Collect. Czech. Chem. Commun. 2004, 69, 703-714

Characterisation of Mutated Proteinases Derived from HIV-Positive Patients: Enzyme Activity, Vitality and Inhibition

Milan Kožíšeka,b, Jana Prejdováa,b, Milan Součeka, Ladislav Machalac, Marie Staňkovác, Marek Linkad, Marie Brůčkovád and Jan Konvalinkaa,b,*

a Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo nám. 2, 166 10 Prague 6, Czech Republic
b Department of Biochemistry, Faculty of Science, Charles University in Prague, Hlavova 2030, 128 43 Prague 2, Czech Republic
c AIDS Center at the Clinic of Infectious Diseases, Faculty Clinic Bulovka, Budínova 2, 180 00 Prague 8, Czech Republic
d National Institutes of Public Health, National Reference Laboratory on AIDS, Šrobárova 48, 100 42 Prague 10, Czech Republic


HIV protease (PR) specifically cleaves viral polyproteins to yield infectious progeny virus particles. Inactivation of PR leads to loss of virus infectivity and PR thus became an attractive pharmaceutic target. Indeed, seven protease inhibitors (PI) have been approved for clinical use to date. However, emerging resistant viral variants with reduced sensitivity to PIs become a major obstacle to successful control of viral replication. We have previously reported the design, testing and structural analysis of a pseudopeptide inhibitor, QF34, which efficiently inhibits a wide variety of PR variants. In a clinical study, we have monitored more than 100 HIV-positive patients in the Czech Republic undergoing highly active antiretroviral therapy including PI. In this paper we describe kinetic characterisation of two highly resistant PR species isolated from these patients. The mutated proteases accumulated as much as 14 amino acid exchanges and develop resistance to saquinavir, ritonavir, indinavir and nelfinavir with vitality value up to 150. Kinetic analyses revealed that second-generation PI lopinavir and QF34 retained their subnanomolar potency against both multidrug resistant PR variants. These results suggest a route to the design of PIs capable of inhibiting a variety of resistant PR mutants.

Keywords: Enzyme inhibitors; Enzyme kinetics; HIV protease; Aspartic proteases; Viral resistance; Antiviral drugs; AIDS; Peptidomimetics; Pseudopeptides.

References: 33 live references.