Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 34
Filtrar
1.
Proc Natl Acad Sci U S A ; 120(42): e2220029120, 2023 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-37812700

RESUMO

Voltage-gated potassium channels (Kv) are tetrameric membrane proteins that provide a highly selective pathway for potassium ions (K+) to diffuse across a hydrophobic cell membrane. These unique voltage-gated cation channels detect changes in membrane potential and, upon activation, help to return the depolarized cell to a resting state during the repolarization stage of each action potential. The Kv3 family of potassium channels is characterized by a high activation potential and rapid kinetics, which play a crucial role for the fast-spiking neuronal phenotype. Mutations in the Kv3.1 channel have been shown to have implications in various neurological diseases like epilepsy and Alzheimer's disease. Moreover, disruptions in neuronal circuitry involving Kv3.1 have been correlated with negative symptoms of schizophrenia. Here, we report the discovery of a novel positive modulator of Kv3.1, investigate its biophysical properties, and determine the cryo-EM structure of the compound in complex with Kv3.1. Structural analysis reveals the molecular determinants of positive modulation in Kv3.1 channels by this class of compounds and provides additional opportunities for rational drug design for the treatment of associated neurological disorders.


Assuntos
Neurônios , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Humanos , Neurônios/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Canais de Potássio/metabolismo , Potenciais de Ação/fisiologia , Proteínas de Membrana/metabolismo
2.
Protein Expr Purif ; 215: 106406, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-37995943

RESUMO

The baculovirus expression system is a powerful and widely used method to generate large quantities of recombinant protein. However, challenges exist in workflows utilizing either liquid baculovirus stocks or the Titerless Infected-Cells Preservation and Scale-Up (TIPS) method, including the time and effort to generate baculoviruses, screen for protein expression and store large numbers of baculovirus stocks. To mitigate these challenges, we have developed a streamlined, hybrid workflow which utilizes high titer liquid virus stocks for rapid plate-based protein expression screening, followed by a TIPS-based scale-up for larger protein production efforts. Additionally, we have automated each step in this screening workflow using a custom robotic system. With these process improvements, we have significantly reduced the time, effort and resources required to manage large baculovirus generation and expression screening campaigns.


Assuntos
Baculoviridae , Triagem , Fluxo de Trabalho , Baculoviridae/genética , Baculoviridae/metabolismo , Proteínas Recombinantes , Vetores Genéticos
3.
Toxicol Appl Pharmacol ; 474: 116601, 2023 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-37321326

RESUMO

Two potent and selective KRASG12D inhibitors, ERAS-4693 and ERAS-5024, were generated as possible clinical candidates to treat patients harboring G12D mutations in solid tumors. Both molecules exhibited strong anti-tumor activity in the KRASG12D mutant PDAC xenograft mouse models while ERAS-5024 also showed tumor growth inhibition when administered on an intermittent dosing regimen. Acute dose-limiting toxicity consistent with an allergic reaction was observed for both molecules shortly after administration at doses just above those which demonstrated anti-tumor activity, indicative of a narrow therapeutic index. A series of studies were subsequently conducted to identify a common underlying mechanism for the observed toxicity, including CETSA® (Cellular Thermal Shift Assay) as well as several functional off-target screens. Both ERAS-4693 and ERAS-5024 were identified to agonize MRGPRX2 which has been linked to pseudo-allergic reactions. In vivo toxicologic characterization of both molecules included repeat-dose studies in the rat and dog. Dose-limiting toxicities were observed in both species with ERAS-4693 and ERAS-5024 and plasma exposure levels at the maximum tolerated doses were generally below that which caused strong anti-tumor activity, supporting the initial observation of a narrow therapeutic index. Additional overlapping toxicities included a reduction in reticulocytes and clinical pathological changes suggestive of an inflammatory response. Furthermore, increases in plasma histamine were observed in dogs administered ERAS-5024, supporting the hypothesis that MRGPRX2 agonism may be the cause of the pseudo-allergic reaction. This work highlights the importance of balancing both the safety and efficacy of KRASG12D inhibitors as this class of molecules begins to enter clinical development.


Assuntos
Hipersensibilidade , Neoplasias Pancreáticas , Humanos , Camundongos , Ratos , Animais , Cães , Proteínas Proto-Oncogênicas p21(ras)/genética , Neoplasias Pancreáticas/patologia , Mutação , Proteínas do Tecido Nervoso , Receptores de Neuropeptídeos/genética , Receptores Acoplados a Proteínas G/genética
4.
Protein Expr Purif ; 179: 105796, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33221505

RESUMO

TREM2 has been identified by genomic analysis as a potential and novel target for the treatment of Alzheimer's disease. To enable structure-based screening of potential small molecule therapeutics, we sought to develop a robust crystallization platform for the TREM2 Ig-like domain. A systematic set of constructs containing the structural chaperone, maltose binding protein (MBP), fused to the Ig domain of TREM2, were evaluated in parallel expression and purification, followed by crystallization studies. Using protein crystallization and high-resolution diffraction as a readout, a MBP-TREM2 Ig fusion construct was identified that generates reproducible protein crystals diffracting at 2.0 Å, which makes it suitable for soaking of potential ligands. Importantly, analysis of crystal packing interfaces indicates that most of the surface of the TREM2 Ig domain is available for small molecule binding. A proof of concept co-crystallization study with a small library of fragments validated potential utility of this system for the discovery of new TREM2 therapeutics.


Assuntos
Cristalização/métodos , Glicoproteínas de Membrana , Chaperonas Moleculares , Receptores Imunológicos , Proteínas Recombinantes de Fusão , Humanos , Proteínas Ligantes de Maltose/química , Proteínas Ligantes de Maltose/genética , Proteínas Ligantes de Maltose/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Receptores Imunológicos/química , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
5.
N Engl J Med ; 374(1): 54-61, 2016 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-26698910

RESUMO

In a patient who had metastatic anaplastic lymphoma kinase (ALK)-rearranged lung cancer, resistance to crizotinib developed because of a mutation in the ALK kinase domain. This mutation is predicted to result in a substitution of cysteine by tyrosine at amino acid residue 1156 (C1156Y). Her tumor did not respond to a second-generation ALK inhibitor, but it did respond to lorlatinib (PF-06463922), a third-generation inhibitor. When her tumor relapsed, sequencing of the resistant tumor revealed an ALK L1198F mutation in addition to the C1156Y mutation. The L1198F substitution confers resistance to lorlatinib through steric interference with drug binding. However, L1198F paradoxically enhances binding to crizotinib, negating the effect of C1156Y and resensitizing resistant cancers to crizotinib. The patient received crizotinib again, and her cancer-related symptoms and liver failure resolved. (Funded by Pfizer and others; ClinicalTrials.gov number, NCT01970865.).


Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Lactamas Macrocíclicas/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Mutação , Inibidores de Proteínas Quinases/uso terapêutico , Pirazóis/uso terapêutico , Piridinas/uso terapêutico , Receptores Proteína Tirosina Quinases/genética , Aminopiridinas , Quinase do Linfoma Anaplásico , Sítios de Ligação , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/secundário , Crizotinibe , Feminino , Humanos , Lactamas , Falência Hepática/etiologia , Neoplasias Hepáticas/secundário , Neoplasias Pulmonares/genética , Pessoa de Meia-Idade , Estrutura Molecular , Pirimidinas/uso terapêutico , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Sulfonas/uso terapêutico
6.
Proc Natl Acad Sci U S A ; 112(11): 3493-8, 2015 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-25733882

RESUMO

Oncogenic c-ros oncogene1 (ROS1) fusion kinases have been identified in a variety of human cancers and are attractive targets for cancer therapy. The MET/ALK/ROS1 inhibitor crizotinib (Xalkori, PF-02341066) has demonstrated promising clinical activity in ROS1 fusion-positive non-small cell lung cancer. However, emerging clinical evidence has shown that patients can develop resistance by acquiring secondary point mutations in ROS1 kinase. In this study we characterized the ROS1 activity of PF-06463922, a novel, orally available, CNS-penetrant, ATP-competitive small-molecule inhibitor of ALK/ROS1. In vitro, PF-06463922 exhibited subnanomolar cellular potency against oncogenic ROS1 fusions and inhibited the crizotinib-refractory ROS1(G2032R) mutation and the ROS1(G2026M) gatekeeper mutation. Compared with crizotinib and the second-generation ALK/ROS1 inhibitors ceritinib and alectinib, PF-06463922 showed significantly improved inhibitory activity against ROS1 kinase. A crystal structure of the PF-06463922-ROS1 kinase complex revealed favorable interactions contributing to the high-affinity binding. In vivo, PF-06463922 showed marked antitumor activity in tumor models expressing FIG-ROS1, CD74-ROS1, and the CD74-ROS1(G2032R) mutation. Furthermore, PF-06463922 demonstrated antitumor activity in a genetically engineered mouse model of FIG-ROS1 glioblastoma. Taken together, our results indicate that PF-06463922 has potential for treating ROS1 fusion-positive cancers, including those requiring agents with CNS-penetrating properties, as well as for overcoming crizotinib resistance driven by ROS1 mutation.


Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Lactamas Macrocíclicas/farmacologia , Mutação/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Pirazóis/farmacologia , Piridinas/farmacologia , Aminopiridinas , Animais , Carcinogênese/efeitos dos fármacos , Carcinogênese/patologia , Proliferação de Células/efeitos dos fármacos , Crizotinibe , Cristalografia por Raios X , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Glioma/patologia , Humanos , Lactamas , Lactamas Macrocíclicas/química , Camundongos , Modelos Moleculares , Transdução de Sinais/efeitos dos fármacos
7.
N Engl J Med ; 368(25): 2395-401, 2013 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-23724914

RESUMO

Crizotinib, an inhibitor of anaplastic lymphoma kinase (ALK), has also recently shown efficacy in the treatment of lung cancers with ROS1 translocations. Resistance to crizotinib developed in a patient with metastatic lung adenocarcinoma harboring a CD74-ROS1 rearrangement who had initially shown a dramatic response to treatment. We performed a biopsy of a resistant tumor and identified an acquired mutation leading to a glycine-to-arginine substitution at codon 2032 in the ROS1 kinase domain. Although this mutation does not lie at the gatekeeper residue, it confers resistance to ROS1 kinase inhibition through steric interference with drug binding. The same resistance mutation was observed at all the metastatic sites that were examined at autopsy, suggesting that this mutation was an early event in the clonal evolution of resistance. (Funded by Pfizer and others; ClinicalTrials.gov number, NCT00585195.).


Assuntos
Adenocarcinoma/genética , Resistência a Medicamentos/genética , Neoplasias Pulmonares/genética , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Pirazóis/uso terapêutico , Piridinas/uso terapêutico , Translocação Genética , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/patologia , Crizotinibe , Evolução Fatal , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Pessoa de Meia-Idade , Mutação , Conformação Proteica , Proteínas Tirosina Quinases/química , Proteínas Proto-Oncogênicas/química , Relação Estrutura-Atividade
8.
ACS Med Chem Lett ; 14(10): 1351-1357, 2023 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-37849557

RESUMO

KRAS G12D mutation has been found in approximately 45% of pancreatic ductal adenocarcinoma (PDAC) cases, making it an attractive therapeutic target. Through structure-based drug design, a series of potent and selective KRAS G12D inhibitors were designed. The lead compound, ERAS-5024, inhibited ERK1/2 phosphorylation and cell proliferation in three-dimensional Cell-Titer Glo assays in AsPC-1 PDAC cells with single-digit nanomolar potency and caused tumor regression in the in vivo efficacy studies. We describe here the details of the design and synthesis program that led to the discovery of ERAS-5024.

9.
Mol Cancer Ther ; 21(1): 3-15, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34737197

RESUMO

Protein arginine methyltransferase 5 (PRMT5) overexpression in hematologic and solid tumors methylates arginine residues on cellular proteins involved in important cancer functions including cell-cycle regulation, mRNA splicing, cell differentiation, cell signaling, and apoptosis. PRMT5 methyltransferase function has been linked with high rates of tumor cell proliferation and decreased overall survival, and PRMT5 inhibitors are currently being explored as an approach for targeting cancer-specific dependencies due to PRMT5 catalytic function. Here, we describe the discovery of potent and selective S-adenosylmethionine (SAM) competitive PRMT5 inhibitors, with in vitro and in vivo characterization of clinical candidate PF-06939999. Acquired resistance mechanisms were explored through the development of drug resistant cell lines. Our data highlight compound-specific resistance mutations in the PRMT5 enzyme that demonstrate structural constraints in the cofactor binding site that prevent emergence of complete resistance to SAM site inhibitors. PRMT5 inhibition by PF-06939999 treatment reduced proliferation of non-small cell lung cancer (NSCLC) cells, with dose-dependent decreases in symmetric dimethyl arginine (SDMA) levels and changes in alternative splicing of numerous pre-mRNAs. Drug sensitivity to PF-06939999 in NSCLC cells associates with cancer pathways including MYC, cell cycle and spliceosome, and with mutations in splicing factors such as RBM10. Translation of efficacy in mouse tumor xenograft models with splicing mutations provides rationale for therapeutic use of PF-06939999 in the treatment of splicing dysregulated NSCLC.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , S-Adenosilmetionina/metabolismo , Animais , Apoptose , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Resistência a Medicamentos , Feminino , Humanos , Neoplasias Pulmonares/patologia , Camundongos
10.
Nat Commun ; 12(1): 815, 2021 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-33547286

RESUMO

Narcolepsy type 1 (NT1) is a chronic neurological disorder that impairs the brain's ability to control sleep-wake cycles. Current therapies are limited to the management of symptoms with modest effectiveness and substantial adverse effects. Agonists of the orexin receptor 2 (OX2R) have shown promise as novel therapeutics that directly target the pathophysiology of the disease. However, identification of drug-like OX2R agonists has proven difficult. Here we report cryo-electron microscopy structures of active-state OX2R bound to an endogenous peptide agonist and a small-molecule agonist. The extended carboxy-terminal segment of the peptide reaches into the core of OX2R to stabilize an active conformation, while the small-molecule agonist binds deep inside the orthosteric pocket, making similar key interactions. Comparison with antagonist-bound OX2R suggests a molecular mechanism that rationalizes both receptor activation and inhibition. Our results enable structure-based discovery of therapeutic orexin agonists for the treatment of NT1 and other hypersomnia disorders.


Assuntos
Aminopiridinas/química , Azepinas/química , Antagonistas dos Receptores de Orexina/química , Receptores de Orexina/química , Peptídeos/química , Medicamentos Indutores do Sono/química , Sulfonamidas/química , Triazóis/química , Aminopiridinas/metabolismo , Azepinas/metabolismo , Sítios de Ligação , Clonagem Molecular , Microscopia Crioeletrônica , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Simulação de Dinâmica Molecular , Antagonistas dos Receptores de Orexina/metabolismo , Receptores de Orexina/agonistas , Receptores de Orexina/metabolismo , Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Medicamentos Indutores do Sono/metabolismo , Sulfonamidas/metabolismo , Triazóis/metabolismo
11.
J Med Chem ; 64(1): 644-661, 2021 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-33356246

RESUMO

The phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling pathway is a frequently dysregulated pathway in human cancer, and PI3Kα is one of the most frequently mutated kinases in human cancer. A PI3Kα-selective inhibitor may provide the opportunity to spare patients the side effects associated with broader inhibition of the class I PI3K family. Here, we describe our efforts to discover a PI3Kα-selective inhibitor by applying structure-based drug design (SBDD) and computational analysis. A novel series of compounds, exemplified by 2,2-difluoroethyl (3S)-3-{[2'-amino-5-fluoro-2-(morpholin-4-yl)-4,5'-bipyrimidin-6-yl]amino}-3-(hydroxymethyl)pyrrolidine-1-carboxylate (1) (PF-06843195), with high PI3Kα potency and unique PI3K isoform and mTOR selectivity were discovered. We describe here the details of the design and synthesis program that lead to the discovery of 1.


Assuntos
Desenho de Fármacos , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Inibidores de Fosfoinositídeo-3 Quinase/química , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Animais , Linhagem Celular , Cromatografia Líquida de Alta Pressão/métodos , Cristalografia por Raios X , Humanos , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética/métodos , Camundongos , Estrutura Molecular , Inibidores de Fosfoinositídeo-3 Quinase/síntese química , Ratos , Espectrometria de Massas por Ionização por Electrospray/métodos
12.
Structure ; 14(2): 321-30, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16472751

RESUMO

The Eph receptor tyrosine kinases and their ligands, the ephrins, regulate numerous biological processes in developing and adult tissues and have been implicated in cancer progression and in pathological forms of angiogenesis. We report the crystal structure of the EphB4 receptor in complex with a highly specific antagonistic peptide at a resolution of 1.65 angstroms. The peptide is situated in a hydrophobic cleft of EphB4 corresponding to the cleft in EphB2 occupied by the ephrin-B2 G-H loop, consistent with its antagonistic properties. Structural analysis identifies several residues within the EphB4 binding cleft that likely determine the ligand specificity of this receptor, while isothermal titration calorimetry experiments with truncated forms of the peptide define the amino acid residues of the peptide that are critical for receptor binding. These studies reveal structural features that will aid drug discovery initiatives to develop EphB4 antagonists for therapeutic applications.


Assuntos
Efrina-B2/química , Modelos Moleculares , Peptídeos/química , Receptor EphB4/química , Sítios de Ligação , Calorimetria , Cristalografia por Raios X , Humanos , Estrutura Terciária de Proteína , Receptor EphB4/antagonistas & inibidores , Receptor EphB4/metabolismo , Termodinâmica
13.
ACS Med Chem Lett ; 9(9): 878-883, 2018 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-30258534

RESUMO

Structure-based drug design (SBDD) is commonly leveraged in rational drug design. Usually, ligand and binding site atomic coordinates from crystallographic data are exploited to optimize potency and selectivity. In addition to traditional, static views of proteins and ligands, we propose using normalized B-factors to study protein dynamics as a part of the drug optimization process. A retrospective case study of crizotinib and lorlatinib bound to both c-ros oncogene 1 kinase (ROS1) and anaplastic lymphoma kinase (ALK) L1196M related normalized B-factors to differences in binding affinity. This analysis showed that ligand binding can have protein-stabilizing effects that start near the ligand but propagate through nearby residues and structural waters to more distal motifs. The potential opportunities for analyzing normalized B-factors in SBDD are also discussed.

14.
ACS Med Chem Lett ; 9(9): 872-877, 2018 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-30258533

RESUMO

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase that can become oncogenic by activating mutations or overexpression. Full kinetic characterization of both phosphorylated and nonphosphorylated wildtype and mutant ALK kinase domain was done. Our structure-based drug design programs directed at ALK allowed us to interrogate whether X-ray crystallography data could be used to support the hypothesis that activation of ALK by mutation occurs due to increased protein dynamics. Crystallographic B-factors were converted to normalized B-factors, which allowed analysis of wildtype ALK, ALK-C1156Y, and ALK-L1196M. This data suggests that mobility of the P-loop, αC-helix, and activation loop (A-loop) may be important in catalytic activity increases, with or without phosphorylation. Both molecular dynamics simulations and hydrogen-deuterium exchange experimental data corroborated the normalized B-factors data.

15.
J Med Chem ; 61(3): 650-665, 2018 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-29211475

RESUMO

A new series of lactam-derived EZH2 inhibitors was designed via ligand-based and physicochemical-property-based strategies to address metabolic stability and thermodynamic solubility issues associated with previous lead compound 1. The new inhibitors incorporated an sp3 hybridized carbon atom at the 7-position of the lactam moiety present in lead compound 1 as a replacement for a dimethylisoxazole group. This transformation enabled optimization of the physicochemical properties and potency compared to compound 1. Analysis of relationships between calculated log D (clogD) values and in vitro metabolic stability and permeability parameters identified a clogD range that afforded an increased probability of achieving favorable ADME data in a single molecule. Compound 23a exhibited the best overlap of potency and pharmaceutical properties as well as robust tumor growth inhibition in vivo and was therefore advanced as a development candidate (PF-06821497). A crystal structure of 23a in complex with the three-protein PRC2 complex enabled understanding of the key structural features required for optimal binding.


Assuntos
Desenho de Fármacos , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Isoquinolinas/farmacologia , Isoquinolinas/farmacocinética , Administração Oral , Disponibilidade Biológica , Linhagem Celular Tumoral , Humanos , Isoquinolinas/administração & dosagem , Isoquinolinas/química , Modelos Moleculares , Conformação Molecular
16.
Elife ; 62017 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-29185984

RESUMO

Polycomb repressive complex 2 (PRC2) is a key chromatin modifier responsible for methylation of lysine 27 in histone H3. PRC2 has been shown to interact with thousands of RNA species in vivo, but understanding the physiological function of RNA binding has been hampered by the lack of separation-of-function mutants. Here, we use comprehensive mutagenesis and hydrogen deuterium exchange mass spectrometry (HDX-MS) to identify critical residues for RNA interaction in PRC2 core complexes from Homo sapiens and Chaetomium thermophilum, for which crystal structures are known. Preferential binding of G-quadruplex RNA is conserved, surprisingly using different protein elements. Key RNA-binding residues are spread out along the surface of EZH2, with other subunits including EED also contributing, and missense mutations of some of these residues have been found in cancer patients. The unusual nature of this protein-RNA interaction provides a paradigm for other epigenetic modifiers that bind RNA without canonical RNA-binding motifs.


Assuntos
Aminoácidos/genética , Aminoácidos/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Quadruplex G , RNA/metabolismo , Chaetomium/enzimologia , Análise Mutacional de DNA , Proteína Potenciadora do Homólogo 2 de Zeste/química , Humanos , Espectrometria de Massas , Ligação Proteica
17.
Nat Commun ; 7: 11384, 2016 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-27122193

RESUMO

Polycomb repressive complex 2 (PRC2) mediates gene silencing through chromatin reorganization by methylation of histone H3 lysine 27 (H3K27). Overexpression of the complex and point mutations in the individual subunits of PRC2 have been shown to contribute to tumorigenesis. Several inhibitors of the PRC2 activity have shown efficacy in EZH2-mutated lymphomas and are currently in clinical development, although the molecular basis of inhibitor recognition remains unknown. Here we report the crystal structures of the inhibitor-bound wild-type and Y641N PRC2. The structures illuminate an important role played by a stretch of 17 residues in the N-terminal region of EZH2, we call the activation loop, in the stimulation of the enzyme activity, inhibitor recognition and the potential development of the mutation-mediated drug resistance. The work presented here provides new avenues for the design and development of next-generation PRC2 inhibitors through establishment of a structure-based drug design platform.


Assuntos
Antineoplásicos/química , Inibidores Enzimáticos/química , Complexo Repressor Polycomb 2/antagonistas & inibidores , Complexo Repressor Polycomb 2/química , Resistencia a Medicamentos Antineoplásicos , Proteína Potenciadora do Homólogo 2 de Zeste/química , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Humanos , Modelos Moleculares , Mutação , Neoplasias/genética , Neoplasias/metabolismo , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo
18.
Assay Drug Dev Technol ; 13(4): 235-40, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-26065559

RESUMO

Conformational remodeling of chromatin in cells is known to alter gene expression. The histone code hypothesis postulates that multiple modifications present on histone tails can regulate gene expression both through direct effects on chromatin compaction as well as through recruitment of unique complexes that signal specific downstream functions. Histone methylation is an important component of the histone code, and the dysregulation of histone methylation in disease makes methyltransferases and demethylases viable targets for drug discovery. We developed a biochemical assay platform, which takes advantage of the fact that protein methyltransferases (PMTs) all utilize the cofactor S-Adenosyl-L-methionine (SAM) as the methyl donor. The platform utilizes the High-throughput Mass Spectrometry (MS) technology to measure SAM and the S-Adenosyl-L-homocysteine product in a label-free manner. The platform has all the advantages of a label-free system coupled with the benefit of substrate agnostic measurements making it an ideal setup for PMT biochemical studies and drug discovery. In addition, MS is ideally suited for detecting multiple modification events within the same substrate. The ability to adjust the detection to monitor the methyl acceptor product allows for real-time measurements of multiple product species simultaneously, a distinct advantage over other commonly used assay formats.


Assuntos
Ensaios Enzimáticos/métodos , Ensaios de Triagem em Larga Escala , Espectrometria de Massas , Proteína-Arginina N-Metiltransferases/análise , Radiometria/métodos , Sítios de Ligação , Humanos , Proteína-Arginina N-Metiltransferases/metabolismo
19.
Protein Sci ; 23(10): 1332-40, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25043846

RESUMO

PI3Kα remains an attractive target for the development of anticancer targeted therapy. A number of p110α crystal structures in complex with the nSH2-iSH2 fragment of p85 regulatory subunit have been reported, including a few small molecule co-crystal structures, but the utilization of this crystal form is limited by low diffraction resolution and a crystal packing artifact that partially blocks the ATP binding site. Taking advantage of recent data on the functional characterization of the lipid binding properties of p110α, we designed a set of novel constructs allowing production of isolated stable p110α subunit missing the Adapter Binding Domain and lacking or featuring a modified C-terminal lipid binding motif. While this protein is not catalytically competent to phosphorylate its substrate PIP2, it retains ligand binding properties as indicated by direct binding studies with a pan-PI3Kα inhibitor. Additionally, we determined apo and PF-04691502 bound crystal structures of the p110α (105-1048) subunit at 2.65 and 2.85 Å, respectively. Comparison of isolated p110α(105-1048) with the p110α/p85 complex reveals a high degree of structural similarity, which validates suitability of this catalytically inactive p110α for iterative SBDD. Importantly, this crystal form of p110α readily accommodates the binding of noncovalent inhibitor by means of a fully accessible ATP site. The strategy presented here can be also applied to structural studies of other members of PI3KIA family.


Assuntos
Fosfatidilinositol 3-Quinases/química , Fosfatidilinositol 3-Quinases/genética , Fosfolipídeos/metabolismo , Piridonas/química , Piridonas/farmacologia , Pirimidinas/química , Pirimidinas/farmacologia , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Domínio Catalítico , Classe I de Fosfatidilinositol 3-Quinases , Clonagem Molecular , Cristalização , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Inibidores de Fosfoinositídeo-3 Quinase , Estrutura Terciária de Proteína , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/química , Subunidades Proteicas/genética , Relação Estrutura-Atividade , Ressonância de Plasmônio de Superfície
20.
Biosci Rep ; 34(2)2014 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-27919031

RESUMO

The PKN (protein kinase N) family of Ser/Thr protein kinases regulates a diverse set of cellular functions, such as cell migration and cytoskeletal organization. Inhibition of tumour PKN activity has been explored as an oncology therapeutic approach, with a PKN3-targeted RNAi (RNA interference)-derived therapeutic agent in Phase I clinical trials. To better understand this important family of kinases, we performed detailed enzymatic characterization, determining the kinetic mechanism and lipid sensitivity of each PKN isoform using full-length enzymes and synthetic peptide substrate. Steady-state kinetic analysis revealed that PKN1-3 follows a sequential ordered Bi-Bi kinetic mechanism, where peptide substrate binding is preceded by ATP binding. This kinetic mechanism was confirmed by additional kinetic studies for product inhibition and affinity of small molecule inhibitors. The known lipid effector, arachidonic acid, increased the catalytic efficiency of each isoform, mainly through an increase in kcat for PKN1 and PKN2, and a decrease in peptide KM for PKN3. In addition, a number of PKN inhibitors with various degrees of isoform selectivity, including potent (Ki<10 nM) and selective PKN3 inhibitors, were identified by testing commercial libraries of small molecule kinase inhibitors. This study provides a kinetic framework and useful chemical probes for understanding PKN biology and the discovery of isoform-selective PKN-targeted inhibitors.


Assuntos
Trifosfato de Adenosina/química , Ácido Araquidônico/química , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/química , Inibidores de Proteínas Quinases/química , Catálise , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Cinética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA