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2019 Summer Student Fellows

Student Fellow: Mark Smendik, Chemistry
Faculty Mentor: Dr. Daniel Adsmond, Arts, Sciences and Education, Physical Sciences
Project:  Using Carboxylic Acids to Induce Sulfameter Polymorphism

In our research, we studied polymorphs of sulfameter, an antibacterial drug. Polymorphs, which are different crystal packing arrangements of molecules, are of interest because they have differences in critical physical properties such as solubility in the bloodstream and shelf life. The most stable and most commonly observed polymorph of sulfameter is referred to as form I. In previous work, while attempting to grow co-crystals from solutions of sulfameter and benzilic acid, it was discovered that instead of obtaining the desired co-crystals, crystals of a less stable polymorph (form III) of the drug appeared. We wondered whether sulfameter could be induced to crystallize in form III even when adding a much smaller amount of benzylic acid. The goal of our work this past summer was to determine the smallest amount of acid required to induce the growth of form III from solution. Initially we grew crystals by evaporation of solutions containing equal amounts sulfameter and carboxylic acid. We then repeated the experiment multiple times, each time cutting the amount of acid until we were down to 1/100th the amount of drug. We analyzed the products using infrared spectroscopy (IR) to determine which form of the drug we had obtained. We discovered that under fast evaporation (2-4 days) the crystals obtained were usually a mix of both form I and form III, but under slow evaporation (9-24 days) we were more likely to obtain pure crystals. During our research we expected to eventually find a point where we added too little acid for form III crystals to form. We were surprised to find that form III appeared even when there was a hundred times more sulfameter than acid; although with trace amounts of acid, occasionally mixes of both sulfameter form I and III appeared. All together we studied three different acids: benzilic, hydrocinnamic, and 2-nitrobenzoic acid. All three acids produced similar results, although benzylic acid tended to create the purest crystals. Hydrocinnamic acid tended to create mixtures of both forms at higher concentrations of acid. Finally, trace amounts of 2-nitrobenzoic acid were sometimes trapped in the crystals.

Student Fellow: Tasha Vincent, Biochemistry
Faculty Mentor: Dr. Daniel Adsmond, Arts, Sciences and Education, Physical Sciences
Project: An Infrared and Crystallographic Study of the Phases in Competition with Ternary Cocrystals

Two or more different kinds of molecules can come together in the same crystal when there is an unusually strong attraction between them. Binary cocrystals, containing two different kinds of molecules in the same crystal, are common but the design and synthesis of ternary cocrystals, containing three different kinds of molecules in the same crystal, is much more challenging. Attempting to grow a ternary cocrystal from solution often results in two of the components forming a binary cocrystal leaving the third component still dissolved in solution. In practice, merely changing the solvent or the ratios of components to favor the ternary cocrystal is frequently an ineffective strategy. Therefore, the purpose of our research is to more easily form cocrystals by removing the solvent from the experimental procedure. We needed to reproduce known ternary and competing binary cocrystals from solution and confirm their composition by H1-NMR spectroscopy. By infrared spectroscopy, we established a fingerprint for comparison with the products of the solventless experiments. Then we aimed to form binary and ternary cocrystals in absence of solvent by simply grinding together a mixture of the two or three components, grinding them with a drop of solvent, or by melting them together. By comparing the infrared spectra to those of the confirmed cocrystals obtained from solution will determined the identity of the solventless products. Using carboxyphenols, acridine and aminopyrimidines we recreated 6 known binary cocrystals and 4 known ternary cocrystals in the absence of solvent. We discovered 11 binary cocrystal phases not previously obtained from solution. The technique of liquid assisted grinding was the most successful in reproducing our solution results.

Student Fellow: Joshua Matson, Biology/Pre-Pharmacy
Faculty Mentor: Dr. Felix Amissah, Pharmacy, Pharmaceutical Sciences
Project: Preliminary Investigation of the Pharmacological Effects of Dual EGFR and Aurora Kinase Inhibitors on A549 Cells

The American Cancer Society has estimated that 228,000 new cases of lung cancer will occur in 2019. A subset of these cases will be non-small cell lung cancers (NSCLC) that harbor mutated proteins involved in the uncontrolled regulation of KRAS signaling. These cases present a challenge to treat as previous investigations in directly targeting the continuously active KRAS proteins have not resulted in clinically useful drugs. Therefore, alternative approaches are required to regulate the abnormal KRASactivity for targeted treatment of these NSCLC cases. Both inhibition of epidermal growth factor receptor (EGFR) and Aurora Kinases A and B (AURKA and AURKB) have been shown to reduce NSCLC cell growth and proliferation. Inhibition of Aurora Kinases have been reported to indirectly inhibit KRAS activity leading to a decrease in cell division. In this project, we investigated the ability of EMRC4, a novel compound designed to inhibit EGFR, AURKA, and AURKB, to suppress NSCLC cell growth and proliferation. The effect of EMRC4 on the viability of KRAS mutant (A549) cells and EGFR mutant (NCI-H1975) cells, was determined and compared to reference drugs (Gefitinib, Alisertib and Barasertib). The project also identified the effect of EMRC4 on colony formation and expression of KRAS in A549 cells. Treatment of the lung cancer cells with EMRC4, resulted in concentration-dependent cell death in both A549 and NCI-H1975 cells. Colony forming assays also showed EMRC4 prevented regrowth of treated cells in a similar manner. Western Blot analysis indicated that EMRC4 reduced the expression of KRAS and AURKA protein levels relative to beta-actin in A549 cells. In addition, EMRC4 induced the phosphorylation of MAP kinase (ERK 1/2) supporting the concept of developing dual EGFR and Aurora kinase inhibitors as potentially useful anticancer agents.

Student Fellow: Jonathan Kendall, Optometry
Faculty Mentor: Dr. Alison Jenerou, Optometry
Project: Dynamic Visual Acuity Measurements:  Stationary vs Running

Background: Dynamic Visual Acuity (DVA) describes an individual’s ability to see a moving object. Previous studies have shown a post exercise related increase in detection DVA while others have shown no change in acuity post exercise. In addition, the faster the target of intent is moving the more recognition DVA declines. In studies involving moving subjects, biking or walking, Static Visual Acuity (SVA) generally decreased with increased subject speeds. The purpose of this study was to compare DVA and SVA before, during, and after running on a treadmill to determine how DVA and SVA changed.

Methods: Two acuity charts were designed, each using Sloan optotypes of LogMAR sizes. The SVA chart was similar in appearance to a standard Bailey-Lovie chart. The DVA chart was round, with lines of optotypes radiating from the center and affixed to a Bernell rotator trainer. While stationary, subjects were directed to read lines from the static chart followed by lines on the dynamic chart at 16rpm and 24rpm. Following a warmup, subjects ran on a treadmill until achieving a heart rate between 70-85% of the maximum recommended heart rate for their age. The subjects then repeated the sequence of acuity tests while running and again after stopping and allowing their heart rates to drop below 100 beats per minute.

Results:  While stationary and running, DVA performance was worse than SVA. Compared to being stationary, running worsened dynamic visual acuity at both rotation speeds (16rpm and 24rpm). There was no difference in performance for the different rotation speeds.

Conclusion: While running had a statistically significant negative affect on DVA, clinically it was approximately one letter of difference. The research did not demonstrate a post exercise boost in acuity for static or dynamic acuity post run.

Student Fellow: Kaitlin Assaad, Pharmacy
Faculty Mentor: Dr. Sonali Kurup, Pharmacy, Pharmaceutical Sciences
Project: Design and Synthesis of Novel 4-Substituted Pyrrolo [2, 3-d] Pyrimidines as Dual EGFR/AURKB Inhibitors

Epidermal growth factor receptor kinase (EGFR) inhibitors are approved in the treatment of lung, head and neck, and colorectal cancers. However, resistance to EGFR inhibitors has developed rapidly due to EGFR mutations and redundant signaling through mutant KRAS leading to treatment failures. Aurora kinase A (AURKA) and aurora kinase B (AURKB) are mitosis-related kinases that play a role in cell division and sustain processes that support tumor progression. Simultaneous inhibition of both EGFR and AURK have shown synergistic effects in the inhibition of tumor progression and in overcoming resistance to EGFR inhibitors. Compounds 1 and 2 incorporating a pyrrolo[2,3-d]pyridimidine scaffold were previously identified as dual inhibitors of EGFR and AURK. Compound 1 demonstrated sub-micromolar EGFR (0.44 uM) and single-digit micromolar AURKB (1.5 uM) inhibition. Compound 2 demonstrated single-digit micromolar EGFR (5.2 uM) and AURKA (3.4 uM) inhibition. Compound 3 demonstrated dual AURKA/AURKB inhibition with single-digit micromolar AURKA (1.2 uM) and sub-micromolar AURKB (0.47 uM) inhibition. This study describes the design and synthesis of compounds 4-8 as novel analogs of 1-3 and dual EGFR/AURK inhibitors. The main focus of this project is to optimally functionalize the pyrrolo[2,3-d]pyridimidine scaffold for improved potency and efficacy as dual EGFR/AURK inhibitors. Compounds 4-8 were synthesized via nucleophilic aromatic substitution and standard peptide coupling protocols using 4-chloropyrrolo[2,3-d]pyridimidine as the starting material. Reaction progress was monitored via thin-layer chromatography, and compounds were purified via flash chromatography. Compounds 4-8 and intermediates 9-12 were confirmed using nuclear magnetic resonance (NMR) and mass spectroscopy. Future research will focus on the evaluation of the effects of compounds 4-8 on EGFR, AURKA and AURKB inhibition, and as potential anticancer agents.

Student Fellow: Molly Von Seggern, Pharmacy
Faculty Mentor: Dr. Jennifer Lamberts, Pharmacy, Pharmaceutical Sciences
Effect of Naltrexone on Neuronal Cell Death

Parkinson’s disease (PD) is the fastest growing neurodegenerative disease in prevalence, disability, and death, and these numbers are likely to continue increasing due to industrialization, an aging population, and a general decrease in smoking.  Neuroinflammation is one pathogenetic component of PD, and it has the potential to be a major therapeutic target in treating the disease.  Naltrexone, which is an opioid antagonist indicated for opioid dependence, has anti-inflammatory properties at low doses, and has been suggested to reduce neuroinflammation in PD.  As a preliminary model for the cell death that occurs with neuroinflammation, we used the neurotoxin rotenone to induce cell death in murine N2a neuroblastoma cells.  We propose that low dose naltrexone will protect N2a cells from rotenone-induced injury.  To test our hypothesis, we grew N2a cells on two separate plates and differentiated the cells using serum deprivation and dbcAMP plus N2 neuronal supplement.  Cells were treated with either 10 uM rotenone only, 10 uM naltrexone only, 10 uM rotenone and 10 uM naltrexone, or buffer as the control.  One plate was stained with an antibody against βIII-tubulin, a cytoskeletal protein, and a marker for cell nuclei. The other plate was imaged without staining, and confluence was measured by taking the average percent confluence of 10 random coordinates in each well.  Based on these preliminary results, naltrexone appeared to protect the N2a cells from the damage done by the rotenone treatment.  Future experiments will aim to create cellular models of inflammation-induced neuronal injury using conditioned media from activated microglial cells, which will more accurately represent neuronal injury in PD.  These models will be treated with naltrexone to look at its effects on neuronal cell death.

Student Fellow:  Katelyn Brown, Biotechnology
Faculty Mentor: Dr. Eric Nybo, Pharmacy, Pharmaceutical Sciences
Project: Metabolic Engineering for Production of Novel Tetracenomycins

Streptomyces olivaceus produces the anthracycline anticancer agent elloramycin, which features an 8-O-glycosidically linked L-rhamnose sugar. Previously, the biosynthetic genes for production of elloramycin were cloned onto cosmid cos16F4 and heterologously expressed in Streptomyces lividans TK24 to produce the aglycone 8-demethyl tetracenomycin C (8-DMTC). Interestingly, cos16F4 lacks the biosynthetic genes to synthesize TDP-L-rhamnose, however, expression of a “sugar plasmid” encoding TDP-L-rhamnose biosynthesis in S. lividans (cos16F4) results in production of elloramycin. ElmGT has been shown to be a TDP-deoxysugar promiscuous glycosyltransferase responsible for the transfer of >20 different deoxysugar substrates to 8-DMTC, resulting in production of novel elloramycins with diversified anticancer activities. In this work, we optimized a heterologous polyketide glycosylation pathway via balancing of TDP-deoxysugar donor and polyketide acceptor substrates. First, we overexpressed the Streptomyces coelicolor acetyl-CoA carboxylase (accA2BE) genes to enhance malonyl-CoA production. We hypothesized that this would result in greater carbon flux towards synthesis of 8-DMTC. Secondly, we engineered S. lividans (cos16F4) with “sugar plasmids” for production of TDP-D-olivose, TDP-L-olivose, and TDP-L-rhamnose to determine production of glycosylated analogues. For this purpose, we expressed variant genes encoding TDP-D-glucose synthase and TDP-D-glucose- 4,6-dehydratase (desIII-desIV, mtmD-mtmE, and oleS-oleE) to evaluate their differing in vivo kinetic properties. Third, we cloned the optimal gene combinations in high-copy number (e.g. pWHM3-oriT) and low-copy number (e.g. pENTG1) vectors to determine the effect of gene dosage on polyketide glycosylation. This production platform opens the door for glycodiversification of other anthracycline natural products for the synthesis of improved analogs.

Student Fellow: Carlye Szarowicz, Biotechnology
Faculty Mentor: Dr. Schuyler Pike, Arts, Sciences and Education, Biological Sciences
Project: MALDI-TOF MS Method Development for Elucidation of Cannabinoid Receptors

Research on the endocannabinoid system (ES) is important for further elucidation of major regulatory functions and metabolic processes in the body. The ES is a biological signaling system comprised of cannabinoid receptors and their endogenous ligands known as endocannabinoids. Endocannabinoids are lipid-based neurotransmitters characterized by their ability to bind and activate membrane-anchored cannabinoid receptors. These receptors include cannabinoid receptor 1 (CB1), predominantly found in the central nervous system, and cannabinoid receptor 2 (CB2), primarily abundant in the immune system. The two most characterized endocannabinoids that bind CB1 and CB2 are 2-archyidonoylglycerol (2-AG) and N-arachidonoylethanolamide (anandamide, AEA). In the immune system, AEA regulates cytokine production, cell activation, and cell migration. The specific mechanisms of action, however, are still being elucidated, including the discovery of unknown receptors. To this end, the goal of this project is to develop a method using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identity CB1 and CB2 in immune cells. To implement this, the MALDI-TOF MS was calibrated using a standard calibration kit. After obtaining an incomplete CB1 peptide, CB1 yielded the accurate molecular weight with a detection limit of approximately 500 fmol/μL. C18 columns were then utilized to separate proteins and to determine the effect of AEA addition on elution time. Lastly, white blood cells were collected from laboratory rats and analyzed by MALDI-TOF MS for the presence of CB1. This method will be utilized in future studies to identify novel receptors of endogenous AEA in white blood cells.

Student Fellow: Matthew Swanson, Biotechnology
Faculty Mentor: Dr. Schuyler Pike, Arts, Sciences and Education, Biological Sciences
Project: Quantitative LC-MS/MS Method Development for Mithramycin

Mithramycin (MTM), also known as plicamycin, is an RNA synthesis inhibitor with anticancer activity. Recent studies have shown it to be effective for pediatric cancers such as Ewing sarcoma. However, MTM is also hepatotoxic, and there is a need to develop drug dosing regimens that yield serum drug concentrations that are effective while minimizing hepatic impact. Accordingly, a Shimadzu 8040 liquid chromatographer – mass spectrometer (LC-MS/MS) was used to elute a peak for detection of mass ionization. This allowed for a quantitative method to be developed for MTM over a broad range. The method was optimized to address MTM carryover in the autosampler focusing on a purge protocol with successive non-polar solvent washes. After autosampler purge development, 0.2% formic acid in both water and methanol were finalized as the aqueous and organic solvents, respectively, with a flow rate of 1ml/min and sample component separation accomplished through a C18 Phenomenex Prodigy 5u ODS-3V 100A 4.6mm x 200mm column. Subsequent work optimized the multiple reaction monitoring (MRM) program, tracking the precursor ion 1083.35 in negative mode which produced precursor to product fragments of: 1083.35>935.3, 1083.35>1039.5, and 1083.35>269.1. A final optimization was performed to determine the optimum interface voltage, dwell time, and distance between the ESI probe and corona needle of the mass spec. The method was found to have a quantifiable range of 1 ng/mL to above 50,000 ng/mL.

Student Fellow: Logan Rosneck, Biochemistry
Faculty Mentor: Dr. Luis Rivera, Arts, Sciences and Education, Physical Sciences
Project: Molecular Dynamics Thermal Partial-Denturation of the Amyloid Precursor Protein's C99 Transmembrane Domain

Every protein is characterized by a unique three-dimensional structure known as its native conformation. Proteopathy refers to a class of related diseases characterized by abnormalities in protein structure and function. Alzheimer’s Disease (AD) remains one of the most prevalent proteopathies today. This neurodegenerative disease is stimulated by overproduction of β-amyloid (Aβ) protein. Aβ aggregates to form hard, insoluble plaques in the brain, thereby exhibiting the pathology associated with AD. The amyloid precursor protein (APP) is a small helical transmembrane protein crucial for understanding AD. Membrane tethered aspartyl protease, β-secretase, cleaves APP, resulting in a 99-residue C-terminal fragment (C99) which is further processed by γ-secretase along its transmembrane domain (TMD) to generate Aβ. The AMBER molecular dynamics (MD) software was used to study the thermodynamic denaturation pathway of APP along its C99 TMD. This peptide was modeled in both an aqueous solution and membrane-mimetic environment. The aqueous system was heated from 300 K to 400 K using 50 K increments. The membrane-mimetic system was heated from 300 K to 500 K using 50 K increments. Constant temperatures were maintained at each 50 K increment during prolonged 5.0 ns production simulations. Partial denaturation in both systems occurred within the precise region γ-secretase would otherwise bind, a result which indicates this region is an important target for drug development and therapeutic treatment of AD. Thermal melting of the C99 TMD occurred around 330 K. Denaturation in the membrane-mimetic environment proved more thermodynamically stable than in aqueous solution, a result which could be extrapolated to a generalized class of integral proteins. Overall, this research provided computational support to existing experimental studies evaluating the melting temperature of APP, and revealed important information pertaining to the unfolding pathway of a peptide central to our understanding of AD.

Student Fellow: Aidan Reynolds, Biochemistry
Faculty Mentor: Dr. Luis Rivera, Arts, Sciences and Education, Physical Sciences
Project: Structural Survey of Zinc Finger Protein NEMO via Molecular Dynamics Simulations

Metalloproteins are proteins that contain metal ions and account for nearly half of all proteins in biology. They are responsible for catalyzing some of the most difficult and important cellular functions. Zinc-containing metalloproteins or “zinc fingers” are the second most abundant metalloprotein within cells. The synthesis of these zinc-containing proteins begins with the creation of a polypeptide chain which then folds itself into a conformation that optimizes its binding affinity to a zinc ion, followed by the subsequent binding of the zinc ion. The zinc finger protein NEMO was studied due to its biological importance as a regulatory zinc finger in the NF-kB signaling pathway which is responsible for initiating cell death via apoptosis or necrosis in response to oncogenesis. In this study, AMBER molecular dynamics software was used to examine the thermostability of NEMO prior to accepting a zinc ion to illustrate the duration that NEMO can maintain an optimal structure for zinc-binding before denaturing. NEMO was modeled with and without its zinc ion using the Zinc AMBER Force Field and solvated with water molecules. Simulations for NEMO with its zinc ion were run at 350K for 2 ns. Simulations for NEMO without its zinc ion were run at 350K for 2 ns, 350K for 5 ns, and at physiological conditions for 5 nanoseconds. Simulations involving NEMO with its zinc ion bound showed no significant denaturation due to the flexibility of the tetrahedral coordination of the zinc ion at its core, whereas NEMO without its zinc ion showed significant denaturation and loss of function at 350K. At 310K, NEMO without its zinc ion denatured within 0.6 ns, losing all affinity for zinc. This indicates that for NEMO to form in physiological conditions, it must bind to a zinc ion within 0.6 ns before it denatures irreversibly and loses affinity for zinc and function. The results of this study are key to further examining the synthesis of NEMO as well as other zinc-containing small proteins by illustrating how quickly the zinc ion must bind to create a functional protein.

Student Fellow: Cole McGowen, Biology – Environmental
Faculty Mentor: Dr. Anne Spain, Arts, Sciences and Education, Biological Sciences
Project: Characterization of the Diversity and Ecology of the Plant and Microbial Communities in a Central Michigan Bog

Sphagnum-dominated peat bogs are a common occurrence among the numerous types of ecosystems in the Northern Hemisphere, and many can be found right here in Michigan. Bogs are classified as extreme, “desert like” environments due to the high levels of acidity, low levels of available nutrients, low temperatures, and low abundance of flora and fauna. Despite these conditions, peat bogs play a crucial ecological role housing and storing organic carbon and greenhouse gasses. Unfortunately, the microbial communities of peat bogs are understudied, and the roles of these microbes are largely unknown. The diversity and abundance of plant specimens located in peat bog ecosystems varies on the available nutrients and the acidity of the water. Due to these parameters, some plant species have evolved interesting characteristics in order to survive. This experiment aimed to characterize both the microbial and plant communities in a bog of Central Michigan (located near Rodney, MI) to then later be used to determine and understand the roles of each species found. From three different locations, plant species were identified, and peat and water samples were collected in triplicate. Water samples were filter sterilized on site and used for pH and anion analysis. DNA was extracted from the peat and used for PCR with primers specialized to copy the 16S rRNA gene (a common gene used to determine species identity) from bacteria within the sample. Gel Electrophoresis of the PCR product showed that the correct gene fragment was copied from all nine samples. Purified PCR products and water samples were shipped to The University of Oklahoma for sequencing and anion analysis, respectively. Plant community analyses revealed that the plant with the highest abundance between all sample locations is the Common Spike Rush, accounting for 47% of the total vegetative abundance. From this experiment, we will have a better understanding of what species of microbes and plants are typically found in peat bogs and the overall role they have in making the peat bog ecosystem the unique, yet ecologically important environment they are today.