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
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.
- 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
Zika and Dengue viruses have attracted substantial attention from researchers in light of recent outbreaks of Dengue fever and increases in cases of congenital microcephaly in areas with Zika incidence. This review summarizes the current state of knowledge about Zika and Dengue proteases. These enzymes have several interesting features: 1) NS3 serine protease requires the activating co-factor NS2B, which is anchored in the membrane of the endoplasmic reticulum; 2) NS2B displays extensive conformational dynamics; 3) NS3 is a multidomain protein with proteolytic, NTPase, RNA 5' triphosphatase and helicase activity and has many protein-protein interaction partners; 4) NS3 is autoproteolytically released from its precursor. Attempts to design tight-binding and specific active-site inhibitors are complicated by the facts that the substrate pocket of the NS2B-NS3 protease is flat and the active-site ligands are charged. The ionic character of potential active-site inhibitors negatively influences their cell permeability. Possibilities to block cis-autoprocessing of the protease precursor have recently been considered. Additionally, potential allosteric sites on NS2B-NS3 proteases have been identified and allosteric compounds have been designed to impair substrate binding and/or block the NS2B-NS3 interaction. Such compounds could be specific to viral proteases, without off-target effects on host serine proteases, and could have favorable pharmacokinetic profiles. This review discusses various groups of inhibitors of these proteases according to their mechanisms of action and chemical structures.
- MeSH
- alosterické místo MeSH
- antivirové látky chemie farmakologie MeSH
- dengue farmakoterapie virologie MeSH
- infekce virem zika farmakoterapie virologie MeSH
- inhibitory proteas chemie farmakologie MeSH
- katalytická doména MeSH
- kinetika MeSH
- konformace proteinů MeSH
- lidé MeSH
- racionální návrh léčiv * MeSH
- serinové endopeptidasy chemie metabolismus MeSH
- virus dengue účinky léků enzymologie MeSH
- virus zika účinky léků enzymologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy 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
Multitemplate polymerase chain reaction (PCR) is used for preparative and analytical applications in diagnostics and research. Classical PCR and qPCR are two basic setups with many possible experimental modifications. Classical PCR is a method of choice to obtain enough material for subsequent sophisticated applications such as construction of libraries for next-generation sequencing or high-throughput screening. Sequencing and Single Nucleotide Primer Extension (SNuPE) employ one-strand synthesis and represent a distinct variant of analytical DNA synthesis. In all these applications, maintaining the initial ratio of templates and avoiding underestimation of minority templates is desired. Here, we demonstrate that different templates can amplify independently at low template concentrations (typical in qPCR setups, in which the polymerase concentration is usually several orders of magnitude higher than the template concentration). However, rare templates can be diluted in an effort to keep DNA amplification in the exponential phase, or template concentration can be biased by differences in amplification efficiency. Moreover, amplification of templates present in low concentrations is more vulnerable to stochastic events that lead to proportional changes in the product ratio, as well as by incomplete amplification leading to chimera formation. These undesired effects can be compensated for by using highly processive polymerases with high and equal affinity to different primer-template complexes. Novel enhanced polymerases are desired. With increasing concentration of a primer-template of interest, the system becomes more deterministic. Nevertheless, marked deviation from independent exponential amplification occurs when the total template concentration starts to approach the polymerase concentration. The primer-template complexes compete for enzyme molecules, and the amount of products grows arithmetically-the system starts to obey Michaelis-Menten kinetics. Synthesis of rare products in a multitemplate mixture can run more easily under the detection limit in such conditions, although it would be unequivocally detectable in a single template assay. When fishing out rare template variants, the best processive polymerases should be used to decrease both amplification and detection limits. The possibility of stochastic events, should be taken into account to correctly interpret the obtained data.
Since new and dangerous influenza virus strains, such as H5N1 “avian flu” and more recently the swine-origin H1N1 “swine flu”, are constantly evolving, the need for effective anti-influenza drugs is pressing. It is becoming clear that the emergence of drug-resistant viruses will be a major potential problem in future efforts to control influenza virus infection. Moreover, development of vaccines against new influenza strains takes several months, and their production capacity is limited. Thus, new classes of anti-influenza drugs are highly sought after. This review focuses mainly on novel strategies, including targeting viral entry into host cells, inhibition of viral transcription and genome replication, and targeting of the NS1 influenza protein. Another approach involves viral RNA silencing by siRNAs or by antisense oligonucleotides. Inhibitors of viral neuraminidase have been the most successful approach in influenza virus breakdown to date. Viral maturation can also be blocked by inhibition of hemagglutinin-processing cellular proteinases. Compounds modifying the host cell immune response have also been reported. Design of specific compounds universally active against all viral variants with a reduced potential for the emergence of drug-resistant mutants is the main challenge in anti-influenza drug development, and the goals in this field are discussed here. A review with 140 references.
BACKGROUND: The introduction of HIV proteinase inhibitors (PIs) as anti-AIDS drugs resulted in decreased mortality and prolonged life expectancy of HIV-positive patients. However, rapid selection of drug-resistant HIV variants is a common complication in patients undergoing highly active anti-retroviral therapy (HAART). Thus, monitoring of clinical resistance development is indispensable for rational pharmacotherapy. OBJECTIVE: We present a non-infectious cell-based assay for drug resistance quantification of HIV proteinase (PR) - an important target of HAART. STUDY DESIGN: Previously, we showed [Lindsten K, Uhlikova T, Konvalinka J, Masucci MG, Dantuma NP. Cell-based fluorescence assay for human immunodeficiency virus type 1 protease activity. Antimicrob Agents Chemother 2001;45:2616-22] that the expression of a fusion protein (GFP-PR), comprised of HIV-1 proteinase wild-type artificial precursor (PR) and green fluorescent protein (GFP), in transiently transfected tissue culture cells depends on the presence of PR-specific inhibitors (PIs). Here we show that in the GFP-PR reporter the HIV wild-type PR can be replaced by a drug-resistant HIV PR mutant, yielding a simple and biologically relevant tool for the quantitative analysis of drug-resistant HIV PR mutants susceptibility to HIV proteinase inhibitors. RESULTS: We cloned a set of GFP-PR reporters, some of which possess a simple, well-defined drug-resistant PR mutant (G48V L90M, V82A, A71V V82T I84V, D30N, K45I); another four complex PR mutants were obtained from patients undergoing HAART. The results were compared with genotyping and enzyme kinetics data. Furthermore, we designed a single inhibitor concentration experiment setup for easy evaluation of drug resistance profiles for mutants of interest. The resistance profiles clearly demonstrate the importance of succession of individual drugs during the treatment for drug resistance development. CONCLUSION: We show that the GFP-PR assay might serve as a non-infectious, rapid, cheap, and reliable alternative to the currently used phenotypic assays.
- MeSH
- biotest metody MeSH
- fenotyp MeSH
- financování organizované MeSH
- fluorescenční mikroskopie MeSH
- fluorometrie MeSH
- HeLa buňky MeSH
- HIV-proteasa genetika metabolismus účinky léků MeSH
- hodnotící studie jako téma MeSH
- inhibitory HIV-proteasy farmakologie MeSH
- kinetika MeSH
- klonování DNA MeSH
- lidé MeSH
- monoklonální protilátky metabolismus MeSH
- mutace MeSH
- plazmidy MeSH
- průtoková cytometrie MeSH
- rekombinantní fúzní proteiny metabolismus MeSH
- reportérové geny MeSH
- transfekce MeSH
- virová léková rezistence MeSH
- virové geny MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- western blotting MeSH
- zelené fluorescenční proteiny metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- srovnávací studie MeSH
