(686f) HINT1 Regulated Supramolecular Assembly of Nucleoside Phosphoramidate Pro-Gelators

The non-covalent association of molecules is a ubiquitous feature of life. Protein scaffolds, components of the extracellular matrix, and even duplexed nucleic acids all undergo supramolecular assembly. To generate synthetic self-assembled nanostructures, a wide variety of small molecule motifs have been developed that utilize hydrogen bonding, electrostatics, and hydrophobic interactions to contribute to the assembly process. Growing interest in the regulation of supramolecular assembly has produced a variety of responsiveness motifs. The governing effect of these motifs is exerted through physical and chemical cues such as ultrasound, temperature, pH, reduction and oxidation, and enzymatic responsiveness. Enzymes are of interest for the regulation of supramolecular interactions as they allow the mimicry of biological systems. They are catalytic in nature, able to be regulated by various biomolecules and ligands, and are biocompatible.

To create a responsive and tunable self-assembly system, we have developed Histidine Triad Nucleotide Binding Protein 1 (HINT1) responsive nucleoside phosphoramidate pro-gelators (PPGs). HINT1 has been well characterized as a nucleoside phosphoramidase and acyl-adenylate hydrolase. Utilizing this enzymatic activity in the regulation of assembly, nucleoside phosphoramidate moieties were chemically conjugated to self-assembling peptides to inhibit supramolecular interactions and hydrogelation. HINT1 catalytic activity removes the blocking effect of nucleoside phosphoramidates and allows released peptides to self-assemble into supramolecular hydrogels. Characterization of these materials with oscillatory rheometry has revealed that PPG substrates have differing gelation kinetics depending on nucleobase identity. Additionlly, transmission electron microscopy has yielded data demonstrating different PPGs giving rise to morphologically different nanofiber structures after HINT1 activation. We are utilizing chemical biological tools such as small molecule inhibitors, catalytically dead HINT1 mutants, and slow substrates to investigate the role of HINT1 active site and catalytic activity on the regulation of PPG assembly. Inhibitors have been shown to block HINT1 activity on PPG substrates, and Hint1 H112N catalytically dead mutant has been shown to be unable to activate self-assembly. Nucleoside thiophosphoramidate substrates have been demonstrated to stabilize the covalent adenylated active site intermediate generated during the HINT1 catalytic cycle. We are currently synthesizing slow PPG substrates bearing a thiophosphoramidate group to further tune the kinetic activation of these substrates for self-assembly. Our goal is to develop an adaptable system for the construction of biologically responsive materials that may be assembled in situ in response to HINT1 activity.