2013 REU Students

Kristen Biernat

Mentor:  Brian Bachmann
Department:  Chemistry
Home Institution:  Ursinus College

“Investigating Actinomycete Biodiversity in Hypogean Environments by 16s rDNA Analysis”

Microbial natural products have been a vital source of drugs in the pharmaceutical industry. However, drug discovery efforts have shifted away from natural products due to high rediscovery rates and the growth of combinatorial chemistry. Despite this de-emphasis, the vast majority of microbial diversity remains unexplored and represents an untapped resource for therapeutic agents. Hypogean ecosystems, such as caves, are ideal locations to search for biodiversity with the potential to produce novel natural products. Bacteria in these hypercompetitive environments are likely to yield a variety of defensive, signaling, and predatory compounds with antibiotic properties. The actinomycetes, a class of gram-positive bacteria, are known producers of antibiotics, making them suitable targets for exploring the secondary metabolite potential of hypogean microbes. In this work, actinomycetes strains isolated from four caves were identified based on 16s rRNA gene analysis to investigate their diversity in hypogean environments. Actinomycetes were isolated using ex situ plate dilution methods as well as in situ traps selective for their characteristic mycelia growth. Preliminary results indicate that the majority of the isolates belong to the Streptomyces genus. The relatedness among the isolated Streptomyces strains will be determined. Future work will entail screening rare actinomycetes for novel natural products with therapeutic properties.

Davon Carter

Mentor:  Ned Porter Lab
Department: Chemistry
Home Institution:  Xavier University, Louisiana

“Smith Lemli Opitz Syndrome Lipid Toxicity and Metabolite Investigation”

Oxygen is one of the most essential elements for metabolism and energy production for the large majority of life forms on Earth. Despite the life-sustaining properties a so called ‘Oxygen Paradox’ exists, where some reactions involving oxygen can give rise to reactive oxygen species (ROS) which subsequently lead to substantial damages in cells, tissues, and organs. Autoxidation of polyunsaturated fatty acids (PUFAs) and sterols is one of the consequences of ROS generation. Interest in lipid peroxidation and autoxidation have increased in recent decades due to the realization that these processes are involved extensively in the pathophysiology of common diseases including atherosclerosis, asthma, and neurodegenerative disorders such as Alzheimer’s disease and Parkinson’s disease. Peroxidation products of sterols, known as oxysterols, also exhibit biological activities such as cytotoxicity, regulation of cholesterol homeostasis, and suppression of immune system response. Oxysterols can be formed not only by peroxidation of cholesterol but also from biosynthetic intermediates in the cholesterol biosynthetic pathway, especially when elevated levels of these compounds are present such as 7-dehydrocholesterol (7-DHC) in Smith-Lemli-Opitz Syndrome (SLOS). A selection of oxysterols derived from 7-DHC. My research primary consist of indentifing novel metabolites of 7-DHC using mass spectrometry, in addition to, investigating the cytotoxicity of these 7-DHC metabolites in Neuro-2A cells (mouse neural crest-derived cell line) by montoring cell viability.

Brandan Dotson

Mentor:  Gary Sulikowski 
Department:  Chemistry
Home Institution:  Xavier University, Louisiana

“Synthesis and Click Chemistry of 9-Hydroxymethylbicyclo [6.1.0]nonyne for Bioorthogonal Labeling and Three-Dimensional Imaging of Living Cells”

Bioorthogonal labeling has proven particularly useful for the study of biomolecules in their native environment because of a highly selective cycloaddition reaction between an abiotic tag and chemical probe. This process is achieved through “click chemistry,” a rapid and efficient way to connect molecules together. However current methods involve the use of toxic metal catalysts that are not suitable for living systems. Recent studies have shown that azides and nitrones react readily with cyclooctynes in a metal-free strain-promoted cycloaddition reaction. Based on known reactivity studies, 9-hydroxymethylbicyclo[6.1.0]nonyne (BCN) and its analogues form a class of versatile cyclooctynes for bioconjugation by combining stability with high reactivity. These compounds are expected to have a broad range of applications that require highly efficient metal-free conjugation of separate molecular entities.

Matthew Draelos

Mentor:  Gary Sulikowski 
Department:  Chemistry
Home Institution:  North Carolina State University

“Probing of bacterial pathogenesis by activation of heme-sensing in Staphylococcus aereus and the synthesis of coelichelin”

Multi-drug resistance in bacteria has necessitated the identification of novel targets by which to combat bacterial infection. Notably, bacteria often recruit essential nutrients from infected host tissue to sustain the infection, with metal recruitment being identified as a particularly important component of pathogenesis. Therefore, targeting metal acquisition homeostasis has been proposed as a therapeutically viable approach to combating bacterial infection. We investigated bacterial metal recruitment and metal homeostasis by two means. First, we synthesized and screened analogs of VU0043981, a small molecule identified as activating the heme-sensing pathway of Staphylococcus aereus. Notably, two analogs of VU0043981 replacing the 4-fluoro benzylamine with a 3-methyl benzylamine and a 2-methyl benzylamine, respectively, were identified as good probes of the heme-sensing pathway of S. aereus due to higher activation of the heme-sensing pathway and lower toxicity than the parent VU0043981 compound. Second, we synthesized the novel natural product coelichelin, a hypothetical metal chelating metabolite from Streptomyces coelicolor. The synthesis of coelichelin provided a means by which to probe bacterial metal recruitment in S. coelicolor, a non-infectious model system. The synthesis of coelichelin was proposed from three amino acid derived fragments: a formylated hydroxylamine L-ornithine, a threonine acetonide, and a cyclized L-ornithine with a hydroxylamine functionalization. These fragments were synthesized and coupled via two peptide bonds to arrive at the novel natural product.

Anna Elleman

Mentor:  Larry Marnett 
Department:  Biochemistry
Home Institution:  University of California, Berkeley

“Des-Sulfonylmethyl Analogues of Rofecoxib as Potent and Selective COX‑1 Inhibitors”

Purpose⎯Cyclooxygenase-1 (COX-1) is an attractive target for molecular imaging of ovarian cancer because it is overexpressed in the early stages of disease and plays a role in tumor progression. Thus, we sought to (i) synthesize and evaluate a series of fluorine-containing, rofecoxib-based compounds as COX-1-selective inhibitors, and (ii) 18F-label the lead compound for detection of COX-1 expressing ovarian cancers by positron emission topography (PET). Results⎯Rofecoxib is a potent, time-dependent inhibitor of COX-2. As deletion of the sulfonylmethyl group of rofecoxib produces a weak COX-1 inhibitor, we decided to explore functionalization at that position. Thirty-seven compounds were synthesized and analyzed for COX-1 inhibitory activity and 3-(3-fluorophenyl)-4-(4-methoxyphenyl)furan-2(5H)-one (1) emerged as the most promising compound when tested against both purified COX-1 (IC50 = 0.36 μM) and COX-1 expressed in an ovarian cancer cell line (OVCAR-3; IC50 = 0.18 μM). Compound 1 did not inhibit COX-2. A molecular modeling (docking) study in COX-1 suggested that the fluoro group of compound 1 interacts with Ser-530 through a halogen bond, the 4-methoxy group forms a hydrophobic interaction with Ile-523 in the side pocket of the enzyme, and the furanone ring projects towards the constriction site comprised of Tyr-355 and Arg-120. Conclusions⎯We synthesized a series of fluorinated rofecoxib analogs and identified a lead compound (1) that is a selective and potent inhibitor of COX-1. The in vitro properties and modeling data suggest that 18F-1 will be a useful PET probe for early detection of COX-1 expressed in neoplastic diseases, such as ovarian cancer.

Francisco Guevara

Mentor:  David Wright
Department:  Chemistry
Home Institution:  University of Puerto Rico

“Fluorescenct Aptamer-Based Molecular Beacon with High Specificity Toward pfLDH Protein of Plasmodium falciparum”

Malaria is a disease caused by protists that belong to the Plasmodium genus; it is known to propagate in animals and humans circulatory system causing a wide range of symptoms and even death. Under-developed countries are the most affected by this parasite do to the limited access to diagnostic technology. A rapid and efficient diagnostic device is of great interest for low resource countries, to prevent continued expansion of this disease. Our lab is developing a molecular beacon with specificity toward the molecular biomarker, lactate dehydrogenase (pfLDH) of the parasite. The molecular beacon consists of two main regions, a DNA aptamer that recognizes a conserved region in the LDH protein and a small peptide sequence (AHHAHHAAD)₂ conjugated with an iridium fluorescent emitting probe (Ir(ppy)₂(H₂O)₂+). We have shown previous that this iridium probe selectively binds histidine to elicit a long-lived fluorescent signal. Using a monoclonal antibody that recognizes the pfLDH protein and the molecular beacon created, we can develop an efficient, long lasting and accessible ELISA-based assay suitable for low-resource areas.

Megumi Ito

Mentor:  Gary Sulikowski
Department:  Chemistry
Home Institution:  University of California, Berkeley

"Progress toward the total synthesis of Apoptolidinone C"

Natural products provide a large pool of biologically active molecules, a subset of which holds great promise as potent therapeutics. The activity of many natural products is elucidated by structure activity relationship studies where portions of the molecule are modified and the resulting changes in bioactivity are noted. Apoptolidin A is a macrolide known to be cytotoxic to specific cancerous cell lines and therefore is of interest as a therapeutic lead. However, its mechanism of action currently is unknown and must be defined before further development toward its application in medicine. Apoptolidin is glycoslylated at multiple sites and its glycosylation state has been found to have strong effects on its activity. In order to pursue structure activity relationship studies of apoptolidin, we are carrying out a total synthesis of the macrocycle to afford the aglycone, which can glycosylated to create a panel of apoptolidin analogues and employed in biological studies.

Rachel Kloecker

Mentor:  Sandra Rosenthal
Department:  Chemistry
Home Institution:  Allegheny College

“Differentiation of Neuro 2A cells to increase DAT expression” 

The dopamine transporter (DAT) is a membrane protein responsible for the reuptake of dopamine from neuronal synapses and has been linked to disorders including schizophrenia and Parkinson’s disease. Therefore, DAT is of high interest for drug studies and has been successfully labeled in transfected HEK cells using ligand-conjugated quantum dots. However, a more realistic model of naturally occurring DAT activity in neuronal cells is desired. In this study, we attempted to increase DAT expression by differentiating mouse Neuro 2A (N2A) neuroblastoma cells into dopaminergic neurons using dibutyryl cyclic adenosine monophosphate (dbcAMP). Fluorescence microscopy using IDT444 ligand-conjugated quantum dot assays demonstrated more labeling in dbcAMP-treated cells compared to controls. However, results varied depending on the percentage of differentiated cells per colony. Further experiments would include quantitation of the fluorescence using flow cytometry or monoclonal growth of selected cells to create a homozygous culture.

Kevin Koenders

Mentor:  Charles Hong
Department:  Medicine
Home Institution:  University of Rochester
 

“Synthesis of Selective Inhibitors of Phosphodiesterase-4 (PDE4)”

Phosphodiesterase-4 (PDE4) is a member of the cyclic nucleotide phosphodiesterase (PDE) superfamily responsible for the hydrolysis of cAMP1, a signaling mediator of a wide variety of cellular processes important to the central nervous system, inflammation, and respiratory physiology. Since PDE4 regulates cAMP concentration, it is considered an important therapeutic target. Recently, the laboratory of Dr. Charles Hong discovered the compound eggmanone based on its inhibitory effects on the hedgehog (Hh) signaling in vitro and in vivo. Target identification studies indicate that eggmanone functions by inhibiting PDE4, revealing a novel role of this enzyme in the hedgehog signaling downstream of the smoothened receptor. This is clinically relevant because, while most small molecule hedgehog inhibitors developed as anti-cancer agents are smoothened antagonists, many tumors already harbor mutations in the smoothened receptor that confer drug resistance. Therefore, drugs like eggmanone which targets a downstream Hh component may be useful for treating tumors resistant to smoothened inhibitors. My project involved synthesizing eggmanone analogs with improved biological and physical properties with regard to PDE4 inhibition. We synthesized libraries of related structures focusing on the derivatization of a secondary amine. Synthesis of the analogs involved a few different reactions that placed different functional groups including amide, sulfonamide, and reductive amination reactions. Each of the reactions also allowed for attachment of new functionalities with withdrawing or donating properties. After a pure product was made, in vivo and in vitro testing of the compounds was done. In vivo testing was performed using a zebrafish model, where particular phenotypes resembling that of the original hit eggmanone likely corresponded to inhibitor interaction with the enzyme. In vitro testing was done using a cell-based luciferase assay in which inhibition could be quantified using luminescence. In conclusion, novel PDE4 inhibitors with enhanced pharmacological properties may prove to be valuable as anti-cancer agents that selectively target the hedgehog signaling pathway.

Rhyan Martin

Mentor:  John McLean
Department:  Chemistry
Home Institution:  Georgetown College

“Development of a Collision Cross Section Database for Small Molecule Metabolites Using Ion-Mobility Mass Spectrometry (IM-MS)”

Metabolite structure and function are an essential part of drug discovery and development. Characterizing these molecules by ion-mobility mass spectrometry (IM-MS) provides structural information of the molecules based on the two-dimensional conformational space occupied within ion mobility drift cells. Gas-phase molecular collision cross section (CCS) within an ion-mobility drift cell can be described as the surface area of the molecule in which collision with gas molecules is possible. A set of standard primary metabolites were grouped based on solubility. The metabolites were analyzed using two different electrospray ionization (ESI)—IM-MS platforms: a prototype electrostatic field IM-MS instrument and an electrodynamic field IM-MS instrument. Mass-to-charge (m/z) values of the detected analytes were selected using commercial software which two-dimensionally plotted m/z and drift time through the ion mobility cell. Collision cross sections for the set of common small molecule metabolites were derived. A database of CCS values was obtained from both platforms using nitrogen buffer gas. We hypothesize a metabolite collision cross section database will give predictive power in characterizing the gas-phase confirmation space of unknown metabolites and assist in the future identification of these compounds, in particular when integrated with existing metabolite databases such as METLIN.

Bianca Ramirez

Mentor:  Gary Sulikowski
Department:  Chemistry
Home Institution:  Texas A&M

“Identification and Evaluation of Potential Circadian Rhythm Period Modifiers”

Within a 24-hour period, the body undergoes many physiological and metabolic changes that are all controlled by an intrinsic circadian timing system, the circadian clock. On a cellular level, the circadian clock controls daily oscillations in gene expression, which in turn, oscillate biological processes such as sleeping patterns, body temperature, and hormone secretion. A high throughput screen identified two scaffolds as potent period modifiers of the circadian rhythm. Through examination of structure activity relationship (SAR), analogs were identified that enhanced activity for the period shortening scaffold. The period shortening scaffold is currently being modified for photoaffinity labeling studies in order to identify its molecular target. Different structural analogs failed to improve activity for the period lengthening small molecule. Because the period lengthening scaffold is suspected to affect reactive oxygen species (ROS) in the cells, analogs which would delete the ability to cycle reactive oxygen species are being synthesized. If these analogs fail to induce any period effect in cells we will investigate the role of our period lengthening scaffold and ROS cycling in the circadian rhythm.

Tyree Ratcliff

Mentor:  Jens Meiler
Department:  Chemistry
Home Institution:  William Cary University

“Quantitative Structure Activity Relationship modeling for identification of C/EBP homologous protein (CHOP) inhibitors”

C/EBP homologous protein (CHOP) is an enhancer binding protein that regulates the unfolded protein response (UPR). UPR is a cellular stress response that occurs in the endoplasmic reticulum, due to an accumulation of unfolded or misfolded protein. UPR appears in a number of metabolic and cognitive disorders, such as diabetes, Alzheimer’s disease, and Parkinson’s. A high-throughput screening (HTS) assay, which is publically available through PubChem, identified active small molecule inhibitors of CHOP. These experimental actives were previously confirmed through confirmation screens that validated active compounds and counter-screens that validated that active compounds were selective for the given protein. Through ligand-based computer-aided drug discovery campaign, quantitative structure activity relationship (QSAR) models were developed using the experimental data as a knowledge base. The machine learning algorithm, an artificial neural network, was trained and applied to distinguish experimental active from inactive compounds. These neural networks emulate the human brain's ability to recognize patterns. The predictive power of the QSAR model was evaluated by means of the receiver operating characteristic (ROC) curve and enrichment. The ROC curve is a graph of the ANN’s ability to distinguish the true positive hits from true negative hits. Another metric that was used to evaluate the ANNs was enrichment, which is the rate of positives predictive values over the hit rate. The final cross-validated model achieved an area under the ROC curve of 79% and an enrichment of 50 fold. Through virtual screening of a large, commercially available compound library a chemically diverse set of active compounds were predicted and ranked representing potential new hit compounds of CHOP inhibitors. These hits were filtered by scaffold diversity and predicted activity for subsequent acquisition and experimental validation. Since the crystal structure of CHOP has yet to be determined, we used QUARK, a computer algorithm for ab initio protein folding and structure prediction, to obtain models. Currently the ligand binding sites are unknown for CHOP. Using these protein models and other low resolution techniques, we were able to identify possible ligand binding sites for further hit compound validation through ligand docking studies.

Ryan Reyes 

Mentor:  John McLean
Department:  Chemistry
Home Institution:  University of Arkansas, Monticello

“Enhancing Biomolecular Class Separation in Complex Sample Analysis Using Ion Mobility-Mass Spectrometry (IM-MS)”

Ion mobility mass spectrometry (IM-MS) is an emerging technique for addressing sample complexity in the field of bioanalytical chemistry.  With the advent of commercially available traveling wave ion mobility-mass spectrometry (TWIMS) instrumentation it is necessary to investigate the optimal instrumental parameters necessary for the study of complex biological samples.  Based on a mechanistic understanding of TWIMS, wave amplitude and velocity settings are known to affect measurement precision and resolving power differentially for different biomolecular classes of compounds.  In an attempt to determine a set of instrument parameters for optimal sample characterization, a set of carbohydrate, peptide, and quaternary ammonium salt standards were analyzed, and collisional cross section (CCS) values were obtained.  CCS describes the structural information inherent to IM-MS as it defines the apparent surface area of an ion.  The optimal experimental conditions were further validated for application to complex biological samples through the analysis of head and neck squamous cell carcinoma (HNSCC) cell line samples which contain a diverse representation of different biochemical classes.  Conclusions drawn in this study can be expanded on with further investigation into improving TWIMS resolution and accuracy by studying other factors known to enhance experimental precision and accuracy (e.g. IM drift gas pressure and composition), as well as the exploration of optimal conditions for other biologically relevant molecular classes such as lipids and oligonucleotides.