RESUMO
Cyclin-dependent kinases (CDKs) are historic therapeutic targets implicated in tumorigenic events due to their critical involvement in the cell cycle phase. However, selectivity has proven to be a bottleneck, causing repeated failures. Previously, we reported CR6-interacting factor 1 (CRIF1), acting as a cell cycle negative regulator through interaction with CDK2. In the current report, we identified the CRIF1-CDK2 interaction interface by in silico studies and shortlisted interface inhibitors through virtual screening on CRIF1 using 40â¯678 drug-like compounds. These compounds were tested by cell proliferation assay, and four of these molecules were found to selectively inhibit the proliferation of osteosarcoma (OS) cell lines, but do not affect normal bone mesenchymal stem cells (BMSC). A binding study reveals significant affinities of the inhibitors on CRIF1. More importantly, treatment of the OS cells with a combination of ionizing radiation (IR) and the best-performing inhibitors remarkably increased IR inhibition potential from 19.9% to 59.6%. This occurred by selectively promoting G2/M arrest and apoptosis related to CDK2 overactivation in OS cells but not in BMSC and was supported by significant CDK2 phosphorylation modifications. Knocking down of CRIF1 by siRNA treatment showed similar effects to the interface inhibitors. Together we substantiate the identification of novel lead molecules, which may provide a new treatment to overcome selectivity issues and enhance the radiosensitivity of tumor cells, opening a conceptually novel strategy of CDK-targeting for different cancer types.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Radiossensibilizantes/farmacologia , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/efeitos da radiação , Proteínas de Ciclo Celular/química , Linhagem Celular Tumoral , Quinase 2 Dependente de Ciclina/química , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Modelos Moleculares , Ligação Proteica/efeitos dos fármacos , Conformação ProteicaRESUMO
Zika virus (ZIKV) is an emerging mosquito-borne virus recently linked to intrauterine growth restriction including abnormal fetal brain development. The recent outbreak of ZIKV reached pandemic level resulting in an alarming public health emergency. At present, there is limited understanding of the infectious mechanism and no approved therapy. Nonstructural protein 5 is essential for capping and replication of viral RNA and comprises a methyltransferase (MTase) and RNA dependent RNA polymerase domain. Here we used molecular modeling to obtain the structure of ZIKV MTase and molecular docking to identify the additional hydrophobic region uniquely conserved in flavivirus MTase that can be used as a druggable site. Subsequently, a virtual screening with a library of 28â¯341 compounds identified 10 best hits showing decisive contacts with the MTase. In vitro efficacy analysis of these compounds against ZIKV, by plaque reduction assay, has confirmed four of the top scored ligands (Life Chemicals ID: F3043-0013, F0922-0796, F1609-0442, and F1750-0048) having EC50 (50% effective concentration) values of 4.8 ± 2.3, 12.5 ± 7.4, 17.5 ± 8.4, and 17.6 ± 3.1 µM respectively, identifying lead compounds for anti-ZIKV drug development.
Assuntos
Descoberta de Drogas , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Metiltransferases/antagonistas & inibidores , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/farmacologia , Zika virus/enzimologia , Avaliação Pré-Clínica de Medicamentos , Metiltransferases/química , Modelos Moleculares , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha betaRESUMO
Human type 1 17ß-hydroxysteroid dehydrogenase (17ß-HSD1),a member of the short-chain dehydrogenase/reductase family, catalyzes the last step in the bioactivation of the most potent estrogen estradiol with high specificity and is thus involved in estrogen-dependent diseases. As an oxidoreductase, 17ß-HSD1 can utilize both triphosphate and diphosphate cofactors in reaction at the molecular level, but more specific with triphosphate cofactor. The NADPH is much higher than NADP+ in living cells leading to preliminary reduction action. The enzyme also showed substrate-induced inhibition unprecedented in other members of 17ß-HSDs. Our previous study elucidated the structural mechanism of substrate inhibition is due to the reversely bound estrone (E1) in the substrate-binding pocket of the enzyme resulting in a dead-end complex. However, the effect of the cofactor preference on the substrate inhibition of the enzyme is not yet clear. In the present study, we solved the ternary crystal structures of 17ß-HSD1 in complex with E1 and cofactor analog NAD+ . Combined with molecular dynamics simulation using the enzyme with NADH/NADPH and different oriented E1 (normally oriented, E1N; reversely oriented, E1R), such ternary structure provides a complete picture of enzyme-substrate-cofactor interactions. The results reveal that different cofactors and substrate binding mode affect the allosteric effect between the two subunits of the enzyme. And the results from MD simulations confirmed that His221 plays a key role in the formation of dead-end complex in NADPH complex, and the absence of stable interaction between His221 and E1R in the NADH complex should be the main reason for its lack of substrate inhibition.
Assuntos
17-Hidroxiesteroide Desidrogenases , NAD , Humanos , 17-Hidroxiesteroide Desidrogenases/metabolismo , Sítios de Ligação , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Estrogênios , NAD/metabolismo , NADP/metabolismo , Ligação Proteica , Especificidade por SubstratoRESUMO
Paclitaxel (taxol), a chemotherapeutic agent, remains the standard of care for the lethal triple-negative breast cancer (TNBC). However, over 50% of TNBC patients become resistant to chemotherapy and, to date, no solution is available. CR6-interacting factor 1 (CRIF1) is reported to act as a negative regulator of the cell cycle by interacting with cyclin-dependent kinase 2 (CDK2). In our study, two selective CRIF1-CDK2 interface inhibitors were used to investigate whether they could exert anti-proliferative activity on the TNBC cell lines. When combined with taxol treatment, these two inhibitors can advance the cells from G0/G1 to S and G2/M phases, producing irreparable damage to the cells, which then undergo apoptosis. Moreover, they enhanced the reduction in cell proliferation induced by taxol in TNBC cells, thereby improving sensitivity to taxol in these cell lines. Importantly, the inhibitors did not regulate the cell cycle in normal cells, indicating their high selectivity towards TNBC cells. Overall, the resistance to the anti-proliferative effects induced by taxol can be significantly reduced by the combined treatment with selective CRIF1-CDK2 interface inhibitors, making a conceptual advance in the CDK-related cancer treatment.
RESUMO
The global spread of COVID-19 constitutes the most dangerous pandemic to emerge during the last one hundred years. About seventy-nine million infections and more than 1.7 million death have been reported to date, along with destruction of the global economy. With the uncertainty evolved by alarming level of genome mutations, coupled with likelihood of generating only a short lived immune response by the vaccine injections, the identification of antiviral drugs for direct therapy is the need of the hour. Strategies to inhibit virus infection and replication focus on targets such as the spike protein and non-structural proteins including the highly conserved RNA-dependent-RNA-polymerase, nucleotidyl-transferases, main protease and papain-like proteases. There is also an indirect option to target the host cell recognition systems such as angiotensin-converting enzyme 2 (ACE2), transmembrane protease, serine 2, host cell expressed CD147, and the host furin. A drug search strategy consensus in tandem with analysis of currently available information is extremely important for the rapid identification of anti-viral. An unprecedented display of cooperation among the scientific community regarding SARS-CoV-2 research has resulted in the accumulation of an enormous amount of literature that requires curation. Drug repurposing and drug combinations have drawn tremendous attention for rapid therapeutic application, while high throughput screening and virtual searches support de novo drug identification. Here, we examine how certain approved drugs targeting different viruses can play a role in combating this new virus and analyze how they demonstrate efficacy under clinical assessment. Suggestions on repurposing and de novo strategies are proposed to facilitate the fight against the COVID-19 pandemic.
Assuntos
Antivirais/farmacologia , Tratamento Farmacológico da COVID-19 , COVID-19 , SARS-CoV-2 , COVID-19/epidemiologia , Desenvolvimento de Medicamentos/métodos , Reposicionamento de Medicamentos/métodos , Humanos , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/fisiologia , Resultado do Tratamento , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/genética , Internalização do Vírus/efeitos dos fármacosRESUMO
The opportunistic pathogen Pseudomonas aeruginosa is a leading cause of nosocomial infections, which are becoming increasingly difficult to treat due to antibiotic resistance. Polyphosphate (polyP) plays a key role in P. aeruginosa virulence, stress response, and antibiotic tolerance, suggesting an attractive drug target. Here, we show that the small molecule gallein disrupts polyphosphate homeostasis by inhibiting all members of both polyphosphate kinase (PPK) families (PPK1 and PPK2) encoded by P. aeruginosa, demonstrating dual-specificity PPK inhibition for the first time. Inhibitor treatment phenocopied ppk deletion to reduce cellular polyP accumulation and attenuate biofilm formation, motility, and pyoverdine and pyocyanin production. Most importantly, gallein attenuated P. aeruginosa virulence in a Caenorhabditis elegans infection model and synergized with antibiotics while exhibiting negligible toxicity toward the nematodes or HEK293T cells, suggesting our discovery of dual-specificity PPK inhibitors as a promising starting point for the development of new antivirulence therapeutics. IMPORTANCE Many priority bacterial pathogens such as P. aeruginosa encode both PPK1 and PPK2 enzymes to maintain polyphosphate homeostasis. While PPK1 and PPK2 have distinct structures and catalytic mechanisms, they are both capable of synthesizing and consuming polyphosphate; thus, PPK2 enzymes can compensate for the loss of PPK1 and vice versa. In this study, we identified the small molecule gallein as a dual-specificity inhibitor of both PPK1 and PPK2 enzyme families in P. aeruginosa. Inhibitor treatment reduced cellular polyP in wild-type (WT), Δppk1, and Δppk2 strains to levels that were on par with the Δppk1 Δppk2A Δppk2B Δppk2C knockout control. Treatment also attenuated biofilm formation, motility, toxin production, and virulence to a similar extent, thereby elucidating a hitherto-undocumented role of PPK2 enzymes in P. aeruginosa virulence phenotypes. This work therefore establishes PPK2s, in addition to PPK1, as valuable drug targets in P. aeruginosa and provides a favorable starting molecule for future inhibitor design efforts.
Assuntos
Antibacterianos/farmacologia , Inibidores Enzimáticos/farmacologia , Fosfotransferases (Aceptor do Grupo Fosfato)/antagonistas & inibidores , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/patogenicidade , Xantenos/farmacologia , Animais , Antibacterianos/uso terapêutico , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/microbiologia , Inibidores Enzimáticos/uso terapêutico , Células HEK293 , Humanos , Fenótipo , Fosfotransferases (Aceptor do Grupo Fosfato)/classificação , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/enzimologia , Virulência/efeitos dos fármacos , Xantenos/uso terapêuticoRESUMO
The pandemic outbreak of a new coronavirus (CoV), SARS-CoV-2, has captured the world's attention, demonstrating that CoVs represent a continuous global threat. As this is a highly contagious virus, it is imperative to understand RNA-dependent-RNA-polymerase (RdRp), the key component in virus replication. Although the SARS-CoV-2 genome shares 80% sequence identity with severe acute respiratory syndrome SARS-CoV, their RdRps and nucleotidyl-transferases (NiRAN) share 98.1% and 93.2% identity, respectively. Sequence alignment of six coronaviruses demonstrated higher identity among their RdRps (60.9%-98.1%) and lower identity among their Spike proteins (27%-77%). Thus, a 3D structural model of RdRp, NiRAN, non-structural protein 7 (nsp7), and nsp8 of SARS-CoV-2 was generated by modeling starting from the SARS counterpart structures. Furthermore, we demonstrate the binding poses of three viral RdRp inhibitors (Galidesivir, Favipiravir, and Penciclovir), which were recently reported to have clinical significance for SARS-CoV-2. The network of interactions established by these drug molecules affirms their efficacy to inhibit viral RNA replication and provides an insight into their structure-based rational optimization for SARS-CoV-2 inhibition.
Assuntos
Betacoronavirus/enzimologia , Nucleotidiltransferases/química , RNA Polimerase Dependente de RNA/química , Adenina/análogos & derivados , Adenina/química , Adenina/metabolismo , Adenosina/análogos & derivados , Amidas/química , Amidas/metabolismo , Antivirais/química , Antivirais/metabolismo , Betacoronavirus/isolamento & purificação , Sítios de Ligação , COVID-19 , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Humanos , Simulação de Acoplamento Molecular , Nucleotidiltransferases/metabolismo , Pandemias , Pneumonia Viral/epidemiologia , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Estrutura Terciária de Proteína , Pirazinas/química , Pirazinas/metabolismo , Pirrolidinas/química , Pirrolidinas/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , SARS-CoV-2RESUMO
Human 17ß-hydroxysteroid dehydrogenase type 1 (17ß-HSD1) catalyses the last step in estrogen activation and is thus involved in estrogen-dependent diseases (EDDs). Unlike other 17ß-HSD members, 17ß-HSD1 undergoes a significant substrate-induced inhibition that we have previously reported. Here we solved the binary and ternary crystal structures of 17ß-HSD1 in complex with estrone (E1) and cofactor analog NADP+ , demonstrating critical enzyme-substrate-cofactor interactions. These complexes revealed a reversely bound E1 in 17ß-HSD1 that provides the basis of the substrate inhibition, never demonstrated in estradiol complexes. Structural analysis showed that His221 is the key residue responsible for the reorganization and stabilization of the reversely bound E1, leading to the formation of a dead-end complex, which exists widely in NADP(H)-preferred enzymes for the regulation of their enzymatic activity. Further, a new inhibitor is proposed that may inhibit 17ß-HSD1 through the formation of a dead-end complex. This finding indicates a simple mechanism of enzyme regulation in the physiological background and introduces a pioneer inhibitor of 17ß-HSD1 based on the dead-end inhibition model for efficiently targeting EDDs. DATABASES: Coordinates and structure factors of 17ß-HSD1-E1 and 17ß-HSD1-E1-NADP+ have been deposited in the Protein Data Bank with accession code 6MNC and 6MNE respectively. ENZYMES: 17ß-hydroxysteroid dehydrogenase type 1 (17ß-HSD1) EC 1.1.1.62.
Assuntos
17-Hidroxiesteroide Desidrogenases/química , Estrona/química , NADP/química , Conformação Proteica , 17-Hidroxiesteroide Desidrogenases/genética , Sequência de Aminoácidos/genética , Sítios de Ligação/genética , Catálise , Cristalografia por Raios X , Inibidores Enzimáticos/química , Estrogênios/química , Estrogênios/genética , Humanos , Modelos Moleculares , Oxirredução , Ligação Proteica/genética , Especificidade por SubstratoRESUMO
Since the first major outbreak of Zika virus (ZIKV) in 2007, ZIKV is spreading explosively through South and Central America, and recent reports in highly populated developing countries alarm the possibility of a more catastrophic outbreak. ZIKV infection in pregnant women leads to embryonic microcephaly and Guillain-Barré syndrome in adults. At present, there is limited understanding of the infectious mechanism, and no approved therapy has been reported. Despite the withdrawal of public health emergency, the WHO still considers the ZIKV as a highly significant and long-term public health challenge that the situation has to be addressed rapidly. Non-structural protein 5 is essential for capping and replication of viral RNA and comprises a methyltransferase and RNA-dependent RNA polymerase (RdRp) domain. We used molecular modeling to obtain the structure of ZIKV RdRp, and by molecular docking and phylogeny analysis, we here demonstrate the potential sites for drug screening. Two metal binding sites and an NS3-interacting region in ZIKV RdRp are demonstrated as potential drug screening sites. The docked structures reveal a remarkable degree of conservation at the substrate binding site and the potential drug screening sites. A phylogeny-based approach is provided for an emergency preparedness, where similar class of ligands could target phylogenetically related proteins.
Assuntos
RNA Polimerase Dependente de RNA/metabolismo , Proteínas Virais/metabolismo , Zika virus/enzimologia , Sítios de Ligação , Surtos de Doenças , Desenho de Fármacos , Humanos , Simulação de Acoplamento Molecular , Filogenia , Estrutura Terciária de Proteína , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/classificação , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais/antagonistas & inibidores , Zika virus/metabolismo , Infecção por Zika virus/epidemiologia , Infecção por Zika virus/patologia , Infecção por Zika virus/virologiaRESUMO
Aminoacyl-tRNA synthetases are essential components in protein biosynthesis. Arginyl-tRNA synthetase (ArgRS) belongs to the small group of aminoacyl-tRNA synthetases requiring cognate tRNA for amino acid activation. The crystal structure of Escherichia coli (Eco) ArgRS has been solved in complex with tRNAArg at 3.0-Å resolution. With this first bacterial tRNA complex, we are attempting to bridge the gap existing in structure-function understanding in prokaryotic tRNAArg recognition. The structure shows a tight binding of tRNA on the synthetase through the identity determinant A20 from the D-loop, a tRNA recognition snapshot never elucidated structurally. This interaction of A20 involves 5 amino acids from the synthetase. Additional contacts via U20a and U16 from the D-loop reinforce the interaction. The importance of D-loop recognition in EcoArgRS functioning is supported by a mutagenesis analysis of critical amino acids that anchor tRNAArg on the synthetase; in particular, mutations at amino acids interacting with A20 affect binding affinity to the tRNA and specificity of arginylation. Altogether the structural and functional data indicate that the unprecedented ArgRS crystal structure represents a snapshot during functioning and suggest that the recognition of the D-loop by ArgRS is an important trigger that anchors tRNAArg on the synthetase. In this process, A20 plays a major role, together with prominent conformational changes in several ArgRS domains that may eventually lead to the mature ArgRS:tRNA complex and the arginine activation. Functional implications that could be idiosyncratic to the arginine identity of bacterial ArgRSs are discussed.
Assuntos
Arginina-tRNA Ligase/química , Arginina-tRNA Ligase/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Ligases/química , Ligases/metabolismo , RNA de Transferência de Arginina/metabolismo , Arginina-tRNA Ligase/genética , Sítios de Ligação , Cristalografia por Raios X , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Ligases/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ligação Proteica , Conformação Proteica , RNA Bacteriano , RNA de Transferência de Arginina/químicaRESUMO
Aminoacyl-tRNA synthetase:transfer RNA (aaRS:tRNA) systems became recently essential targets in molecular medicine, because perturbed recognition of cognate tRNAs by aaRSs and poor precision in tRNA aminoacylation do not guarantee accurate protein biosynthesis, thus leading to diseases. Sets of identity determinants situated at particular zones of tRNA are responsible for functional accuracy. Recent work in X-ray crystallography has revealed various snapshots of aaRS:ligand complexes which represent the stages required for aminoacylation. Here we focus on a small group of class I aaRSs conserved in evolution, the ArgRSs, GluRSs, GlnRSs, and atypical LysRSs found mostly in Archaea and in a few Bacteria, that catalyze amino acid activation only in the presence of their cognate tRNAs. Structural and functional features of these aaRSs, ranked in subclass Ib, together with their peculiar mode of tRNA recognition and identity expression are reviewed and compared. Strategies to inhibit class Ib aaRS:tRNA aminoacylation systems, their dysfunction leading to human diseases, and the implications for pharmacology are outlined.
Assuntos
Aminoacil-tRNA Sintetases/antagonistas & inibidores , Aminoacil-tRNA Sintetases/metabolismo , Aminoacil-tRNA Sintetases/química , Catálise , Cristalografia por Raios X , Humanos , Modelos Moleculares , Especificidade por SubstratoRESUMO
Glucoamylases, containing starch-binding domains (SBD), have a wide range of scientific and industrial applications. Random mutagenesis and DNA shuffling of the gene encoding a starch-binding domain have resulted in only minor improvements in the affinities of the corresponding protein to their ligands, whereas circular permutation of the RoCBM21 substantially improved its binding affinity and selectivity towards longer-chain carbohydrates. For the study reported herein, we used a standard soluble ligand (amylose EX-I) to characterize the functional and structural aspects of circularly permuted RoCBM21 (CP90). Site-directed mutagenesis and the analysis of crystal structure reveal the dimerisation and an altered binding path, which may be responsible for improved affinity and altered selectivity of this newly created starch-binding domain. The functional and structural characterization of CP90 suggests that it has significant potential in industrial applications.
Assuntos
Glucana 1,4-alfa-Glucosidase/química , Glucana 1,4-alfa-Glucosidase/metabolismo , Mutação , Multimerização Proteica , Rhizopus/enzimologia , Amilose/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Glucana 1,4-alfa-Glucosidase/genética , Ligantes , Modelos Moleculares , Mutagênese Sítio-Dirigida , Estrutura Quaternária de ProteínaRESUMO
Proteins containing starch-binding domains (SBDs) are used in a variety of scientific and technological applications. A circularly permutated SBD (CP90) with improved affinity and selectivity toward longer-chain carbohydrates was synthesized, suggesting that a new starch-binding protein may be developed for specific scientific and industrial applications.
Assuntos
Proteínas de Fluorescência Verde/química , Ligantes , Amido/química , Amilose/química , Eletroforese em Gel de Poliacrilamida , Proteínas de Fluorescência Verde/metabolismo , Modelos Moleculares , Ligação Proteica , Estrutura Terciária de Proteína , Amido/metabolismo , Especificidade por SubstratoRESUMO
Pyruvate phosphate dikinase (PPDK) is the key enzyme essential for the glycolytic pathway in most common and perilous parasite Entamoeba histolytica. Inhibiting the function of this enzyme could control the wide spread of intestinal infections caused by Entamoeba histolytica in humans. With this objective, we modeled the three dimensional structure of the PPDK protein. We used templates with 51% identity and 67% similarity to employ homology-modeling approach. Stereo chemical quality of protein structure was validated by protein structure validation program PROCHECK and VERIFY3D. Experimental proof available in literature along with the in silico studies indicated Lys21, Arg91, Asp323, Glu325 and Gln337 to be the probable active sites in the target protein. Virtual screening was carried out using the genetic docking algorithm GOLD and a consensus scoring function X-Score to substantiate the prediction. The small molecule libraries (ChemDivision database, Diversity dataset, Kinase inhibitor database) were used for screening process. Along with the high scoring results, the interaction studies provided promising ligands for future experimental screening to inhibit the function of PPDK in Entamoeba histolytica. Further, the phylogeny study was carried out to assess the possibility of using the proposed ligands as inhibitors in related pathogens.