RESUMO
Monoubiquitination of histones H2B-K120 (H2BK120ub) and H2A-K119 (H2AK119ub) play opposing roles in regulating transcription and chromatin compaction. H2BK120ub is a hallmark of actively transcribed euchromatin, while H2AK119ub is highly enriched in transcriptionally repressed heterochromatin. Whereas H2BK120ub is known to stimulate the binding or activity of various chromatin-modifying enzymes, this post-translational modification (PTM) also interferes with the binding of several proteins to the nucleosome H2A/H2B acidic patch via an unknown mechanism. Here, we report cryoEM structures of an H2BK120ub nucleosome showing that ubiquitin adopts discrete positions that occlude the acidic patch. Molecular dynamics simulations show that ubiquitin remains stably positioned over this nucleosome region. By contrast, our cryoEM structures of H2AK119ub nucleosomes show ubiquitin adopting discrete positions that minimally occlude the acidic patch. Consistent with these observations, H2BK120ub, but not H2AK119ub, abrogates nucleosome interactions with acidic patch-binding proteins RCC1 and LANA, and single-domain antibodies specific to this region. Our results suggest a mechanism by which H2BK120ub serves as a gatekeeper to the acidic patch and point to distinct roles for histone H2AK119 and H2BK120 ubiquitination in regulating protein binding to nucleosomes.
RESUMO
Lipoprotein diacylglyceryl transferase (Lgt) catalyzes the first step in the biogenesis of Gram-negative bacterial lipoproteins which play crucial roles in bacterial growth and pathogenesis. We demonstrate that Lgt depletion in a clinical uropathogenic Escherichia coli strain leads to permeabilization of the outer membrane and increased sensitivity to serum killing and antibiotics. Importantly, we identify G2824 as the first-described Lgt inhibitor that potently inhibits Lgt biochemical activity in vitro and is bactericidal against wild-type Acinetobacter baumannii and E. coli strains. While deletion of a gene encoding a major outer membrane lipoprotein, lpp, leads to rescue of bacterial growth after genetic depletion or pharmacologic inhibition of the downstream type II signal peptidase, LspA, no such rescue of growth is detected after Lgt depletion or treatment with G2824. Inhibition of Lgt does not lead to significant accumulation of peptidoglycan-linked Lpp in the inner membrane. Our data validate Lgt as a novel antibacterial target and suggest that, unlike downstream steps in lipoprotein biosynthesis and transport, inhibition of Lgt may not be sensitive to one of the most common resistance mechanisms that invalidate inhibitors of bacterial lipoprotein biosynthesis and transport. IMPORTANCE As the emerging threat of multidrug-resistant (MDR) bacteria continues to increase, no new classes of antibiotics have been discovered in the last 50 years. While previous attempts to inhibit the lipoprotein biosynthetic (LspA) or transport (LolCDE) pathways have been made, most efforts have been hindered by the emergence of a common mechanism leading to resistance, namely, the deletion of the gene encoding a major Gram-negative outer membrane lipoprotein lpp. Our unexpected finding that inhibition of Lgt is not susceptible to lpp deletion-mediated resistance uncovers the complexity of bacterial lipoprotein biogenesis and the corresponding enzymes involved in this essential outer membrane biogenesis pathway and potentially points to new antibacterial targets in this pathway.
Assuntos
Escherichia coli/metabolismo , Lipoproteínas/metabolismo , Transferases/metabolismo , Animais , Antibacterianos/farmacologia , Ácido Aspártico Endopeptidases , Proteínas de Bactérias , Escherichia coli/genética , Feminino , Deleção de Genes , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Camundongos , Peptidoglicano/metabolismo , Transferases/química , Transferases/genética , Escherichia coli Uropatogênica/genética , Escherichia coli Uropatogênica/metabolismoRESUMO
Discovery of novel classes of Gram-negative antibiotics with activity against multi-drug resistant infections is a critical unmet need. As an essential member of the lipoprotein biosynthetic pathway, lipoprotein signal peptidase II (LspA) is an attractive target for antibacterial drug discovery, with the natural product inhibitor globomycin offering a modestly-active starting point. Informed by structure-based design, the globomycin depsipeptide was optimized to improve activity against E. coli. Backbone modifications, together with adjustment of physicochemical properties, afforded potent compounds with good in vivo pharmacokinetic profiles. Optimized compounds such as 51 (E. coli MIC 3.1 µM) and 61 (E. coli MIC 0.78 µM) demonstrate broad spectrum activity against gram-negative pathogens and may provide opportunities for future antibiotic discovery.
Assuntos
Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Peptídeos/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Relação Dose-Resposta a Droga , Testes de Sensibilidade Microbiana , Estrutura Molecular , Peptídeos/síntese química , Peptídeos/química , Relação Estrutura-AtividadeRESUMO
Gram-negative bacteria express a diverse array of lipoproteins that are essential for various aspects of cell growth and virulence, including nutrient uptake, signal transduction, adhesion, conjugation, sporulation, and outer membrane protein folding. Lipoprotein maturation requires the sequential activity of three enzymes that are embedded in the cytoplasmic membrane. First, phosphatidylglycerol:prolipoprotein diacylglyceryl transferase (Lgt) recognizes a conserved lipobox motif within the prolipoprotein signal sequence and catalyzes the addition of diacylglycerol to an invariant cysteine. The signal sequence is then cleaved by signal peptidase II (LspA) to give an N-terminal S-diacylglyceryl cysteine. Finally, apolipoprotein N-acyltransferase (Lnt) catalyzes the transfer of the sn-1-acyl chain of phosphatidylethanolamine to this N-terminal cysteine, generating a mature, triacylated lipoprotein. Although structural studies of Lgt and LspA have yielded significant mechanistic insights into this essential biosynthetic pathway, the structure of Lnt has remained elusive. Here, we present crystal structures of wild-type and an active-site mutant of Escherichia coli Lnt. The structures reveal a monomeric eight-transmembrane helix fold that supports a periplasmic carbon-nitrogen hydrolase domain containing a Cys-Glu-Lys catalytic triad. Two lipids are bound at the active site in the structures, and we propose a putative phosphate recognition site where a chloride ion is coordinated near the active site. Based on these structures and complementary cell-based, biochemical, and molecular dynamics approaches, we propose a mechanism for substrate engagement and catalysis by E. coli Lnt.
Assuntos
Aciltransferases/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Lipoproteínas/metabolismo , Acilação , Aciltransferases/química , Sítios de Ligação , Domínio Catalítico , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/química , Mutação , Conformação ProteicaRESUMO
Activating mutations in KRAS and BRAF are found in more than 30% of all human tumours and 40% of melanoma, respectively, thus targeting this pathway could have broad therapeutic effects. Small molecule ATP-competitive RAF kinase inhibitors have potent antitumour effects on mutant BRAF(V600E) tumours but, in contrast to mitogen-activated protein kinase kinase (MEK) inhibitors, are not potent against RAS mutant tumour models, despite RAF functioning as a key effector downstream of RAS and upstream of MEK. Here we show that ATP-competitive RAF inhibitors have two opposing mechanisms of action depending on the cellular context. In BRAF(V600E) tumours, RAF inhibitors effectively block the mitogen-activated protein kinase (MAPK) signalling pathway and decrease tumour growth. Notably, in KRAS mutant and RAS/RAF wild-type tumours, RAF inhibitors activate the RAF-MEK-ERK pathway in a RAS-dependent manner, thus enhancing tumour growth in some xenograft models. Inhibitor binding activates wild-type RAF isoforms by inducing dimerization, membrane localization and interaction with RAS-GTP. These events occur independently of kinase inhibition and are, instead, linked to direct conformational effects of inhibitors on the RAF kinase domain. On the basis of these findings, we demonstrate that ATP-competitive kinase inhibitors can have opposing functions as inhibitors or activators of signalling pathways, depending on the cellular context. Furthermore, this work provides new insights into the therapeutic use of ATP-competitive RAF inhibitors.
Assuntos
Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Neoplasias/patologia , Inibidores de Proteínas Quinases/farmacologia , Quinases raf/antagonistas & inibidores , Quinases raf/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Benzamidas/farmacologia , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Difenilamina/análogos & derivados , Difenilamina/farmacologia , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Indenos/farmacologia , Indóis/farmacologia , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Multimerização Proteica , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/química , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas c-raf/deficiência , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas Proto-Oncogênicas p21(ras) , Pirazóis/farmacologia , Sulfonamidas/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases raf/química , Quinases raf/genética , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
Herein we describe the design of a novel series of ATP competitive B-Raf inhibitors via structure-based methods. These 3-N-methylquinazoline-4(3H)-one based inhibitors exhibit both excellent cellular potency and striking B-Raf selectivity. Optimization led to the identification of compound 16, a potent, selective and orally available agent with excellent pharmacokinetic properties and robust tumor growth inhibition in xenograft studies. Our work also demonstrates that by replacing an aryl amide with an aryl sulfonamide, a multikinase inhibitor such as AZ-628, can be converted to a selective B-Raf inhibitor, a finding that should have broad application in kinase drug discovery.
Assuntos
Desenho de Fármacos , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Quinazolinas/síntese química , Quinazolinas/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Cristalografia por Raios X , Ativação Enzimática/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Estrutura Molecular , Inibidores de Proteínas Quinases/química , Quinazolinas/química , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
During tumor development, promoter CpG islands (CGIs) that are normally silenced by Polycomb repressive complexes (PRCs) become DNA hypermethylated. The molecular mechanism by which de novo DNA methyltransferase(s) catalyze CpG methylation at PRC-regulated regions remains unclear. Here we report a cryo-EM structure of the DNMT3A long isoform (DNMT3A1) N-terminal region in complex with a nucleosome carrying PRC1-mediated histone H2A lysine 119 monoubiquitination (H2AK119Ub). We identify regions within the DNMT3A1 N-terminus that bind H2AK119Ub and the nucleosome acidic patch. This bidentate interaction is required for effective DNMT3A1 engagement with H2AK119Ub-modified chromatin in cells. Furthermore, aberrant redistribution of DNMT3A1 to Polycomb target genes inhibits their transcriptional activation during cell differentiation and recapitulates the cancer-associated DNA hypermethylation signature. This effect is rescued by disruption of the DNMT3A1-acidic patch interaction. Together, our analyses reveal a binding interface critical for countering promoter CGI DNA hypermethylation, a major molecular hallmark of cancer.
RESUMO
This Letter details the synthesis and evaluation of imidazo[4,5-b]pyridines as inhibitors of B-Raf kinase. These compounds bind in a DFG-in, αC-helix out conformation of B-Raf, which is a binding mode associated with significant kinase selectivity. Structure-activity relationship studies involved optimization of the ATP-cleft binding region of these molecules, and led to compound 23, an inhibitor with excellent enzyme/cell potency, and kinase selectivity.
Assuntos
Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Purinas/química , Purinas/farmacologia , Animais , Células CACO-2 , Desenho de Fármacos , Humanos , Camundongos , Simulação de Acoplamento Molecular , Proteínas Proto-Oncogênicas B-raf/química , Proteínas Proto-Oncogênicas B-raf/metabolismo , Purinas/farmacocinética , Ratos , Relação Estrutura-AtividadeRESUMO
The maintenance of gene expression patterns during metazoan development is achieved by the actions of Polycomb group (PcG) complexes. An essential modification marking silenced genes is monoubiquitination of histone H2A lysine 119 (H2AK119Ub) deposited by the E3 ubiquitin ligase activity of the non-canonical Polycomb Repressive Complex 1. The Polycomb Repressive Deubiquitinase (PR-DUB) complex cleaves monoubiquitin from histone H2A lysine 119 (H2AK119Ub) to restrict focal H2AK119Ub at Polycomb target sites and to protect active genes from aberrant silencing. BAP1 and ASXL1, subunits that form active PR-DUB, are among the most frequently mutated epigenetic factors in human cancers, underscoring their biological importance. How PR-DUB achieves specificity for H2AK119Ub to regulate Polycomb silencing is unknown, and the mechanisms of most of the mutations in BAP1 and ASXL1 found in cancer have not been established. Here we determine a cryo-EM structure of human BAP1 bound to the ASXL1 DEUBAD domain in complex with a H2AK119Ub nucleosome. Our structural, biochemical, and cellular data reveal the molecular interactions of BAP1 and ASXL1 with histones and DNA that are critical for remodeling the nucleosome and thus establishing specificity for H2AK119Ub. These results further provide a molecular explanation for how >50 mutations in BAP1 and ASXL1 found in cancer can dysregulate H2AK119Ub deubiquitination, providing new insight into understanding cancer etiology. One Sentence Summary: We reveal the molecular mechanism of nucleosomal H2AK119Ub deubiquitination by human BAP1/ASXL1.
RESUMO
Histone H2A lysine 119 (H2AK119Ub) is monoubiquitinated by Polycomb repressive complex 1 and deubiquitinated by Polycomb repressive deubiquitinase complex (PR-DUB). PR-DUB cleaves H2AK119Ub to restrict focal H2AK119Ub at Polycomb target sites and to protect active genes from aberrant silencing. The PR-DUB subunits (BAP1 and ASXL1) are among the most frequently mutated epigenetic factors in human cancers. How PR-DUB establishes specificity for H2AK119Ub over other nucleosomal ubiquitination sites and how disease-associated mutations of the enzyme affect activity are unclear. Here, we determine a cryo-EM structure of human BAP1 and the ASXL1 DEUBAD in complex with a H2AK119Ub nucleosome. Our structural, biochemical, and cellular data reveal the molecular interactions of BAP1 and ASXL1 with histones and DNA that are critical for restructuring the nucleosome and thus establishing specificity for H2AK119Ub. These results further provide a molecular explanation for how >50 mutations in BAP1 and ASXL1 found in cancer can dysregulate H2AK119Ub deubiquitination, providing insight into understanding cancer etiology.
Assuntos
Proteínas de Drosophila , Neoplasias , Humanos , Histonas/genética , Nucleossomos , Lisina , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Proteínas do Grupo Polycomb/genética , Proteínas de Drosophila/genética , Neoplasias/genética , Proteínas Repressoras/genética , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
In nucleosomes, histone N-terminal tails exist in dynamic equilibrium between free/accessible and collapsed/DNA-bound states. The latter state is expected to impact histone N-termini availability to the epigenetic machinery. Notably, H3 tail acetylation (e.g. K9ac, K14ac, K18ac) is linked to increased H3K4me3 engagement by the BPTF PHD finger, but it is unknown if this mechanism has a broader extension. Here, we show that H3 tail acetylation promotes nucleosomal accessibility to other H3K4 methyl readers, and importantly, extends to H3K4 writers, notably methyltransferase MLL1. This regulation is not observed on peptide substrates yet occurs on the cis H3 tail, as determined with fully-defined heterotypic nucleosomes. In vivo, H3 tail acetylation is directly and dynamically coupled with cis H3K4 methylation levels. Together, these observations reveal an acetylation 'chromatin switch' on the H3 tail that modulates read-write accessibility in nucleosomes and resolves the long-standing question of why H3K4me3 levels are coupled with H3 acetylation.
Assuntos
Cromatina , Histonas , Histonas/metabolismo , Nucleossomos , Metilação , AcetilaçãoRESUMO
Cell potent inhibitors of B-Raf(V600E) that bind to the kinase in the DFG-out conformation are reported. These compounds utilize the hinge-binding group and lipophilic linker from a previously disclosed series of B-Raf(V600E) inhibitors that bind to the kinase in an atypical DFG-in, αC-helix-out conformation. This new series demonstrates that DFG-out kinase inhibitors can be rationally designed from related inhibitors which utilize an unconventional binding mode. Kinase selectivity profiles are compared. The pattern of kinase selectivity was found to be determined by the feature of the inhibitor which extends into the back pocket of the kinase and leads to the kinase conformation, rather than by the hinge-binding group or other minor modifications.
Assuntos
Desenho de Fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Pirazóis/farmacologia , Piridinas/farmacologia , Animais , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Estabilidade de Medicamentos , Humanos , Camundongos , Microssomos/química , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas B-raf/metabolismo , Pirazóis/síntese química , Pirazóis/química , Piridinas/síntese química , Piridinas/química , Ratos , Relação Estrutura-AtividadeRESUMO
A single crystal was obtained of a lead B-Raf(V600E) inhibitor with low aqueous solubility. The X-ray crystal structure revealed hydrogen-bonded head-to-tail dimers formed by the pyrazolopyridine and sulfonamide groups of a pair of molecules. This observation suggested a medicinal chemistry strategy to disrupt crystal packing and reduce the high crystal lattice energy of alternative inhibitors. Both a bulkier group at the interface of the dimer and an out-of-plane substituent were required to decrease the compound's melting point and increase aqueous solubility. These substituents were selected based on previously developed structure-activity relationships so as to concurrently maintain good enzymatic and cellular activity against B-Raf(V600E).
Assuntos
Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Pirazóis/farmacologia , Piridinas/farmacologia , Cristalografia por Raios X , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas B-raf/metabolismo , Pirazóis/síntese química , Pirazóis/química , Piridinas/síntese química , Piridinas/química , Solubilidade , Relação Estrutura-Atividade , Água/químicaRESUMO
Herein we describe a novel series of ATP competitive B-Raf inhibitors based on the pyrazolo[1,5-a]pyrimidine scaffold. These inhibitors exhibit both excellent cellular potency and striking B-Raf selectivity. Optimization led to the identification of compound 17, a potent, selective and orally available agent with improved physicochemical and pharmacokinetic properties.
Assuntos
Inibidores Enzimáticos/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Pirazóis/farmacologia , Pirimidinas/farmacologia , Animais , Físico-Química , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Camundongos , Modelos Moleculares , Estrutura Molecular , Proteínas Proto-Oncogênicas B-raf/metabolismo , Pirazóis/síntese química , Pirazóis/química , Pirimidinas/síntese química , Pirimidinas/química , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Herein we describe the discovery of a novel series of ATP competitive B-Raf inhibitors via structure based drug design (SBDD). These pyridopyrimidin-7-one based inhibitors exhibit both excellent cellular potency and striking B-Raf selectivity. Optimization led to the identification of compound 17, a potent, selective and orally available agent with excellent pharmacokinetic properties and robust tumor growth inhibition in xenograft studies.
Assuntos
Descoberta de Drogas , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Pirimidinonas/farmacologia , Administração Oral , Disponibilidade Biológica , Cristalografia por Raios X , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacocinética , Pirimidinonas/química , Pirimidinonas/farmacocinéticaRESUMO
A novel series of spirochromane pan-Akt inhibitors is reported. SAR optimization furnished compounds with improved enzyme potencies and excellent selectivity over the related AGC kinase PKA. Attempted replacement of the phenol hinge binder provided compounds with excellent Akt enzyme and cell activities but greatly diminished selectivity over PKA.
Assuntos
Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Sítios de Ligação , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Relação Estrutura-AtividadeRESUMO
Structure-activity relationships around a novel series of B-Raf(V600E) inhibitors are reported. The enzymatic and cellular potencies of inhibitors derived from two related hinge-binding groups were compared and3-methoxypyrazolopyridine proved to be superior. The 3-alkoxy group of lead B-Raf(V600E) inhibitor 1 was extended and minimally affected potency. The propyl sulfonamide tail of compound 1, which occupies the small lipophilic pocket formed by an outward shift of the αC-helix, was expanded to a series of arylsulfonamides. X-ray crystallography revealed that this lipophilic pocket unexpectedly enlarges to accommodate the bulkier aryl group.
Assuntos
Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Pirazóis/farmacologia , Piridinas/farmacologia , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas B-raf/metabolismo , Pirazóis/síntese química , Pirazóis/química , Piridinas/síntese química , Piridinas/química , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
We describe the design and synthesis of novel bicyclic spiro sulfonamides as potent Akt inhibitors. Through structure-based rational design, we have successfully improved PKA selectivity of previously disclosed spirochromanes. Representative compounds showed favorable Akt potency while exhibiting up to 1000-fold selectivity against PKA.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Compostos de Espiro/química , Sulfonamidas/química , Sítios de Ligação , Simulação por Computador , Cristalografia por Raios X , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Avaliação Pré-Clínica de Medicamentos , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/farmacologiaRESUMO
The discovery and optimization of a series of pyrrolopyrimidine based protein kinase B (Pkb/Akt) inhibitors discovered via HTS and structure based drug design is reported. The compounds demonstrate potent inhibition of all three Akt isoforms and knockdown of phospho-PRAS40 levels in LNCaP cells and tumor xenografts.
Assuntos
Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Pirimidinas/química , Pirróis/química , Proteínas Adaptadoras de Transdução de Sinal , Animais , Sítios de Ligação , Linhagem Celular , Cristalografia por Raios X , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Fosfoproteínas/deficiência , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirimidinas/síntese química , Pirimidinas/farmacologia , Pirróis/síntese química , Pirróis/farmacologia , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Herein we report the discovery and synthesis of a novel series of dihydrothieno- and dihydrofuropyrimidines (2 and 3) as potent pan Akt inhibitors. Utilizing previous SAR and analysis of the amino acid sequences in the binding site we have designed inhibitors displaying increased PKA and general kinase selectivity with improved tolerability compared to the progenitor pyrrolopyrimidine (1). A representative dihydrothieno compound (34) was advanced into a PC3-NCI prostate mouse tumor model in which it demonstrated a dose-dependent reduction in tumor growth and stasis when dosed orally daily at 200 mg/kg.