A Nanoparticle Approach to the Inhibition of Protein-Protein Binding

Abstract

This study explores the functionalization of generation 2.5 Polyamidoamine (PAMAM) dendrimers with various amino acids, including Tyrosine, Phenylalanine, Alanine and Valine, and evaluates their ability to stabilize iron oxide magnetic nanoparticles (Fe₃O₄ MNPs) and bind to cytochrome-c. The primary goal was to enhance the binding affinity and selectivity of MNPs using amino acid-functionalized dendrimers for potential applications in protein interaction inhibition. The functionalization of dendrimers was confirmed through ¹H NMR, 13C NMR, IR and mass spectroscopy, and the resulting functionalized MNPs were characterized by dynamic light scattering (DLS) and TEM, showing greatly improved stability compared to unfunctionalized systems. The binding affinities of these functionalized MNPs were tested against cytochrome-c, revealing that Tyrosine-functionalized PAMAM dendrimers exhibited the highest binding affinity (53%) due to the presence of aromatic rings and hydroxyl groups, which facilitated π-π stacking and hydrogen bonding. Valine-functionalized systems also showed strong binding (38%) driven by hydrophobic interactions, while phenylalanine functionalized MNPs demonstrated moderate binding (26%) due to the absence of hydroxyl groups and limited hydrogen bonding capacity. Alanine-functionalized MNPs exhibited intermediate binding (29%) due to the presence of an additional carboxylic acid group, which contributed to strengthening the binding interactions. This extra carboxyl group increased the overall binding affinity by providing additional sites for electrostatic interactions, hydrogen bonding, and other non-covalent forces, thereby enhancing both the stability and specificity of the binding. The unfunctionalized PAMAM G3.5 dendrimer system served as a control, showing only minimal binding (12%). These findings highlight the potential of amino acid functionalized dendrimers to enhance MNP stability and selectivity for protein binding, offering a promising strategy for inhibiting protein-protein interactions in disease-related applications. In the third chapter, we explore the functionalization of graphene oxide (GO) with monomeric and oligomeric glutamic acid to enhance its potential for enzyme inhibition, particularly targeting α-chymotrypsin. GO was synthesized via the modified Tour method, ensuring improved safety and efficiency over traditional methods. The functionalization process aimed to increase the surface carboxylic acid groups of GO through the attachment of glutamic acid, enabling stronger electrostatic interactions and improved protein binding capabilities. Two approaches were employed: monomeric glutamic acid functionalization, involving the use of diester-protected glutamic acid, and oligomeric functionalization, where unprotected glutamic acid facilitated the formation of glutamic acid chains on the GO surface. Comprehensive characterization was performed using Fourier-transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), and X-ray diffraction (XRD) to confirm the successful attachment of glutamic acid and examine structural changes. Results showed that oligomeric functionalization led to higher uniformity and amide bond formation, enhancing protein interaction. This functionalized GO exhibited promising potential as a platform for enzyme inhibition, showing selectivity and binding efficiency towards α-chymotrypsin, with implications for therapeutic and biochemical applications.