Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 12 de 12
Filtrar
1.
Biochemistry ; 62(14): 2147-2160, 2023 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-37403936

RESUMO

Werner syndrome protein (WRN) is a multifunctional enzyme with helicase, ATPase, and exonuclease activities that are necessary for numerous DNA-related transactions in the human cell. Recent studies identified WRN as a synthetic lethal target in cancers characterized by genomic microsatellite instability resulting from defects in DNA mismatch repair pathways. WRN's helicase activity is essential for the viability of these high microsatellite instability (MSI-H) cancers and thus presents a therapeutic opportunity. To this end, we developed a multiplexed high-throughput screening assay that monitors exonuclease, ATPase, and helicase activities of full-length WRN. This screening campaign led to the discovery of 2-sulfonyl/sulfonamide pyrimidine derivatives as novel covalent inhibitors of WRN helicase activity. The compounds are specific for WRN versus other human RecQ family members and show competitive behavior with ATP. Examination of these novel chemical probes established the sulfonamide NH group as a key driver of compound potency. One of the leading compounds, H3B-960, showed consistent activities in a range of assays (IC50 = 22 nM, KD = 40 nM, KI = 32 nM), and the most potent compound identified, H3B-968, has inhibitory activity IC50 ∼ 10 nM. These kinetic properties trend toward other known covalent druglike molecules. Our work provides a new avenue for screening WRN for inhibitors that may be adaptable to different therapeutic modalities such as targeted protein degradation, as well as a proof of concept for the inhibition of WRN helicase activity by covalent molecules.


Assuntos
Neoplasias , Síndrome de Werner , Humanos , Exodesoxirribonucleases/genética , RecQ Helicases/genética , RecQ Helicases/metabolismo , Ensaios de Triagem em Larga Escala , Instabilidade de Microssatélites , Helicase da Síndrome de Werner/metabolismo
2.
J Biol Chem ; 298(11): 102539, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36179791

RESUMO

Recent studies have reported that the peroxisome proliferator-activated receptor gamma (PPARγ) pathway is activated in approximately 40% of patients with muscle-invasive bladder cancer. This led us to investigate pharmacological repression of PPARγ as a possible intervention strategy. Here, we characterize PPARγ antagonists and inverse agonists and find that the former behave as silent ligands, whereas inverse agonists (T0070907 and SR10221) repress downstream PPARγ target genes leading to growth inhibition in bladder cancer cell lines. To understand the mechanism, we determined the ternary crystal structure of PPARγ bound to T0070907 and the corepressor (co-R) peptide NCOR1. The structure shows that the AF-2 helix 12 (H12) rearranges to bind inside the ligand-binding domain, where it forms stabilizing interactions with the compound. This dramatic movement in H12 unveils a large interface for co-R binding. In contrast, the crystal structure of PPARγ bound to a SR10221 analog shows more subtle structural differences, where the compound binds and pushes H12 away from the ligand-binding domain to allow co-R binding. Interestingly, we found that both classes of compound promote recruitment of co-R proteins in biochemical assays but with distinct conformational changes in H12. We validate our structural models using both site-directed mutagenesis and chemical probes. Our findings offer new mechanistic insights into pharmacological modulation of PPARγ signaling.


Assuntos
PPAR gama , Neoplasias da Bexiga Urinária , Humanos , PPAR gama/metabolismo , Ligantes , Benzamidas/farmacologia
3.
J Biol Chem ; 290(2): 762-74, 2015 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-25391653

RESUMO

Cancer-associated point mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) confer a neomorphic enzymatic activity: the reduction of α-ketoglutarate to d-2-hydroxyglutaric acid, which is proposed to act as an oncogenic metabolite by inducing hypermethylation of histones and DNA. Although selective inhibitors of mutant IDH1 and IDH2 have been identified and are currently under investigation as potential cancer therapeutics, the mechanistic basis for their selectivity is not yet well understood. A high throughput screen for selective inhibitors of IDH1 bearing the oncogenic mutation R132H identified compound 1, a bis-imidazole phenol that inhibits d-2-hydroxyglutaric acid production in cells. We investigated the mode of inhibition of compound 1 and a previously published IDH1 mutant inhibitor with a different chemical scaffold. Steady-state kinetics and biophysical studies show that both of these compounds selectively inhibit mutant IDH1 by binding to an allosteric site and that inhibition is competitive with respect to Mg(2+). A crystal structure of compound 1 complexed with R132H IDH1 indicates that the inhibitor binds at the dimer interface and makes direct contact with a residue involved in binding of the catalytically essential divalent cation. These results show that targeting a divalent cation binding residue can enable selective inhibition of mutant IDH1 and suggest that differences in magnesium binding between wild-type and mutant enzymes may contribute to the inhibitors' selectivity for the mutant enzyme.


Assuntos
Descoberta de Drogas , Inibidores Enzimáticos/química , Isocitrato Desidrogenase/química , Neoplasias/tratamento farmacológico , Sítio Alostérico , Cristalografia por Raios X , Metilação de DNA/genética , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/uso terapêutico , Escherichia coli , Regulação Neoplásica da Expressão Gênica , Humanos , Isocitrato Desidrogenase/antagonistas & inibidores , Isocitrato Desidrogenase/biossíntese , Isocitrato Desidrogenase/genética , Magnésio/química , Proteínas Mutantes/química , Proteínas Mutantes/genética , Neoplasias/genética , Neoplasias/patologia , Conformação Proteica
4.
Nat Commun ; 12(1): 4491, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301950

RESUMO

Intron selection during the formation of prespliceosomes is a critical event in pre-mRNA splicing. Chemical modulation of intron selection has emerged as a route for cancer therapy. Splicing modulators alter the splicing patterns in cells by binding to the U2 snRNP (small nuclear ribonucleoprotein)-a complex chaperoning the selection of branch and 3' splice sites. Here we report crystal structures of the SF3B module of the U2 snRNP in complex with spliceostatin and sudemycin FR901464 analogs, and the cryo-electron microscopy structure of a cross-exon prespliceosome-like complex arrested with spliceostatin A. The structures reveal how modulators inactivate the branch site in a sequence-dependent manner and stall an E-to-A prespliceosome intermediate by covalent coupling to a nucleophilic zinc finger belonging to the SF3B subunit PHF5A. These findings support a mechanism of intron recognition by the U2 snRNP as a toehold-mediated strand invasion and advance an unanticipated drug targeting concept.


Assuntos
DNA/genética , Íntrons/genética , Piranos/metabolismo , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Compostos de Espiro/metabolismo , Spliceossomos/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , DNA/química , DNA/metabolismo , Humanos , Lactonas/química , Lactonas/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Piranos/química , Pironas/química , Pironas/metabolismo , Ribonucleoproteína Nuclear Pequena U2/química , Compostos de Espiro/química , Spliceossomos/ultraestrutura
5.
ACS Med Chem Lett ; 12(1): 93-98, 2021 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-33488969

RESUMO

Fibroblast growth factor receptors (FGFR) 2 and 3 have been established as drivers of numerous types of cancer with multiple drugs approved or entering late stage clinical trials. A limitation of current inhibitors is vulnerability to gatekeeper resistance mutations. Using a combination of targeted high-throughput screening and structure-based drug design, we have developed a series of aminopyrazole based FGFR inhibitors that covalently target a cysteine residue on the P-loop of the kinase. The inhibitors show excellent activity against the wild-type and gatekeeper mutant versions of the enzymes. Further optimization using SAR analysis and structure-based drug design led to analogues with improved potency and drug metabolism and pharmacokinetics properties.

6.
Sci Rep ; 8(1): 15907, 2018 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-30349061

RESUMO

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

7.
Sci Rep ; 8(1): 14124, 2018 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-30237436

RESUMO

Phosphorylation of Pseudomonas aeruginosa lipopolysaccharide (LPS) is important for maintaining outer membrane integrity and intrinsic antibiotic resistance. We solved the crystal structure of the LPS heptose kinase WaaP, which is essential for growth of P. aeruginosa. WaaP was structurally similar to eukaryotic protein kinases and, intriguingly, was complexed with acylated-acyl carrier protein (acyl-ACP). WaaP produced by in vitro transcription-translation was insoluble unless acyl-ACP was present. WaaP variants designed to perturb the acyl-ACP interaction were less stable in cells and exhibited reduced kinase function. Mass spectrometry identified myristyl-ACP as the likely physiological binding partner for WaaP in P. aeruginosa. Together, these results demonstrate that acyl-ACP is required for WaaP protein solubility and kinase function. To the best of our knowledge, this is the first report describing acyl-ACP in the role of a cofactor necessary for the production and stability of a protein partner.


Assuntos
Proteína de Transporte de Acila/metabolismo , Proteínas de Bactérias/metabolismo , Lipopolissacarídeos/metabolismo , Pseudomonas aeruginosa/metabolismo , Acilação
8.
Cell Chem Biol ; 25(6): 705-717.e11, 2018 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-29628435

RESUMO

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 Cultivadas
9.
Structure ; 11(6): 703-13, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12791258

RESUMO

Choline kinase catalyzes the ATP-dependent phosphorylation of choline, the first committed step in the CDP-choline pathway for the biosynthesis of phosphatidylcholine. The 2.0 A crystal structure of a choline kinase from C. elegans (CKA-2) reveals that the enzyme is a homodimeric protein with each monomer organized into a two-domain fold. The structure is remarkably similar to those of protein kinases and aminoglycoside phosphotransferases, despite no significant similarity in amino acid sequence. Comparisons to the structures of other kinases suggest that ATP binds to CKA-2 in a pocket formed by highly conserved and catalytically important residues. In addition, a choline binding site is proposed to be near the ATP binding pocket and formed by several structurally flexible loops.


Assuntos
Colina Quinase/química , Estrutura Terciária de Proteína , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Caenorhabditis elegans/enzimologia , Cálcio/metabolismo , Cristalografia por Raios X , Dimerização , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Dobramento de Proteína , Estrutura Quaternária de Proteína , Alinhamento de Sequência
10.
Biochim Biophys Acta ; 1648(1-2): 33-42, 2003 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-12758145

RESUMO

Choline kinase is the first enzymatic step in the CDP-choline pathway for phosphatidylcholine biosynthesis. The genome of the nematode, Caenorhabditis elegans, contains seven genes that appear likely to encode choline and/or ethanolamine kinases. We cloned five and expressed four of these genes, and purified or partially purified three of the encoded enzymes. All expressed proteins had choline kinase activity; those that most closely resemble the mammalian choline kinases were the most active. CKA-2, a very active form, was purified to near homogeneity. The K(m) values for CKA-2 were 1.6 and 2.4 mM for choline and ATP, respectively, and k(cat) was 74 s(-1). CKA-2 was predominantly a homodimer as assessed by glycerol gradient sedimentation and dynamic light scattering. CKB-2, which was less similar to mammalian choline kinases, had K(m) values for choline and ATP of 13 and 0.7 mM, and k(cat) was 3.8 s(-1). Both of these highly purified enzymes required magnesium, had very alkaline pH optima, and were much more active with choline as substrate than with ethanolamine. These results provide a foundation for future studies on the structure and function of choline kinases, as well as studies on the genetic analysis of the function of the multiple isoforms in this organism.


Assuntos
Caenorhabditis elegans/enzimologia , Colina Quinase/genética , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/genética , Colina/metabolismo , Colina Quinase/isolamento & purificação , Colina Quinase/metabolismo , Clonagem Molecular , Códon de Terminação , Isoenzimas/genética , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Cinética , Magnésio/metabolismo , Lipídeos de Membrana/metabolismo , Dados de Sequência Molecular , Fosfatidilcolinas/metabolismo , Biossíntese de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência
11.
mBio ; 2(4)2011.
Artigo em Inglês | MEDLINE | ID: mdl-21810964

RESUMO

Gram-negative outer membrane (OM) integrity is maintained in part by Mg(2+) cross-links between phosphates on lipid A and on core sugars of adjacent lipopolysaccharide (LPS) molecules. In contrast to other Gram-negative bacteria, waaP, encoding an inner-core kinase, could not be inactivated in Pseudomonas aeruginosa. To examine this further, expression of the kinases WaaP or WapP/WapQ/PA5006 was placed under the control of the arabinose-regulated pBAD promoter. Growth of these strains was arabinose dependent, confirming that core phosphorylation is essential in P. aeruginosa. Transmission electron micrographs of kinase-depleted cells revealed marked invaginations of the inner membrane. SDS-PAGE of total LPS from WaaP-depleted cells showed accumulation of a fast-migrating band. Mass spectrometry (MS) analysis revealed that LPS from these cells exhibits a unique truncated core consisting of two 3-deoxy-d-manno-octulosonic acids (Kdo), two l-glycero-d-manno-heptoses (Hep), and one hexose but completely devoid of phosphates, indicating that phosphorylation by WaaP is necessary for subsequent core phosphorylations. MS analysis of lipid A from WaaP-depleted cells revealed extensive 4-amino-4-deoxy-l-arabinose modification. OM prepared from these cells by Sarkosyl extraction of total membranes or by sucrose density gradient centrifugation lacked truncated LPS. Instead, truncated LPS was detected in the inner membrane fractions, consistent with impaired transport/assembly of this species into the OM. IMPORTANCE Gram-negative bacteria have an outer membrane (OM) comprised of a phospholipid inner leaflet and a lipopolysaccharide (LPS) outer leaflet. The OM protects cells from toxic molecules and is important for survival during infection. The LPS core kinase gene waaP can be deleted in several Gram-negative bacteria but not in Pseudomonas aeruginosa. We used a controlled-expression system to deplete WaaP directly in P. aeruginosa cells, which halted growth. WaaP depletion also caused gross changes in cell morphology and led to the accumulation of an aberrant LPS lacking several core sugars and all core phosphates. The aberrant LPS failed to reach the OM, suggesting that WaaP is essential in P. aeruginosa because it is required to produce the full-length LPS that is recognized by the OM transport/assembly machinery in this organism. Therefore, WaaP may constitute a good target for the development of novel antipseudomonal agents.


Assuntos
Membrana Celular/metabolismo , Lipopolissacarídeos/biossíntese , Lipopolissacarídeos/química , Fosfatos/metabolismo , Pseudomonas aeruginosa/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transporte Biológico , Membrana Celular/química , Membrana Celular/genética , Lipopolissacarídeos/metabolismo , Fosfotransferases/genética , Fosfotransferases/metabolismo , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/genética
12.
Mol Microbiol ; 51(2): 471-81, 2004 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-14756787

RESUMO

The genomes of Treponema denticola and Treponema pallidum contain a gene, licCA, which is predicted to encode a fusion protein containing choline kinase and CTP:phosphocholine cytidylyltransferase activities. Because both organisms have been reported to contain phosphatidylcholine, this raises the possibility that they use a CDP-choline pathway for the biosynthesis of phosphatidylcholine. This report shows that phosphatidylcholine is a major phospholipid in T. denticola, accounting for 35-40% of total phospholipid. This organism readily incorporated [14C]choline into phosphatidylcholine, indicating the presence of a choline-dependent biosynthetic pathway. The licCA gene was cloned, and recombinant LicCA had choline kinase and CTP:phosphocholine cytidylyltransferase activity. The licCA gene was disrupted in T. denticola by erythromycin cassette mutagenesis, resulting in a viable mutant. This disruption completely blocked incorporation of either [14C]choline or 32Pi into phosphatidylcholine. The rate of production of another phospholipid in T. denticola, phosphatidylethanolamine, was elevated considerably in the licCA mutant, suggesting that the elevated level of this lipid compensated for the loss of phosphatidylcholine in the membranes. Thus it appears that T. denticola does contain a licCA-dependent CDP-choline pathway for phosphatidylcholine biosynthesis.


Assuntos
Colina Quinase/genética , Colina-Fosfato Citidililtransferase/genética , Citidina Difosfato Colina/metabolismo , Fosfatidilcolinas/biossíntese , Treponema/genética , Treponema/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Colina Quinase/química , Colina Quinase/metabolismo , Colina-Fosfato Citidililtransferase/química , Colina-Fosfato Citidililtransferase/metabolismo , Clonagem Molecular , Primers do DNA , DNA Bacteriano/química , DNA Bacteriano/genética , Cinética , Dados de Sequência Molecular , Mutagênese , Plasmídeos/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Treponema/crescimento & desenvolvimento , Treponema pallidum/genética , Treponema pallidum/crescimento & desenvolvimento , Treponema pallidum/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA