The Influence of I47A Mutation on Reduced
Susceptibility to the Protease
Inhibitor Lopinavir
Klára Šašková1,2, Milan Kožíšek1,2
, Jiří Brynda1,4, Martin Lepšík1, Ladislav Machala3
and Jan Konvalinka1,2*
1 Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech
Republic, Flemingovo n. 2, 16610 Praha 6, Czech Republic
2 Department of Biochemistry, School of Science, Charles University, Hlavova 8, 128 43
Praha 2, Czech Republic
3 AIDS Center at the Clinic of Infectious Diseases, Faculty Clinic Bulovka, Budinova 2, CZ-
12000 Praha 8, Czech Republic
4 Institute of Molecular Genetics, Academy of Science of the Czech Republic, Flemingovo
n.2, 166 10 Praha 6, Czech Republic
* to whom the correspondence should be
sent. E-mail: konval@uochb.cas.cz,
fax: +420 220 183 578
The introduction of protease inhibitors
(PI) in mid 90´s led to decreased mortality and prolonged life expectancy of
HIV-positive patients. However, the selection pressure of a virostatic leads to
rapid selection of viral variants resistant towards a specific inhibitor.
Different PIs select different patterns of mutations. Lopinavir (LPV) is a second generation PI that
was rationally designed to inhibit resistnant PR species. Its resistance
profile has not been clearly defined yet. Mutations at 11 amino acid positions
in protease (PR) coding region were identified as associated with reduced
susceptibility (lopinavir mutation score). The higher number of mutations accumulated at these
positions, the bigger the resistance to LPV. However, individual mutations
resulting in LPV resistance are very rare. Recently, mutation I47A has been
identified to confer high–level resistance.
In this work, we set to identify the relative contributions of I47A mutation and others from the lopinavir mutation score. We designed a panel of resistant PR species containing I47A substitution in the backround of the wild–type PR and also in the backround of a highly resistant specimen from a patient. Individual recombinant PRs were expressed, purified and characterized and their inhibition profiles were analysed with a panel of PIs. The X-ray analysis of the protease containing I47A mutation in complex of LPV showed the loss of van der Waals interactions of Ala47 with lopinavir phenoxyacetyl moiety. Futhermore, energy calculations confirmed weaker affinity of LPV and the decrease of both electrostatic and van der Waals interactions.