The generally accepted catalytic mechanism for ALDOA is shown in Physique S1. while CF2 substitution slightly increases the binding free energy, which matches the experimental measurement. In addition, we found that NDB with two CF2 insertions, the strongest binder, is entropically driven, while others including NDA with one CF2 insertion are all enthalpically driven. This work provides insights into the mechanisms underlying protein-phosphate binding and enhances the capability RECA of applying computational and theoretical frameworks to model, predict and design diagnostic strategies targeting malignancy. Graphical Abstract Introduction For most cells, glycolysis is critical for generating energy and supplying metabolic intermediates for cellular biomass. One of the hallmarks of malignancy is the altered metabolism preferential dependence on glycolysis in an oxygen-independent manner instead of oxidative phosphorylation, known as the Warburg effect.1 Recently, a novel feed-forward mechanism for hypoxic malignancy has been identified. While HIF-1 upregulates transcription of glycolytic enzymes, the glycolysis under inadequate oxygen supply, in turn, increases HIF1a transcriptional activity and stimulates tumor growth.2 (Physique 1). Tumor glycolysis has been actively analyzed and serves as a potential target for malignancy therapy.3C4 Open in a separate window Determine 1. Columbianadin Glycolysis functions as a feed-forward mechanism for HIF-1 action. A leading candidate for this target is the fructose-bisphosphate Columbianadin aldolase A (ALDOA), a central enzyme in glycolysis.5 ALDOA is responsible for transforming fructose-1,6-biphosphate (FDP) into glyceraldehyde 3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP). The generally accepted catalytic mechanism for ALDOA is usually shown in Physique S1. The reaction proceeds with the formation of a Schiff base intermediate between LYS229 of the active site and the carbonyl group of the substrate FDP.6C7 The inhibition of ALDOA has been shown to block the glycolysis, decrease HIF-1 activity and break the feed-forward loop mechanism in cells. Thus aldolase A has potential customers for controlling malignancy proliferation.2, 8C10 Aldolase inhibitors have been designed to mimic the substrate of FDP by probing the nature of the active site.6, 11 General principles of drug design involve keeping the strong electrostatic interactions with residues in the active site while maintaining hydrophobic interactions Columbianadin in the linkage. Aldolase A has been co-crystallized with naphthalene-2,6-diyl bisphosphate (ND1), an active site substrate-mimetic. Physique 2 shows the 2D structure of the ND1 and highlights the key residues in the binding pocket. Figure S2 shows the same graphic enlarged. H-bonds have been found between the two negatively charged phosphate groups and the polar and positive charged residues including SER35, SER38, SER271, LYS229 and LYS107 as well as the neutral GLY272 and GLY302. These interactions include binding to both residues backbone O and N, and sidechain ?OH and ?NH2. Besides, hydrophobic interactions are marked in red including LEU270, ALA31, ASP33, and TRY301. Note that negatively charged Asp33 interacts with the naphthalene ring, not the phosphate groups. Although ND1 is usually a potent inhibitor, with two polar phosphate groups, it is easy to be hydrolyzed and hard to deliver in vivo. Open in a separate window Physique 2. 2D plot of the binding pocket of ND1 in crystal structure2 generated using LigPlot+. Left: Key residues include LYS107, SER35, SER38, SER271, GLY272, GLY302, LEU270, ALA31, ASP33, and Try301. Right: Intermolecular interactions around negatively charged phosphate groups are marked in green with distances while those hydrophobic ones involved aromatic systems are marked in reddish. These interactions include binding to both residues backbone O and N, and sidechains ?OH and ?NH2. Molecular dynamics (MD) simulations are a powerful tool for understanding the driving forces underlying molecular acknowledgement, accelerating drug discovery, and guiding molecular design.12C18 Classical force fields such as AMBER19, CHARMM20, OPLS-AA21, or GROMOS22 are Columbianadin computationally efficient and sufficiently accurate for many applications.14, 23C25 However, for highly charged species like phosphates-containing ligands, the actual charge distributions of atoms and their changes in response to the Columbianadin environments electric field is complicated and challenging to model and simulate.26C32 Recently, polarizable force fields have.