Inflammation has been linked to neuropathic pain — pain resulting from injury to the somatosensory system — through upregulation of pro-inflammatory cytokines and endogenous messengers which increase local neurons’ sensitivity to painful stimuli. Additionally, epigenetic mechanisms can modulate inflammation by regulating the expression of pro- and anti-inflammatory cytokines, correlated through high levels of histone acetylation near their promoters. Histone deacetylases (HDACs) and Bromodomain and Extraterminal (BET) proteins which either remove or bind to acetylated histones, are attractive therapeutic targets for a wide range of inflammation-driven diseases. Simultaneous targeting of these proteins has emerged as a new avenue, especially for neuropathic pain. While new, SUM52 has been recently tested as a fusion of BET and HDAC inhibitors, iBET762 and Vorinostat. Thus, the goal of this study is to add to the library of dual HDAC/BET inhibitors with a 1,2,3-triazole-based inhibitor developed in the Pomerantz lab, maintaining pre-fusion potency. For this study, the BET inhibitor NV1127 and the HDAC inhibitor based on Vorinostat have been designed and are being synthesized. These inhibitors will then be linked together using either a Copper-Catalyzed Alkyne-Azide Click (CuAAC) reaction or a peptide coupling reaction. Further biophysical analysis will be used to assess inhibitor potency and bioactivity, and cell-based work will inform target engagement and efficacy. Progress towards these goals will be presented.

Ceramides are a subclass of sphingolipids which consist of a sphingoid backbone along with a fatty acid chain of varying lengths, attached at the amino group. Ceramides serve a crucial role in various cellular processes like apoptosis, senescence, autophagy and ER stress. Several studies have demonstrated the accumulation of ceramide during apoptosis. Ceramides that accumulate at the mitochondria activate B-cell lymphoma 2 associated X protein (BAX). This pro-apoptotic protein facilitates mitochondrial outer membrane permeabilization which leads to the release of cytochrome c as well as activation of caspase-9 and other downstream caspases, ultimately resulting in cell death. Our objective is to elucidate the changes in the sphingolipid family during apoptosis. We will use a human lung fibroblast cell line, MRC-5, as the model system to study lipid changes in apoptosis, induced by doxorubicin treatment. Apoptotic activity will be characterized using MTT-based viability assays and Western blotting. We will carry out a targeted Liquid Chromatography-Quadruple Time of Flight-Mass Spectrometry-based (LC-QToF-MS) lipidomics approach and compare the sphingolipid levels in apoptotic and control cells. The results of these experiment will reveal the regulation of cellular ceramide and other sphingolipids in doxorubicin-induced apoptosis in lung fibroblast cells.

Silica is one of the most investigated packing materials in High-Performance Liquid Chromatography (HPLC) column technology due to its favorable chemical and physical properties. Traditionally, the inorganic silica support is chemically modified at the surface to introduce chemistries for chromatographic applications. However, the application of stationary phases attached with siloxane bonds to inorganic silica for chromatographic separations is limited due to poor hydrolytic stability at low (< pH 2) and high (> pH 8) pH levels. Superficially porous silica is ideal for analytical separations because it has adequate loading capacity and a shorter flow path through the particle. The incorporation of radially oriented pores (ROP) further improves separation efficiency. In this study, the development of superficially porous silica with an organo-silica shell and ROP was investigated as a potentially highly hydrolytically stable packing material. This was achieved by incorporating propyl azide functionalities into the otherwise inorganic silica shell to add organic character. Additionally, the azide groups on the surface could be used to attach a stationary phase to the particle surface using the well-established Cu(I)-catalyzed azide−alkyne cycloaddition (CuAAC) reaction, commonly known as click chemistry, instead of conventional salinization to obtain improved hydrolytic stability. These particles will be packed into stainless steel columns and studied for hydrolytic stability under extreme pH conditions.

Malate Dehydrogenases have either a dimer or a tetrameric quaternary structure, and subunit interactions are involved in both activity and regulation.. Despite the importance of understanding the molecular mechanisms of such interactions in allosteric drug design, little is known at the molecular level of how the subunits communicate the presence of a ligand on one subunit to another in the quaternary structure. Plasmodium falciparum (the parasite mainly responsible for malaria has a homotetrameric malate dehydrogenase in a dimer of dimer type structure with three types of interface- A-B, A-C, and A-D interfaces. The A-B interface resembles that found in dimeric MDHs. Analysis of the A-B interface shows a unique feature in the first region of the multipart interface. A conserved Glutamate, E18, in the middle of an alpha helix that makes contact with the equivalent helix across the interface. MM-GBSA analysis suggests that E18 interactions are antagonistic. We hypothesize the polarity of the local environment of E18, governed by 4 conserved residues Q11, I15, L19, and L22 modulates E18 interactions across the interface and governs ligand-induced subunit interactions. We constructed site-directed mutants of each, Q11I, I15Q, L19N, and L22N. and the resultant proteins expressed, purified, and characterized using enzyme kinetics and size exclusion chromatography. All four had significantly lower specific activities than wildtype, with Q11I having the highest turnover number, approximately 8% of the wildtype. Size exclusion chromatography showed that the Q11I mutation caused a shift in quaternary structure from a simple tetramer structure to a tetramer-dimer-monomer equilibrium. The L19N mutation shifts the equilibrium almost entirely to the monomer with minimal indication of tetramer or dimer forms.