RESUMO
Ponatinib (AP24534) was previously identified as a pan-BCR-ABL inhibitor that potently inhibits the T315I gatekeeper mutant, and has advanced into clinical development for the treatment of refractory or resistant CML. In this study, we explored a novel series of five and six membered monocycles as alternate hinge-binding templates to replace the 6,5-fused imidazopyridazine core of ponatinib. Like ponatinib, these monocycles are tethered to pendant toluanilides via an ethynyl linker. Several compounds in this series displayed excellent in vitro potency against both native BCR-ABL and the T315I mutant. Notably, a subset of inhibitors exhibited desirable PK and were orally active in a mouse model of T315I-driven CML.
Assuntos
Alcinos/síntese química , Alcinos/farmacologia , Compostos de Anilina/síntese química , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Tolueno/síntese química , Administração Oral , Alcinos/química , Compostos de Anilina/química , Compostos de Anilina/farmacologia , Animais , Ciclização , Modelos Animais de Doenças , Proteínas de Fusão bcr-abl/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Camundongos , Modelos Moleculares , Estrutura Molecular , Mutação , Ratos , Relação Estrutura-Atividade , Tolueno/química , Tolueno/farmacologiaRESUMO
Novel N(9)-arenethenyl purines, optimized potent dual Src/Abl tyrosine kinase inhibitors, are described. The key structural feature is a trans vinyl linkage at N(9) on the purine core which projects hydrophobic substituents into the selectivity pocket at the rear of the ATP site. Their synthesis was achieved through a Horner-Wadsworth-Emmons reaction of N(9)-phosphorylmethylpurines and substituted benzaldehydes or Heck reactions between 9-vinyl purines and aryl halides. Most compounds are potent inhibitors of both Src and Abl kinase, and several possess good oral bioavailability.
Assuntos
Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Purinas/química , Purinas/farmacologia , Quinases da Família src/antagonistas & inibidores , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Inibidores do Crescimento/química , Inibidores do Crescimento/farmacologia , Humanos , Células K562 , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas c-abl/fisiologia , RatosRESUMO
Src tyrosine kinase was the first gene product shown to have an essential function in bone using recombinant DNA technology after its expression was knocked out in mice approximately 15 years ago. Since then, our understanding of the regulation of bone catabolism has advanced significantly with the identification of other key enzymes that regulate osteoclast formation, activation, and survival after their knockout in mice or recognition of mutations in them in humans. This led to the discovery or development of specific inhibitors of some of these key enzymes, including Src, as proof-of-concept lead compounds or potential clinical candidates for the prevention of diseases associated with increased bone resorption, such as osteoporosis and metastatic bone disease. Although bisphosphonates have been prescribed with proven and improving efficacy for the prevention of bone loss for >30 years, adverse effects, such as upper gastrointestinal tract symptoms, and the requirement to take them at least 2 hours before food have limited patient compliance. Thus, with growing knowledge of the pathways regulating osteoclast function and the appreciation that some of these are active also in tumor cells, drug companies have made efforts to identify small-molecular lead compounds for development into new therapeutic agents for the prevention of bone loss with efficacy that matches or supersedes that of bisphosphonates. In this article, we review our current understanding of the signaling pathways that regulate osteoclast formation, activation, and survival with specific reference to the role of Src tyrosine kinase and downstream signaling and highlight in a variety of models of increased bone resorption the effects of Src kinase inhibitors that have been targeted to bone to limit potential adverse effects on other cells.
Assuntos
Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/secundário , Reabsorção Óssea/prevenção & controle , Osteoclastos/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Quinases da Família src/metabolismo , Animais , Diferenciação Celular , Humanos , Camundongos , Osteoclastos/citologia , Osteoclastos/enzimologia , Inibidores de Proteínas Quinases/química , Transdução de Sinais/fisiologia , Relação Estrutura-Atividade , Quinases da Família src/efeitos dos fármacosRESUMO
Understanding of the regulation of bone catabolism has advanced significantly over the past two decades with the identification of key enzymes that regulate osteoclast formation, activation, and survival following their knockout in mice or recognition of mutations in humans. This led to the discovery of specific inhibitors of some of these key enzymes as proof-of-concept lead compounds or potential clinical candidates for the prevention of osteoporosis and other diseases associated with increased bone resorption. Bisphosphonates have been the major therapeutic agents prescribed for the prevention of bone loss in a variety of pathologic conditions for over 30 years. More potent amino bisphosphonates have increased efficacy than earlier drugs, but side effects such as upper gastrointestinal symptoms and the requirement to take them at least 2 h before food have limited patient compliance. This, coupled with the growing knowledge of the pathways regulating osteoclast function, has driven efforts to identify small molecular lead compounds that could be developed into new therapeutic agents with efficacy that matches or supersedes that of bisphosphonates for the prevention of bone loss. In this article, we review briefly the effects of specific inhibitors of bone resorption that have been developed to date and highlight in a variety of models of increased bone resorption the effects of Src kinase inhibitors that have been targeted to bone to limit potential unwanted side effects on other cells.
Assuntos
Doenças Ósseas/tratamento farmacológico , Osso e Ossos/metabolismo , Inibidores Enzimáticos/uso terapêutico , Reabsorção Óssea , Catepsina K , Catepsinas/antagonistas & inibidores , Humanos , Osteoclastos/fisiologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Sirolimo/antagonistas & inibidores , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidoresRESUMO
c-Src is frequently activated in human malignancies, including colon, breast, and pancreatic carcinomas. Several recent studies have shown that activation of Src family kinases leads to tumor progression and metastasis by increasing cellular migration and invasion, promoting cell growth and survival, and deregulating expression of proangiogenic molecules. Therefore, selective inhibitors of Src are being developed for cancer therapy. In this study, we characterize the biological effects of the novel ATP-based Src family kinase inhibitor, AP23846, in tumor cells with high Src activity. As a lead compound, AP23846 is a potent c-Src kinase inhibitor (IC50 approximately 0.5 nmol/L in vitro, approximately 10-fold more potent than PP2, the most widely used commercially available Src family kinase inhibitor). At concentrations of 1 micromol/L, AP23846 led to complete Src inhibition for 48 hours in cells. No cytotoxicity was observed under these conditions, although proliferation rates were slower. Therefore, this was an excellent inhibitor to examine Src-regulated signaling pathways in tumor cells. AP23846 reduced cellular migration, vascular endothelial growth factor, and interleukin-8 in a dose-dependent fashion in pancreatic adenocarcinoma cells grown in vitro. Correspondingly, cell culture supernatants from L3.6pl pancreatic adenocarcinoma cells pretreated with AP23846 failed to promote migration of hepatic endothelial cells in vitro and failed to support angiogenesis into gel foams implanted s.c. in mice in vivo. These results suggest that Src inhibitors affect biological properties of tumor progression and may be useful as cancer therapeutic agents in more advanced disease.
Assuntos
Trifosfato de Adenosina/análogos & derivados , Inibidores Enzimáticos/farmacologia , Interleucina-8/metabolismo , Neoplasias/metabolismo , Neovascularização Patológica/prevenção & controle , Fator A de Crescimento do Endotélio Vascular/metabolismo , Quinases da Família src/antagonistas & inibidores , Trifosfato de Adenosina/farmacologia , Animais , Sequência de Bases , Western Blotting , Linhagem Celular Tumoral , Movimento Celular , Primers do DNA , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoprecipitação , Camundongos , Camundongos Endogâmicos C3H , Neoplasias/irrigação sanguínea , Fosforilação , RNA Interferente PequenoRESUMO
Bone-targeted Src tyrosine kinase (STK) inhibitors have recently been developed for the treatment of osteoporosis and cancer-related bone diseases. The concept of bone targeting derives from bisphosphonates, and from the evolution of such molecules in terms of therapeutic efficacy for the treatment of bone disorders. Interestingly, some of the earliest bisphosphonates were recognized for their ability to inhibit calcium carbonate precipitation (scaling) by virtue of their affinity to chelate calcium. This chelating property was subsequently exploited in the development of bisphosphonate analogs as inhibitors of the bone-resorbing cells known as osteoclasts, giving rise to breakthrough medicines, such as Fosamax (for the treatment of osteoporosis) and Zometa (for the treatment of osteoporosis and bone metastases). Relative to these milestone achievements, there is a tremendous opportunity to explore beyond the limited chemical space (functional group diversity) of such bisphosphonates to design novel bone-targeting moieties, which may be used to develop other classes of promising small-molecule drugs affecting different biological pathways. Here, we review studies focused on bone-targeted inhibitors of STK, a key enzyme in osteoclast-dependent bone resorption. Two strategies are described relative to bone-targeted STK inhibitor drug discovery: (i) the development of novel Src homology (SH)-2 inhibitors incorporating non-hydrolyzable phosphotyrosine mimics and exhibiting molecular recognition and bone-targeting properties, leading to the in vivo-effective lead compound AP-22408; and (ii) the development of novel ATP-based Src kinase inhibitors incorporating bone-targeting moieties, leading to the in vivo-effective lead compound AP-23236. In summary, AP-22408 and AP-23236, which differ mechanistically by virtue of blocking Src-dependent non-catalytic or catalytic activities in osteoclasts, exemplify ARIAD Pharmaceuticals' structure-based design of novel bone-targeted lead compounds, successfully achieving in vivo proof-of-concept and providing the framework for the next-generation molecules that have further advanced, in terms of preclinical studies, for the treatment of osteoporosis and related bone diseases, including osteolytic bone metastases.
Assuntos
Osso e Ossos/efeitos dos fármacos , Osso e Ossos/enzimologia , Difosfonatos/farmacologia , Fenóis/farmacologia , Purinas/farmacologia , Quinases da Família src/antagonistas & inibidores , Animais , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/enzimologia , Reabsorção Óssea/tratamento farmacológico , Reabsorção Óssea/enzimologia , Difosfonatos/uso terapêutico , Desenho de Fármacos , Humanos , Modelos Moleculares , Osteoclastos/efeitos dos fármacos , Osteoclastos/enzimologia , Osteoporose/tratamento farmacológico , Osteoporose/enzimologia , Fenóis/uso terapêutico , Conformação Proteica , Purinas/uso terapêutico , Relação Estrutura-Atividade , Domínios de Homologia de src , Quinases da Família src/químicaRESUMO
The structural and functional characterization of Src homology-2 (SH2) domains and their relationship to catalytic proteins (e.g., kinases, phosphatases, and lipases) or non-catalytic proteins (e.g., upstream adapters, and downstream transcription factors) has significantly impacted our understanding of signal transduction pathways and the identification of promising therapeutic targets for drug discovery. Such SH2-containing proteins are known to be intimately involved in the regulation of a number of cellular processes, including growth, mitogenesis, motility, metabolism, and gene transcription. Molecular recognition and biochemical selectivity exists for various SH2 domains based on their binding to phosphotyrosine (pTyr) and contiguous C-terminal amino acids of cognate protein 'partners' in a sequence-dependent manner (i.e., -pTyr-AA(1)-AA(2)-AA(3)-) which result in the formation of signal transduction protein complexes in cells. In recent years, drug discovery efforts have advanced peptidomimetic and nonpeptide inhibitors of such protein-protein interactions based on mimicking pTyr-containing peptide ligands as well as SH2 structure-based de novo design of nonpeptide templates that can capture key binding sites on the target protein. Noteworthy are peptidomimetic and nonpeptide inhibitors of Src, Lck, Grb2, PI-3K, and Zap70 from pioneering efforts that led to the first examples of cellularly and in vivo active SH2 inhibitors. This mini-review highlights key achievements in SH2 inhibitor drug discovery with an emphasis on peptidomimetic and nonpeptide lead compounds in terms of structure-based design, key chemical and biological properties, and proof-of-concept studies relative to further defining the role(s) of SH2 domains in signal transduction processes, cellular functions, and in vivo disease models.
Assuntos
Mimetismo Molecular , Peptídeos/farmacologia , Domínios de Homologia de src/efeitos dos fármacos , Conformação Molecular , Peptídeos/química , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
In the treatment of chronic myeloid leukemia (CML) with BCR-ABL kinase inhibitors, the T315I gatekeeper mutant has emerged as resistant to all currently approved agents. This report describes the structure-guided design of a novel series of potent pan-inhibitors of BCR-ABL, including the T315I mutation. A key structural feature is the carbon-carbon triple bond linker which skirts the increased bulk of Ile315 side chain. Extensive SAR studies led to the discovery of development candidate 20g (AP24534), which inhibited the kinase activity of both native BCR-ABL and the T315I mutant with low nM IC(50)s, and potently inhibited proliferation of corresponding Ba/F3-derived cell lines. Daily oral administration of 20g significantly prolonged survival of mice injected intravenously with BCR-ABL(T315I) expressing Ba/F3 cells. These data, coupled with a favorable ADME profile, support the potential of 20g to be an effective treatment for CML, including patients refractory to all currently approved therapies.
Assuntos
Antineoplásicos/síntese química , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Imidazóis/síntese química , Inibidores de Proteínas Quinases/síntese química , Piridazinas/síntese química , Administração Oral , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Barreira Hematoencefálica/metabolismo , Linhagem Celular Tumoral , Cristalografia por Raios X , Ensaios de Seleção de Medicamentos Antitumorais , Proteínas de Fusão bcr-abl/genética , Imidazóis/farmacocinética , Imidazóis/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/mortalidade , Camundongos , Camundongos SCID , Modelos Moleculares , Mutação , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/farmacologia , Piridazinas/farmacocinética , Piridazinas/farmacologia , RatosRESUMO
Inhibition of BCR-ABL by imatinib induces durable responses in many patients with chronic myeloid leukemia (CML), but resistance attributable to kinase domain mutations can lead to relapse and a switch to second-line therapy with nilotinib or dasatinib. Despite three approved therapeutic options, the cross-resistant BCR-ABL(T315I) mutation and compound mutants selected on sequential inhibitor therapy remain major clinical challenges. We report design and preclinical evaluation of AP24534, a potent, orally available multitargeted kinase inhibitor active against T315I and other BCR-ABL mutants. AP24534 inhibited all tested BCR-ABL mutants in cellular and biochemical assays, suppressed BCR-ABL(T315I)-driven tumor growth in mice, and completely abrogated resistance in cell-based mutagenesis screens. Our work supports clinical evaluation of AP24534 as a pan-BCR-ABL inhibitor for treatment of CML.
Assuntos
Proteínas de Fusão bcr-abl/antagonistas & inibidores , Imidazóis/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Piridazinas/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cristalografia por Raios X , Proteínas de Fusão bcr-abl/química , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Imidazóis/química , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Camundongos , Camundongos SCID , Modelos Moleculares , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-abl/química , Proteínas Proto-Oncogênicas c-abl/genética , Proteínas Proto-Oncogênicas c-abl/metabolismo , Piridazinas/química , Transdução de Sinais/efeitos dos fármacosRESUMO
A novel series of potent dual Src/Abl kinase inhibitors based on a 9-(arenethenyl)purine core has been identified. Unlike traditional dual Src/Abl inhibitors targeting the active enzyme conformation, these inhibitors bind to the inactive, DFG-out conformation of both kinases. Extensive SAR studies led to the discovery of potent and orally bioavailable inhibitors, some of which demonstrated in vivo efficacy. Once-daily oral administration of inhibitor 9i (AP24226) significantly prolonged the survival of mice injected intravenously with wild type Bcr-Abl expressing Ba/F3 cells at a dose of 10 mg/kg. In a separate model, oral administration of 9i to mice bearing subcutaneous xenografts of Src Y527F expressing NIH 3T3 cells elicited dose-dependent tumor shrinkage with complete tumor regression observed at the highest dose. Notably, several inhibitors (e.g., 14a, AP24163) exhibited modest cellular potency (IC50 = 300-400 nM) against the Bcr-Abl mutant T315I, a variant resistant to all currently marketed therapies for chronic myeloid leukemia.
Assuntos
Desenho de Fármacos , Inibidores de Proteínas Quinases/síntese química , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Purinas/síntese química , Quinases da Família src/antagonistas & inibidores , Animais , Feminino , Humanos , Células K562 , Camundongos , Células NIH 3T3 , Conformação Proteica , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-abl/química , Purinas/farmacologia , Ratos , Relação Estrutura-Atividade , Quinases da Família src/químicaRESUMO
Targeted disruption of the pp60(src) (Src) gene has implicated this tyrosine kinase in osteoclast-mediated bone resorption and as a therapeutic target for the treatment of osteoporosis and other bone-related diseases. Here, we describe structure activity relationships of a novel series of carbon-linked, 2-substituted purines that led to the identification of AP23451 as a potent inhibitor of Src tyrosine kinase with antiresorptive activity in vivo. AP23451 features the use of an arylphosphinylmethylphosphinic acid moiety which confers bone-targeting properties to the molecule, thereby increasing local concentrations of the inhibitor to actively resorbing osteoclasts at the bone interface. AP23451 exhibited an IC50 = 68 nm against Src kinase; an X-ray crystal structure of the molecule complexed with Src detailed the molecular interactions responsible for its Src inhibition. In vivo, AP23451 demonstrated a dose-dependent decrease in PTH-induced hypercalcemia. Moreover, AP23517, a structurally and biochemically similar molecule with comparable activity (IC50 = 73 nm) except devoid of the bone-targeting element, demonstrated significantly reduced in vivo efficacy, suggesting that Src activity was necessary but not sufficient for in vivo activity in this series of compounds.
Assuntos
Adenina/análogos & derivados , Reabsorção Óssea/tratamento farmacológico , Organofosfonatos/farmacologia , Purinas/farmacologia , Quinases da Família src/antagonistas & inibidores , Adenina/química , Adenina/farmacologia , Cristalografia por Raios X , Sistemas de Liberação de Medicamentos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Hipercalcemia , Concentração Inibidora 50 , Estrutura Molecular , Organofosfonatos/química , Osteoporose/tratamento farmacológico , Hormônio Paratireóideo/farmacologia , Ácidos Fosfínicos , Purinas/síntese química , Relação Estrutura-Atividade , Quinases da Família src/químicaRESUMO
Mutation in the ABL kinase domain is the principal mechanism of imatinib resistance in patients with chronic myelogenous leukemia. Many mutations favor active kinase conformations that preclude imatinib binding. Because the active forms of ABL and SRC resemble one another, we tested two dual SRC-ABL kinase inhibitors, AP23464 and PD166326, against 58 imatinib-resistant (IM(R)) BCR/ABL kinase variants. Both compounds potently inhibit most IM(R) variants, and in vitro drug selection demonstrates that active (AP23464) and open (PD166326) conformation-specific compounds are less susceptible to resistance than imatinib. Combinations of inhibitors suppressed essentially all resistance mutations, with the notable exception of T315I. Guided by mutagenesis studies and molecular modeling, we designed a series of AP23464 analogues to target T315I. The analogue AP23846 inhibited both native and T315I variants of BCR/ABL with submicromolar potency but showed nonspecific cellular toxicity. Our data illustrate how conformational dynamics of the ABL kinase accounts for the activity of dual SRC-ABL inhibitors against IM(R)-mutants and provides a rationale for combining conformation specific inhibitors to suppress resistance.
Assuntos
Resistência a Medicamentos/fisiologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-abl/metabolismo , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Benzamidas , Proteínas de Fusão bcr-abl , Humanos , Mesilato de Imatinib , Modelos Moleculares , Estrutura Molecular , Mutação , Piperazinas/química , Piperazinas/metabolismo , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Estrutura Terciária de Proteína , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas c-abl/genética , Piridinas/química , Piridinas/metabolismo , Pirimidinas/química , Pirimidinas/metabolismo , Quinases da Família src/genéticaRESUMO
The tyrosine kinase pp60src (Src) is the prototypical member of a family of proteins that participate in a broad array of cellular signal transduction processes, including cell growth, differentiation, survival, adhesion, and migration. Abnormal Src family kinase (SFK) signaling has been linked to several disease states, including osteoporosis and cancer metastases. Src has thus emerged as a molecular target for the discovery of small-molecule inhibitors that regulate Src kinase activity by binding to the ATP pocket within the catalytic domain. Here, we present crystal structures of the kinase domain of Src in complex with two purine-based inhibitors: AP23451, a small-molecule inhibitor designed to inhibit Src-dependent bone resorption, and AP23464, a small-molecule inhibitor designed to inhibit the Src-dependent metastatic spread of cancer. In each case, a trisubstituted purine template core was elaborated using structure-based drug design to yield a potent Src kinase inhibitor. These structures represent early examples of high affinity purine-based Src family kinase-inhibitor complexes, and they provide a detailed view of the specific protein-ligand interactions that lead to potent inhibition of Src. In particular, the 3-hydroxyphenethyl N9 substituent of AP23464 forms unique interactions with the protein that are critical to the picomolar affinity of this compound for Src. The comparison of these new structures with two relevant kinase-inhibitor complexes provides a structural basis for the observed kinase inhibitory selectivity. Further comparisons reveal a concerted induced-fit movement between the N- and C-terminal lobes of the kinase that correlates with the affinity of the ligand. Binding of the most potent inhibitor, AP23464, results in the largest induced-fit movement, which can be directly linked to interactions of the hydrophenethyl N9 substituent with a region at the interface between the two lobes. A less pronounced induced-fit movement is also observed in the Src-AP23451 complex. These new structures illustrate how the combination of structural, computational, and medicinal chemistry can be used to rationalize the process of developing high affinity, selective tyrosine kinase inhibitors as potential therapeutic agents.
Assuntos
Adenina/análogos & derivados , Desenho de Fármacos , Inibidores Enzimáticos/química , Organofosfonatos/química , Purinas/química , Relação Estrutura-Atividade , Quinases da Família src/química , Adenina/química , Adenina/metabolismo , Adenina/farmacologia , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Osso e Ossos/metabolismo , Carbono/química , Domínio Catalítico/efeitos dos fármacos , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Quinase 2 Dependente de Ciclina/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Ligantes , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/antagonistas & inibidores , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/química , Modelos Moleculares , Neoplasias/tratamento farmacológico , Nitrogênio/química , Organofosfonatos/metabolismo , Organofosfonatos/farmacologia , Conformação Proteica/efeitos dos fármacos , Purinas/síntese química , Purinas/farmacologia , Pirimidinas/química , Especificidade por Substrato , Quinases da Família src/antagonistas & inibidoresRESUMO
Oncogenic mutations of the Kit receptor tyrosine kinase occur in several types of malignancy. Juxtamembrane domain mutations are common in gastrointestinal stromal tumors, whereas mutations in the kinase activation loop, most commonly D816V, are seen in systemic mastocytosis and acute myelogenous leukemia. Kit activation-loop mutants are insensitive to imatinib mesylate and have been largely resistant to targeted inhibition. We determined the sensitivities of both Kit mutant classes to the adenosine triphosphate (ATP)-based inhibitors AP23464 and AP23848. In cell lines expressing activation-loop mutants, low-nM concentrations of AP23464 inhibited phosphorylation of Kit and its downstream targets Akt and signal transducer and activator of transcription 3 (STAT3). This was associated with cell-cycle arrest and apoptosis. Wild-type Kit-and juxtamembrane-mutant-expressing cell lines required considerably higher concentrations for equivalent inhibition, suggesting a therapeutic window in which cells harboring D816V Kit could be eliminated without interfering with normal cellular function. Additionally, AP23464 did not disrupt normal hematopoietic progenitor-cell growth at concentrations that inhibited activation-loop mutants of Kit. In a murine model, AP23848 inhibited activation-loop mutant Kit phosphorylation and tumor growth. Thus, AP23464 and AP23848 potently and selectively target activation-loop mutants of Kit in vitro and in vivo and could have therapeutic potential against D816V-expressing malignancies.
Assuntos
Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Linfócitos B/citologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/imunologia , Linhagem Celular Tumoral , Regulação Leucêmica da Expressão Gênica , Humanos , Técnicas In Vitro , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Camundongos , Mutagênese , Fosforilação/efeitos dos fármacos , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-kit/química , Transdução de Sinais/efeitos dos fármacosRESUMO
A series of novel nonpeptide inhibitors of the pp60(c-Src) (Src) SH2 domain is described that exploit multifunctional group replacement of the phenylphosphate moiety of phosphotyrosine (pTyr). Relative to an x-ray structure of citrate complexed to the pTyr binding site of the Src SH2 domain, these nonpeptide ligands illustrate the systematic replacement of the phosphate group by multiple nonhydrolyzable, mono- or dianionic functionalities. Specifically, several phenylalanine (Phe) analogs incorporating key 4' and 3' substituents were synthesized and incorporated into a bicyclic benzamide template previously reported (W. C. Shakespeare et al., Proceedings of the National Academy of Science USA, 2000, Vol. 97, pp. 9373-9378). These pTyr mimetics included 4',3'-diphosphono-Phe (Dpp), 4',3'-dicarboxymethyloxy-Phe (Dcp), and 4'-phosphono-3'-carboxymethyloxy-Phe (Cpp). Noteworthy were nonpeptide inhibitors 8-11 that were 5- to 10-fold more potent than the cognate tetrapeptide ligand Ac-pTyr-Glu-Glu-Ile-NH(2) in binding to the Src SH2 domain.
Assuntos
Peptídeos/química , Fosfotirosina/química , Proteínas Proto-Oncogênicas pp60(c-src)/antagonistas & inibidores , Domínios de Homologia de src , Sítios de Ligação , Materiais Biomiméticos/química , Materiais Biomiméticos/farmacologia , Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Modelos Moleculares , Peptídeos/farmacologia , Relação Estrutura-AtividadeRESUMO
Src tyrosine kinase is a therapeutic target for bone diseases that has been validated by gene knockout studies. Furthermore, in vitro cellular studies implicate that Src has a positive regulatory role in osteoclasts and a negative regulatory role in osteoblasts. The potential use of Src inhibitors for osteoporosis therapy has been previously shown by novel bone-targeted ligands of the Src SH2 (e.g., AP22408) and non-bone-targeted, ATP-based inhibitors of Src kinase. Significant to this study, compounds 2-12 exemplify novel analogues of known pyrrolopyrimidine and pyrazolopyrimidine template-based Src kinase inhibitors that incorporate bone-targeting group modifications designed to provide tissue (bone) selectivity and diminished side effects. Accordingly, we report here the structure-based design, synthetic chemistry and biological testing of these compounds and proof-of-concept studies thereof.
Assuntos
Trifosfato de Adenosina/análogos & derivados , Desenho de Fármacos , Osteoporose/tratamento farmacológico , Pirimidinas/síntese química , Quinases da Família src/antagonistas & inibidores , Animais , Doenças Ósseas/tratamento farmacológico , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Humanos , Concentração Inibidora 50 , Modelos Moleculares , Purinas/síntese química , Purinas/farmacologia , Pirimidinas/farmacologia , Relação Estrutura-AtividadeRESUMO
Novel bone-targeted 2,6,9-trisubstituted purine template-based inhibitors of Src tyrosine kinase are described. Drug design studies of known purine compounds revealed that both positions-2 and -6 were suitable for incorporating bone-seeking moieties. A variety of bone-targeting groups with different affinity to hydroxyapatite were utilized in the study. Compound 3d was determined to be a potent Src inhibitor and was quite selective against a panel of other protein kinases.
Assuntos
Doenças Ósseas/tratamento farmacológico , Purinas/síntese química , Quinases da Família src/antagonistas & inibidores , Trifosfato de Adenosina/análogos & derivados , Animais , Sistemas de Liberação de Medicamentos , Desenho de Fármacos , Durapatita/metabolismo , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Humanos , Concentração Inibidora 50 , Purinas/farmacologia , Relação Estrutura-AtividadeRESUMO
The deregulated, oncogenic tyrosine kinase Bcr-Abl causes chronic myeloid leukemia (CML). Imatinib mesylate (Gleevec, STI571), a Bcr-Abl kinase inhibitor, selectively inhibits proliferation and promotes apoptosis of CML cells. Despite the success of imatinib mesylate in the treatment of CML, resistance is observed, particularly in advanced disease. The most common imatinib mesylate resistance mechanism involves Bcr-Abl kinase domain mutations that impart varying degrees of drug insensitivity. AP23464, a potent adenosine 5'-triphosphate (ATP)-based inhibitor of Src and Abl kinases, displays antiproliferative activity against a human CML cell line and Bcr-Abl-transduced Ba/F3 cells (IC(50) = 14 nM; imatinib mesylate IC(50) = 350 nM). AP23464 ablates Bcr-Abl tyrosine phosphorylation, blocks cell cycle progression, and promotes apoptosis of Bcr-Abl-expressing cells. Biochemical assays with purified glutathione S transferase (GST)-Abl kinase domain confirmed that AP23464 directly inhibits Abl activity. Importantly, the low nanomolar cellular and biochemical inhibitory properties of AP23464 extend to frequently observed imatinib mesylate-resistant Bcr-Abl mutants, including nucleotide binding P-loop mutants Q252H, Y253F, E255K, C-terminal loop mutant M351T, and activation loop mutant H396P. AP23464 was ineffective against mutant T315I, an imatinib mesylate contact residue. The potency of AP23464 against imatinib mesylate-refractory Bcr-Abl and its distinct binding mode relative to imatinib mesylate warrant further investigation of AP23464 for the treatment of CML.
Assuntos
Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Proteínas de Fusão bcr-abl/genética , Mutação/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Trifosfato de Adenosina/química , Aminoácidos/genética , Aminoácidos/metabolismo , Apoptose/efeitos dos fármacos , Benzamidas , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Inibidores Enzimáticos/química , Proteínas de Fusão bcr-abl/química , Proteínas de Fusão bcr-abl/metabolismo , Regulação Neoplásica da Expressão Gênica , Células HL-60 , Humanos , Mesilato de Imatinib , Concentração Inibidora 50 , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Proteínas do Leite/metabolismo , Modelos Moleculares , Proteínas Nucleares/metabolismo , Fosforilação/efeitos dos fármacos , Fosfotirosina/metabolismo , Piperazinas/química , Piperazinas/farmacologia , Estrutura Terciária de Proteína , Piridonas/química , Piridonas/farmacologia , Pirimidinas/química , Pirimidinas/farmacologia , Fator de Transcrição STAT5 , Transativadores/metabolismoRESUMO
The design of bone-targeted pyrido[2,3-d]pyrimidin-7-ones as Src tyrosine kinase inhibitors is described. Leveraging SAR from known compounds and using structure-based methods, we were able to rapidly incorporate bone binding components, which maintained, and even increased potency against the target enzyme. Compound 4 displayed a high affinity for hydroxyapatite, a major constituent of bone, and demonstrated antiresoprtive activity in our cell-based assay.