RESUMO
Casein kinase 1 alpha (CK1α) is a serine/threonine kinase with numerous functions, including regulating the Wnt/ß-catenin and p53 pathways. CK1α has a well-established role in inhibiting the p53 tumor suppressor by binding to MDMX and stimulating MDMX-p53 interaction. MDMX purified from cells contains near-stoichiometric amounts of CK1α, suggesting that MDMX may in turn regulate CK1α function. We present evidence that MDMX is a potent competitive inhibitor of CK1α kinase activity (Ki = 8 nM). Depletion of MDMX increases CK1α activity and ß-catenin S45 phosphorylation, whereas ectopic MDMX expression inhibits CK1α activity and ß-catenin phosphorylation. The MDMX acidic domain and zinc finger are necessary and sufficient for binding and inhibition of CK1α. P53 binding to MDMX disrupts an intramolecular auto-regulatory interaction and enhances its ability to inhibit CK1α. P53-null mice expressing the MDMXW200S/W201G mutant, defective in CK1α binding, exhibit reduced Wnt/ß-catenin target gene expression and delayed tumor development. Therefore, MDMX is a physiological inhibitor of CK1α and has a role in modulating cellular response to Wnt signaling. The MDMX-CK1α interaction may account for certain p53-independent functions of MDMX.
Assuntos
Caseína Quinase Ialfa/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Via de Sinalização Wnt , Células A549 , Animais , Caseína Quinase Ialfa/genética , Proteínas de Ciclo Celular/genética , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Estimating the actual extent of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is challenging because virus test positivity data undercount the actual number and proportion of persons infected. SARS-CoV-2 seroprevalence is a marker of past SARS-CoV-2 infection regardless of presence or severity of symptoms and therefore is a robust biomarker of infection period prevalence. We estimated SARS-CoV-2 seroprevalence among residents of Hillsborough County, Florida, USA, to determine factors independently associated with SARS-CoV-2 antibody status overall and among asymptomatic antibody-positive persons. Among 867 participants, SARS-CoV-2 period prevalence (October 2020-March 2021) was 19.5% (asymptomatic seroprevalence was 8%). Seroprevalence was 2-fold higher than reported SARS-CoV-2 virus test positivity. Factors related to social distancing (e.g., essential worker status, not practicing social distancing, contact with a virus-positive person, and length of contact exposure time) were consistently associated with seroprevalence but did not differ by time since suspected or known infection (<6 months vs. >6 months).
Assuntos
COVID-19 , SARS-CoV-2 , Anticorpos Antivirais , Florida/epidemiologia , Humanos , Pandemias , Estudos SoroepidemiológicosRESUMO
Based on a previously reported 1,4-dihydropyridinebutyrolactone virtual screening hit, nine lactone ring-opened ester and seven amide analogs were prepared. The analogs were designed to provide interactions with residues at the entrance of the ZA loop of the testis-specific bromodomain (ZA) channel to enhance the affinity and selectivity for the bromodomain and extra-terminal (BET) subfamily of bromodomains. Compound testing by AlphaScreen showed that neither the affinity nor the selectivity of the ester and lactam analogs was improved for BRD4-1 and the first bromodomain of the testis-specific bromodomain (BRDT-1). The esters retained affinity comparable to the parent compound, whereas the affinity for the amide analogs was reduced 10-fold. A representative benzyl ester analog was found to retain high selectivity for BET bromodomains as shown by a BROMOscan. X-ray analysis of the allyl ester analog in complex with BRD4-1 and BRDT-1 revealed that the ester side chain is located next to the ZA loop and solvent exposed.
Assuntos
Proteínas Nucleares , Fatores de Transcrição , Humanos , Masculino , Amidas/farmacologia , Proteínas de Ciclo Celular , Ésteres/farmacologia , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Relação Estrutura-Atividade , Lactonas/químicaRESUMO
A monocarboxylic inhibitor was designed and synthesized to disrupt the protein-protein interaction (PPI) between GRB2 and phosphotyrosine-containing proteins. Biochemical characterizations show compound 7 binds with the Src homology 2 (SH2) domain of GRB2 and is more potent than EGFR1068 phosphopeptide 14-mer. X-ray crystallographic studies demonstrate compound 7 occupies the GRB2 binding site for phosphotyrosine-containing sequences and reveal key structural features for GRB2-inhibitor binding. This compound with a -1 formal charge offers a new direction for structural optimization to generate cell-permeable inhibitors for this key protein target of the aberrant Ras-MAPK signaling cascade.
Assuntos
Ácidos Carboxílicos/farmacologia , Proteína Adaptadora GRB2/antagonistas & inibidores , Ácidos Carboxílicos/síntese química , Ácidos Carboxílicos/química , Relação Dose-Resposta a Droga , Proteína Adaptadora GRB2/metabolismo , Humanos , Estrutura Molecular , Relação Estrutura-Atividade , Domínios de Homologia de src/efeitos dos fármacosRESUMO
Gene specific recruitment of bromodomain-containing proteins to chromatin is affected by post-translational acetylation of lysine on histones. Whereas interactions of the bromodomain with acetylation patterns of native histones (H2A, H2B, H3, and H4) have been well characterized, the motif for recognition for histone variants H2A.Z I and H2A.Z II by bromodomains has yet to be fully investigated. Elucidating these molecular mechanisms is crucial for understanding transcriptional regulation in cellular processes involved in both development and disease. Here, we have used protein-observed fluorine NMR to fully characterize the affinities of H2A.Z I and II acetylation patterns for BPTF's bromodomain and found the diacetylated mark of lysine 7 and 13 on H2A.Z II to have the strongest interaction with K7ac preferentially engaging the binding site. We further examined the selectivity of H2A.Z histones against a variety of bromodomains, revealing that the bromodomain of CECR2 binds with the highest affinity and specificity for acetylated H2A.Z I over isoform II. These results support a possible role for different H2A.Z transcriptional activation mechanisms that involve recruitment of chromatin remodeling complexes.
Assuntos
Histonas/metabolismo , Ressonância Magnética Nuclear Biomolecular , Nucleossomos/metabolismo , Fatores de Transcrição/metabolismo , Acetilação , Histonas/química , Histonas/genética , Humanos , Nucleossomos/química , Processamento de Proteína Pós-Traducional , Fatores de Transcrição/química , Ativação TranscricionalRESUMO
WEE2 oocyte meiosis inhibiting kinase is a well-conserved oocyte specific kinase with a dual regulatory role during meiosis. Active WEE2 maintains immature, germinal vesicle stage oocytes in prophase I arrest prior to the luteinizing hormone surge and facilitates exit from metaphase II arrest at fertilization. Spontaneous mutations at the WEE2 gene locus in women have been linked to total fertilization failure indicating that selective inhibitors to this kinase could function as non-hormonal contraceptives. Employing co-crystallization with WEE1 G2 checkpoint kinase inhibitors, we revealed the structural basis of action across WEE kinases and determined type I inhibitors were not selective to WEE2 over WEE1. In response, we performed in silico screening by FTMap/FTSite and Schrodinger SiteMap analysis to identify potential allosteric sites, then used an allosterically biased activity assay to conduct high-throughput screening of a 26 000 compound library containing scaffolds of known allosteric inhibitors. Resulting hits were validated and a selective inhibitor that binds full-length WEE2 was identified, designated GPHR-00336382, along with a fragment-like inhibitor that binds the kinase domain, GPHR-00355672. Additionally, we present an in vitro testing workflow to evaluate biological activity of candidate WEE2 inhibitors including; (1) enzyme-linked immunosorbent assays measuring WEE2 phosphorylation activity of cyclin dependent kinase 1 (CDK1; also known as cell division cycle 2 kinase, CDC2), (2) in vitro fertilization of bovine ova to determine inhibition of metaphase II exit, and (3) cell-proliferation assays to look for off-target effects against WEE1 in somatic (mitotic) cells.
Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Anticoncepcionais Femininos/administração & dosagem , Meiose/efeitos dos fármacos , Inibidores de Proteínas Quinases/administração & dosagem , Proteínas Tirosina Quinases/antagonistas & inibidores , Animais , Bovinos , Proliferação de Células/efeitos dos fármacos , Simulação por Computador , Humanos , Oócitos/efeitos dos fármacos , Oócitos/metabolismoRESUMO
Bromodomain-containing proteins regulate transcription through protein-protein interactions with chromatin and serve as scaffolding proteins for recruiting essential members of the transcriptional machinery. One such protein is the bromodomain and PHD-containing transcription factor (BPTF), the largest member of the nucleosome remodeling complex, NURF. Despite an emerging role for BPTF in regulating a diverse set of cancers, small molecule development for inhibiting the BPTF bromodomain has been lacking. Here we cross-validate three complementary biophysical assays to further the discovery of BPTF bromodomain inhibitors for chemical probe development: two direct binding assays (protein-observed 19F (PrOF) NMR and surface plasmon resonance (SPR)) and a competitive inhibition assay (AlphaScreen). We first compare the assays using three small molecules and acetylated histone peptides with reported affinity for the BPTF bromodomain. Using SPR with both unlabeled and fluorinated BPTF, we further determine that there is a minimal effect of 19F incorporation on ligand binding for future PrOF NMR experiments. To guide medicinal chemistry efforts towards chemical probe development, we subsequently evaluate two new BPTF inhibitor scaffolds with our suite of biophysical assays and rank-order compound affinities which could not otherwise be determined by PrOF NMR. Finally, we cocrystallize a subset of small molecule inhibitors and present the first published small molecule-protein structures with the BPTF bromodomain. We envision the biophysical assays described here and the structural insights from the crystallography will guide researchers towards developing selective and potent BPTF bromodomain inhibitors.
Assuntos
Proteínas do Tecido Nervoso/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Antígenos Nucleares/química , Fenômenos Biofísicos , Espectroscopia de Ressonância Magnética , Proteínas do Tecido Nervoso/química , Domínios Proteicos , Ressonância de Plasmônio de Superfície , Fatores de Transcrição/químicaRESUMO
Upon binding to thalidomide and other immunomodulatory drugs, the E3 ligase substrate receptor cereblon (CRBN) promotes proteosomal destruction by engaging the DDB1-CUL4A-Roc1-RBX1 E3 ubiquitin ligase in human cells but not in mouse cells, suggesting that sequence variations in CRBN may cause its inactivation. Therapeutically, CRBN engagers have the potential for broad applications in cancer and immune therapy by specifically reducing protein expression through targeted ubiquitin-mediated degradation. To examine the effects of defined sequence changes on CRBN's activity, we performed a comprehensive study using complementary theoretical, biophysical, and biological assays aimed at understanding CRBN's nonprimate sequence variations. With a series of recombinant thalidomide-binding domain (TBD) proteins, we show that CRBN sequence variants retain their drug-binding properties to both classical immunomodulatory drugs and dBET1, a chemical compound and targeting ligand designed to degrade bromodomain-containing 4 (BRD4) via a CRBN-dependent mechanism. We further show that dBET1 stimulates CRBN's E3 ubiquitin-conjugating function and degrades BRD4 in both mouse and human cells. This insight paves the way for studies of CRBN-dependent proteasome-targeting molecules in nonprimate models and provides a new understanding of CRBN's substrate-recruiting function.
Assuntos
Proteínas Culina/metabolismo , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Proteólise , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Azepinas/farmacologia , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Sequência Conservada , Humanos , Fatores Imunológicos/metabolismo , Fatores Imunológicos/farmacologia , Lenalidomida/farmacologia , Ligantes , Camundongos , Sondas Moleculares , Proteínas Nucleares/efeitos dos fármacos , Proteínas Nucleares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Linfócitos T/metabolismo , Talidomida/análogos & derivados , Talidomida/metabolismo , Talidomida/farmacologia , Fatores de Transcrição/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Triazóis/farmacologia , Ubiquitina/metabolismoRESUMO
The p53 inhibitor MDMX is controlled by multiple stress signaling pathways. Using a proteolytic fragment release (PFR) assay, we detected an intramolecular interaction in MDMX that mechanistically mimics the interaction with p53, resulting in autoinhibition of MDMX. This mimicry is mediated by a hydrophobic peptide located in a long disordered central segment of MDMX that has sequence similarity to the p53 transactivation domain. NMR spectroscopy was used to show this hydrophobic peptide interacts with the N-terminal domain of MDMX in a structurally analogous manner to p53. Mutation of two critical tryptophan residues in the hydrophobic peptide disrupted the intramolecular interaction and increased p53 binding, providing further evidence for mechanistic mimicry. The PFR assay also revealed a second intramolecular interaction between the RING domain and central region that regulates MDMX nuclear import. These results establish the importance of intramolecular interactions in MDMX regulation, and validate a new assay for the study of intramolecular interactions in multidomain proteins with intrinsically disordered regions.
Assuntos
Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Sequência de Aminoácidos , Sítios de Ligação/genética , Western Blotting , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Humanos , Interações Hidrofóbicas e Hidrofílicas , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Mimetismo Molecular , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/química , Proteínas Nucleares/genética , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteólise , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-mdm2/química , Proteínas Proto-Oncogênicas c-mdm2/genética , Homologia de Sequência de Aminoácidos , Triptofano/química , Triptofano/genética , Triptofano/metabolismo , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genéticaRESUMO
Meiotic recombination ensures faithful segregation of homologous chromosomes during meiosis and generates genetic diversity in gametes. MEIOB (meiosis specific with OB domains), a meiosis-specific single-stranded DNA-binding homolog of replication protein A1 (RPA1), is essential for meiotic recombination. Here, we investigated the molecular mechanisms of MEIOB by characterizing its binding partners spermatogenesis associated 22 (SPATA22) and RPA. We find that MEIOB and SPATA22 form an obligate complex and contain defined interaction domains. The interaction between these two proteins is unusual in that nearly any deletion in the binding domains abolishes the interaction. Strikingly, a single residue D383 in MEIOB is indispensable for the interaction. The MEIOB/SPATA22 complex interacts with the RPA heterotrimeric complex in a collaborative manner. Furthermore, MEIOB and SPATA22 are recruited to induced DNA double-strand breaks (DSBs) together but not alone. These results demonstrate the cooperative property of the MEIOB-SPATA22 complex in its interaction with RPA and recruitment to DSBs.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Quebras de DNA de Cadeia Simples , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Meiose/genética , Meiose/fisiologia , Proteína de Replicação A/metabolismo , Animais , Ácido Aspártico/metabolismo , Células Cultivadas , Quebras de DNA de Cadeia Dupla , Imunoprecipitação , Masculino , Camundongos , Modelos MolecularesRESUMO
The molecular chaperone Hsp90 is regulated by co-chaperones such as p50Cdc37, which recruits a wide selection of client protein kinases. Targeted disruption of the Hsp90-p50Cdc37 complex by protein-protein interaction (PPI) inhibitors has emerged as an alternative strategy to treat diseases characterized by aberrant Hsp90 activity. Using isothermal microcalorimetry, ELISA and GST-pull down assays we evaluated reported Hsp90 inhibitors and nucleotides for their ability to inhibit formation of the human Hsp90ß-p50Cdc37 complex, reconstituted in vitro from full-length proteins. Hsp90 inhibitors, including the proposed PPI inhibitors gedunin and H2-gamendazole, did not affect the interaction of Hsp90 with p50Cdc37 in vitro. Phosphorylation of Hsp90 and p50Cdc37 by casein kinase 2 (CK2) did not alter the thermodynamic signature of complex formation. However, the phosphorylated complex was vulnerable to disruption by ADP (IC50 = 32 µM), while ATP, AMPPNP and Hsp90 inhibitors remained largely ineffective. The differential inhibitory activity of ADP suggests that phosphorylation by CK2 primes the complex for dissociation in response to a drop in ATP/ADP levels. The approach applied herein provides robust assays for a comprehensive biochemical evaluation of potential effectors of the Hsp90-p50Cdc37 complex, such as phosphorylation by a kinase or the interaction with small molecule ligands.
Assuntos
Caseína Quinase II/metabolismo , Proteínas de Ciclo Celular/metabolismo , Chaperoninas/metabolismo , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Nucleotídeos/farmacologia , Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Calorimetria , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Cinética , Modelos Moleculares , Fosforilação/efeitos dos fármacos , Mapas de Interação de Proteínas/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Bibliotecas de Moléculas Pequenas/farmacologia , TermodinâmicaRESUMO
Terreic acid is a metabolite with antibiotic properties produced by the fungus Aspergillus terreus, but its cellular target remains unknown. We recently reported that terreic acid inactivates the bacterial cell wall biosynthetic enzyme MurA in vitro by covalent reaction with residue Cys115 in a similar manner as the MurA-specific antibiotic fosfomycin. To address if terreic acid also targets MurA in vivo, we conducted antibacterial studies using selected E. coli strains in parallel with fosfomycin. While overexpression of MurA conferred resistance to fosfomycin, it did not protect cells treated with terreic acid. Furthermore, flow cytometry revealed that the antibiotic action of terreic acid appears to be primarily bacteriostatic, as opposed to the bactericidal action observed for fosfomycin. Combined, the data suggest that MurA is not the molecular target of terreic acid and that the antibiotic activity of terreic acid proceeds through a different mechanism of action. The methodology applied here provides a reliable and convenient tool to rapidly assess the potential of newly discovered in vitro inhibitors to target residue Cys115 of MurA in the cell.
Assuntos
Alquil e Aril Transferases/antagonistas & inibidores , Antibacterianos/farmacologia , Fosfomicina/farmacologia , Aspergillus , Proliferação de Células/efeitos dos fármacos , Farmacorresistência Bacteriana , Escherichia coli/citologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Escherichia coli/genética , Mutação , Quinonas/farmacologiaRESUMO
The UNC-51-like kinase-1 (ULK1) is one of the central upstream regulators of the autophagy pathway, represents a key target for the development of molecular probes to abrogate autophagy and explore potential therapeutic avenues. Here we report the discovery, structure-activity and structure-property relationships of selective, potent, and cell-active ULK1/2 inhibitors based on a 7-azaindole scaffold. Using structure-based drug design, we have developed a series of analogs with excellent binding affinity and biochemical activity against ULK1/2 (IC50 < 25 nM). The validation of cellular target engagement for these compounds was achieved through the employment of the ULK1 NanoBRET intracellular kinase assay. Notably, we have successfully solved the crystal structure of the lead compound, MR-2088, bound to the active site of ULK1. Moreover, the combination treatment of MR-2088 with known KRASâRAFâMEKâERK pathway inhibitors, such as trametinib, showed promising synergistic effect in vitro using H2030 (KRASG12C) cell lines. Lastly, our findings underscore MR-2088's potential to inhibit starvation/stimuli-induced autophagic flux, coupled with its suitability for in vivo studies based on its pharmacokinetic properties.
Assuntos
Indóis , Proteínas Proto-Oncogênicas p21(ras) , Indóis/farmacologia , Autofagia , Linhagem CelularRESUMO
Chemical discovery efforts commonly target individual protein domains. Many proteins, including the EP300/CBP histone acetyltransferases (HATs), contain several targetable domains. EP300/CBP are critical gene-regulatory targets in cancer, with existing high potency inhibitors of either the catalytic HAT domain or protein-binding bromodomain (BRD). A domain-specific inhibitory approach to multidomain-containing proteins may identify exceptional-responding tumor types, thereby expanding a therapeutic index. Here, we discover that targeting EP300/CBP using the domain-specific inhibitors, A485 (HAT) or CCS1477 (BRD) have different effects in select tumor types. Group 3 medulloblastoma (G3MB) cells are especially sensitive to BRD, compared with HAT inhibition. Structurally, these effects are mediated by the difluorophenyl group in the catalytic core of CCS1477. Mechanistically, bromodomain inhibition causes rapid disruption of genetic dependency networks that are required for G3MB growth. These studies provide a domain-specific structural foundation for drug discovery efforts targeting EP300/CBP and identify a selective role for the EP300/CBP bromodomain in maintaining genetic dependency networks in G3MB.
Assuntos
Proteína p300 Associada a E1A , Redes Reguladoras de Genes , Meduloblastoma , Humanos , Meduloblastoma/genética , Meduloblastoma/tratamento farmacológico , Meduloblastoma/metabolismo , Meduloblastoma/patologia , Proteína p300 Associada a E1A/metabolismo , Proteína p300 Associada a E1A/genética , Proteína p300 Associada a E1A/antagonistas & inibidores , Linhagem Celular Tumoral , Redes Reguladoras de Genes/efeitos dos fármacos , Animais , Domínios Proteicos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Camundongos , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/tratamento farmacológico , Neoplasias Cerebelares/metabolismo , Neoplasias Cerebelares/patologia , Antineoplásicos/farmacologiaRESUMO
The enzyme MurA has been an established antibiotic target since the discovery of fosfomycin, which specifically inhibits MurA by covalent modification of the active site residue Cys-115. Early biochemical studies established that Cys-115 also covalently reacts with substrate phosphoenolpyruvate (PEP) to yield a phospholactoyl adduct, but the structural and functional consequences of this reaction remained obscure. We captured and depicted the Cys-115-PEP adduct of Enterobacter cloacae MurA in various reaction states by X-ray crystallography. The data suggest that cellular MurA predominantly exists in a tightly locked complex with UDP-N-acetylmuramic acid (UNAM), the product of the MurB reaction, with PEP covalently attached to Cys-115. The uniqueness and rigidity of this "dormant" complex was previously not recognized and presumably accounts for the failure of drug discovery efforts toward the identification of novel and effective MurA inhibitors. We demonstrate that recently published crystal structures of MurA from various organisms determined by different laboratories were indeed misinterpreted and actually contain UNAM and covalently bound PEP. The Cys-115-PEP adduct was also captured in vitro during the reaction of free MurA and substrate UDP-N-acetylglucosamine or isomer UDP-N-acetylgalactosamine. The now available series of crystal structures allows a comprehensive view of the reaction cycle of MurA. It appears that the covalent reaction of MurA with PEP fulfills dual functions by tightening the complex with UNAM for the efficient feedback regulation of murein biosynthesis and by priming the PEP molecule for instantaneous reaction with substrate UDP-N-acetylglucosamine.
Assuntos
Alquil e Aril Transferases/química , Alquil e Aril Transferases/metabolismo , Enterobacter cloacae/enzimologia , Fosfoenolpiruvato/metabolismo , Alquil e Aril Transferases/genética , Cristalografia por Raios X , Adutos de DNA/metabolismo , Enterobacter cloacae/genética , Ativação Enzimática/fisiologia , Escherichia coli/genética , Ácidos Murâmicos/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato/fisiologiaRESUMO
The anti-apoptotic Bcl-2 family of proteins, including Bcl-2, Bcl-X(L) and Mcl-1, are well-validated drug targets for cancer treatment. Several small molecules have been designed to interfere with Bcl-2 and its fellow pro-survival family members. While ABT-737 and its orally active analog ABT-263 are the most potent and specific inhibitors to date that bind Bcl-2 and Bcl-X(L) with high affinity but have a much lower affinity for Mcl-1, they are not very effective as single agents in certain cancer types because of elevated levels of Mcl-1. Accordingly, compounds that specifically target Mcl-1 may overcome this resistance. In this study, we identified and characterized the natural product marinopyrrole A as a novel Mcl-1-specific inhibitor and named it maritoclax. We found that maritoclax binds to Mcl-1, but not Bcl-X(L), and is able to disrupt the interaction between Bim and Mcl-1. Moreover, maritoclax induces Mcl-1 degradation via the proteasome system, which is associated with the pro-apoptotic activity of maritoclax. Importantly, maritoclax selectively kills Mcl-1-dependent, but not Bcl-2- or Bcl-X(L)-dependent, leukemia cells and markedly enhances the efficacy of ABT-737 against hematologic malignancies, including K562, Raji, and multidrug-resistant HL60/VCR, by â¼60- to 2000-fold at 1-2 µM. Taken together, these results suggest that maritoclax represents a new class of Mcl-1 inhibitors, which antagonizes Mcl-1 and overcomes ABT-737 resistance by targeting Mcl-1 for degradation.
Assuntos
Compostos de Bifenilo/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Leucemia/tratamento farmacológico , Nitrofenóis/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Sulfonamidas/farmacologia , Animais , Resistencia a Medicamentos Antineoplásicos/genética , Células HL-60 , Humanos , Células Jurkat , Células K562 , Leucemia/genética , Leucemia/metabolismo , Camundongos , Proteína de Sequência 1 de Leucemia de Células Mieloides , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Pirróis , Proteína bcl-X/antagonistas & inibidores , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMO
(-)-Tulipalin B and (+)-6-tuliposide B were confirmed to inhibit MurA in vitro. However, contrary to fosfomycin, these compounds showed potent inhibitory activities against MurA overexpressing Escherichia coli, especially in the presence of UDP-GlcNAc. These observations suggest that these compounds induced bacterial cell death not through a MurA malfunction, but in such a MurA-mediated indirect manner as the inhibition of other Mur enzymes.
Assuntos
4-Butirolactona/análogos & derivados , Alquil e Aril Transferases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Glucosídeos/farmacologia , Hidroxibutiratos/farmacologia , 4-Butirolactona/farmacologia , Alquil e Aril Transferases/genética , Escherichia coli/genética , Escherichia coli/metabolismoRESUMO
Photoreactive fragment-like probes have been applied to discover target proteins that constitute novel cellular vulnerabilities and to identify viable chemical hits for drug discovery. Through forming covalent bonds, functionalized probes can achieve stronger target engagement and require less effort for on-target mechanism validation. However, the design of probe libraries, which directly affects the biological target space that is interrogated, and effective target prioritization remain critical challenges of such a chemical proteomic platform. In this study, we designed and synthesized a diverse panel of 20 fragment-based probes containing natural product-based privileged structural motifs for small-molecule lead discovery. These probes were fully functionalized with orthogonal diazirine and alkyne moieties and used for protein crosslinking in live lung cancer cells, target enrichment via "click chemistry," and subsequent target identification through label-free quantitative liquid chromatography-tandem mass spectrometry analysis. Pair-wise comparison with a blunted negative control probe and stringent prioritization via individual cross-comparisons against the entire panel identified glutathione S-transferase zeta 1 (GSTZ1) as a specific and unique target candidate. DepMap database query, RNA interference-based gene silencing, and proteome-wide tyrosine reactivity profiling suggested that GSTZ1 cooperated with different oncogenic alterations by supporting survival signaling in refractory non-small cell lung cancer cells. This finding may form the basis for developing novel GSTZ1 inhibitors to improve the therapeutic efficacy of oncogene-directed targeted drugs. In summary, we designed a novel fragment-based probe panel and developed a target prioritization scheme with improved stringency, which allows for the identification of unique target candidates, such as GSTZ1 in refractory lung cancer.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Proteômica , Neoplasias Pulmonares/tratamento farmacológico , Proteínas , Glutationa , Glutationa Transferase/metabolismoRESUMO
Although cyclin-dependent kinase 2 (CDK2) is a validated target for both cancer and contraception, developing a CDK2 inhibitor with exquisite selectivity has been challenging due to the structural similarity of the ATP-binding site, where most kinase inhibitors bind. We previously discovered an allosteric pocket in CDK2 with the potential to bind a selective compound and then discovered and structurally confirmed an anthranilic acid scaffold that binds this pocket with high affinity. These allosteric inhibitors are selective for CDK2 over structurally similar CDK1 and show contraceptive potential. Herein, we describe the screening and optimization that led to compounds like EF-4-177 with nanomolar affinity for CDK2. EF-4-177 is metabolically stable, orally bioavailable, and significantly disrupts spermatogenesis, demonstrating this series' therapeutic potential. This work details the discovery of the highest affinity allosteric CDK inhibitors reported and shows promise for this series to yield an efficacious and selective allosteric CDK2 inhibitor.
Assuntos
Anticoncepcionais Masculinos , Masculino , Humanos , Animais , Camundongos , Quinase 2 Dependente de Ciclina , Relação Estrutura-Atividade , Anticoncepcionais Masculinos/farmacologia , Contagem de Espermatozoides , Sêmen/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/químicaRESUMO
Reported here are the synthesis and in vitro evaluation of a series of 26 retinoic acid analogs based on dihydronaphthalene and chromene scaffolds using a transactivation assay. Chromene amide analog 21 was the most potent and selective retinoic acid receptor α antagonist identified from this series. In vitro evaluation indicated that 21 has favorable physicochemical properties and a favorable pharmacokinetic PK profile in vivo with significant oral bioavailability, metabolic stability, and testes exposure. Compound 21 was evaluated for its effects on spermatogenesis and disruption of fertility in a mouse model. Oral administration of compound 21 at low doses showed reproducibly characteristic albeit modest effects on spermatogenesis, but no effects on fertility were observed in mating studies. The inhibition of spermatogenesis could not be enhanced by raising the dose and lengthening the duration of dosing. Thus, 21 may not be a good candidate to pursue further for effects on male fertility.