RESUMEN
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.
Asunto(s)
Resistencia a Medicamentos/fisiología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-abl/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-abl/metabolismo , Familia-src Quinasas/antagonistas & inhibidores , Familia-src Quinasas/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Benzamidas , Proteínas de Fusión bcr-abl , Humanos , Mesilato de Imatinib , Modelos Moleculares , Estructura Molecular , Mutación , Piperazinas/química , Piperazinas/metabolismo , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/metabolismo , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas c-abl/genética , Piridinas/química , Piridinas/metabolismo , Pirimidinas/química , Pirimidinas/metabolismo , Familia-src Quinasas/genéticaRESUMEN
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.
Asunto(s)
Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Inhibidores Enzimáticos/farmacología , Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Proteínas de Fusión bcr-abl/genética , Mutación/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adenosina Trifosfato/química , Aminoácidos/genética , Aminoácidos/metabolismo , Apoptosis/efectos de los fármacos , Benzamidas , Ciclo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Inhibidores Enzimáticos/química , Proteínas de Fusión bcr-abl/química , Proteínas de Fusión bcr-abl/metabolismo , Regulación Neoplásica de la Expresión Génica , Células HL-60 , Humanos , Mesilato de Imatinib , Concentración 50 Inhibidora , Células K562 , Leucemia Mielógena Crónica BCR-ABL Positiva/enzimología , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Proteínas de la Leche/metabolismo , Modelos Moleculares , Proteínas Nucleares/metabolismo , Fosforilación/efectos de los fármacos , Fosfotirosina/metabolismo , Piperazinas/química , Piperazinas/farmacología , Estructura Terciaria de Proteína , Piridonas/química , Piridonas/farmacología , Pirimidinas/química , Pirimidinas/farmacología , Factor de Transcripción STAT5 , Transactivadores/metabolismoRESUMEN
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.
Asunto(s)
Huesos/efectos de los fármacos , Huesos/enzimología , Difosfonatos/farmacología , Fenoles/farmacología , Purinas/farmacología , Familia-src Quinasas/antagonistas & inhibidores , Animales , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/enzimología , Resorción Ósea/tratamiento farmacológico , Resorción Ósea/enzimología , Difosfonatos/uso terapéutico , Diseño de Fármacos , Humanos , Modelos Moleculares , Osteoclastos/efectos de los fármacos , Osteoclastos/enzimología , Osteoporosis/tratamiento farmacológico , Osteoporosis/enzimología , Fenoles/uso terapéutico , Conformación Proteica , Purinas/uso terapéutico , Relación Estructura-Actividad , Dominios Homologos src , Familia-src Quinasas/químicaRESUMEN
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.
Asunto(s)
Adenosina Trifosfato/análogos & derivados , Diseño de Fármacos , Osteoporosis/tratamiento farmacológico , Pirimidinas/síntesis química , Familia-src Quinasas/antagonistas & inhibidores , Animales , Enfermedades Óseas/tratamiento farmacológico , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Humanos , Concentración 50 Inhibidora , Modelos Moleculares , Purinas/síntesis química , Purinas/farmacología , Pirimidinas/farmacología , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Enfermedades Óseas/tratamiento farmacológico , Purinas/síntesis química , Familia-src Quinasas/antagonistas & inhibidores , Adenosina Trifosfato/análogos & derivados , Animales , Sistemas de Liberación de Medicamentos , Diseño de Fármacos , Durapatita/metabolismo , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Humanos , Concentración 50 Inhibidora , Purinas/farmacología , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Enfermedades Óseas/tratamiento farmacológico , Resorción Ósea/prevención & control , Pirimidinonas/síntesis química , Familia-src Quinasas/antagonistas & inhibidores , Animales , Resorción Ósea/tratamiento farmacológico , Simulación por Computador , Dentina/metabolismo , Diseño de Fármacos , Durapatita/metabolismo , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Concentración 50 Inhibidora , Osteoclastos/efectos de los fármacos , Pirimidinonas/farmacología , Conejos , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Péptidos/química , Fosfotirosina/química , Proteínas Proto-Oncogénicas pp60(c-src)/antagonistas & inhibidores , Dominios Homologos src , Sitios de Unión , Materiales Biomiméticos/química , Materiales Biomiméticos/farmacología , Diseño de Fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Modelos Moleculares , Péptidos/farmacología , Relación Estructura-ActividadRESUMEN
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.