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
Binary (nucleotide-protein dimer and hexamer complexes) and ternary (nucleotide-protein-inhibitor complexes) p97 complexes were subjected to molecular dynamics simulations in an attempt to further our understanding of the p97 protein oligomer domain stability and, more importantly, of the recently reported diverse molecular mechanisms of inhibition including allosteric, ATP-competitive and covalent inhibitors. Analysis of stable states following equilibration phases indicated a higher intrinsic stability of the homohexamer as opposed to the dimer, and of N-D1 domains as opposed to the D2 domain. The molecular dynamics of the proposed allosteric binding model reproduced important molecular interactions identified experimentally with high frequency throughout the trajectory. Observed conformational changes occurring in the D2 nucleotide binding site provided a novel bind-rearrange-react hypothesis of stepwise molecular events involved in the specific covalent inhibitor mode of action.
RESUMO
Chordomas are rare bone tumors with no approved therapy. These tumors express several activated tyrosine kinase receptors, which prompted attempts to treat patients with tyrosine kinase inhibitors. Although clinical benefit was observed in phase II clinical trials with imatinib and sorafenib, and sporadically also with EGFR inhibitors, therapies evaluated to date have shown modest activity. With the goal of identifying new drugs with immediate therapeutic potential for chordoma patients, we collected clinically approved drugs and other advanced inhibitors of MET, PDGFRß, and EGFR tyrosine kinases, and assessed their antiproliferative activity against a panel of chordoma cell lines. Chordoma cell lines were not responsive to MET and PDGFRß inhibitors. U-CH1 and UM-Chor1 were sensitive to all EGFR inhibitors, whereas the remaining cell lines were generally insensitive to these drugs. Afatinib was the only EGFR inhibitor with activity across the chordoma panel. We then investigated the molecular mechanisms behind the responses observed and found that the antiproliferative IC50s correlate with the unique ability of afatinib to promote degradation of EGFR and brachyury, an embryonic transcription factor considered a key driver of chordoma. Afatinib displayed potent antitumor efficacy in U-CH1, SF8894, CF322, and CF365 chordoma tumor models in vivo In the panel analyzed, high EGFR phosphorylation and low AXL and STK33 expression correlated with higher sensitivity to afatinib and deserve further investigation as potential biomarkers of response. These data support the use of afatinib in clinical trials and provide the rationale for the upcoming European phase II study on afatinib in advanced chordoma. Mol Cancer Ther; 17(3); 603-13. ©2017 AACR.
Assuntos
Afatinib/uso terapêutico , Neoplasias Ósseas/tratamento farmacológico , Cordoma/tratamento farmacológico , Receptores ErbB/antagonistas & inibidores , Proteínas Fetais/antagonistas & inibidores , Proteínas com Domínio T/antagonistas & inibidores , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cordoma/genética , Cordoma/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Proteínas Fetais/genética , Proteínas Fetais/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos Nus , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/genéticaRESUMO
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
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
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
Valosine containing protein (VCP), also known as p97, is a member of AAA ATPase family that is involved in several biological processes and plays a central role in the ubiquitin-mediated degradation of misfolded proteins. VCP is an ubiquitously expressed, highly abundant protein and has been found overexpressed in many tumor types, sometimes associated with poor prognosis. In this respect, VCP has recently received a great deal of attention as a potential new target for cancer therapy. In this paper, the discovery and structure-activity relationships of alkylsulfanyl-1,2,4-triazoles, a new class of potent, allosteric VCP inhibitors, are described. Medicinal chemistry manipulation of compound 1, identified via HTS, led to the discovery of potent and selective inhibitors with submicromolar activity in cells and clear mechanism of action at consistent doses. This represents a first step toward a new class of potential anticancer agents.
Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Proteínas de Ciclo Celular/antagonistas & inibidores , Triazóis/farmacologia , Adenosina Trifosfatases/química , Regulação Alostérica , Proteínas de Ciclo Celular/química , Humanos , Neoplasias/patologia , Relação Estrutura-Atividade , Triazóis/síntese química , Triazóis/química , Proteína com ValosinaRESUMO
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
Solubility data for 930 diverse compounds have been analyzed using linear Partial Least Square (PLS) and nonlinear PLS methods, Continuum Regression (CR), and Neural Networks (NN). 1D and 2D descriptors from MOE package in combination with E-state or ISIS keys have been used. The best model was obtained using linear PLS for a combination between 22 MOE descriptors and 65 ISIS keys. It has a correlation coefficient (r2) of 0.935 and a root-mean-square error (RMSE) of 0.468 log molar solubility (log S(w)). The model validated on a test set of 177 compounds not included in the training set has r2 0.911 and RMSE 0.475 log S(w). The descriptors were ranked according to their importance, and at the top of the list have been found the 22 MOE descriptors. The CR model produced results as good as PLS, and because of the way in which cross-validation has been done it is expected to be a valuable tool in prediction besides PLS model. The statistics obtained using nonlinear methods did not surpass those got with linear ones. The good statistic obtained for linear PLS and CR recommends these models to be used in prediction when it is difficult or impossible to make experimental measurements, for virtual screening, combinatorial library design, and efficient leads optimization.
RESUMO
The relationship of rotatable bond count (N(rot)) and polar surface area (PSA) with oral bioavailability in rats was examined for 434 Pharmacia compounds and compared with an earlier report from Veber et al. (J. Med. Chem. 2002, 45, 2615). N(rot) and PSA were calculated with QikProp or Cerius2. The resulting correlations depended on the calculation method and the therapeutic class within the data superset. These results underscore that such generalizations must be used with caution.
Assuntos
Disponibilidade Biológica , Preparações Farmacêuticas/química , Preparações Farmacêuticas/metabolismo , Administração Oral , Animais , Estrutura Molecular , Preparações Farmacêuticas/administração & dosagem , Ratos , Relação Estrutura-AtividadeRESUMO
A number of computational approaches are being proposed for an early optimization of ADME (absorption, distribution, metabolism and excretion) properties to increase the success rate in drug discovery. The present study describes the development of an in silico model able to estimate, from the three-dimensional structure of a molecule, the stability of a compound with respect to the human cytochrome P450 (CYP) 3A4 enzyme activity. Stability data were obtained by measuring the amount of unchanged compound remaining after a standardized incubation with human cDNA-expressed CYP3A4. The computational method transforms the three-dimensional molecular interaction fields (MIFs) generated from the molecular structure into descriptors (VolSurf and Almond procedures). The descriptors were correlated to the experimental metabolic stability classes by a partial least squares discriminant procedure. The model was trained using a set of 1800 compounds from the Pharmacia collection and was validated using two test sets: the first one including 825 compounds from the Pharmacia collection and the second one consisting of 20 known drugs. This model correctly predicted 75% of the first and 85% of the second test set and showed a precision above 86% to correctly select metabolically stable compounds. The model appears a valuable tool in the design of virtual libraries to bias the selection toward more stable compounds.