RESUMO
Vaccinia-related kinase 1 (VRK1) and the δ and ε isoforms of casein kinase 1 (CK1) are linked to various disease-relevant pathways. However, the lack of tool compounds for these kinases has significantly hampered our understanding of their cellular functions and therapeutic potential. Here, we describe the structure-based development of potent inhibitors of VRK1, a kinase highly expressed in various tumor types and crucial for cell proliferation and genome integrity. Kinome-wide profiling revealed that our compounds also inhibit CK1δ and CK1ε. We demonstrate that dihydropteridinones 35 and 36 mimic the cellular outcomes of VRK1 depletion. Complementary studies with existing CK1δ and CK1ε inhibitors suggest that these kinases may play overlapping roles in cell proliferation and genome instability. Together, our findings highlight the potential of VRK1 inhibition in treating p53-deficient tumors and possibly enhancing the efficacy of existing cancer therapies that target DNA stability or cell division.
Assuntos
Inibidores de Proteínas Quinases , Proteínas Serina-Treonina Quinases , Pteridinas , Humanos , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/síntese química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Pteridinas/farmacologia , Pteridinas/química , Pteridinas/síntese química , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proliferação de Células/efeitos dos fármacos , Relação Estrutura-Atividade , Caseína Quinase Idelta/antagonistas & inibidores , Caseína Quinase Idelta/metabolismo , Caseína Quinase 1 épsilon/antagonistas & inibidores , Caseína Quinase 1 épsilon/metabolismo , Linhagem Celular TumoralRESUMO
The dual-specificity protein kinase MKK3 has been implicated in tumor cell proliferation and survival, yet its precise role in cancer remains inconclusive. A critical step in elucidating the kinase's involvement in disease biology is the identification of potent, cell-permeable kinase inhibitors. Presently, MKK3 lacks a dedicated tool compound for these purposes, along with validated methods for the facile screening, identification, and optimization of inhibitors. In this study, we have developed a TR-FRET-based enzymatic assay for the detection of MKK3 activity in vitro and a BRET-based assay to assess ligand binding to this enzyme within intact human cells. These assays were instrumental in identifying hit compounds against MKK3 that share a common chemical scaffold, sourced from a library of bioactive kinase inhibitors. Initial hits were subsequently expanded through the synthesis of novel analogs. The resulting structure-activity relationship (SAR) was rationalized using molecular dynamics simulations against a homology model of MKK3. We expect our findings to expedite the development of novel, potent, selective, and bioactive inhibitors, thus facilitating investigations into MKK3's role in various cancers.
Assuntos
Neoplasias , Pirimidinas , Humanos , MAP Quinase Quinase 3 , Pirimidinas/química , Relação Estrutura-Atividade , Fosforilação , Proliferação de Células , Inibidores de Proteínas Quinases/químicaRESUMO
Here, we report a bioluminescence resonance energy transfer (BRET) assay as a novel way to investigate the binding of unlabeled ligands to the human transient receptor potential mucolipin 1 (hTRPML1), a lysosomal ion channel involved in several genetic diseases and cancer progression. This novel BRET assay can be used to determine equilibrium and kinetic binding parameters of unlabeled compounds to hTRPML1 using intact human-derived cells, thus complementing the information obtained using functional assays based on ion channel activation. We expect this new BRET assay to expedite the identification and optimization of cell-permeable ligands that interact with hTRPML1 within the physiologically relevant environment of lysosomes.
Assuntos
Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Canais de Potencial de Receptor Transitório , Humanos , Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Ligantes , Lisossomos/metabolismo , Canais de Potencial de Receptor Transitório/metabolismoRESUMO
Epithelial calcium channel TRPV6 plays vital roles in calcium homeostasis, and its dysregulation is implicated in multifactorial diseases, including cancers. Here, we study the molecular mechanism of selective nanomolar-affinity TRPV6 inhibition by (4-phenylcyclohexyl)piperazine derivatives (PCHPDs). We use x-ray crystallography and cryo-electron microscopy to solve the inhibitor-bound structures of TRPV6 and identify two types of inhibitor binding sites in the transmembrane region: (i) modulatory sites between the S1-S4 and pore domains normally occupied by lipids and (ii) the main site in the ion channel pore. Our structural data combined with mutagenesis, functional and computational approaches suggest that PCHPDs plug the open pore of TRPV6 and convert the channel into a nonconducting state, mimicking the action of calmodulin, which causes inactivation of TRPV6 channels under physiological conditions. This mechanism of inhibition explains the high selectivity and potency of PCHPDs and opens up unexplored avenues for the design of future-generation biomimetic drugs.
Assuntos
Canais de Cálcio , Canais de Cátion TRPV , Cálcio/metabolismo , Canais de Cálcio/química , Calmodulina/metabolismo , Microscopia Crioeletrônica , Humanos , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/metabolismoRESUMO
Herein we report the first photoswitchable inhibitor of Transient Receptor Potential Vanilloid 6 (TRPV6), a selective calcium channel involved in a number of diseases and in cancer progression. By surveying analogs of a previously reported TRPV6 inhibitor appended with a phenyl-diazo group, we identified a compound switching between a weak TRPV6 inhibitor in its dark, E-diazo stereoisomer (Z/E = 3:97, IC50 â« 10 µM) and a potent inhibitor as the Z-diazo stereoisomer accessible reversibly by UV irradiation at λ = 365 nm (Z/E = 3:1, IC50 = 1.7 ± 0.4 µM), thereby allowing precise spatiotemporal control of inhibition. This new tool compound should be useful to deepen our understanding of TRPV6.