The parasite Plasmodium falciparum is responsible for the major world scourge malaria, a disease that affects 3.3 billion people worldwide. The development of new drugs is critical because of the diminished effectiveness of current antimalarial agents mainly due to parasitic resistance, side effects and cost. Molecular docking was used to explore structural motifs responsible for the interactions between triose phosphate isomerase (TPI), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and aldolase (ALD) from human and Plasmodium cells with 8 novel sufonylamide derivatives. All the ligands modeled, interact with all three enzymes in the micromolar range. The top ligand (sulfaE) shows a 70-fold increase in selective binding to pfTPI compared to hTPI (dissociation constant-KI of 7.83 μM and 0.177 μM for hTPI and pfTPI respectively), on par with antimalarial drug chloroquine.ALD and GAPDH form complexes with similar binding sites, comprising amino acids of similar chemical properties and polarities. Human TPI and pfTPI bind sulfonamide derivatives using two distinct binding sites and residues. Key residues at the dimer interface of pfTPI (VAL44, SER45, TYR48, GLN64, ASN65, VAL78) form a tight pocket with favorable polar contacts. The affinity with TPI is the most specific, stable, and selective suggesting pfTPI is a candidate for development of antimalarial drugs.
Global mapping data and the World Health Organization indicate that malaria continues to be a major challenge with about 3.3 billion people at risk of exposure (Kehr and Guerra) [
The main causative agent of malaria, the Plasmodium parasite, places a more than 100 fold demand on glucose requirements for parasitized red bloods relative to uninfected host cells [
It is therefore essential to determine whether such differences and other unique structural motifs can be used to develop inhibitors that selectively target Plasmodium parasite enzymes, without harming the human host cells. In this study we have used molecular modeling tools to determine the binding affinity and binding modes of three glycolytic enzymes; triosephosphate isomerase (TPI), aldolase (ALD), and glyceraldehyde phosphate dehydrogenase (GAPDH) with 8 novel sulfonamide ligand derivatives. This study will use molecular modeling to identify selective differences in molecular recognition patches observed in the interactions of three glycolytic enzymes from humans and the Plasmodium parasites with sulfonamide derivatives. The specific questions we address include: 1) Which glycolytic enzymes show a strong and selective affinity with the novel ligands? 2) What are structural differences between the binding domains and residues responsible for interactions? 3) How do the binding affinities of new analogues compare with common antimalarial agents? 4) Which sulfonamide derivatives boost selectivity, based on the binding affinities and dissociation constants?
The glycolytic pathway is the catabolic process involving a series of enzymes that converts one molecule of glucose into two molecules of pyruvate with the release of ATP for required by cells (Scheme 1).
This study will focus on three glycolytic enzymes that catalyse steps 4, 5 and 6 of the glycolytic pathway (Scheme 1). Fructose-1,6-bisphosphate aldolase (ALD) is a homotetrameric enzyme that catalyses the aldol cleavage of fructose-1,6-bisphosphate (F1,
Scheme 1. The glycolytic pathway; metabolic breaking down of glucose to lactate.
6BP) to two triose phosphates, glyceraldehyde-3-phosphate or glyceraldehyde and dihydroxyacetone phosphate (Scheme 1) [
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays an important role in glycolysis and gluconeogenesis by reversibly catalysing the oxidation and phosphorylation of glyceraldehydes-3-phosphate (G3P) to the first energy-rich intermediate 1,3- bisphosphoglycerate (1,3BPG) in glycolysis (Scheme 1) [
TPI is a key dimeric enzyme that speeds up the final investment phase of glycolysis (Scheme 1). The X-ray crystal structures of hTPI and pfTPI have been determined to atomic resolution and were also downloaded from the RCSB Protein Data Bank, with accession codes 4POC (Roland, 2015) and 2VFI, respectively (Gayathri, 2009). HTPI and PFTPI share a sequence identity of 42%. Despite the 58% difference in sequence
identity, the three dimensional structure of both molecules have similar structural folds. For example, a root mean square deviation (RMSD) of 0.825 Å is obtained when both enzymes are structurally aligned (
Aniline and sulfonamide based drugs like sulfanilamide have historically been used to treat bacterial and yeast infections. This is because they target enzymatic reactions in the foliate metabolic pathway producing cellular cofactors important for amino acids and DNA synthesis. For example, sulfadoxine has been shown to interfere with the foliate metabolism, by inhibiting the enzyme dihydropteroate synthase [
The three-dimensional structures of the enzymes used were refined prior to docking using Pymol and Chimera [
where Ki (dissociation constant, and i indicates it is also an inhibition constant)
In these simulations, 2 × 106 energy evaluations between the sulfonamide derivatives were used to identify binding sites and amino acid residues involved in formation of enzyme ligand complexes. All the sulfonamide ligands screened successfully docked to the three glycolytic enzymes with varying binding affinities. We also wanted to determine whether any of these ligands showed a selective affinity tothe human glycolytic enzymes (ALD, GAPDH and TPI) compared to Plasmodium enzymes. The binding energy difference between the complex and free enzyme and ligands was used to determine strength of interaction (
The relative binding energy data suggest that the sulfonamide derivatives seem to interact more strongly with human aldolase compared to Plasmodium aldolase (
The dissociation constants or inhibition constants (Ki) usually indicate the concentration at which binding domains of the enzyme is half filled. In general, a small dissociation constant is an indication of a tightly bound complex. Larger bars signify weak binding, while bars of similar sizes indicate no preference in binding one enzyme over the other. The dissociation constants again show that Plasmodium ALD does not form strong complexes with the sulfonamide derivatives. For example, SulfaE binds human ALD with a Ki of 0.197 uM and 1.64 uM for pfALD (
All the glycolytic enzymes modeled are docked with the sulfonamide derivatives. Aldolase and GAPDH however, do not seem to form complexes that discriminate between human and Plasmodium enzymes. The interaction affinities and dissociations constants do suggest that there is potential of selective binding with TPI enzymes. Due to structural similarity of many glycolytic enzymes, the significance of this observation can be explained by the nature of complexes formed and the residues responsible for interactions. All the sulfonamide ligands screened interact with each enzyme using similar binding sides and residues (
In general, there are two main sites that these ligands use to interact with the glycolytic enzymes. These include an allosteric binding side occurring at the dimer interface or between monomers (Site D), and a site proximal to the active site or cofactor-binding site of the enzyme (site A). Amongst the 100 complexes analyzed for SulfaE docking calculations, site D seems to be the preferred site of binding in hALD, pfALD, hGAPDH, and pfGAPDH (
common in Aldolase, however, the pfALD also shows about 38 % of docked complexes in the site B, with about 5% docked complexes for hALD (
There is a significant difference in distribution of docked complexes amongst the binding sites D and A in the interactions between all the sulfonamide drug derivatives with hTPI or pfTPI. These differences can explain the selectivity observed in the binding energies and dissociation constants. In docking simulations involving pfTPI, more of the complexes are formed using site D, while site A is preferred in hTPI docking solutions (
The major goal of this study was to computationally determine whether the novel fluorinated sulfonamide ligands selectively interact and hence inhibit glycolytic enzymes of Plasmodium cells as opposed to humans. The broad similarity in structure of glycolytic enzymes amongst different species, has generally limited their use as molecular receptors or targets in the development of antimalarial therapies [
In this study we provide data that shows that sulfonamide ligands are suitable candidates to selectively target glycolytic enzymes. The data involving rigid enzyme targets reveal that all the sulfonamide ligands screened, bind the three glycolytic enzymes (ALD, GAPDH, TPI) with high affinity. The dissociation constants observed for these interactions also fall in the micromolar range, comparable to many current drugs in the market (ref). While these affinities are strong, the interactions with ALD and GAPDH are not selective (
ALD and GAPDH from the both species interact with the ligands using similar binding sites, and the residues all have similar chemical properties (size and polarity) (
The docking binding affinities of the new sulfonamide analogues also compare with affinities of current antimalarial drugs like chloroquine (
Eliminating the debilitating effects of the disease malaria remains a major concern of the WHO and many countries in the world. Many antimalarial agents (e.g. Chloroquine) have been used for a number of years to fight malaria without any clear-cut mode of action. The reemergence of parasitic resistance has continued to spur research
for more effective analogues. In this study, we have shown that eight novel sulfonamide ligands screened can successfully dock to the three glycolytic enzymes ALD, GAPDH and TPI from humans and Plasmodium falciparum. The docking results suggest that glycolytic enzymes generally used two binding sites; site D (the dimer or quaternary structure interface) and site A (site proximal to active site or cofactor binding site). In interactions involving ALD and GAPDH similar binding sites and amino acid residues are responsible for complex formation in the enzymes from both species. The molecular docking results suggest that triose phosphate isomerase (TPI) is a good target for the development of new antimalarial agents. Specifically, we have determined that the current sulfonamide ligands interact with pfTPI and hTPI using different very binding domains. This dimer interface binding site is different from the TPI active site, and may suggest the possibility of noncompetitive or uncompetitive inhibition. SulfaE, SulfaH and SulfaC also stand out as key molecules can be fine-tuned to take advantage of differences in binding domains and residues between pfTPI and HTPI. These sulfonamide derivatives showed an enhancement in dissociation constants on par or greater than some known antimalarial agents. The micromolar range affinity of sulfonamide derivatives is comparable to the affinity of many current antimalarial agents. The sulfonamide derivatives can thus serve as pharmacophores for the development of novel antimalarial drugs. The rigid nature docking is one limitation of this study because dynamic effects of binding during complex formation are not accounted for. Further investigations using molecular dynamics simulations are underway to dynamically test the stability and hydrogen bonding network responsible for complexes. Specifically, it will be important to understand how the flexibility and dynamic motions of both ligand and enzyme will affect the interactions using molecular dynamics simulations. The specificity of binding and inhibitory effects of these ligands also have to be tested using kinetic experiments associated with the pfTPI dimer interface.
This work was supported with funds from the GGC STEM Minigrant, and VPASA Seed Fund Program at Georgia Gwinnett College.
Forlemu, N., Watkins, P. and Sloop, J. (2017) Molecular Docking of Selective Binding Affinity of Sulfonamide Derivatives as Potential Antimalarial Agents Targeting the Glycolytic Enzymes: GAPDH, Aldolase and TPI. Open Journal of Biophysics, 7, 41-57. http://dx.doi.org/10.4236/ojbiphy.2017.71004