RESUMO
Spencer-Smith et al. (2022)1 investigate multiple functions of the BRAF cysteine-rich domain (CRD), finding distinct classes of RASopathy-associated BRAF mutations and unique features among RAF paralogs that may contribute to the spectrum of mutations observed in disease.
Assuntos
Proteínas Proto-Oncogênicas B-raf , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Mutação , Domínios ProteicosRESUMO
Kinases are highly regulated enzymes with diverse mechanisms controlling their catalytic output. Over time, chemical discovery efforts for kinases have produced ATP-competitive compounds, allosteric regulators, irreversible binders, and highly specific inhibitors. These distinct classes of small molecules have revealed many novel aspects about kinase-mediated signaling, and some have progressed from simple tool compounds into clinically validated therapeutics. This review explores several small-molecule inhibitors for kinases highlighting elaborate mechanisms by which kinase function is modulated. A complete surprise of targeted kinase drug discovery has been the finding of ATP-competitive inhibitors that behave as agonists, rather than antagonists, of their direct kinase target. These studies hint at a connection between ATP-binding site occupancy and networks of communication that are independent of kinase catalysis. Indeed, kinase inhibitors that induce changes in protein localization, protein-protein interactions, and even enhancement of catalytic activity of the target kinase have been found. The relevance of these findings to the therapeutic efficacy of kinase inhibitors and to the future identification of new classes of drug targets is discussed.
Assuntos
Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Biologia Computacional , Descoberta de Drogas , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Mutação , Neoplasias/enzimologia , Neoplasias/genética , Conformação Proteica , Proteínas Quinases/genética , Alinhamento de SequênciaRESUMO
The RAS-mitogen-activated protein kinase (MAPK) pathway includes KSR, RAF, MEK and the phospho-regulatory sensor 14-3-3. Specific assemblies among these components drive various diseases and likely dictate efficacy for numerous targeted therapies, including allosteric MEK inhibitors (MEKi). However, directly measuring drug interactions on physiological RAS-MAPK complexes in live cells has been inherently challenging to query and therefore remains poorly understood. Here we present a series of NanoBRET-based assays to quantify direct target engagement of MEKi on MEK1 and higher-order MEK1-bound complexes with ARAF, BRAF, CRAF, KSR1 and KSR2 in the presence and absence of 14-3-3 in living cells. We find distinct MEKi preferences among these complexes that can be compiled to generate inhibitor binding profiles. Further, these assays can report on the influence of the pathogenic BRAF-V600E mutant on MEKi binding. Taken together, these approaches can be used as a platform to screen for compounds intended to target specific complexes in the RAS-MAPK cascade.
Assuntos
Bioensaio , Proteínas Proto-Oncogênicas B-raf , Proteínas Proto-Oncogênicas B-raf/genética , Sistema de Sinalização das MAP Quinases , Inibidores de Proteínas Quinases/farmacologiaRESUMO
The MAPK/ERK kinase MEK is a shared effector of the frequent cancer drivers KRAS and BRAF that has long been pursued as a drug target in oncology1, and more recently in immunotherapy2,3 and ageing4. However, many MEK inhibitors are limited owing to on-target toxicities5-7 and drug resistance8-10. Accordingly, a molecular understanding of the structure and function of MEK within physiological complexes could provide a template for the design of safer and more effective therapies. Here we report X-ray crystal structures of MEK bound to the scaffold KSR (kinase suppressor of RAS) with various MEK inhibitors, including the clinical drug trametinib. The structures reveal an unexpected mode of binding in which trametinib directly engages KSR at the MEK interface. In the bound complex, KSR remodels the prototypical allosteric pocket of the MEK inhibitor, thereby affecting binding and kinetics, including the drug-residence time. Moreover, trametinib binds KSR-MEK but disrupts the related RAF-MEK complex through a mechanism that exploits evolutionarily conserved interface residues that distinguish these sub-complexes. On the basis of these insights, we created trametiglue, which limits adaptive resistance to MEK inhibition by enhancing interfacial binding. Our results reveal the plasticity of an interface pocket within MEK sub-complexes and have implications for the design of next-generation drugs that target the RAS pathway.
Assuntos
Quinases de Proteína Quinase Ativadas por Mitógeno/química , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Piridonas/química , Piridonas/farmacologia , Pirimidinonas/química , Pirimidinonas/farmacologia , Sequência de Aminoácidos , Animais , Sítios de Ligação/efeitos dos fármacos , Humanos , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Modelos Moleculares , Ligação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Especificidade por Substrato , Quinases raf/química , Quinases raf/metabolismoRESUMO
Deregulation of the Ras-mitogen activated protein kinase (MAPK) pathway is an early event in many different cancers and a key driver of resistance to targeted therapies. Sustained signalling through this pathway is caused most often by mutations in K-Ras, which biochemically favours the stabilization of active RAF signalling complexes. Kinase suppressor of Ras (KSR) is a MAPK scaffold that is subject to allosteric regulation through dimerization with RAF. Direct targeting of KSR could have important therapeutic implications for cancer; however, testing this hypothesis has been difficult owing to a lack of small-molecule antagonists of KSR function. Guided by KSR mutations that selectively suppress oncogenic, but not wild-type, Ras signalling, we developed a class of compounds that stabilize a previously unrecognized inactive state of KSR. These compounds, exemplified by APS-2-79, modulate KSR-dependent MAPK signalling by antagonizing RAF heterodimerization as well as the conformational changes required for phosphorylation and activation of KSR-bound MEK (mitogen-activated protein kinase kinase). Furthermore, APS-2-79 increased the potency of several MEK inhibitors specifically within Ras-mutant cell lines by antagonizing release of negative feedback signalling, demonstrating the potential of targeting KSR to improve the efficacy of current MAPK inhibitors. These results reveal conformational switching in KSR as a druggable regulator of oncogenic Ras, and further suggest co-targeting of enzymatic and scaffolding activities within Ras-MAPK signalling complexes as a therapeutic strategy for overcoming Ras-driven cancers.
Assuntos
Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Oncogenes/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Quinazolinas/farmacologia , Proteínas ras/antagonistas & inibidores , Alelos , Regulação Alostérica/efeitos dos fármacos , Linhagem Celular , Estabilidade Enzimática/efeitos dos fármacos , Humanos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/química , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Modelos Moleculares , Mutação , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/genética , Neoplasias/metabolismo , Oncogenes/genética , Fosforilação/efeitos dos fármacos , Ligação Proteica , Conformação Proteica/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/genética , Piridonas/farmacologia , Pirimidinonas/farmacologia , Quinases raf/química , Quinases raf/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
Pseudokinases play important roles in signal transduction and cellular processes similar to those of catalytically competent kinases. However, pseudokinase pharmacological tractability and conformational space accessibility are poorly understood. Pseudokinases have only recently been suggested to adopt "inactive" conformations or interact with conformation-specific kinase inhibitors (e.g., type II compounds). In this work, the heavily substituted pseudokinase STRADα, which possesses a DFG â GLR substitution in the catalytic site that permits nucleotide binding while impairing divalent cation coordination, is used as a test case to demonstrate the potential applicability of conformation-specific, type II compounds to pseudokinase pharmacology. Integrated structural modeling is employed to generate a "GLR-out" conformational ensemble. Likely interacting type II compounds are identified through virtual screening against this ensemble model. Biophysical validation of compound binding is demonstrated through protein thermal stabilization and ATP competition. Localization of a top-performing compound through surface methylation strongly suggests that STRADα can adopt the "GLR-out" conformation and interact with compounds that comply with the standard type II pharmacophore. These results suggest that, despite a loss of catalytic function, some pseudokinases, including STRADα, may retain the conformational switching properties of conventional protein kinases.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/química , Trifosfato de Adenosina/química , Humanos , Domínios Proteicos , Estabilidade ProteicaRESUMO
Aberrant phospho-signaling is a hallmark of cancer. We investigated kinase-substrate regulation of 33,239 phosphorylation sites (phosphosites) in 77 breast tumors and 24 breast cancer xenografts. Our search discovered 2134 quantitatively correlated kinase-phosphosite pairs, enriching for and extending experimental or binding-motif predictions. Among the 91 kinases with auto-phosphorylation, elevated EGFR, ERBB2, PRKG1, and WNK1 phosphosignaling were enriched in basal, HER2-E, Luminal A, and Luminal B breast cancers, respectively, revealing subtype-specific regulation. CDKs, MAPKs, and ataxia-telangiectasia proteins were dominant, master regulators of substrate-phosphorylation, whose activities are not captured by genomic evidence. We unveiled phospho-signaling and druggable targets from 113 kinase-substrate pairs and cascades downstream of kinases, including AKT1, BRAF and EGFR. We further identified kinase-substrate-pairs associated with clinical or immune signatures and experimentally validated activated phosphosites of ERBB2, EIF4EBP1, and EGFR. Overall, kinase-substrate regulation revealed by the largest unbiased global phosphorylation data to date connects driver events to their signaling effects.
Assuntos
Neoplasias da Mama/metabolismo , Proteínas Quinases/metabolismo , Feminino , Humanos , Fosforilação , Transdução de SinaisRESUMO
Synthetic tailoring of approved drugs for new indications is often difficult, as the most appropriate targets may not be readily apparent, and therefore few roadmaps exist to guide chemistry. Here, we report a multidisciplinary approach for accessing novel target and chemical space starting from an FDA-approved kinase inhibitor. By combining chemical and genetic modifier screening with computational modeling, we identify distinct kinases that strongly enhance ('pro-targets') or limit ('anti-targets') whole-animal activity of the clinical kinase inhibitor sorafenib in a Drosophila medullary thyroid carcinoma (MTC) model. We demonstrate that RAF-the original intended sorafenib target-and MKNK kinases function as pharmacological liabilities because of inhibitor-induced transactivation and negative feedback, respectively. Through progressive synthetic refinement, we report a new class of 'tumor calibrated inhibitors' with unique polypharmacology and strongly improved therapeutic index in fly and human MTC xenograft models. This platform provides a rational approach to creating new high-efficacy and low-toxicity drugs.
Assuntos
Carcinoma Neuroendócrino/metabolismo , Carcinoma/metabolismo , Drosophila/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Neoplasias da Glândula Tireoide/metabolismo , Animais , Animais Geneticamente Modificados , Linhagem Celular Tumoral , Movimento Celular , Modelos Animais de Doenças , Desenho de Fármacos , Feminino , Células HCT116 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Simulação de Acoplamento Molecular , Transplante de Neoplasias , Isoformas de Proteínas , Proteínas Proto-Oncogênicas c-raf/metabolismo , Transdução de Sinais , Sorafenibe/farmacologiaRESUMO
Drosophila provides an inexpensive and quantitative platform for measuring whole animal drug response. A complementary approach is virtual screening, where chemical libraries can be efficiently screened against protein target(s). Here, we present a unique discovery platform integrating structure-based modeling with Drosophila biology and organic synthesis. We demonstrate this platform by developing chemicals targeting a Drosophila model of Medullary Thyroid Cancer (MTC) characterized by a transformation network activated by oncogenic dRetM955T. Structural models for kinases relevant to MTC were generated for virtual screening to identify unique preliminary hits that suppressed dRetM955T-induced transformation. We then combined features from our hits with those of known inhibitors to create a 'hybrid' molecule with improved suppression of dRetM955T transformation. Our platform provides a framework to efficiently explore novel kinase inhibitors outside of explored inhibitor chemical space that are effective in inhibiting cancer networks while minimizing whole body toxicity.
Assuntos
Antineoplásicos/farmacologia , Carcinoma Neuroendócrino , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases , Neoplasias da Glândula Tireoide , Animais , Carcinoma Neuroendócrino/enzimologia , Carcinoma Neuroendócrino/metabolismo , Biologia Computacional/métodos , Drosophila , Modelos Biológicos , Neoplasias Experimentais/enzimologia , Neoplasias Experimentais/metabolismo , Proteínas Quinases/efeitos dos fármacos , Proteínas Quinases/metabolismo , Neoplasias da Glândula Tireoide/enzimologia , Neoplasias da Glândula Tireoide/metabolismoRESUMO
The complexity of cancer has led to recent interest in polypharmacological approaches for developing kinase-inhibitor drugs; however, optimal kinase-inhibition profiles remain difficult to predict. Using a Ret-kinase-driven Drosophila model of multiple endocrine neoplasia type 2 and kinome-wide drug profiling, here we identify that AD57 rescues oncogenic Ret-induced lethality, whereas related Ret inhibitors imparted reduced efficacy and enhanced toxicity. Drosophila genetics and compound profiling defined three pathways accounting for the mechanistic basis of efficacy and dose-limiting toxicity. Inhibition of Ret plus Raf, Src and S6K was required for optimal animal survival, whereas inhibition of the 'anti-target' Tor led to toxicity owing to release of negative feedback. Rational synthetic tailoring to eliminate Tor binding afforded AD80 and AD81, compounds featuring balanced pathway inhibition, improved efficacy and low toxicity in Drosophila and mammalian multiple endocrine neoplasia type 2 models. Combining kinase-focused chemistry, kinome-wide profiling and Drosophila genetics provides a powerful systems pharmacology approach towards developing compounds with a maximal therapeutic index.