Crystallographic, molecular dynamics, and enzymatic studies of multi-drug resistant HIV-1 protease and implications for structure based drug design (project 1); crystallographic studies of human myelin protein zero (project 2)

Under drug selective pressure, emerging mutations render HIV-1 protease drug resistant, leading to antiretroviral therapy failure. The multidrug-resistant isolate 769 (MDR769) of HIV-1 protease, with resistance mutations at residues 10, 36, 46, 54, 62, 63, 71, 82, 84, 90, was selected for the present study to investigate the drug resistance issue.;Ten additional mutations were introduced into to MDR769 HIV-1 protease to study the structural influences introduced by these mutations. We obtained crystal structures of four variants (I10V, A82F, A82S and A82T) of MDR769 HIV-1 protease. All these mutations failed to further open the flaps and further expand the active site cavity of MDR769 HIV-1 protease, which was characterized by wide open flaps and expanded active site cacity. The conserved flaps and active site cavity, despite the introduction of additional mutations, indicated that the MDR769 HIV-1 protease represented the end stage form of HIV-1 protease. In addition, these crystal structures provided the first structure based evidence for the mutation induced conformational changes in the 80s loops of the HIV-1 protease apo-enzyme, although the flap and active site cavity were not changed dramatically. The alternate conformations of Pro81 (proline switch) in the I10V mutant and the side chain of Phe82 with flipped-out conformation in A82F mutant showed distorted S1/S1' binding pockets that caused loss of contacts and unstable binding of the inhibitors. Similarly, the mutants A82S and A82T showed distortion in the S1/S1' binding pockets due to local changes in the electrostatics caused by the mutation from non-polar to polar residues.;Molecular mechanics studies performed to understand the wide-open nature of the MDR769 HIV-1 protease flaps showed that the MDR protease exhibited a state of conformational rigidity with respect to the flap closure compared to that of the wild type protease. This suggested that the accumulation of mutations changed the structure of the MDR HIV-1 protease and resulted in a cumulative steric hindrance during the flap closure. Our studies showed that modeling a substrate (Gag-Capsid) into the active site cavity of the MDR protease did not result in flap closure. Since flap closure was crucial in protease inhibitor binding, the conformational rigidity of MDR protease might represent a novel mechanism for multidrug-resistance of the MDR protease. In addition, our molecular dynamics simulation revealed the realignment of the substrate peptide in the MDR769 HIV-1 protease, making it less accessible to the Asp25 and Asp125 amino acid residues in the active site. This finding indirectly indicated the reduced catalytic activity of MDR769 HIV-1 protease in substrate cleavage compared to that of the WT HIV-1 protease.;The IC50 values of the FDA approved HIV-1 protease inhibitors and the library of reduced CA/p2 peptide analogs were measured. The results indicated that the reduced peptide analogs bind to the MDR769 HIV-1 protease and WT HIV-1 protease with equal affinity, while the FDA approved inhibitors showed reduced binding affinity to the MDR769 HIV-1 protease compared to that of WT HIV-1 protease. The enzyme measurements demonstrated that lopinavir was the least resistant HIV-1 protease inhibitor and that reduced peptide P1'F was comparable to FDA approved inhibitors from the aspect of the inhibitory activity against HIV-1 protease.;Based on these studies, a library of potential drug candidates against HIV-1 protease was proposed to overcome the drug resistance issue. These drug candidates are being synthesized in the lab and will be evaluated in the future.;Project 2. The wild type extracellular domain of human myelin protein zero (hP0ex) fused with maltose binding protein (MBP) was crystallized to investigate the molecular mechanism of Charcot-Marie-Tooth disease subtype 1B. Based on the wild type structure of the extracellular domain of the human myelin protein zero, five clinically important mutants were further structurally investigated in details. The molecular pathology was proposed for these mutants individually. The relationship between amyloidosis disease and CMT1B is being further explored. From a technical point of view, the WT hP0ex MBP fusion structure was another example of crystallographic studies facilited by the presence of a purification and crystallization protein tag, the maltose binding protein.