A molecular dynamics-based algorithm for evaluating the glycosaminoglycan mimicking potential of synthetic, homogenous, sulfated small molecules

<div><p>Glycosaminoglycans (GAGs) are key natural biopolymers that exhibit a range of biological functions including growth and differentiation. Despite this multiplicity of function, natural GAG sequences have not yielded drugs because of problems of heterogeneity and synthesis. Recently, several homogenous non-saccharide glycosaminoglycan mimetics (NSGMs) have been reported as agents displaying major therapeutic promise. Yet, it remains unclear whether sulfated NSGMs structurally mimic sulfated GAGs. To address this, we developed a three-step molecular dynamics (MD)-based algorithm to compare sulfated NSGMs with GAGs. In the first step of this algorithm, parameters related to the range of conformations sampled by the two highly sulfated molecules as free entities in water were compared. The second step compared identity of binding site geometries and the final step evaluated comparable dynamics and interactions in the protein-bound state. Using a test case of interactions with fibroblast growth factor-related proteins, we show that this three-step algorithm effectively predicts the GAG structure mimicking property of NSGMs. Specifically, we show that two unique dimeric NSGMs mimic hexameric GAG sequences in the protein-bound state. In contrast, closely related monomeric and trimeric NSGMs do not mimic GAG in either the free or bound states. These results correspond well with the functional properties of NSGMs. The results show for the first time that appropriately designed sulfated NSGMs can be good structural mimetics of GAGs and the incorporation of a MD-based strategy at the NSGM library screening stage can identify promising mimetics of targeted GAG sequences.</p></div

Comparison of NSGMs and HS06 bound to FGF2–FGFR1 using EED and MVEE as parameters.

<p>The spatial equivalence of NSGMs to HS06 sequences containing IdoA2S in ²S_O and ¹C₄ conformations was evaluated from the EED (A) and MVEE (B) calculated for each MD frame in FGF2–FGFR1 bound form. The orientation of NSGMs (G2.1-pink, G2.2-blue, stick representation) and HS06 (green, ball & stick representation) in FGF2 binding pocket with interacting residues (single letter code) colored by atom-type.</p

Structures of heparan sulfate (HS) sequences of varying lengths (HS02 to HS08) and non-saccharide glycosaminoglycan mimetics (NSGMs) G1.1, G2.1, G2.2 and G4.1.

<p>G1.1 can be considered as monomeric NSGM; G2.1 and G2.2 are approximately dimeric and G4.1 can be thought of as tetrameric NSGM.</p

Overview of the algorithm developed for assessing GAG structure mimicking potential of NSGMs.

<p>We have developed an algorithm consisting of three sequential legs. The first leg is to identify GAG chain length mimicking potential in free solution using end-to-end distance (EED) and minimum volume enclosing ellipsoid (MVEE) as two parameters. The second leg identifies whether the desired NSGMs bind to the same protein binding site as the target GAG. The third leg attempts assessing the mimicking potential in the protein-bound form with explicit solvent MD simulations. In this comparison is made to GAG sequence using EED, MVEE, inter-molecular hydrogen bonds and the total binding free energy parameters.</p

The consistency of intermolecular hydrogen bonds across the MD simulation (final 10 ns) for FGF2–FGFR1 residues.

<p>The occurrence of inter-molecular hydrogen bonds between FGF2–FGFR1 residues (shown on y-axis) and small molecule ligands (NSGMs and HS06) are shown for each frame of the final 10 ns of the MD run. A) FGF2–FGFR1–G2.1 complex; B) FGF2–FGFR1–G2.2 complex; C) FGF2–FGFR1–HSO6(²S_O) complex; D) FGF2–FGFR1–HS06(¹C₄) complex.</p

The consistency of intermolecular hydrogen bonds across the MD simulation (final 10 ns) for FGF2 residues.

<p>The occurrence of inter-molecular hydrogen bonds between FGF2 residues (shown on y-axis) and small molecule ligands (NSGMs and HS06) are shown for each frame of the final 10 ns of the MD run. A) FGF2–G2.1 complex; B) FGF2–G2.2 complex; C) FGF2–HSO6(²S_O) complex; D) FGF2–HS06(¹C₄) complex.</p

MD-based structural equivalence of NSGMs to HS06 in free solution using minimum volume enclosing ellipsoid (MVEE) as one of the comparable parameters.

<p>A) shows MVEE of each MD frame for all four NSGMs; B) shows MVEE for HS06 sequence containing IdoA2S in ²S_O and ¹C₄ conformations; and C) compiles the results across the MD simulation in a box plot.</p

Comparison of NSGMs and HS06 bound to FGF2 in the acidic binding pocket using EED and MVEE as parameters.

<p>The spatial equivalence of NSGMs to HS06 sequences containing IdoA2S in ²S_O and ¹C₄ conformations was evaluated from the EED (A) and MVEE (B) calculated for each MD frame in FGF2 bound form. The orientation of NSGMs (G2.1-pink, G2.2-blue, stick representation) and HS06 (green, ball & stick representation) in FGF2 binding pocket with interacting residues (single letter code) colored by atom-type.</p

Molecular dynamics (MD)-based structural equivalence of NSGMs to HS06 in free solution using end-to-end distance (EED) as one of the comparable parameters.

<p>A) shows EED of each MD frame for all four NSGMs; B) shows EED for HS06 sequence containing IdoA2S in ²S_O and ¹C₄ conformations; and C) compiles the results across the MD simulation in a box plot.</p

Comparison of NSGMs and HS06 in the protein bound state using overall average inter-molecular hydrogen bond occupancy (A) and total binding free energy (B).

<p>A) shows the stability of bound NSGMs and HS06 based on average residue-level, inter-molecular hydrogen bond occupancy; B) shows the stability of the bound NSGMs and HS06 based on total binding free energy ΔG (in simulated kcal/mol, error bars shows standard deviation). See text for details.</p