Discovery and development of HIV-protease inhibitors Save Many major physiological processes depend on regulation of proteolytic enzyme activity and there can be dramatic consequences when equilibrium between an enzyme and its substrates is disturbed. In this prospective, the discovery of small-molecule ligandslike protease inhibitorsthat can modulate catalytic activities has an enormous therapeutic effect.
Second-generation Non-peptidic inhibitor In comparison to HIV-1, few crystal structures of the viral protease PR in complex with the inhibitors have been studied for HIV-2 [ 17 ]; moreover, the currently approved PIs are essentially designed for HIV-1, and their association with the HIV-2 viral protease had not been thoroughly characterized.
To date, a limited number of studies tested the effect of protease inhibitors against HIV-2, either in enzymatic or phenotypic susceptibility assays [ 181920212223 ].
Many factors can influence the results obtained, such as the stability and level of purity of the protease in enzymatic experiments, the type of cells and methods used in cell culture assays, as well as the viral strain.
Therefore, the use of a standardized protocol that examines the efficacy of the inhibitor on the same viral enzyme in kinetic and cell culture assays will greatly aid in the determination of a reliable, accurate and comparable IC50 values.
For this purpose, we have developed an HIV-2 protease modular cassette system [ 24 ], which enables the study of the susceptibility of the viral protease to a panel of commercially available PIs, in comparative enzymatic and cell culture experiments.
The viral protease is a homodimeric aspartyl protease, composed of two identical subunits that are 99 amino acids each [ 25 ].
The enzyme is crucial for the viral life-cycle by processing the viral Gag and Gag-Pro-Pol viral polyproteins, leading to the formation of mature, infection-competent virions.
As a consequence of amino acid substitution s in the substrate binding pocket or in a nearby site, the binding affinity of the inhibitors may become substantially reduced, leading to failure in blocking the viral protease [ 282930 ].
Of the treatment-associated mutations observed in HIV-2 protease, I54M and L90M have been shown to be implicated in the reduced susceptibility to certain protease inhibitors in phenotypic assays [ 1820 ], but as far as we know, their effects have never been characterized in association with all of the inhibitors, neither enzymatically nor in cell culture.
Evidence suggests that resistance to inhibitors may either be a natural phenomenon, as a result of polymorphism in the protease sequence, or may occur selectively under treatment pressure, in sites usually comparable to those found in the HIV-1 protease [ 183132 ]. Substitution of the wild-type amino acid by methionine Met introduces a longer side chain, resulting in a shift of the main chain atoms relative to their original positions, in addition to the formation of new van der Waals forces and hydrophobic interactions with the neighboring residues.
I54M and L90M single mutations were not found to have a disruptive effect on the secondary or tertiary structure [ 33 ]. Ile is positioned in the flap region of the protease, but it is not exposed to the active site cavity. It forms hydrophobic interactions with neighboring flap and loop residues.
Substitution of Ile to Met has been found to confer resistance to the protease against certain inhibitors in both HIV-1 and -2 [ 3435 ].
Leu on the other hand, is present in the hydrophobic core of the protease, near the catalytic aspartate residues; but outside of the active site.
Substitution of this residue with Met also results in resistance to some inhibitors, again affecting both viruses [ 183637 ]. Even though these mutations do not occur in the active site; as already mentioned, it is thought that substitution of the native amino acid to methionine may result in the displacement of certain amino acids, with an overall impact of altering the pocket binding site and the stability of the dimer—inhibitor association, leading to decreased efficacy or resistance to the inhibitor s [ 3638 ].
Results and Discussion 2.
In Vitro Kinetic Assays In our in vitro analysis, following the expression, purification of the protease and confirmation of its activity, kinetic parameters of the enzyme were then determined for both the wild-type and double mutant protease.
As for the double mutant protease, values were: These values suggest that the mutations caused a substantial increase of the Km value without affecting the turnover number.
Inhibition profiling assays were performed using the high-performance liquid chromatography HPLC method in triplicate measurements.Researchers are investigating the use of protease inhibitors developed for HIV treatment as anti-protozoals for use against malaria and gastrointestinal protozoal infections: A combination of ritonavir and lopinavir was found to have some effectiveness against Giardia infection.
The scientific investigation of the life cycle of HIV has been an area of intense research since the first description of the virus in In order to gain entry to the intracellular human machinery, which all viruses require for replication, the virus must fuse with the human cell membrane.
This systematic review and meta-analysis tries to determine whether there is an association between the use of protease inhibitors (PIs) and the incidence of diabetes mellitus (DM) and/or metabolic syndrome (MS) in HIV-infected patients.
Protease inhibitors block the activity of the protease enzyme, which HIV uses to break up large polyproteins into the smaller pieces required for assembly of new viral particles.
While HIV can still replicate in the presence of protease inhibitors, the resulting virions are immature and unable to . Background. There is little data on the safety of combining radiation therapy and human immunodeficiency virus (HIV) protease inhibitors to treat cancers in HIV-positive patients.
HIV protease inhibitors (HIV PIs) have been reported with radiosensitizing activities in HNSCC cells, but the underlying cellular/molecular mechanisms remain unclear.
Our previous study has shown that HIV PIs induce cell apoptosis via activation of endoplasmic reticulum (ER) stress.