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.

• Klíčová slova
• 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
• antivirové látky * farmakologie   chemie   MeSH
• COVID-19 virologie   MeSH
• farmakoterapie COVID-19 * MeSH
• HEK293 buňky MeSH
• inhibitory proteas * farmakologie   chemie   MeSH
• koronavirové proteasy 3C * metabolismus   genetika   antagonisté a inhibitory   chemie   MeSH
• lidé MeSH
• mutace MeSH
• objevování léků * MeSH
• SARS-CoV-2 * enzymologie   účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 3C-like proteinase, SARS-CoV-2 MeSH Prohlížeč
• antivirové látky * MeSH
• inhibitory proteas * MeSH
• koronavirové proteasy 3C * 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
• antivirové látky farmakologie   terapeutické užití   MeSH
• COVID-19 * MeSH
• HIV infekce * farmakoterapie   MeSH
• lidé MeSH
• SARS-CoV-2 MeSH
• virové proteasy MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• antivirové látky MeSH
• nirmatrelvir and ritonavir drug combination MeSH Prohlížeč
• virové proteasy 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.

• Klíčová slova
• Human Immunodeficiency Virus (HIV), Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), activation, adenoviruses, autoprocessing, flaviviruses, herpesviruses, precursor, protease,
• MeSH
• antivirové látky farmakologie   MeSH
• Flavivirus účinky léků   metabolismus   MeSH
• Herpesviridae účinky léků   metabolismus   MeSH
• HIV-1 účinky léků   MeSH
• inhibitory virových proteáz farmakologie   MeSH
• lidé MeSH
• lidské adenoviry účinky léků   metabolismus   MeSH
• SARS-CoV-2 účinky léků   metabolismus   MeSH
• virové nemoci farmakoterapie   MeSH
• virové proteasy biosyntéza   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• antivirové látky MeSH
• inhibitory virových proteáz MeSH
• virové proteasy MeSH

Drug repositioning is a successful approach in medicinal research. It significantly simplifies the long-term process of clinical drug evaluation, since the drug being tested has already been approved for another condition. One example of drug repositioning involves cardiac glycosides (CGs), which have, for a long time, been used in heart medicine. Moreover, it has been known for decades that CGs also have great potential in cancer treatment and, thus, many clinical trials now evaluate their anticancer potential. Interestingly, heart failure and cancer are not the only conditions for which CGs could be effectively used. In recent years, the antiviral potential of CGs has been extensively studied, and with the ongoing SARS-CoV-2 pandemic, this interest in CGs has increased even more. Therefore, here, we present CGs as potent and promising antiviral compounds, which can interfere with almost any steps of the viral life cycle, except for the viral attachment to a host cell. In this review article, we summarize the reported data on this hot topic and discuss the mechanisms of antiviral action of CGs, with reference to the particular viral life cycle phase they interfere with.

• Klíčová slova
• COVID-19, Na+/K+-ATPase, cardiac steroids, coronavirus, digitoxin, digoxin, drug repurposing, lanatoside C, ouabain, virus entry,
• MeSH
• antivirové látky farmakologie   terapeutické užití   MeSH
• COVID-19 MeSH
• digitoxin MeSH
• digoxin MeSH
• internalizace viru účinky léků   MeSH
• lidé MeSH
• nádory farmakoterapie   MeSH
• ouabain MeSH
• pandemie MeSH
• přehodnocení terapeutických indikací léčivého přípravku metody   MeSH
• replikace viru účinky léků   MeSH
• SARS-CoV-2 MeSH
• sodíko-draslíková ATPasa MeSH
• srdeční glykosidy metabolismus   terapeutické užití   MeSH
• srdeční selhání farmakoterapie   virologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• antivirové látky MeSH
• digitoxin MeSH
• digoxin MeSH
• ouabain MeSH
• sodíko-draslíková ATPasa MeSH
• srdeční glykosidy MeSH

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 regarding precursor inhibition is available. Here, we describe a cell-based assay addressing precursor inhibition. We used a reporter molecule containing the transframe (TFP) and p6* peptides, PR, and N-terminal fragment of reverse transcriptase flanked by the fluorescent proteins mCherry and EGFP on its N- and C- termini, respectively. The level of FRET between EGFP and mCherry indicates the amount of unprocessed reporter, allowing specific monitoring of precursor inhibition. The inhibition can be quantified by flow cytometry. Additionally, two microscopy techniques confirmed that the reporter remains unprocessed within individual cells upon inhibition. We tested darunavir, atazanavir and nelfinavir and their combinations against wild-type PR. Shedding light on an inhibitor's ability to act on non-mature forms of PR may aid novel strategies for next-generation drug design.

• MeSH
• atazanavir sulfát farmakologie   MeSH
• buněčné linie MeSH
• darunavir farmakologie   MeSH
• fluorescenční barviva MeSH
• HIV-1 enzymologie   MeSH
• inhibitory HIV-proteasy farmakologie   MeSH
• látky proti HIV farmakologie   MeSH
• lidé MeSH
• nelfinavir farmakologie   MeSH
• proteinové prekurzory antagonisté a inhibitory   MeSH
• proteolýza MeSH
• průtoková cytometrie MeSH
• rezonanční přenos fluorescenční energie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• atazanavir sulfát MeSH
• darunavir MeSH
• fluorescenční barviva MeSH
• inhibitory HIV-proteasy MeSH
• látky proti HIV MeSH
• nelfinavir MeSH
• proteinové prekurzory MeSH

HIV protease (PR) is required for proteolytic maturation in the late phase of HIV replication and represents a prime therapeutic target. The regulation and kinetics of viral polyprotein processing and maturation are currently not understood in detail. Here we design, synthesize, validate and apply a potent, photodegradable HIV PR inhibitor to achieve synchronized induction of proteolysis. The compound exhibits subnanomolar inhibition in vitro. Its photolabile moiety is released on light irradiation, reducing the inhibitory potential by 4 orders of magnitude. We determine the structure of the PR-inhibitor complex, analyze its photolytic products, and show that the enzymatic activity of inhibited PR can be fully restored on inhibitor photolysis. We also demonstrate that proteolysis of immature HIV particles produced in the presence of the inhibitor can be rapidly triggered by light enabling thus to analyze the timing, regulation and spatial requirements of viral processing in real time.

• MeSH
• aminokumariny chemická syntéza   farmakologie   MeSH
• časové faktory MeSH
• fotolýza MeSH
• HEK293 buňky MeSH
• HIV-1 účinky léků   fyziologie   účinky záření   MeSH
• HIV-proteasa chemie   metabolismus   MeSH
• inhibitory HIV-proteasy chemická syntéza   farmakologie   MeSH
• karbamáty chemická syntéza   farmakologie   MeSH
• kinetika MeSH
• lidé MeSH
• molekulární modely MeSH
• proteinové prekurzory antagonisté a inhibitory   chemie   metabolismus   MeSH
• proteolýza účinky léků   MeSH
• replikace viru MeSH
• světlo MeSH
• valin analogy a deriváty   chemická syntéza   farmakologie   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aminokumariny MeSH
• HIV-proteasa MeSH
• inhibitory HIV-proteasy MeSH
• karbamáty MeSH
• p55 gag precursor protein, Human immunodeficiency virus 1 MeSH Prohlížeč
• proteinové prekurzory MeSH
• thiazol-5-ylmethyl (5-(2-amino-3-methylbutanamido)-3-hydroxy-1,6-diphenylhexan-2-yl)carbamate MeSH Prohlížeč
• valin MeSH