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1.
J Mol Biol ; 430(17): 2857-2872, 2018 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-29864443

RESUMO

REV1 is a DNA damage tolerance protein and encodes two ubiquitin-binding motifs (UBM1 and UBM2) that are essential for REV1 functions in cell survival under DNA-damaging stress. Here we report the first solution and X-ray crystal structures of REV1 UBM2 and its complex with ubiquitin, respectively. Furthermore, we have identified the first small-molecule compound, MLAF50, that directly binds to REV1 UBM2. In the heteronuclear single quantum coherence NMR experiments, peaks of UBM2 but not of UBM1 are significantly shifted by the addition of ubiquitin, which agrees to the observation that REV1 UBM2 but not UBM1 is required for DNA damage tolerance. REV1 UBM2 interacts with hydrophobic residues of ubiquitin such as L8 and L73. NMR data suggest that MLAF50 binds to the same residues of REV1 UBM2 that interact with ubiquitin, indicating that MLAF50 can compete with the REV1 UBM2-ubiquitin interaction orthosterically. Indeed, MLAF50 inhibited the interaction of REV1 UBM2 with ubiquitin and prevented chromatin localization of REV1 induced by cisplatin in U2OS cells. Our results structurally validate REV1 UBM2 as a target of a small-molecule inhibitor and demonstrate a new avenue to targeting ubiquitination-mediated protein interactions with a chemical tool.


Assuntos
Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Éteres Fenílicos/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Ubiquitina/química , Ubiquitina/metabolismo , Sequência de Aminoácidos , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Cromatina/química , Cristalografia por Raios X , DNA/química , DNA/metabolismo , Dano ao DNA , Humanos , Modelos Moleculares , Proteínas Nucleares/efeitos dos fármacos , Nucleotidiltransferases/efeitos dos fármacos , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Células Tumorais Cultivadas , Ubiquitina/efeitos dos fármacos , Ubiquitinação
2.
Bioorg Med Chem ; 26(9): 2345-2353, 2018 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-29598900

RESUMO

REV1 protein is a mutagenic DNA damage tolerance (DDT) mediator and encodes two ubiquitin-binding motifs (i.e., UBM1 and UBM2) that are essential for the DDT function. REV1 interacts with K164-monoubiquitinated PCNA (UbPCNA) in cells upon DNA-damaging stress. By using AlphaScreen assays to detect inhibition of REV1 and UbPCNA protein interactions along with an NMR-based strategy, we identified small-molecule compounds that inhibit the REV1/UbPCNA interaction and that directly bind to REV1 UBM2. In cells, one of the compound prevented recruitment of REV1 to PCNA foci on chromatin upon cisplatin treatment, delayed removal of UV-induced cyclopyrimidine dimers from nuclei, prevented UV-induced mutation of HPRT gene, and diminished clonogenic survival of cells that were challenged by cyclophosphamide or cisplatin. This study demonstrates the potential utility of a small-molecule REV1 UBM2 inhibitor for preventing DDT.


Assuntos
Dano ao DNA/efeitos dos fármacos , DNA/química , Proteínas Nucleares/metabolismo , Nucleotidiltransferases/metabolismo , Piperazinas/farmacologia , Antígeno Nuclear de Célula em Proliferação/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Cisplatino/farmacologia , DNA/efeitos da radiação , Humanos , Hipoxantina Fosforribosiltransferase/genética , Lisina/química , Mutagênese , Ressonância Magnética Nuclear Biomolecular , Proteínas Nucleares/química , Nucleotidiltransferases/química , Piperazinas/síntese química , Antígeno Nuclear de Célula em Proliferação/química , Ligação Proteica/efeitos dos fármacos , Ubiquitinação , Raios Ultravioleta
3.
Bioorg Med Chem ; 24(18): 4339-4346, 2016 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-27448776

RESUMO

DNA interstrand crosslink (ICL) repair (ICLR) has been implicated in the resistance of cancer cells to ICL-inducing chemotherapeutic agents. Despite the clinical significance of ICL-inducing chemotherapy, few studies have focused on developing small-molecule inhibitors for ICLR. The mammalian DNA polymerase ζ, which comprises the catalytic subunit REV3L and the non-catalytic subunit REV7, is essential for ICLR. To identify small-molecule compounds that are mechanistically capable of inhibiting ICLR by targeting REV7, high-throughput screening and structure-activity relationship (SAR) analysis were performed. Compound 1 was identified as an inhibitor of the interaction of REV7 with the REV7-binding sequence of REV3L. Compound 7 (an optimized analog of compound 1) bound directly to REV7 in nuclear magnetic resonance analyses, and inhibited the reactivation of a reporter plasmid containing an ICL in between the promoter and reporter regions. The normalized clonogenic survival of HeLa cells treated with cisplatin and compound 7 was lower than that for cells treated with cisplatin only. These findings indicate that a small-molecule inhibitor of the REV7/REV3L interaction can chemosensitize cells by inhibiting ICLR.


Assuntos
Antineoplásicos/farmacologia , Reparo do DNA , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas Mad2/antagonistas & inibidores , Pirimidinonas/farmacologia , Tiofenos/farmacologia , Antineoplásicos/síntese química , Cisplatino/farmacologia , DNA Polimerase Dirigida por DNA , Resistencia a Medicamentos Antineoplásicos , Células HeLa , Humanos , Ligação Proteica , Pirimidinonas/síntese química , Relação Estrutura-Atividade , Tiofenos/síntese química
4.
Bioorg Med Chem ; 24(5): 1071-8, 2016 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-26833244

RESUMO

DNA interstrand crosslinks (ICLs) represent physical obstacles to advancing replication forks and transcription complexes. A range of ICL-inducing agents have successfully been incorporated into cancer therapeutics. While studies have adopted UVA-activated psoralens as model ICL-inducing agents for investigating ICL repair, direct detection of the lesion has often been tempered by tagging the psoralen scaffold with a relatively large reporter group that may perturb the biological activity of the parent psoralen. Here a minimally-modified psoralen probe was prepared featuring a small alkyne handle suitable for click chemistry. The psoralen probe, designated 8-propargyloxypsoralen (8-POP), can be activated by UVA in vitro to generate ICLs that are susceptible to post-labeling with an azide-tagged fluorescent reporter via a copper-catalyzed reaction. A modified alkaline comet assay demonstrated that UVA-activated 8-POP proficiently generated ICLs in cells. Cellular 8-POP-DNA lesions were amenable to click-mediated ligation to fluorescent reporters in situ, which permitted their detection and quantitation by fluorescence microscopy and flow cytometry. Small molecule DNA repair inhibitors to 8-POP-treated cells attenuated the removal of 8-POP-DNA lesions, validating 8-POP as an appropriate probe for investigating cellular ICL repair. The post-labeling strategy applied in this study is inexpensive, rapid and highly modular in nature with the potential for multiple applications in DNA repair studies.


Assuntos
Química Click , Adutos de DNA/análise , Reparo do DNA , Ficusina/química , Corantes Fluorescentes/química , Alcinos/química , Azidas/química , Cobre/química , Células HeLa , Humanos , Raios Ultravioleta
5.
Bioorg Med Chem ; 23(21): 6912-21, 2015 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-26462050

RESUMO

Cells have evolved complex biochemical pathways for DNA interstrand crosslink (ICL) removal. Despite the chemotherapeutic importance of ICL repair, there have been few attempts to identify which mechanistic DNA repair inhibitor actually inhibits ICL repair. To identify such compounds, a new and robust ICL repair assay was developed using a novel plasmid that contains synthetic ICLs between a CMV promoter region that drives transcription and a luciferase reporter gene, and an SV40 origin of replication and the large T antigen (LgT) gene that enables self-replication in mammalian cells. In a screen against compounds that are classified as inhibitors of DNA repair or synthesis, the reporter generation was exquisitely sensitive to ribonucleotide reductase (RNR) inhibitors such as gemcitabine and clofarabine, but not to inhibitors of PARP, ATR, ATM, Chk1, and others. The effect was observed also by siRNA downregulation of RNR. Moreover, the reporter generation was also particularly sensitive to 3-AP, a non-nucleoside RNR inhibitor, but not significantly sensitive to DNA replication stressors, suggesting that the involvement of RNR in ICL repair is independent of incorporation of a nucleotide RNR inhibitor into DNA to induce replication stress. The reporter generation from a modified version of the plasmid that lacks the LgT-SV40ori motif was also adversely affected by RNR inhibitors, further indicating a role for RNR in ICL repair that is independent of DNA replication. Intriguingly, unhooking of cisplatin-ICL from nuclear DNA was significantly inhibited by low doses of gemcitabine, suggesting an unidentified functional role for RNR in the process of ICL unhooking. The assay approach could identify other molecules essential for ICLR in quantitative and flexible manner.


Assuntos
Inibidores Enzimáticos/química , Ribonucleotídeo Redutases/metabolismo , Antígenos Transformantes de Poliomavirus/genética , Linhagem Celular Tumoral , Cisplatino/química , Ensaio Cometa , Reparo do DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Desoxicitidina/análogos & derivados , Desoxicitidina/química , Desoxicitidina/metabolismo , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Genes Reporter , Células HEK293 , Humanos , Plasmídeos/genética , Plasmídeos/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ribonucleosídeo Difosfato Redutase/antagonistas & inibidores , Ribonucleosídeo Difosfato Redutase/genética , Ribonucleosídeo Difosfato Redutase/metabolismo , Ribonucleotídeo Redutases/antagonistas & inibidores , Ubiquitinação , Gencitabina
6.
Proc Natl Acad Sci U S A ; 112(17): 5272-9, 2015 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-25825779

RESUMO

For many E3 ligases, a mobile RING (Really Interesting New Gene) domain stimulates ubiquitin (Ub) transfer from a thioester-linked E2∼Ub intermediate to a lysine on a remotely bound disordered substrate. One such E3 is the gigantic, multisubunit 1.2-MDa anaphase-promoting complex/cyclosome (APC), which controls cell division by ubiquitinating cell cycle regulators to drive their timely degradation. Intrinsically disordered substrates are typically recruited via their KEN-box, D-box, and/or other motifs binding to APC and a coactivator such as CDH1. On the opposite side of the APC, the dynamic catalytic core contains the cullin-like subunit APC2 and its RING partner APC11, which collaborates with the E2 UBCH10 (UBE2C) to ubiquitinate substrates. However, how dynamic RING-E2∼Ub catalytic modules such as APC11-UBCH10∼Ub collide with distally tethered disordered substrates remains poorly understood. We report structural mechanisms of UBCH10 recruitment to APC(CDH1) and substrate ubiquitination. Unexpectedly, in addition to binding APC11's RING, UBCH10 is corecruited via interactions with APC2, which we visualized in a trapped complex representing an APC(CDH1)-UBCH10∼Ub-substrate intermediate by cryo-electron microscopy, and in isolation by X-ray crystallography. To our knowledge, this is the first structural view of APC, or any cullin-RING E3, with E2 and substrate juxtaposed, and it reveals how tripartite cullin-RING-E2 interactions establish APC's specificity for UBCH10 and harness a flexible catalytic module to drive ubiquitination of lysines within an accessible zone. We propose that multisite interactions reduce the degrees of freedom available to dynamic RING E3-E2∼Ub catalytic modules, condense the search radius for target lysines, increase the chance of active-site collision with conformationally fluctuating substrates, and enable regulation.


Assuntos
Ciclossomo-Complexo Promotor de Anáfase/química , Subunidade Apc1 do Ciclossomo-Complexo Promotor de Anáfase/química , Subunidade Apc11 do Ciclossomo-Complexo Promotor de Anáfase/química , DNA Helicases/química , Proteínas de Ligação a DNA/química , Enzimas de Conjugação de Ubiquitina/química , Ubiquitina/química , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Subunidade Apc1 do Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Subunidade Apc11 do Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Cristalografia por Raios X , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo
7.
Bioorg Med Chem ; 22(22): 6333-43, 2014 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-25438756

RESUMO

Proliferating cell nuclear antigen (PCNA) assumes an indispensable role in supporting cellular DNA replication and repair by organizing numerous protein components of these pathways via a common PCNA-interacting sequence motif called a PIP-box. Given the multifunctional nature of PCNA, the selective inhibition of PIP-box-mediated interactions may represent a new strategy for the chemosensitization of cancer cells to existing DNA-directed therapies; however, promiscuous blockage of these interactions may also be universally deleterious. To address these possibilities, we utilized a chemical strategy to irreversibly block PIP-box-mediated interactions. Initially, we identified and validated PCNA methionine 40 (M40) and histidine 44 (H44) as essential residues for PCNA/PIP-box interactions in general and, more specifically, for efficient PCNA loading onto chromatin within cells. Next, we created a novel small molecule incorporating an electrophilic di-chloro platinum moiety that preferentially alkylated M40 and H44 residues. The compound, designated T2Pt, covalently cross-linked wild-type but not M40A/H44A PCNA, irreversibly inhibited PCNA/PIP-box interactions, and mildly alkylated plasmid DNA in vitro. In cells, T2Pt persistently induced cell cycle arrest, activated ATR-Chk1 signaling and modestly induced DNA strand breaks, features typical of cellular replication stress. Despite sustained activation of the replication stress response by the compound and its modestly genotoxic nature, T2Pt demonstrated little activity in clonogenic survival assays as a single agent, yet sensitized cells to cisplatin. The discovery of T2Pt represents an original effort directed at the development of irreversible PCNA inhibitors and sets the stage for the discovery of analogues more selective for PCNA over other cellular nucleophiles.


Assuntos
Compostos Organoplatínicos/química , Antígeno Nuclear de Célula em Proliferação/química , Sítios de Ligação , Carbamatos/síntese química , Carbamatos/química , Carbamatos/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Dano ao DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Polarização de Fluorescência , Células HeLa , Humanos , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Compostos Organoplatínicos/síntese química , Compostos Organoplatínicos/farmacologia , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
8.
Tuberculosis (Edinb) ; 92(2): 173-81, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22209468

RESUMO

The anti-mycobacterial activities of nine series of dicarboxyl and tricarboxyl dendritic amphiphiles with one alkyl, two alkyl, and cholestanyl tails against Mycobacterium abscessus, Mycobacterium avium, Mycobacterium chelonae, Mycobacterium marinum and Mycobacterium smegmatis have been measured. The dendritic amphiphiles overcame the limited aqueous solubility of natural long-chain fatty acids, alcohols, and amines to enable profiling the susceptibilities of the different mycobacterial species to the physicochemical properties of these amphiphiles. Several dendritic amphiphiles showed strong anti-mycobacterial activity with high critical micelle concentrations and low hemolytic activities thereby offering platforms for the development of antibiotics of higher activity against nontuberculous mycobacteria.


Assuntos
Antibacterianos/farmacologia , Dendrímeros/farmacologia , Micobactérias não Tuberculosas/efeitos dos fármacos , Antibacterianos/química , Biofilmes/efeitos dos fármacos , Dendrímeros/química , Avaliação Pré-Clínica de Medicamentos/métodos , Farmacorresistência Bacteriana , Hemólise/efeitos dos fármacos , Humanos , Fluidez de Membrana/efeitos dos fármacos , Micelas , Testes de Sensibilidade Microbiana/métodos , Mycobacterium avium/efeitos dos fármacos , Mycobacterium avium/crescimento & desenvolvimento , Mycobacterium avium/fisiologia , Micobactérias não Tuberculosas/classificação , Micobactérias não Tuberculosas/crescimento & desenvolvimento , Micobactérias não Tuberculosas/fisiologia , Especificidade da Espécie , Relação Estrutura-Atividade , Temperatura
9.
Bioorg Med Chem ; 19(9): 2918-26, 2011 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-21493076

RESUMO

Homologous dicarboxyl dendritic amphiphiles-RCONHC(CH(3))(CH(2)CH(2)COOH)(2), 4(n); and ROCONHC(CH(3))(CH(2)CH(2)COOH)(2), 5(n), where R=n-C(n)H(2)(n)(+1) and n=13-22 carbon atoms-were synthesized. Critical micelle concentrations (CMCs) in aqueous triethanolamine solutions and at pH 7.4 were measured along with hemolytic activity (effective concentrations, EC(10)) in phosphate-buffered saline (PBS). LogCMC showed a linear dependence on chain length (n); the longest chain in each series had the lowest CMC-in triethanolamine: 4(21), 180µM and 5(22), 74µM and at pH 7.4: 4(21), 78µM and 5(22), 33µM. These two series, 4(n) and 5(n), and three series of homologous tricarboxyl dendritic amphiphiles-RCONHC(CH(2)CH(2)COOH)(3), 1(n); ROCONHC(CH(2)CH(2)COOH)(3), 2(n); RNHCONHC(CH(2)CH(2)COOH)(3), 3(n), where R=n-C(n)H(2)(n)(+1) and n=13-22 carbon atoms-were tested for growth inhibition of Staphylococcus aureus strain ATCC 6358 and methicillin-resistant S. aureus (MRSA) strain ATCC 43330 by microdilution in 0.1-strength brain heart infusion broth (BHIB). Amphiphiles 4(19), 4(21), 5(18), and 5(20) showed the strongest antibacterial activity (2.2-3.4µg/mL) against S. aureus (vancomycin, MIC=0.25µg/mL). These four plus 1(21), 2(20), 2(22), and 3(20) exhibited the strongest antibacterial activity (1.7-6.8µg/mL) against MRSA (vancomycin, MIC=0.25µg/mL). The MICs of these amphiphiles against six clinical MRSA were similar to those against the ATCC strain. In PBS, EC(10)s of the most active homologues ranged from 7 to 18µg/mL and 18 to 220µg/mL for di- and tricarboxyl dendritic amphiphiles, respectively. To assess the potential safety of using dendritic amphiphiles as drugs, measurements of micellar and hemolytic properties were conducted in the same medium (full-strength BHIB) that was used for antibacterial activity. The CMCs (9-36µg/mL, ∼18-72µM) of ten amphiphiles were measured by microdilution (log2 progression) with dye-covered beads. The EC(10)s were similar to those in PBS. The MICs of most amphiphiles (14-72µg/mL) and vancomycin (1.1-2.2µg/mL) against both S. aureus and MRSA increased significantly compared to the MICs measured in 0.1-strength BHIB. The one exception, 5(18), had an MIC against S. aureus of 1.1µg/mL compared to vancomycin (2.2µg/mL). With CMC (9-18µg/mL) and EC(10) (16µg/mL) values higher than the MIC, 5(18) was discovered as a lead for further development.


Assuntos
Antibacterianos/química , Dendrímeros/química , Ácidos Heptanoicos/química , Micelas , Antibacterianos/síntese química , Antibacterianos/farmacologia , Dendrímeros/farmacologia , Hemólise , Ácidos Heptanoicos/síntese química , Ácidos Heptanoicos/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Staphylococcus aureus/efeitos dos fármacos
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