RESUMO
We explore mechanisms that enable cancer cells to tolerate PI3K or Akt inhibitors. Prolonged treatment of breast cancer cells with PI3K or Akt inhibitors leads to increased expression and activation of a kinase termed SGK3 that is related to Akt. Under these conditions, SGK3 is controlled by hVps34 that generates PtdIns(3)P, which binds to the PX domain of SGK3 promoting phosphorylation and activation by its upstream PDK1 activator. Furthermore, under conditions of prolonged PI3K/Akt pathway inhibition, SGK3 substitutes for Akt by phosphorylating TSC2 to activate mTORC1. We characterise 14h, a compound that inhibits both SGK3 activity and activation in vivo, and show that a combination of Akt and SGK inhibitors induced marked regression of BT-474 breast cancer cell-derived tumours in a xenograft model. Finally, we present the kinome-wide analysis of mRNA expression dynamics induced by PI3K/Akt inhibition. Our findings highlight the importance of the hVps34-SGK3 pathway and suggest it represents a mechanism to counteract inhibition of PI3K/Akt signalling. The data support the potential of targeting both Akt and SGK as a cancer therapeutic.
Assuntos
Carcinogênese , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Complexos Multiproteicos/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Modelos Animais de Doenças , Feminino , Xenoenxertos , Humanos , Alvo Mecanístico do Complexo 1 de RapamicinaRESUMO
Bryostatins with modified C17-C27 fragments have not been widely studied. The synthesis of 20,20-difluorinated analogues was therefore investigated. Such substitution would inhibit dehydration involving the C19-hydroxyl group and stabilise the ring-closed hemiacetal tautomers. Following preliminary studies, allyldifluorination was used to prepare difluorinated alkenols. Oxidation followed by stereoselective Wittig reactions of the resulting α,α-difluorinated ketones gave (E)-α,ß-unsaturated esters that were taken through to complete syntheses of 2-hydroxytetrahydropyrans corresponding to C17-C27 fragments of 20,20-difluorinated bryostatin. These compounds showed modest binding to protein kinase Cα isozyme. Attempts were also undertaken to synthesise macrocyclic 20,20-difluorinated analogues. During preliminary studies, allyldifluorination was carried out using a 2-alkyl-3-bromo-1,1-difluoropropene.
Assuntos
Briostatinas/química , Briostatinas/síntese química , Halogenação , Briostatinas/metabolismo , Técnicas de Química Sintética , Modelos Moleculares , Conformação Molecular , Proteína Quinase C/metabolismoRESUMO
Protein phosphorylation lies at the heart of cell signalling, and somatic mutation(s) in kinases drives and sustains a multitude of human diseases, including cancer. The human protein kinase superfamily (the kinome) encodes approximately 50 'pseudokinases', which were initially predicted to be incapable of dynamic cell signalling when compared with canonical enzymatically active kinases. This assumption was supported by bioinformatics, which showed that amino acid changes at one or more key loci, making up the nucleotide-binding site or phosphotransferase machinery, were conserved in multiple vertebrate and non-vertebrate pseudokinase homologues. Protein kinases are highly attractive targets for drug discovery, as evidenced by the approval of almost 30 kinase inhibitors in oncology, and the successful development of the dual JAK1/2 (Janus kinase 1/2) inhibitor ruxolitinib for inflammatory indications. However, for such a large (>550) protein family, a remarkable number have still not been analysed at the molecular level, and only a surprisingly small percentage of kinases have been successfully targeted clinically. This is despite evidence that many are potential candidates for the development of new therapeutics. Indeed, several recent reports confirm that disease-associated pseudokinases can bind to nucleotide co-factors at concentrations achievable in the cell. Together, these findings suggest that drug targeting using either ATP-site or unbiased ligand-discovery approaches should now be attempted using the validation technology currently employed to evaluate their classic protein kinase counterparts. In the present review, we discuss members of the human pseudokinome repertoire, and catalogue somatic amino acid pseudokinase mutations that are emerging as the depth and clinical coverage of the human cancer pseudokinome expand.
Assuntos
Proteínas de Neoplasias/metabolismo , Neoplasias/enzimologia , Proteínas Quinases/metabolismo , Proteoma/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Loci Gênicos , Humanos , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/classificação , Proteínas de Neoplasias/genética , Neoplasias/tratamento farmacológico , Neoplasias/genética , Nitrilas , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Quinases/classificação , Proteínas Quinases/genética , Proteoma/antagonistas & inibidores , Proteoma/classificação , Proteoma/genética , Pirazóis/uso terapêutico , PirimidinasRESUMO
The human Tribbles (TRB)-related pseudokinases are CAMK (calcium/calmodulin-dependent protein kinase)-related family members that have evolved a series of highly unusual motifs in the 'pseudocatalytic' domain. In canonical kinases, conserved amino acids bind to divalent metal ions and align ATP prior to efficient phosphoryl-transfer to substrates. However, in pseudokinases, atypical residues give rise to diverse and often unstudied biochemical and structural features that are thought to be central to cellular functions. TRB proteins play a crucial role in multiple signalling networks and overexpression confers cancer phenotypes on human cells, marking TRB pseudokinases out as a novel class of drug target. In the present paper, we report that the human pseudokinase TRB2 retains the ability to both bind and hydrolyse ATP weakly in vitro. Kinase activity is metal-independent and involves a catalytic lysine residue, which is conserved in TRB proteins throughout evolution alongside several unique amino acids in the active site. A similar low level of autophosphorylation is also preserved in the closely related human TRB3. By employing chemical genetics, we establish that the nucleotide-binding site of an 'analogue-sensitive' (AS) TRB2 mutant can be targeted with specific bulky ligands of the pyrazolo-pyrimidine (PP) chemotype. Our analysis confirms that TRB2 retains low levels of ATP binding and/or catalysis that is targetable with small molecules. Given the significant clinical successes associated with targeting of cancer-associated kinases with small molecule inhibitors, it is likely that similar approaches will be useful for further evaluating the TRB pseudokinases, with the translation of this information likely to furnish new leads for drug discovery.
Assuntos
Trifosfato de Adenosina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Modelos Moleculares , Proteínas Mutantes/metabolismo , Proteínas de Neoplasias/metabolismo , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Substituição de Aminoácidos , Biocatálise , Proteínas Quinases Dependentes de Cálcio-Calmodulina , Domínio Catalítico , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Sequência Conservada , Humanos , Hidrólise , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lisina/química , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Fosforilação , Mutação Puntual , Conformação Proteica , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Alinhamento de SequênciaRESUMO
Tribbles (TRIB) proteins are pseudokinase mediators of eukaryotic signalling that have evolved important roles in lipoprotein metabolism, immune function and cellular differentiation and proliferation. In addition, an evolutionary-conserved modulation of PI3K/AKT signalling pathways highlights them as novel and rather unusual pharmaceutical targets. The three human TRIB family members are uniquely defined by an acidic pseudokinase domain containing a 'broken' α C-helix and a MEK (MAPK/ERK)-binding site at the end of the putative C-lobe and a distinct C-terminal peptide motif that interacts directly with a small subset of cellular E3 ubiquitin ligases. This latter interaction drives proteasomal-dependent degradation of networks of transcription factors, whose rate of turnover determines the biological attributes of individual TRIB family members. Defining the function of individual Tribs has been made possible through evaluation of individual TRIB knockout mice, siRNA/overexpression approaches and genetic screening in flies, where the single TRIB gene was originally described 15 years ago. The rapidly maturing TRIB field is primed to exploit chemical biology approaches to evaluate endogenous TRIB signalling events in intact cells. This will help define how TRIB-driven protein-protein interactions and the atypical TRIB ATP-binding site, fit into cellular signalling modules in experimental scenarios where TRIB-signalling complexes remain unperturbed. In this mini-review, we discuss how small molecules can reveal rate-limiting signalling outputs and functions of Tribs in cells and intact organisms, perhaps serving as guides for the development of new drugs. We predict that appropriate small molecule TRIB ligands will further accelerate the transition of TRIB pseudokinase analysis into the mainstream of cell signalling.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteínas de Ciclo Celular/metabolismo , Descoberta de Drogas/métodos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Repressoras/metabolismo , Sequência de Aminoácidos , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Dados de Sequência Molecular , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Homologia de Sequência de Aminoácidos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
Protein kinase-like domains that lack conserved residues known to catalyse phosphoryl transfer, termed pseudokinases, have emerged as important signalling domains across all kingdoms of life. Although predicted to function principally as catalysis-independent protein-interaction modules, several pseudokinase domains have been attributed unexpected catalytic functions, often amid controversy. We established a thermal-shift assay as a benchmark technique to define the nucleotide-binding properties of kinase-like domains. Unlike in vitro kinase assays, this assay is insensitive to the presence of minor quantities of contaminating kinases that may otherwise lead to incorrect attribution of catalytic functions to pseudokinases. We demonstrated the utility of this method by classifying 31 diverse pseudokinase domains into four groups: devoid of detectable nucleotide or cation binding; cation-independent nucleotide binding; cation binding; and nucleotide binding enhanced by cations. Whereas nine pseudokinases bound ATP in a divalent cation-dependent manner, over half of those examined did not detectably bind nucleotides, illustrating that pseudokinase domains predominantly function as non-catalytic protein-interaction modules within signalling networks and that only a small subset is potentially catalytically active. We propose that henceforth the thermal-shift assay be adopted as the standard technique for establishing the nucleotide-binding and catalytic potential of kinase-like domains.
Assuntos
Janus Quinase 2/química , Janus Quinase 2/classificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Receptor ErbB-3/química , Receptor ErbB-3/classificação , Sequência de Aminoácidos , Animais , Linhagem Celular , Humanos , Insetos , Janus Quinase 2/genética , Dados de Sequência Molecular , Ligação Proteica/fisiologia , Receptor ErbB-3/genéticaRESUMO
Metastatic uveal melanoma (UM) is invariably fatal, usually within a year of diagnosis. There are currently no effective therapies, and clinical studies employing kinase inhibitors have so far demonstrated limited success. This is despite common activating mutations in GNAQ/11 genes, which trigger signalling pathways that might predispose tumours to a variety of targeted drugs. In this study, we have profiled kinome expression network dynamics in various human ocular melanomas. We uncovered a shared transcriptional profile in human primary UM samples and across a variety of experimental cell-based models. The poor overall response of UM cells to FDA-approved kinase inhibitors contrasted with much higher sensitivity to the bromodomain inhibitor JQ1, a broad transcriptional repressor. Mechanistically, we identified a repressed FOXM1-dependent kinase subnetwork in JQ1-exposed cells that contained multiple cell cycle-regulated protein kinases. Consistently, we demonstrated vulnerability of UM cells to inhibitors of mitotic protein kinases within this network, including the investigational PLK1 inhibitor BI6727. We conclude that analysis of kinome-wide signalling network dynamics has the potential to reveal actionable drug targets and inhibitors of potential therapeutic benefit for UM patients.
Assuntos
Perfilação da Expressão Gênica , Melanoma/genética , Terapia de Alvo Molecular , Proteínas Quinases/metabolismo , Neoplasias Uveais/genética , Azepinas/farmacologia , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Biologia Computacional , Regulação para Baixo/efeitos dos fármacos , Proteína Forkhead Box M1/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos dos fármacos , Humanos , Melanoma/patologia , Proteínas Nucleares/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Transcriptoma/genética , Triazóis/farmacologia , Neoplasias Uveais/patologiaRESUMO
A major challenge associated with biochemical and cellular analysis of pseudokinases is a lack of target-validated small-molecule compounds with which to probe function. Tribbles 2 (TRIB2) is a cancer-associated pseudokinase with a diverse interactome, including the canonical AKT signaling module. There is substantial evidence that human TRIB2 promotes survival and drug resistance in solid tumors and blood cancers and therefore is of interest as a therapeutic target. The unusual TRIB2 pseudokinase domain contains a unique cysteine-rich C-helix and interacts with a conserved peptide motif in its own carboxyl-terminal tail, which also supports its interaction with E3 ubiquitin ligases. We found that TRIB2 is a target of previously described small-molecule protein kinase inhibitors, which were originally designed to inhibit the canonical kinase domains of epidermal growth factor receptor tyrosine kinase family members. Using a thermal shift assay, we discovered TRIB2-binding compounds within the Published Kinase Inhibitor Set (PKIS) and used a drug repurposing approach to classify compounds that either stabilized or destabilized TRIB2 in vitro. TRIB2 destabilizing agents, including the covalent drug afatinib, led to rapid TRIB2 degradation in human AML cancer cells, eliciting tractable effects on signaling and survival. Our data reveal new drug leads for the development of TRIB2-degrading compounds, which will also be invaluable for unraveling the cellular mechanisms of TRIB2-based signaling. Our study highlights that small molecule-induced protein down-regulation through drug "off-targets" might be relevant for other inhibitors that serendipitously target pseudokinases.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias/enzimologia , Afatinib/farmacologia , Alelos , Inibidores Enzimáticos/farmacologia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Células HeLa , Humanos , Ligação Proteica , Domínios Proteicos , Transdução de Sinais , Bibliotecas de Moléculas Pequenas , Células U937RESUMO
Acquired resistance to targeted kinase inhibitors is a well-documented clinical problem that is potentially fatal for patients to whom a suitable back-up is not available. However, protein kinase alleles that promote resistance to inhibitors can be exploited experimentally as gold-standards for "on"- and "off"-target validation strategies and constitute a powerful resource for assessing the ability of new or combined therapies to override resistance. Clinical resistance to kinase inhibitors is an evident in all tyrosine kinase-driven malignancies, where high rates of mutation drive tumor evolution toward the insidious drug-resistant (DR) state through a variety of mechanisms. Unfortunately, this problem is likely to intensify in the future as the number of target kinases, approved inhibitors, and clinical indications increase. To empower the analysis of resistance in kinases, we have validated a bioinformatic, structural, and cellular workflow for designing and evaluating resistance at key mutational hotspots among kinome members. In this chapter, we discuss how mutation of amino acids in the gatekeeper and hinge-loop regions (collectively termed the "resistance tetrad") and the DFG motif represent an effective approach for generating panels of DR kinase alleles for chemical genetics and biological target validation.