Plants produce pigments called flavonoids that are synthesized (de novo) primarily from the amino acid phenylalanine. Many natural products such as herb preparations for medicinal use contain flavonoids and so these diverse structural compounds have become potential lead compounds for a variety of illnesses ranging from specific cancers to gout. Recently, Biotechnology has been utilized through bioorganic engineering to manufacture flavonoid and modified flavonoids for medicinal applications. A growing number of protein-flavonoid complexes have been crystallized and their structures solved in the last decade. A summary of the protein-flavonoid complexes with medicinal relevance is presented herein. A detailed analysis of selected protein-flavonoid complexes is provided. The goal is to provide insights for modifications to known flavonoids that could be biomanufactured to generate more specific and efficient binding of flavonoids to protein targets with medicinal relevance.

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Cyclooxygenase‐2 (COX‐2) is an enzyme that plays a critical role in the formation of biological mediators called prostanoids. These prostanoids consist of prostaglandins that are a class of signaling molecules, which are responsible for the inflammation and pain processes that occur as an anticipated immune response in our body. COX‐2 inhibitors are a type of anti‐inflammatory drugs (NSAIDs) that directly select, target and block the COX‐2 enzyme. Blocking this enzyme prevents the production of those chemical stimuli that cause inflammation (pain and swelling), arthritis and fever. A few common examples of COX‐2 inhibitors drugs are acetylsalicylic acid (common name: aspirin), celecoxib (brand name: Celebrex) and rofecoxib (brand name: Vioxx). However, since December 2011, Celebrex is the only COX‐2 inhibitor currently used in the United States because data from clinical trials revealed that other medicines such as Rofecoxib (Vioxx) and valdecoxib (Bextra) increased the risk of heart attacks and strokes with long‐term use. Aspirin as a safe alternative is an anti‐inflammatory drug that inhibits the COX‐2 enzyme. COX‐2 triggers inflammation as an innate physiological response. Thus, in blocking COX‐2, aspirin performs as a potent analgesic, anti‐inflammatory and antipyretic. In addition, aspirin blocks COX‐1 the key enzymes in thromboxane TXA2 (a blood‐clotting protein). This is why aspirin is universal over the counter blood thinner or antiplatelet drug. As an irreversible inhibitor, aspirin inhibits the production of prostaglandins and thromboxanes due to its irreversible inactivation of the (COX) enzyme. This action is a result of the ability of aspirin to act on COX‐2 as an acetylating agent with its acetyl group covalently bonded to a serine residue in the active site of the COX enzyme. The goal of this computational study is to fully characterize the aspirin binding mechanism and binding mode in the COX‐2 protein. PDB code 5F1A crystal structure of COX‐2 bound to salicylic acid was utilized since at present, there is no structure solved of the aspirin‐COX‐2 complex. Results of docking with Swiss Dock program indicate that there are two possible binding sites for aspirin in COX‐2. One predicted binding site overlays with Salicylic acid. The other predicted binding site is approximately 8–10 angstroms away within a helix‐turn‐helix structural domain which may be a novel binding site for aspirin. Details of the predicted binding modes, involving amino acids specifically interacting with COX‐2 are represented. More specifically, amino acids of COX‐2 have been identified forming H‐bonds ≤ 3.75 angstroms to aspirin oxygens. Amino acids with hydrophobic interactions < 5.00 angstroms distance from docked aspirin have also been identified. Preliminary results are discussed, including site‐directed mutagenesis studies to confirm the identity of the binding site.

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Human Fructose 1,6 Bisphosphatase (FBPase), an enzyme which serves as the control point in the gluconeogenesis pathway, was selected as a protein target for this study. FBPase is a putative target for drug development to combat Type II Diabetes. The FBPase protein structure has previously been solved (Zhang et al., 1995), and coordinates were readily available (PDB code IFTA) and downloaded for docking studies from the Rutgers Consortium of Structural Biology (RCSB) Protein Data Bank (PDB). The potential inhibitory molecules (IBS Natural Products Catalog) were downloaded from the ZINC Database (Irwin et al., 2012) in pdbqt file format. AutoDock Vina was selected as the ideal docking program for FBPase as determined by comparing co‐crystallized ligands to docked ligand positions. Selected compounds were chosen and docked into the FBPase active site and allosteric binding sites. Following the identification of promising theoretical binding constants for each molecule, molecular dynamics studies were performed for the FBPase protein target in the absence and presence of these promising molecules. Studies were caeried out using a NAMD/VMD (Nanoscale Molecular Dynamics with Visual Molecular Dynamics) system. NAMD implemented with CHARMM foce field is known to be highly efficient in simulating large systems for molecular motions. Dissociation constants (Ki) for the protein‐ligand complexes were calculated through NAMD runs, and respective conformational changes in the FBPase binding pockets were observed. Binding pockets were evaluated based on two factors: 1) pocket shape, and 2) pocket volume. Laboratory‐based site‐directed mutagenesis results revealed various levels of activation/inhibition of the enzyme as a result of mutations, an outcome with genetic implications for disease. Mutant enzymes resisted attempts at crystallization so molecular dynamics (MD) studies were performed on models of mutants. Focused on the active site of allosteric binding sites, further analysis was performed with key residues (virtually mutated) to initiate activation or inhibition of the enzyme (retention in T‐state or transition to R‐state) using NAMD. Resulting models of FBPase after MD runs shed light on possible interfacial structural changes produced in response to these mutations. Structural alterations in the models were observed in the active site, AMP (adenosine monophosphate) allosteric binding site, dimer interface allosteric binding site, and the interface between the active site and the AMP binding site. The signal from one part of the molecule is trasmittedto another part of the molecule through a known hydrogen‐bonding network (a sequential allosteric transition) vconnecting the active sites to the AMP allosteric binding sites. Novel hydrophobic networks were identified to connect the active sites to the allosteric binding sites using the NAMD protocol.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.