Doctor of Philosophy (Ph.D.)
Degree Granting Department
Wayne C. Guida, Ph.D.
Wesley Brooks, Ph.D.
Kenyon Daniel, Ph.D.
Jianfeng Cai, Ph.D.
Abdul Malik, Ph.D.
ApoE4,, cocrystals, Cruzain, Proteasome, STIM1
Calcium ions have important roles in cellular processes including intracellular signaling, protein folding, enzyme activation and initiation of programmed cell death. Cells maintain low levels of calcium in their cytosol in order to regulate these processes. When activation of calcium-dependent processes is needed, cells can release calcium stored in the endoplasmic reticulum (ER) into the cytosol to initiate the processes. This can also initiate formation of plasma membrane channels that allow entry of additional calcium from the extracellular milieu. The change in calcium levels is referred to as calcium flux. A key protein involved in initiation of calcium flux is Stromal Interaction Molecule 1 (STIM1), which has recently been identified as a sensor of ER calcium levels. STIM1 is an ER transmembrane protein that is activated by a drop in ER calcium levels. Upon activation, STIM1 oligomerizes with a plasma membrane protein, ORA1, to form calcium-selective plasma membrane channels. Dysregulation of calcium flux has been reported in cancers, autoimmune diseases and other diseases. STIM1 is a promising target in drug discovery due to its key role early in calcium flux. Here we review the involvement and importance of STIM1 in diseases and we discuss STIM1 as a viable target for drug discovery using computational chemistry methods to rapidly identify new molecules to target STIM1. Herein, computational techniques were used to understand the mechanistic role of STIM1 and virtual screening is in process to discover potential inhibitors of STIM1 activity. Also mutational analysis on STIM1 was performed computationally to see the effect it had on the protein computationally.
It has been found that tumor cells and tissues, compared to normal cells, have higher levels of copper and possibly other metal ions. This presents a potential vulnerability of tumor cells that can serve as a physiological difference between cancer cells and normal cells and allows design of compounds that selectively target tumor cells while sparing normal cells. Recently we have identified compounds that have potential to inhibit the proteasome in tumor cells and induce cell death by mobilizing endogenous tumor copper resulting in in cellulo activation of the compound. These compounds hence act as pro-drugs, becoming active drugs in tumor cells with high copper content but remaining essentially inactive in normal cells, thereby greatly reducing adverse effects in patients. Such use would be of significant benefit in early detection and treatment of cancers, in particular, aggressive cancers such as pancreatic cancer which is usually not detected until it has reached an advanced stage. Six compounds were identified following virtual screening of the NCI Diversity Set with our proteasome computer model followed by confirmation with a biochemical assay that showed significant inhibition of the proteasome by the compounds in the presence of copper ions. In a dose response assay, NSC 37408 (6, 7-dihydroxy-1-benzofuran-3-one), our best compound, exhibited an IC50 of 3µM in the presence of 100 nM copper.
Chagas’ Disease, a parasitic disease caused by the parasite Trypanosma Cruzi, is endemic to Latin America. The disease manifests itself in a short acute phase and a long chronic phase. Current treatments are effective only in the acute phase and are not used in the chronic phase due to toxicity of the drugs. Hence a new drug discovery approach was chosen for this disease. Cruzain is the major etiologic enzyme involved in the disease and is only present in the parasite. It is also an enzyme expressed by the parasite in both phases. Herein, a novel peptoid library containing hydromethylketones was constructed and screened against a virtual structure of cruzain. The peptoids thus found through this drug discovery effort can be used as potential drug candidates against cruzain. Computational techniques will help achieve a high degree of specificity and aid in proposing assays for determining compounds with high activity
Alzheimer disease is the most common form of dementia. Its pathogenesis incorporates many potential targets for treatment. Among the targets identified, Apolipoprotein E4 (apoE4) is especially interesting due to its catalytic role in the degradation and clearance of amyloid beta (Aβ), a risk factor for Alzheimer disease. ApoE exists in 3 isoforms which directly impact its functionality in the body. There are characteristic structural differences between them. In ApoE4 ionic interactions exist between Arg-61 and Glu-255 residues, unlike the other isoforms. Hence interruption of this interaction by inhibitors may change the structure of apoE4 to a more linear structure as observed in the other isoforms. Virtual screening of the NCI diversity set on an energy minimized protein virtual structure was performed to identify potential small molecule inhibitors and to gain further understanding of interactions that can be targeted to inhibit this protein. From the top ligands in the NCI diversity set, a peptide library was designed to target the protein.
Previous research has indicated that liquid assisted grinding (LAG) is efficient and reliable for cocrystal formation when compared to solvent crystallization and dimethyl formamide is the best solvent for grinding. Herein, we report the comparison of four screening processes: Slurry, solvent crystallization, LAG and dry grinding. Thirty-eight crystal forms containing the Narom··· COOH, Narom···OH supramolecular heterosynthons were screened in the process, and it was observed that slurry methodology is as efficient and reliable in forming cocrystals as solution crystallization. Twenty-four new crystal forms were also isolated herein. LAG was found to be more efficient as compared to dry grinding and was successful in the formation of twenty-five crystal forms of the thirty-eight screened. Dimethyl formamide still remains the best solvent for LAG. All our slurry experiments were performed in water and it was found that water can be used reliably for this method for compounds within a wide range of solubility, thereby increasing the versatility and usability of this method for future screening procedures.
Scholar Commons Citation
Mukherjee, Sreya, "Applications of Molecular Modelling and Structure Based Drug Design in Drug Discovery" (2016). Graduate Theses and Dissertations.