Mutant protease

In recent times, HIV protease with mutations that confer resistance to protease inhibitors have been examined (10). This assay works for these mutant proteases as well. However, typically these mutant proteases have reduced catalytic efficiency with this substrate and require higher (10-fold or more) enzyme concentrations to see activity. 

A reasonable prediction is that the substrate specificity of the mutant protease would resemble that of trypsin. The mutant enzyme would be predicted to hydrolyze peptide bonds that follow either lysine or arginine in the sequence (i.e., peptide bonds whose carbonyl groups are from either lysine or arginine). 

Except for a very few studies most of the work so far has been done using SAR data of wild-type protease. Further QSAR and molecular modeling studies on prodrug derivatives, wild-type vs. mutant SAR data and lateral validation of these models via comparative analysis can provide useful insight. Such studies can highlight the differences and similarity, if any, in their mechanism of interaction with wild-type and mutant protease receptor. 

The continued derivation now shifts to the more specific problem of a ligand binding to wild type HIV-1 protease and its mutants. In this case P will be now be called and P will be called P,, y, where WT and MU stand for wild type and mutant protease respectively. HIV-1 protease is composed of two noncovalently associated structurally identical monomers, the active site contains two conserved catalytic aspartic acid residues, one from each monomer. Furthermore, the active site is C2 symmetric. Any active site mutation will result in structural effects in two different locations, one on each monomer, therefore, mutation does not break the C2 symmetry. As a result of these considerations, we can assume that the symmetry numbers for P,. and P are the same. This will also hold for LP, and LP. The ratio of symmetry numbers will then be equal to unity and the natural logarithm then zeros out the first term of equation (15). 

Sprang, S., et al. The three-dimensional structure of Asn ° mutant of trypsin role of Asp ° in serine protease catalysis. Science 237 905-909, 1987. 

Until recently, the catalytic role of Asp ° in trypsin and the other serine proteases had been surmised on the basis of its proximity to His in structures obtained from X-ray diffraction studies, but it had never been demonstrated with certainty in physical or chemical studies. As can be seen in Figure 16.17, Asp ° is buried at the active site and is normally inaccessible to chemical modifying reagents. In 1987, however, Charles Craik, William Rutter, and their colleagues used site-directed mutagenesis (see Chapter 13) to prepare a mutant trypsin with an asparagine in place of Asp °. This mutant trypsin possessed a hydrolytic activity with ester substrates only 1/10,000 that of native trypsin, demonstrating that Asp ° is indeed essential for catalysis and that its ability to immobilize and orient His is crucial to the function of the catalytic triad. 

A final but important consideration is viral mutation. Certain mutant HIV strains are resistant to one or more of the protease inhibitors, and even for patients who respond initially to protease inhibitors it is possible that mutant viral forms may eventually thrive in the infected individual. The search for new and more effective protease inhibitors is ongoing. 

Chen, Z., Li, Y, Schock, H. B., et al., 1995. Three-dimensional structure of a mutant HIV-1 protease displaying cros.s-resi.stance to all protease inhibitors in clinical txiAs. Journal of Biological Chemistry 270 21433-21436. 

J ,3J ,4J ,5J )-2,5-bis(benzyloxy)-3,4-dihydroxy-Nd -bis (lS)-2-methyl-l-[(methylamino)carbonyl]propyl hexanediamide is a C2-symmetric HIV-1 protease inhibitor [29]. Derivatization in the para positions of the benzyl-oxy groups via microwave-assisted Stille reaction on the corresponding di-brominated inhibitor smoothly yielded the desired heteroarylated derivatives (Scheme 10). Interestingly, the 1,3-thiazole derivative showed a higher antiviral activity on the wild type virus than the lead compound. The activity remained at the same level in the presence of seriun. Unfortimately, a low activity was observed on mutants. 

Allaire M, Chernaia MM, Malcolm BA, James MN (1994) Picomaviral 3C cysteine proteinases have a fold similar to chymotrypsin-Kke serine proteinases. Nature 369 72-76 Altman MD, Nalivaika EA, Prabu-Jeyabalan M, Schiffer CA, Tidor B (2008) Computational design and experimental study of tighter binding peptides to an inactivated mutant of HIV-1 protease. Proteins 70 678-694... 

A general mechanism of resistance is reducing the affinity of the antiretroviral compound for its mutant target protein. Resistance mutations associated with reduced affinity are observed during treatment failure with a fusion inhibitor, nonnucleoside reverse transcriptase inhibitors (NNRTl), integrase inhibitor, and protease inhibitors as reviewed in Chaps. 3,4, 6, and 7 (Hazuda et al. 2007 Hsiou et al. 2001 King et al. 2002 Mink et al. 2005). 

Changes in human immunodeficiency vims type 1 Gag at positions 1449 and P453 are linked to I50V protease mutants in vivo and cause reduction of sensitivity to amprenavir and improved viral fitness in vitro. J Virol 76 7398-7406... 

The development of resistance against HCV NS3/4 protease inhibitors will become a major challenge for the clinical use of these new compounds. Clinical trials of telaprevir (VX-950) have shown that mutations at different positions are rapidly selected (Sarrazin et al. 2005). In vitro studies indicate that cells bearing repUcons with those mutations are associated with different levels of resistance to telaprevir (< 10-fold change to >40-fold change in sensitivity). However, telaprevir-resistant mutants remain susceptible to interferon-a, at least in the replicon system. Likewise, replicon mutants that are resistant to boceprevir are still sensitive to interferon-a (Tong et al. 2006). 

The Rieske protein in mitochondrial bci complexes is assembled when the protein is incorporated into the complex. The Rieske protein is encoded in the nucleus and synthesized in the cytosol with a mitochondrial targeting presequence, which is required to direct the apoprotein to the mitochondrial matrix. The C-terminus is then targeted back to the outside of the inner mitochondrial membrane where the Rieske cluster is assembled. In addition, the presequence is removed and the protein is processed to its mature size after the protein is inserted into the bci complex. In mammals, the presequence is cleaved in a single step by the core proteins 1 and 2, which are related to the general mitochondrial matrix processing protease (MPP) a and (3 subunits the bovine heart presequence is retained as a 8.0 kDa subunit of the complex (42, 107). In Saccharomyces cerevis-iae, processing occurs in two steps Initially, the yeast MPP removes 22 amino acid residues to convert the precursor to the intermediate form, and then the mitochondrial intermediate protease (MIP) removes 8 residues after the intermediate form is in the bci complex (47). Cleavage by MIP is independent of the assembly of the Rieske cluster Conversion of the intermediate to the mature form was observed in a yeast mutant that did not assemble any Rieske cluster (35). However, in most mutants where the assembly of the Rieske cluster is prevented, the amount of Rieske protein is drastically reduced, most likely because of instability (35, 44). 

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