Abstract:HIV-1 protease (PR) is a homodimeric enzyme that is autocatalytically cleaved from the Gag-Pol precursor. Known PR inhibitors bind the mature enzyme several orders of magnitude more strongly than the PR precursor. Inhibition of PR at the precursor level, however, may stop the process at its rate-limiting step before the proteolytic cascade is initiated. Due to its structural heterogeneity, limited solubility and autoprocessing, the PR precursor is difficult to access by classical methods, and limited knowledge… Show more
“…Darunavir demonstrated the most potent inhibition; indinavir and saquinavir were similar showing submicromolar IC 50 s; tipranavir was the weakest at suppressing precursor autoprocessing. Our data is consistent with other report showing that various PIs exhibit diverse efficacies at inhibition precursor autoprocessing with low micromolar IC 50 s 31,39 .Figure 2Quantification of precursor autoprocessing. ( A – D ) Detection of precursor autoprocessing by western blotting.…”
Section: Resultssupporting
confidence: 92%
“…This assay allows examination of precursor autoprocessing reactions inside of mammalian cells, which is different from the in vitro assay using the recombinant p6*-PR purified out of E. coli inclusion body followed by protein re-folding 29,36,37 . With our assay, we have demonstrated that the currently available HIV- 1 protease inhibitors (PIs) are much less effective at suppressing precursor-mediated autoprocessing than inhibiting mature protease activity 33,34,38 , which is consistent with other reports 29–31,39 , confirming that the precursor is enzymatically different from the mature PR. Additionally, we reported that precursor autoprocessing is a context-dependent process such that different fusion precursors carrying different tags and/or mutations outside the PR coding region released autoprocessing products with distinct enzymatic properties 34,38 .…”
HIV-1 protease autoprocessing liberates the free mature protease from its Gag-Pol polyprotein precursor through a series of highly regulated autoproteolysis reactions. Herein, we report the development and validation (Z’ ≥ 0.50) of a cell-based functional assay for high-throughput screening (HTS) of autoprocessing inhibitors using fusion precursors in combination with AlphaLISA (amplified luminescent proximity homogeneous assay ELISA). Through pilot screening of a collection of 130 known protease inhibitors, the AlphaLISA assay confirmed all 11 HIV protease inhibitors in the library capable of suppressing precursor autoprocessing at low micromolar concentrations. Meanwhile, other protease inhibitors had no impact on precursor autoprocessing. We next conducted HTS of ~23,000 compounds but found no positive hits. Such high selectivity is advantageous for large-scale HTS campaigns and as anticipated based on assay design because a positive hit needs simultaneously to be nontoxic, cell permeable, and inhibiting precursor autoprocessing. Furthermore, AlphaLISA quantification of fusion precursors carrying mutations known to cause resistance to HIV protease inhibitors faithfully recapitulated the reported resistance, suggesting that precursor autoprocessing is a critical step contributing to drug resistance. Taken together, this reported AlphaLISA platform will provide a useful tool for drug discovery targeting HIV-1 protease autoprocessing and for quantification of PI resistance.
“…Darunavir demonstrated the most potent inhibition; indinavir and saquinavir were similar showing submicromolar IC 50 s; tipranavir was the weakest at suppressing precursor autoprocessing. Our data is consistent with other report showing that various PIs exhibit diverse efficacies at inhibition precursor autoprocessing with low micromolar IC 50 s 31,39 .Figure 2Quantification of precursor autoprocessing. ( A – D ) Detection of precursor autoprocessing by western blotting.…”
Section: Resultssupporting
confidence: 92%
“…This assay allows examination of precursor autoprocessing reactions inside of mammalian cells, which is different from the in vitro assay using the recombinant p6*-PR purified out of E. coli inclusion body followed by protein re-folding 29,36,37 . With our assay, we have demonstrated that the currently available HIV- 1 protease inhibitors (PIs) are much less effective at suppressing precursor-mediated autoprocessing than inhibiting mature protease activity 33,34,38 , which is consistent with other reports 29–31,39 , confirming that the precursor is enzymatically different from the mature PR. Additionally, we reported that precursor autoprocessing is a context-dependent process such that different fusion precursors carrying different tags and/or mutations outside the PR coding region released autoprocessing products with distinct enzymatic properties 34,38 .…”
HIV-1 protease autoprocessing liberates the free mature protease from its Gag-Pol polyprotein precursor through a series of highly regulated autoproteolysis reactions. Herein, we report the development and validation (Z’ ≥ 0.50) of a cell-based functional assay for high-throughput screening (HTS) of autoprocessing inhibitors using fusion precursors in combination with AlphaLISA (amplified luminescent proximity homogeneous assay ELISA). Through pilot screening of a collection of 130 known protease inhibitors, the AlphaLISA assay confirmed all 11 HIV protease inhibitors in the library capable of suppressing precursor autoprocessing at low micromolar concentrations. Meanwhile, other protease inhibitors had no impact on precursor autoprocessing. We next conducted HTS of ~23,000 compounds but found no positive hits. Such high selectivity is advantageous for large-scale HTS campaigns and as anticipated based on assay design because a positive hit needs simultaneously to be nontoxic, cell permeable, and inhibiting precursor autoprocessing. Furthermore, AlphaLISA quantification of fusion precursors carrying mutations known to cause resistance to HIV protease inhibitors faithfully recapitulated the reported resistance, suggesting that precursor autoprocessing is a critical step contributing to drug resistance. Taken together, this reported AlphaLISA platform will provide a useful tool for drug discovery targeting HIV-1 protease autoprocessing and for quantification of PI resistance.
“…Results showed that Darunavir, Amprenavir, Rimantadine, Saquinavir, Tipranavir and Indinavir were more effective in targeting the twelve SARS-CoV-2 proteins and their complexes (Tables 12 , 13 ). Darunavir is a nonpeptidic benzenesulfonamide inhibitor that targets active site of HIV-1 protease [ 38 , 39 ]. Amprenavir is a hydroxyethylamine sulfonamide derivative that inhibits HIV-1 protease [ 40 , 41 ].…”
Background
Novel Coronavirus disease 2019 or COVID-19 has become a threat to human society due to fast spreading and increasing mortality. It uses vertebrate hosts and presently deploys humans. Life cycle and pathogenicity of SARS-CoV-2 have already been deciphered and possible drug target trials are on the way.
Results
The present study was aimed to analyze Non-Structural Proteins that include conserved enzymes of SARS-CoV-2 like papain-like protease, main protease, Replicase, RNA-dependent RNA polymerase, methyltransferase, helicase, exoribonuclease and endoribonucleaseas targets to all known drugs. A bioinformatic based web server Drug ReposeER predicted several drug binding motifs in these analyzed proteins. Results revealed that anti-viral drugs Darunavir,Amprenavir, Rimantadine and Saquinavir were the most potent to have 3D-drug binding motifs that were closely associated with the active sites of the SARS-CoV-2 enzymes .
Conclusions
Repurposing of the antiviral drugs Darunavir, Amprenavir, Rimantadine and Saquinavir to treat COVID-19 patients could be useful that can potentially prevent human mortality.
Graphic abstract
“…The anti-HIV-1 and anti-HSV-1 activity and cytotoxicity in MT-4 cells and Vero cells, respectively, were determined as previously described [44,45]. Three-fold dilution of the compounds were tested in triplicate, using HIV-1 (laboratory strain NL4-3) and HSV-1 (strain HF), respectively.…”
(1) Background: To compare the effect of selected triterpenoids with their structurally resembling derivatives, designing of the molecular ribbons was targeted to develop compounds with selectivity in their pharmacological effects. (2) Methods: In the synthetic procedures, Huisgen 1,3-dipolar cycloaddition was applied as a key synthetic step for introducing a 1,2,3-triazole ring as a part of a junction unit in the molecular ribbons. (3) Results: The antimicrobial activity, antiviral activity, and cytotoxicity of the prepared compounds were studied. Most of the molecular ribbons showed antimicrobial activity, especially on Staphylococcus aureus, Pseudomonas aeruginosa, and Enterococcus faecalis, with a 50–90% inhibition effect (c = 25 µg·mL−1). No target compound was effective against HSV-1, but 8a displayed activity against HIV-1 (EC50 = 50.6 ± 7.8 µM). Cytotoxicity was tested on several cancer cell lines, and 6d showed cytotoxicity in the malignant melanoma cancer cell line (G-361; IC50 = 20.0 ± 0.6 µM). Physicochemical characteristics of the prepared compounds were investigated, namely a formation of supramolecular gels and a self-assembly potential in general, with positive results achieved with several target compounds. (4) Conclusions: Several compounds of a series of triterpenoid molecular ribbons showed better pharmacological profiles than the parent compounds and displayed certain selectivity in their effects.
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