Heterocyclic Substitutions Greatly Improve Affinity and Stability of Folic Acid towards FRα. an In Silico Insight

Abstract

Folate receptor alpha (FRα) is known as a biological marker for many cancers due to its overexpression in cancerous epithelial tissue. The folic acid (FA) binding affinity to the FRα active site provides a basis for designing more specific targets for FRα. Heterocyclic rings have been shown to interact with many receptors and are important to the metabolism and biological processes within the body. Nineteen FA analogs with substitution with various heterocyclic rings were designed to have higher affinity toward FRα. Molecular docking was used to study the binding affinity of designed analogs compared to FA, methotrexate (MTX), and pemetrexed (PTX). Out of 19 FA analogs, analogs with a tetrazole ring (FOL03) and benzothiophene ring (FOL08) showed the most negative binding energy and were able to interact with ASP81 and SER174 through hydrogen bonds and hydrophobic interactions with amino acids of the active site. Hence, 100 ns molecular dynamics (MD) simulations were carried out for FOL03, FOL08 compared to FA, MTX, and PTX. The root mean square deviation (RMSD) and root mean square fluctuation (RMSF) of FOL03 and FOL08 showed an apparent convergence similar to that of FA, and both of them entered the binding pocket (active site) from the pteridine part, while the glutamic part was stuck at the FRα pocket entrance during the MD simulations. Molecular mechanics Poisson-Boltzmann surface accessible (MM-PBSA) and H-bond analysis revealed that FOL03 and FOL08 created more negative free binding and electrostatic energy compared to FA and PTX, and both formed stronger H-bond interactions with ASP81 than FA with excellent H-bond profiles that led them to become bound tightly in the pocket. In addition, pocket volume calculations showed that the volumes of active site for FOL03 and FOL08 inside the FRα pocket were smaller than the FA–FRα system, indicating strong interactions between the protein active site residues with these new FA analogs compared to FA during the MD simulations.

Conjugated β-Cyclodextrin Enhances the Affinity of Folic Acid towards FRα: Molecular Dynamics Study

“…ASP81 form two strong H-bonds with FA-βCD; one with a pteridine ring at N5 at a distance of 2.01Å and the other with N7 (1.84 Å) (Figure 7b). This observation is consistent with previous studies that showed that ASP81 interacted with the pteridine ring and is considered as a key contributor to the high folate affinity [26,27,30]. In FA-FRα (Figure 7a), two H-bonds were also observed between the pteridine ring with ASP81 (1.93 Å and 2.15 Å).…”

“…It is noted that the βCD did not enter the binding site, and only formed an interaction with the amino acid at the surface of FRα such as LYS19. Interestingly, conjugation to βCD resulted in the FA to elude its H-bonds interaction between its glutamic acid moiety with LYS136, GLY137, TRP138, and TRP140, a behaviour observed in our previous study [27]. In addition, Figure 3b shows that the residue TYR85 lost its π-π stacking interaction with the pteridine ring from the FA structure of the FA-βCD.…”

“…H-bonds are essential for protein folding and protein-ligand interactions [32,33]. It is well known that ASP81 is the key residue in the FRα binding site, playing a critical role in increasing ligand binding affinity and anchoring the FA pteridine region deep within the site [27,30,34]. Specifically, the crystal structure used in this study demonstrated that ASP81 formed strong H-bonds with N1 and N2 atoms in the pteridine ring of FA.…”

“…Some regions of the protein are omitted to facilitate visualization. As with the FA-FRα complex (Figures 2 and 3a), the folate moiety of the FRα-FA-βCD complex was also stabilized by polar amino acids such as ASP81, TYR60, ARG103, HIS135, and nonpolar residues such as TRP102 and TRP171 [27]. The main interactions of these residues were with pteroate moiety (pteridine ring with PABA (p-amino benzoic acid)) (Figure 3b).…”

“…This observation is not surprising as the apo-FRα was built from the FA-bound complex [26]. Removal of FA from the holo form (folate complex) might result in the transition from one biological trafficking state to another in the system, resulting in higher RMSF especially in regions where the ligands are bound [26,27], which in this case is the folate-binding pocket [26]. The FRα-FA-βCD system reached a stable and equilibrated dynamical state relatively quick, i.e., in less than 10 ns, and continued to fluctuate (protein backbone) within a stable conformational ensemble with an average RMSD value of 1.9 Å throughout the 100 ns simulations.…”