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1.
Nucleic Acids Res ; 51(14): 7288-7313, 2023 08 11.
Artículo en Inglés | MEDLINE | ID: mdl-37378433

RESUMEN

We have conducted a detailed transcriptomic, proteomic and phosphoproteomic analysis of CDK8 and its paralog CDK19, alternative enzymatic components of the kinase module associated with transcriptional Mediator complex and implicated in development and diseases. This analysis was performed using genetic modifications of CDK8 and CDK19, selective CDK8/19 small molecule kinase inhibitors and a potent CDK8/19 PROTAC degrader. CDK8/19 inhibition in cells exposed to serum or to agonists of NFκB or protein kinase C (PKC) reduced the induction of signal-responsive genes, indicating a pleiotropic role of Mediator kinases in signal-induced transcriptional reprogramming. CDK8/19 inhibition under basal conditions initially downregulated a small group of genes, most of which were inducible by serum or PKC stimulation. Prolonged CDK8/19 inhibition or mutagenesis upregulated a larger gene set, along with a post-transcriptional increase in the proteins comprising the core Mediator complex and its kinase module. Regulation of both RNA and protein expression required CDK8/19 kinase activities but both enzymes protected their binding partner cyclin C from proteolytic degradation in a kinase-independent manner. Analysis of isogenic cell populations expressing CDK8, CDK19 or their kinase-inactive mutants revealed that CDK8 and CDK19 have the same qualitative effects on protein phosphorylation and gene expression at the RNA and protein levels, whereas differential effects of CDK8 versus CDK19 knockouts were attributable to quantitative differences in their expression and activity rather than different functions.


Asunto(s)
Quinasas Ciclina-Dependientes , Complejo Mediador , Humanos , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , Complejo Mediador/genética , Complejo Mediador/metabolismo , Fosforilación , Proteómica , ARN/metabolismo
2.
J Biol Chem ; 298(9): 102336, 2022 09.
Artículo en Inglés | MEDLINE | ID: mdl-35931111

RESUMEN

Mitochondrial chelatable iron contributes to the severity of several injury processes, including ischemia/reperfusion, oxidative stress, and drug toxicity. However, methods to measure this species in living cells are lacking. To measure mitochondrial chelatable iron in living cells, here we synthesized a new fluorescent indicator, mitoferrofluor (MFF). We designed cationic MFF to accumulate electrophoretically in polarized mitochondria, where a reactive group then forms covalent adducts with mitochondrial proteins to retain MFF even after subsequent depolarization. We also show in cell-free medium that Fe2+ (and Cu2+), but not Fe3+, Ca2+, or other biologically relevant divalent cations, strongly quenched MFF fluorescence. Using confocal microscopy, we demonstrate in hepatocytes that red MFF fluorescence colocalized with the green fluorescence of the mitochondrial membrane potential (ΔΨm) indicator, rhodamine 123 (Rh123), indicating selective accumulation into the mitochondria. Unlike Rh123, mitochondria retained MFF after ΔΨm collapse. Furthermore, intracellular delivery of iron with membrane-permeant Fe3+/8-hydroxyquinoline (FeHQ) quenched MFF fluorescence by ∼80% in hepatocytes and other cell lines, which was substantially restored by the membrane-permeant transition metal chelator pyridoxal isonicotinoyl hydrazone. We also show FeHQ quenched the fluorescence of cytosolically coloaded calcein, another Fe2+ indicator, confirming that Fe3+ in FeHQ undergoes intracellular reduction to Fe2+. Finally, MFF fluorescence did not change after addition of the calcium mobilizer thapsigargin, which shows MFF is insensitive to physiologically relevant increases of mitochondrial Ca2+. In conclusion, the new sensor reagent MFF fluorescence is an indicator of mitochondrial chelatable Fe2+ in normal hepatocytes with polarized mitochondria as well as in cells undergoing loss of ΔΨm.


Asunto(s)
Colorantes Fluorescentes , Quelantes del Hierro , Mitocondrias , Animales , Calcio/metabolismo , Cationes Bivalentes/análisis , Células Cultivadas , Fluorescencia , Colorantes Fluorescentes/química , Quelantes del Hierro/análisis , Ratones , Mitocondrias/química , Proteínas Mitocondriales/química , Oxiquinolina/química , Rodamina 123 , Tapsigargina/farmacología
3.
Bioorg Med Chem Lett ; 26(15): 3754-60, 2016 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-27297568

RESUMEN

Inhibition of cyclin dependent kinase 2 (CDK2) in complex with cyclin A in G1/S phase of the cell cycle has been shown to promote selective apoptosis of cancer cells through the E2F1 pathway. An alternative approach to catalytic inhibition is to target the substrate recruitment site also known as the cyclin binding groove (CBG) to generate selective non-ATP competitive inhibitors. The REPLACE strategy has been applied to identify fragment alternatives and substituted benzoic acid derivatives were evaluated as a promising scaffold to present appropriate functionality to mimic key peptide determinants. Fragment Ligated Inhibitory Peptides (FLIPs) are described which potently inhibit both CDK2/cyclin A and CDK4/cyclin D1 and have preliminary anti-tumor activity. A structural rationale for binding was obtained through molecular modeling further demonstrating their potential for further development as next generation non ATP competitive CDK inhibitors.


Asunto(s)
Benzamidas/farmacología , Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Peptidomiméticos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Benzamidas/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Quinasa 2 Dependiente de la Ciclina/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Peptidomiméticos/química , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Relación Estructura-Actividad
4.
Bioorg Med Chem Lett ; 26(22): 5573-5579, 2016 11 15.
Artículo en Inglés | MEDLINE | ID: mdl-27769620

RESUMEN

Using molecular modeling and rationally designed structural modifications, the multi-target structure-activity relationship for a series of ranitidine analogs has been investigated. Incorporation of a variety of isosteric groups indicated that appropriate aromatic moieties provide optimal interactions with the hydrophobic and π-π interactions with the peripheral anionic site of the AChE active site. The SAR of a series of cyclic imides demonstrated that AChE inhibition is increased by additional aromatic rings, where 1,8-naphthalimide derivatives were the most potent analogs and other key determinants were revealed. In addition to improving AChE activity and chemical stability, structural modifications allowed determination of binding affinities and selectivities for M1-M4 receptors and butyrylcholinesterase (BuChE). These results as a whole indicate that the 4-nitropyridazine moiety of the JWS-USC-75IX parent ranitidine compound (JWS) can be replaced with other chemotypes while retaining effective AChE inhibition. These studies allowed investigation into multitargeted binding to key receptors and warrant further investigation into 1,8-naphthalimide ranitidine derivatives for the treatment of Alzheimer's disease.


Asunto(s)
Acetilcolinesterasa/metabolismo , Inhibidores de la Colinesterasa/química , Inhibidores de la Colinesterasa/farmacología , Nootrópicos/química , Nootrópicos/farmacología , Ranitidina/análogos & derivados , Ranitidina/farmacología , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/enzimología , Enfermedad de Alzheimer/metabolismo , Animales , Butirilcolinesterasa/metabolismo , Inhibidores de la Colinesterasa/síntesis química , Humanos , Ratones , Modelos Moleculares , Naftalimidas/síntesis química , Naftalimidas/química , Naftalimidas/farmacología , Nootrópicos/síntesis química , Ranitidina/síntesis química , Receptor Muscarínico M1/metabolismo , Receptor Muscarínico M4/metabolismo , Relación Estructura-Actividad
5.
Protein Expr Purif ; 113: 8-16, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-25956535

RESUMEN

Bacterial expression of human proteins continues to present a critical challenge in protein crystallography and drug design. While human cyclin A constructs have been extensively characterized in complex with cyclin dependent kinase 2 (CDK2), efforts to express the monomeric human cyclin A2 in Escherichia coli in a stable form, without the kinase subunit, have been laden with technical difficulties, including solubility, yield and purity. Here, optimized conditions are described with the aim of generating for first time, sufficient quantities of human recombinant cyclin A2 in a soluble and active form for crystallization and ligand characterization purposes. The studies involve implementation of a His-tagged heterologous expression system under conditions of auto-induction and mediated by molecular chaperone-expressing plasmids. A high yield of human cyclin A2 was obtained in natively folded and soluble form, through co-expression with groups of molecular chaperones from E. coli in various combinations. A one-step affinity chromatography method was utilized to purify the fusion protein products to homogeneity, and the biological activity confirmed through ligand-binding affinity to inhibitory peptides, representing alternatives for the key determinants of the CDK2 substrate recruitment site on the cyclin regulatory subunit. As a whole, obtaining the active cyclin A without the CDK partner (referred to as monomeric in this work) in a straightforward and facile manner will obviate protein--production issues with the CDK2/cyclin A complex and enable drug discovery efforts for non-ATP competitive CDK inhibition through the cyclin groove.


Asunto(s)
Ciclina A2/química , Ciclina A2/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Ciclina A2/genética , Ciclina A2/aislamiento & purificación , Escherichia coli/genética , Humanos , Chaperonas Moleculares/genética , Unión Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Solubilidad
6.
Bioorg Med Chem ; 22(1): 616-22, 2014 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-24286762

RESUMEN

In order to develop non-ATP competitive CDK2/cyclin A inhibitors, the REPLACE strategy has been applied to generate fragment alternatives for the N-terminal tetrapeptide of the cyclin binding motif (HAKRRLIF) involved in substrate recruitment prior to phosphotransfer. The docking approach used for the prediction of small molecule mimics for peptide determinants was validated through reproduction of experimental binding modes of known inhibitors and provides useful information for evaluating binding to protein-protein interaction sites. Further to this, potential arginine isosteres predicted using the validated LigandFit docking method were ligated to the truncated C-terminal peptide, RLIF using solid phase synthesis and evaluated in a competitive binding assay. After testing, identified fragments were shown to represent not only appropriate mimics for a critical arginine residue but also to interact effectively with a minor hydrophobic pocket present in the binding groove. Further evaluation of binding modes was undertaken to optimize the potency of these compounds. Through further application of the REPLACE strategy in this study, peptide-small molecule hybrid CDK2 inhibitors were identified that are more drug-like and suitable for further optimization as anti-tumor therapeutics.


Asunto(s)
Arginina/metabolismo , Quinasa 2 Dependiente de la Ciclina/química , Ciclinas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Descubrimiento de Drogas , Humanos , Relación Estructura-Actividad
7.
Cell Chem Biol ; 2024 Jul 09.
Artículo en Inglés | MEDLINE | ID: mdl-39043186

RESUMEN

Lysyl oxidase (LOX) is upregulated in highly stiff aggressive tumors, correlating with metastasis, resistance, and worse survival; however, there are currently no potent, safe, and orally bioavailable small molecule LOX inhibitors to treat these aggressive desmoplastic solid tumors in clinics. Here we discovered bi-thiazole derivatives as potent LOX inhibitors by robust screening of drug-like molecules combined with cell/recombinant protein-based assays. Structure-activity relationship analysis identified a potent lead compound (LXG6403) with ∼3.5-fold specificity for LOX compared to LOXL2 while not inhibiting LOXL1 with a competitive, time- and concentration-dependent irreversible mode of inhibition. LXG6403 shows favorable pharmacokinetic properties, globally changes ECM/collagen architecture, and reduces tumor stiffness. This leads to better drug penetration, inhibits FAK signaling, and induces ROS/DNA damage, G1 arrest, and apoptosis in chemoresistant triple-negative breast cancer (TNBC) cell lines, PDX organoids, and in vivo. Overall, our potent and tolerable bi-thiazole LOX inhibitor enhances chemoresponse in TNBC, the deadliest breast cancer subtype.

8.
J Clin Invest ; 134(10)2024 Mar 28.
Artículo en Inglés | MEDLINE | ID: mdl-38546787

RESUMEN

Mediator kinases CDK19 and CDK8, pleiotropic regulators of transcriptional reprogramming, are differentially regulated by androgen signaling, but both kinases are upregulated in castration-resistant prostate cancer (CRPC). Genetic or pharmacological inhibition of CDK8 and CDK19 reverses the castration-resistant phenotype and restores the sensitivity of CRPC xenografts to androgen deprivation in vivo. Prolonged CDK8/19 inhibitor treatment combined with castration not only suppressed the growth of CRPC xenografts but also induced tumor regression and cures. Transcriptomic analysis revealed that Mediator kinase inhibition amplified and modulated the effects of castration on gene expression, disrupting CRPC adaptation to androgen deprivation. Mediator kinase inactivation in tumor cells also affected stromal gene expression, indicating that Mediator kinase activity in CRPC molded the tumor microenvironment. The combination of castration and Mediator kinase inhibition downregulated the MYC pathway, and Mediator kinase inhibition suppressed a MYC-driven CRPC tumor model even without castration. CDK8/19 inhibitors showed efficacy in patient-derived xenograft models of CRPC, and a gene signature of Mediator kinase activity correlated with tumor progression and overall survival in clinical samples of metastatic CRPC. These results indicate that Mediator kinases mediated androgen-independent in vivo growth of CRPC, supporting the development of CDK8/19 inhibitors for the treatment of this presently incurable disease.


Asunto(s)
Quinasa 8 Dependiente de Ciclina , Quinasas Ciclina-Dependientes , Neoplasias de la Próstata Resistentes a la Castración , Inhibidores de Proteínas Quinasas , Ensayos Antitumor por Modelo de Xenoinjerto , Masculino , Humanos , Animales , Neoplasias de la Próstata Resistentes a la Castración/patología , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/genética , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/enzimología , Ratones , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , Quinasa 8 Dependiente de Ciclina/antagonistas & inhibidores , Quinasa 8 Dependiente de Ciclina/genética , Quinasa 8 Dependiente de Ciclina/metabolismo , Línea Celular Tumoral , Inhibidores de Proteínas Quinasas/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Microambiente Tumoral/efectos de los fármacos
9.
ACS Chem Biol ; 18(7): 1642-1652, 2023 07 21.
Artículo en Inglés | MEDLINE | ID: mdl-37433100

RESUMEN

Polo-like kinase 1 (PLK1) is an essential protein kinase with multiple roles in mitotic progression. PLK1 consists of a kinase domain (KD) and a phosphopeptide-binding polobox domain (PBD), which is responsible for substrate recognition and subcellular localization. The regulation of PLK1 involves an autoinhibitory conformation in which KD and PBD interact. Our previous work identified PBD-binding molecules termed abbapolins that inhibit the cellular phosphorylation of a PLK1 substrate and induce the loss of intracellular PLK1. Here, we describe a comparison of the abbapolin activity with that of KD inhibitors to gain insight into conformational features of PLK1. As measured by a cellular thermal shift assay, abbapolins produce ligand-induced thermal stabilization of PLK1. In contrast, KD inhibitors decreased the soluble PLK1, suggesting that catalytic-site binding causes a less thermally stable PLK1 conformation. Binding measurements with full-length PLK1 and a KD inhibitor also demonstrated a conformational change. Interestingly, the cellular consequences of KD versus PBD engagement contrast as KD binding causes the accumulation of intracellular PLK1, whereas PBD binding produces a striking loss of nuclear PLK1. These data are consistent with the relief of autoinhibited PLK1 by KD binders; an explanation for these observations is presented using structures for the catalytic domain and full-length PLK1 predicted by AlphaFold. Collectively, the results highlight an underappreciated aspect of targeting PLK1, namely, conformational perturbations induced by KD versus PBD binding. In addition to their significance for PBD-binding ligands, these observations have implications for the development of ATP-competitive PLK1 inhibitors because catalytic inhibitors may conversely promote PLK1 noncatalytic functions, which may explain their lack of clinical efficacy to date.


Asunto(s)
Proteínas de Ciclo Celular , Proteínas Serina-Treonina Quinasas , Humanos , Proteínas de Ciclo Celular/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/química , Adenosina Trifosfato , Células HeLa , Quinasa Tipo Polo 1
10.
Expert Opin Drug Discov ; 18(1): 65-81, 2023 01.
Artículo en Inglés | MEDLINE | ID: mdl-36524399

RESUMEN

INTRODUCTION: Polo Like Kinase 1 (PLK1) is a key regulator of mitosis and its overexpression is frequently observed in a wide variety of human cancers, while often being associated with poor survival rates. Therefore, it is considered a potential and attractive target for cancer therapeutic development. The Polo like kinase family is characterized by the presence of a unique C terminal polobox domain (PBD) involved in regulating kinase activity and subcellular localization. Among the two functionally essential, druggable sites with distinct properties that PLK1 offers, targeting the PBD presents an alternative approach for therapeutic development. AREAS COVERED: Significant progress has been made in progressing from the peptidic PBD inhibitors first identified, to peptidomimetic and recently drug-like small molecules. In this review, the rationale for targeting the PBD over the ATP binding site is discussed, along with recent progress, challenges, and outlook. EXPERT OPINION: The PBD has emerged as a viable alternative target for the inhibition of PLK1, and progress has been made in using compounds to elucidate mechanistic aspects of activity regulation and in determining roles of the PBD. Studies have resulted in proof of concept of in vivo efficacy suggesting promise for PBD binders in clinical development.


Asunto(s)
Proteínas de Ciclo Celular , Neoplasias , Humanos , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Neoplasias/tratamiento farmacológico , Diseño de Fármacos , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/química
11.
Eur J Med Chem ; 227: 113926, 2022 Jan 05.
Artículo en Inglés | MEDLINE | ID: mdl-34735919

RESUMEN

Polo-like kinase 1 (PLK1) is a serine/threonine-protein kinase involved in cell cycle regulation and mitotic progression. Studies have shown that PLK1 is upregulated in many tumors and high levels are adversely related to a poor prognosis. Knocking down or inhibiting PLK1 results in synthetic lethality in PTEN deficient prostate tumors and Kras mutant colorectal tumors, further validating PLK1 as an oncotarget. Substrate recognition by PLK1 occurs through the Polo-Box Domain (PBD), which is a phospho-peptide binding site also responsible for subcellular localization. Much effort has been directed to target this kinase therapeutically through the ATP-binding site, and a few such inhibitors have advanced to clinical trials however with limited clinical efficacy. Moreover, it has been shown that a point mutation in PLK1 (C67V) confers dramatic cellular resistance to catalytic site inhibitors. An alternative approach to target PLK1 potently and selectively is through the PBD to block its protein-protein interactions. Through the REPLACE strategy, for converting peptide inhibitors into more drug-like non peptidic compounds, a PBD targeting compound series ("ABBAs"), has been identified and the key determinants of potency and selectivity elucidated through structure-activity relationship studies. In cellular experiments, the ABBAs were shown to lead to profound effects on the cell cycle, to inhibit tumor proliferation and overcome resistance of cells expressing the PLK1 C67V mutant to ATP-based inhibitors. These non-ATP competitive inhibitors of PLK1 were also used chemical biology probes to investigate the gene regulatory effects of PLK1, known to act on transcription factors such as p53.


Asunto(s)
Adenosina Trifosfato/farmacología , Antineoplásicos/farmacología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Adenosina Trifosfato/síntesis química , Adenosina Trifosfato/química , Antineoplásicos/síntesis química , Antineoplásicos/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Ligandos , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Relación Estructura-Actividad , Quinasa Tipo Polo 1
12.
J Med Chem ; 65(4): 3420-3433, 2022 02 24.
Artículo en Inglés | MEDLINE | ID: mdl-35114084

RESUMEN

Senexins are potent and selective quinazoline inhibitors of CDK8/19 Mediator kinases. To improve their potency and metabolic stability, quinoline-based derivatives were designed through a structure-guided strategy based on the simulated drug-target docking model of Senexin A and Senexin B. A library of quinoline-Senexin derivatives was synthesized to explore the structure-activity relationship (SAR). An optimized compound 20a (Senexin C) exhibits potent CDK8/19 inhibitory activity with high selectivity. Senexin C is more metabolically stable and provides a more sustained inhibition of CDK8/19-dependent cellular gene expression when compared with the prototype inhibitor Senexin B. In vivo pharmacokinetic (PK) and pharmacodynamic (PD) evaluation using a novel tumor-based PD assay showed good oral bioavailability of Senexin C with a strong tumor-enrichment PK profile and tumor-PD marker responses. Senexin C inhibits MV4-11 leukemia growth in a systemic in vivo model with good tolerability.


Asunto(s)
Antineoplásicos/farmacología , Antineoplásicos/farmacocinética , Neoplasias del Colon/tratamiento farmacológico , Quinasa 8 Dependiente de Ciclina/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/farmacocinética , Animales , Antineoplásicos/uso terapéutico , Disponibilidad Biológica , Línea Celular Tumoral , Humanos , Leucemia/tratamiento farmacológico , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Simulación del Acoplamiento Molecular , Estructura Molecular , Inhibidores de Proteínas Quinasas/toxicidad , Quinolinas , Relación Estructura-Actividad , Especificidad por Sustrato , Ensayos Antitumor por Modelo de Xenoinjerto
13.
J Med Chem ; 64(14): 9916-9925, 2021 07 22.
Artículo en Inglés | MEDLINE | ID: mdl-34210138

RESUMEN

PLK1, polo-like kinase 1, is a central player regulating mitosis. Inhibition of the subcellular localization and kinase activity of PLK1 through the PBD, polo-box domain, is a viable alternative to ATP-competitive inhibitors, for which the development of resistance and inhibition of related PLK family members are concerns. We describe novel nonpeptidic PBD-binding inhibitors, termed abbapolins, identified through successful application of the REPLACE strategy and demonstrate their potent antiproliferative activity in prostate tumors and other cell lines. Furthermore, abbapolins show PLK1-specific binding and inhibitory activity, as measured by a cellular thermal shift assay and an ability to block phosphorylation of TCTP, a validated target of PLK1-mediated kinase activity. Additional evidence for engagement of PLK1 was obtained through the unique observation that abbapolins induce PLK1 degradation in a manner that closely matches antiproliferative activity. Moreover, abbapolins demonstrate antiproliferative activity in cells that are dramatically resistant to ATP-competitive PLK1 inhibitors.


Asunto(s)
Antineoplásicos/farmacología , Ácido Benzoico/farmacología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Ácido Benzoico/síntesis química , Ácido Benzoico/química , Proteínas de Ciclo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Células HeLa , Humanos , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad , Proteína Tumoral Controlada Traslacionalmente 1 , Quinasa Tipo Polo 1
14.
Oncogene ; 39(21): 4155-4169, 2020 05.
Artículo en Inglés | MEDLINE | ID: mdl-32269299

RESUMEN

The RAF kinases activated by RAS GTPases regulate cell growth and division by signal transduction through the ERK cascade and mutations leading to constitutive activity are key drivers of human tumors, as are upstream activators including RAS and receptor tyrosine kinases. The development of first-generation RAF inhibitors, including vemurafenib (VEM) and dabrafenib led to initial excitement due to high response rates and profound regression of malignant melanomas carrying BRAFV600E mutations. The excitement about these unprecedented response rates, however, was tempered by tumor unresponsiveness through both intrinsic and acquired drug-resistance mechanisms. In recent years much insight into the complexity of the RAS-RAF axis has been obtained and inactivation and signal transduction mechanisms indicate that RAF dimerization is a critical step in multiple cellular contexts and plays a key role in resistance. Both homo- and hetero-dimerization of BRAF and CRAF can modulate therapeutic response and disease progression in patients treated with ATP-competitive inhibitors and are therefore highly clinically significant. Ten years after the definition of the RAF dimer interface (DIF) by crystallography, this review focuses on the implications of RAF kinase dimerization in signal transduction and for drug development, both from a classical ATP-competitive standpoint and from the perspective of new therapeutic strategies including inhibiting dimer formation. A structural perspective of the DIF, how dimerization impacts inhibitor activation and the structure-based design of next-generation RAF kinase inhibitors with unique mechanisms of action is presented. We also discuss potential fields of application for DIF inhibitors, ranging from non-V600E oncoproteins and BRAF fusions to tumors driven by aberrant receptor tyrosine kinase or RAS signaling.


Asunto(s)
Descubrimiento de Drogas , Melanoma , Mutación Missense , Inhibidores de Proteínas Quinasas/uso terapéutico , Multimerización de Proteína/efectos de los fármacos , Proteínas Proto-Oncogénicas B-raf , Transducción de Señal/efectos de los fármacos , Sustitución de Aminoácidos , Humanos , Melanoma/tratamiento farmacológico , Melanoma/enzimología , Melanoma/genética , Melanoma/patología , Multimerización de Proteína/genética , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Transducción de Señal/genética , Proteínas ras/genética , Proteínas ras/metabolismo
15.
ChemMedChem ; 15(12): 1058-1066, 2020 06 17.
Artículo en Inglés | MEDLINE | ID: mdl-32232973

RESUMEN

The polo-box domain (PBD) of PLK1 determines mitotic substrate recognition and subcellular localization. Compounds that target PLK1 selectively are required due to the tumor-suppressor roles of PLK3. A structure-activity analysis of the PBD phosphopeptide binding motif has identified potent peptides that delineate the determinants required for mimicry by nonpeptidic inhibitors and provide insights into the structural basis for the selectivity of inhibitors for the PLK1 PBD. Fragment-ligated inhibitory peptides (FLIPs) obtained through REPLACE have been optimized to enhance in vitro binding and a systematic analysis of selectivity for PLK1 vs PLK3 has been carried out for peptides and peptidomimetics. Furthermore, these more drug-like non-ATP-competitive inhibitors had on-target engagement in a cellular context, as evidenced by stabilization of PLK1 in a thermal-shift assay and by inhibition of the phosphorylation of TCTP, a target of PLK1. Investigation in cells expressing a mutant PLK1 showed that these cells are sensitive to PBD inhibitors but dramatically resistant to clinically investigated ATP-competitive compounds. These results further validate targeting the PBD binding site in the move towards PLK1 inhibitors that are active against tumors resistant to ATP inhibitors.


Asunto(s)
Proteínas de Ciclo Celular/antagonistas & inhibidores , Péptidos/farmacología , Peptidomiméticos/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Supresoras de Tumor/metabolismo , Proteínas de Ciclo Celular/metabolismo , Humanos , Simulación del Acoplamiento Molecular , Estructura Molecular , Células PC-3 , Péptidos/química , Péptidos/metabolismo , Peptidomiméticos/química , Peptidomiméticos/metabolismo , Unión Proteica , Dominios Proteicos , Proteínas Proto-Oncogénicas/metabolismo , Relación Estructura-Actividad , Proteína Tumoral Controlada Traslacionalmente 1 , Quinasa Tipo Polo 1
16.
BMC Mol Biol ; 10: 27, 2009 Mar 27.
Artículo en Inglés | MEDLINE | ID: mdl-19327148

RESUMEN

BACKGROUND: The homologous recombination (HR) pathway is vital for maintaining genomic integrity through the restoration of double-stranded breaks and interstrand crosslinks. The RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3) are essential for this process in vertebrates, and the RAD51D paralog is unique in that it participates in both HR repair and telomere maintenance. RAD51D is also known to directly interact with the RAD51C and XRCC2 proteins. Rad51d splice variants have been reported in mouse and human tissues, supportive of a role for alternative splicing in HR regulation. The present study evaluated the interaction of the Rad51d splice isoform products with RAD51C and XRCC2 and their expression patterns. RESULTS: Yeast-2-hybrid analysis was used to determine that the Mus musculus Rad51d splice variant product RAD51DDelta7b (deleted for residues 219 through 223) was capable of interacting with both RAD51C and XRCC2 and that RAD51D+int3 interacted with XRCC2. In addition, the linker region (residues 54 through 77) of RAD51D was identified as a region that potentially mediates binding with XRCC2. Cellular localization, detected by EGFP fusion proteins, demonstrated that each of the splice variant products tested was distributed throughout the cell similar to the full-length protein. However, none of the splice variants were capable of restoring resistance of Rad51d-deficient cell lines to mitomycin C. RT-PCR expression analysis revealed that Rad51dDelta3 (deleted for exon 3) and Rad51dDelta5 (deleted for exon 5)transcripts display tissue specific expression patterns with Rad51dDelta3 being detected in each tissue except ovary and Rad51dDelta5 not detected in mammary gland and testis. These expression studies also led to the identification of two additional Rad51d ubiquitously expressed transcripts, one deleted for both exon 9 and 10 and one deleted for only exon 10. CONCLUSION: These results suggest Rad51d alternative splice variants potentially modulate mechanisms of HR by sequestering either RAD51C or XRCC2.


Asunto(s)
Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Secuencia de Aminoácidos , Animales , Mapeo Cromosómico , Proteínas de Unión al ADN/metabolismo , Prueba de Complementación Genética , Ratones , Isoformas de Proteínas/química , Estructura Terciaria de Proteína , Recombinasa Rad51/metabolismo , Recombinación Genética/fisiología , Eliminación de Secuencia
17.
J Med Chem ; 62(8): 3886-3897, 2019 04 25.
Artículo en Inglés | MEDLINE | ID: mdl-30977659

RESUMEN

Despite the clinical success of BRAF inhibitors like vemurafenib in treating metastatic melanoma, resistance has emerged through "paradoxical MEK/ERK signaling" where transactivation of one protomer occurs as a result of drug inhibition of the other partner in the activated dimer. The importance of the dimerization interface in the signaling potential of wild-type BRAF in cells expressing oncogenic Ras has recently been demonstrated and proposed as a site of therapeutic intervention in targeting cancers resistant to adenosine triphosphate competitive drugs. The proof of concept for a structure-guided approach targeting the dimerization interface is described through the design and synthesis of macrocyclic peptides that bind with high affinity to BRAF and that block paradoxical signaling in malignant melanoma cells occurring through this drug target. The lead compounds identified are type-IV kinase inhibitors and represent an ideal framework for conversion into next-generation BRAF inhibitors through macrocyclic drug discovery.


Asunto(s)
Diseño de Fármacos , Inhibidores de Proteínas Quinasas/síntesis química , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Línea Celular , Dimerización , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Quinasas Quinasa Quinasa PAM/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Simulación de Dinámica Molecular , Péptidos/química , Péptidos/metabolismo , Péptidos/farmacología , Unión Proteica , Inhibidores de Proteínas Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/metabolismo , Relación Estructura-Actividad
18.
Drug Discov Today ; 13(19-20): 875-81, 2008 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-18639646

RESUMEN

The increase in understanding of the events of cell growth and division has enabled the development of pharmacological agents inhibiting key regulatory proteins, with the cyclin dependent kinases (CDKs) representing a major area of interest. Owing to multiple CDK variants having cell cycle and transcriptional regulatory roles and difficulties in generating selective inhibitors, the prospects for drug discovery and development are complex. Numerous CDK inhibitors with differing mechanistic profiles are currently being preclinically and clinically evaluated but have not as of yet resulted in a drug approval. The major issues of CDK inhibition related to current understanding from genetic studies and also from observed anti-tumor efficacy of representative compounds are discussed in this review.


Asunto(s)
Antineoplásicos/farmacología , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Animales , Ciclo Celular/efectos de los fármacos , Ciclo Celular/fisiología , Quinasas Ciclina-Dependientes/genética , Evaluación Preclínica de Medicamentos , Humanos
19.
Curr Opin Chem Biol ; 11(5): 494-502, 2007 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-17936059

RESUMEN

The identification of novel therapeutic targets and characterization of their 3D structures is increasing at a dramatic rate. Computational screening methods continue to be developed and improved as credible and complementary alternatives to high-throughput biochemical compound screening (HTS). While the majority of drug candidates currently being developed have been found using HTS methods, high-throughput docking and pharmacophore-based searching algorithms are gaining acceptance and becoming a major source of lead molecules in drug discovery. Refinements and optimization of high-throughput docking methods have lead to improvements in reproducing experimental data and in hit rates obtained, validating their use in hit identification. In parallel with virtual screening methods, concomitant developments in cheminformatics including identification, design and manipulation of drug-like small molecule libraries have been achieved. Herein, currently used in silico screening techniques and their utility on a comparative and target dependent basis is discussed.


Asunto(s)
Simulación por Computador , Evaluación Preclínica de Medicamentos/métodos , Algoritmos , Biología Computacional , Ligandos , Unión Proteica
20.
Cell Chem Biol ; 25(9): 1107-1116.e4, 2018 09 20.
Artículo en Inglés | MEDLINE | ID: mdl-30017915

RESUMEN

The polo kinase family are important oncology targets that act in regulating entry into and progression through mitosis. Structure-guided discovery of a new class of inhibitors of Polo-like kinase 1 (PLK1) catalytic activity that interact with Cys67 of the ATP binding site is described. Compounds containing the benzothiazole N-oxide scaffold not only bind covalently to this residue, but are reversible inhibitors through the formation of Meisenheimer complexes. This mechanism of kinase inhibition results in compounds that can target PLK1 with high selectivity, while avoiding issues with irreversible covalent binding and interaction with other thiol-containing molecules in the cell. Due to renewed interest in covalent drugs and the plethora of potential drug targets, these represent prototypes for the design of kinase inhibitory compounds that achieve high specificity through covalent interaction and yet still bind reversibly to the ATP cleft, a strategy that could be applied to avoid issues with conventional covalent binders.


Asunto(s)
Adenosina Trifosfato/metabolismo , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/metabolismo , Diseño de Fármacos , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/metabolismo , Benzotiazoles/química , Benzotiazoles/farmacología , Sitios de Unión/efectos de los fármacos , Dominio Catalítico/efectos de los fármacos , Proteínas de Ciclo Celular/química , Descubrimiento de Drogas , Células HeLa , Humanos , Simulación del Acoplamiento Molecular , Proteínas Serina-Treonina Quinasas/química , Proteínas Proto-Oncogénicas/química , Pteridinas/química , Pteridinas/farmacología , Quinasa Tipo Polo 1
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