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1.
Proc Natl Acad Sci U S A ; 115(13): 3249-3254, 2018 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-29531094

RESUMO

For cancer cells to survive and proliferate, they must escape normal immune destruction. One mechanism by which this is accomplished is through immune suppression effected by up-regulation of indoleamine 2,3-dioxygenase (IDO1), a heme enzyme that catalyzes the oxidation of tryptophan to N-formylkynurenine. On deformylation, kynurenine and downstream metabolites suppress T cell function. The importance of this immunosuppressive mechanism has spurred intense interest in the development of clinical IDO1 inhibitors. Herein, we describe the mechanism by which a class of compounds effectively and specifically inhibits IDO1 by targeting its apo-form. We show that the in vitro kinetics of inhibition coincide with an unusually high rate of intrinsic enzyme-heme dissociation, especially in the ferric form. X-ray crystal structures of the inhibitor-enzyme complexes show that heme is displaced from the enzyme and blocked from rebinding by these compounds. The results reveal that apo-IDO1 serves as a unique target for inhibition and that heme lability plays an important role in posttranslational regulation.


Assuntos
Inibidores Enzimáticos/farmacologia , Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Indolamina-Pirrol 2,3,-Dioxigenase/química , Apoproteínas/química , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Células HeLa , Heme/metabolismo , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Concentração Inibidora 50 , Mioglobina/química
2.
Bioorg Med Chem Lett ; 28(4): 732-736, 2018 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-29398543

RESUMO

A novel series of o-phenylenediamine-based inhibitors of indoleamine 2,3-dioxygenase (IDO) has been identified. IDO is a heme-containing enzyme, overexpressed in the tumor microenvironment of many cancers, which can contribute to the suppression of the host immune system. Synthetic modifications to a previously described diarylether series resulted in an additional degree of molecular diversity which was exploited to afford compounds that demonstrated significant potency in the HeLa human cervical cancer IDO1 assay. .


Assuntos
Inibidores Enzimáticos/farmacologia , Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Fenilenodiaminas/farmacologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Células HeLa , Humanos , Microssomos Hepáticos/metabolismo , Fenilenodiaminas/síntese química , Fenilenodiaminas/química , Fenilenodiaminas/metabolismo , Relação Estrutura-Atividade
4.
Bioorg Med Chem Lett ; 22(18): 5995-8, 2012 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-22902653

RESUMO

A series of aminothiazoles that are potent inhibitors of LIM kinases 1 and 2 is described. Appropriate choice of substituents led to molecules with good selectivity for either enzyme. An advanced member of the series was shown to effectively interfere with the phosphorylation of the LIM kinases substrate cofilin. Consistent with the important role of the LIM kinases in regulating cytoskeletal structure, treated cells displayed dramatically reduced F-actin content.


Assuntos
Fatores de Despolimerização de Actina/metabolismo , Quinases Lim/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Tiazóis/farmacologia , Linhagem Celular , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Humanos , Quinases Lim/metabolismo , Modelos Moleculares , Estrutura Molecular , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Relação Estrutura-Atividade , Tiazóis/síntese química , Tiazóis/química
5.
Bioorg Med Chem Lett ; 20(5): 1744-8, 2010 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-20153189
6.
Bioorg Med Chem Lett ; 20(3): 853-6, 2010 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-20060717

RESUMO

To selectively target doxorubicin (Dox) to tumor tissue and thereby improve the therapeutic index and/or efficacy of Dox, matrix metalloproteinases (MMP) activated peptide-Dox prodrugs were designed and synthesized by coupling MMP-cleavable peptides to Dox. Preferred conjugates were good substrates for MMPs, poor substrates for neprilysin, an off-target proteinase, and stable in blood ex vivo. When administered to mice with HT1080 xenografts, conjugates, such as 19, preferentially released Dox in tumor relative to heart tissue and prevented tumor growth with less marrow toxicity than Dox.


Assuntos
Antineoplásicos/química , Doxorrubicina/análogos & derivados , Descoberta de Drogas , Metaloproteinases da Matriz/química , Pró-Fármacos/química , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Descoberta de Drogas/métodos , Humanos , Metaloproteinases da Matriz/farmacologia , Camundongos , Pró-Fármacos/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
7.
J Comput Aided Mol Des ; 19(2): 111-22, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16075305

RESUMO

Cyclin-dependent kinases (CDKs) play a key role in regulating the cell cycle. The cyclins, their activating agents, and endogenous CDK inhibitors are frequently mutated in human cancers, making CDKs interesting targets for cancer chemotherapy. Our aim is the discovery of selective CDK4/cyclin D1 inhibitors. An ATP-competitive pyrazolopyrimidinone CDK inhibitor was identified by HTS and docked into a CDK4 homology model. The resulting binding model was consistent with available SAR and was validated by a subsequent CDK2/inhibitor crystal structure. An iterative cycle of chemistry and modeling led to a 70-fold improvement in potency. Small substituent changes resulted in large CDK4/CDK2 selectivity changes. The modeling revealed that selectivity is largely due to hydrogen-bonded interactions with only two kinase residues. This demonstrates that small differences between enzymes can efficiently be exploited in the design of selective inhibitors.


Assuntos
CDC2-CDC28 Quinases/antagonistas & inibidores , Ciclina A/antagonistas & inibidores , Ciclina D1/antagonistas & inibidores , Quinases Ciclina-Dependentes/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Pirimidinonas/farmacologia , Sequência de Aminoácidos , CDC2-CDC28 Quinases/química , Quinase 2 Dependente de Ciclina , Quinase 4 Dependente de Ciclina , Quinases Ciclina-Dependentes/química , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Ligações de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas/química , Pirimidinonas/química , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
8.
J Med Chem ; 47(24): 5894-911, 2004 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-15537345

RESUMO

Using a high-throughput screening strategy, a series of 1-aryl-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-ones was identified that inhibit the cyclin-dependent kinase (CDK) 4/cyclin D1 complex-mediated phosphorylation of a protein substrate with IC(50)s in the low micromolar range. On the basis of preliminary structure-activity relationships (SAR), a model was proposed in which these inhibitors occupy the ATP-binding site of the enzyme, forming critical hydrogen bonds to the same residue (Val96) to which the amino group in ATP is presumed to bind. X-ray diffraction studies on a later derivative bound to CDK2 support this binding mode. Iterative cycles of synthesis and screening lead to a novel series of potent, CDK2-selective 6-(arylmethyl)pyrazolopyrimidinones. Placement of a hydrogen-bond donor in the meta-position on the 6-arylmethyl group resulted in approximately 100-fold increases in CDK4 affinity, giving ligands that were equipotent inhibitors of CDK4 and CDK2. These compounds exhibit antiproliferative effects in the NCI HCT116 and other cell lines. The potency of these antiproliferative effects is enhanced in anilide derivatives and translates into tumor growth inhibition in a mouse xenograft model.


Assuntos
Quinases Ciclina-Dependentes/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Pirazóis/síntese química , Pirimidinas/síntese química , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Sítios de Ligação , Linhagem Celular Tumoral , Células Cultivadas , Cristalografia por Raios X , Ciclina D1/antagonistas & inibidores , Ciclina D1/metabolismo , Quinase 4 Dependente de Ciclina , Quinases Ciclina-Dependentes/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Camundongos , Modelos Moleculares , Estrutura Molecular , Fosforilação , Proteínas Proto-Oncogênicas/metabolismo , Pirazóis/química , Pirazóis/farmacologia , Pirimidinas/química , Pirimidinas/farmacologia , Relação Estrutura-Atividade , Transplante Heterólogo
10.
J Biol Chem ; 278(30): 27820-7, 2003 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-12738779

RESUMO

The MMP-11 proteinase, also known as stromelysin-3, probably plays an important role in human cancer because MMP-11 is frequently overexpressed in human tumors and MMP-11 levels affect tumorogenesis in mice. Unlike other MMPs, however, human MMP-11 does not cleave extracellular matrix proteins, such as collagen, laminin, fibronectin, and elastin. To help identify physiologic MMP-11 substrates, a phage display library was used to find peptide substrates for MMP-11. One class of peptides containing 26 members had the consensus sequence A(A/Q)(N/A) downward arrow (L/Y)(T/V/M/R)(R/K), where downward arrow denotes the cleavage site. This consensus sequence was similar to that for other MMPs, which also cleave peptides containing Ala in position 3, Ala in position 1, and Leu/Tyr in position 1', but differed from most other MMP substrates in that proline was rarely found in position 3 and Asn was frequently found in position 1. A second class of peptides containing four members had the consensus sequence G(G/A)E downward arrow LR. Although other MMPs also cleave peptides with these residues, other MMPs prefer proline at position 3 in this sequence. In vitro assays with MMP-11 and representative peptides from both classes yielded modest kcat/Km values relative to values found for other MMPs with their preferred peptide substrates. These reactions also showed that peptides with proline in position 3 were poor substrates for MMP-11. A structural basis for the lower kcat/Km values of human MMP-11, relative to other MMPs, and poor cleavage of position 3 proline substrates by MMP-11 is provided. Taken together, these findings explain why MMP-11 does not cleave most other MMP substrates and predict that MMP-11 has unique substrates that may contribute to human cancer.


Assuntos
Metaloendopeptidases/química , Biblioteca de Peptídeos , Sítios de Ligação , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Matriz Extracelular/metabolismo , Vetores Genéticos , Humanos , Cinética , Metaloproteinase 11 da Matriz , Metaloproteinases da Matriz Associadas à Membrana , Metaloendopeptidases/metabolismo , Metaloendopeptidases/fisiologia , Modelos Moleculares , Neoplasias/metabolismo , Peptídeos/química , Prolina/química , Ligação Proteica , Estrutura Terciária de Proteína , Especificidade por Substrato
11.
J Med Chem ; 45(24): 5224-32, 2002 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-12431050

RESUMO

We disclose a novel series of indenopyrazole-based cyclin-dependent kinase (CDK) inhibitors. Kinetic experiments confirmed our initial molecular modeling studies that the compounds are competitive with respect to adenosine 5'-triphosphate (ATP) and bind in the kinase ATP pocket. A unique combination of active pharmacophores led us to a series of semicarbazide-based inhibitors that are highly potent against CDK2 and CDK4 while maintaining selectivity against other relevant serine/threonine kinases. These compounds were active against a transformed human colon cancer cell line (HCT116) while maintaining an acceptable margin of activity against a normal fibroblast cell line. The compounds were found to be highly protein bound in our cell-based assay with the exception of 11k, which maintained a reasonable level of activity in the presence of human plasma proteins.


Assuntos
CDC2-CDC28 Quinases , Quinases Ciclina-Dependentes/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Morfolinas/síntese química , Proteínas Proto-Oncogênicas , Pirazóis/síntese química , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Antineoplásicos/toxicidade , Proteínas Sanguíneas/metabolismo , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Quinase 2 Dependente de Ciclina , Quinase 4 Dependente de Ciclina , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/toxicidade , Humanos , Cinética , Modelos Moleculares , Morfolinas/química , Morfolinas/farmacologia , Ligação Proteica , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirazóis/química , Pirazóis/farmacologia , Pirazóis/toxicidade , Relação Estrutura-Atividade , Células Tumorais Cultivadas
12.
J Med Chem ; 45(24): 5233-48, 2002 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-12431051

RESUMO

The identification of indeno[1,2-c]pyrazol-4-ones as inhibitors of cyclin-dependent kinases (CDKs) has led to the discovery of a series of novel and potent compounds. Herein, we report the effects of substitutions at C3 of the indeno[1,2-c]pyrazol-4-one core with alkyls, heterocycles, and substituted phenyls. Substitutions at the para position of the phenyl ring at C3 were generally well-tolerated; however, larger groups were generally inactive. For alkyls directly attached to C3, longer chain substituents were not tolerated; however, shorter alkyl groups and cyclic alkyls were acceptable. In general, the heterocycles at C3 gave the most potent analogues. One such heterocycle, 24j, was examined in detail and was determined to have a biological profile consistent with CDK inhibition. An X-ray crystal structure of one of the alkyl compounds, 13q, complexed with CDK2 was determined and showed the inhibitor residing in the adenosine 5'-triphosphate pocket of the enzyme.


Assuntos
CDC2-CDC28 Quinases , Quinases Ciclina-Dependentes/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Proteínas Proto-Oncogênicas , Pirazóis/síntese química , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose , Divisão Celular/efeitos dos fármacos , Cristalografia por Raios X , Quinase 2 Dependente de Ciclina , Quinase 4 Dependente de Ciclina , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Cinética , Modelos Moleculares , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirazóis/química , Pirazóis/farmacologia , Relação Estrutura-Atividade , Células Tumorais Cultivadas
13.
Biochemistry ; 41(41): 12552-61, 2002 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-12369847

RESUMO

The bacterial cell wall is a polymer consisting of alternating N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) units, cross-linked via peptides appended to MurNAc. The final steps in the formation of cell wall, also referred to as murein, are catalyzed by high-molecular-weight, class A penicillin-binding proteins (PBPs). These bifunctional enzymes catalyze both glycosyltransfer, to form the carbohydrate backbone of murein, and transpeptidation, to form the interstrand peptide linkages. Using PBP1b from Eschericia coli, an in vitro kinetic characterization of the glycosyltransfer reaction was carried out. Initial studies with unlabeled substrate (Lipid II) revealed that activity is strongly influenced by DMSO, as well as metal and detergent. In addition, a continuous fluoresence assay was developed and used to determine the effect of pH on the reaction. A single basic residue was titrated, with a pK(a) of 7.0. Taken together, these data suggest a mechanism for PBP1b where the glycosyltransfer reaction is catalyzed by the concerted effect of an active site base to deprotonate the glycosyl acceptor and a divalent metal to assist departure of the leaving group of the glycosyl donor.


Assuntos
Proteínas de Bactérias , Proteínas de Transporte/química , Proteínas de Escherichia coli/química , Glicosiltransferases/química , Hexosiltransferases , Muramilpentapeptídeo Carboxipeptidase/química , Peptidoglicano Glicosiltransferase , Peptidil Transferases/química , D-Ala-D-Ala Carboxipeptidase Tipo Serina , Proteínas de Transporte/análise , Cromatografia Líquida de Alta Pressão , Compostos de Dansil/química , Ativação Enzimática , Proteínas de Escherichia coli/análise , Fluorometria/métodos , Glicosiltransferases/análise , Cinética , Complexos Multienzimáticos/análise , Complexos Multienzimáticos/química , Muramilpentapeptídeo Carboxipeptidase/análise , Ressonância Magnética Nuclear Biomolecular , Proteínas de Ligação às Penicilinas , Peptidil Transferases/análise , Proteínas Recombinantes/análise , Proteínas Recombinantes/química , Especificidade por Substrato
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