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1.
J Biol Chem ; 292(43): 17963-17974, 2017 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-28860188

RESUMO

Aberrant activation of matrix metalloproteinases (MMPs) is a common feature of pathological cascades observed in diverse disorders, such as cancer, fibrosis, immune dysregulation, and neurodegenerative diseases. MMP-9, in particular, is highly dynamically regulated in several pathological processes. Development of MMP inhibitors has therefore been an attractive strategy for therapeutic intervention. However, a long history of failed clinical trials has demonstrated that broad-spectrum MMP inhibitors have limited clinical utility, which has spurred the development of inhibitors selective for individual MMPs. Attaining selectivity has been technically challenging because of sequence and structural conservation across the various MMPs. Here, through a biochemical and structural screening paradigm, we have identified JNJ0966, a highly selective compound that inhibited activation of MMP-9 zymogen and subsequent generation of catalytically active enzyme. JNJ0966 had no effect on MMP-1, MMP-2, MMP-3, MMP-9, or MMP-14 catalytic activity and did not inhibit activation of the highly related MMP-2 zymogen. The molecular basis for this activity was characterized as an interaction of JNJ0966 with a structural pocket in proximity to the MMP-9 zymogen cleavage site near Arg-106, which is distinct from the catalytic domain. JNJ0966 was efficacious in reducing disease severity in a mouse experimental autoimmune encephalomyelitis model, demonstrating the viability of this therapeutic approach. This discovery reveals an unprecedented pharmacological approach to MMP inhibition, providing an opportunity to improve selectivity of future clinical drug candidates. Targeting zymogen activation in this manner may also allow for pharmaceutical exploration of other enzymes previously viewed as intractable drug targets.


Assuntos
Precursores Enzimáticos/antagonistas & inibidores , Precursores Enzimáticos/química , Metaloproteinase 9 da Matriz/química , Inibidores de Metaloproteinases de Matriz/química , Regulação Alostérica , Animais , Células COS , Domínio Catalítico , Chlorocebus aethiops , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Humanos , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Domínios Proteicos
2.
J Med Chem ; 48(4): 909-12, 2005 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-15715460

RESUMO

HDM2 binds to an alpha-helical transactivation domain of p53, inhibiting its tumor suppressive functions. A miniaturized thermal denaturation assay was used to screen chemical libraries, resulting in the discovery of a novel series of benzodiazepinedione antagonists of the HDM2-p53 interaction. The X-ray crystal structure of improved antagonists bound to HDM2 reveals their alpha-helix mimetic properties. These optimized molecules increase the transcription of p53 target genes and decrease proliferation of tumor cells expressing wild-type p53.


Assuntos
Benzodiazepinas/síntese química , Proteínas Nucleares/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteína Supressora de Tumor p53/agonistas , Benzodiazepinas/química , Benzodiazepinas/farmacologia , Sítios de Ligação , Linhagem Celular Tumoral , Técnicas de Química Combinatória , Cristalografia por Raios X , Humanos , Modelos Moleculares , Mimetismo Molecular , Estrutura Molecular , Proteínas Proto-Oncogênicas c-mdm2 , Estereoisomerismo , Relação Estrutura-Atividade , Proteína Supressora de Tumor p53/biossíntese
3.
Protein Eng Des Sel ; 28(10): 385-93, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26275855

RESUMO

A number of classes of proteins have been engineered for high stability using consensus sequence design methods. Here we describe the engineering of a novel albumin binding domain (ABD) three-helix bundle protein. The resulting engineered ABD molecule, called ABDCon, is expressed at high levels in the soluble fraction of Escherichia coli and is highly stable, with a melting temperature of 81.5°C. ABDCon binds human, monkey and mouse serum albumins with affinity as high as 61 pM. The solution structure of ABDCon is consistent with the three-helix bundle design and epitope mapping studies enabled a precise definition of the albumin binding interface. Fusion of a 10 kDa scaffold protein to ABDCon results in a long terminal half-life of 60 h in mice and 182 h in cynomolgus monkeys. To explore the link between albumin affinity and in vivo exposure, mutations were designed at the albumin binding interface of ABDCon yielding variants that span an 11 000-fold range in affinity. The PK properties of five such variants were determined in mice in order to demonstrate the tunable nature of serum half-life, exposure and clearance with variations in albumin binding affinity.


Assuntos
Albuminas/metabolismo , Sequência Consenso , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacocinética , Sequência de Aminoácidos , Animais , Escherichia coli/genética , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Estabilidade Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo
4.
J Biomol Screen ; 17(5): 629-40, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22496098

RESUMO

Endocannabinoids such as 2-arachidonylglycerol (2-AG) are ligands for cannabinoid receptors that contribute to the transmission and modulation of pain signals. The antinociceptive effect of exogenous 2-AG suggests that inhibition of monoglyceride lipase (MGLL), the enzyme responsible for degrading 2-AG and arresting signaling, may be a target for pain modulation. Here we describe the characterization of MGLL ligands following a high-throughput screening campaign. Ligands were discovered using ThermoFluor, a label-free affinity-based screening tool that measures ligand binding via modulation of protein thermal stability. A kinetic fluorescent assay using the substrate 4-methylcoumarin butyrate was used to counterscreen confirmed HTS positives. A comparison of results from binding and inhibition assays allowed elucidation of compound mechanism of action. We demonstrate the limit of each technology and the benefits of using orthogonal assay techniques in profiling compounds.


Assuntos
Domínio Catalítico/efeitos dos fármacos , Ensaios Enzimáticos/métodos , Inibidores Enzimáticos/farmacologia , Monoacilglicerol Lipases/antagonistas & inibidores , Ácidos Araquidônicos/química , Endocanabinoides , Inibidores Enzimáticos/química , Glicerídeos/química , Ensaios de Triagem em Larga Escala , Humanos , Hidrólise , Concentração Inibidora 50 , Cinética , Monoacilglicerol Lipases/química , Monoacilglicerol Lipases/metabolismo , Ligação Proteica , Solubilidade , Especificidade por Substrato
5.
Protein Sci ; 20(4): 670-83, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21308848

RESUMO

A high-resolution structure of a ligand-bound, soluble form of human monoglyceride lipase (MGL) is presented. The structure highlights a novel conformation of the regulatory lid-domain present in the lipase family as well as the binding mode of a pharmaceutically relevant reversible inhibitor. Analysis of the structure lacking the inhibitor indicates that the closed conformation can accommodate the native substrate 2-arachidonoyl glycerol. A model is proposed in which MGL undergoes conformational and electrostatic changes during the catalytic cycle ultimately resulting in its dissociation from the membrane upon completion of the cycle. In addition, the study outlines a successful approach to transform membrane associated proteins, which tend to aggregate upon purification, into a monomeric and soluble form.


Assuntos
Monoacilglicerol Lipases/antagonistas & inibidores , Monoacilglicerol Lipases/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ácidos Araquidônicos/química , Ácidos Araquidônicos/metabolismo , Moduladores de Receptores de Canabinoides/química , Moduladores de Receptores de Canabinoides/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Endocanabinoides , Glicerídeos/química , Glicerídeos/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Monoacilglicerol Lipases/genética , Monoacilglicerol Lipases/metabolismo , Mutagênese Sítio-Dirigida , Ligação Proteica , Eletricidade Estática
6.
Bioorg Med Chem Lett ; 16(12): 3310-4, 2006 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-16600594
8.
Bioorg Med Chem Lett ; 12(3): 491-5, 2002 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-11814826

RESUMO

A study of the S1 binding of lead 5-methylthiothiophene amidine 3, an inhibitor of urokinase-type plasminogen activator, was undertaken by the introduction of a variety of substituents at the thiophene 5-position. The 5-alkyl substituted and unsubstituted thiophenes were prepared using organolithium chemistry. Heteroatom substituents were introduced at the 5-position using a novel displacement reaction of 5-methylsulfonylthiophenes and the corresponding oxygen or sulfur anions. Small alkyl group substitution at the 5-position provided inhibitors equipotent with but possessing improved solubility.


Assuntos
Amidinas/síntese química , Amidinas/farmacologia , Inibidores de Serina Proteinase/síntese química , Inibidores de Serina Proteinase/farmacologia , Tiofenos/síntese química , Tiofenos/farmacologia , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Alquilação , Indicadores e Reagentes , Compostos de Lítio/química , Ligação Proteica , Relação Estrutura-Atividade , Tiazóis/síntese química , Tiazóis/farmacologia
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