distinct from its natural peptide substrates. A biophysical screen identifies drug-like small molecules that displace the hit peptide from 14-3-3ε, providing starting points for structure-guided development. Our findings exemplify "protein interference," an approach using evolutionarily diverse, natural peptides to rapidly identify, validate, and develop chemical probes against MMIs essential for complex cellular phenotypes.


Assuntos
Descoberta de Drogas , Proteína Forkhead Box O3/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Células Cultivadas , Feminino , Proteína Forkhead Box O3/genética , Proteína Forkhead Box O3/metabolismo , Genes Supressores de Tumor/efeitos dos fármacos , Humanos , Biblioteca de Peptídeos , Fosforilação , Bibliotecas de Moléculas Pequenas/química
2.
J Med Chem ; 63(23): 14700-14723, 2020 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-33297683

RESUMO

PI3Kδ inhibitors have been approved for B-cell malignancies like CLL, small lymphocytic lymphoma, and so forth. However, currently available PI3Kδ inhibitors are nonoptimal, showing weakness against at least one of the several important properties: potency, isoform selectivity, and/or pharmacokinetic profile. To come up with a PI3Kδ inhibitor that overcomes all these deficiencies, a pharmacophoric expansion strategy was employed. Herein, we describe a systematic transformation of a "three-blade propeller" shaped lead, 2,3-disubstituted quinolizinone 11, through a 1,2-disubstituted quinolizinone 20 to a novel "four-blade propeller" shaped 1,2,3-trisubstituted quinolizinone 34. Compound 34 has excellent potency, isoform selectivity, metabolic stability across species, and exhibited a favorable pharmacokinetic profile. Compound 34 also demonstrated a differentiated efficacy profile in human germinal center B and activated B cell-DLBCL cell lines and xenograft models. Compound 34 qualifies for further evaluation as a candidate for monotherapy or in combination with other targeted agents in DLBCLs and other forms of iNHL.


Assuntos
Antineoplásicos/uso terapêutico , Classe I de Fosfatidilinositol 3-Quinases/uso terapêutico , Neoplasias Hematológicas/tratamento farmacológico , Inibidores de Fosfoinositídeo-3 Quinase/uso terapêutico , Quinolizinas/uso terapêutico , Animais , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Classe I de Fosfatidilinositol 3-Quinases/síntese química , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Classe I de Fosfatidilinositol 3-Quinases/farmacocinética , Cães , Descoberta de Drogas , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos SCID , Simulação de Acoplamento Molecular , Estrutura Molecular , Inibidores de Fosfoinositídeo-3 Quinase/síntese química , Inibidores de Fosfoinositídeo-3 Quinase/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/farmacocinética , Quinolizinas/síntese química , Quinolizinas/metabolismo , Quinolizinas/farmacocinética , Células RAW 264.7 , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de Xenoenxerto
3.
ChemMedChem ; 14(18): 1620-1632, 2019 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-31334915

RESUMO

The tandem BRCT domains (tBRCT) of BRCA1 engage phosphoserine-containing motifs in target proteins to propagate intracellular signals initiated by DNA damage, thereby controlling cell cycle arrest and DNA repair. Recently, we identified Bractoppin, the first small-molecule inhibitor of the BRCA1 tBRCT domain, which selectively interrupts BRCA1-mediated cellular responses evoked by DNA damage. Here, we combine structure-guided chemical elaboration, protein mutagenesis and cellular assays to define the structural features responsible for Bractoppin's activity. Bractoppin fails to bind mutant forms of BRCA1 tBRCT bearing K1702A, a key residue mediating phosphopeptide recognition, or F1662R or L1701K that adjoin the pSer-recognition site. However, the M1775R mutation, which engages the Phe residue in the consensus phosphopeptide motif pSer-X-X-Phe, does not affect Bractoppin binding, confirming a binding mode distinct from the substrate phosphopeptide binding. We explored these structural features through structure-guided chemical elaboration and characterized structure-activity relationships (SARs) in biochemical assays. Two analogues, CCBT2088 and CCBT2103 were effective in abrogating BRCA1 foci formation and inhibiting G2 arrest induced by irradiation of cells. Collectively, our findings reveal structural features underlying the activity of a novel inhibitor of phosphopeptide recognition by the BRCA1 tBRCT domain, providing fresh insights to guide the development of inhibitors that target protein-protein interactions.


Assuntos
Proteína BRCA1/antagonistas & inibidores , Imidazóis/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Proteína BRCA1/isolamento & purificação , Proteína BRCA1/metabolismo , Relação Dose-Resposta a Droga , Imidazóis/síntese química , Imidazóis/química , Modelos Moleculares , Estrutura Molecular , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade
4.
Cell Chem Biol ; 25(6): 677-690.e12, 2018 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-29606576

RESUMO

Intracellular signals triggered by DNA breakage flow through proteins containing BRCT (BRCA1 C-terminal) domains. This family, comprising 23 conserved phosphopeptide-binding modules in man, is inaccessible to small-molecule chemical inhibitors. Here, we develop Bractoppin, a drug-like inhibitor of phosphopeptide recognition by the human BRCA1 tandem (t)BRCT domain, which selectively inhibits substrate binding with nanomolar potency in vitro. Structure-activity exploration suggests that Bractoppin engages BRCA1 tBRCT residues recognizing pSer in the consensus motif, pSer-Pro-Thr-Phe, plus an abutting hydrophobic pocket that is distinct in structurally related BRCT domains, conferring selectivity. In cells, Bractoppin inhibits substrate recognition detected by Förster resonance energy transfer, and diminishes BRCA1 recruitment to DNA breaks, in turn suppressing damage-induced G2 arrest and assembly of the recombinase, RAD51. But damage-induced MDC1 recruitment, single-stranded DNA (ssDNA) generation, and TOPBP1 recruitment remain unaffected. Thus, an inhibitor of phosphopeptide recognition selectively interrupts BRCA1 tBRCT-dependent signals evoked by DNA damage.


Assuntos
Proteína BRCA1/metabolismo , Fosfopeptídeos/metabolismo , Transdução de Sinais , Proteína BRCA1/isolamento & purificação , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Humanos , Simulação de Dinâmica Molecular , Estrutura Molecular , Fosfopeptídeos/análise , Fosfopeptídeos/antagonistas & inibidores , Domínios Proteicos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade
5.
EMBO J ; 25(17): 4050-60, 2006 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-16932745

RESUMO

Stabilization and maturation of synapses are important for development and function of the nervous system. Previous studies have implicated cholesterol-rich lipid microdomains in synapse stabilization, but the underlying mechanisms remain unclear. We found that cholesterol stabilizes clusters of synaptic acetylcholine receptors (AChRs) in denervated muscle in vivo and in nerve-muscle explants. In paralyzed muscles, cholesterol triggered maturation of nerve sprout-induced AChR clusters into pretzel shape. Cholesterol treatment also rescued a specific defect in AChR cluster stability in cultured src(-/-);fyn(-/-) myotubes. Postsynaptic proteins including AChRs, rapsyn, MuSK and Src-family kinases were strongly enriched in lipid microdomains prepared from wild-type myotubes. Microdomain disruption by cholesterol-sequestering methyl-beta-cyclodextrin disassembled AChR clusters and decreased AChR-rapsyn interaction and AChR phosphorylation. Amounts of microdomains and enrichment of postsynaptic proteins into microdomains were decreased in src(-/-);fyn(-/-) myotubes but rescued by cholesterol treatment. These data provide evidence that cholesterol-rich lipid microdomains and SFKs act in a dual mechanism in stabilizing the postsynapse: SFKs enhance microdomain-association of postsynaptic components, whereas microdomains provide the environment for SFKs to maintain interactions and phosphorylation of these components.


Assuntos
Colesterol/fisiologia , Microdomínios da Membrana/fisiologia , Junção Neuromuscular/fisiologia , Receptores Colinérgicos/metabolismo , Sinapses/fisiologia , Animais , Células Cultivadas , Colesterol/metabolismo , Microdomínios da Membrana/metabolismo , Camundongos , Fibras Musculares Esqueléticas/fisiologia , Proteínas Musculares/metabolismo , Junção Neuromuscular/ultraestrutura , Fosforilação , Proteínas Proto-Oncogênicas c-fyn/genética , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Sinapses/ultraestrutura , Quinases da Família src/genética , Quinases da Família src/metabolismo
6.
J Neurosci ; 25(45): 10479-93, 2005 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-16280586

RESUMO

Postnatal stabilization and maturation of the postsynaptic membrane are important for development and function of the neuromuscular junction (NMJ), but the underlying mechanisms remain poorly characterized. We examined the role of Src-family kinases (SFKs) in vivo. Electroporation of kinase-inactive Src constructs into soleus muscles of adult mice caused NMJ disassembly: acetylcholine receptor (AChR)-rich areas became fragmented; the topology of nerve terminal, AChRs, and synaptic nuclei was disturbed; and occasionally nerves started to sprout. Electroporation of kinase-overactive Src produced similar but milder effects. We studied the mechanism of SFK action using cultured src(-/-);fyn(-/-) myotubes, focusing on clustering of postsynaptic proteins, their interaction with AChRs, and AChR phosphorylation. Rapsyn and the utrophin-glycoprotein complex were recruited normally into AChR-containing clusters by agrin in src(-/-);fyn(-/-) myotubes. But after agrin withdrawal, clusters of these proteins disappeared rapidly in parallel with AChRs, revealing that SFKs are of general importance in postsynaptic stability. At the same time, AChR interaction with rapsyn and dystrobrevin and AChR phosphorylation decreased after agrin withdrawal from mutant myotubes. Unexpectedly, levels of rapsyn protein were increased in src(-/-);fyn(-/-) myotubes, whereas rapsyn-cytoskeleton interactions were unaffected. The overall cytoskeletal link of AChRs was weak but still strengthened by agrin in mutant cells, consistent with the normal formation but decreased stability of AChR clusters. These data show that correctly balanced activity of SFKs is critical in maintaining adult NMJs in vivo. SFKs hold the postsynaptic apparatus together through stabilization of AChR-rapsyn interaction and AChR phosphorylation. In addition, SFKs control rapsyn levels and AChR-cytoskeletal linkage.


Assuntos
Citoesqueleto/metabolismo , Junção Neuromuscular/metabolismo , Receptores Colinérgicos/metabolismo , Quinases da Família src/fisiologia , Agrina/farmacologia , Animais , Bungarotoxinas/metabolismo , Proteínas de Transporte/metabolismo , Células Cultivadas , Disbindina , Distroglicanas/metabolismo , Proteínas Associadas à Distrofina/metabolismo , Eletroporação/métodos , Regulação da Expressão Gênica/fisiologia , Proteínas de Fluorescência Verde/metabolismo , Imageamento Tridimensional/métodos , Immunoblotting/métodos , Imuno-Histoquímica/métodos , Técnicas In Vitro , Camundongos , Microscopia Confocal/métodos , Modelos Biológicos , Fibras Musculares Esqueléticas/fisiologia , Proteínas Musculares/metabolismo , Mutagênese/fisiologia , Proteínas de Neurofilamentos/metabolismo , Junção Neuromuscular/efeitos dos fármacos , Fosforilação , Proteínas Proto-Oncogênicas c-fyn/deficiência , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Interferência de RNA/fisiologia , Sinaptofisina/metabolismo , Fatores de Tempo , Utrofina/metabolismo , Quinases da Família src/deficiência
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