RESUMO
Spleen tyrosine kinase (SYK) is a non-receptor cytosolic kinase. Due to its pivotal role in B cell receptor and Fc-receptor signaling, inhibition of SYK has been targeted in a variety of disease areas. Herein, we report the optimization of a series of potent and selective SYK inhibitors, focusing on improving metabolic stability, pharmacokinetics and hERG inhibition. As a result, we identified 30, which exhibited no hERG activity but unfortunately was poorly absorbed in rats and mice. We also identified a SYK chemical probe, 17, which exhibits excellent potency at SYK, and an adequate rodent PK profile to support in vivo efficacy/PD studies.
Assuntos
Indazóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Quinase Syk/antagonistas & inibidores , Animais , Sítios de Ligação , Células CACO-2 , Cristalografia por Raios X , Canal de Potássio ERG1/antagonistas & inibidores , Humanos , Indazóis/síntese química , Indazóis/metabolismo , Indazóis/farmacocinética , Camundongos , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Ligação Proteica , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacocinética , Ratos Wistar , Relação Estrutura-Atividade , Quinase Syk/química , Quinase Syk/metabolismoRESUMO
The stability of the Wnt pathway transcription factor beta-catenin is tightly regulated by the multi-subunit destruction complex. Deregulated Wnt pathway activity has been implicated in many cancers, making this pathway an attractive target for anticancer therapies. However, the development of targeted Wnt pathway inhibitors has been hampered by the limited number of pathway components that are amenable to small molecule inhibition. Here, we used a chemical genetic screen to identify a small molecule, XAV939, which selectively inhibits beta-catenin-mediated transcription. XAV939 stimulates beta-catenin degradation by stabilizing axin, the concentration-limiting component of the destruction complex. Using a quantitative chemical proteomic approach, we discovered that XAV939 stabilizes axin by inhibiting the poly-ADP-ribosylating enzymes tankyrase 1 and tankyrase 2. Both tankyrase isoforms interact with a highly conserved domain of axin and stimulate its degradation through the ubiquitin-proteasome pathway. Thus, our study provides new mechanistic insights into the regulation of axin protein homeostasis and presents new avenues for targeted Wnt pathway therapies.
Assuntos
Proteínas Repressoras/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tanquirases/antagonistas & inibidores , Proteínas Wnt/antagonistas & inibidores , Proteína Axina , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Proteômica , Proteínas Repressoras/química , Tanquirases/metabolismo , Transcrição Gênica/efeitos dos fármacos , Ubiquitina/metabolismo , Ubiquitinação , Proteínas Wnt/metabolismo , beta Catenina/antagonistas & inibidores , beta Catenina/metabolismoRESUMO
Lactic acid transport is a key process maintaining glycolytic flux in tumors. Inhibition of this process will result in glycolytic shutdown, impacting on cell growth and survival and thus has been pursued as a therapeutic approach for cancers. Using a cell-based screen in a MCT4-dependent cell line, we identified and optimized compounds for their ability to inhibit the efflux of intracellular lactic acid with good physical and pharmacokinetic properties. To deconvolute the mechanism of lactic acid efflux inhibition, we have developed three assays to measure cellular target engagement. Specifically, we synthesized a biologically active photoaffinity probe (IC50 < 10 nM), and using this probe, we demonstrated selective engagement of MCT4 of our parent molecule through a combination of confocal microscopy and in-cell chemoproteomics. As an orthogonal assay, the cellular thermal shift assay (CETSA) confirmed binding to MCT4 in the cellular system. Comparisons of lactic acid efflux potencies in cells with differential expression of MCT family members further confirmed that the optimized compounds inhibit the efflux of lactic acid through the inhibition of MCT4. Taken together, these data demonstrate the power of orthogonal chemical biology methods to determine cellular target engagement, particularly for proteins not readily amenable to traditional biophysical methods.
Assuntos
Biologia , Ácido Láctico , Ácido Láctico/metabolismo , Transporte Biológico , Linhagem Celular Tumoral , Proliferação de CélulasRESUMO
Cyclin-dependent-kinases (CDKs) are members of the serine/threonine kinase family and are highly regulated by cyclins, a family of regulatory subunits that bind to CDKs. CDK9 represents one of the most studied examples of these transcriptional CDKs. CDK9 forms a heterodimeric complex with its regulatory subunit cyclins T1, T2 and K to form the positive transcription elongation factor b (P-TEFb). This complex regulates transcription via the phosphorylation of RNA polymerase II (RNAPolII) on Ser-2, facilitating promoter clearance and transcription elongation and thus remains an attractive therapeutic target. Herein, we have utilized classical affinity purification chemical proteomics, kinobeads assay, compressed CEllular Thermal Shift Assay (CETSA)-MS and Limited Proteolysis (LiP) to study the selectivity, target engagement and downstream mechanistic insights of a CDK9 tool compound. The above experiments highlight the value of quantitative mass spectrometry approaches to drug discovery, specifically proteome wide target identification and selectivity profiling. The approaches utilized in this study unanimously indicated that the CDK family of kinases are the main target of the compound of interest, with CDK9, showing the highest target affinity with remarkable consistency across approaches. We aim to provide guidance to the scientific community on the available chemical biology/proteomic tools to study advanced lead molecules and to highlight pros and cons of each technology while describing our findings in the context of the CDKs biology.
Assuntos
Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Proteômica , Linhagem Celular Tumoral , Fracionamento Químico , Quinase 9 Dependente de Ciclina/genética , Quinase 9 Dependente de Ciclina/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Espectrometria de MassasRESUMO
Deregulation of the PRC2 complex, comprised of the core subunits EZH2, SUZ12, and EED, drives aberrant hypermethylation of H3K27 and tumorigenicity of many cancers. Although inhibitors of EZH2 have shown promising clinical activity, preclinical data suggest that resistance can be acquired through secondary mutations in EZH2 that abrogate drug target engagement. To address these limitations, we have designed several hetero-bifunctional PROTACs (proteolysis-targeting chimera) to efficiently target EED for elimination. Our PROTACs bind to EED (pKD â¼ 9.0) and promote ternary complex formation with the E3 ubiquitin ligase. The PROTACs potently inhibit PRC2 enzyme activity (pIC50 â¼ 8.1) and induce rapid degradation of not only EED but also EZH2 and SUZ12 within the PRC2 complex. Furthermore, the PROTACs selectively inhibit proliferation of PRC2-dependent cancer cells (half maximal growth inhibition [GI50] = 49-58 nM). In summary, our data demonstrate a therapeutic modality to target PRC2-dependent cancer through a PROTAC-mediated degradation mechanism.
Assuntos
Complexo Repressor Polycomb 2/metabolismo , Proteólise/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Estrutura Molecular , Complexo Repressor Polycomb 2/antagonistas & inibidores , Relação Estrutura-AtividadeRESUMO
Spinal muscular atrophy (SMA), a rare neuromuscular disorder, is the leading genetic cause of death in infants and toddlers. SMA is caused by the deletion or a loss of function mutation of the survival motor neuron 1 (SMN1) gene. In humans, a second closely related gene SMN2 exists; however it codes for a less stable SMN protein. In recent years, significant progress has been made toward disease modifying treatments for SMA by modulating SMN2 pre-mRNA splicing. Herein, we describe the discovery of LMI070/branaplam, a small molecule that stabilizes the interaction between the spliceosome and SMN2 pre-mRNA. Branaplam (1) originated from a high-throughput phenotypic screening hit, pyridazine 2, and evolved via multiparameter lead optimization. In a severe mouse SMA model, branaplam treatment increased full-length SMN RNA and protein levels, and extended survival. Currently, branaplam is in clinical studies for SMA.
Assuntos
Encéfalo/efeitos dos fármacos , Canal de Potássio ERG1/metabolismo , Atrofia Muscular Espinal/tratamento farmacológico , Piridazinas/química , Administração Oral , Animais , Encéfalo/metabolismo , Linhagem Celular , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Canal de Potássio ERG1/antagonistas & inibidores , Humanos , Camundongos Endogâmicos C57BL , Neurônios Motores/efeitos dos fármacos , Atrofia Muscular Espinal/genética , Piridazinas/farmacologia , Relação Quantitativa Estrutura-Atividade , Splicing de RNA , Ratos Sprague-Dawley , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Proteína 2 de Sobrevivência do Neurônio Motor/genéticaRESUMO
Cyclin-dependent kinase (CDK) 12 knockdown via siRNA decreases the transcription of DNA-damage-response genes and sensitizes BRCA wild-type cells to poly(ADP-ribose) polymerase (PARP) inhibition. To recapitulate this effect with a small molecule, we sought a potent, selective CDK12 inhibitor. Crystal structures and modeling informed hybridization between dinaciclib and SR-3029, resulting in lead compound 5 [(S)-2-(1-(6-(((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl)amino)-9-ethyl-9H-purin-2-yl)piperidin-2-yl)ethan-1-ol]. Further structure-guided optimization delivered a series of selective CDK12 inhibitors, including compound 7 [(S)-2-(1-(6-(((6,7-difluoro-1H-benzo[d]imidazol-2-yl)methyl)amino)-9-isopropyl-9H-purin-2-yl)piperidin-2-yl)ethan-1-ol]. Profiling of this compound across CDK9, 7, 2, and 1 at high ATP concentration, single-point kinase panel screening against 352 targets at 0.1â µm, and proteomics via kinase affinity matrix technology demonstrated the selectivity. This series of compounds inhibits phosphorylation of Ser2 on the C-terminal repeat domain of RNA polymeraseâ II, consistent with CDK12 inhibition. These selective compounds were also acutely toxic to OV90 as well as THP1 cells.
Assuntos
Benzimidazóis/química , Compostos Bicíclicos Heterocíclicos com Pontes/química , Quinases Ciclina-Dependentes/antagonistas & inibidores , Piperidinas/síntese química , Purinas/química , Compostos de Piridínio/química , Benzimidazóis/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cristalização , Óxidos N-Cíclicos , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Desenho de Fármacos , Humanos , Indolizinas , Cinética , Fosforilação , Piperidinas/farmacologia , Ligação Proteica , Purinas/farmacologia , Compostos de Piridínio/farmacologia , RNA Polimerase II/metabolismo , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Activating KRAS mutations are major oncogenic drivers in multiple tumor types. Synthetic lethal screens have previously been used to identify targets critical for the survival of KRAS mutant cells, but their application to drug discovery has proven challenging, possibly due in part to a failure of monolayer cultures to model tumor biology. Here, we report the results of a high-throughput synthetic lethal screen for small molecules that selectively inhibit the growth of KRAS mutant cell lines in soft agar. Chemoproteomic profiling identifies the target of the most KRAS-selective chemical series as dihydroorotate dehydrogenase (DHODH). DHODH inhibition is shown to perturb multiple metabolic pathways. In vivo preclinical studies demonstrate strong antitumor activity upon DHODH inhibition in a pancreatic tumor xenograft model.
Assuntos
Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Pirimidinas/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Di-Hidro-Orotato Desidrogenase , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Camundongos , Camundongos SCID , Mutação , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/genética , Pirimidinas/química , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Células Tumorais CultivadasRESUMO
Toll-like receptor (TLR) signaling is a key component of innate immunity. Aberrant TLR activation leads to immune disorders via dysregulation of cytokine production, such as IL-12/IL-23. Herein, we identify and characterize PIKfyve, a lipid kinase, as a critical player in TLR signaling using apilimod as an affinity tool. Apilimod is a potent small molecular inhibitor of IL-12/IL-23 with an unknown target and has been evaluated in clinical trials for patients with Crohn's disease or rheumatoid arthritis. Using a chemical genetic approach, we show that it binds to PIKfyve and blocks its phosphotransferase activity, leading to selective inhibition of IL-12/IL-23p40. Pharmacological or genetic inactivation of PIKfyve is necessary and sufficient for suppression of IL-12/IL-23p40 expression. Thus, we have uncovered a phosphoinositide-mediated regulatory mechanism that controls TLR signaling.
Assuntos
Interleucina-12/antagonistas & inibidores , Interleucina-23/antagonistas & inibidores , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Transdução de Sinais/efeitos dos fármacos , Receptores Toll-Like/metabolismo , Triazinas/farmacologia , Animais , Linhagem Celular , Citocinas/metabolismo , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Hidrazonas , Camundongos , Morfolinas/metabolismo , Ligação Proteica , Pirimidinas , Especificidade por Substrato , Triazinas/metabolismoRESUMO
Tankyrase 1 and 2 have been shown to be redundant, druggable nodes in the Wnt pathway. As such, there has been intense interest in developing agents suitable for modulating the Wnt pathway in vivo by targeting this enzyme pair. By utilizing a combination of structure-based design and LipE-based structure efficiency relationships, the core of XAV939 was optimized into a more stable, more efficient, but less potent dihydropyran motif 7. This core was combined with elements of screening hits 2, 19, and 33 and resulted in highly potent, selective tankyrase inhibitors that are novel three pocket binders. NVP-TNKS656 (43) was identified as an orally active antagonist of Wnt pathway activity in the MMTV-Wnt1 mouse xenograft model. With an enthalpy-driven thermodynamic signature of binding, highly favorable physicochemical properties, and high lipophilic efficiency, NVP-TNKS656 is a novel tankyrase inhibitor that is well suited for further in vivo validation studies.
Assuntos
Acetamidas/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Pirimidinonas/farmacologia , Tanquirases/antagonistas & inibidores , Acetamidas/administração & dosagem , Acetamidas/química , Administração Oral , Animais , Área Sob a Curva , Disponibilidade Biológica , Inibidores Enzimáticos/administração & dosagem , Camundongos , Modelos Moleculares , Pirimidinonas/administração & dosagem , Pirimidinonas/química , Relação Estrutura-AtividadeRESUMO
Iterative cross-coupling is a highly efficient and versatile strategy for modular construction in organic synthesis, though this has historically been demonstrated solely in the context of C-C bond formation. A C-N cross-coupling of haloarene methyliminodiacetic acid (MIDA) boronates with a wide range of aromatic and aliphatic amines is reported. Successful cross-coupling of aliphatic amines was realized only through protective enolization of the MIDA group. This reaction paradigm was subsequently utilized to achieve a one-pot C-N/C-C cross-coupling sequence.