RESUMO
We previously demonstrated that natural product-inspired 3,4-dihydropyrrolo[1,2-a]pyrazin-1(2H)-ones derivatives delivered potent and selective PIM kinases inhibitors however with non-optimal ADME/PK properties and modest oral bioavailability. Herein, we describe a structure-based scaffold decoration and a stereoselective approach to this chemical class. The synthesis, structure-activity relationship studies, chiral analysis, and pharmacokinetic data of compounds from this inhibitor class are presented herein. Compound 20c demonstrated excellent potency on PIM1 and PIM2 with exquisite kinases selectivity and PK properties that efficiently and dose-dependently promoted c-Myc degradation and appear to be promising lead compounds for further development.
Assuntos
Alcaloides , Antineoplásicos , Alcaloides/farmacologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-pim-1/química , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
In this article we describe the identification of unprecedented ATP-competitive ChoKα inhibitors starting from initial hit NMS-P830 that binds to ChoKα in an ATP concentration-dependent manner. This result is confirmed by the co-crystal structure of NMS-P830 in complex with Δ75-ChoKα. NMS-P830 is able to inhibit ChoKα in cells resulting in the reduction of intracellular phosphocholine formation. A structure-based medicinal chemistry program resulted in the identification of selective compounds that have good biochemical activity, solubility and metabolic stability and are suitable for further optimization. The ChoKα inhibitors disclosed in this article demonstrate for the first time the possibility to inhibit ChoKα with ATP-competitive compounds.
Assuntos
Trifosfato de Adenosina/antagonistas & inibidores , Colina Quinase/antagonistas & inibidores , Cicloexanos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Trifosfato de Adenosina/metabolismo , Colina Quinase/metabolismo , Cicloexanos/síntese química , Cicloexanos/química , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Relação Estrutura-AtividadeRESUMO
NGR-hTNF is a therapeutic agent for a solid tumor that specifically targets angiogenic tumor blood vessels, through the NGR motif. Its activity has been assessed in several clinical studies encompassing tumors of different histological types. The drug's activity is based on an improved permeabilization of newly formed tumor vasculature, which favors intratumor penetration of chemotherapeutic agents and leukocyte trafficking. This work investigated the binding and the signaling properties of the NGR-hTNF, to elucidate its mechanism of action. The crystal structure of NGR-hTNF and modeling of its interaction with TNFR suggested that the NGR region is available for binding to a specific receptor. Using 2D TR-NOESY experiments, this study confirmed that the NGR-peptides binds to a specific CD13 isoform, whose expression is restricted to tumor vasculature cells, and to some tumor cell lines. The interaction between hTNF or NGR-hTNF with immobilized TNFRs showed similar kinetic parameters, whereas the competition experiments performed on the cells expressing both TNFR and CD13 showed that NGR-hTNF had a higher binding affinity than hTNF. The analysis of the NGR-hTNF-triggered signal transduction events showed a specific impairment in the activation of pro-survival pathways (Ras, Erk and Akt), compared to hTNF. Since a signaling pattern identical to NGR-hTNF was obtained with hTNF and NGR-sequence given as distinct molecules, the inhibition observed on the survival pathways was presumably due to a direct effect of the NGR-CD13 engagement on the TNFR signaling pathway. The reduced activation of the pro survival pathways induced by NGR-hTNF correlated with the increased caspases activation and reduced cell survival. This study demonstrates that the binding of the NGR-motif to CD13 determines not only the homing of NGR-hTNF to tumor vessels, but also the increase in its antiangiogenic activity.
Assuntos
Inibidores da Angiogênese/farmacologia , Neoplasias/irrigação sanguínea , Oligopeptídeos/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Inibidores da Angiogênese/química , Linhagem Celular Tumoral , Cristalografia por Raios X , Células Endoteliais da Veia Umbilical Humana , Humanos , Modelos Moleculares , Oligopeptídeos/química , Proteínas Recombinantes de Fusão/química , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/químicaRESUMO
VCP (also known as p97 or Cdc48p in yeast) is an AAA(+) ATPase regulating endoplasmic reticulum-associated degradation. After high-throughput screening, we developed compounds that inhibit VCP via different mechanisms, including covalent modification of an active site cysteine and a new allosteric mechanism. Using photoaffinity labeling, structural analysis and mutagenesis, we mapped the binding site of allosteric inhibitors to a region spanning the D1 and D2 domains of adjacent protomers encompassing elements important for nucleotide-state sensing and ATP hydrolysis. These compounds induced an increased affinity for nucleotides. Interference with nucleotide turnover in individual subunits and distortion of interprotomer communication cooperated to impair VCP enzymatic activity. Chemical expansion of this allosteric class identified NMS-873, the most potent and specific VCP inhibitor described to date, which activated the unfolded protein response, interfered with autophagy and induced cancer cell death. The consistent pattern of cancer cell killing by covalent and allosteric inhibitors provided critical validation of VCP as a cancer target.
Assuntos
Acetanilidas/farmacologia , Adenosina Trifosfatases/antagonistas & inibidores , Antineoplásicos/farmacologia , Benzotiazóis/farmacologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Acetanilidas/química , Adenosina Trifosfatases/metabolismo , Regulação Alostérica/efeitos dos fármacos , Antineoplásicos/química , Benzotiazóis/química , Proteínas de Ciclo Celular/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Inibidores Enzimáticos/química , Humanos , Modelos Moleculares , Estrutura Molecular , Neoplasias/metabolismo , Relação Estrutura-Atividade , Proteína com ValosinaRESUMO
Compound 1, a hit from the screening of our chemical collection displaying activity against JAK2, was deconstructed for SAR analysis into three regions, which were explored. A series of compounds was synthesized leading to the identification of the potent and orally bioavailable JAK2 inhibitor 16 (NMS-P830), which showed an encouraging tumour growth inhibition in SET-2 xenograft tumour model, with evidence for JAK2 pathway suppression demonstrated by in vivo pharmacodynamic effects.
Assuntos
Amidas/síntese química , Antineoplásicos/síntese química , Janus Quinase 2/antagonistas & inibidores , Leucemia Megacarioblástica Aguda/tratamento farmacológico , Inibidores de Proteínas Quinases/síntese química , Pirróis/síntese química , Amidas/farmacologia , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Expressão Gênica , Ensaios de Triagem em Larga Escala , Humanos , Janus Quinase 2/química , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Leucemia Megacarioblástica Aguda/enzimologia , Leucemia Megacarioblástica Aguda/genética , Leucemia Megacarioblástica Aguda/patologia , Células Progenitoras de Megacariócitos/efeitos dos fármacos , Células Progenitoras de Megacariócitos/enzimologia , Células Progenitoras de Megacariócitos/patologia , Camundongos , Camundongos Nus , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Inibidores de Proteínas Quinases/farmacologia , Pirróis/farmacologia , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We report herein the discovery, structure guided design, synthesis and biological evaluation of a novel class of JAK2 inhibitors. Optimization of the series led to the identification of the potent and orally bioavailable JAK2 inhibitor 28 (NMS-P953). Compound 28 displayed significant tumour growth inhibition in SET-2 xenograft tumour model, with a mechanism of action confirmed in vivo by typical modulation of known biomarkers, and with a favourable pharmacokinetic and safety profile.
Assuntos
Antineoplásicos/farmacologia , Janus Quinase 2/antagonistas & inibidores , Neoplasias Experimentais/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Pirróis/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Janus Quinase 2/metabolismo , Camundongos , Camundongos SCID , Modelos Moleculares , Estrutura Molecular , Neoplasias Experimentais/patologia , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Pirimidinas/síntese química , Pirimidinas/química , Pirróis/síntese química , Pirróis/química , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
A novel series of PIM inhibitors was derived from a combined effort in natural product-inspired library generation and screening. The novel pyrrolo[1,2-a]pyrazinones initial hits are inhibitors of PIM isoforms with IC50 values in the low micromolar range. The application of a rational optimization strategy, guided by the determination of the crystal structure of the complex in the kinase domain of PIM1 with compound 1, led to the discovery of compound 15a, which is a potent PIM kinases inhibitor exhibiting excellent selectivity against a large panel of kinases, representative of each family. The synthesis, structure-activity relationship studies, and pharmacokinetic data of compounds from this inhibitor class are presented herein. Furthermore, the cellular activities including inhibition of cell growth and modulation of downstream targets are also described.
Assuntos
Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Pirazinas/química , Pirazinas/farmacologia , Descoberta de Drogas , Humanos , Simulação de Acoplamento Molecular , Inibidores de Proteínas Quinases/síntese química , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-pim-1/química , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Pirazinas/síntese químicaRESUMO
The generation of novel chemotypes in support of our oncology research projects expanded in recent years from a canonical design of kinase-targeted compound libraries to a broader interpretation of purinome-targeted libraries (PTL) addressing the specificity of cancer relevant targets such as kinases and ATPases. Successful screening of structurally diverse ATP-binding targets requires compound libraries covering multiple design elements, which may include phosphate surrogate moieties in ATPase inhibitors or far reaching lipophilic residues stabilizing inactive kinase conformations. Here, we exemplify the design and preparation of drug-like combinatorial libraries and report significantly enhanced screening performance on purinomic targets. We compared overall hit rates of PTL with a simultaneously tested unbiased collection of 200,000 compounds and found consistent superiority of the targeted libraries in all cases. We also analyzed the performance of the largest targeted libraries in comparison with each other and often found striking differences in how a specific target responds to various chemotypes and to whole collections.
Assuntos
Trifosfato de Adenosina/análogos & derivados , Técnicas de Química Combinatória/métodos , Desenho de Fármacos , Bibliotecas de Moléculas Pequenas/análise , Bibliotecas de Moléculas Pequenas/farmacologia , Sítios de Ligação , Bases de Dados como Assunto , Ensaios de Triagem em Larga Escala , Modelos Moleculares , Inibidores de Proteínas Quinases/análise , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Pirazóis/química , Padrões de ReferênciaRESUMO
Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase involved in the development of several human cancers and, as a result, is a recognized target for the development of small-molecule inhibitors for the treatment of ALK-positive malignancies. Here, we present the crystal structures of the unphosphorylated human ALK kinase domain in complex with the ATP competitive ligands PHA-E429 and NVP-TAE684. Analysis of these structures provides valuable information concerning the specific characteristics of the ALK active site as well as giving indications about how to obtain selective ALK inhibitors. In addition, the ALK-KD-PHA-E429 structure led to the identification of a potential regulatory mechanism involving a link made between a short helical segment immediately following the DFG motif and an N-terminal two-stranded beta-sheet. Finally, mapping of the activating mutations associated with neuroblastoma onto our structures may explain the roles these residues have in the activation process.
Assuntos
Modelos Moleculares , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/química , Pirimidinas/química , Quinase do Linfoma Anaplásico , Animais , Linhagem Celular , Inibidores Enzimáticos/química , Humanos , Estrutura Terciária de Proteína , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Receptores Proteína Tirosina Quinases , SpodopteraRESUMO
Cdc7 is an essential kinase that promotes DNA replication by activating origins of replication. Here, we characterized the potent Cdc7 inhibitor PHA-767491 (1) in biochemical and cell-based assays, and we tested its antitumor activity in rodents. We found that the compound blocks DNA synthesis and affects the phosphorylation of the replicative DNA helicase at Cdc7-dependent phosphorylation sites. Unlike current DNA synthesis inhibitors, PHA-767491 prevents the activation of replication origins but does not impede replication fork progression, and it does not trigger a sustained DNA damage response. Treatment with PHA-767491 results in apoptotic cell death in multiple cancer cell types and tumor growth inhibition in preclinical cancer models. To our knowledge, PHA-767491 is the first molecule that directly affects the mechanisms controlling initiation as opposed to elongation in DNA replication, and its activities suggest that Cdc7 kinase inhibition could be a new strategy for the development of anticancer therapeutics.
Assuntos
Antineoplásicos/farmacologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Replicação do DNA/efeitos dos fármacos , DNA/efeitos dos fármacos , Piperidonas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirróis/farmacologia , Animais , Antineoplásicos/química , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , DNA/biossíntese , Relação Dose-Resposta a Droga , Fibroblastos/efeitos dos fármacos , Células HeLa , Humanos , Camundongos , Camundongos Nus , Camundongos SCID , Componente 2 do Complexo de Manutenção de Minicromossomo , Estrutura Molecular , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/química , Fosforilação , Piperidonas/química , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Pirróis/química , Ratos , Bibliotecas de Moléculas Pequenas , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We have recently reported CDK inhibitors based on the 6-substituted pyrrolo[3,4-c]pyrazole core structure. Improvement of inhibitory potency against multiple CDKs, antiproliferative activity against cancer cell lines and optimization of the physico-chemical properties led to the identification of highly potent compounds. Compound 31 (PHA-793887) showed good efficacy in the human ovarian A2780, colon HCT-116 and pancreatic BX-PC3 carcinoma xenograft models and was well tolerated upon daily treatments by iv administration. It was identified as a drug candidate for clinical evaluation in patients with solid tumors.
Assuntos
Antineoplásicos/química , Quinases Ciclina-Dependentes/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Pirazóis/química , Pirróis/química , Animais , Antineoplásicos/síntese química , Antineoplásicos/farmacocinética , Sítios de Ligação , Linhagem Celular Tumoral , Cristalografia por Raios X , Quinases Ciclina-Dependentes/metabolismo , Células HCT116 , Humanos , Injeções Intravenosas , Camundongos , Camundongos Nus , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacocinética , Pirazóis/síntese química , Pirazóis/farmacocinética , Pirróis/síntese química , Pirróis/farmacocinética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Poly(ADP-ribose) polymerase-1 (PARP-1) is an enzyme involved in signaling and repair of DNA single strand breaks. PARP-1 employs NAD+ to modify substrate proteins via the attachment of poly(ADP-ribose) chains. PARP-1 is a well established target in oncology, as testified by the number of marketed drugs (e.g., Lynparza, Rubraca, Zejula, and Talzenna) used for the treatment of ovarian, breast, and prostate tumors. Efforts in investigating an uncharted region of the previously identified isoindolinone carboxamide series delivered (S)-13 (NMS-P515), a potent inhibitor of PARP-1 both in biochemical (K d: 0.016 µM) and cellular (IC50: 0.027 µM) assays. Cocrystal structure allowed explaining NMS-P515 stereospecific inhibition of the target. After having ruled out potential loss of enantiopurity in vitro and in vivo, NMS-P515 was synthesized in an asymmetric fashion. NMS-P515 ADME profile and its antitumor activity in a mouse xenograft cancer model render the compound eligible for further optimization.
RESUMO
Activated ALK and ROS1 tyrosine kinases, resulting from chromosomal rearrangements, occur in a subset of non-small cell lung cancers (NSCLC) as well as other tumor types and their oncogenic relevance as actionable targets has been demonstrated by the efficacy of selective kinase inhibitors such as crizotinib, ceritinib, and alectinib. More recently, low-frequency rearrangements of TRK kinases have been described in NSCLC, colorectal carcinoma, glioblastoma, and Spitzoid melanoma. Entrectinib, whose discovery and preclinical characterization are reported herein, is a novel, potent inhibitor of ALK, ROS1, and, importantly, of TRK family kinases, which shows promise for therapy of tumors bearing oncogenic forms of these proteins. Proliferation profiling against over 200 human tumor cell lines revealed that entrectinib is exquisitely potent in vitro against lines that are dependent on the drug's pharmacologic targets. Oral administration of entrectinib to tumor-bearing mice induced regression in relevant human xenograft tumors, including the TRKA-dependent colorectal carcinoma KM12, ROS1-driven tumors, and several ALK-dependent models of different tissue origins, including a model of brain-localized lung cancer metastasis. Entrectinib is currently showing great promise in phase I/II clinical trials, including the first documented objective responses to a TRK inhibitor in colorectal carcinoma and in NSCLC. The drug is, thus, potentially suited to the therapy of several molecularly defined cancer settings, especially that of TRK-dependent tumors, for which no approved drugs are currently available. Mol Cancer Ther; 15(4); 628-39. ©2016 AACR.
Assuntos
Antineoplásicos/farmacologia , Benzamidas/farmacologia , Indazóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Quinase do Linfoma Anaplásico , Animais , Benzamidas/química , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Modelos Animais de Doenças , Ativação Enzimática/efeitos dos fármacos , Humanos , Indazóis/química , Imageamento por Ressonância Magnética , Masculino , Camundongos , Camundongos Transgênicos , Mortalidade , Inibidores de Proteínas Quinases/química , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Translocação Genética , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase responsible for the development of different tumor types. Despite the remarkable clinical activity of crizotinib (Xalkori), the first ALK inhibitor approved in 2011, the emergence of resistance mutations and of brain metastases frequently causes relapse in patients. Within our ALK drug discovery program, we identified compound 1, a novel 3-aminoindazole active on ALK in biochemical and in cellular assays. Its optimization led to compound 2 (entrectinib), a potent orally available ALK inhibitor active on ALK-dependent cell lines, efficiently penetrant the blood-brain barrier (BBB) in different animal species and highly efficacious in in vivo xenograft models. Moreover, entrectinib resulted to be strictly potent on the closely related tyrosine kinases ROS1 and TRKs recently found constitutively activated in several tumor types. Entrectinib is currently undergoing phase I/II clinical trial for the treatment of patients affected by ALK-, ROS1-, and TRK-positive tumors.
Assuntos
Antineoplásicos/farmacologia , Benzamidas/farmacologia , Descoberta de Drogas , Indazóis/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Administração Oral , Quinase do Linfoma Anaplásico , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Benzamidas/administração & dosagem , Benzamidas/química , Barreira Hematoencefálica/efeitos dos fármacos , Western Blotting , Permeabilidade da Membrana Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cristalização , Cristalografia por Raios X , Cães , Humanos , Indazóis/administração & dosagem , Indazóis/química , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , Camundongos SCID , Microssomos Hepáticos/efeitos dos fármacos , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Ratos , Ratos Wistar , Receptor trkA/antagonistas & inibidores , Receptor trkB/antagonistas & inibidores , Receptor trkC/antagonistas & inibidores , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Kynurenic acid (KA) is an endogenous glutamate receptor antagonist at the level of the different ionotropic glutamate receptors. One of the enzymes responsible for the production of KA, kynurenine aminotransferase I (KATI), also catalyses the reversible transamination of glutamine to oxoglutaramic acid (GTK, EC 2.6.1.15). The enzyme exists in a cytosolic and in a mitochondrial form because of the presence of two different KATI mRNAs coding for a protein respectively with and without leader sequence targeting the protein into mitochondria. We have cloned from a phage library of rat kidney cDNA four new KATI cDNAs containing different 5' untranslated regions (UTRs). One of the transcripts (+14KATI cDNA) contains an alternative site of initiation of translation. The tissue distribution of the different transcripts was studied by RT-PCR. The study demonstrated that several KATI mRNAs are constitutively expressed in ubiquitous manner, while +14KATI mRNA is present only in kidney. The translational efficiency of the different transcripts was studied in vitro and enzymatic activities were measured in transiently transfected Cos-1 cells. Each KATI mRNA exhibits a different in vitro translational efficiency, which corresponds to different levels of KAT enzymatic activity in transfected cells. Both findings correlate with the predicted accessibility of the ribosomal binding sites of the different mRNAs. The structure of the rat KATI/GTK gene was also studied. The expression of several KATI mRNAs with different 5'UTRs represents an interesting example of transcriptional/translational control on the expression of pyridoxal phosphate (PLP)-dependent aminotransferases.
Assuntos
Regulação da Expressão Gênica , Liases/biossíntese , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Transaminases/biossíntese , Regiões 5' não Traduzidas , Animais , Sítios de Ligação , Northern Blotting , Células COS , Clonagem Molecular , Cosmídeos , DNA/metabolismo , DNA Complementar/metabolismo , Biblioteca Gênica , Rim/metabolismo , Liases/genética , Modelos Genéticos , Hibridização de Ácido Nucleico , Isoformas de Proteínas , Coelhos , Ratos , Proteínas Recombinantes/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Distribuição Tecidual , Transaminases/genética , Transcrição Gênica , TransfecçãoRESUMO
PARP inhibitors are an exciting new class of antineoplastic drugs that have been proven to be efficacious as single agents in cancer settings with inherent DNA repair defects, as well as in combination with DNA-damaging chemotherapeutics. Currently, they are designed to target the catalytic domain of PARP-1, the most studied member of the family, with a key role in the DNA-damage repair process. Because PARP inhibitors are substrate (NAD(+)) competitors, there is a need for a deeper understanding of their cross-reactivity. This is particularly relevant for PARP-2, the PARP-1 closest homologue, for which an embryonic lethal phenotype has been observed in double knockout mice. In this study, we describe the development and validation of binding assays based on fluorescence polarization (FP) and surface plasmon resonance (SPR) techniques. PARP-1, PARP-2, PARP-3, and TNKS-1 FP displacement assays are set up by employing ad hoc synthesized probes. These assays are suitable for high-throughput screening (HTS) and selectivity profiling, thus allowing the identification of NAD(+)binding site selective inhibitors. The PARP-1 and PARP-2 complementary SPR binding assays confirm displacement data and the in-depth inhibitor characterization. Moreover, these formats have the potential to be broadly applicable to other members of the PARP family.
Assuntos
Polarização de Fluorescência/métodos , Ensaios de Triagem em Larga Escala/métodos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Ressonância de Plasmônio de Superfície/métodos , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Humanos , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/metabolismo , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reprodutibilidade dos Testes , Tanquirases/genética , Tanquirases/metabolismoRESUMO
The NTRK1 gene encodes Tropomyosin-related kinase A (TRKA), the high-affinity Nerve Growth Factor Receptor. NTRK1 was originally isolated from a colorectal carcinoma (CRC) sample as component of a somatic rearrangement (TPM3-NTRK1) resulting in expression of the oncogenic chimeric protein TPM3-TRKA, but there has been no subsequent report regarding the relevance of this oncogene in CRC. The KM12 human CRC cell line expresses the chimeric TPM3-TRKA protein and is hypersensitive to TRKA kinase inhibition. We report the detailed characterization of the TPM3-NTRK1 genomic rearrangement in KM12 cells and through a cellular screening approach, the identification of NMS-P626, a novel highly potent and selective TRKA inhibitor. NMS-P626 suppressed TPM3-TRKA phosphorylation and downstream signaling in KM12 cells and showed remarkable antitumor activity in mice bearing KM12 tumors. Finally, using quantitative reverse transcriptase PCR and immunohistochemistry (IHC) we identified the TPM3-NTRK1 rearrangement in a CRC clinical sample, therefore suggesting that this chromosomal translocation is indeed a low frequency recurring event in CRC and that such patients might benefit from therapy with TRKA kinase inhibitors.
Assuntos
Inibidores de Proteínas Quinases/farmacologia , Receptor trkA/antagonistas & inibidores , Receptor trkA/metabolismo , Tropomiosina/metabolismo , Animais , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Imunoprecipitação , Técnicas In Vitro , Camundongos , Ligação Proteica/efeitos dos fármacosRESUMO
Valosine-containing protein (VCP), also known as p97 or cdc48 in yeast, is a highly abundant protein belonging to the AAA ATPase family involved in a number of essential cellular functions, including ubiquitin-proteasome mediated protein degradation, Golgi reassembly, transcription activation, and cell cycle control. Altered expression of VCP has been detected in many cancer types sometimes associated with poor prognosis. Furthermore, VCP mutations are causative of some neurodegenerative disorders. In this paper we report the discovery, synthesis, and structure-activity relationships of substituted 2-aminopyrimidines, representing a new class of reversible VCP inhibitors. This class of compounds, identified in a HTS campaign against recombinant VCP, has been progressively expanded and manipulated to increase biochemical potency and gain cellular activity.
Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Proteínas de Ciclo Celular/antagonistas & inibidores , Proliferação de Células/efeitos dos fármacos , Descoberta de Drogas , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Pirimidinas/química , Pirimidinas/farmacologia , Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Células HCT116 , Humanos , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Proteína com ValosinaRESUMO
The activity of the cyclin-dependent kinaseâ 9 (CDK9) is critical for HIV-1 Tat-mediated transcription and represents a promising target for antiviral therapy. Here we present computational studies that, along with preliminary synthetic efforts, allowed us to identify and characterize a new class of nontoxic anti-CDK9 chemotypes based on the 2-phenylquinazolinone scaffold. Inhibition of CDK9 translated into the ability to interfere selectively with Tat-mediated transactivation of the viral promoter and in the inhibition of HIV-1 reactivation from latently infected cells, with the most potent derivative 37 (2-(4-aminophenyl)-7-chloroquinazolin-4(3H)-one) showing an IC50 value of 4.0â µM. Because the herein reported 2-phenylquinazolinones are merely fragments, they are largely optimizable, paving the way to derivatives with improved potency.