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Emergence of newer variants of SARS-CoV-2 underscores the need for effective antivirals to complement the vaccination program in managing COVID-19. The multi-functional papain-like protease (PLpro) of SARS-CoV-2 is an essential viral protein that not only regulates the viral replication but also modulates the host immune system, making it a promising therapeutic target. To this end, we developed an in vitro interferon stimulating gene 15 (ISG15)-based Förster resonance energy transfer (FRET) assay and screened the National Cancer Institute (NCI) Diversity Set VI compound library, which comprises 1584 small molecules. Subsequently, we assessed the PLpro enzymatic activity in the presence of screened molecules. We identified three potential PLpro inhibitors, namely, NSC338106, 651084, and 679525, with IC50 values in the range from 3.3 to 6.0 µM. These molecules demonstrated in vitro inhibition of the enzyme activity and exhibited antiviral activity against SARS-CoV-2, with EC50 values ranging from 0.4 to 4.6 µM. The molecular docking of all three small molecules to PLpro suggested their specificity towards the enzyme's active site. Overall, our study contributes promising prospects for further developing potential antivirals to combat SARS-CoV-2 infection.
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Antivirales , Proteasas Similares a la Papaína de Coronavirus , Citocinas , Ensayos Analíticos de Alto Rendimiento , SARS-CoV-2 , Ubiquitinas , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/enzimología , Antivirales/farmacología , Antivirales/química , Humanos , Ensayos Analíticos de Alto Rendimiento/métodos , Proteasas Similares a la Papaína de Coronavirus/antagonistas & inhibidores , Proteasas Similares a la Papaína de Coronavirus/química , Proteasas Similares a la Papaína de Coronavirus/metabolismo , Citocinas/metabolismo , Ubiquitinas/metabolismo , Ubiquitinas/química , Ubiquitinas/antagonistas & inhibidores , Simulación del Acoplamiento Molecular , Tratamiento Farmacológico de COVID-19 , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/química , Proteasas 3C de Coronavirus/antagonistas & inhibidores , Proteasas 3C de Coronavirus/metabolismo , Proteasas 3C de Coronavirus/química , Transferencia Resonante de Energía de Fluorescencia , COVID-19/virologíaRESUMEN
Despite a major threat to the public health in tropical and subtropical regions, dengue virus (DENV) infections are untreatable. Therefore, efforts are needed to investigate cost-effective therapeutic agents that could cure DENV infections in future. The NS2B-NS3 protease encoded by the genome of DENV is considered a critical target for the development of anti-dengue drugs. The objective of the current study was to find out a specific inhibitor of the NS2B-NS3 proteases from all four serotypes of DENV. To begin with, nine plant extracts with a medicinal history were evaluated for their role in inhibiting the NS2B-NS3 proteases by Fluorescence Resonance Energy Transfer (FRET) assay. Among the tested extracts, Punica granatum was found to be the most effective one. The metabolic profiling of this extract revealed the presence of several active compounds, including ellagic acid, punicalin and punicalagin, which are well-established antiviral agents. Further evaluation of IC50 values of these three antiviral molecules revealed punicalagin as the most potent anti-NS2B-NS3 protease drug with IC50 of 0.91 ± 0.10, 0.75 ± 0.05, 0.42 ± 0.03, 1.80 ± 0.16 µM against proteases from serotypes 1, 2, 3 and 4, respectively. The docking studies demonstrated that these compounds interacted at the active site of the enzyme, mainly with His and Ser residues. Molecular dynamics simulations analysis also showed the structural stability of the NS2B-NS3 proteases in the presence of punicalagin. In summary, this study concludes that the punicalagin can act as an effective inhibitor against NS2B-NS3 proteases from all four serotypes of DENV.Communicated by Ramaswamy H. Sarma.
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SARS-CoV-2 pandemic in the end of 2019 led to profound consequences on global health and economy. Till producing successful vaccination strategies, the healthcare sectors suffered from the lack of effective therapeutic agents that could control the spread of infection. Thus, academia and the pharmaceutical sector prioritise SARS-CoV-2 antiviral drug discovery. Here, we exploited previous reports highlighting the anti-SARS-CoV-2 activities of isatin-based molecules to develop novel triazolo-isatins for inhibiting main protease (Mpro) of the virus, a crucial enzyme for its replication in the host cells. Particularly, sulphonamide 6b showed promising inhibitory activity with an IC50= 0.249 µM. Additionally, 6b inhibited viral cell proliferation with an IC50 of 4.33 µg/ml, and was non-toxic to VERO-E6 cells (CC50 = 564.74 µg/ml) displaying a selectivity index of 130.4. In silico analysis of 6b disclosed its ability to interact with key residues in the enzyme active site, supporting the obtained in vitro findings.
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COVID-19 , Isatina , Humanos , SARS-CoV-2 , Sulfanilamida , Sulfonamidas/farmacologíaRESUMEN
INTRODUCTION: The objective of the present study was to evaluate and compare the validity and utility of two fully automated ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) activity assays for clinical diagnostic decision-making and to compare their performance. METHODS: Two automated ADAMTS13 activity assays (Werfen HemosIL® AcuStar ADAMTS13 Activity, Technoclone Technofluor ADAMTS13 Activity) were compared with a manual FRET assay (BioMedica ACTIFLUOR ADAMTS13 Activity). The following samples were used: 13 acute phase TTP (thrombotic thrombocytopenic purpura) samples from 11 different patients, one sample from a patient with congenital ADAMTS13 deficiency, 16 samples from control patients, three follow-up samples from TTP patients in long-term remission and one sample from a patient with stem cell transplantation related thrombotic microangiopathy (TMA). The WHO 1st International Standard for ADAMTS13 and several dilutions of normal plasma with ADAMTS13-depleted normal plasma were also tested. Statistical analysis included descriptive statistics, sensitivity and specificity, Passing & Bablok regression and Bland-Altman plot. RESULTS: The quantitative comparison between the HemosIL® (x) and Technofluor (y) methods showed a strong correlation (Pearson r = 0.98, n = 49). When considering an ADAMTS13 activity of <10% as a hallmark for the diagnosis of TTP, two fully automated assays were both able to identify all TTP- and non-TTP-samples correctly, resulting in sensitivities and specificities of 100%. CONCLUSION: Both fully automated ADAMTS13 activity assays showed a good diagnostic performance and quantitative correlation among themselves, discriminating reliably between TTP- and non-TTP-patients.
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Trasplante de Células Madre Hematopoyéticas , Púrpura Trombocitopénica Trombótica , Microangiopatías Trombóticas , Humanos , Proteínas ADAM/metabolismo , Proteína ADAMTS13 , Transferencia Resonante de Energía de Fluorescencia , Púrpura Trombocitopénica Trombótica/diagnósticoRESUMEN
Membrane type 1 matrix metalloproteinase (MT1-MMP) has been shown to be crucial for tumor angiogenesis, invasion, and metastasis, and thus MT1-MMP is a high priority target for potential cancer therapies. To properly evaluate MT1-MMP inhibitors, a screening protocol is desired by which enzyme activity can be quantified in a tumor microenvironment-like model system. In the present study, we applied a fluorogenic, collagen model triple-helical substrate to quantify MT1-MMP activity for tumor spheroids embedded in a collagen hydrogel. The substrate was designed to be MT1-MMP selective and to possess fluorescent properties compatible with cell-based assays. The proteolysis of the substrate correlated to glioma spheroid invasion. In turn, the application of either small molecule or protein-based MMP inhibitors reduced proteolytic activity and glioma spheroid invasion. The presence of MT1-MMP in glioma spheroids was confirmed by western blotting. Thus, spheroid invasion was dependent on MT1-MMP activity, and inhibitors of MT1-MMP and invasion could be conveniently screened in a high-throughput format. The combination of the fluorogenic, triple-helical substrate, the three-dimensional tumor spheroids embedded in collagen, and Hit-Pick software resulted in an easily adaptable in vivo-like tumor microenvironment for rapidly processing inhibitor potential for anti-cancer use.
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Elevated expression of anti-apoptotic proteins, such as Bcl-2 and Mcl-1 contributes to poor prognosis and resistance to current treatment modalities in multiple cancers. Here, we report the design, synthesis and characterization of benzimidazole chalcone and flavonoid scaffold-derived bicyclic compounds targeting both Bcl-2 and Mcl-1 by optimizing the structural differences in the binding sites of both these proteins. Initial docking screen of Bcl-2 and Mcl-1 with pro-apoptotic protein Bim revealed possible hits with optimal binding energies. All the optimized bicyclic compounds were screened for their in vitro cytotoxic activity against two oral cancer cell lines (AW8507 and AW13516) which express high levels of Bcl-2 and Mcl-1. Compound 4d from the benzimidazole chalcone series and compound 6d from the flavonoid series exhibited significant cytotoxic activity (IC50 7.12 µM and 17.18 µM, respectively) against AW13516 cell line. Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) analysis further demonstrated that compound 4d and compound 6d could effectively inhibit the Bcl-2 and Mcl-1 proteins by displacing their BH3 binding partners. Both compounds exhibited potent activation of canonical pathway of apoptosis evident from appearance of cleaved Caspase-3 and PARP. Further, treatment of oral cancer cells with the inhibitors induced dissociation of the BH3 only protein Bim from Mcl-1 and Bak from Bcl-2 but failed to release Bax from Bcl-xL thereby confirming the nature of compounds as BH3-mimetics selectively targeting Bcl-2 and Mcl-1. Our study thus identifies bicyclic compounds as promising candidates for anti-apoptotic Bcl-2/Mcl-1 dual inhibitors with a potential for further development.
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Chalconas , Neoplasias de la Boca , Humanos , Chalconas/farmacología , Proteína bcl-X/metabolismo , Proteína bcl-X/farmacología , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Proteína Destructora del Antagonista Homólogo bcl-2/farmacología , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Reguladoras de la Apoptosis/farmacología , Apoptosis , Compuestos Bicíclicos con Puentes/farmacología , Bencimidazoles/farmacología , Línea Celular TumoralRESUMEN
Several strains and species of lactic acid bacteria (LAB) are widely used in fermented foods, including dairy products and also as probiotics, because of their contribution to various health benefits in humans. Sortase enzymes decorate the bacterial cell wall with different surface proteins and pili for facilitating the interactions with host and environment for the colonization and beneficial effects. While the sortases and sortase anchored proteins from pathogens have been the prime focus of the research in the past, sortases from many non-pathogenic bacteria, including LAB strains, have attracted attention for their potential applications in vaccine delivery and other clinical interventions. Here, we report the purification and functional characterization of two sortases (housekeeping SrtA and pilus-specific SrtC) from a probiotic Lactococcus lactis. The purified sortases were found to be active against the putative LPXTG motif-based peptide substrates, albeit with differences. The in-silico analysis provides insights into the residues involved in substrate binding and specificity. Overall, this study sheds new light on the aspects of structure, substrate specificity, and function of sortases from non-pathogenic bacteria, which may have physiological ramifications as well as their applications in sortase-mediated protein bioconjugation.
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Aminoaciltransferasas , Proteínas Bacterianas , Cisteína Endopeptidasas , Lactococcus lactis , Probióticos , Aminoaciltransferasas/metabolismo , Proteínas Bacterianas/metabolismo , Cisteína Endopeptidasas/metabolismo , Lactococcus lactis/enzimología , Especificidad por SustratoRESUMEN
BACKGROUND: Hepatitis C virus infection is the main cause of liver ailments across the globe. Several HCV genotypes have been identified in different parts of the world. Effective drugs for combating HCV infections are available but not affordable, particularly to infected individuals from resource-limited countries. Hence, cost-effective drugs need to be developed against important HCV drug targets. As Citrus fruits naturally contain bioactive compounds with antiviral activities, the current study was designed to identify antiviral inhibitors from Citrus fruit extracts against an important drug target, NS3 protease, of HCV genotype 3a which is found predominantly in South Asian countries. METHODS: The full-length NS3 protease alone and the NS3 protease domain in fusion with the cognate NS4A cofactor were expressed in Escherichia coli, and purified by chromatographic techniques. Using the purified protein as a drug target, Citrus extracts were evaluated in a FRET assay, and active ingredients, identified using ESI-MS/MS, were docked to observe the interaction with active site residues of NS3. The best interacting compound was further confirmed through the FRET assay as the inhibitor of NS3 protease. RESULTS: Fusion of the NS3 protease domain to the NS4A cofactor significantly improved the purification yield, and NS3-NS4A was functionally more active than the full-length NS3 alone. The purified protein (NS3-NS4A) was successfully employed in a validated FRET assay to evaluate 14 Citrus fruit extracts, revealing that the mesocarp extract of Citrus paradisi, and whole fruit extracts of C. sinesis, C. aurantinum, and C. reticulata significantly inhibited the protease activity of HCV NS3 protease (IC50 values of 5.79 ± 1.44 µg/mL, 37.19 ± 5.92 µg/mL, 42.62 ± 6.89 µg/mL, and 57.65 ± 3.81 µg/mL, respectively). Subsequent ESI-MSn analysis identified a flavonoid, hesperidin, abundantly present in all the afore-mentioned Citrus extracts. Importantly, docking studies suggested that hesperidin interacts with active site residues, and acts as a potent inhibitor of NS3 protease, exhibiting an IC50 value of 11.34 ± 3.83 µg/mL. CONCLUSIONS: A FRET assay was developed using NS3-NS4A protease, which was successfully utilized for the evaluation of Citrus fruit extracts. Hesperidin, a compound present in the Citrus extracts, was identified as the main flavonoid, which can serve as a cost-effective potent inhibitor of NS3 protease, and could be developed as a drug for antiviral therapy against HCV genotype 3a.
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Citrus , Hepatitis C , Hesperidina , Genotipo , Hesperidina/farmacología , Péptido Hidrolasas/genética , Extractos Vegetales/farmacología , Serina Endopeptidasas/química , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Espectrometría de Masas en Tándem , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismoRESUMEN
Protein-protein interactions play pivotal roles in life, and the protein interaction affinity confers specific protein interaction events in physiology or pathology. Förster resonance energy transfer (FRET) has been widely used in biological and biomedical research to detect molecular interactions in vitro and in vivo. The FRET assay provides very high sensitivity and efficiency. Several attempts have been made to develop the FRET assay into a quantitative measurement for protein-protein interaction affinity in the past. However, the progress has been slow due to complicated procedures or because of challenges in differentiating the FRET signal from other direct emission signals from donor and receptor. This review focuses on recent developments of the quantitative FRET analysis and its application in the determination of protein-protein interaction affinity (KD), either through FRET acceptor emission or donor quenching methods. This paper mainly reviews novel theatrical developments and experimental procedures rather than specific experimental results. The FRET-based approach for protein interaction affinity determination provides several advantages, including high sensitivity, high accuracy, low cost, and high-throughput assay. The FRET-based methodology holds excellent potential for those difficult-to-be expressed proteins and for protein interactions in living cells.
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Transferencia Resonante de Energía de Fluorescencia/métodos , Mapeo de Interacción de Proteínas/métodos , Proteínas/química , Algoritmos , Cinética , Modelos Teóricos , Unión Proteica , Proteínas/metabolismo , SolucionesRESUMEN
Antiviral treatments inhibiting Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication may represent a strategy complementary to vaccination to fight the ongoing Coronavirus disease 19 (COVID-19) pandemic. Molecules or extracts inhibiting the SARS-CoV-2 chymotripsin-like protease (3CLPro) could contribute to reducing or suppressing SARS-CoV-2 replication. Using a targeted approach, we identified 17 plant products that are included in current and traditional cuisines as promising inhibitors of SARS-CoV-2 3CLPro activity. Methanolic extracts were evaluated in vitro for inhibition of SARS-CoV-2 3CLPro activity using a quenched fluorescence resonance energy transfer (FRET) assay. Extracts from turmeric (Curcuma longa) rhizomes, mustard (Brassica nigra) seeds, and wall rocket (Diplotaxis erucoides subsp. erucoides) at 500 µg mL-1 displayed significant inhibition of the 3CLPro activity, resulting in residual protease activities of 0.0%, 9.4%, and 14.9%, respectively. Using different extract concentrations, an IC50 value of 15.74 µg mL-1 was calculated for turmeric extract. Commercial curcumin inhibited the 3CLPro activity, but did not fully account for the inhibitory effect of turmeric rhizomes extracts, suggesting that other components of the turmeric extract must also play a main role in inhibiting the 3CLPro activity. Sinigrin, a major glucosinolate present in mustard seeds and wall rocket, did not have relevant 3CLPro inhibitory activity; however, its hydrolysis product allyl isothiocyanate had an IC50 value of 41.43 µg mL-1. The current study identifies plant extracts and molecules that can be of interest in the search for treatments against COVID-19, acting as a basis for future chemical, in vivo, and clinical trials.
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Alzheimer's disease is one of the most common neurodegenerative disorder afflicting a large mass of population. BACE-1 (ß-secretase) is an aspartyl protease of the amyloidogenic pathway considered responsible for Alzheimer's disease (AD). Since it catalyzes the rate-limiting step of Aß-42 production from amyloid precursor protein (APP), its inhibition is considered a viable therapeutic strategy. We have reported the design of small molecular weight compounds supposed to be blood brain permeable as BACE-1 inhibitors. The clue for the design of this series is drawn from the previously designed series from our research group. OBJECTIVE: Design and synthesis of 2,4,6-substituted pyrimidine derivatives has been reported. In vitro FRET-based screening of synthesized derivatives was performed to evaluate the BACE-1 inhibition profile. METHODS: Based on the docking simulation studies, a library of derivatives was designed, synthesized and evaluated for BACE-1 inhibition in-vitro. The docking studies were performed on Glide (Schrodinger suite) and Molegro virtual docker. Theoretical toxicity was predicted using Osiris Property Explorer. The synthesized compounds were tested for BACE-1 inhibition using in vitro assay based on Fluorescence Resonance Energy Transfer technique. The percent inhibition was calculated as a measure of activity. RESULTS: The designed compounds revealed strong interactions with the desired amino acids of BACE-1 active sites. The aromatic rings placed at the fourth and sixth position of the pyrimidine ring occupied S1 and S3 substrate-binding clefts while the amino group formed hydrogen bonding interactions with Asp32 and Asp228. In silico data ensured that the compounds were orally bioavailable and brain permeable. The in vitro testing showed that the compounds inhibited BACE-1 at 10µM concentration. CONCLUSION: Compounds substituted with m-benzyloxy on one aromatic ring and o,p-di-chloro on another aromatic ring displayed maximum BACE-1 inhibition. Compound 2.13A displayed high docking score and was found to be most potent with IC50 of 6.92µM. The series displayed a good correlation between the docking score and BACE-1 inhibition profile.
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Enfermedad de Alzheimer , Enfermedad de Alzheimer/tratamiento farmacológico , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Ácido Aspártico Endopeptidasas/metabolismo , Dominio Catalítico , Humanos , Pirimidinas/farmacologíaRESUMEN
Antiapoptotic members of B-cell leukemia/lymphoma-2 (BCL-2) family proteins are one of the overexpressed proteins in cancer cells that are oncogenic targets. As such, targeting of BCL-2 family proteins raises hopes for new therapeutic discoveries. Thus, we used multistep screening and filtering approaches that combine structure and ligand-based drug design to identify new, effective BCL-2 inhibitors from a small molecule database (Specs SC), which includes more than 210,000 compounds. This database is first filtered based on binary "cancer-QSAR" model constructed with 886 training and 167 test set compounds and common 26 toxicity quantitative structure-activity relationships (QSAR) models. Predicted non-toxic compounds are considered for target-driven studies. Here, we applied two different approaches to filter and select hit compounds for further in vitro biological assays and human cell line experiments. In the first approach, a molecular docking and filtering approach is used to rank compounds based on their docking scores and only a few top-ranked molecules are selected for further long (100-ns) molecular dynamics (MD) simulations and in vitro tests. While docking algorithms are promising in predicting binding poses, they can be less prone to precisely predict ranking of compounds leading to decrease in the success rate of in silico studies. Hence, in the second approach, top-docking poses of each compound filtered through QSAR studies are subjected to initially short (1 ns) MD simulations and their binding energies are calculated via molecular mechanics generalized Born surface area (MM/GBSA) method. Then, the compounds are ranked based on their average MM/GBSA energy values to select hit molecules for further long MD simulations and in vitro studies. Additionally, we have applied text-mining approaches to identify molecules that contain "indol" phrase as many of the approved drugs contain indole and indol derivatives. Around 2700 compounds are filtered based on "cancer-QSAR" model and are then docked into BCL-2. Short MD simulations are performed for the top-docking poses for each compound in complex with BCL-2. The complexes are again ranked based on their MM/GBSA values to select hit molecules for further long MD simulations and in vitro studies. In total, seven molecules are subjected to biological activity tests in various human cancer cell lines as well as Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) assay. Inhibitory concentrations are evaluated, and biological activities and apoptotic potentials are assessed by cell culture studies. Four molecules are found to be limiting the proliferation capacity of cancer cells while increasing the apoptotic cell fractions.
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PPARγ is a pharmacological target in inflammatory and metabolic diseases. Upon agonistic treatment or following antagonism, binding of co-factors is altered, which consequently affects PPARγ-dependent transactivation as well as its DNA-independent properties. Therefore, establishing techniques to characterize these interactions is an important issue in living cells. Methods: Using the FRET pair Clover/mRuby2, we set up a flow cytometry-based FRET assay by analyzing PPARγ1 binding to its heterodimerization partner RXRα. Analyses of PPARγ-reporter and co-localization studies by laser-scanning microscopy validated this system. Refining the system, we created a new readout to distinguish strong from weak interactions, focusing on PPARγ-binding to the co-repressor N-CoR2. Results: We observed high FRET in cells expressing Clover-PPARγ1 and mRuby2-RXRα, but no FRET when cells express a mRuby2-RXRα deletion mutant, lacking the PPARγ interaction domain. Focusing on the co-repressor N-CoR2, we identified in HEK293T cells the new splice variant N-CoR2-ΔID1-exon. Overexpressing this isoform tagged with mRuby2, revealed no binding to Clover-PPARγ1, nor in murine J774A.1 macrophages. In HEK293T cells, binding was even lower in comparison to N-CoR2 constructs in which domains established to mediate interaction with PPARγ binding are deleted. These data suggest a possible role of N-CoR2-ΔID1-exon as a dominant negative variant. Because binding to N-CoR2-mRuby2 was not altered following activation or antagonism of Clover-PPARγ1, we determined the effect of pharmacological treatment on FRET intensity. Therefore, we calculated flow cytometry-based FRET efficiencies based on our flow cytometry data. As with PPARγ antagonism, PPARγ agonist treatment did not prevent binding of N-CoR2. Conclusion: Our system allows the close determination of protein-protein interactions with a special focus on binding intensity, allowing this system to characterize the role of protein domains as well as the effect of pharmacological agents on protein-protein interactions.
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Citometría de Flujo/métodos , Transferencia Resonante de Energía de Fluorescencia/métodos , PPAR gamma/metabolismo , Animales , Dimerización , Células HEK293 , Humanos , Ratones , Co-Represor 1 de Receptor Nuclear/química , Co-Represor 1 de Receptor Nuclear/genética , Co-Represor 1 de Receptor Nuclear/metabolismo , PPAR gamma/química , PPAR gamma/genética , Unión Proteica , Dominios Proteicos , Receptor alfa X Retinoide/química , Receptor alfa X Retinoide/genética , Receptor alfa X Retinoide/metabolismoRESUMEN
A robust, fluorescence-based analysis and discovery platform is described for bacterial A-site binders. The assay relies on an incorporated isomorphic fluorescent uridine analog, which substitutes the A-site's U1406 and serves as a FRET donor to an A-site bound coumarin-labeled aminoglycoside that serves as the FRET acceptor. Binding efficiency of unlabeled A-site ligands can be determined by competition experiments, where the acceptor-labeled aminoglycoside is displaced. The replacement efficiency is gauged by the concentration-dependent loss of the sensitized FRET acceptor's signal with concomitant restoration of the donor's emission. Plotting the relative emission intensity of both the donor and acceptor as a function of ligand concentration followed by fitting of the data points to a dose-response curve yields IC50 values, one possible measure of the antibiotic potency of new A-site binders.
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Aminoglicósidos/metabolismo , Cumarinas/metabolismo , Transferencia Resonante de Energía de Fluorescencia/métodos , Fluorescencia , ARN Bacteriano/metabolismo , Uridina/metabolismo , Aminoglicósidos/química , Cumarinas/química , Ligandos , ARN Bacteriano/química , Uridina/químicaRESUMEN
The transcription factor nuclear factor of activated T cells (NFAT) has a key role in both T cell activation and tolerance and has emerged as an important target of immune modulation. NFAT directs the effector arm of the immune response in the presence of activator protein-1 (AP-1), and T cell anergy/exhaustion in the absence of AP-1. Envisioning a strategy for selective modulation of the immune response, we designed a FRET-based high-throughput screen to identify compounds that disrupt the NFAT:AP-1:DNA complex. We screened â¼202,000 small organic compounds and identified 337 candidate inhibitors. We focus here on one compound, N-(3-acetamidophenyl)-2-[5-(1H-benzimidazol-2-yl)pyridin-2-yl]sulfanylacetamide (Compound 10), which disrupts the NFAT:AP-1 interaction at the composite antigen-receptor response element-2 site without affecting the binding of NFAT or AP-1 alone to DNA. Compound 10 binds to DNA in a sequence-selective manner and inhibits the transcription of the Il2 gene and several other cyclosporin A-sensitive cytokine genes important for the effector immune response. This study provides proof-of-concept that small molecules can inhibit the assembly of specific DNA-protein complexes, and opens a potential new approach to treat human diseases where known transcription factors are deregulated.
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Acetamidas/farmacología , Expresión Génica/efectos de los fármacos , Factores de Transcripción NFATC/antagonistas & inhibidores , Factor de Transcripción AP-1/antagonistas & inhibidores , Citocinas/metabolismo , ADN/metabolismo , Escherichia coli , Ensayos Analíticos de Alto Rendimiento , Factores de Transcripción NFATC/metabolismo , Prueba de Estudio Conceptual , Bibliotecas de Moléculas Pequeñas , Factor de Transcripción AP-1/metabolismoRESUMEN
Newly identified, nontypable Haemophilus influenzae (H. influenza) strains represent a serious threat to global health. Due to the increasing prevalence of antibiotic resistance, virulence factors have emerged as potential therapeutic targets that would be less likely to promote resistance. IgA1 proteases are secreted virulence factors of many Gram-negative human pathogens. These enzymes play important roles in tissue invasion as well as evasion of the immune response, yet there has been limited work on pharmacological inhibitors. Here, we report the discovery of the first small molecule, nonpeptidic inhibitors of H. influenzae IgA1 proteases. We screened over 47â¯000 compounds in a biochemical assay using recombinant protease and identified a hit compound with micromolar potency. Preliminary structure-activity relationships produced additional inhibitors, two of which showed improved inhibition and selectivity for IgA protease over other serine proteases. We further showed dose-dependent inhibition against four different IgA1 protease variants collected from clinical isolates. These data support further development of IgA protease inhibitors as potential therapeutics for antibiotic-resistant H. influenza strains. The newly discovered inhibitors also represent valuable probes for exploring the roles of these proteases in bacterial colonization, invasion, and infection of mucosal tissues.
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Antivirales/síntesis química , Haemophilus influenzae/enzimología , Inhibidores de Serina Proteinasa/síntesis química , Bibliotecas de Moléculas Pequeñas/síntesis química , Antivirales/química , Antivirales/farmacología , Biología Computacional , Relación Dosis-Respuesta a Droga , Variación Genética , Haemophilus influenzae/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento , Serina Endopeptidasas/química , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/farmacología , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Relación Estructura-Actividad , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Virales/metabolismo , Factores de Virulencia/antagonistas & inhibidores , Factores de Virulencia/química , Factores de Virulencia/genéticaRESUMEN
In this chapter, we describe techniques to detect ubiquitination events occurring in vivo. We focus on methodologies capable of preserving and detecting target protein ubiquitination in physiological conditions, without overexpressing a tagged version of ubiquitin. We provide detailed protocols for mammalian and Drosophila melanogaster systems using linkage-specific antibodies against ubiquitin. We analyze immunoblotting and immunofluorescence approaches as well as an ELISA-based quantitative approach, which allows comparing more samples and conditions. Finally, we describe the use of in vitro ubiquitination assays with purified proteins that allows a direct analysis of the molecular mechanisms behind specific E3 activity.
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Ensayo de Inmunoadsorción Enzimática/métodos , Técnica del Anticuerpo Fluorescente/métodos , Ubiquitinación , Animales , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Células HeLa , Humanos , Immunoblotting/métodos , Poliubiquitina/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Human Atg4 homologs are cysteine proteases, which play key roles in the macroautophagy/autophagy process by cleaving Atg8 homologs for conjugation to lipid membranes and for deconjugation of Atg8 homologs from membranes. Expression of ATG4B is significantly increased in colorectal cancer cells compared to normal cells, suggesting that ATG4B may be important for cancer biology. Inhibition of ATG4B may reduce the autophagy activity, thereby sensitizing cancer cells to therapeutic agents. Thus, developing specific and potent ATG4B inhibitors for research as well as for potential therapeutic uses is highly needed. In this study, we integrated in silico screening and in vitro assays to discover a potent ATG4B inhibitor, named S130, from a noncommercial library. This chemical binds to ATG4B with strong affinity and specifically suppresses the activity of ATG4B but not other proteases. S130 did not cause the impairment of autophagosome fusion, nor did it result in the dysfunction of lysosomes. Instead, S130 might attenuate the delipidation of LC3-II on the autolysosomes to suppress the recycling of LC3-I, which normally occurs after LC3-II cleavage by ATG4B. Intriguingly, S130 induced cell death, which was accompanied with autophagy stress and could be further exacerbated by nutrient deprivation. Such cytotoxicity could be partially reversed by enhancing ATG4B activity. Finally, we found that S130 was distributed in tumor tissues in vivo and was also effective in arresting the growth of colorectal cancer cells. Thus, this study indicates that ATG4B is a potential anticancer target and S130 might be a novel small-molecule candidate for future cancer therapy.
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Apoptosis/efectos de los fármacos , Proteínas Relacionadas con la Autofagia/antagonistas & inhibidores , Autofagia/efectos de los fármacos , Neoplasias Colorrectales/patología , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Proteínas Relacionadas con la Autofagia/metabolismo , Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/ultraestructura , Cisteína Endopeptidasas/metabolismo , Femenino , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Ratones Endogámicos BALB C , Ratones Desnudos , Proteínas Asociadas a Microtúbulos/metabolismo , Unión Proteica/efectos de los fármacosRESUMEN
Chikungunya virus (CHIKV), a mosquito-borne pathogenic alphavirus is a growing public health threat. No vaccines or antiviral drug is currently available in the market for chikungunya treatment. nsP2pro, the viral cysteine protease, carries out an essential function of nonstructural polyprotein processing and forms four nonstructural proteins (nsPs) that makes the replication complex, hence constitute a promising drug target. In this study, crystal structure of nsP2pro has been determined at 2.59â¯Å, which reveals that the protein consists of two subdomains: an N-terminal protease subdomain and a C-terminal methyltransferase subdomain. Structural comparison of CHIKV nsP2pro with structures of other alphavirus nsP2 advances that the substrate binding cleft is present at the interface of two subdomains. Additionally, structure insights revealed that access to the active site and substrate binding cleft is blocked by a flexible interdomain loop in CHIKV nsP2pro. This loop contains His548, the catalytic residue, and Trp549 and Asn547, the residues predicted to bind substrate. Interestingly, mutation of Asn547 leads to three-fold increase in Km confirming that Asn547 plays important role in substrate binding and recognition. This study presents the detailed molecular analysis and signifies the substrate specificity residues of CHIKV nsP2pro, which will be beneficial for structure-based drug design and optimization of CHIKV protease inhibitors.
Asunto(s)
Virus Chikungunya/química , Proteasas de Cisteína/química , Proteínas no Estructurales Virales/química , Antivirales/farmacología , Dominio Catalítico/efectos de los fármacos , Virus Chikungunya/efectos de los fármacos , Cristalografía por Rayos X/métodos , Diseño de Fármacos , Inhibidores de Proteasas/farmacología , Especificidad por Sustrato/efectos de los fármacosRESUMEN
Chikungunya virus (CHIKV), a mosquito-borne pathogenic virus that reemerged and caused epidemic in the Indian Ocean island of La Réunion, is a potential public health threat. Currently there is no antiviral drug or vaccine commercially available for the treatment of chikungunya fever, which necessitates the urge for an effective antiviral therapy for chikungunya treatment. In the present study, a FRET based protease assay was used to analyze the proteolytic activity of chikungunya nsP2 protease (CHIKV nsP2pro) - an essential viral enzyme, with fluorogenic substrate peptide. This protease assay was used to assess the inhibitory activity of Pep-I (MMsINC® database ID MMs03131094) and Pep-II (MMsINC® database ID MMs03927237), peptidomimetic compounds identified in a previous study by our group. Both compounds inhibited CHIKV nsP2pro with half maximal inhibition concentration (IC50) values of â¼34⯵M and â¼42⯵M, respectively. Kinetic studies showed that the inhibition constant (Ki) value is 33.34⯱â¯2.53⯵M for Pep-I and 45.89⯱â¯4.38⯵M for Pep-II. Additionally, these two compounds significantly inhibited CHIKV replication in BHK-21â¯cells at concentrations much lower than their cytotoxic concentrations. Intriguingly, these compounds did not show inhibitory effect on Sindbis virus. This suggests that Pep-I and Pep-II compounds identified as CHIKV nsP2 substrate peptidomimetics, specifically inhibit CHIKV replication.