Individuals with poor control of HIV replication while on cART represent a highrisk subgroup

lly similar binding orientation within CYP2A6. To further explore the molecular factors that determine the binding affinity and binding energy of 8-MOP with CYP2A6 mutants, we conducted detailed molecular docking of the compound to the CYP2A6 active site using the CDOCKER module in DS 3.5. Fig. 4 shows the structural overview of CYP2A6 we constructed based on the published wild type crystal structure with locations of the six mutations investigated shown. In order to validate docking reliability of our constructed model, 8-MOP was re-docked to the binding site of CYP2A6 and the docked conformation PubMed ID: corresponding to the lowest energy was chosen as the most probable binding conformation. As suggested by Yano et. al., CYP2A6 has hydrophobic active site with one Inhibition of CYP2A6 Alleles by GW 5074 cost 8-Methoxypsoralen and Fig. 8 illustrate the overall change in the geometry of the protein tertiary structures and the active sites whereas 30, 28 and 28 sites could be searched for CYP2A615, CYP2A616, CYP2A621 and CYP2A622 respectively. The mutations have caused changes in the active site volume. CYP2A61 showed a volume of 89.56 A3 but the volume was increased in three mutants. This enlarged volume has resulted in 8-MOP adopting different binding orientation as well as losing the H bonding with Asn297 as discussed above. Unlike the other three mutants, the volume for CYP2A616 was determined to be 88.25 A3 which was similar to the wild type. Interestingly, this allele has IC50 and CDIE values closest to the wild type, indicating that, similar to CYP2A61, its active site has assumed a smaller and 5 Inhibition of CYP2A6 Alleles by 8-Methoxypsoralen more compact topology which has allowed a tighter packing interaction and binding with 8-MOP. As discussed above, the R203S mutation in this allele could have induced geometric changes other than expanding cavity volume that have caused the change in 8-MOP orientation within the binding cavity and the loss of H bond leading to reduced affinity observed. When all these in silico data are considered together, the docking data presented are consistent with the in vitro data and support the notion that mutations have caused detrimental effect on 8-MOP binding to CYP2A6. Variant CYP2A615 showed the largest IC50 and significant larger Km implying the detrimental effect of K194E substitution in both 8-MOP and coumarin binding. This is supported by our H bond formation CYP protein CYP2A61 CYP2A615 CYP2A616 CYP2A621 CYP2A622 CDOCKER Interaction Energy 229.17 216.00 220.49 28.82 219.60 Bond number 1 0 0 0 0 Residue involved in bonding Asn297 – Distance between 8-MOP carbonyl oxygen and Asn297 1.869 4.625 6.157 5.328 5.717 doi:10.1371/journal.pone.0086230.t002 6 Inhibition of CYP2A6 Alleles by 8-Methoxypsoralen docking data that showed enlarged active site volume and loss of H bond. Although this residue is located adjacently to helix F which partially embraces SRS-2, this amino acid substitution could possibly disrupt the access channel and binding affinity of ligands and thus affecting the access and binding of 8-MOP and coumarin at the putative active site of CYP2A6. Minimum effects on ligand binding observed in CYP2A616 indicate the lesser detrimental effect of R203S in this variant as compared to mutations in the other three alleles. This is also supported by our docking data that showed minimum change in active site volume. From the numerous molecular modeling and site-directed mutagenesis studies on CYP2A6 thus far

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