The inclusion bodies were solubilized in 8 M urea and the protease was refolded by dialysis against 0

The inclusion bodies were solubilized in 8 M urea and the protease was refolded by dialysis against 0.05 M sodium phosphate buffer (0.05 M Na2HPO4, 0.005 M EDTA, 0.3 M NaCl, and 0.001 M DTT, pH 7.3). sliding mechanism by causing a redistribution of van der Waals interactions in the hydrophobic core in PRG48T/L89M-SQV. Our mechanism for PRG48T/L89M-SQV drug resistance proposes that a defective hydrophobic sliding mechanism results in altered conformational dynamics of the protease. As a consequence, the protease is unable to achieve a fully closed conformation that results in an expanded active site and weaker inhibitor binding. Human immunodeficiency computer virus type 1 (HIV-1) remains a serious global health concern. In 2012, 35.3 million people were living with HIV/AIDS worldwide and 1.6 million people died from the disease.2 The use of highly active antiretroviral therapy (HAART) that involves combinations of reverse transcriptase and protease inhibitors can lead to a reduction in viral weight to nearly undetectable levels in infected individuals.3,4 However, the major challenge limiting current therapy is the rapid evolution of drug resistance resulting from the high mutation rate caused by the absence of a proofreading function in HIV reverse transcriptase.5 Consequently, there is a continuing need for next generation PIs with efficacy against drug resistant strains of HIV. This work will add to the growing amount of information on resistance mechanisms with an aim toward new drug development. This study examines the effect of drug resistant mutations on HIV-1 protease, which is usually involved in the processing of the Gag and Gag-Pol viral polyproteins. These processing events allow the computer virus to efficiently form new virion particles and infect new host cells.6 Consequently, PR is a valuable drug target since inhibition of PR activity results in immature noninfectious virions.7,8 We utilized the Stanford University HIV Drug Resistance Database to determine novel drug resistant mutations that may develop in PR in response BNS-22 to ritonavir boosted protease inhibitor therapy. An analysis of the database facilitated the determination of a previously uncharacterized, SQV/RTV resistant variant, Gly48Thr/Leu89Met (PRG48T/L89M). Residue Gly48 is located in the flaps of the protease and contributes to the formation of the S2/S2 and S3/S3 binding pouches of the enzyme;9 however, residue Leu89 does not make contact with the inhibitor directly. Instead, residue Leu89 is located in the hydrophobic core of PR which is usually distal to the active site. While the effect of main mutations on inhibitor binding can be more easily rationalized because those amino acids make direct contact with the inhibitor, many PR mutations are secondary and are found outside of the active site. How these mutations transmit their deleterious effect on inhibitor binding in the active site is less obvious.10 Several studies suggest that secondary mutations interfere with the conformational equilibrium between the open and closed forms of PR.10?12 Since PIs are rigid and are designed to bind the closed conformation, mutations that shift the conformational equilibrium of PR to the open form may result in weaker PI binding.10 Mutations of both Gly48 and Leu89 result in PR drug resistance. Gly48Val, a primary mutation, occurs in response to SQV treatment and less often from IDV and LPV treatment13?16 and confers high-level resistance to SQV, intermediate-level resistance to ATV, and low-level resistance to NFV, IDV, and LPV.17?19 Gly48Met occurs in patients who have received multiple PIs and results in a similar resistance profile as Gly48Val.17,20?22 Gly48Ala/Ser/Thr/Gln/Leu are extremely rare PR mutations23 that occur primarily in viruses containing multiple PI-resistance mutations and appear to have comparable but weaker effects on PI susceptibility than do Gly48Val and Gly48Met.18 Leu89Val, a secondary mutation, is an accessory mutation and is located outside the active site in the hydrophobic core of PR and occurs in response to treatment with IDV, NFV, FPV, and DRV. It reduces susceptibility to these inhibitors. Several groups have investigated.Future work on the development of allosteric inhibitors that work in conjunction with FDA approved PIs to drive the conformational equilibrium of a drug resistance protease to the closed form may provide an alternative approach to addressing the problem of HIV drug resistance.69 Alternatively, another approach to overcome drug resistance may be the design of drugs that bind the open conformation such as compounds like metallacarboranes and pyrrolidine-based inhibitors.70 Glossary Abbreviations:SQVsaquinavirAPVamprenavirATZatazanavirLPVlopinavirDRVdarunavirRTVritonavirTPVtipranavirIDVindinavirNFVnelfinavirFPVfosamprenavirPRproteasePRWTHIV-1 protease without stabilizing Keratin 18 antibody mutationsPRWT-SQVWT HIV-1 protease without stabilizing mutations bound with saquinavir PDB code 1HXBPRG48T/L89Mapo HIV-1 protease Gly48Thr/Leu89MetPRG48T/L89M-SQVHIV-1 protease Gly48Thr/Leu89Met bound with saquinavirPIprotease inhibitorHIV-1human immunodeficiency virus type-1HAARThighly antiretroviral therapyvdwvan der WaalDIQdecahydroisoquinolineMDmolecular dynamicsRMSFroot-mean-square fluctuationSnsubsites of the PR active sitePnPositions of a ligand bound in the active site of PRfulcrum(amino acids 11C22)elbows(amino acids 39C57)cantilever(amino acids 58C78) Funding Statement National Institutes of Health, United States Supporting Information Available Additional structural and molecular dynamic simulation data. and weaker interatomic flap interactions. We also show that the Leu89Met mutation disrupts the hydrophobic sliding mechanism by causing a redistribution of van der Waals interactions in the hydrophobic core in PRG48T/L89M-SQV. Our mechanism for PRG48T/L89M-SQV drug resistance proposes that a defective hydrophobic sliding mechanism results in modified conformational dynamics of the protease. As a consequence, the protease is unable to achieve a fully closed conformation that results in an expanded active site and weaker inhibitor binding. Human immunodeficiency virus type 1 (HIV-1) remains a serious global health concern. In 2012, 35.3 million people were living with HIV/AIDS worldwide and 1.6 million people died from the disease.2 The use of highly active antiretroviral therapy (HAART) that involves combinations of reverse transcriptase and protease inhibitors can lead to a reduction in viral load to nearly undetectable levels in infected individuals.3,4 However, the major challenge limiting current therapy is the rapid evolution of drug resistance resulting from the high mutation rate caused by the absence of a proofreading function in HIV reverse transcriptase.5 Consequently, there is a continuing need for next generation PIs with efficacy against drug resistant strains of HIV. This work will add to the growing amount of information on BNS-22 resistance mechanisms with an aim toward new drug development. This study examines the effect of drug resistant mutations on HIV-1 protease, which is involved in the processing of the Gag and Gag-Pol viral polyproteins. These processing events allow the virus to efficiently form new virion particles and infect new host cells.6 Consequently, PR is a valuable drug target since inhibition of PR activity results in immature noninfectious virions.7,8 We utilized the Stanford University HIV Drug Resistance Database to determine novel drug resistant mutations that may develop in PR in response to ritonavir boosted protease inhibitor therapy. An analysis of the database facilitated the determination of a previously uncharacterized, SQV/RTV resistant variant, Gly48Thr/Leu89Met (PRG48T/L89M). Residue Gly48 is located in the flaps of the protease and contributes to the formation of the S2/S2 and S3/S3 binding pockets of the enzyme;9 however, residue Leu89 does not make contact with the inhibitor directly. Instead, residue Leu89 is located in the hydrophobic core of PR which is distal to the active site. While the effect of primary mutations on inhibitor binding can be more easily rationalized because those amino acids make direct contact with the inhibitor, many PR mutations are secondary and are found outside of the active site. How these mutations BNS-22 transmit their deleterious effect on inhibitor binding in the active site is less clear.10 Several studies suggest that secondary mutations interfere with the conformational equilibrium between the open and closed forms of PR.10?12 Since PIs are rigid and are designed to bind the closed conformation, mutations that shift the conformational equilibrium of PR to the open form may result in weaker PI binding.10 Mutations of both Gly48 and Leu89 result in PR drug resistance. Gly48Val, a primary mutation, occurs in response to SQV treatment and less often from IDV and LPV treatment13?16 and confers high-level resistance to SQV, intermediate-level resistance to ATV, and low-level resistance to NFV, IDV, and LPV.17?19 Gly48Met occurs in patients who have received multiple PIs and results in a similar resistance profile as Gly48Val.17,20?22 Gly48Ala/Ser/Thr/Gln/Leu are extremely rare PR mutations23 that occur primarily in viruses containing multiple PI-resistance mutations and appear to have similar but weaker effects on PI susceptibility than do Gly48Val and Gly48Met.18 Leu89Val, a secondary mutation, is an accessory mutation and is located outside the active site in the hydrophobic core of PR and occurs in response.