The determination of the effect(s) of solute carrier family 22-member 2 (SLC22A2) haplotype variants on drug binding via molecular dynamic simulation systems

Abstract

Single nucleotide polymorphisms detected in the solute carrier member family-22 has been shown to result in a variable response in the treatment of type 2 diabetes mellitus with Metformin. This study predicted a three-dimensional protein structure for the SLC22A2 protein sequence using AlphaFold 2 and modelled five haplotypes within SLC22A2 protein structure observed in the Xhosa population of South Africa. The protein models were used to determine the effect(s) of haplotype variations on the transport function of Metformin and 10 other drugs by the SLC22A2 protein. Molecular dynamic simulation studies, molecular docking and interaction analysis of the five SLC22A2 haplotypes were performed in complex with the ligand 5RE in a POPC lipid bilayer to understand the mechanism of drug binding. Weakest binding free energy was found between 5RE and haplotype 1. Molecular docking studies indicated the top binding ligands as well as Metformin to bind inside the transport channel in all haplotypes increasing the probability of Metformin inhibition during co-administration of drugs. Metformin showed reduced binding affinity and number of interactions compared to the top four binding molecules. Molecular dynamic simulation analysis indicated that haplotypes 1, 3 and 4 were less stable than 2 and 5. The findings suggest haplotypes 4 and 5 having stronger preference for large inhibitor molecule binding in the active site and this could result in haplotypes 4 and 5 demonstrating reduced Metformin clearance via the SLC22A2 transporter during co-administration of drugs. The current study is the first to investigate the potential effect(s) of haplotype variation on the protein structure of SLC22A2 to assess its ability to transport Metformin in an indigenous South African population.

Figure 1

Pairwise protein sequence alignments of SLC22A2 variants: (a) rs316019 (A270S), (b) rs8177516 (R400C) and (c) rs8177517 (K432Q) (variant position indicated by red arrow).

Figure 2

3D structure of SLC22A2 protein embedded in the POPC lipid bilayer membrane: the SLC22A2 protein structure (coloured by secondary structure) in complex with the 5RE inhibitor (shown as spheres in magenta) phosphate lipid head groups shown as orange spheres.

Figure 3

3D structure of SLC22A2 in complex with 5RE generated by AlphaFold 2: the three mutated residues identified in the South African Xhosa population are shown as sticks (coloured deep-teal) and the 5RE inhibitor in magenta. Solid black arrows point to the locations of the mutations identified to characterise the haplotypes existing in the study population.

Figure 4

Statistical analysis of haplotypes over 200 ns simulation. (A) The backbone RMSD deviation of SLC22A2-5RE in the five observed haplotypes [ordinate is RMSD (nm) and the abscissa is time (ns)]. (B) RMSF deviation for protein residues for the five haplotype systems over the last 100 ns. Regions of high flexibility are boxed in red and labelled as R1, 2, 3 and 4 on the figure.

Figure 5

Area per lipid fluctuation for the five haplotype systems over 200 ns.

Figure 6

Ligand overlay for haplotype 1 SLC22A2 protein. (A) Image was generated using Pymol with the first line anti-diabetic drug, Metformin, shown in blue. The four top binding ligands Isavuconazole, Dolutegravir, Glibenclamide, and Gliclazide are shown in deep teal, grey, hot-pink and green respectively. The 5RE inhibitor is indicated in magenta. (B,C) 2D PoseView illustration, showing interacting residues for Metformin and the 5RE inhibitor respectively—dotted black lines denote hydrogen bonds, solid green lines denote hydrophobic interactions and dotted green lines denote π stacking &/or aromatic interactions.

Figure 7

Ligand overlay for haplotype 2 SLC22A2 protein. (A) Image was generated using Pymol with the first line anti-diabetic drug, Metformin, shown in blue. The four top binding ligands Isavuconazole, Dolutegravir, Glibenclamide, and Gliclazide are shown in deep teal, grey, hot-pink and green respectively. The 5RE inhibitor is indicated in magenta. (B,C) 2D PoseView illustration, showing interacting residues for Metformin and the 5RE inhibitor respectively—dotted black lines denote hydrogen bonds, solid green lines denote hydrophobic interactions and dotted green lines denote π stacking &/or aromatic interactions.

Figure 8

Ligand overlay for haplotype 3 SLC22A2 protein. (A) Image was generated using Pymol with the first line anti-diabetic drug, Metformin, shown in blue. The four top binding ligands Dolutegravir, Glibenclamide, Vandetanib and Isavuconazole are shown in grey, hot-pink, orange and deep teal respectively. The 5RE inhibitor is indicated in magenta. (B,C) 2D PoseView illustration, showing interacting residues for Metformin and the 5RE inhibitor respectively—dotted black lines denote hydrogen bonds, solid green lines denote hydrophobic interactions and dotted green lines denote π stacking &/or aromatic interactions.

Figure 9

Ligand overlay for haplotype 4 SLC22A2 protein. (A) Image was generated using Pymol with the first line anti-diabetic drug, Metformin, shown in blue. The four top binding ligands Isavuconazole, Dolutegravir, Glibenclamide, and Vandetanib are shown in deep teal, grey, hot-pink, and orange respectively. The 5RE inhibitor is indicated in magenta. (B,C) 2D PoseView illustration, showing interacting residues for Metformin and the 5RE inhibitor respectively—dotted black lines denote hydrogen bonds, solid green lines denote hydrophobic interactions and dotted green lines denote π stacking &/or aromatic interactions.

Figure 10

Ligand overlay for haplotype 5 SLC22A2 protein. (A) Image was generated using Pymol with the first line anti-diabetic drug, Metformin, shown in blue. The four top binding ligands Dolutegravir, Glibenclamide, Isavuconazole and Ranolazine are shown in grey, hot-pink, deep teal, and yellow respectively. The 5RE inhibitor is indicated in magenta. (B,C) 2D PoseView illustration, showing interacting residues for Metformin and the 5RE inhibitor respectively—dotted black lines denote hydrogen bonds, solid green lines denote hydrophobic interactions and dotted green lines denote π stacking &/or aromatic interactions.