The role and effects of the IL-33/ST2 pathway in disease is still unknown. The work described here centers upon two important members of the IL-33/ST2 axis: pro-IL-33 and the sST2 receptor. The first portion of the work focuses on the ligand pro-IL-33, an important signaling and transcriptional regulator for the IL-33/ST2 system whose structure and biophysical characteristics have yet to be elucidated due to lack of a high expression system. This need was addressed through the iterative testing and eventual purification of an Eschericia coli based expression system utilizing a His-SUMO construct coupled with autoinduction. The second portion of the work centers upon the role of glycosylations on the sST2 receptor. To address the role of glycosylations in the IL-33/ST2 axis, site specific occupancy was analyzed on the sST2 receptor revealing occupancy of 7 of 8 possible sites present. Subsequent enzymatic based characterization of these sites revealed a mixed population of tri- and tetra- antennary complex glycans present. The effects of these glycans were probed through the implementation of a novel add-on to the computational processing program SMOG2 to enable interpretation and simulation using GROMACS. Analysis of the trajectories revealed that N-linked glycosylations reduce dynamics present in sST2 in a global and local manner; and stabilize the structure of an unstructured loop present from Tyr34 to Val65 of the sST2 receptor when bound to IL-33. As such, this work lays the foundation for further exploration into the function and internal regulation abilities of the IL-33/ST2 axis

Histone deacetylase inhibitors (HDACi) target abnormal epigenetic states associated with a variety of pathologies, including cancer. Here, the development of a prodrug of the canonical broad-spectrum HDACi suberoylanilide hydroxamic acid (SAHA) is described. Although hydroxamic acids are utilized universally in the development of metalloenzyme inhibitors, they are considered to be poor pharmacophores with reduced activity in vivo. We developed a prodrug of SAHA by appending a promoiety, sensitive to thiols, to the hydroxamic acid warhead (termed SAHA-TAP). After incubation of SAHA-TAP with an HDAC, the thiol of a conserved HDAC cysteine residue becomes covalently tagged with the promoiety, initiating a cascade reaction that leads to the release of SAHA. Mass spectrometry and enzyme kinetics experiments validate that the cysteine residue is covalently appended with the TAP promoiety. SAHA-TAP demonstrates cytotoxicity activity against various cancer cell lines. This strategy represents an original prodrug design with a dual mode of action for HDAC inhibition.