Introduction The introduction of drug resistance during antiretroviral therapy is a key concern for HIV individuals. to protease inhibitor treatment [1 2 The current HIV-1 protease inhibitors are designed having a hydroxyl group to mimic the transition state of the substrate’s scissile peptide relationship. Due to the structural similarity of inhibitors the mutations in HIV-1 protease are commonly associated with cross-resistance to the additional inhibitors [3]. The medical multi-drug resistant (MDR) HIV-1 strain 769 was isolated by Palmer et al. from individuals faltering protease inhibitor-containing antiretroviral regimens and the protease of strain 769 MDR 769 is definitely resistant to all protease inhibitors tested [4]. As observed in the crystal framework previously resolved by our group the flaps of MDR 769 are further aside set alongside the length of wild-type (WT) HIV-1 protease flaps [5]. The threonine mutation at residue 82 from the MDR HIV-1 protease (MDR 769 82T) alters the hydrophobicity from the P1 and P1′ binding storage compartments and may further improve cross-drug level of resistance. The uncomplexed MDR 769 82T crystal framework adopts the wide-open flap conformation as reported previously in MDR 769 [5] [6]. Based on virologic response research darunavir and tipranavir present an increased hereditary hurdle to level of resistance [7]. Both inhibitors have been used to treat patients infected with protease inhibitor-resistant viral strains and have effectively inhibited a range of MDR protease isolates [8 9 10 Based on the rating function of the HIV Drug Resistance Database (http://hivdb.stanford.edu) MDR 769 82T has low resistance to darunavir and large resistance to tipranavir as well as the other seven protease inhibitors [1 2 The structural study of the inhibitor-bound MDR HIV-1 protease facilitates the understanding of drug resistance mechanisms. The aim of this study is to test the in vitro inhibitory potency of C 75 supplier darunavir and tipranavir against MDR 769 82T and to determine the mechanism of overcoming resistance by analyzing the binding conformation important contacts and the stability of inhibitor-protease complexes. The protease inhibition assays demonstrate the decreased susceptibility of MDR 769 82T to all the tested inhibitors and confirm that 82T seriously enhances drug resistance. Compared to additional protease inhibitors the higher resistance barrier of C 75 supplier darunavir is due to maintaining BMP15 main chain hydrogen bonds by inhibitor flexibility while the higher resistance barrier of tipranavir is due to limited flap binding. 2 Materials and methods 2.1 Protein expression and purification Table 1 lists the protein sequences of MDR 769 MDR 769 82T and WT (NL4-3) HIV-1 protease. Active MDR 769 and MDR 769 82T and inactive MDR769 82T genes were codon C 75 supplier optimized for E. coli manifestation with the software DNA 2.0 [11] synthesized by GENEART Inc. (Regensburg Germany) and C 75 supplier put into the pET21b plasmid. The inactive MDR 769 82T protease experienced an active site mutation D25N to remove catalytic activity. To prevent auto-proteolyses the Q7K mutation was launched into the active MDR genes. The protein manifestation purification and refolding methods were explained earlier [12]. The proteases prepared for crystallization were concentrated to 1 1.5 mg/ml using Amicon concentrators with 5 kDa molecular mass cut-off (Millipore Corporation Billerica MA). 2.2 Protease inhibition assays The HIV-1 protease inhibitors requested from your NIH AIDS Study and Reference Reagent Program and HIV-1 protease Forster Resonance Energy Transfer (FRET) substrate I purchased from AnaSpec Inc. (Fremont CA) were used in the half-maximal inhibitory concentration (IC50) determination experiments. The fluorescence emitted by substrate cleavages was monitored with a microplate reader (SpectraMax M5 Molecular Devices Sunnyvale CA) at a 340 nm excitation wavelength with an emission wavelength of 490 nm. The HIV-1 protease reaction buffer was adjusted to pH 4.7 [0.1 M sodium acetate 1 M sodium chloride 1 mM ethylenediaminetetraacetic acid (EDTA) 1 mM DTT 10 dimethylsulfoxide (DMSO) and 1 mg/ml bovine serum albumin (BSA)]. In the reaction buffer containing 5 μM FRET substrate the concentration of all proteases used in enzyme assays was adjusted to a substrate cleavage velocity of 5 Relative Fluorescence Units (RFU)/min. The final HIV-1 protease concentration was approximately 7 nM. The protease inhibitor was serially diluted in DMSO from 10 μM to 0.013 nM. The active proteases and inhibitors were pre-incubated at 37°C for 20 min prior to fluorescence.