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1.
Bioorg Med Chem Lett ; 25(21): 4812-4819, 2015 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-26195137

RESUMO

The IC50 of a beta-secretase (BACE-1) lead compound was improved ∼200-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-Atividade
2.
J Biol Chem ; 286(13): 11218-25, 2011 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-21247903

RESUMO

The receptor tyrosine kinase c-Met is implicated in oncogenesis and is the target for several small molecule and biologic agents in clinical trials for the treatment of cancer. Binding of the hepatocyte growth factor to the cell surface receptor of c-Met induces activation via autophosphorylation of the kinase domain. Here we describe the structural basis of c-Met activation upon autophosphorylation and the selective small molecule inhibiton of autophosphorylated c-Met. MK-2461 is a potent c-Met inhibitor that is selective for the phosphorylated state of the enzyme. Compound 1 is an MK-2461 analog with a 20-fold enthalpy-driven preference for the autophosphorylated over unphosphorylated c-Met kinase domain. The crystal structure of the unbound kinase domain phosphorylated at Tyr-1234 and Tyr-1235 shows that activation loop phosphorylation leads to the ejection and disorder of the activation loop and rearrangement of helix αC and the G loop to generate a viable active site. Helix αC adopts a orientation different from that seen in activation loop mutants. The crystal structure of the complex formed by the autophosphorylated c-Met kinase domain and compound 1 reveals a significant induced fit conformational change of the G loop and ordering of the activation loop, explaining the selectivity of compound 1 for the autophosphorylated state. The results highlight the role of structural plasticity within the kinase domain in imparting the specificity of ligand binding and provide the framework for structure-guided design of activated c-Met inhibitors.


Assuntos
Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/química , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/química , Animais , Linhagem Celular , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Fosforilação , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Spodoptera , Relação Estrutura-Atividade , c-Mer Tirosina Quinase
3.
J Virol ; 84(15): 7625-33, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20484498

RESUMO

HIV/AIDS continues to be a menace to public health. Several drugs currently on the market have successfully improved the ability to manage the viral burden in infected patients. However, new drugs are needed to combat the rapid emergence of mutated forms of the virus that are resistant to existing therapies. Currently, approved drugs target three of the four major enzyme activities encoded by the virus that are critical to the HIV life cycle. Although a number of inhibitors of HIV RNase H activity have been reported, few inhibit by directly engaging the RNase H active site. Here, we describe structures of naphthyridinone-containing inhibitors bound to the RNase H active site. This class of compounds binds to the active site via two metal ions that are coordinated by catalytic site residues, D443, E478, D498, and D549. The directionality of the naphthyridinone pharmacophore is restricted by the ordering of D549 and H539 in the RNase H domain. In addition, one of the naphthyridinone-based compounds was found to bind at a second site close to the polymerase active site and non-nucleoside/nucleotide inhibitor sites in a metal-independent manner. Further characterization, using fluorescence-based thermal denaturation and a crystal structure of the isolated RNase H domain reveals that this compound can also bind the RNase H site and retains the metal-dependent binding mode of this class of molecules. These structures provide a means for structurally guided design of novel RNase H inhibitors.


Assuntos
Inibidores Enzimáticos/metabolismo , Transcriptase Reversa do HIV/antagonistas & inibidores , Transcriptase Reversa do HIV/química , HIV-1/efeitos dos fármacos , Naftiridinas/metabolismo , Ribonuclease H do Vírus da Imunodeficiência Humana/antagonistas & inibidores , Ribonuclease H do Vírus da Imunodeficiência Humana/química , Sítios de Ligação , Domínio Catalítico , Cátions/metabolismo , Cristalografia por Raios X , HIV , Transcriptase Reversa do HIV/metabolismo , HIV-1/química , Humanos , Metais/metabolismo , Modelos Moleculares , Ligação Proteica , Estrutura Terciária de Proteína , Ribonuclease H do Vírus da Imunodeficiência Humana/metabolismo
4.
Biochemistry ; 48(21): 4488-96, 2009 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-19284778

RESUMO

BACE-1 (beta-site amyloid precursor protein cleaving enzyme), a prominent target in Alzheimer's disease drug discovery efforts, was surveyed using Tethering technology to discover small molecule fragment ligands that bind to the enzyme active site. Screens of a library of >15000 thiol-containing fragments versus a panel of BACE-1 active site cysteine mutants under redox-controlled conditions revealed several novel amine-containing fragments that could be selectively captured by subsets of the tethering sites. For one such hit class, defined by a central aminobenzylpiperidine (ABP) moiety, X-ray crystal structures of BACE mutant-disulfide conjugates revealed that the fragment bound by engaging both catalytic aspartates with hydrogen bonds. The affinities of ABP fragments were improved by structure-guided chemistry, first for conjugation as thiol-containing fragments and then for stand-alone, noncovalent inhibition of wild-type (WT) BACE-1 activity. Crystallography confirmed that the inhibitors bound in exactly the same mode as the disulfide-conjugated fragments that were originally selected from the screen. The ABP ligands represent a new type of nonpeptidic BACE-1 inhibitor motif that has not been described in the aspartyl protease literature and may serve as a starting point for the development of BACE-1-directed Alzheimer's disease therapeutics.


Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Descoberta de Drogas/métodos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Secretases da Proteína Precursora do Amiloide/química , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Biocatálise , Domínio Catalítico , Cisteína , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/metabolismo , Humanos , Ligantes , Modelos Moleculares , Conformação Molecular , Mutação , Peptídeos/química , Piperidinas/química , Piperidinas/metabolismo , Relação Estrutura-Atividade
5.
J Med Virol ; 80(12): 2053-63, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19040279

RESUMO

The development of new HIV inhibitors with distinct resistance profiles is essential in order to combat the development of multi-resistant viral strains. A drug discovery program based on the identification of compounds that are active against drug-resistant viruses has produced PL-100, a novel potent protease inhibitor (PI) that incorporates a lysine-based scaffold. A selection for resistance against PL-100 in cord blood mononuclear cells was performed, using the laboratory-adapted IIIb strain of HIV-1, and it was shown that resistance appears to develop slower against this compound than against amprenavir, which was studied as a control. Four mutations in protease (PR) were selected after 25 weeks: two flap mutations (K45R and M46I) and two novel active site mutations (T80I and P81S). Site-directed mutagenesis revealed that all four mutations were required to develop low-level resistance to PL-100, which is indicative of the high genetic barrier of the compound. Importantly, these mutations did not cause cross-resistance to currently marketed PIs. In contrast, the P81S mutation alone caused hypersensitivity to two other PIs, saquinavir (SQV) and nelfinavir (NFV). Analysis of p55Gag processing showed that a marked defect in protease activity caused by mutation P81S could only be compensated when K45R and M46I were present. These data correlated well with the replication capacity (RC) of the mutant viruses as measured by a standard viral growth assay, since only viruses containing all four mutations approached the RC of wild type virus. X-ray crystallography provided insight on the structural basis of the resistance conferred by the identified mutations.


Assuntos
Carbamatos/farmacologia , Farmacorresistência Viral , Inibidores da Protease de HIV/farmacologia , Protease de HIV/genética , HIV-1/efeitos dos fármacos , HIV-1/genética , Mutação de Sentido Incorreto , Sulfonamidas/farmacologia , Domínio Catalítico , Células Cultivadas , Furanos , Protease de HIV/química , HIV-1/crescimento & desenvolvimento , Humanos , Leucócitos Mononucleares/virologia , Modelos Moleculares , Mutagênese Sítio-Dirigida , Estrutura Terciária de Proteína
6.
Mol Immunol ; 38(14): 1051-61, 2002 May.
Artigo em Inglês | MEDLINE | ID: mdl-11955597

RESUMO

The gammadelta T cell receptors (TCRs) and alphabeta TCRs are similar in both sequence and structure; however, gammadelta+ and alphabeta+ T cells are not merely similar lymphocytes with subtly different receptors. These cell types differ in several ways, including the types of antigens recognized, the mechanism of antigen presentation and recognition and the mechanism and kinetics of downstream signaling events. gammadelta TCRs can directly recognize antigens in the form of intact proteins or non-peptidic compounds, unlike alphabeta TCRs which recognize peptide antigens bound to major histocompatibility complex molecules (MHC). One of the major classes of human gammadelta+ T cells expresses Vgamma9Vdelta2 TCRs which recognize pyrophosphomonoester, alkylamine and aminobisphosphonate antigens. This review focuses on the recently determined structure of a Vgamma9Vdelta2 TCR, with emphasis on antigen recognition and receptor signaling.


Assuntos
Antígenos/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Transdução de Sinais/imunologia , Animais , Complexo CD3/imunologia , Humanos , Modelos Moleculares , Peptídeos , Estrutura Terciária de Proteína , Receptores de Antígenos de Linfócitos T gama-delta/química , Superantígenos/imunologia
7.
ACS Med Chem Lett ; 6(3): 318-23, 2015 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-25815153

RESUMO

3-Hydroxy-4-pyridinones and 5-hydroxy-4-pyrimidinones were identified as inhibitors of catechol-O-methyltransferase (COMT) in a high-throughput screen. These heterocyclic catechol mimics exhibit potent inhibition of the enzyme and an improved toxicity profile versus the marketed nitrocatechol inhibitors tolcapone and entacapone. Optimization of the series was aided by X-ray cocrystal structures of the novel inhibitors in complex with COMT and cofactors SAM and Mg(2+). The crystal structures suggest a mechanism of inhibition for these heterocyclic inhibitors distinct from previously disclosed COMT inhibitors.

8.
J Med Chem ; 56(6): 2294-310, 2013 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-23379595

RESUMO

This report documents the first example of a specific inhibitor of protein kinases with preferential binding to the activated kinase conformation: 5H-benzo[4,5]cyclohepta[1,2-b]pyridin-5-one 11r (MK-8033), a dual c-Met/Ron inhibitor under investigation as a treatment for cancer. The design of 11r was based on the desire to reduce time-dependent inhibition of CYP3A4 (TDI) by members of this structural class. A novel two-step protocol for the synthesis of benzylic sulfonamides was developed to access 11r and analogues. We provide a rationale for the observed selectivity based on X-ray crystallographic evidence and discuss selectivity trends with additional examples. Importantly, 11r provides full inhibition of tumor growth in a c-Met amplified (GTL-16) subcutaneous tumor xenograft model and may have an advantage over inactive form kinase inhibitors due to equal potency against a panel of oncogenic activating mutations of c-Met in contrast to c-Met inhibitors without preferential binding to the active kinase conformation.


Assuntos
Benzocicloeptenos/metabolismo , Benzocicloeptenos/farmacologia , Descoberta de Drogas , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-met/metabolismo , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Sulfonamidas/metabolismo , Sulfonamidas/farmacologia , Animais , Benzocicloeptenos/química , Linhagem Celular Tumoral , Cães , Ativação Enzimática/efeitos dos fármacos , Feminino , Humanos , Camundongos , Modelos Moleculares , Conformação Proteica , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/química , Ratos , Especificidade por Substrato , Sulfonamidas/química , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Proc Natl Acad Sci U S A ; 102(12): 4240-5, 2005 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-15761054

RESUMO

Although eradicated from nature more than two decades ago, the threat of smallpox has reemerged because of concerns over its use as a biological weapon. We present the structure of the poxvirus L1 protein, a molecule that is conserved throughout the poxvirus family and is nearly identical in vaccinia virus and in variola virus, which causes smallpox. L1 is a myristoylated envelope protein that is a potent target for neutralizing antibodies and an important component of current experimental vaccines. The L1 structure reveals a hydrophobic cavity located adjacent to its N terminus. The cavity would be capable of shielding the myristate moiety, which is essential for virion assembly. The structure of L1 is a step in the elucidation of molecular mechanisms common to all poxviruses that may stimulate the design of safer vaccines and new antipoxvirus drugs.


Assuntos
Poxviridae/química , Proteínas do Envelope Viral/química , Sequência de Aminoácidos , Animais , Anticorpos Antivirais , Sequência de Bases , Cristalografia por Raios X , DNA Viral/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Dissulfetos/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Dados de Sequência Molecular , Ácidos Mirísticos/química , Testes de Neutralização , Poxviridae/genética , Poxviridae/imunologia , Conformação Proteica , Homologia de Sequência de Aminoácidos , Vacina Antivariólica/química , Vacina Antivariólica/genética , Vacina Antivariólica/imunologia , Eletricidade Estática , Vaccinia virus/química , Vaccinia virus/genética , Vaccinia virus/imunologia , Vírus da Varíola/química , Vírus da Varíola/genética , Vírus da Varíola/imunologia , Proteínas do Core Viral/química , Proteínas do Core Viral/genética , Proteínas do Core Viral/imunologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia
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