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1.
J Med Chem ; 67(4): 3112-3126, 2024 02 22.
Artículo en Inglés | MEDLINE | ID: mdl-38325398

RESUMEN

CDK2 is a critical regulator of the cell cycle. For a variety of human cancers, the dysregulation of CDK2/cyclin E1 can lead to tumor growth and proliferation. Historically, early efforts to develop CDK2 inhibitors with clinical applications proved unsuccessful due to challenges in achieving selectivity over off-target CDK isoforms with associated toxicity. In this report, we describe the discovery of (4-pyrazolyl)-2-aminopyrimidines as a potent class of CDK2 inhibitors that display selectivity over CDKs 1, 4, 6, 7, and 9. SAR studies led to the identification of compound 17, a kinase selective and highly potent CDK2 inhibitor (IC50 = 0.29 nM). The evaluation of 17 in CCNE1-amplified mouse models shows the pharmacodynamic inhibition of CDK2, measured by reduced Rb phosphorylation, and antitumor activity.


Asunto(s)
Quinasas Ciclina-Dependientes , Neoplasias , Animales , Humanos , Ratones , Quinasa 2 Dependiente de la Ciclina , Quinasa 4 Dependiente de la Ciclina/metabolismo , Fosforilación , Pirimidinas/farmacología , Pirazoles/química , Pirazoles/metabolismo , Pirazoles/farmacología
2.
J Med Chem ; 65(22): 15433-15442, 2022 11 24.
Artículo en Inglés | MEDLINE | ID: mdl-36356320

RESUMEN

Upregulation of the fibroblast growth factor receptor (FGFR) signaling pathway has been implicated in multiple cancer types, including cholangiocarcinoma and bladder cancer. Consequently, small molecule inhibition of FGFR has emerged as a promising therapy for patients suffering from these diseases. First-generation pan-FGFR inhibitors, while highly effective, suffer from several drawbacks. These include treatment-related hyperphosphatemia and significant loss of potency for the mutant kinases. Herein, we present the discovery and optimization of novel FGFR2/3 inhibitors that largely maintain potency for the common gatekeeper mutants and have excellent selectivity over FGFR1. A combination of meticulous structure-activity relationship (SAR) analysis, structure-based drug design, and medicinal chemistry rationale ultimately led to compound 29, a potent and selective FGFR2/3 inhibitor with excellent in vitro absorption, distribution, metabolism, excretion (ADME), and pharmacokinetics in rat. A pharmacodynamic study of a closely related compound established that maximum inhibition of downstream ERK phosphorylation could be achieved with no significant effect on serum phosphate levels relative to vehicle.


Asunto(s)
Neoplasias , Inhibidores de Proteínas Quinasas , Receptores de Factores de Crecimiento de Fibroblastos , Animales , Ratas , Neoplasias/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/química , Transducción de Señal , Relación Estructura-Actividad , Receptores de Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Receptores de Factores de Crecimiento de Fibroblastos/química , Receptores de Factores de Crecimiento de Fibroblastos/efectos de los fármacos
3.
Nat Commun ; 10(1): 3117, 2019 07 15.
Artículo en Inglés | MEDLINE | ID: mdl-31308367

RESUMEN

The DNA translocation activity of the minichromosome maintenance (MCM) complex powers DNA strand separation of the replication forks of eukaryotes and archaea. Here we illustrate an atomic level mechanism for this activity with a crystal structure of an archaeal MCM hexamer bound to single-stranded DNA and nucleotide cofactors. Sequence conservation indicates this rotary mechanism is fully possible for all eukaryotes and archaea. The structure definitively demonstrates the ring orients during translocation with the N-terminal domain leading, indicating that the translocation activity could also provide the physical basis of replication initiation where a double-hexamer idly encircling double-stranded DNA transforms to single-hexamers that encircle only one strand. In this mechanism, each strand binds to the N-terminal tier of one hexamer and the AAA+ tier of the other hexamer such that one ring pulls on the other, aligning equivalent interfaces to enable each hexamer to pull its translocation strand outside of the opposing hexamer.


Asunto(s)
Replicación del ADN , Proteínas de Mantenimiento de Minicromosoma/química , Sulfolobus solfataricus/genética , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfatasas/fisiología , Cristalografía por Rayos X , ADN de Archaea/química , Proteínas de Mantenimiento de Minicromosoma/fisiología , Translocación Genética
4.
J Mol Biol ; 427(9): 1779-1796, 2015 May 08.
Artículo en Inglés | MEDLINE | ID: mdl-25724843

RESUMEN

The DEAD-box RNA helicase DDX3X is frequently mutated in pediatric medulloblastoma. We dissect how these mutants affect DDX3X function with structural, biochemical, and genetic experiments. We identify an N-terminal extension ("ATP-binding loop", ABL) that is critical for the stimulation of ATP hydrolysis by RNA. We present crystal structures suggesting that the ABL interacts dynamically with ATP and confirming that the interaction occurs in solution by NMR chemical shift perturbation and isothermal titration calorimetry. DEAD-box helicases require interaction between two conserved RecA-like helicase domains, D1 and D2 for function. We use NMR chemical shift perturbation to show that DDX3X interacts specifically with double-stranded RNA through its D1 domain, with contact mediated by residues G302 and G325. Mutants of these residues, G302V and G325E, are associated with pediatric medulloblastoma. These mutants are defective in RNA-stimulated ATP hydrolysis. We show that DDX3X complements the growth defect in a ded1 temperature-sensitive strain of Schizosaccharomyces pombe, but the cancer-associated mutants G302V and G325E do not complement and exhibit protein expression defects. Taken together, our results suggest that impaired translation of important mRNA targets by mutant DDX3X represents a key step in the development of medulloblastoma.


Asunto(s)
Adenosina Trifosfato/metabolismo , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Meduloblastoma/genética , Mutación/genética , ARN/genética , Sitios de Unión , Neoplasias Cerebelosas/genética , Niño , Cristalización , Cristalografía por Rayos X , ARN Helicasas DEAD-box/química , Prueba de Complementación Genética , Humanos , Hidrólisis , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Resonancia Magnética Nuclear Biomolecular , Biosíntesis de Proteínas , Conformación Proteica , Schizosaccharomyces/crecimiento & desarrollo , Schizosaccharomyces/metabolismo
5.
Elife ; 3: e03433, 2014 Sep 29.
Artículo en Inglés | MEDLINE | ID: mdl-25262915

RESUMEN

In a previous Research article (Froelich et al., 2014), we suggested an MCM helicase activation mechanism, but were limited in discussing the ATPase domain because it was absent from the crystal structure. Here we present the crystal structure of a nearly full-length MCM hexamer that is helicase-active and thus has all features essential for unwinding DNA. The structure is a chimera of Sulfolobus solfataricus N-terminal domain and Pyrococcus furiosus ATPase domain. We discuss three major findings: 1) a novel conformation for the A-subdomain that could play a role in MCM regulation; 2) interaction of a universally conserved glutamine in the N-terminal Allosteric Communication Loop with the AAA+ domain helix-2-insert (h2i); and 3) a recessed binding pocket for the MCM ssDNA-binding motif influenced by the h2i. We suggest that during helicase activation, the h2i clamps down on the leading strand to facilitate strand retention and regulate ATP hydrolysis.


Asunto(s)
Proteínas de Mantenimiento de Minicromosoma/química , Multimerización de Proteína , Adenosina Difosfato/química , Adenosina Trifosfatasas/química , Regulación Alostérica , Secuencia de Aminoácidos , Dominio Catalítico , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Pyrococcus furiosus/enzimología , Proteínas Recombinantes de Fusión/química , Sulfolobus solfataricus/enzimología
6.
Elife ; 3: e01993, 2014 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-24692448

RESUMEN

The ring-shaped MCM helicase is essential to all phases of DNA replication. The complex loads at replication origins as an inactive double-hexamer encircling duplex DNA. Helicase activation converts this species to two active single hexamers that encircle single-stranded DNA (ssDNA). The molecular details of MCM DNA interactions during these events are unknown. We determined the crystal structure of the Pyrococcus furiosus MCM N-terminal domain hexamer bound to ssDNA and define a conserved MCM-ssDNA binding motif (MSSB). Intriguingly, ssDNA binds the MCM ring interior perpendicular to the central channel with defined polarity. In eukaryotes, the MSSB is conserved in several Mcm2-7 subunits, and MSSB mutant combinations in S. cerevisiae Mcm2-7 are not viable. Mutant Mcm2-7 complexes assemble and are recruited to replication origins, but are defective in helicase loading and activation. Our findings identify an important MCM-ssDNA interaction and suggest it functions during helicase activation to select the strand for translocation. DOI: http://dx.doi.org/10.7554/eLife.01993.001.


Asunto(s)
Secuencia Conservada , Replicación del ADN/fisiología , ADN de Cadena Simple/metabolismo , Proteínas de Mantenimiento de Minicromosoma/fisiología , Secuencia de Aminoácidos , Cristalografía por Rayos X , ADN de Cadena Simple/química , Proteínas de Mantenimiento de Minicromosoma/química , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Datos de Secuencia Molecular , Conformación Proteica , Pyrococcus furiosus/enzimología , Saccharomyces cerevisiae/enzimología , Homología de Secuencia de Aminoácido
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