RESUMEN
The cell division cycle 25 phosphatases CDC25A, B and C regulate cell cycle transitions by dephosphorylating residues in the conserved glycine-rich loop of CDKs to activate their activity. Here, we present the cryo-EM structure of CDK2-cyclin A in complex with CDC25A at 2.7 Å resolution, providing a detailed structural analysis of the overall complex architecture and key protein-protein interactions that underpin this 86 kDa complex. We further identify a CDC25A C-terminal helix that is critical for complex formation. Sequence conservation analysis suggests CDK1/2-cyclin A, CDK1-cyclin B and CDK2/3-cyclin E are suitable binding partners for CDC25A, whilst CDK4/6-cyclin D complexes appear unlikely substrates. A comparative structural analysis of CDK-containing complexes also confirms the functional importance of the conserved CDK1/2 GDSEID motif. This structure improves our understanding of the roles of CDC25 phosphatases in CDK regulation and may inform the development of CDC25-targeting anticancer strategies.
Asunto(s)
Microscopía por Crioelectrón , Ciclina A , Quinasa 2 Dependiente de la Ciclina , Fosfatasas cdc25 , Fosfatasas cdc25/metabolismo , Fosfatasas cdc25/química , Fosfatasas cdc25/ultraestructura , Fosfatasas cdc25/genética , Quinasa 2 Dependiente de la Ciclina/metabolismo , Quinasa 2 Dependiente de la Ciclina/química , Quinasa 2 Dependiente de la Ciclina/ultraestructura , Humanos , Ciclina A/metabolismo , Ciclina A/química , Unión Proteica , Modelos Moleculares , Secuencia de AminoácidosRESUMEN
Fragment-based drug discovery (FBDD) has brought several drugs to the clinic, notably to target proteins once considered to be challenging, or even undruggable. Screening in FBDD relies upon observing and/or measuring weak (millimolar-scale) binding events using biophysical techniques or crystallographic fragment screening. This latter structural approach provides no information about binding affinity but can reveal binding mode and atomic detail on protein-fragment interactions to accelerate hit-to-lead development. In recent years, high-throughput platforms have been developed at synchrotron facilities to screen thousands of fragment-soaked crystals. However, using accessible manual techniques it is possible to run informative, smaller-scale screens within an academic lab setting. This chapter describes general protocols for home laboratory-scale fragment screening, from fragment soaking through to structure solution and, where appropriate, signposts to background, protocols or alternatives elsewhere.
Asunto(s)
Detección Precoz del Cáncer , Neoplasias , Cristalografía por Rayos X , Descubrimiento de Drogas/métodos , Proteínas , Evaluación Preclínica de Medicamentos/métodosRESUMEN
p27KIP1 (cyclin-dependent kinase inhibitor 1B, p27) is a member of the CIP/KIP family of CDK (cyclin dependent kinase) regulators that inhibit cell cycle CDKs. p27 phosphorylation by CDK1/2, signals its recruitment to the SCFSKP2 (S-phase kinase associated protein 1 (SKP1)-cullin-SKP2) E3 ubiquitin ligase complex for proteasomal degradation. The nature of p27 binding to SKP2 and CKS1 was revealed by the SKP1-SKP2-CKS1-p27 phosphopeptide crystal structure. Subsequently, a model for the hexameric CDK2-cyclin A-CKS1-p27-SKP1-SKP2 complex was proposed by overlaying an independently determined CDK2-cyclin A-p27 structure. Here we describe the experimentally determined structure of the isolated CDK2-cyclin A-CKS1-p27-SKP1-SKP2 complex at 3.4 Å global resolution using cryogenic electron microscopy. This structure supports previous analysis in which p27 was found to be structurally dynamic, transitioning from disordered to nascent secondary structure on target binding. We employed 3D variability analysis to further explore the conformational space of the hexameric complex and uncovered a previously unidentified hinge motion centred on CKS1. This flexibility gives rise to open and closed conformations of the hexameric complex that we propose may contribute to p27 regulation by facilitating recognition with SCFSKP2. This 3D variability analysis further informed particle subtraction and local refinement approaches to enhance the local resolution of the complex.
Asunto(s)
Quinasas CDC2-CDC28 , Proteínas Quinasas Asociadas a Fase-S , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Ciclina A/metabolismo , Microscopía por Crioelectrón , Quinasas Ciclina-Dependientes/metabolismoRESUMEN
Ordered protein phosphorylation by CDKs is a key mechanism for regulating the cell cycle. How temporal order is enforced in mammalian cells remains unclear. Using a fixed cell kinase assay and phosphoproteomics, we show how CDK1 activity and non-catalytic CDK1 subunits contribute to the choice of substrate and site of phosphorylation. Increases in CDK1 activity alter substrate choice, with intermediate- and low-sensitivity CDK1 substrates enriched in DNA replication and mitotic functions, respectively. This activity dependence is shared between Cyclin A- and Cyclin B-CDK1. Cks1 has a proteome-wide role as an enhancer of multisite CDK1 phosphorylation. Contrary to the model of CDK1 as an exclusively proline-directed kinase, we show that Cyclin A and Cks1 enhance non-proline-directed phosphorylation, preferably on sites with a +3 lysine residue. Indeed, 70% of cell-cycle-regulated phosphorylations, where the kinase carrying out this modification has not been identified, are non-proline-directed CDK1 sites.
Asunto(s)
Proteína Quinasa CDC2 , Ciclina A , Animales , Fosforilación , Ciclina A/metabolismo , Consenso , Proteína Quinasa CDC2/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , División Celular , Mitosis , Mamíferos/metabolismoRESUMEN
Aberrant activity of the cyclin-dependent kinase family is frequently noted in a number of diseases identifying them as potential targets for drug development. However, current CDK inhibitors lack specificity owing to the high sequence and structural conservation of the ATP binding cleft across family members, highlighting the necessity of finding novel modes of CDK inhibition. The wealth of structural information regarding CDK assemblies and inhibitor complexes derived from X-ray crystallographic studies has been recently complemented through the use of cryo-electron microscopy. These recent advances have provided insights into the functional roles and regulatory mechanisms of CDKs and their interaction partners. This review explores the conformational malleability of the CDK subunit, the importance of SLiM recognition sites in CDK complexes, the progress made in chemically induced CDK degradation and how these studies can contribute to CDK inhibitor design. Additionally, fragment-based drug discovery can be utilised to identify small molecules that bind to allosteric sites on the CDK surface employing interactions which mimic those of native protein-protein interactions. These recent structural advances in CDK inhibitor mechanisms and in chemical probes which do not occupy the orthosteric ATP binding site can provide important insights for targeted CDK therapies.
RESUMEN
The development of ligands for biological targets is critically dependent on the identification of sites on proteins that bind molecules with high affinity. A set of compounds, called FragLites, can identify such sites, along with the interactions required to gain affinity, by X-ray crystallography. We demonstrate the utility of FragLites in mapping the binding sites of bromodomain proteins BRD4 and ATAD2 and demonstrate that FragLite mapping is comparable to a full fragment screen in identifying ligand binding sites and key interactions. We extend the FragLite set with analogous compounds derived from amino acids (termed PepLites) that mimic the interactions of peptides. The output of the FragLite maps is shown to enable the development of ligands with leadlike potency. This work establishes the use of FragLite and PepLite screening at an early stage in ligand discovery allowing the rapid assessment of tractability of protein targets and informing downstream hit-finding.
Asunto(s)
Proteínas Nucleares , Factores de Transcripción , Ligandos , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Dominios Proteicos , Sitios de Unión , Cristalografía por Rayos X , Péptidos/metabolismo , Unión Proteica , Proteínas de Ciclo Celular/metabolismoRESUMEN
The nonclassical extracellular signal-related kinase 5 (ERK5) mitogen-activated protein kinase pathway has been implicated in increased cellular proliferation, migration, survival, and angiogenesis; hence, ERK5 inhibition may be an attractive approach for cancer treatment. However, the development of selective ERK5 inhibitors has been challenging. Previously, we described the development of a pyrrole carboxamide high-throughput screening hit into a selective, submicromolar inhibitor of ERK5 kinase activity. Improvement in the ERK5 potency was necessary for the identification of a tool ERK5 inhibitor for target validation studies. Herein, we describe the optimization of this series to identify nanomolar pyrrole carboxamide inhibitors of ERK5 incorporating a basic center, which suffered from poor oral bioavailability. Parallel optimization of potency and in vitro pharmacokinetic parameters led to the identification of a nonbasic pyrazole analogue with an optimal balance of ERK5 inhibition and oral exposure.
Asunto(s)
Proteína Quinasa 7 Activada por Mitógenos , Pirroles , Proliferación Celular , Pirroles/farmacologíaRESUMEN
NF-κB-inducing kinase (NIK) is a key enzyme in the noncanonical NF-κB pathway, of interest in the treatment of a variety of diseases including cancer. Validation of NIK as a drug target requires potent and selective inhibitors. The protein contains a cysteine residue at position 444 in the back pocket of the active site, unique within the kinome. Analysis of existing inhibitor scaffolds and early structure-activity relationships (SARs) led to the design of C444-targeting covalent inhibitors based on alkynyl heterocycle warheads. Mass spectrometry provided proof of the covalent mechanism, and the SAR was rationalized by computational modeling. Profiling of more potent analogues in tumor cell lines with constitutively activated NIK signaling induced a weak antiproliferative effect, suggesting that kinase inhibition may have limited impact on cancer cell growth. This study shows that alkynyl heterocycles are potential cysteine traps, which may be employed where common Michael acceptors, such as acrylamides, are not tolerated.
Asunto(s)
Alquinos/farmacología , Cisteína/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Pirimidinas/farmacología , Alquinos/síntesis química , Alquinos/química , Cisteína/química , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Pirimidinas/síntesis química , Pirimidinas/química , Relación Estructura-Actividad , Quinasa de Factor Nuclear kappa BRESUMEN
Inhibition of murine double minute 2 (MDM2)-p53 protein-protein interaction with small molecules has been shown to reactivate p53 and inhibit tumor growth. Here, we describe rational, structure-guided, design of novel isoindolinone-based MDM2 inhibitors. MDM2 X-ray crystallography, quantum mechanics ligand-based design, and metabolite identification all contributed toward the discovery of potent in vitro and in vivo inhibitors of the MDM2-p53 interaction with representative compounds inducing cytostasis in an SJSA-1 osteosarcoma xenograft model following once-daily oral administration.
Asunto(s)
Antineoplásicos/farmacología , Isoindoles/farmacología , Osteosarcoma/tratamiento farmacológico , Multimerización de Proteína/efectos de los fármacos , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Antineoplásicos/síntesis química , Antineoplásicos/metabolismo , Neoplasias Óseas/tratamiento farmacológico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Estabilidad de Medicamentos , Femenino , Humanos , Isoindoles/síntesis química , Isoindoles/metabolismo , Macaca fascicularis , Masculino , Ratones Endogámicos BALB C , Ratones Desnudos , Microsomas Hepáticos/metabolismo , Estructura Molecular , Unión Proteica , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The SCFSKP2 ubiquitin ligase relieves G1 checkpoint control of CDK-cyclin complexes by promoting p27KIP1 degradation. We describe reconstitution of stable complexes containing SKP1-SKP2 and CDK1-cyclin B or CDK2-cyclin A/E, mediated by the CDK regulatory subunit CKS1. We further show that a direct interaction between a SKP2 N-terminal motif and cyclin A can stabilize SKP1-SKP2-CDK2-cyclin A complexes in the absence of CKS1. We identify the SKP2 binding site on cyclin A and demonstrate the site is not present in cyclin B or cyclin E. This site is distinct from but overlapping with features that mediate binding of p27KIP1 and other G1 cyclin regulators to cyclin A. We propose that the capacity of SKP2 to engage with CDK2-cyclin A by more than one structural mechanism provides a way to fine tune the degradation of p27KIP1 and distinguishes cyclin A from other G1 cyclins to ensure orderly cell cycle progression.
Asunto(s)
Ciclina A/química , Quinasa 2 Dependiente de la Ciclina/química , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/química , Puntos de Control de la Fase G1 del Ciclo Celular , Proteínas Quinasas Asociadas a Fase-S/química , Sitios de Unión , Quinasas CDC2-CDC28/química , Quinasas CDC2-CDC28/genética , Quinasas CDC2-CDC28/metabolismo , Ciclina A/genética , Ciclina A/metabolismo , Ciclina E/química , Ciclina E/genética , Ciclina E/metabolismo , Quinasa 2 Dependiente de la Ciclina/genética , Quinasa 2 Dependiente de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Regulación de la Expresión Génica , Células HEK293 , Humanos , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteolisis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Quinasas Asociadas a Fase-S/genética , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transducción de SeñalRESUMEN
Proteins of the cyclin family have divergent sequences and execute diverse roles within the cell while sharing a common fold: the cyclin box domain. Structural studies of cyclins have played a key role in our characterization and understanding of cellular processes that they control, though to date only ten of the 29 CDK-activating cyclins have been structurally characterized by X-ray crystallography or cryo-electron microscopy with or without their cognate kinases. In this review, we survey the available structures of human cyclins, highlighting their molecular features in the context of their cellular roles. We pay particular attention to how cyclin activity is regulated through fine control of degradation motif recognition and ubiquitination. Finally, we discuss the emergent roles of cyclins independent of their roles as cyclin-dependent protein kinase activators, demonstrating the cyclin box domain to be a versatile and generalized scaffolding domain for protein-protein interactions across the cellular machinery.
Asunto(s)
Ciclinas/química , Ciclinas/metabolismo , Animales , Humanos , Modelos Moleculares , Dominios Proteicos , Proteolisis , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Extracellular regulated kinase 5 (ERK5) signalling has been implicated in driving a number of cellular phenotypes including endothelial cell angiogenesis and tumour cell motility. Novel ERK5 inhibitors were identified using high throughput screening, with a series of pyrrole-2-carboxamides substituted at the 4-position with an aroyl group being found to exhibit IC50 values in the micromolar range, but having no selectivity against p38α MAP kinase. Truncation of the N-substituent marginally enhanced potency (â¼3-fold) against ERK5, but importantly attenuated inhibition of p38α. Systematic variation of the substituents on the aroyl group led to the selective inhibitor 4-(2-bromo-6-fluorobenzoyl)-N-(pyridin-3-yl)-1H-pyrrole-2-carboxamide (IC50 0.82⯵M for ERK5; IC50â¯>â¯120⯵M for p38α). The crystal structure (PDB 5O7I) of this compound in complex with ERK5 has been solved. This compound was orally bioavailable and inhibited bFGF-driven Matrigel plug angiogenesis and tumour xenograft growth. The selective ERK5 inhibitor described herein provides a lead for further development into a tool compound for more extensive studies seeking to examine the role of ERK5 signalling in cancer and other diseases.
Asunto(s)
Antineoplásicos/farmacología , Proteína Quinasa 14 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 7 Activada por Mitógenos/antagonistas & inhibidores , Proteínas Nucleares/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Factores de Transcripción/antagonistas & inhibidores , Administración Oral , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Disponibilidad Biológica , Proteínas de Ciclo Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Ratones , Ratones Desnudos , Proteína Quinasa 14 Activada por Mitógenos/metabolismo , Proteína Quinasa 7 Activada por Mitógenos/metabolismo , Estructura Molecular , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Proteínas Nucleares/metabolismo , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/química , Relación Estructura-Actividad , Factores de Transcripción/metabolismoRESUMEN
Identifying ligand binding sites on proteins is a critical step in target-based drug discovery. Current approaches to this require resource-intensive screening of large libraries of lead-like or fragment molecules. Here, we describe an efficient and effective experimental approach to mapping interaction sites using a set of halogenated compounds expressing paired hydrogen-bonding motifs, termed FragLites. The FragLites identify productive drug-like interactions, which are identified sensitively and unambiguously by X-ray crystallography, exploiting the anomalous scattering of the halogen substituent. This mapping of protein interaction surfaces provides an assessment of druggability and can identify efficient start points for the de novo design of hit molecules incorporating the interacting motifs. The approach is illustrated by mapping cyclin-dependent kinase 2, which successfully identifies orthosteric and allosteric sites. The hits were rapidly elaborated to develop efficient lead-like molecules. Hence, the approach provides a new method of identifying ligand sites, assessing tractability and discovering new leads.
Asunto(s)
Halogenación , Sitios de Unión , Cristalografía por Rayos X , Descubrimiento de Drogas/métodos , Evaluación Preclínica de Medicamentos , Ligandos , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
Dysregulation of the cell cycle characterizes many cancer subtypes, providing a rationale for developing cyclin-dependent kinase (CDK) inhibitors. Potent CDK2 inhibitors might target certain cancers in which CCNE1 is amplified. However, current CDK2 inhibitors also inhibit CDK1, generating a toxicity liability. We have used biophysical measurements and X-ray crystallography to investigate the ATP-competitive inhibitor binding properties of cyclin-free and cyclin-bound CDK1 and CDK2. We show that these kinases can readily be distinguished by such inhibitors when cyclin-free, but not when cyclin-bound. The basis for this discrimination is unclear from either inspection or molecular dynamics simulation of ligand-bound CDKs, but is reflected in the contacts made between the kinase N- and C-lobes. We conclude that there is a subtle but profound difference between the conformational energy landscapes of cyclin-free CDK1 and CDK2. The unusual properties of CDK1 might be exploited to differentiate CDK1 from other CDKs in future cancer therapeutic design.
Asunto(s)
Proteína Quinasa CDC2/antagonistas & inhibidores , Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Entropía , Inhibidores de Proteínas Quinasas/farmacología , Proteína Quinasa CDC2/aislamiento & purificación , Proteína Quinasa CDC2/metabolismo , Quinasa 2 Dependiente de la Ciclina/aislamiento & purificación , Quinasa 2 Dependiente de la Ciclina/metabolismo , Humanos , Conformación Molecular , Simulación de Dinámica Molecular , Inhibidores de Proteínas Quinasas/química , Resonancia por Plasmón de SuperficieRESUMEN
Since their characterization as conserved modules that regulate progression through the eukaryotic cell cycle, cyclin-dependent protein kinases (CDKs) in higher eukaryotic cells are now also emerging as significant regulators of transcription, metabolism and cell differentiation. The cyclins, though originally characterized as CDK partners, also have CDK-independent roles that include the regulation of DNA damage repair and transcriptional programmes that direct cell differentiation, apoptosis and metabolic flux. This review compares the structures of the members of the CDK and cyclin families determined by X-ray crystallography, and considers what mechanistic insights they provide to guide functional studies and distinguish CDK- and cyclin-specific activities. Aberrant CDK activity is a hallmark of a number of diseases, and structural studies can provide important insights to identify novel routes to therapy.
Asunto(s)
Quinasas Ciclina-Dependientes/química , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/química , Ciclinas/metabolismo , Animales , Sitios de Unión , Ciclo Celular , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Familia de Multigenes , Unión Proteica , Conformación ProteicaRESUMEN
ATAD2 is an ATPase that is overexpressed in a variety of cancers and associated with a poor patient prognosis. This protein has been suggested to function as a cofactor for a range of transcription factors, including the proto-oncogene MYC and the androgen receptor. ATAD2 comprises an ATPase domain, implicated in chromatin remodelling, and a bromodomain which allows it to interact with acetylated histone tails. Dissection of the functional roles of these two domains would benefit from the availability of selective, cell-permeable pharmacological probes. An in silico evaluation of the 3D structures of various bromodomains suggested that developing small molecule ligands for the bromodomain of ATAD2 is likely to be challenging, although recent reports have shown that ATAD2 bromodomain ligands can be identified. We report a structure-guided fragment-based approach to identify lead compounds for ATAD2 bromodomain inhibitor development. Our findings indicate that the ATAD2 bromodomain can accommodate fragment hits (Mr < 200) that yield productive structure-activity relationships, and structure-guided design enabled the introduction of selectivity over BRD4.
Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas/antagonistas & inhibidores , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/metabolismo , Diseño de Fármacos , Proteínas Nucleares/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Factores de Transcripción/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas/química , Proteínas de Ciclo Celular , Diseño Asistido por Computadora , Proteínas de Unión al ADN/química , Humanos , Ligandos , Simulación del Acoplamiento Molecular , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Proteínas Nucleares/química , Unión Proteica , Dominios Proteicos/efectos de los fármacos , Proto-Oncogenes Mas , Factores de Transcripción/químicaRESUMEN
Selective recruitment of protein kinases to the Hsp90 system is mediated by the adaptor co-chaperone Cdc37. We show that assembly of CDK4 and CDK6 into protein complexes is differentially regulated by the Cdc37-Hsp90 system. Like other Hsp90 kinase clients, binding of CDK4/6 to Cdc37 is blocked by ATP-competitive inhibitors. Cdc37-Hsp90 relinquishes CDK6 to D3- and virus-type cyclins and to INK family CDK inhibitors, whereas CDK4 is relinquished to INKs but less readily to cyclins. p21CIP1 and p27KIP1 CDK inhibitors are less potent than the INKs at displacing CDK4 and CDK6 from Cdc37. However, they cooperate with the D-type cyclins to generate CDK4/6-containing ternary complexes that are resistant to cyclin D displacement by Cdc37, suggesting a molecular mechanism to explain the assembly factor activity ascribed to CIP/KIP family members. Overall, our data reveal multiple mechanisms whereby the Hsp90 system may control formation of CDK4- and CDK6-cyclin complexes under different cellular conditions.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Chaperoninas/metabolismo , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 6 Dependiente de la Ciclina/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Aminopiridinas/química , Aminopiridinas/metabolismo , Bencimidazoles/metabolismo , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Chaperoninas/antagonistas & inhibidores , Chaperoninas/genética , Ciclina D/metabolismo , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Proteínas HSP90 de Choque Térmico/genética , Humanos , Concentración 50 Inhibidora , Cinética , Piperazinas/química , Piperazinas/metabolismo , Unión Proteica , Purinas/química , Purinas/metabolismo , Piridinas/química , Piridinas/metabolismo , Resonancia por Plasmón de SuperficieRESUMEN
The cell fate-determining roles played by members of the cyclin-dependent protein kinase (CDK) family explain why their dysregulation can promote proliferative diseases, and identify them as potential targets for drug discovery in oncology and beyond. After many years of research, the first efficacious CDK inhibitors have now been registered for clinical use in a defined segment of breast cancer. Research is underway to identify inhibitors with appropriate CDK-inhibitory profiles to recapitulate this success in other disease settings. Here, we review the structural data that illustrate the interactions and properties that confer upon inhibitors affinity and/or selectivity toward different CDK family members. We conclude that where CDK inhibitors display selectivity, that selectivity derives from exploiting active site sequence peculiarities and/or from the capacity of the target CDK(s) to access conformations compatible with optimizing inhibitor-target interactions.
Asunto(s)
Antineoplásicos/farmacología , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Descubrimiento de Drogas/métodos , Simulación del Acoplamiento Molecular , Proteínas de Neoplasias/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Antineoplásicos/síntesis química , Dominio Catalítico , Quinasas Ciclina-Dependientes/química , Quinasas Ciclina-Dependientes/metabolismo , Humanos , Terapia Molecular Dirigida , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Neoplasias/genética , Neoplasias/patología , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Inhibidores de Proteínas Quinasas/síntesis química , Relación Estructura-ActividadRESUMEN
Purines and related heterocycles substituted at C-2 with 4'-sulfamoylanilino and at C-6 with a variety of groups have been synthesized with the aim of achieving selectivity of binding to CDK2 over CDK1. 6-Substituents that favor competitive inhibition at the ATP binding site of CDK2 were identified and typically exhibited 10-80-fold greater inhibition of CDK2 compared to CDK1. Most impressive was 4-((6-([1,1'-biphenyl]-3-yl)-9H-purin-2-yl)amino) benzenesulfonamide (73) that exhibited high potency toward CDK2 (IC50 0.044 µM) but was â¼2000-fold less active toward CDK1 (IC50 86 µM). This compound is therefore a useful tool for studies of cell cycle regulation. Crystal structures of inhibitor-kinase complexes showed that the inhibitor stabilizes a glycine-rich loop conformation that shapes the ATP ribose binding pocket and that is preferred in CDK2 but has not been observed in CDK1. This aspect of the active site may be exploited for the design of inhibitors that distinguish between CDK1 and CDK2.