RESUMO
In contrast to studies on class I histone deacetylase (HDAC) inhibitors, the elucidation of the molecular mechanisms and therapeutic potential of class IIa HDACs (HDAC4, HDAC5, HDAC7 and HDAC9) is impaired by the lack of potent and selective chemical probes. Here we report the discovery of inhibitors that fill this void with an unprecedented metal-binding group, trifluoromethyloxadiazole (TFMO), which circumvents the selectivity and pharmacologic liabilities of hydroxamates. We confirm direct metal binding of the TFMO through crystallographic approaches and use chemoproteomics to demonstrate the superior selectivity of the TFMO series relative to a hydroxamate-substituted analog. We further apply these tool compounds to reveal gene regulation dependent on the catalytic active site of class IIa HDACs. The discovery of these inhibitors challenges the design process for targeting metalloenzymes through a chelating metal-binding group and suggests therapeutic potential for class IIa HDAC enzyme blockers distinct in mechanism and application compared to current HDAC inhibitors.
Assuntos
Inibidores de Histona Desacetilases/química , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Zinco/química , Linhagem Celular Tumoral , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Inibidores de Histona Desacetilases/síntese química , Histona Desacetilases/genética , Humanos , Modelos Moleculares , Estrutura Molecular , Compostos Organometálicos/síntese química , Compostos Organometálicos/química , Compostos Organometálicos/farmacologia , Oxidiazóis/química , Relação Estrutura-Atividade , Zinco/metabolismoRESUMO
High-throughput screening for inhibitors of the human metalloprotease, methionine aminopeptidase-2 (MetAP2), identified a potent class of 3-anilino-5-benzylthio-1,2,4-triazole compounds. Efficient array and interative synthesis of triazoles led to rapid SAR development around the aniline, benzylthio, and triazole moeities. Evaluation of these analogs in a human MetAP2 enzyme assay led to the identification of several inhibitors with potencies in the 50-100 picomolar range. The deleterious effects on inhibitor potency by methylation of the anilino-triazole nitrogens, as well as the X-ray crystal structure of triazole 102 bound in the active site of MetAP2, confirm the key interactions between the triazole nitrogens, the active site cobalt atoms, and the His-231 side-chain. The structure has also provided a rationale for interpreting SAR within the triazole series. Key aniline (2-isopropylphenyl) and sulfur substituents (furanylmethyl) identified in the SAR studies led to the identification of potent inhibitors (103 and 104) of endothelial cell proliferation. Triazoles 103 and 104 also exhibited dose-dependent activity in an aortic ring tissue model of angiogenesis highlighting the potential utility of MetAP2 inhibitors as anticancer agents.
Assuntos
Aminopeptidases/antagonistas & inibidores , Inibidores da Angiogênese/síntese química , Furanos/síntese química , Metaloendopeptidases/antagonistas & inibidores , Tiazóis/síntese química , Tiofenos/síntese química , Triazóis/síntese química , Inibidores da Angiogênese/química , Inibidores da Angiogênese/farmacologia , Animais , Aorta Torácica/efeitos dos fármacos , Capilares/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Endotélio Vascular/citologia , Furanos/química , Furanos/farmacologia , Técnicas In Vitro , Masculino , Modelos Moleculares , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Tiazóis/química , Tiazóis/farmacologia , Tiofenos/química , Tiofenos/farmacologia , Triazóis/química , Triazóis/farmacologiaRESUMO
The SAR development is described for a series of N-acyl pyrrolidine inhibitors of the Hepatitis C virus RNA-dependent RNA polymerase, NS5B, from tractable Delta21 enzyme inhibitors to an example with antiviral activity in a cellular assay (HCV replicon).
Assuntos
Antivirais/farmacologia , Química Farmacêutica/métodos , Hepacivirus/química , Hepacivirus/genética , Pirrolidinas/antagonistas & inibidores , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Replicon/genética , Proteínas não Estruturais Virais/farmacologia , Antivirais/química , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Concentração Inibidora 50 , Modelos Químicos , Conformação Molecular , RNA Viral/química , Proteínas não Estruturais Virais/química , Replicação Viral/efeitos dos fármacosRESUMO
Recently, we disclosed a new class of HCV polymerase inhibitors discovered through high-throughput screening (HTS) of the GlaxoSmithKline proprietary compound collection. This interesting class of 3-(1,1-dioxo-2H-1,2,4-benzothiadiazin-3-yl)-4-hydroxy-2(1H)-quinolinones potently inhibits HCV polymerase enzymatic activity and inhibits the ability of the subgenomic HCV replicon to replicate in Huh-7 cells. This report will focus on the structure-activity relationships (SAR) of substituents on the quinolinone ring, culminating in the discovery of 1-(2-cyclopropylethyl)-3-(1,1-dioxo-2H-1,2,4-benzothiadiazin-3-yl)-6-fluoro-4-hydroxy-2(1H)-quinolinone (130), an inhibitor with excellent potency in biochemical and cellular assays possessing attractive molecular properties for advancement as a clinical candidate. The potential for development and safety assessment profile of compound 130 will also be discussed.
Assuntos
Antivirais/síntese química , Benzotiadiazinas/síntese química , Hepacivirus/enzimologia , Quinolonas/síntese química , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Tiadiazinas/síntese química , Animais , Antivirais/química , Antivirais/farmacologia , Benzotiadiazinas/química , Benzotiadiazinas/farmacologia , Disponibilidade Biológica , Proteínas Sanguíneas/metabolismo , Linhagem Celular , Cristalografia por Raios X , Cães , Genótipo , Meia-Vida , Hepacivirus/genética , Macaca fascicularis , Modelos Moleculares , Estrutura Molecular , Mutação , Ligação Proteica , Quinolonas/química , Quinolonas/farmacologia , RNA Polimerase Dependente de RNA/química , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Tiadiazinas/química , Tiadiazinas/farmacologiaRESUMO
Inhibitors of human methionine aminopeptidase type 2 (hMetAP2) are of interest as potential treatments for cancer. A new class of small molecule reversible inhibitors of hMetAP2 was discovered and optimized, the 4-aryl-1,2,3-triazoles. Compound 24, a potent inhibitor of cobalt-activated hMetAP2, also inhibits human and mouse endothelial cell growth. Using a mouse matrigel model, this reversible hMetAP2 inhibitor was also shown to inhibit angiogenesis in vivo.
Assuntos
Aminopeptidases/antagonistas & inibidores , Inibidores da Angiogênese/síntese química , Metaloendopeptidases/antagonistas & inibidores , Triazóis/síntese química , Aminopeptidases/química , Inibidores da Angiogênese/química , Inibidores da Angiogênese/farmacologia , Animais , Sítios de Ligação , Disponibilidade Biológica , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Cobalto/metabolismo , Colágeno , Cristalografia por Raios X , Combinação de Medicamentos , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Endotélio Vascular/citologia , Ativação Enzimática , Humanos , Laminina , Metaloendopeptidases/química , Camundongos , Modelos Moleculares , Estrutura Molecular , Proteoglicanas , Ratos , Relação Estrutura-Atividade , Triazóis/química , Triazóis/farmacologiaRESUMO
HTS of the compound collection for inhibition of the HCV RNA dependent RNA polymerase identified two 168 member N-acyl pyrrolidine combinatorial mixture hits. Deconvolution and expansion of these mixtures by solid phase synthesis to establish initial SAR and identify a potent inhibitor is reported.
Assuntos
Hepacivirus/enzimologia , Pirrolidinas/química , Pirrolidinas/farmacologia , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Técnicas de Química Combinatória , Estrutura MolecularRESUMO
The hepatitis C virus (HCV) NS5B protein encodes an RNA-dependent RNA polymerase (RdRp), the primary catalytic enzyme of the HCV replicase complex. We established a biochemical RNA synthesis assay, using purified recombinant NS5B lacking the C-terminal 21 amino acid residues, to identify potential polymerase inhibitors from a high throughput screen of the GlaxoSmithKline proprietary compound collection. The benzo-1,2,4-thiadiazine compound 1 was found to be a potent, highly specific inhibitor of NS5B. This agent interacts directly with the viral polymerase and inhibits RNA synthesis in a manner noncompetitive with respect to GTP. Furthermore, in the absence of an in vitro-reconstituted HCV replicase assay employing viral and host proteins, the ability of compound 1 to inhibit NS5B-directed viral RNA replication was determined using the Huh7 cell-based HCV replicon system. Compound 1 reduced viral RNA in replicon cells with an IC(50) of approximately 0.5 microm, suggesting that the inhibitor was able to access the perinuclear membrane and inhibit the polymerase activity in the context of a replicase complex. Preliminary structure-activity studies on compound 1 led to the identification of a modified inhibitor, compound 4, showing an improvement in both biochemical and cell-based potency. Lastly, data are presented suggesting that these compounds interfere with the formation of negative and positive strand progeny RNA by a similar mode of action. Investigations are ongoing to assess the potential utility of such agents in the treatment of chronic HCV disease.