Darunavir and tipranavir are two inhibitors that are active against multi-drug resistant (MDR) HIV-1 protease variants. In this study, the in vitro inhibitory efficacy was tested against a MDR HIV-1 protease variant, MDR 769 82T, containing the drug resistance mutations of 46L/54V/82T/84V/90M. Crystallographic and enzymatic studies were performed to examine the mechanism of resistance and the relative maintenance of potency. The key findings are as follows: (i) The MDR protease exhibits decreased susceptibility to all nine HIV-1 protease inhibitors approved by the US Food and Drug Administration (FDA), among which darunavir and tipranavir are the most potent; (ii) the threonine 82 mutation on the protease greatly enhances drug resistance by altering the hydrophobicity of the binding pocket; (iii) darunavir or tipranavir binding facilitates closure of the wide-open flaps of the MDR protease; and (iv) the remaining potency of tipranavir may be preserved by stabilizing the flaps in the inhibitor-protease complex while darunavir maintains its potency by preserving protein main chain hydrogen bonds with the flexible P2 group. These results could provide new insights into drug design strategies to overcome multi-drug resistance of HIV-1 protease variants. (C) 2011 Elsevier Inc. All rights reserved.