Enabling Technology to Screen and Quantify Sialylated Structures for Activity Against Viral Enzymes and Receptors

Sialylated glycans are involved in complex regulation and signaling and play a critical role in disease. In the virus life cycle, receptor binding and sialic acid cleavage to facilitate release can compete and are therefore delicately balanced. While monovalent binding with a single sialylated ligand is weak (KD

~mM), hemagglutinin forms a trimer, which enables multivalent binding. Multivalent binding involving more than 1 ligand leads to strong binding (KD ~nM). This switch between multivalent and monovalent binding allows the hemagglutinin to bind to the host with high affinity that is easily reversed following

internalization, replication, by cleavage of only some of the sialylated ligands. Neuraminidase inhibitors for influenza (Tamiflu, Relenza, and Rapivab) block the neuraminidase cleavage in some infections. The development of therapeutic strategies to block hemagglutinin binding with small molecule sialylated

inhibitors has been limited. Several analytical barriers to the analysis of sialic acids and sialylated compounds have challenged research in this area. The long term objective of this project is to bridge this gap with enabling technology through two key innovations. A new screening approach for enzymes

and receptors is introduced through the use of thermally reversible nanogels. With this new strategy, the sialic acid structures that interact with enzymes or receptors are identified through capillary electrophoresis. This work is based on rapid in-line exoglycosidase reactions facilitated with patterned

nanogels. A new capillary electrophoresis-mass spectrometry interface based on acousto-mechanical energy is introduced to enable coupling both techniques without concern for voltage or flow rate. Aim 1 creates a new functional screening tool for enzyme inhibition and reduces both the amount of enzyme

and the time to evaluate a neuraminidase preparation. The biocompatibility, automation, and low reagent and sample requirements are harnessed in Aim 2 to establish a quantitative screening tool to select and evaluate enzyme inhibition of sialylated structures that interact strongly with the receptor

binding domain of the hemagglutinin protein. The full power of label-free structural identification of capillary electrophoresis interfaced to mass spectrometry outlined in Aim 3 leverages the unprecedented gains in signal with electrically assisted vibrational sharp edge ionization, overcoming barriers of current analytical technologies for the analysis of sialylated glycans. The proposed activities

are significant because the low cost, speed, and automation of the separation-based microscale assays yield previously unattainable information about sialylation fundamental to mitigating viral infections. These new tools address challenges associated with chemical analyses of sialylated structures to

leverage the role of sialylation in viral infections; thereby, providing researchers the means to combat viral diseases and advance human health.