Peptidomimetic and Non- Peptidomimetic Derivatives as Possible SARS-CoV-2 Main Protease (Mpro) Inhibitors

To design novel inhibitors of the SARS-CoV-2 main protease (Mpro), we investigated the binding mode of the recently reported α-ketoamide inhibitors of this enzyme. Following, we utilized in-silico screening to identify 168 peptidomimetic and non-peptidomimetic compounds that are high probability Mpro binding candidates. The compounds were synthesized in 5 to 10 mg for initial screening for their potential inhibition of Mpro using Fluorescence Resonance Energy Transfer (FRET) assay. The study was conducted using the main protease, MBP-tagged (SARS-CoV-2) Assay Kit (BPS Bioscience, #79955-2), and the fluorescence due to enzymatic cleavage of substrate measured using BMG LABTECH CLARIOstar™, a fluorescent microplate reader, with an excited/emission wavelength of 360 nm/460 nm, respectively. The FRET assay showed 29 compounds to exhibit lower fluorescence compared to the positive control, indicating inhibitory activity, with three of the compounds exhibiting over 50% enzymatic inhibition. The assay average scores were plotted as dose inhibition curves using variable parameter nonlinear regression to calculate the IC50 values. To design more potent inhibitors, an in-silico molecular docking simulation using the SARS-CoV-2 Mpro crystal structure was conducted to investigate on a molecular level the key binding residues at the active site, as well as the possible binding modes and affinity of the lead inhibitors. Additionally, an in-silico study of the compounds\u27 molecular properties and physicochemical profiles was performed to predict their pharmacokinetic properties and assess their suitability as potential orally active drug candidates.https://scholarscompass.vcu.edu/gradposters/1139/thumbnail.jp

Characterization of the Escherichia coli pyridoxal 5'-phosphate homeostasis protein (YggS): Role of lysine residues in PLP binding and protein stability

The pyridoxal 5'-phosphate (PLP) homeostasis protein (PLPHP) is a ubiquitous member of the COG0325 family with apparently no catalytic activity. Although the actual cellular role of this protein is unknown, it has been observed that mutations of the PLPHP encoding gene affect the activity of PLP-dependent enzymes, B6 vitamers and amino acid levels. Here we report a detailed characterization of the Escherichia coli ortholog of PLPHP (YggS) with respect to its PLP binding and transfer properties, stability, and structure. YggS binds PLP very tightly and is able to slowly transfer it to a model PLP-dependent enzyme, serine hydroxymethyltransferase. PLP binding to YggS elicits a conformational/flexibility change in the protein structure that is detectable in solution but not in crystals. We serendipitously discovered that the K36A variant of YggS, affecting the lysine residue that binds PLP at the active site, is able to bind PLP covalently. This observation led us to recognize that a number of lysine residues, located at the entrance of the active site, can replace Lys36 in its PLP binding role. These lysines form a cluster of charged residues that affect protein stability and conformation, playing an important role in PLP binding and possibly in YggS function

Protein Structure and Interaction: The Role of Aromatic Residues in Protein Structure and Interactions Between Pyridoxine 5\u27-Phosphate Oxidase/Dopa Decarboxylase

Naturally developed proteins are capable of carrying out a wide variety of molecular functions due to their highly precise three-dimensional structures, which are determined by their genetically encoded sequences of amino acids. A thorough knowledge of protein structures and interactions at the atomic level will enable researchers to get a deep foundational understanding of the molecular interactions and enzymatic processes required for cells, resulting in more effective therapeutic interventions. This dissertation intends to use structural knowledge from solved protein structures for two distinct objectives. In the first project, we conducted a bioinformatics structural analysis of experimental protein structures using our novel paradigm 3D Interaction Homology . The three-dimensional structure of biological macromolecules, particularly proteins, provides us with a better understanding of protein interactions and functions, enabling us to establish hypotheses about how to modulate, regulate, or modify their functions. Therefore, the Kellogg lab proposed a new paradigm for the building and refinement of protein structure models using 3D hydropathic maps. To accomplish this goal, we have been characterizing the hydropathic interaction residue environments by compiling a database of residue type- and backbone angle-dependent 3D maps. In this work, 3D hydropathic interaction maps feature of the HINT (Hydropathic INTeraction) program enabled calculation and visualization of the 3D hydropathic environments of the three aromatic amino acid residues. We have shown that these 3D maps are information rich descriptors of preferred conformations, interaction types and energetics, and solvent accessibility. We calculated and analyzed sidechain-to-environment 3D maps for over 70,000 phenylalanine, tyrosine, and tryptophan residues. Moreover, significant and occurrence of some special non-covalent interactions (π-π and π-cation) were calculated and analyzed. This recognition of even these subtle interactions in the 3D hydropathic environment maps is key support for our interaction homology paradigm of protein structure elucidation and possibly prediction. In the second project, we aimed to investigate the physical interaction between a vitamin B6-salvage enzyme, pyridoxine-5\u27 phosphate oxidase (PNPO), and a vitamin B6-dependent enzyme, dopa decarboxylase (DDC), employing different approaches, including molecular modeling, biophysical, enzyme kinetics, and site-directed mutagenesis studies. PLP, the active vitamer of B6, serves as a cofactor for approximately 180 B6-dependent (PLP-dependent) enzymes and play crucial roles on many of cellular functions, e.g., heme, amino acid, neurotransmitter, DNA/RNA biosynthesis. Vitamin B6 deficiency is suspected to contribute to several pathologies, e.g., seizures, autism, schizophrenia, epilepsy, and Alzheimer’s disease. High levels of vitamin B6 are also linked to neurotoxic effects due in part to potential toxicity by free PLP in the cell. Therefore, the cellular content of free PLP is kept very low. Understanding the role of this vitamin in these pathologies requires knowledge on its metabolism and regulation, and subsequent transfer to dozens of apo-B6 enzymes. We hypothesize that the transfer of PLP from the donor PNPO salvage enzyme to the acceptor apo-B6 enzyme DDC requires that both enzymes form a complex to offer an efficient and protected means of delivery of the highly reactive PLP. Knowledge of the 3D protein structures of PNPO and DDC (in both active state or holo-form and inactive state or apo-form) enabled us to undertake protein-protein docking and molecular dynamics simulations studies to predict the most likely near-native structure of the complex. The physical binding between PNPO and DDC were experimentally characterized using fluorescence polarization (FP), surface plasmon resonance (SPR), and isothermal calorimetry (ITC) techniques. The dissociation constants (KD) was observed to be in low micromolar range. Expectedly, interactions between PNPO and apoDDC was found to be about 3-fold stronger than interactions between PNPO and holoDDC, with KD values of 0.92 ± 0.07 μM and 2.59 ± 0.11 μM, respectively. PLP transfer studies were carried out to demonstrate that PLP is capable of transferring from PNPO and activating the apoDDC. Site mutation investigations of critical residues identified by computational/modeling studies to be important in protein-protein interaction were carried out but showed negligible effect on the complex formation

Elucidating the Interaction between Pyridoxine 5′-Phosphate Oxidase and Dopa Decarboxylase: Activation of B6-Dependent Enzyme

Pyridoxal 5′-phosphate (PLP), the active form of vitamin B6, serves as a cofactor for scores of B6-dependent (PLP-dependent) enzymes involved in many cellular processes. One such B6 enzyme is dopa decarboxylase (DDC), which is required for the biosynthesis of key neurotransmitters, e.g., dopamine and serotonin. PLP-dependent enzymes are biosynthesized as apo-B6 enzymes and then converted to the catalytically active holo-B6 enzymes by Schiff base formation between the aldehyde of PLP and an active site lysine of the protein. In eukaryotes, PLP is made available to the B6 enzymes through the activity of the B6-salvage enzymes, pyridoxine 5′-phosphate oxidase (PNPO) and pyridoxal kinase (PLK). To minimize toxicity, the cell keeps the content of free PLP (unbound) very low through dephosphorylation and PLP feedback inhibition of PNPO and PLK. This has led to a proposed mechanism of complex formation between the B6-salvage enzymes and apo-B6 enzymes prior to the transfer of PLP, although such complexes are yet to be characterized at the atomic level, presumably due to their transient nature. A computational study, for the first time, was used to predict a likely PNPO and DDC complex, which suggested contact between the allosteric PLP tight-binding site on PNPO and the active site of DDC. Using isothermal calorimetry and/or surface plasmon resonance, we also show that PNPO binds both apoDDC and holoDDC with dissociation constants of 0.93 ± 0.07 μM and 2.59 ± 0.11 μM, respectively. Finally, in the presence of apoDDC, the tightly bound PLP on PNPO is transferred to apoDDC, resulting in the formation of about 35% holoDDC