Most cited article - PubMed ID 29992979
Inhibition of the precursor and mature forms of HIV-1 protease as a tool for drug evaluation
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro) autocatalytically releases itself out of the viral polyprotein to form a fully active mature dimer in a manner that is not fully understood. Here, we introduce several tools to help elucidate differences between cis (intramolecular) and trans (intermolecular) proteolytic processing and to evaluate inhibition of precursor Mpro. We found that many mutations at the P1 position of the N-terminal autoprocessing site do not block cis autoprocessing but do inhibit trans processing. Notably, substituting the WT glutamine at the P1 position with isoleucine retains Mpro in an unprocessed precursor form that can be purified and further studied. We also developed a cell-based reporter assay suitable for compound library screening and evaluation in HEK293T cells. This assay can detect both overall Mpro inhibition and the fraction of uncleaved precursor form of Mpro through separable fluorescent signals. We observed that inhibitory compounds preferentially block mature Mpro. Bofutrelvir and a novel compound designed in-house showed the lowest selectivity between precursor and mature Mpro, indicating that inhibition of both forms may be possible. Additionally, we observed positive modulation of precursor activity at low concentrations of inhibitors. Our findings help expand understanding of the SARS-CoV-2 viral life cycle and may facilitate development of strategies to target precursor form of Mpro for inhibition or premature activation of Mpro.
- Keywords
- Förster resonance energy transfer (FRET), SARS-CoV-2 main protease, activation, autoprocessing, cell-based assay, fluorescence cross-correlation spectroscopy (FCCS), fluorescence life-time imaging, inhibitor, maturation, nsp5, precursor, protease, virus,
- MeSH
- Antiviral Agents * pharmacology chemistry MeSH
- COVID-19 virology MeSH
- COVID-19 Drug Treatment * MeSH
- HEK293 Cells MeSH
- Protease Inhibitors * pharmacology chemistry MeSH
- Coronavirus 3C Proteases * metabolism genetics antagonists & inhibitors chemistry MeSH
- Humans MeSH
- Mutation MeSH
- Drug Discovery * MeSH
- SARS-CoV-2 * enzymology drug effects MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- 3C-like proteinase, SARS-CoV-2 MeSH Browser
- Antiviral Agents * MeSH
- Protease Inhibitors * MeSH
- Coronavirus 3C Proteases * MeSH
Some medically important viruses-including retroviruses, flaviviruses, coronaviruses, and herpesviruses-code for a protease, which is indispensable for viral maturation and pathogenesis. Viral protease inhibitors have become an important class of antiviral drugs. Development of the first-in-class viral protease inhibitor saquinavir, which targets HIV protease, started a new era in the treatment of chronic viral diseases. Combining several drugs that target different steps of the viral life cycle enables use of lower doses of individual drugs (and thereby reduction of potential side effects, which frequently occur during long term therapy) and reduces drug-resistance development. Currently, several HIV and HCV protease inhibitors are routinely used in clinical practice. In addition, a drug including an inhibitor of SARS-CoV-2 main protease, nirmatrelvir (co-administered with a pharmacokinetic booster ritonavir as Paxlovid®), was recently authorized for emergency use. This review summarizes the basic features of the proteases of human immunodeficiency virus (HIV), hepatitis C virus (HCV), and SARS-CoV-2 and discusses the properties of their inhibitors in clinical use, as well as development of compounds in the pipeline.
- MeSH
- Antiviral Agents pharmacology therapeutic use MeSH
- COVID-19 * MeSH
- HIV Infections * drug therapy MeSH
- Humans MeSH
- SARS-CoV-2 MeSH
- Viral Proteases MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
- Names of Substances
- Antiviral Agents MeSH
- nirmatrelvir and ritonavir drug combination MeSH Browser
- Viral Proteases MeSH
Viral proteases are indispensable for successful virion maturation, thus making them a prominent drug target. Their enzyme activity is tightly spatiotemporally regulated by expression in the precursor form with little or no activity, followed by activation via autoprocessing. These cleavage events are frequently triggered upon transportation to a specific compartment inside the host cell. Typically, precursor oligomerization or the presence of a co-factor is needed for activation. A detailed understanding of these mechanisms will allow ligands with non-canonical mechanisms of action to be designed, which would specifically modulate the initial irreversible steps of viral protease autoactivation. Binding sites exclusive to the precursor, including binding sites beyond the protease domain, can be exploited. Both inhibition and up-regulation of the proteolytic activity of viral proteases can be detrimental for the virus. All these possibilities are discussed using examples of medically relevant viruses including herpesviruses, adenoviruses, retroviruses, picornaviruses, caliciviruses, togaviruses, flaviviruses, and coronaviruses.
- Keywords
- Human Immunodeficiency Virus (HIV), Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), activation, adenoviruses, autoprocessing, flaviviruses, herpesviruses, precursor, protease,
- MeSH
- Antiviral Agents pharmacology MeSH
- Flavivirus drug effects metabolism MeSH
- Herpesviridae drug effects metabolism MeSH
- HIV-1 drug effects MeSH
- Viral Protease Inhibitors pharmacology MeSH
- Humans MeSH
- Adenoviruses, Human drug effects metabolism MeSH
- SARS-CoV-2 drug effects metabolism MeSH
- Virus Diseases drug therapy MeSH
- Viral Proteases biosynthesis metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
- Names of Substances
- Antiviral Agents MeSH
- Viral Protease Inhibitors MeSH
- Viral Proteases MeSH
(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.
