Research

Our research focuses on multivalent interactions on cell surface, which is essential for biological recognition (ex. pathogen-host interaction) and signal transduction (ex. G-protein coupled receptor drug development). These interactions generally involve with the simultaneous binding of multiple ligands to oligomeric protein receptor, such as lectins, or receptors in proximity within lipid microdomains on cell surface. One promising strategy to study this complicated process is through controlled presentation of the multiple ligand on a scaffold with the ligand spacing to match the spatial requirement of target receptor oligomers that is generally in the scale of nanometers. Therefore, the investigation toward understanding and manipulation of cell surface receptors would require the combination organic synthesis, biomolecule interaction and nanotechnology. We exploit molecular scaffold to precisely control the ligands position to probe the probe and manipulate protein oligomers.

Development of a microarray system to determine spatial specificity of protein

The development of multivalent ligands usually requires the oligomeric structure of target protein so that the scaffold can be designed accordingly. However, it takes a lot of time and effort to obtain the crystal structure of protein oligomer. An efficient approach to rapidly determine the favorable ligand spacing for a given protein oligomer would greatly help the development of inhibitors. While microarray system has been widely used to characterize protein interactions due to its minimal protein amount requirement and high-throughput capability, it has a lot potential for this purpose. Unfortunately, one major difficulty is that the ligand spacing on surface cannot be controlled precisely by traditional immobilization approach that a wide distribution in the ligand spacings is resulted to give poor spatial selectivity.

To solve this problem, we improved traditional microarray system by introducing polyproline helix, which forms well-documented polyproline helix II structure (PPII) in aqueous solution. By controlling the peptide synthesis sequence, we can control the locations of the ligands precisely to adjust the ligand spacing to test the target protein oligomers.

The stable attachment of the polyproline scaffold on surface is another essential issue for the novel microarray design and we exploited fluorous interaction to allow non-covalent immobilization. The design of fluorous anchor on scaffold not only allowed the orientation of helix on surface to be controlled, but also stabilized the helix structure due to the unique symmetry of PPII. To test the capability of the microarray system, we synthesized fluorous polyproline peptide with oligomannose ligand separated by 9, 18 and 27 Å to test HIV broadly neutralizing mAb 2G12 to measure the surface dissociation constant KD. The results indicated the 27 Å ligand spacing is the favorite for 2G12 binding and in good agreement with the binding site that separated by 31 Å from structural studies. We further synthesized polyproline soluble polyproline peptide carrying the ligands with the same spacings to test as inhibitors in solution. The results also conclude the 27 Å scaffold as the most potent inhibitor among the different scaffolds made. The finding demonstrated the ligand spacing can be controlled on this microarray system for characterize protein oligomer and provide a potent inhibitor design. As the characterization on microarray does not require the target protein oligomeric structure, this could be employed as a strategy for rapid development of potent and selective inhibitors.

For more information: ACS Appl Mater Interfaces. 2017, 9, 41691-41699.

DOI: 10.1021/acsami.7b13200

Development of dimeric inhibitor for lectin LecA

LecA is a lectin from Pseudomonas aeruginosa, a serious threat of nosocomial infection for immune-compromised patients. LecA, a tetrameric galactose binding lectin, represents a promising pharmaceutical target as it contributed to infection and antibiotic resistance. We develop a series of inhibitors base on changing 1) the anomeric aromatic moiety of galactosides, and 2) the variable distance between the two galactoside ligands on polyproline scaffolds.

To efficiently change the two variables for search for the most potent inhibitor, we used copper (I) catalyzed azide-alkyne cycloaddition (CuAAC) reaction to conjugate the ligand and the peptide scaffold. Therefore, polyproline peptides were installed with alkynyl groups at the desired locations that were controlled by peptide synthesis. This convenient strategy allowed us to rapidly identify a potent LecA inhibitor with a nanomolar range dissociation constant measured by surface plasmon resonance. The series of glycopeptide inhibitors also provide information of the structure-activity relationship and identify the suitable distance between ligand to target LecA. One of the interesting galactose analog binding was explained by molecular docking.

For more information: Chem Asian J. 2018, 13, 686-700.

DOI: 10.1002/asia.201701724

Asialoglycoprotein receptor and RNAi delivery to hepatoma cells

Asialoglycoprotein is a galactose/GalNAc binding receptor that almost exclusively expressed on hepatocyte and even at a higher level for hepatoma cells. We employed this unique feature for to improve the efficiency for selective delivery to hepatoma cells by using galactose and GalNAc analogs. In collaboration with Yunching Chen’s group in NTHU, the efficiency of galactoside analog decorated nanoparticle carrying RNAi for delivery was evaluated. The nanoparticles were further tested in vivo to show anti-cancer effect on a mice model.

For more information: Biomacromolecules 2018, 19, 2330-2339.

DOI: 10.1021/acs.biomac.8b00358