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

High-pressure methods have become an interesting tool of investigation of structural stability of proteins. They are used to study protein unfolding, but dissociation of oligomeric proteins can be addressed this way, too. HIV-1 protease, although an interesting object of biophysical experiments, has not been studied at high pressure yet. In this study HIV-1 protease is investigated by high pressure (up to 600 MPa) fluorescence spectroscopy of either the inherent tryptophan residues or external 8-anilino-1-naphtalenesulfonic acid at 25°C. A fast concentration-dependent structural transition is detected that corresponds to the dimer-monomer equilibrium. This transition is followed by a slow concentration independent transition that can be assigned to the monomer unfolding. In the presence of a tight-binding inhibitor none of these transitions are observed, which confirms the stabilizing effect of inhibitor. High-pressure enzyme kinetics (up to 350 MPa) also reveals the stabilizing effect of substrate. Unfolding of the protease can thus proceed only from the monomeric state after dimer dissociation and is unfavourable at atmospheric pressure. Dimer-destabilizing effect of high pressure is caused by negative volume change of dimer dissociation of -32.5 mL/mol. It helps us to determine the atmospheric pressure dimerization constant of 0.92 μM. High-pressure methods thus enable the investigation of structural phenomena that are difficult or impossible to measure at atmospheric pressure.

• MeSH
• Anilino Naphthalenesulfonates metabolism   MeSH
• Atmospheric Pressure MeSH
• Darunavir metabolism   MeSH
• Dimerization MeSH
• Spectrometry, Fluorescence MeSH
• HIV Protease chemistry   metabolism   MeSH
• HIV Protease Inhibitors metabolism   MeSH
• Kinetics MeSH
• Protein Conformation MeSH
• Humans MeSH
• Models, Molecular MeSH
• Protein Multimerization MeSH
• Protein Folding * MeSH
• Protein Stability drug effects   MeSH
• Thermodynamics MeSH
• Tryptophan metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 1-anilino-8-naphthalenesulfonate MeSH Browser
• Anilino Naphthalenesulfonates MeSH
• Darunavir MeSH
• HIV Protease MeSH
• HIV Protease Inhibitors MeSH
• p16 protease, Human immunodeficiency virus 1 MeSH Browser
• Tryptophan MeSH

The design, development and clinical success of HIV protease inhibitors represent one of the most remarkable achievements of molecular medicine. This review describes all nine currently available FDA-approved protease inhibitors, discusses their pharmacokinetic properties, off-target activities, side-effects, and resistance profiles. The compounds in the various stages of clinical development are also introduced, as well as alternative approaches, aiming at other functional domains of HIV PR. The potential of these novel compounds to open new way to the rational drug design of human viruses is critically assessed.

• Keywords
• HAART, HIV protease, alternative inhibitors, pharmacokinetic boosting, protease dimerization, protease inhibitors, resistance development,
• Publication type
• Journal Article MeSH