Enzymes and Coenzymes Commons^™

Protein Allostery Probed By Ligands Across Sites, Virgil A. Woods

Dissertations, Theses, and Capstone Projects

Allostery is a pervasive regulatory principle throughout biology, yet the structural pathways by which distal inputs alter active‑site chemistry within protein structures remain incompletely defined and exploited. This dissertation uses protein tyrosine phosphatase 1B (PTP1B) as a tractable model to map those pathways with complementary experimental and computational tools. I integrate high‑resolution hydrogen-deuterium exchange mass spectrometry (HDX-MS), room-temperature crystallography, NMR, steady-state kinetics, crystallographic pseudo-ensembles, and machine-learning-guided ligand discovery. The working premise is that regulation reflects redistribution within a conformational ensemble rather than a binary switch. That orthogonal perturbations by small molecules, mutations, and protein partners can be used to both …

Selective And Brain-Penetrant Acss2 Inhibitors Target Breast Cancer Brain Metastatic Cells, Emily Esquea, Lorela Ciraku, Riley Young, Jessica Merzy, Alexandra Talarico, Nusaiba Ahmed, Mangalam Karuppiah, Anna Ramesh, Adam Chatoff, Claudia Crispim, Adel Rashad, Simon Cocklin, Nathaniel Snyder, Joris Beld, Nicole Simone, Mauricio Reginato, Alexej Dick

Kimmel Cancer Center Faculty Papers

Breast cancer brain metastasis (BCBM) typically results in an end-stage diagnosis and is hindered by a lack of brain-penetrant drugs. Tumors in the brain rely on the conversion of acetate to acetyl-CoA by the enzyme acetyl-CoA synthetase 2 (ACSS2), a key regulator of fatty acid synthesis and protein acetylation. Here, we used a computational pipeline to identify novel brain-penetrant ACSS2 inhibitors combining pharmacophore-based shape screen methodology with absorption, distribution, metabolism, and excretion (ADME) property predictions. We identified compounds AD-5584 and AD-8007 that were validated for specific binding affinity to ACSS2. Treatment of BCBM cells with AD-5584 and AD-8007 leads to …

Identification And Characterization Of Novel, Small Molecule Inhibitors Of Spermine Oxidase, Amelia Bryn Furbish

MUSC Theses and Dissertations

The major intracellular polyamines spermine and spermidine are abundant endogenous compounds that are essential for cellular growth and development. Dysregulation of polyamine metabolism has been implicated as a key mechanism of injury across multiple forms of clinically challenging pathologies. Of the enzymes within the polyamine pathway, the catabolic enzyme spermine oxidase (SMOX) is of particular interest as it is subject to induction in response to infection, neuronal excitotoxicity, ischemia, and oxidative stress. In addition to the loss of radical scavenging capabilities associated with spermine depletion, catabolism of spermine by SMOX results in the production of toxic byproducts, including H_{2 …}

Development Of A Chemical Biology Approach To Uncover The Influence Of Sequence Variations On Ces1 Activity In Live Cells, Samuel James Knebel

Masters Theses

Drug metabolism is the biochemical process of modifying drugs to detoxify and remove them through enzymatic transformations. These biotransformation’s occur primarily in the liver and are critical to understanding how pharmaceutical compounds are chemically altered inside the human body. Human carboxylesterases (CESs) catalyze the hydrolysis of esters, amides, thioesters, and carbamates. CES-mediated hydrolysis plays an important role in the metabolism of many drugs including the first FDA approved antiviral treatment for COVID-19, remdesivir (Veklury), the seizure control medication rufinamide (Banzel), and the flu antiviral drug oseltamivir (Tamiflu). CES activity is known to be influenced by a variety of factors including …

Targeted Therpeutics For Prostate Cancer, Behind The Scenes Of Psma & Cysteine Protease Trapping Mechanism, Alireza Basiri

Theses & Dissertations

In an attempt to increase the residence time of PSMA-targeted theranostics inside the prostate cancer cells and tumors, we designed and prepared a CPTA-PSMATA resembling PSMA-617 by adding a well-known cysteine protease inhibitor (E-64) to the conjugate. Lysosomal proteases, including cysteine, serine, and aspartic families of hydrolytic enzymes, are expressed in high concentrations in the lysosomes and are responsible for the catabolism of proteins inside the cell [165, 278]. We proposed that after successful internalization of the PSMA-targeted conjugates into the cells, they will be conveyed to the endolysosomes. In these endolysosomal compartments, the E-64 moiety will covalently bind to …

Editorial: Adenylyl Cyclase Isoforms As Potential Drug Targets, Tarsis F. Brust, Rennolds S. Ostrom, Val J. Watts

Pharmacy Faculty Articles and Research

Editorial on the Research Topic Adenylyl cyclase isoforms as potential drug targets

Effects Of Chronic Cirrhosis Induced By Intraperitoneal Thioacetamide Injection On The Protein Content And Michaelis–Menten Kinetics Of Cytochrome P450 Enzymes In The Rat Liver Microsomes, Devaraj Venkatapura Chandrashekar, Barent N. Dubois, Mamunur Rashid, Reza Mehvar

Pharmacy Faculty Articles and Research

Chronic intraperitoneal injection of thioacetamide (TAA) in rats has been used as an animal model of human cirrhosis to study the effects of the disease on drug metabolism. However, TAA inhibits P450 enzymes directly and independently of cirrhosis. We investigated the effects of chronic cirrhosis in rats, induced by 10 weeks of intraperitoneal TAA, on the P450 enzymes after a 10-day washout period to eliminate TAA. Liver histology and serum biomarkers of hepatic function confirmed cirrhosis in all animals. Microsomal total P450 content, P450 reductase activity and ethoxycoumarin O-deethylase activity, a general marker of P450 activity, were significantly reduced by …

Characterizing And Overcoming Resistance To Aminomethylspectinomycins In Gram-Negative Bacteria, Nisha Das

Theses and Dissertations (ETD)

Spectinomycin (SPC) is a broad-spectrum aminocyclitol antibiotic. Its use in agriculture has led to widespread resistance in enteric bacteria, necessitating the development of more effective analogs. Aminomethyl spectinomycins (amSPC) are modified spectinomycins with increased potency against many bacterial species. These species include Legionella pneumophila, which harbors a chromosomally encoded aminoglycoside modifying enzyme (AME). In this study, we follow up on this observation and examine the extent to which the amSPCs are substrates for AMEs through adenylation (ANTs) and phosphorylation (APH). APH(9)-Ia and ANT(3")(9) were expressed in E. coli BL21(DE3) and purified using the Ni-affinity chromatography. The ability of AMEs to …

Uplc-Ms/Ms Analysis Of The Michaelis-Menten Kinetics Of Cyp3a-Mediated Midazolam 1′- And 4-Hydroxylation In Rat Brain Microsomes, Devaraj Venkatapura Chandrashekar, Barent Dubois, Reza Mehvar

Pharmacy Faculty Articles and Research

Midazolam (MDZ) is a short-acting benzodiazepine with rapid onset of action, which is metabolized by CYP3A isoenzymes to two hydroxylated metabolites, 1′-hydroxymidazolam and 4-hydroxymidazolam. The drug is also commonly used as a marker of CYP3A activity in the liver microsomes. However, the kinetics of CYP3A-mediated hydroxylation of MDZ in the brain, which contains much lower CYP content than the liver, have not been reported. In this study, UPLC-MS/MS and metabolic incubation methods were developed and validated for simultaneous measurement of low concentrations of both hydroxylated metabolites of MDZ in brain microsomes. Different concentrations of MDZ (1–500 µM) were incubated with …

Differential Effect Of Proinflammatory Cytokines On Corneal And Conjunctival Epithelial Cell Mucins And Glycocalyx, Kiumars Shamloo, Priya Mistry, Ashley Barbarino, Christopher Ross, Vishal Jhanji

Pharmacy Faculty Articles and Research

Purpose: Ocular surface mucins and glycocalyx are critical for providing ocular hydration as well lubrication and repelling pathogens or allergens. Elevated levels of tear proinflammatory cytokines in dry eye may have detrimental effect on mucins and glycocalyx. The present study tested the effect of proinflammatory cytokines IL-6, TNF-α, and IFN-γ on membrane-tethered mucins expression, glycocalyx, and viability of ocular surface epithelial cells.

Methods: Stratified cultures of human corneal and conjunctival epithelial cells were exposed to different concentrations of IL-6, TNF-α, and IFN-γ for 24 hours. The mucins gene and protein expressions were quantified by real-time polymerase chain reaction …

The Pharmabiotic For Phenylketonuria: Development Of A Novel Therapeutic, Chloé Elizabeth Lebegue

Senior Theses

Phenylketonuria, now known as phenylalanine hydroxylase (PAH) deficiency, is a genetic disorder of metabolism affecting approximately one in every 15,000 infants born in the United States. Patients have nonfunctional PAH enzyme secondary to one or more genetic mutations. The enzyme deficit results in destructive supraphysiologic blood phenylalanine levels upon consumption of the essential dietary amino acid phenylalanine. Current standards of care mitigate signs and symptoms of the disorder, but do not approach a cure. The methods for creating a prototype pharmabiotic as an innovative treatment strategy for PAH deficiency are described herein.

DNA molecular cloning techniques were utilized to engineer …

Glycosaminoglycan Lyases In The Preparation Of Oligosaccharides, Alhumaidi B. Alabbas

Theses and Dissertations

Glycosaminoglycans are heterogeneous polysaccharides that mediate important biological functions. There has been considerable interest in deciphering the precise GAG sequences that are responsible for protein interactions. In fact, several GAG oligosaccharides have been discovered to date as targeting proteins with higher level of specificity. Yet, it has been difficult to develop GAG oligosaccharides as drugs. One of the key reasons for this state of art is that GAG synthesis is extremely challenging and is highly structure-specific. Thus, much of the biology and pharmacology of GAG remains unknown and unexploited to date.

An alternative approach is to prepare GAG oligosaccharides using …

Chemoenzymatic Studies To Enhance The Chemical Space Of Natural Products, Jhong-Min Chen

Theses and Dissertations--Pharmacy

Natural products provide some of the most potent anticancer agents and offer a template for new drug design or improvement with the advantage of an enormous chemical space. The overall goal of this thesis research is to enhance the chemical space of two natural products in order to generate novel drugs with better in vivo bioactivities than the original natural products.

Polycarcin V (PV) is a gilvocarcin-type antitumor agent with similar structure and comparable bioactivity with the principle compound of this group, gilvocarcin V (GV). Modest modifications of the polyketide-derived tetracyclic core of GV had been accomplished, but the most …

Rescuing Acetylcholinesterase From Nerve Agent Inhibition: Protein Dynamics Driven Drug Discovery, Aiyana M. Emigh, Brian Bennion

STAR Program Research Presentations

Severe morbidity and mortality consequences result from irreversible inhibition of human acetylcholinesterase by organophosphates (OPs). Oxime-based reactivators are currently the only available treatments but lack efficacy in the central nervous system (CNS) where the most damage occurs. Computational docking and molecular dynamics (MD) simulations reveal complex structural barriers that may reduce oxime efficacy. These results may guide future drug designs of more effective countermeasures.

Novel Approaches For Designing 5'-O-Ester Prodrugs Of 3'-Azido-2'3'-Dideoxythymidine (Azt), Keykavous Parang, Leonard I. Wiebe, Edward E. Knaus

Pharmacy Faculty Articles and Research

3'-Azido-2'3'-dideoxythymidine (AZT, 1, zidovudine, RetrovirTM) is used to treat patients with human immunodeficiency virus (HIV) infection. AZT, after conversion to AZT-5'-triphosphate (AZT-TP) by cellular enzymes, inhibits HIV-reverse transcriptase (HIV-RT). The major clinical limitations of AZT are due to clinical toxicities that include bone marrow suppression, hepatic abnormalities and myopathy, absolute dependence on host cell kinase-mediated activation which leads to low activity, limited brain uptake, a sort half-life of about one hour in plasma that dictates frequent administration to maintain therapeutic drug levels, low potential for metabolic activation and/or high susceptibility to catabolism, and the rapid development of resistance by HIV-1. …