Studies on the Mechanism and Inhibitor Discovery Based on the Structures of Two Catalytic Enzymes: HpKDO8PS and NSDHL

Abstract

Structure-based drug design (SBDD) is the technique to design compounds whose chemical structures are fitted into the three dimensional structure of a protein and to optimize those hits' into clinical candidates. The knowledge of the protein structure can help to accelerate drug development and make it more cost-effective. For development novel antibiotics and lipid-lowering agents, KDO8PS [KDO8P (3-deoxy-D-manno-octulosonate-8-phosphate) synthase] from Helicobacter pylori (HpKDO8PS) and NSDHL [NAD(P) dependent steroid dehydrogenase-like protein (sterol-4-α-carboxylate 3-dehydrogenase, decarboxylating)] from Homo sapiens were selected and detailed studies have been conducted on the protein structures and biophysical properties. HpKDO8PS is the enzyme that catalyzes the condensation reaction between arabinose 5-phosphate (A5P) and phosphoenolpyruvate (PEP) to synthesize KDO8P, the precursor of the 8-carbon sugar 3-deoxy-D-manno-octulosonate (KDO). The crystal structure of HpKDO8PS was determined alone and within various complexes, revealing an extra helix (HE) that is absent in the structures of KDO8PS from other organisms. In contrast to the metal coordination of the KDO8PS enzyme from Aquifex aeolicus, HpKDO8PS is specifically coordinated with Cd2+ or Zn2+ ions, and isothermal titration calorimetry (ITC) and differential scanning fluorimetry (DSF) revealed that Cd2+ thermally stabilizes the protein structure more efficiently than Zn2+. In the substrate-bound structure, water molecules play a key role in fixing residues in the proper configuration to achieve a compact structure. Using the structures of HpKDO8PS and API [arabinose 5-phosphate (A5P) and phosphoenolpyruvate (PEP) bisubstrate inhibitor], 21 compounds showing potential HpKDO8PS-binding properties were generated via in silico virtual screening. The binding capacity to HpKDO8PS of three compounds (avicularin, hyperin, and MC181) was confirmed through saturation transfer difference (STD) experiments, and binding mode of each compound was identified by combining competition experiments and docking simulation analysis. Hyperin was confirmed to bind to the A5P binding site, primarily via hydrophilic interaction, whereas MC181 bound to both the PEP and A5P binding sites through hydrophilic and hydrophobic interactions. These results were consistent with the epitope mapping by STD. The results are expected to provide clues for the development of HpKDO8PS inhibitors. NSDHL is one of the enzymes in the cholesterol biosynthesis that catalyzes NAD+-dependent oxidative decarboxylation of the C4 methyl groups from 4α-carboxysterol to produce the corresponding 3-keto, C4-decarboxylated products. To determine the NSDHL crystal structure, the protein was purified, and crystallized. However, structural determination of NSDHL was not successful, due to the weak anomalous signals from the SeMet data and the difficulty in indexing the data. Alternatively, homology modeling has been used to generate the 3D structure and identify the catalytic key residues of NSDHL. They adopt a Rossmann fold with six α-helices and parallel five β-strands. By comparing the NSDHL models with other Rossmann folds, the active site and the coenzyme binding site could be suggested. The coenzyme binding region is notably well conserved. Mutants causing genetic disorders (CHILD, CKS) were produced by site-directed mutagenesis and they were purified. A correlation between the mutants and thermal stability was investigated using DSF. The mutants are significantly less stable than the wild-type protein supporting the hypothesis that mutations can affect NSDHL folding and thermal stability. Also, ITC was performed to investigate the thermodynamics of the protein-coenzymes. The Kd values from the experiments revealed that NSDHL prefers NAD(H) to NADP(H) for its enzyme reaction. In addition, the STD spectra for NAD and NADH gave information of their binding modes to NSDHL. The spectra indicated NSDHL-coenzymes interaction. To understand better the relationship of the coenzyme binding modes and the disease-causing mutants, STD experiments of NAD+ or NADH with G205S and K232Δ NSDHL were also performed. The mutants showed lower affinities to NADH compared to the wild-type protein. Also, the result showed slightly different binding modes of the coenzymes. These findings support the possibility that changes in binding modes could be relevant in certain disease states found in CHILD and CKS.