Ab initio study of aspartic and glutamic acid: supplementary evidence for structural requirements at position 9 for glucagon activity

Abstract Our previous work established that position 9 aspartic acid in glucagon was a critical residue for transduction of the hormone response. An uncoupling of the binding interaction from adenylate cyclase activation was demonstrated by the observation that amino acid replacements at position 9 resulted in peptides that had no measurable adenylate cyclase activity yet were still recognized by the glucagon receptor. It was also later shown that His' played a major role in activation, and it was suggested that an electrostatic interaction between the aspartic acid carboxylate and the histidine imidazole occurred as part of the activation mechanism. This did not preclude intermolecular interactions of this aspartic acid with other residues within the receptor binding site. The observation that a conservative substitution of glutamic acid for aspartic acid at position 9 was sufficient to result in the potent antagonist, des-His'd[Glu']glucagon amide, implied that even glutamic acid possessed the minimum properties necessary for inhibition, and that the precise position of the carboxyl group at position 9 in glucagon was an absolute requirement for full agonist activity. The present investigation was conducted with ab initio calculations and molecular modeling to shed some light on the source of this phenomenon

A Proline-Based Neuraminidase Inhibitor: DFT Studies on the Zwitterion Conformation, Stability and Formation

The designs of potent neuraminidase (NA) inhibitors are an efficient way to deal with the recent “2009 H1N1” influenza epidemic. In this work, density functional calculations were employed to study the conformation, stability and formation of the zwitterions of 5-[(1R,2S)-1-(acetylamino)-2-methoxy-2-methylpentyl]-4-[(1Z)-1-propenyl]-(4S,5R)-d-proline (BL), a proline-based NA inhibitor. Compared to proline, the zwitterion stability of BL is enhanced by 1.76 kcal mol−1 due to the introduction of functional groups. However, the zwitterion of BL will not represent a local minimum on the potential energy surface until the number of water molecules increases up to two (n = 2). With the addition of two and three water molecules, the energy differences between the zwitterions and corresponding canonical isomers were calculated at 3.13 and −1.54 kcal mol−1, respectively. The zwitterions of BL are mainly stabilized by the H-bonds with the water molecules, especially in the case of three water molecules where the carboxyl-O atoms are largely coordination-saturated by three H-bonds of medium strengths, causing the zwitterion stability even superior to the canonical isomer. With the presence of two and three water molecules, the energy barriers for the conversion processes from the canonical isomers to the zwitterions are equal to 4.96 and 3.13 kcal mol−1, respectively. It indicated that the zwitterion formation is facile to take place with addition of two molecules and further facilitated by more water molecules. Besides, the zwitterion formation of BL is finished in a single step, different from other NA inhibitors. Owing to the above advantages, BL is a good NA inhibitor candidate and more attention should be paid to explorations of BL-based drugs

Bioprocess development for universal influenza vaccines based on inactivated split chimeric and mosaic hemagglutinin viruses

Seasonal influenza viruses account for 1 billion infections worldwide every year, including 3–5 million cases of severe illness and up to 650,000 deaths. The effectiveness of current influenza virus vaccines is variable and relies on the immunodominant hemagglutinin (HA) and to a lesser extent on the neuraminidase (NA), the viral surface glycoproteins. Efficient vaccines that refocus the immune response to conserved epitopes on the HA are needed to tackle infections by influenza virus variants. Sequential vaccination with chimeric HA (cHA) and mosaic HA (mHA) constructs has proven to induce immune responses to the HA stalk domain and conserved epitopes on the HA head. In this study, we developed a bioprocess to manufacture cHA and mHA inactivated split vaccines and a method to quantify HA with a prefusion stalk based on a sandwich enzyme-linked immunosorbent assay. Virus inactivation with beta-propiolactone (βPL) and splitting with Triton X-100 yielded the highest amount of prefusion HA and enzymatically active NA. In addition, the quantity of residual Triton X-100 and ovalbumin (OVA) was reduced to very low levels in the final vaccine preparations. The bioprocess shown here provides the basis to manufacture inactivated split cHA and mHA vaccines for pre-clinical research and future clinical trials in humans, and can also be applied to produce vaccines based on other influenza viruses