RESUMO
Drug discovery relies on efficient identification of small-molecule leads and their interactions with macromolecular targets. However, understanding how chemotypes impact mechanistically important conformational states often remains secondary among high-throughput discovery methods. Here, we present a conformational discovery pipeline integrating time-resolved, high-throughput small-angle X-ray scattering (TR-HT-SAXS) and classic fragment screening applied to allosteric states of the mitochondrial import oxidoreductase apoptosis-inducing factor (AIF). By monitoring oxidized and X-ray-reduced AIF states, TR-HT-SAXS leverages structure and kinetics to generate a multidimensional screening dataset that identifies fragment chemotypes allosterically stimulating AIF dimerization. Fragment-induced dimerization rates, quantified with time-resolved SAXS similarity analysis (kVR), capture structure-activity relationships (SAR) across the top-ranked 4-aminoquinoline chemotype. Crystallized AIF-aminoquinoline complexes validate TR-SAXS-guided SAR, supporting this conformational chemotype for optimization. AIF-aminoquinoline structures and mutational analysis reveal active site F482 as an underappreciated allosteric stabilizer of AIF dimerization. This conformational discovery pipeline illustrates TR-HT-SAXS as an effective technology for targeting chemical leads to important macromolecular states.
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Mre11 forms the core of the multifunctional Mre11-Rad50-Nbs1 (MRN) complex that detects DNA double-strand breaks (DSBs), activates the ATM checkpoint kinase, and initiates homologous recombination (HR) repair of DSBs. To define the roles of Mre11 in both DNA bridging and nucleolytic processing during initiation of DSB repair, we combined small-angle X-ray scattering (SAXS) and crystal structures of Pyrococcus furiosus Mre11 dimers bound to DNA with mutational analyses of fission yeast Mre11. The Mre11 dimer adopts a four-lobed U-shaped structure that is critical for proper MRN complex assembly and for binding and aligning DNA ends. Further, mutations blocking Mre11 endonuclease activity impair cell survival after DSB induction without compromising MRN complex assembly or Mre11-dependant recruitment of Ctp1, an HR factor, to DSBs. These results show how Mre11 dimerization and nuclease activities initiate repair of DSBs and collapsed replication forks, as well as provide a molecular foundation for understanding cancer-causing Mre11 mutations in ataxia telangiectasia-like disorder (ATLD).
Assuntos
Proteínas Arqueais/metabolismo , Reparo do DNA , DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/metabolismo , Pyrococcus furiosus/química , Proteínas Arqueais/química , Proteínas Arqueais/genética , Cristalografia por Raios X , DNA/química , Quebras de DNA de Cadeia Dupla , Análise Mutacional de DNA , Dimerização , Endodesoxirribonucleases/química , Endodesoxirribonucleases/genética , Exodesoxirribonucleases/química , Exodesoxirribonucleases/genética , Modelos Moleculares , Espalhamento a Baixo Ângulo , Schizosaccharomyces/genética , Técnicas do Sistema de Duplo-Híbrido , Difração de Raios XRESUMO
MRE11 within the MRE11-RAD50-NBS1 (MRN) complex acts in DNA double-strand break repair (DSBR), detection, and signaling; yet, how its endo- and exonuclease activities regulate DSBR by nonhomologous end-joining (NHEJ) versus homologous recombination (HR) remains enigmatic. Here, we employed structure-based design with a focused chemical library to discover specific MRE11 endo- or exonuclease inhibitors. With these inhibitors, we examined repair pathway choice at DSBs generated in G2 following radiation exposure. While nuclease inhibition impairs radiation-induced replication protein A (RPA) chromatin binding, suggesting diminished resection, the inhibitors surprisingly direct different repair outcomes. Endonuclease inhibition promotes NHEJ in lieu of HR, while exonuclease inhibition confers a repair defect. Collectively, the results describe nuclease-specific MRE11 inhibitors, define distinct nuclease roles in DSB repair, and support a mechanism whereby MRE11 endonuclease initiates resection, thereby licensing HR followed by MRE11 exonuclease and EXO1/BLM bidirectional resection toward and away from the DNA end, which commits to HR.
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Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Proteínas de Ligação a DNA/metabolismo , Inibidores Enzimáticos/química , Fase G2 , Reparo de DNA por Recombinação , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Raios gama/efeitos adversos , Humanos , Proteína Homóloga a MRE11 , Proteína de Replicação A/genética , Proteína de Replicação A/metabolismoRESUMO
The post-translational modification of DNA repair and checkpoint proteins by ubiquitin and small ubiquitin-like modifier (SUMO) critically orchestrates the DNA damage response (DDR). The ubiquitin ligase RNF4 integrates signaling by SUMO and ubiquitin, through its selective recognition and ubiquitination of SUMO-modified proteins. Here, we define a key new determinant for target discrimination by RNF4, in addition to interaction with SUMO. We identify a nucleosome-targeting motif within the RNF4 RING domain that can bind DNA and thereby enables RNF4 to selectively ubiquitinate nucleosomal histones. Furthermore, RNF4 nucleosome-targeting is crucially required for the repair of TRF2-depleted dysfunctional telomeres by 53BP1-mediated non-homologous end joining.
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Reparo do DNA , Proteínas Nucleares/metabolismo , Proteínas Nucleares/ultraestrutura , Nucleossomos/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/ultraestrutura , Motivos de Aminoácidos , Animais , Linhagem Celular , Proteínas Cromossômicas não Histona/metabolismo , Cristalografia por Raios X , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Técnicas de Inativação de Genes , Camundongos , Proteínas Nucleares/genética , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologia , Telômero/efeitos dos fármacos , Telômero/genética , Proteína 2 de Ligação a Repetições Teloméricas/genética , Fatores de Transcrição/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53 , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases , UbiquitinaçãoRESUMO
The Mre11 complex (Mre11-Rad50-Nbs1 or MRN) binds double-strand breaks where it interacts with CtIP/Ctp1/Sae2 and ATM/Tel1 to preserve genome stability through its functions in homology-directed repair, checkpoint signaling and telomere maintenance. Here, we combine biochemical, structural and in vivo functional studies to uncover key properties of Mre11-W243R, a mutation identified in two pediatric cancer patients with enhanced ataxia telangiectasia-like disorder. Purified human Mre11-W243R retains nuclease and DNA binding activities in vitro. X-ray crystallography of Pyrococcus furiosus Mre11 indicates that an analogous mutation leaves the overall Mre11 three-dimensional structure and nuclease sites intact but disorders surface loops expected to regulate DNA and Rad50 interactions. The equivalent W248R allele in fission yeast allows Mre11 to form an MRN complex that efficiently binds double-strand breaks, activates Tel1/ATM and maintains telomeres; yet, it causes hypersensitivity to ionizing radiation and collapsed replication forks, increased Rad52 foci, defective Chk1 signaling and meiotic failure. W248R differs from other ataxia telangiectasia-like disorder analog alleles by the reduced stability of its interaction with Rad50 in cell lysates. Collective results suggest a separation-of-function mutation that disturbs interactions amongst the MRN subunits and Ctp1 required for DNA end processing in vivo but maintains interactions sufficient for Tel1/ATM checkpoint and telomere maintenance functions.
Assuntos
Ataxia Telangiectasia/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas de Ligação a DNA/genética , Exodesoxirribonucleases/genética , Mutação de Sentido Incorreto , Proteínas de Schizosaccharomyces pombe/genética , Sequência de Aminoácidos , Antígenos Nucleares/genética , Proteínas Arqueais/química , Proteínas Arqueais/genética , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/metabolismo , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/química , Endodesoxirribonucleases/genética , Exodesoxirribonucleases/química , Exodesoxirribonucleases/metabolismo , Humanos , Autoantígeno Ku , Proteína Homóloga a MRE11 , Meiose , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Serina-Treonina Quinases/metabolismo , Pyrococcus furiosus/enzimologia , Proteína Rad52 de Recombinação e Reparo de DNA/análise , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Homeostase do Telômero , Proteínas Supressoras de Tumor/metabolismoRESUMO
Chemical probes are invaluable tools for investigating essential biological processes. Understanding how small-molecule probes engage biomolecular conformations is critical to developing their functional selectivity. High-throughput solution X-ray scattering is well-positioned to profile target-ligand complexes during probe development, bringing conformational insight and selection to traditional ligand binding assays. Access to high-quality synchrotron SAXS datasets and high-throughput data analysis now allows routine academic users to incorporate conformational information into small-molecule development pipelines. Here we describe a general approach for benchmarking and preparing HT-SAXS chemical screens from small fragment libraries. Using the allosteric oxidoreductase Apoptosis-Inducing Factor (AIF) as an exemplary system, we illustrate how HT-SAXS efficiently identifies an allosteric candidate among hits of a microscale thermophoresis ligand screen. We discuss considerations for pursuing HT-SAXS chemical screening with other systems of interest and reflect on advances to extend screening throughput and sensitivity.
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Oxirredutases , Síncrotrons , Difração de Raios X , Ligantes , Espalhamento a Baixo ÂnguloRESUMO
Despite the popular use of dietary supplements during conventional cancer treatments, their impacts on the efficacies of prevalent immunotherapies, including immune-checkpoint therapy (ICT), are unknown. Surprisingly, our analyses of electronic health records revealed that ICT-treated patients with cancer who took vitamin E (VitE) had significantly improved survival. In mouse models, VitE increased ICT antitumor efficacy, which depended on dendritic cells (DC). VitE entered DCs via the SCARB1 receptor and restored tumor-associated DC functionality by directly binding to and inhibiting protein tyrosine phosphatase SHP1, a DC-intrinsic checkpoint. SHP1 inhibition, genetically or by VitE treatment, enhanced tumor antigen cross-presentation by DCs and DC-derived extracellular vesicles (DC-EV), triggering systemic antigen-specific T-cell antitumor immunity. Combining VitE with DC-recruiting cancer vaccines or immunogenic chemotherapies greatly boosted ICT efficacy in animals. Therefore, combining VitE supplement or SHP1-inhibited DCs/DC-EVs with DC-enrichment therapies could substantially augment T-cell antitumor immunity and enhance the efficacy of cancer immunotherapies. SIGNIFICANCE: The impacts of nutritional supplements on responses to immunotherapies remain unexplored. Our study revealed that dietary vitamin E binds to and inhibits DC checkpoint SHP1 to increase antigen presentation, prime antitumor T-cell immunity, and enhance immunotherapy efficacy. VitE-treated or SHP1-silenced DCs/DC-EVs could be developed as potent immunotherapies. This article is highlighted in the In This Issue feature, p. 1599.
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Vacinas Anticâncer , Neoplasias , Animais , Vacinas Anticâncer/uso terapêutico , Células Dendríticas , Imunoterapia , Camundongos , Neoplasias/tratamento farmacológico , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Vitamina E/metabolismoRESUMO
We present a Chemistry and Structure Screen Integrated Efficiently (CASSIE) approach (named for Greek prophet Cassandra) to design inhibitors for cancer biology and pathogenesis. CASSIE provides an effective path to target master keys to control the repair-replication interface for cancer cells and SARS CoV-2 pathogenesis as exemplified here by specific targeting of Poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribose glycohydrolase ARH3 macrodomains plus SARS CoV-2 nonstructural protein 3 (Nsp3) Macrodomain 1 (Mac1) and Nsp15 nuclease. As opposed to the classical massive effort employing libraries with large numbers of compounds against single proteins, we make inhibitor design for multiple targets efficient. Our compact, chemically diverse, 5000 compound Goldilocks (GL) library has an intermediate number of compounds sized between fragments and drugs with predicted favorable ADME (absorption, distribution, metabolism, and excretion) and toxicological profiles. Amalgamating our core GL library with an approved drug (AD) library, we employ a combined GLAD library virtual screen, enabling an effective and efficient design cycle of ranked computer docking, top hit biophysical and cell validations, and defined bound structures using human proteins or their avatars. As new drug design is increasingly pathway directed as well as molecular and mechanism based, our CASSIE approach facilitates testing multiple related targets by efficiently turning a set of interacting drug discovery problems into a tractable medicinal chemistry engineering problem of optimizing affinity and ADME properties based upon early co-crystal structures. Optimization efforts are made efficient by a computationally-focused iterative chemistry and structure screen. Thus, we herein describe and apply CASSIE to define prototypic, specific inhibitors for PARG vs distinct inhibitors for the related macrodomains of ARH3 and SARS CoV-2 Nsp3 plus the SARS CoV-2 Nsp15 RNA nuclease.
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COVID-19 , Ácidos Nucleicos , Síndrome Respiratória Aguda Grave , Reparo do DNA , Humanos , Simulação de Acoplamento Molecular , SARS-CoV-2RESUMO
DNA double-strand break (DSB) repair is initiated by MRE11 nuclease for both homology-directed repair (HDR) and alternative end joining (Alt-EJ). Here, we found that GRB2, crucial to timely proliferative RAS/MAPK pathway activation, unexpectedly forms a biophysically validated GRB2-MRE11 (GM) complex for efficient HDR initiation. GRB2-SH2 domain targets the GM complex to phosphorylated H2AX at DSBs. GRB2 K109 ubiquitination by E3 ubiquitin ligase RBBP6 releases MRE11 promoting HDR. RBBP6 depletion results in prolonged GM complex and HDR defects. GRB2 knockout increased MRE11-XRCC1 complex and Alt-EJ. Reconstitution with separation-of-function GRB2 mutant caused HDR deficiency and synthetic lethality with PARP inhibitor. Cell and cancer genome analyses suggest biomarkers of low GRB2 for noncanonical HDR deficiency and high MRE11 and GRB2 expression for worse survival in HDR-proficient patients. These findings establish GRB2's role in binding, targeting, and releasing MRE11 to promote efficient HDR over Alt-EJ DSB repair, with implications for genome stability and cancer biology.
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Cancer will directly affect the lives of over one-third of the population. The DNA Damage Response (DDR) is an intricate system involving damage recognition, cell cycle regulation, DNA repair, and ultimately cell fate determination, playing a central role in cancer etiology and therapy. Two primary therapeutic approaches involving DDR targeting include: combinatorial treatments employing anticancer genotoxic agents; and synthetic lethality, exploiting a sporadic DDR defect as a mechanism for cancer-specific therapy. Whereas, many DDR proteins have proven "undruggable", Fragment- and Structure-Based Drug Discovery (FBDD, SBDD) have advanced therapeutic agent identification and development. FBDD has led to 4 (with â¼50 more drugs under preclinical and clinical development), while SBDD is estimated to have contributed to the development of >200, FDA-approved medicines. Protein X-ray crystallography-based fragment library screening, especially for elusive or "undruggable" targets, allows for simultaneous generation of hits plus details of protein-ligand interactions and binding sites (orthosteric or allosteric) that inform chemical tractability, downstream biology, and intellectual property. Using a novel high-throughput crystallography-based fragment library screening platform, we screened five diverse proteins, yielding hit rates of â¼2-8% and crystal structures from â¼1.8 to 3.2 Å. We consider current FBDD/SBDD methods and some exemplary results of efforts to design inhibitors against the DDR nucleases meiotic recombination 11 (MRE11, a.k.a., MRE11A), apurinic/apyrimidinic endonuclease 1 (APE1, a.k.a., APEX1), and flap endonuclease 1 (FEN1).
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Descoberta de Drogas , Preparações Farmacêuticas , Cristalografia por Raios X , Dano ao DNA , Reparo do DNARESUMO
Arrival of the novel SARS-CoV-2 has launched a worldwide effort to identify both pre-approved and novel therapeutics targeting the viral proteome, highlighting the urgent need for efficient drug discovery strategies. Even with effective vaccines, infection is possible, and at-risk populations would benefit from effective drug compounds that reduce the lethality and lasting damage of COVID-19 infection. The CoV-2 MacroD-like macrodomain (Mac1) is implicated in viral pathogenicity by disrupting host innate immunity through its mono (ADP-ribosyl) hydrolase activity, making it a prime target for antiviral therapy. We therefore solved the structure of CoV-2 Mac1 from non-structural protein 3 (Nsp3) and applied structural and sequence-based genetic tracing, including newly determined A. pompejana MacroD2 and GDAP2 amino acid sequences, to compare and contrast CoV-2 Mac1 with the functionally related human DNA-damage signaling factor poly (ADP-ribose) glycohydrolase (PARG). Previously, identified targetable features of the PARG active site allowed us to develop a pharmacologically useful PARG inhibitor (PARGi). Here, we developed a focused chemical library and determined 6 novel PARGi X-ray crystal structures for comparative analysis. We applied this knowledge to discovery of CoV-2 Mac1 inhibitors by combining computation and structural analysis to identify PARGi fragments with potential to bind the distal-ribose and adenosyl pockets of the CoV-2 Mac1 active site. Scaffold development of these PARGi fragments has yielded two novel compounds, PARG-345 and PARG-329, that crystallize within the Mac1 active site, providing critical structure-activity data and a pathway for inhibitor optimization. The reported structural findings demonstrate ways to harness our PARGi synthesis and characterization pipeline to develop CoV-2 Mac1 inhibitors targeting the ADP-ribose active site. Together, these structural and computational analyses reveal a path for accelerating development of antiviral therapeutics from pre-existing drug optimization pipelines.
Assuntos
Antivirais/química , Proteases Semelhantes à Papaína de Coronavírus/metabolismo , Inibidores Enzimáticos/química , Glicosídeo Hidrolases/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/química , Xantinas/química , Sequência de Aminoácidos , Antivirais/farmacologia , Domínio Catalítico , Cristalografia por Raios X , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Humanos , Modelos Moleculares , Domínios Proteicos , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-Atividade , Xantinas/farmacologia , Tratamento Farmacológico da COVID-19RESUMO
Human uracil DNA-glycosylase (UDG) is the prototypic and first identified DNA glycosylase with a vital role in removing deaminated cytosine and incorporated uracil and 5-fluorouracil (5-FU) from DNA. UDG depletion sensitizes cells to high APOBEC3B deaminase and to pemetrexed (PEM) and floxuridine (5-FdU), which are toxic to tumor cells through incorporation of uracil and 5-FU into DNA. To identify small-molecule UDG inhibitors for pre-clinical evaluation, we optimized biochemical screening of a selected diversity collection of >3,000 small-molecules. We found aurintricarboxylic acid (ATA) as an inhibitor of purified UDG at an initial calculated IC50 < 100 nM. Subsequent enzymatic assays confirmed effective ATA inhibition but with an IC50 of 700 nM and showed direct binding to the human UDG with a KD of <700 nM. ATA displays preferential, dose-dependent binding to purified human UDG compared to human 8-oxoguanine DNA glycosylase. ATA did not bind uracil-containing DNA at these concentrations. Yet, combined crystal structure and in silico docking results unveil ATA interactions with the DNA binding channel and uracil-binding pocket in an open, destabilized UDG conformation. Biologically relevant ATA inhibition of UDG was measured in cell lysates from human DLD1 colon cancer cells and in MCF-7 breast cancer cells using a host cell reactivation assay. Collective findings provide proof-of-principle for development of an ATA-based chemotype and "door stopper" strategy targeting inhibitor binding to a destabilized, open pre-catalytic glycosylase conformation that prevents active site closing for functional DNA binding and nucleotide flipping needed to excise altered bases in DNA.
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Reparo do DNA , Uracila-DNA Glicosidase , Domínio Catalítico , Citidina Desaminase , Dano ao DNA , Humanos , Antígenos de Histocompatibilidade Menor , Uracila , Uracila-DNA Glicosidase/genética , Uracila-DNA Glicosidase/metabolismoRESUMO
Poly(ADP-ribose)ylation (PARylation) by PAR polymerase 1 (PARP1) and PARylation removal by poly(ADP-ribose) glycohydrolase (PARG) critically regulate DNA damage responses; yet, conflicting reports obscure PARG biology and its impact on cancer cell resistance to PARP1 inhibitors. Here, we found that PARG expression is upregulated in many cancers. We employed chemical library screening to identify and optimize methylxanthine derivatives as selective bioavailable PARG inhibitors. Multiple crystal structures reveal how substituent positions on the methylxanthine core dictate binding modes and inducible-complementarity with a PARG-specific tyrosine clasp and arginine switch, supporting inhibitor specificity and a competitive inhibition mechanism. Cell-based assays show selective PARG inhibition and PARP1 hyperPARylation. Moreover, our PARG inhibitor sensitizes cells to radiation-induced DNA damage, suppresses replication fork progression and impedes cancer cell survival. In PARP inhibitor-resistant A172 glioblastoma cells, our PARG inhibitor shows comparable killing to Nedaplatin, providing further proof-of-concept that selectively inhibiting PARG can impair cancer cell survival.
Assuntos
Replicação do DNA/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Glicosídeo Hidrolases/antagonistas & inibidores , Neoplasias/genética , Bibliotecas de Moléculas Pequenas/farmacologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Inibidores Enzimáticos/química , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Humanos , Neoplasias/enzimologia , Neoplasias/metabolismo , Neoplasias/fisiopatologia , Poli ADP Ribosilação/efeitos dos fármacos , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Bibliotecas de Moléculas Pequenas/químicaRESUMO
For inhibitor design, as in most research, the best system is question dependent. We suggest structurally defined allostery to design specific inhibitors that target regions beyond active sites. We choose systems allowing efficient quality structures with conformational changes as optimal for structure-based design to optimize inhibitors. We maintain that evolutionarily related targets logically provide molecular avatars, where this Sanskrit term for descent includes ideas of functional relationships and of being a physical embodiment of the target's essential features without requiring high sequence identity. Appropriate biochemical and cell assays provide quantitative measurements, and for biomedical impacts, any inhibitor's activity should be validated in human cells. Specificity is effectively shown empirically by testing if mutations blocking target activity remove cellular inhibitor impact. We propose this approach to be superior to experiments testing for lack of cross-reactivity among possible related enzymes, which is a challenging negative experiment. As an exemplary avatar system for protein and DNA allosteric conformational controls, we focus here on developing separation-of-function inhibitors for meiotic recombination 11 nuclease activities. This was achieved not by targeting the active site but rather by geometrically impacting loop motifs analogously to ribosome antibiotics. These loops are neighboring the dimer interface and active site act in sculpting dsDNA and ssDNA into catalytically competent complexes. One of our design constraints is to preserve DNA substrate binding to geometrically block competing enzymes and pathways from the damaged site. We validate our allosteric approach to controlling outcomes in human cells by reversing the radiation sensitivity and genomic instability in BRCA mutant cells.
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Desenho de Fármacos , Proteína Homóloga a MRE11/antagonistas & inibidores , Regulação Alostérica , Sequência de Aminoácidos , Endonucleases/antagonistas & inibidores , Endonucleases/metabolismo , Evolução Molecular , Exonucleases/antagonistas & inibidores , Exonucleases/metabolismo , Humanos , Proteína Homóloga a MRE11/genética , Proteína Homóloga a MRE11/metabolismo , Conformação Proteica , Sensibilidade e Especificidade , Alinhamento de Sequência , Análise de Sequência de ProteínaRESUMO
H/D and guest-exchange ion-molecule reactions have been used as a new tool to elucidate the operation of multiple hydrogen bonding in gas-phase complexes formed between phosphonate cavitands and ethyl-substituted ammonium ions.
RESUMO
The influence of the spacer arm on the interaction between agarose and a supported ligand was investigated through molecular dynamics for a combination of several spacers. The spacers differ for degree of hydrophobicity, length, and chemical composition, which was varied through insertion of thio, ether, and CH(2) groups. Agarose was modeled through a modified Glycam force field, whose parameters were determined through ab initio calculations. The structural model of agarose used for the calculations was obtained through MD studies of the conformational evolution of several agarose single and double helixes. The simulations showed that a modification of the spacer properties could determine a change of the stable structure of the ligand with respect to the support. In particular, if the spacer is hydrophilic and rigid, the favored structure is with extended spacer and solvated ligand. Either increasing the spacer length, and thus its flexibility, or decreasing its solvation free energy, which corresponds to diminishing its affinity for water, rapidly leads to a conformational change in which the ligand adsorbs on agarose. Interestingly, we found that if the spacer is long and hydrophilic, a third metastable structure, in which the spacer is sandwiched between the ligand and agarose, is possible. Simulations of several ligands adsorbed on neighboring sites on agarose showed that if the support is not held fixed through restraints, the interaction force between vicinal ligands is sufficient to determine a major conformational change of the system.
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Topoisomerase II creates a double-strand break intermediate with topoisomerase covalently coupled to the DNA via a 5'-phosphotyrosyl bond. These intermediate complexes can become cytotoxic protein-DNA adducts and DSB repair at these lesions requires removal of topoisomerase II. To analyse removal of topoisomerase II from genomic DNA we adapted the trapped in agarose DNA immunostaining assay. Recombinant MRE11 from 2 sources removed topoisomerase IIα from genomic DNA in vitro, as did MRE11 immunoprecipitates isolated from A-TLD or K562 cells. Basal topoisomerase II complex levels were very high in A-TLD cells lacking full-length wild type MRE11, suggesting that MRE11 facilitates the processing of topoisomerase complexes that arise as part of normal cellular metabolism. In K562 cells inhibition of MRE11, PARP or replication increased topoisomerase IIα and ß complex levels formed in the absence of an anti-topoisomerase II drug.
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Mre11 nuclease plays a central role in the repair of cytotoxic and mutagenic DNA double-strand breaks. As X-ray structural information has been available only for the Pyrococcus furiosus enzyme (PfMre11), the conserved and variable features of this nuclease across the domains of life have not been experimentally defined. Our crystal structure and biochemical studies demonstrate that TM1635 from Thermotoga maritima, originally annotated as a putative nuclease, is an Mre11 endo/exonuclease (TmMre11) and the first such structure from eubacteria. TmMre11 and PfMre11 display similar overall structures, despite sequence identity in the twilight zone of only approximately 20%. However, they differ substantially in their DNA-specificity domains and in their dimeric organization. Residues in the nuclease domain are highly conserved, but those in the DNA-specificity domain are not. The structural differences likely affect how Mre11 from different organisms recognize and interact with single-stranded DNA, double-stranded DNA and DNA hairpin structures during DNA repair. The TmMre11 nuclease active site has no bound metal ions, but is conserved in sequence and structure with the exception of a histidine that is important in PfMre11 nuclease activity. Nevertheless, biochemical characterization confirms that TmMre11 possesses both endonuclease and exonuclease activities on single-stranded and double-stranded DNA substrates, respectively.
Assuntos
Proteínas de Bactérias/química , Reparo do DNA , DNA de Cadeia Simples/química , DNA/química , Endodesoxirribonucleases/química , Exodesoxirribonucleases/química , Thermotoga maritima/enzimologia , Sequência de Aminoácidos , Cristalografia por Raios X , DNA/genética , DNA de Cadeia Simples/genética , Endodesoxirribonucleases/genética , Exodesoxirribonucleases/genética , Modelos Químicos , Dados de Sequência Molecular , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
The mitogen-activated protein (MAP) kinase protein family has a critical role in cellular signaling events, with MAP kinase p38alpha acting in inflammatory processes and being an important drug discovery target. MAP kinase drug design efforts have focused on small-molecule inhibitors of the ATP catalytic site, which exhibit dose-limiting adverse effects. Therefore, characterizing other potential sites that bind substrates, inhibitors, or allosteric effectors is of great interest. Here, we present the crystal structure of human p38alpha MAP kinase, which has a lead compound bound both in the active site and in the lipid-binding site of the C-terminal cap. This C-terminal cap is formed from an extension to the kinase fold, unique to the MAP kinase and cyclin-dependent kinase families and glycogen synthase kinase 3. Binding of this lead, 4-[3-(4-fluorophenyl)-1H-pyrazol-4-yl]pyridine, to wild-type p38alpha induces movement of the C-terminal cap region, creating a hydrophobic pocket centered around residue Trp197. Computational analysis of this C-terminal domain pocket indicates notable flexibility for potentially binding different-shaped compounds, including lipids, oxidized arachidonic acid species such as leukotrienes, and small-molecule effectors. Furthermore, our structural results defining the open p38alpha C-lobe pocket provide a detailed framework for the design of novel small molecules with affinities comparable to active-site binders: to bind and potentially modulate the shape and activity of p38alpha in predetermined ways. Moreover, these results and analyses of p38alpha suggest strategies for designing specific binding compounds applicable to other MAP kinases, as well as the cyclin-dependent kinase family and glycogen synthase kinase 3beta that also utilize the C-terminal insert in their interactions.
Assuntos
Proteína Quinase 14 Ativada por Mitógeno/química , Sítios de Ligação , Simulação por Computador , Cristalografia por Raios X , Humanos , Modelos Moleculares , Estrutura Terciária de ProteínaRESUMO
Structures and properties of nonbonding interactions involving guanidinium-functionalized hosts and carboxylate substrates were investigated by a combination of ab initio and molecular dynamics approaches. The systems under study are on one hand intended to be a model of the arginine-anion bond, so often observed in proteins and nucleic acids, and on the other to provide an opportunity to investigate the influence of molecular structure on the formation of supramolecular complexes in detail. Use of DFT calculations, including extended basis sets and implicit water treatment, allowed us to determine minimum-energy structures and binding enthalpies that compared well with experimental data. Intermolecular forces were found to be mostly due to electrostatic interactions through three hydrogen bonds, one of which is bifurcate, and are sufficiently strong to induce a conformational change in the ligand consisting of a rotation of about 180 degrees around the guanidiniocarbonylpyrrole axis. Free binding energies of the complexes were evaluated through MD simulations performed in the presence of explicit water molecules by use of the molecular mechanics Poisson-Boltzmann solvent accessible surface area (MM-PBSA) and linear interaction energy (LIE) approaches. LIE energies were in quantitative agreement with experimental data. A detailed analysis of the MD simulations revealed that the complexes cannot be described in terms of a single binding structure, but that they are characterized by a significant internal mobility responsible for several low-energy metastable structures.