PROTEIN TARGET
HIV-1 & HIV-2 Protease
HIV-1 and HIV-2 proteases are the targets of saquinavir. These protease targets are critical for the assembly and maturation of infectious viruses. When they are inhibited by saquinavir, the production of mature virus proteins will stop. These targets are likely to develop resistance to saquinavir.
HIV-1 PROTEASE STRUCTURE
WILD TYPE HIV-1 PROTEASE WITH SAQUINAVIR INSIDE ITS ACTIVE SITE.
The dimer of HIV-1 protease is shown in green and orange, and the inhibitor Saquinavir inside the active site is shown in pink.
PDB: 3OXC
HIV-1 PROTEASE RIBBON STRUCTURE.
The monomers making up the dimer are shown in green and orange.
PDB: 1DMP
PROTEIN TARGET - HIV PROTEASES
STRUCTURE
HIV protease is an aspartyl protease with two identical monomers composed of 99 residues.(2) These two monomers give it a dimer quaternary structure. Each monomer is made from many β-sheets and one α-helix, and they are connected by long, flexible loops and a few turns. The active site is made by both monomers at their interface. The active site is stabilized by the β-sheets and its entrance is protected by two flexible β-hairpin flaps. The flaps will open for substrates to enter the active site. Within each monomer, there is a “fireman’s grip” – D25-T26-G27 residues – that also stabilizes the active site, and the D25 in this motif is critical for catalysis.(1) The two aspartic acid residues, one from each monomer, carry out the primary catalytic activity of the enzyme by activating water molecules for peptide bond cleavage.(1,2)
Compared to cellular proteases such as pepsin, HIV retroviral protease is much smaller in size. It is also different from the cellular protease because of the dimer’s unusual symmetric arrangement. The two monomers in HIV retroviral protease are identical, whereas for nonviral proteases, the two domains are similar in shape but not sequence.(3)
FUNCTION
As a protease, the HIV protease is mainly responsible for processing viral proteins via cleavage of peptide bonds by hydrolysis. HIV-1 protease is involved in Gag proteolysis, which is part of forming a mature virion. If it is inhibited, the viral particles released by the host will be non-infectious.(4) This enzyme cleaves at nine different processing sites on the Gag and Gag-Pol proteins, activating the immature form of the target peptides for assembly and maturation of new infectious viruses.(2) The fragments of the Gag protein are used in many ways, such as structural support and RNA stabilization.(1)
HIV-1 PROTEASE AND VIRUS MATURATION
Why is HIV protease essential for viral infection?
The production of a mature and infectious virus, generally, has three steps:
Assembly: viral structural proteins and enzymes are packed to form a virion (a virus outside of a cell)
Budding: virion forms a lipid envelope and buds out from the membrane
Maturation: virion become infectious through structural changes of select proteins packed inside
A protein called Gag, the main component of an HIV-1 virion, is critical for coordinating all three steps, especially maturation. Gag is the target of HIV-1 protease. HIV-1 protease recognizes five cleavage sites on Gag protein (and five sites on the Gag-Pro-Pol) and interacts with residues around scissile bonds. These interactions disrupt the symmetry of HIV-I protease, leading to cleavage. The cleavage and redistribution of Gag change the internal organization of proteins in a virion, freeing the newly formed components of Gag for different uses within the virus and allowing for the production of a mature and infectious viral peptide. Gag proteolysis by HIV-1 protease also triggers other reactions such as condensing the viral genome, forming proteins for structural support, and producing proteins responsible for invading the host cell. Without Gag proteolysis by HIV-1 protease, the virion remains non-infectious.(1)
(Figure is unmodified and distributed under the Creative Commons License: https://en.wikipedia.org/wiki/HIV#/media/File:HIV-replication-cycle-en.svg)
SAQUINAVIR INHIBITS HIV PROTEASE
How does inhibition by saquinavir impair virus infection and replication?
Saquinavir, like many other enzyme inhibitors, inhibits HIV-1 protease's enzyme activity by blocking the active site of the protease. Based on the “peptidomimetic” principle, saquinavir contains a similar structure to the scissile peptide bond, but cannot be cleaved by HIV protease. Thus, saquinavir occupies the active site and prevents entrance and cleavage of Gag proteins.(5) Failure to process Gag and Gag-Pro-Pol proteins results in virions having no protein segments to stabilize viral RNA or provide structural support.(1) Without these important protein segments, the virion is hindered from invading the healthy cells and replicating, effectively reducing virus infection.
More information on the Inhibitor Mechanism of Action page!
HIV Protease develops resistance to saquinavir easily through mutations
HIV PROTEASE
RESISTANCE
TO SAQUINAVIR
Although the HIV-1 protease is very small in size and was believed to have limited ability to develop resistance, HIV-1 protease is actually prone to developing drug resistance through resistant mutations.(6) Studies have shown that there are three major mutations that increases HIV-1 protease resistance to saquinavir: L90M, G48V, and G48V/L90M. These mutations cause HIV-1 protease to have a lower affinity for saquinavir.(8) G48V occurs at the substrate cleft and it prevents saquinavir from binding. The larger side chain of valine comparing to glycine results in greater steric hinderance, thereby stop saquinavir from binding. L90M also has an impact on the substrate cleft of HIV-protease.(9) These two mutations do not disable the enzyme completely when they occur alone, but if both mutations occur in a double mutant, the HIV-1 protease’s affinity for saquinavir is greatly reduced, with an affinity of 1/1000 compared to the wild type protease.(8, 9)
Mutations have been observed in 49 out of the 99 codons in HIV-1 protease. For every replication cycle, there will be up to 10 mutations generated in a protease’s genome.(7) This high plasticity of the viral protein genome is because the enzyme which generates the cDNA of HIV-1 protease, HIV-1 reverse transcriptase, has no proofreading mechanism and thus has a high error rate.(10) The high replication rate of HIV in the host cell means that many mutations will accumulate in the enzyme genome during this non-stop production of HIV-1 protease. In addition, viral recombination of the genome is also highly likely. These three factors contribute to the unusually diverse genome and high error rate of this small protease.(10)