The 57th Annual MIKIW Meeting-in-Miniature

April 12th – 14th, 2019 The University of Kansas School of Pharmacy Lawrence, KS

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Table of Contents Welcome Letter ……………………………………………………………………3

MIKIW History ……………………………………………………………………4

Acknowledgements……………….………………………………………………..6

Schedule………………………….………………………………………………...9

57th Annual MIKIW Meeting Keynote Speaker …...……………………………..12

Keynote Lecture Abstract ………………………….……………………………..12

Oral Presentations and Abstracts …………………...…………………………….13

Poster Presentations (Poster Number, Name, University, Title).………….……...27

Conference Locations ………………………………………….……..…………..31

Maps of Integrated Science Building (ISB) ………………….………………..…32

Poster Abstracts …………………………………………………………………..33

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Welcome to the 57th Annual MIKIW Meeting!

On behalf of the Department of Medicinal Chemistry at The University of Kansas, we welcome you to Lawrence, Kansas and the 57th Annual MIKIW Meeting-in-Miniature! This weekend provides a unique opportunity to reacquaint with old friends and continue to expand your network in a dynamic and collaborative environment. We look forward to hosting such a brilliant group of young scientists. We also thank you for your scientific contributions to the meeting, because without those we would not be here!

Since its inauguration in 1963, this Meeting-in-Miniature has provided graduate students from the universities of Minnesota, Illinois at Chicago, Kansas, and Iowa with the opportunity to participate in a larger scientific community by organizing and hosting the conference. In 2018 the conference expanded as we welcomed The University of Wisconsin-Madison. The location of the meeting rotates between one of the five participating schools each year, with a full cycle completed every five years. In addition to allowing students to present their research, MIKIW allows both faculty and students to exchange ideas, learn about other fields, and expand their network.

The MIKIW Keynote Address has been presented by world-class scientists, including multiple Nobel Prize winners and ACS Division of Medicinal Chemistry Hall of Fame Inductees. This year, the keynote lecture will be presented by Dr. Nicholas Meanwell, Executive Director of Bristol Myers Squibb, who has extensive industrial experience in drug discovery and development of antiviral agents.

We hope that you all enjoy the meeting, encourage others, and learn something new!

Sincerely,

Jacob Sorrentino, Chair

On Behalf of the MIKIW 2019 Organizing Committee

2019 MIKIW Organizing Committee

Angelo Andres Sahishna Phaniraj Yuwen “Alicia” Yin

Additional Members of the 2019 MIKIW Organizing Team

Bo Han Patrick Ross Zechariah De Silva Hans Tchienga Tomas Smith Matt McCurry Shweta Malvankar Manvendra Singh Jacob Immel Andrew Intelli Alonso Rodriguez Ugalde

Faculty Chair: Ryan Altman, Ph.D.

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Brief History of MIKIW

The first Medicinal Chemistry Meeting-in-Miniature was organized by faculty from the Departments of Medicinal Chemistry and Pharmacognosy at the Universities of Minnesota, Illinois at Chicago, Kansas, and Iowa in 1963. Since then, the Departments of Medicinal Chemistry and Pharmacognosy at these universities have alternately hosted the MIKI meeting. MIKI is the longest running and largest regional medicinal chemistry meeting in the U.S., and brings together over 200 graduate students, post-doctoral fellows, and faculty members from the four universities on an annual basis. In 2018, the University of Wisconsin-Madison joined MIKI, giving the conference the new name MIKIW. This year, the graduate students at The University of Kansas are excited to host the 57th Annual MIKIW Meeting and continue this great tradition.

Graduate students from the hosting university organize and moderate the entire meeting, providing the valuable experience of coordinating a professional event. In addition, participating students receive professional experiences by delivering oral and/or poster presentations, as well as vital networking opportunities. Furthermore, these annual meetings also act as a catalyst for sharing and exchanging ideas among students, postdoctoral researchers, and faculty of the different universities. The meeting features a keynote address from a distinguished scientist in academia, industry, or government, who is a leader in the field of medicinal chemistry.

List of Previous Keynote Speakers Year Host Speaker Association 2019 Kansas Dr. Nicholas Meanwell Bristol Myers Squibb 2018 Illinois Dr. Michelle Arkin University of California, San Francisco 2017 Minnesota Dr. Uttam Tambar University of Texas 2016 Iowa Dr. Amy H. Newman NIH-NIDA 2015 Kansas Dr. Bruce D. Roth Genentech Inc. 2014 Illinois Dr. Paul A. Wender Stanford University 2014 Minnesota Dr. Marvin J. Miller University of Notre Dame 2013 Iowa Dr. Heidi E. Hamm Vanderbilt University 2012 Kansas Dr. Dennis C. Liotta Emory University 2011 Illinois Dr. Tomáš Hudlický Brock University 2010 Minnesota Dr. Dale Boger Scripps Research Institute 2009 Iowa Dr. Daniel Kahne Harvard University 2008 Kansas Dr. Albert Padwa Emory University 2007 Illinois Dr. William Fenical University of California, San Diego 2006 Minnesota Dr. Christopher Lipinski Pfizer Pharmaceuticals 2005 Iowa Dr. Kenner Rice National Institutes of Health

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2004 Kansas Dr. C. Dale Poulter University of Utah 2003 Illinois Dr. Richard B. Silverman Northwestern University 2002 Minnesota Dr. Andrew Hamilton Yale University 2001 Iowa Dr. Michael Marletta University of Michigan 2000 Kansas Dr. Roger M. Friedinger Merck Research Laboratories 1999 Illinois Dr. Richard A. Lerner Scripps Research Institute 1998 Minnesota Dr. John Montgomery Biocryst Pharmaceutical, Inc. 1997 Iowa Dr. David Nichols Purdue University 1996 Kansas Dr. Paul Anderson Dupont Merck Pharmaceutical 1995 Illinois Dr. Arthur Patchett Merck Research Laboratories 1994 Minnesota Dr. Daniel Rich University of Wisconsin, Madison 1993 Iowa Dr. Laurence Hurley University of Texas, Austin 1992 Kansas Dr. Julius Rebek Massachusetts Institute of Technology 1991 Illinois Dr. Koji Nakanishi Columbia University 1990 Minnesota Dr. John Katzenellenbogen University of Illinois, Urbana-Champaign 1989 Iowa Dr. Carl Djerassi Stanford University 1988 Kansas Dr. William Roush Indiana University 1987 Illinois Dr Joseph Fried University of Chicago 1986 Minnesota Dr. David Triggle SUNY Buffalo 1985 Iowa Dr. Alan Katritzky University of Florida 1984 Wisconsin Dr. Paul Bartlett University of California, Berkeley 1983 Kansas Dr. Henry Rapoport University of California, Berkeley 1982 Illinois Dr. Harry Wasserman Yale University 1981 Minnesota Dr. Eugene Jorgensen University of California, San Francisco 1980 Iowa Dr. Alan Sartorelli Yale University 1979 Kansas Dr. Albert Meyers Colorado State University 1978 Illinois Dr. Heinz Floss Purdue University 1977 Minnesota Dr. Donald Jerina National Institutes of Health 1976 Iowa Dr. Everett May National Institutes of Health 1975 Kansas Dr. Marjorie Horning Baylor University 1974 Illinois Dr. Arnold Brossi Hoffman-LaRoche 1973 Minnesota Dr. Gertrude Ellion Burroughs-Wellcome 1972 Iowa Dr. Bernard Belleau University of Ottawa 1971 Kansas Dr. Corwin Hansch Pomona College 1970 Illinois Dr. Everett Maynert University of Illinois 1969 Minnesota Dr. Bernard Baker University of California, Santa Barbara 1968 Iowa Dr. Julius Axelrod National Institutes of Health 1967 Kansas Dr. Richard Schowen University of Kansas

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Thanks to our MIKIW 2019 Sponsors! Bronze and Silver Level Sponsors

Sched

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Platinum Level Sponsors

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Gold Level Sponsors

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Schedule

Friday, April 12, 2019 3:00 – 6:00 p.m. • Registration open Springhill Suites » • Hotel check-in - Minnesota and Illinois Quality Inn and Suites » - Iowa Days Inn » - Wisconsin 7:00 p.m. Welcome reception Arterra Event Gallery

Saturday, April 13, 2019 8:00 – 8:40 a.m. Breakfast and poster setup ISB Atrium

8:40 – 9:00 a.m. Opening remarks ISB Auditorium

Oral presentation session 1: ISB Auditorium Moderators: Patrick Ross, Bo Han

9:00 – 9:25 a.m. Yuwen Yin Blake Peterson lab, KU Phorbol Carbamates as Novel Tools for Controlling Cellular Signal Transduction

9:25 – 9:50 a.m. Arturo L. Aguirre Robert Kerns lab, UI Investigating quinazoline-2,4-dione and fluoroquinolone scaffolds for antibiotic activity and metabolic stability

9:50 – 10:15 a.m. Stephanie Blaszczyk Weiping Tang lab, UW-Madison S-Adamantyl Group Directed Site-Selective Acylation and Its Applications in the Streamlined Assembly of Oligosaccharides

10:15 – 10:40 a.m. Kellan T. Passow Daniel Harki lab, UMN The Discovery of 4CIN: Indoles as a Platform for Novel Nucleoside Fluorophore Development

10:40 –11:00 a.m. Break ISB Atrium

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11:00 – noon Keynote address ISB Auditorium

12:00 – 1:30 p.m. Lunch and poster setup ISB Atrium

Oral presentation session 2: ISB Auditorium Moderators: Sahishna Phaniraj, Bo Han

1:30 – 1:55 p.m. Jacob P. Sorrentino Ryan Altman lab, KU Fluorinated Dextromethorphan Analogs to Prevent the Formation of Psychoactive Metabolites

1:55 – 2:20 p.m. Brian P. David Terry Moore lab, UIC Structure-Activity Relationship and Evaluation of Non-electrophilic NRF2 Activators in a Diabetic Mouse Model

2:20 – 2:45 p.m. Muhammad M. Khalifa, Jennifer Golden lab, UW-Madison Convergent Annulative Platform for the Synthesis of Ring-Fused Quinolinones

2:45 – 3:45 p.m. Poster session: Odd numbered posters present ISB Atrium

3:45 – 4:45 p.m. Poster session: Even numbered posters present ISB Atrium

4:45 – 5:15 p.m. Faculty meeting ISB 1160

6:00 – 7:00 p.m. Faculty mixer (open bar) Van Go inc.

7:00 p.m. Formal banquet dinner Van Go inc.

Sunday, April 14, 2019 Check-out of hotel prior to breakfast

8:00 – 8:40 a.m. Breakfast ISB Atrium

Oral presentation session 3: ISB Auditorium Moderators: Patrick Ross, Bo Han

8:40 – 9:05 a.m. Erick J. Carlson Gunda Georg lab, UMN The Discovery and Characterization of Steroidal Blockers of CatSper

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9:05 – 9:30 a.m. Alanna Condren Laura Sanchez lab, UIC Biofilm inhibition alters specialized metabolism and increases virulence in Pseudomonas aeruginosa

9:30 – 9:55 a.m. Sanjay Bhattarai Michael Wolfe lab, KU Substrate transmembrane mimetic inhibitors of gamma-secretase: probes for chemical structural biology

9:55 – 10:20 a.m. Pratik Rajesh Chheda, Robert Kerns lab, UI Allosteric inhibitors of apicoplast DNA polymerase: New antimalarials that bind a novel allosteric binding site

10:20 –10:40 a.m. Break ISB Atrium

Oral presentation session 4: ISB Auditorium Moderators: Tomas Smith, Bo Han

10:40 – 11:05 p.m. Matthew Worth Jiaoyang Jiang lab, UW-Madison Covalent Inhibitors Target O-GlcNAc Transferase in Cells

11:05 – 11:30 p.m. Ozgun Kilic Carston Rick Wagner lab, UMN Nanorings with Engineered Fibronectins for Cancer Therapeutics

11:30 – 11:55 p.m. Katherine E. Zink, Laura Sanchez lab, UIC A novel imaging mass spectrometry method for identifying preventative approaches in high grade serous ovarian cancer

12:00 p.m. • Concluding remarks ISB Atrium • Box lunch served • Buses depart

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KEYNOTE SPEAKER Nicholas A. Meanwell, Ph.D., FRSC Dept. of Chemistry and Molecular Technologies, Bristol-Myers Squibb Research and Development Nicholas A. Meanwell received his B.Sc. in Special Honors chemistry from the University of Sheffield, Sheffield, England in 1976. In 1979, he earned a Ph.D. degree from the University of Sheffield under the direction of Dr. D. Neville Jones. From 1979-1982, Dr. Meanwell conducted post-doctoral training under the supervision of Professor Carl R. Johnson at Wayne State University, Detroit, MI. In 1982, Dr. Meanwell joined Bristol-Myers Squibb where he led teams in the cardiovascular therapeutic area that developed a series selective phosphodiesterase III inhibitors. From 1991-1994, Dr. Meanwell contributed to CNS drug discovery efforts, leading the early phase team that explored the potential of Ca2+-dependent potassium channel modulators, a program that ultimately produced the Maxi K ion channel opener flindokalner (MaxiPostTM), which was advanced into Phase III clinical trials for the treatment of stroke. In 1994, Dr. Meanwell assumed responsibility for antiviral drug discovery chemistry with the objective of establishing programs directed toward the discovery and development of inhibitors of human immunodeficiency virus-1 (HIV-1), hepatitis C virus (HCV) and respiratory viruses, including influenza and respiratory syncytial virus (RSV). Dr. Meanwell is the author/co-author of over 200 publications, review articles, book chapters and editorials and more than 170 meeting abstracts. He is named as an inventor/co-inventor of more than 110 granted U.S. Patents. In 2014, Dr. Meanwell was recognized for outstanding research in the area of HIV/AIDS as a co-recipient of a PhRMA Research and Hope Award for Biopharmaceutical Industry Research for the discovery and development of HIV-1 attachment inhibitors. In 2015, Dr. Meanwell received the 2015 Philip S. Portoghese Journal of Medicinal Chemistry/ACS Division of Medicinal Chemistry Joint Lectureship Award. He was named to the American Chemical Society Medicinal Chemistry Hall of Fame in August 2015. Keynote Lecture: “Inhibitors of HIV-1 Maturation” Abstract: Maturation of the HIV-1 capsid (CA) peptide by HIV-1 protease-mediated cleavage at the CA- SP1 junction is the final step in the formation of infectious virus capsid. A phenotypic screen identified the betulinic acid derivative bevirimat as an inhibitor specific to this step in virion production and this compound demonstrated antiviral effects in Phase 2a clinical trials. However, naturally-occurring polymorphisms proximal to the cleavage site conferred much reduced sensitivity to the effects of bevirimat in vitro which correlated with the clinical response. Guided by assays incorporating the natural polymorphisms, we identified GSK-3532795/BMS-955176 as a second generation HIV-1 maturation inhibitor that exhibits a broader spectrum of antiviral effect in vitro that translated into improved efficacy in clinical trials. In this presentation, we will describe the strategy and tactics adopted by the drug discovery team that led to the discovery of GSK-3532795/BMS-955176, the antiviral and pharmacokinetic profiles of the molecule and the results of early clinical studies.

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Oral Presentations and Abstracts Session 1

Phorbol Carbamates as Novel Tools for Controlling Cellular Signal Transduction Yuwen Yin and Blake Peterson The University of Kansas

Mammalian cells sense the extracellular environment by expressing cell surface receptors that transmit signals across cellular plasma membranes. Some of these receptors trigger cellular signal transduction by activating phospholipase C (PLC) at the inner leaflet of the plasma membrane. When activated, this enzyme cleaves the lipid phosphatidylinositol-4,5-bisphosphate (PIP2) to generate the second messengers inositol triphosphate (IP3) and diacylglycerol (DAG). These second messengers are linked to both normal cellular signaling and pathological signaling cascades that involve the Protein Kinase C (PKC) family of enzymes. Pathologies associated with PKC include cancer and inflammation, and chemical tools that modulate these enzymes are under investigation as therapeutics and molecular probes. One potent class of chemical activators of PKC are the phorbol esters, natural products that mimic DAG to activate PKC. However, because they are esters, these natural products exhibit metabolic liabilities that can limit their utility as molecular probes. As an approach to improve the chemical stability of derivatives of phorbol, we describe here the synthesis of the first phorbol carbamates. The biological properties of phorbol carbamates and efforts to develop new approaches to control PKC-mediated signaling pathways with these compounds will be discussed.

Keywords: Protein Kinase C (PKC), Protein translocation, Signaling

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Investigating Quinazoline-2,4-Dione and Fluoroquinolone Scaffolds for Antibiotic Activity And Metabolic Stability Arturo L. Aguirre and Robert J. Kerns The University of Iowa

Fluoroquinolones are a class of antibiotics used clinically to treat a wide array of bacterial infections. Fluoroquinolones act by forming a ternary complex with bacterial type II topoisomerases (DNA gyrase or topoisomerase IV) and nicked DNA; religation of DNA is subsequently blocked. In ternary complex the keto-acid moiety of the fluoroquinolone is complexed with a divalent magnesium ion, forming a drug- magnesium-water bridge to a serine and/or an aspartate/glutamate residue on helix-4 of the topoisomerase enzyme. Mutation-mediated resistance arises through substitution of the serine or aspartate/glutamate residues, therefore preventing formation of the magnesium-water bridge and reducing stability of the cleaved complex and diminishing the activity of the fluoroquinolone to block DNA religation. Quinazoline-2,4-diones are structurally similar to fluoroquinolones; diones form ternary complex similar to fluoroquinolones, however, these complexes are less stable because the quinazoline-2,4-diones do not form a magnesium-water bridge to helix-4. While diones are therefore less potent at inhibiting topoisomerase function, their non-reliance on the magnesium water bridge generally affords equipotent activity with wild-type and fluoroquinolone-resistant strains of bacteria. We hypothesized that the quinazoline-2,4-dione structural scaffold, along with drug-topoisomerase co-crystal structures, would provide a structural basis upon which to generate novel inhibitors of bacterial type-II topoisomerases that act on wild-type and fluoroquinolone resistant bacteria. Moreover, commercially available fluoroquinolones are known to be metabolically stable, however, the fluoroquinolone scaffold has been modified significantly to warrant metabolic stability studies with both novel fluoroquinolones and quinazoline-2,4-diones. In this presentation, the design, synthesis, and metabolic stability of novel quinazoline-2,4-dione derivatives, will be discussed.

Keywords: Antibiotics, Resistance, Metabolism

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S-Adamantyl Group Directed Site-Selective Acylation and Its Applications in the Streamlined Assembly of Oligosaccharides Stephanie Blaszczyk and Weiping Tang University of Wisconsin

Carbohydrates are structurally diverse and synthetically challenging molecules that play indispensable roles in biological processes, including growth, development, inflammation, infection and cell signaling. Chemists' inability to efficiently synthesize complex carbohydrates hinders biologists' ability to probe how their structural differences affect biological function. As such, the site-selective functionalization of carbohydrates is an active area of research. We previously reported that a cation-lone pair interaction can direct site-selective acylation in O-glycoside trans-1,2-diols using a chiral catalyst. We speculated that we could selectively functionalize S-glycosides via a similar interaction. Surprisingly, the sterically bulky adamantyl substituent of the S-glycoside directed site-selective acylation at the C2 position of S- glycosides through dispersion interactions between the adamantyl C-H bonds and the π-system of the cationic acylated catalyst. This interaction may have broad implications in many other chemical reactions. Because of their stability, chemical orthogonality, and ease of activation for glycosylation, the site- selective acylation of S-glycosides significantly streamlines oligosaccharide synthesis. This methodology will have broad applications in the synthesis of complex carbohydrates for basic research and drug discovery.

Keywords: Carbohydrates, Site-Selective Functionalization, Glycosylation

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The Discovery of 4CIN: Indoles as a Platform for Novel Nucleoside Fluorophore Development Kellan T. Passow and Daniel A. Harki University of Minnesota

Fluorescent chemical probes are an integral part of modern biological studies, where the labeling of a protein, nucleic acid, or small molecule enables a wide range of mechanistic experiments. The Harki lab aims to develop such tools, with a focus on non-natural nucleosides, nucleotides, and oligonucleotides. This talk will focus on the development of novel isomorphic nucleoside fluorophores containing substituted indole nucleobases, including 4-cyanoindole (4CI). Indole nucleobases are an ideal scaffold for this application given their ease of derivatization and nucleoside synthesis. Moreover, their structural tuneability renders them ideal for substituting purine nucleobases in oligonucleotides. We have recently reported (Passow and Harki, Org. Lett., 2018, 20, 4310) the fluorescent nucleoside 4-cyanoindole-2ʹ- deoxyribonucleoside (4CIN), which displays impressive fluorescent properties both as a monomer and in DNA. 4CIN significantly outperforms the literature standard 2-aminopurine nucleoside (2APN) that experiences significant fluorescence quenching when incorporated into DNA. Given the relative simplicity of the 4CI fluorophore, we hypothesized that other substituted indoles would yield useful fluorophores, as well as provide opportunities to install other chemical reactivity. The synthesis and spectroscopic evaluation of these novel nucleoside fluorophores will be presented.

Keywords: Nucleosides, Fluorescence, 4-Cyanoindole

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Session 2

Fluorinated Dextromethorphan Analogs to Prevent the Formation of Psychoactive Metabolites Jacob P. Sorrentino, Brett R. Ambler and Ryan A. Altman The University of Kansas

Aryl-methyl ethers, while present in many bioactive compounds, are subject to rapid O-dealkylation that can generate biologically inactive or toxic metabolites. In this process, P450-mediated oxidation generates a formyl hemi-acetal intermediate that subsequently undergoes hydrolysis to reveal a phenol. To combat this process, the strategic use of fluorinated substituents can raise the energy barrier P450 mediated oxidation, which in turn, can reduced metabolic liabilities. As an example, the cough suppressant dextromethorphan undergoes such a P450 mediated O-dealkylation to provide the psychoactive phenolic metabolite dextrorphan. This metabolite antagonizes the NMDA receptor causing hallucinations, which encourages recreational abuse. To circumvent this undesired metabolism, we have designed, synthesized, and evaluated in vitro and in vivo new fluorinated analogs of dextromethorphan with improved pharmacokinetic and pharmacodynamic profiles. Evaluation of the pharmacodynamic profile revealed that select compounds in the series bound with nanomolar affinity to monoamine transporters that are known targets for treating depression and anxiety disorders, as well as other CNS disease states. These CNS target profiles are more selective than many approved drugs. Moreover, these fluorinated analogs display improved pharmacokinetic profiles relative to dextromethorphan and related analogs currently in clinical trials. Though synthetic access to these compounds has provided sufficient material for initial pharmacological characterization, more efficient syntheses are required for translational proof-of-concept in vivo studies.

Keywords: Dextromethorphan, Fluorination, Metabolism

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Structure-Activity Relationship and Evaluation of Non-electrophilic NRF2 Activators in a Diabetic Mouse Model Brian P. David and Terry W. Moore University of Illinois at Chicago

Chronic, non-healing wounds of the skin are serious medical complications that affect over 6.5 million Americans, especially diabetic patients and older adults. Strikingly, there are almost no pharmaceutical therapies to accelerate healing of chronic wounds. A major contributor to chronic wounds is a prolonged inflammatory state, and reducing inflammation may be a way to accelerate healing of chronic wounds. Unfortunately, many commonly used anti-inflammatory drugs hinder chronic wound healing; thus, new ways of reducing inflammation are needed. An essential factor in wound healing and an important regulator of inflammation, NRF2 is a transcription factor that induces many cytoprotective and antioxidant genes. As seen in other inflammatory disorders, activating NRF2 could be a useful therapeutic strategy to treat chronic skin wounds. Non-covalent NRF2 activators can be developed by inhibiting the interaction of NRF2 with its negative regulator, KEAP1. We have developed a structure-activity relationship around a known, naphthalene-based non-covalent NRF2 activator, to create a NRF2 activator based on an isoquinoline scaffold. This work highlights some of the SAR work of the peripheral aryl rings, in the hopes of increasing metabolic stability, as well as positive charge to balance the overall charge of our lead compounds. The compounds’ affinities are tested in a fluorescence anisotropy assay. Western blot data shows compounds increase protein levels associated with NRF2. Using a mouse model, we show that one of these isoquinolines can decrease the time to wound healing in a mouse model.

Keywords: Wound Healing, Protein-Protein Interaction

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Convergent Annulative Platform for the Synthesis of Ring-Fused Quinolinones Muhammad M. Khalifa, Satish Chandra Philkhana and Jennifer E. Golden University of Wisconsin

The quinolinone motif is a privileged structure associated with a broad spectrum of reported pharmacology. As part of a program aimed at the efficient assembly of novel, bioactive scaffolds, we developed a convergent synthetic platform to construct a diverse set of novel, ring-fused, N- alkylquinolinones. Using enolizable starting materials spanning a wide range of pKa values (pKa ~ 3.3 - 20.7) such as tetramic or tetronic acids, 1,3-diones, or cycloalkanones in combination with a variety of isatoic anhydrides, over 80 desired annulation products were obtained in yields of up to 95%. Reaction efficiency was driven by three main factors: maintaining stability of the anhydride in the presence of base, promoting complete deprotonation of the enolizable partner, and minimizing product-related side reactions. The method was then applied to the synthesis of penicinotam, an insecticidal natural product derivative. The protocol resulted in the shortest and highest yielding total synthesis to date of this - lactam-fused, N-methylquinolinone in 3 steps and 36% overall yield.

Keywords: Methodology Development, Annulation, Privileged Structure

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Session 3

The Discovery and Characterization of Steroidal Blockers of CatSper Erick J. Carlson, Brian T. Gabet, Melissa R. Miller, Polina V. Lishko, Jon E. Hawkinson and Gunda I. Georg University of Minnesota

The cation channel of sperm (CatSper) is the principal entry point for calcium in mature spermatozoa. CatSper knockout mice are completely infertile with no other observable phenotypes, and genomics studies in men have shown its requirement in humans. CatSper is activated by progesterone and prostaglandin E¬1 (PGE¬1). Once bound, these compounds elicit a large calcium influx into the flagellum of the sperm, which propagates towards the head and induces hyperactivated motility (HAM). HAM is required to penetrate the viscous fluid of the upper reproductive tract and the zona pellucida surrounding the egg. By systematically modifying the steroid backbone of progesterone we have discovered three steroidal compounds that block the progesterone-induced calcium influx observed in a fluorescence-based assay. The details of the discovery and characterization of these blockers, as well as differences to t-type calcium channel blockers, will be discussed.

Keywords: Ion Channels, Contraception

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Biofilm Inhibition Alters Specialized Metabolism and Increases Virulence in Pseudomonas Aeruginosa Alanna Condren and Laura Sanchez University of Illinois at Chicago

Biofilm inhibition by exogenous molecules has been an attractive target for the development of novel therapeutics. Previous studies have shown that taurine-conjugated bile acids, such as taurolithocholic acid (TLCA), have biofilm inhibition activity in vitro. However, the mechanism of action of these acids is still unknown. We hypothesize that the presence of taurine-conjugated bile acids creates an environment that induces susceptible bacteria to alter their specialized metabolism, leading to the observed bioactivity. Thus, we have investigated the impacts of TLCA on the clinically relevant pathogen, Pseudomonas aeruginosa strain PA14, to gain a deeper understanding of the mechanism of action of TLCA. Our studies show that TLCA alters P. aeruginosa colony morphology as well as corresponding specialized metabolism and encourages an increase in virulence as observed in a Galleria mellonella (wax moth) model. Using our investigation with P. aeruginosa as a proof of principle for our techniques and as support for our hypothesis, we have begun to examine the same relationship in a less studied model, Vibrio cholerae. Employing tools such as imaging mass spectrometry, we observed altered specialized metabolism of V. cholerae in axenic cultures in agreement with our previous work in P. aeruginosa. Further in vivo studies of Danio reio (zebrafish) intestines have also revealed altered production between different disease states and genders. Moving forward we plan to continue our in vivo studies with zebrafish to determine if V. cholerae infection can be cleared using taurine-conjugated bile acids as a treatment.

Keywords: Biofilms, Pseudomonas Aeruginosa, Mass Spectrometry

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Substrate Transmembrane Mimetic Inhibitors of γ-Secretase: Probes for Chemical Structural Biology Sanjay Bhattarai, Sujan Devkota, Kathleen M. Meneely, Minli Xing and Michael S. Wolfe The University of Kansas

Gamma (γ)-secretase is a membrane-embedded aspartyl protease complex, with presenilin as its catalytic component, that cleaves the transmembrane domains (TMDs) of a wide variety of type I integral membrane proteins. The two most studied substrates of γ-secretase are the Notch1 receptor, important in developmental biology and in many cancers, and the amyloid precursor protein (APP) of Alzheimer’s disease. Thus, γ-secretase inhibitors and modulators have potential as treatments for cancer and dementia (1). Advances in cryo-electron microscopy paved the way to the first detailed structure of the ~230 kDa γ-secretase complex; however, the nature of substrate recognition and the mechanism of hydrolysis in the lipid bilayer are unclear (2). To address these issues, we aimed to develop chemical tools for structural analysis of the γ-secretase complex that would trap the enzyme in its active state and provide a high- resolution picture of substrate recognition. Here we report chimeric peptidomimetic inhibitors of the γ- secretase complex that contain both a helical peptide inhibitor (HPI) that engages the substrate docking exosite and a transition-state analogue inhibitor (TSA) targeted to the active site (3, 4). Since, these two binding site are proximal (5), we designed conjugates containing a 10-residue HPI inhibitor connected through its C-terminus to the N-terminus of a TSA inhibitor through a ω-aminoalkanoic acid linker. This series of hybrid HPI-TSA conjugates with spacers of various lengths has been synthesized by solid-phase from L-amino acids and a tripeptidomimetic building block accessed in multiple steps. These compounds were purified by preparative HPLC with final purity established by LC-MS. Many of these conjugates showed several-fold improved potency over individual HP or TSA inhibitors, and two conjugates with a 10-atom spacer gave the most potent compounds in the series, with near-stoichiometric inhibition. Moreover, enzyme inhibition kinetics and photoaffinity probe displacement experiments demonstrated that both docking site and active site are engaged by the inhibitors. Thus, these conjugates should be important new tools for structural biology, allowing cryo-EM analysis of γ-secretase in complex with a potent inhibitor that mimics the entire TMD of bound substrate. Such studies are underway.

Keywords: Gamma (Γ)-Secretase, Peptidomimetic Inhibitors, Photoaffinity Probe

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Allosteric inhibitors of apicoplast DNA polymerase: New antimalarials that bind a novel allosteric binding site Pratik Rajesh Chheda and Robert J. Kerns The University of Iowa

Plasmodium spp. are the causative agents of malaria, killing nearly 600,000 people each year. Resistance of Plasmodium to current therapies accentuates the need for new drugs that target novel aspects of the parasite’s biology. Parasites in the phylum Apicocomplexa have an unusual organelle; apicoplast, which participates in the biosynthesis of fatty acids, heme, iron-sulfur clusters, and isoprenoids. Any defect in apicoplast metabolism or its failure to replicate leads to the death of the parasite. Additionally, lack of a human counterpart makes apicoplast a promising drug target. The apicoplast genome is replicated by select DNA replication enzymes, of which apicoplast DNA polymerase (apPOL) is unique to the parasite. The apPOLs from P. falciparum and P. vivax have 84% homology, while the most similar human DNA polymerases are the lesion bypass polymerases theta and nu (23 and 22% identity, respectively). Towards identifying inhibitors of apPOL, a high throughput screen of 400 compounds from the Open Malaria Box provided by (Medicines for Malaria Venture (MMV) identified a sub-micromolar inhibitor of apPOL. Our studies indicate that MMV666123 is specific for apPOL, with no inhibition of human DNA Pol or E. coli DNA Pol I. Additionally, being a malaria-box compound substantiates the anti-malarial activity of MMV666123. Presented here are our current design, synthesis, crystallographic, and in vitro evaluation efforts toward understanding the structural requirements of MMV666123 for inhibition of apPOL, identifying the binding site and designing more potent and drug-like apPOL inhibitor derivatives.

Keywords: Antimalarial, Allosteric Inhibitors, DNA Polymerase

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Session 4

Covalent Inhibitors Target O-GlcNAc Transferase in Cells Matthew Worth, Chia-Wei Hu, Arielis Estevez Davila, Dacheng Fan, Hao Li, Dongsheng Zhu and Jiaoyang Jiang University of Wisconsin

O-GlcNAc transferase (OGT) catalyzes the attachment of O-linked β-N-acetylglucosamine (O-GlcNAc) to over 1,000 nucleocytoplasmic proteins. This modification, termed O-GlcNAcylation, regulates proteins involved in a variety of cellular processes including transcription, translation, and signal transduction. Elevated levels of OGT and O-GlcNAcylation have been detected in many diseases including most types of cancer, and O-GlcNAc dysregulation has been linked to tumor progression. However, the paucity of suitable OGT inhibitors has impeded the dissection of OGT function in cells. A number of OGT inhibitors have previously been published, but each suffers from limitations such as toxicity, poor potency, or lack of specificity. This talk presents our design, synthesis and characterization of a new class of OGT covalent inhibitors that target a unique cysteine residue in OGT active site. Our top analogs irreversibly inhibit OGT in vitro, and X-ray crystal structures confirmed the covalent mechanism of inhibition. Cell permeable analogs of these compounds reduced cellular O-GlcNAc levels more effectively than one of the best reported OGT inhibitors. Furthermore, click chemistry and in-gel fluorescence assay have verified OGT as the primary target of our inhibitor in living cells. These inhibitors are expected to be powerful tools to study the biological functions of OGT and O-GlcNAcylation.

Keywords: Carbohydrate, O-Glcnac, Covalent Inhibitor

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Nanorings with Engineered Fibronectins for Cancer Therapeutics Ozgun Kilic and Carston Rick Wagner University of Minnesota

Engineered non-antibody protein scaffolds have gained interest in recent years to overcome the challenges faced with antibody-based drugs. Several scaffolds have shown promise in both preclinical models and clinical trials. One of these promising scaffolds is the tenth type III domain of human fibronectin, which consists of seven β-strands connected with flexible loops. Analogous to the complementarity-determining regions of antibodies, the amino acid sequences of these loop regions can be engineered to provide target- specific binders with high affinity and specificity. One such target, epidermal growth factor receptor (EGFR) is a well-studied cancer biomarker and a promising target for cancer therapeutics.

Our lab has developed Chemically Self-Assembling Nanorings (CSANs) as a versatile platform that enables different therapeutic applications. Multimeric CSANs are formed when a bis-methotrexate dimerizer induces the oligomerization of dihydrofolate reductase-dihydrofolate reductase (DHFR2) fusion proteins. An advantage of the CSAN platform is that it can be easily expanded through fusion of additional protein domains or by chemical modifications to the dimerizer. By fusing an EGFR-targeting fibronectin to the DHFR2 subunits, we formed fibronectin CSANs with both high affinity and high avidity for EGFR- overexpressing tumor cells. Furthermore, we demonstrate that T cells functionalized with EGFR/CD3 bispecific CSANs selectively recognize and lyse EGFR-overexpressing cancer cells.

Keywords: Engineered Proteins, Cancer Immunotherapy

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A Novel Imaging Mass Spectrometry Method for Identifying Preventative Approaches in High Grade Serous Ovarian Cancer Katherine E. Zink and Laura M. Sanchez University of Illinois at Chicago

High grade serous ovarian cancer (HGSOC) is the fifth leading cause of cancer death among women, and its fatality is closely linked to the difficulty in detecting symptoms of early stages. We have developed a novel method to visualize small molecule chemical gradients via imaging mass spectrometry of the primary metastasis of HGSOC. This method requires very little biological material and detects not only molecules relevant to HGSOC but can also localize their tissue or cell of origin. Excitingly, we believe this innovative visualization of exchanged molecules can be modified to other coculture systems to answer a range of biological queries. We have identified that the release of norepinephrine from the ovary is induced in the presence of tumorigenic fallopian tube epithelial (FTE) cells and not healthy FTE or other cell types. Norepinephrine was validated by tandem mass spectrometry and retention time matching with an authentic standard and has previously been implicated in ovarian cancer development. Currently, efforts are focusing on elucidating the signals from the FTE that are inducing the production of NE from the ovary. We have begun to identify the FTE signal responsible for NE release, called factor X, by doing bioactivity guided fractionation of conditioned FTE media to examine whether the ovary response is mediated by small molecules, lipids, or proteins. Interestingly, it appears that the release of NE from the ovary is likely protein-mediated and further proteomics studies will be conducted to identify the nature of protein factor X.

Keywords: Imaging Mass Spectrometry, Ovarian Cancer

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Poster Presentations

Poster Presenting School Abstract Title Number Author 1 Angelo E. KU A Novel Approach for Studying Cellular Signal Andres Transduction Using Artificial Cell Surface Receptors 2 Tomas Joseph KU Discovery of Small Molecule Inhibitors of P- Smith glycoprotein by High-Content Screening with a Fluorescent Mimic of Taxol 3 Jose A. Colina UIC Role of PAX2 in the development of fallopian tube derived high grade serous ovarian cancer 4 Bailin Lei KU Synthesis of Coumarin-Derived Fluorophores that Target Small Molecules to the Endoplasmic Reticulum of Mammalian Cells 5 Malcolm Cole UMN β-lactams and β-lactamases: Novel strategies for Mycobacterium tuberculosis 6 Audra Lemley UIC Non-natural Amino Acid Tags for in vivo Lifetime Extension and Pharmacokinetic Control of Therapeutic Proteins 7 Yiao Wang UMN Prenylated CSANs for reversible cell-cell interactions and anti-cancer Immunotherapy 8 Md Abdullah UMN RAR alpha inhibitors for male contraception 9 Jesse Gordon- UIC Development of a Novel NAMPT Activator as a Blake Potential Alzheimer’s Disease Therapeutic 10 Caitlin C. Jokipii UMN Ethnic Differences in the Metabolism of 1,3-Butadiene Krueger 11 Ellie Mews UMN Engineering anti-EpCAM Fibronectin Based Nanorings for Immunotherapy 12 Joshua C. UI Interrogating Environmental Contaminate Activity Wilkinson Against the Druggable GPCR-ome via PRESTO- TANGO 13 Krishna K. KU Peptide Agonists of The Delta Opioid Recptor (Dor) Sharma With High G Protein Signaling Bias and Improved Drug-Like Characteristics 14 Shuvra Debnath UIC Synthesizing an 18F Probe Of Pet Imaging for Estrogen Receptor Positive (Er+) Breast Cancer 15 Grant Cooling UI Tertiary Fragment Screen into Glutamate Racemase 16 Brian Gabet UMN Development of CatSper Inhibitors as Potential Male Contraceptives 17 Jian Tang UMN Discovery of Allosteric Inhibitors of NF-κB Inducing Kinase (NIK) 18 Nathan Delvaux UI Overcoming the Cell Membrane Barrier in Non-Viral Gene Delivery with Pore-Forming Polyacridine Peptide Conjugates

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19 Hannah Petraitis UIC Nitric Oxide is a regulator of RNA methylation by controlling the expression of FTO. 20 Stephanie UW- S-Adamantyl Group Directed Site-Selective Acylation Blaszczyk Madison and Its Applications in the Streamlined Assembly of Oligosaccharides 21 Gihan C. KU Modular synthesis and reactivity profiling studies of Dissanayake diverse and stereochemically enriched C-, S-, and P- based electrophilic macrocycles. 22 Matthew Worth UW- Covalent Inhibitors Target O-GlcNAc Transferase in Madison Cells 23 Gang Yan UW- Rearrangement of 2-Chloroquinazolin-4(3H)-ones to Madison Afford Cyclic Guanidines with Potential Axial Chirality 24 Amanda Degner UMN Discovery of Novel N-(4-hydroxybenzyl)valine Hemoglobin Adducts in Human Blood. 25 Haibo Xie UW- Development of Novel Small Molecule PCSK9 Madison Modulators 26 McDermott, C. UMN Anthrax Antitoxin Lead Optimization via Bioisosteric M. Replacement and Other In Silico Strategies 27 Zoe M. UMN Incorporation of Agouti-Related Protein (AgRP) Human Koerperich Single Nucleotide Polymorphisms in the AgRP-Derived Macrocyclic Scaffold c[Pro-Arg-Phe-Phe-Asn-Ala-Phe- DPro] Decreases Melanocortin-4 Receptor Antagonist Potency 28 Viena U. KU Intramolecular C–H Functionalization to Electrophilic Thomas Probes: Generation of Novel Triazole-Containing Sultams 29 Alexander K. UMN Chemical Tools to Elucidate Mechanisms of Dopamine Hurben Toxicity in Parkinson’s Disease 30 Julia R. Austin UIC Baicalein is a phytohormone that signals through the progesterone and glucocorticoid receptors 31 Ka Yang UW- Development of Potent HDAC6 Selective Degraders Madison 32 Xiaolei Li UW- Site- and Stereoselective Alkylation of Carbohydrates Madison by OH Insertion 33 Kornelia J. UIC Development of Gamma-Functionalized Hydrocarbon Skowron Stapled Peptides as Inhibitors of the Estrogen Receptor/Coactivator Interaction 34 Saad A Alqarni UIC Targeting KRAS with small molecules at a novel site 35 Jordan Hunt KU Further Exploration of the SAR of Imidazoquinolines; Identification of Potent C7 Substituted Imidazoquinolines 36 Joe O'Brien UI The Mechanistic Role of Metal Ions, Ca2+ and Mg2+, in RGS: G-Protein Interactions 37 Rachel C Knopp UIC Characterizing Calpain-1 Inhibition as a Therapeutic Approach to Neurodegeneration

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38 Jacob Veenstra UIC Evaluating the effect of oregano essential oil and rosemary extract on Sestrin 2 expression in HCT 116 colon cells 39 Zhe Gao KU Identification of a Fluorinated Pyronin Fluorophore that Enables Visible Blue Light to Rapidly Depolarize Mitochondria 40 Kris Tuttle UI Human Hepatic Microsomal Sulfatase Catalyzes the Hydrolysis of Polychlorinated Biphenyl Sulfates 41 Anija mol T KU Probing substrate recognition and proteolytic site Philip selection by γ-secretase with designed helical peptides 42 Zhen Zheng UW- Vaccine-driven pharmacodynamic dissection of Madison ketamine psychoactivity 43 William McCue UMN Structural Exploration of the HA-Binding Groove in CD44 using Disaccharide Mimetics 44 Minli Xing KU Identification of inhibitors of MSI2-RNA interaction through NMR fragment screening 45 Katherine Jones UMN Identification of Selective APOBEC3 Ligands by Mass Spectrometry Fragment Screening 46 Sahishna KU Nanodisc technology for studies of small molecule- Phaniraj membrane interactions 47 Dan Yin UW- DIPTOINDONESIN G AND ITS ANALOGUES AS Madison SELECTIVE MODULATORS OF ESTROGEN RECEPTORS 48 Conrad Fihn UMN Bacterial Histidine Kinase: Development of Novel Catalytic Domain Inhibitors 49 Ganguly, A. KU Phosphate Tether-Mediated Studies Towards the Synthesis of Leustroducsin B and 13-desmethyl lyngbouilloside 50 Josh Shirley UMN Investigation of β-Lactones as Selective Activity-Based Probes for Penicllin-Binding Proteins 51 Samantha A. UMN Next Generation Pyrimidine-Based Photoactivable Kennelly Catch and Release DNA Decoys 52 Manikandan KU Connecting Remote C–H Bond Functionalization and Selvaraju Decarboxylative Coupling Using Simple Amines 53 Amanda C. UIC Modulation of estrogen biosynthesis in estrogen Maldonado receptor positive breast cancer cells by Humulus lupulus and its bioactive compounds 54 Kelsey KU Targeting fluorescent sensors to endoplasmic reticulum Knewtson membranes enables detection of peroxynitrite during phagocytosis 55 Nan Wang UMN Discovery of CDK2 Allosteric Inhibitors for Male Contraception and Cancer Therapy 56 Daniel Zagal UIC NMR SAMPLE DEGASSING CAN AIDS IN THE FULL SPIN ANALYSIS OF NATURAL PRODUCTS

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57 Xianghong Guan UMN Design and Synthesis of Bivalent Inhibitors for Bromodomain and Extra-Terminal （BET）family proteins 58 Zachary Pearson KU Chronochemical Informatics: Small-molecule Libraries at KU 1947-2017 59 Adam Worob UW- Cross-Reactive Antibodies as Theranostic Tools against Madison Synthetic Cannabinoid Intoxication 60 Duo Zhang UI Development of a Broadly Applicable Method for Identification and Analysis of Polychlorinated Biphenyl Sulfates in Human Serum 61 Jiewei Jiang UMN Design and Synthesis of Dihydropyridine Analogs as BRDT Selective Inhibitors for Male Contraception 62 Scott I. Brody UMN Development of an Activity-Based Probe for PLP- Dependent Enzymes in Mtb 63 Taha Y. Taha UIC Histone deacetylases upregulate hepatitis B virus biosynthesis by stabilizing the viral core protein 64 Rachel Crawford UI The biogenic aldehyde 3,4- dihydroxyphenylacetaldehyde is scavenged by carnosine and L-cysteine 65 Eden E. D. UI Studies to improve cellular penetration of Maack quinazolinediones 66 Marianne UIC Nitric Oxide Regulates DNA Methylation in Cancer Palczewski 67 Dacheng Fan UW- Electrophilic probes for deciphering substrate Madison recognition by O-GlcNAc transferase 68 Desire M. Ortiz UIC Synthesis of Novel, FASN-Specific PET Tracers Torres 69 Michael Grillo UMN Discovery of APOBEC3B DNA Cytosine Deaminase Ligands by Protein Observed Fluorine NMR Screening 70 Laura Cooper UIC Investigation into the Mechanism of Action of Selective Estrogen Receptor Modulators in Ebola Virus Disease 71 Garrett Schey UMN A Farnesyltransfersase Mutant with Dual Orthogonality for Enzymatic Labeling 72 Jhewelle N. Fitz- UW- Nucleophilic Reactivity of 2-Alkyl Quinazolinones in Henley Madison the Synthesis of (E)-Benzamidines 73 Brianna Cagle UI PCB-52 and Metabolites: Toxicity to the Dopaminergic System 74 Max Dillenburg UMN Evaluating Histidine Triad Nucleotide Binding Protein 1 (Hint1) as a Target for Pain Therapy 75 Jenna UMN Translesion Synthesis past DNA-Peptide Crosslinks is Thomforde Dependent on Local DNA Sequence Context 76 Evan Alexander UMN Rationally Designed Inhibitors of TIlivalline Biosynthesis in Whole Cell Klebsiella oxytoca 77+ - - Late Entries

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Conference Locations

Lodging/Registration Springhill Suites, One Riverfront Plaza, Lawrence, KS 66044 –UMN, UIC Quality Inn and Suites, 2309 Iowa St, Lawrence, KS 66046 – UI Days Inn, 730 Iowa St, Lawrence, KS 66044 –UW-Madison

Scientific Program The University of Kansas Integrated Science Building (ISB) 1567 Irving Hill Rd, Lawrence, KS 66044

Friday Welcome Reception Arterra Event Gallery, 2161 Quail Creek Dr., Lawrence, KS 66047

Arterra Event Gallery seeks out local vendors and gives part of its annual revenue back to the Lawrence community. A green initiative exists to recycle at both on-site and off-site events. It’s also the location where the KU MDCM holiday reception is held!

Saturday Night Banquet Venue: Van Go inc., 715 New Jersey St., Lawrence, KS 66044

Van Go, Inc. was founded in 1997 by Lynne Green as an arts- based social service and job-training non-profit for at-risk teens. It is nationally recognized with the prestigious awards Coming Up Taller Award (2006), Kansas Governor’s Art Award (2008), KU’s Community Collaboration Award (2013), and a 2016 visit from the National Endowment for the Arts Chair, Jane Chu.

Catering: Maceli’s, 1031 New Hampshire St., Lawrence, KS 66044

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Floor Plan of Integrated Science Building at KU

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Poster #1 A Novel Approach for Studying Cellular Signal Transduction Using Artificial Cell Surface Receptors Angelo E. Andres and Blake R. Peterson University of Kansas Cell surface receptors are integral proteins involved in cellular signal transduction. Unfortunately, in some diseases, such as specific types of cancer and diabetes, missing or mutated receptors are involved in pathogenesis. To study these receptors as well as signal transduction, genetic engineering has been utilized to create permanent edits to genes involved in these processes. Unfortunately, genetic engineering has inherent limitations. These include irreversibility, toxicities related to genetic delivery and undesired genetic modifications, and certain experimental challenges. To overcome these limitations, we seek to develop a novel approach for studying cellular signal transduction using artificial surface receptors. This unprecedented approach is challenging due to the inherent complexity of most cell surface receptors, which are generally capable of rapidly responding to specific extracellular ligands by transmitting extracellular signals across cellular membranes into the interior. For most receptors, these signals are propagated by interactions between receptors and intracellular signaling proteins. To attempt to overcome this challenge, and to learn more about fundamental cellular processes, we seek to create the first synthetic cell surface receptors that control specific signal transduction pathways. As an approach to develop these types of agents, we are synthesizing and evaluating small molecules termed rigid rods which naturally insert unassisted into the plasma membrane of targeted cells. Further coupling of these molecules to molecules capable of passing across the cellular plasma membrane and engaging specific intracellular proteins is proposed to provide a mechanism for activating signal transduction. Studies of model systems and progress towards these goals will be described.

Keywords: signal transduction, receptors

Poster #2 Discovery of Small Molecule Inhibitors of P-glycoprotein by High-Content Screening with a Fluorescent Mimic of Taxol Tomas Joseph Smith and Blake Peterson University of Kansas Drug resistance often limits the effectiveness of cancer chemotherapy. Although this resistance can result from multiple mechanisms, the overexpression of multidrug efflux pumps such as P-glycoprotein (P-GP) often plays a key role. An important chemotherapeutic drug susceptible to this mechanism of resistance is paclitaxel (Taxol). We previously reported the synthesis of novel fluorescent analogues of Taxol that include the coumarin-derived fluorophore Pacific Blue. Unlike other fluorescent taxoids, Pacific blue-Gly-Taxol (PBGT), maintains substantial cytotoxicity, and this probe is a highly efficient substrate of P-GP. Consequently, cotreatment with the small molecule P-GP inhibitor verapamil is necessary to observe binding of PBGT to its target protein beta-tubulin by fluorescence microscopy in many cancer cell lines. When HeLa cervical carcinoma cells are cotreated with PBGT (1 µM) and verapamil (25 µM), cellular fluorescence increases by ~ 10-fold as analyzed by confocal microscopy or flow cytometry. Because of the simplicity and sensitivity of this assay, we envisioned that it could be optimized in a 96-well plate format to provide a method to identify small molecule inhibitors of P-GP. To provide a proof of concept, 1584 diverse compounds obtained from the National Cancer Institute (NCI) were screened using automated pipetting and flow cytometry. The primary screen yielded more than 30 potential hit compounds with {"IsDistinguishedFolder":true,"FolderId":{"Id":"AQMkADgwMzU3MmM0LTU1ADEwLTRkNTktODBlMC0 yNDc0NWU5ZjRjNTYALgAAAwRhyx9EsV9Dvsr1dnqsQkMBAI96RB/OM/5Gpi5zF/CG/1EAAAIBCQAA AA==","ChangeKey":"AQAAABYAAACPekQfzjP+RqYucxfwhv9RAALRWRzN"}}equivalent or of higher inhibitory activity than verapamil (25 µM). These results illustrate the power of this assay to identify novel inhibitors of P-GP and suggest that this approach could improve our understanding of mechanisms of inhibition of this major drug transporter.

Keywords: Screening, Cancer

Poster #3 Role of PAX2 in the development of fallopian tube derived high grade serous ovarian cancer Jose A. Colina, Peter Varughese, Angela Russo and Joanna E. Burdette University of Illinois Ovarian cancer is most lethal gynecological malignancy and the 5th leading cause of cancer deaths among women. The deadliest subtype of the disease is high grade serous ovarian cancer (HGSOC) with an average 5-year-survival rate of 29%. The secretory cells in the fallopian tube epithelium (FTE) can become preneoplastic lesions called secretory cell outgrowths(SCOUTS), which are hypothesized to go on to become HGSOC. PAX2 is a transcription factor that is lost in HGSOC and SCOUTs, suggesting loss of PAX2 is an early event in fallopian tube-derived HGSOC tumorigenesis. In the present study, we developed PAX2shRNA knockdown and CRISPR deletion murine oviductal cell lines (MOE) to model the development of SCOUTs. We used this model to study how it potentiates the FTE for further transformation. RNA-sequencing of PAX2shRNA cells revealed a transcriptional overhaul that recapitulates the transcriptional dysregulation in key genes present in human SCOUTs. Furthermore, analysis of the RNAseq data of our newly created SCOUT model with RNAseq of estrogen stimulated MOE cells revealed remarkable overlap suggesting that loss of PAX2 regulates hormonal responses, a known pathway in gynecological tumorigenesis. Hormone responsiveness of these cells were investigated using ERE-luciferase assays which revealed higher basal hormone activity and sensitivity to hormone treatment. Our SCOUT model also showed increased estrogen receptor, ligand-independent induction of estrogen-responsive-genes, and increased sensitivity to estrogen mediated genotoxicity. This presents the possibility of a known subset of HGSOC precursor lesions having a predisposition for estrogen-induced stimulation in the estrogen-rich environment of the fallopian tube.

Keywords: Ovarian Cancer, Estrogen

Poster #4 Synthesis of Coumarin-Derived Fluorophores that Target Small Molecules to the Endoplasmic Reticulum of Mammalian Cells Bailin Lei, Zhe Gao and Blake R. Peterson University of Kansas The endoplasmic reticulum (ER) is an interconnected network of tubular membranes that extend throughout the cytoplasm of eukaryotic cells. These membranes play critical roles in the processing of secreted and transmembrane proteins. By synthesizing analogues of the coumarin-derived fluorophore Pacific Blue, we identified novel amino coumarins that accumulate in these membranes in living HeLa cells. When this fluorophore was linked to the anticancer drug paclitaxel, one of the most effective treatments for breast, ovarian, and lung cancers, the resulting fluorescent taxoids were found to accumulate in the ER, as imaged by confocal laser scanning microscopy. However, these compounds retain appreciable cytotoxic activity, suggesting that this subcellular targeting strategy might be used to circumvent a major mechanism of resistance to the anticancer effects of paclitaxel. This drug resistance mechanism involves overexpression of P-glycoprotein (P-gp) on cellular plasma membranes, a protein that promotes cellular efflux of this anticancer agent. By enabling analogues of paclitaxel to occupy the extensive network of membranes of the ER, and retain affinity for the target protein beta- tubulin, subcellular targeting to ER membranes may reduce interactions of taxoids with P-gp, providing a novel strategy to circumvent a common mechanism of resistance to this anticancer agent.

Keywords: Coumarin-Derived Fluorophores, endoplasmic reticulum , paclitaxel

Poster #5 β-lactams and β-lactamases: Novel strategies for Mycobacterium tuberculosis Malcolm Cole and Courtney Aldrich University of Minnesota Tuberculosis (TB) is the leading source of infectious disease mortality globally, causing 1.3 million deaths worldwide in 2017. This unresolved burden on global health is further complicated by the growing prevalence of antibiotic-resistant strains, which comprise an estimated 10% of new TB cases and present an urgent need for novel TB therapeutics. β-lactam antibiotics, arguably the most historically successful class of antibiotics, have traditionally been ineffective against M. tuberculosis (Mtb), the causative agent of TB, due to the organism’s inherent expression of β-lactamases, serine hydrolases that destroy the electrophilic β-lactam warhead. Inspired by similar strategies employed for other drug-resistant organisms, we have developed novel β-lactam prodrugs which exploit this inherent β-lactamase activity to achieve selective release of antitubercular compounds. Cleavage of the β-lactam promoiety furnishes elimination of the antitubercular in vivo; functionalization can impart desired characteristics, such as improved bioavailability or selectivity for the tubercular β-lactamase BlaC over β-lactamases expressed by commensal organisms. To supplement this strategy, we have also begun investigations into the mechanisms of β-lactamase expression present in Mtb. Expression of blaC, the gene encoding BlaC, is governed by a poorly-understood regulatory system that can sense β-lactam antibiotics in the cellular microenvironment and trigger increased expression. We are interested in further exploring the role of β- lactam structure in recognition and signaling within this regulatory system.

Keywords: Tuberculosis, infectious disease, small molecules

Poster #6 Non-natural Amino Acid Tags for in vivo Lifetime Extension and Pharmacokinetic Control of Therapeutic Proteins Audra Lemley and Stephen G. DiMagno University of Illinois Protein therapy is quickly becoming a pillar in medicinal chemistry and pharmacognosy. The success of the field is substantiated by more than one hundred therapeutic proteins in clinical use. However, due to the fragility of non-antibody protein therapeutics, they often suffer from short serum half-lives due to their rapid degradation by peptidases. Herein we have developed a library of non-natural amino acids for incorporation via either genetic encoding or post-translational modification. This strategy enhances the circulatory longevity of the proteins for greater efficacy of targeted drug delivery via this small molecule human serum albumin (HSA) binding functionality. The albumin binding affinity of these amino acids varies over a two-decade range, despite the fact that these structures differ only in the number and disposition of their methylene groups. In addition to altering main chain length, a hydrophobic group in the para position on the phenyl ring also plays a vital role in the dissociation constant (KD) with HSA. To evaluate and validate binding constants of the variable small molecules for HSA, surface plasmon resonance and affinity chromatography have been utilized. Recombinant protein engineering is employed to genetically encode the albumin-targeting non-natural amino acids directly into therapeutic proteins using the genetic machinery of E. coli for protein expression. Conversely, maleimide-cysteine chemistry can be exploited to site-specifically append the non-natural amino acid post-translationally to a surface- exposed cysteine on purified protein structure.

Keywords: therapeutic proteins Poster #7 Prenylated CSANs for reversible cell-cell interactions and anti-cancer Immunotherapy Yiao Wang, Carston R. Wagner and Mark D. Distefano University of Minnesota Manipulation of reversible cell-cell interactions is essential for many cell-based therapies, such as anti-cancer immunotherapy and tissue regeneration. Herein, we have developed a self-assembled nanostructure called chemically self-assembled nanorings (CSANs) by preparing the DHFR-DHFR fusion protein and bivalent methotrexate (bisMTX) to efficiently mediate cell-cell interactions. Upon binding to bisMTX, the monomeric DHFR-DHFR protein can be oligomerized into octamers. The fibronectin based targeting element can be fused to the N-terminus of the DHFR-DHFR protein for targeting specific cell types. Meanwhile, the engineered DHFR- DHFR protein can also be prenylated by farnesyltransferase or geranylgeranyltransferase. The consequent prenylated CSANs can universally modify the cell surface through hydrophobic interactions with the cell membrane and therefore empower the modified cells to interact with other cell targets. In this study, we have successfully developed anti-EpCAM and anti-EGFR CSANs for cell-cell interactions. The prenylated CSANs can rapidly modify cell surface with a tunable binding affinity and enable the modified cells to interact with target cells. Upon the treatment of a competitive inhibitor of DHFR, the CSANs can be disassembled, leading to a quick dissociation of cells, which was shown by fluorescent microscopy and flow cytometry. Moreover, the activated PBMCs modified by prenylated anti-cancer CSANs demonstrated selective cytotoxicity to the target cancer cells in the LDH release assay. The prenylated anti-cancer CSANs exhibited great advantages over conventional CAR-T therapy and can be a promising tool for adoptive cell therapy in the future.

Keywords: engineering cell-cell interactions, anti-cancer immunotherapy, protein prenylation

Poster #8 RAR alpha inhibitors for male contraception Md Abdullah Al Noman and Gunda I. Georg University of Minnesota The discovery of small molecule male contraceptive agents is an unmet challenge. Previous drug discovery attempts focused on the male sex hormone testosterone and related analogs. However, because of a number of side effects no agent has been marketed yet. Our group is seeking to discover non-hormonal male contraceptives that are safe, effective, side effect free and above all, reversible. The retinoic acid receptor alpha (RAR alpha) was validated as a target for male contraceptive by classical and chemical genetics approaches. Genetic knockout of RAR alpha inhibited sperm maturation and induced infertility, and chemical inhibition of with a pan-RAR inhibitor lead to reversible infertility in mice. Our goal is to develop a potent, selective, and reversible inhibitor of RAR alpha as a drug candidate for male contraception. We designed and synthesized about a hundred compounds based on several scaffolds aided by structural information and used a luciferase-reporter cell assay to evaluate RAR inhibitory activity and selectivity. A number of compounds showed single-digit nanomolar IC50 values for the inhibition of RAR alpha and one highly RAR alpha-selective compound demonstrated reversible contraceptive effects in a mouse model. Modification of active compounds is ongoing to obtain additional potent and selective inhibitors with suitable pharmacokinetic properties.

Keywords: contraceptive, RAR alpha inhibitor

Poster #9 Development of a Novel NAMPT Activator as a Potential Alzheimer’s Disease Therapeutic Jesse Gordon-Blake and Gregory Thatcher University of Illinois Therapies directly targeting amyloid¬β (Aβ) and hyperphosphorylated tau in Alzheimer’s disease (AD) have been unsuccessful, so an alternative drug target is needed. One potential strategy to prevent or reverse the course of the disease is to target the NAD/NAMPT pathway. NAD levels decline with age and thus negatively affect NAD- dependent enzymes and cellular energy pathways crucial to neuron function. In combination with protein misfolding associated AD pathophysiology, NAD depletion can drive disease progression. An efficient way to increase NAD is to activate NAMPT, which catalyzes the rate limiting step in NAD biosynthesis. Activating NAMPT will increase NAD levels and support neuroprotective pathways that will prevent neurodegenerative progression. To test this hypothesis, we employed a high throughput screen (HTS) of 2000 compounds measuring turnover of NAM, the substrate for NAMPT. This produced two hits that activated NAMPT in the nanomolar range. Analogs of hit 1 were synthesized to optimize its pharmacokinetics. We next measured neuroprotection against oxygen-glucose deprivation (OGD), an in vitro model of ischemia-reperfusion injury in stroke that simulates some of the toxicity present in AD. An analog, KB-49, showed concentration-dependent neuroprotection on SH-SY5Y cells at various treatment paradigms. Following these results, we hope to optimize this compound for improved potency, in vivo stability, and blood-brain barrier penetration. Additionally, a second HTS will be performed to find NAMPT activating scaffolds with superior performance.

Keywords: NAMPT, Alzheimers

Poster #10 Ethnic Differences in the Metabolism of 1,3-Butadiene Caitlin C. Jokipii Krueger, Guru Madugundu, Yesha Patel, Steven Carmella, Daniel Stram, Loic LeMerchand, Stephen Hecht and Natalia Tretyakova University of Minnesota 1,3-butadiene (BD) is a carcinogen abundant in tobacco smoke. BD is metabolically activated by cytochrome P450 monooxygenases to reactive epoxides including 3,4-epoxy-1-butene (EB). EB can undergo detoxification by glutathione S-transferase θ-1 (GSTT1) to form the EB-glutathione (EB-GSH) conjugate, which can be further metabolized to form monohydroxy-3-butenyl mercapturic acid (MHBMA). In a previous study, we found that the order of MHBMA levels among ethnic groups is consistent with their respective lung cancer risk and can be partially explained by GSTT1 genotype. If not detoxified, these epoxides can form a range of covalent DNA adducts at nucleophilic sites of DNA, leading to mutations and cancer. Adducts formed at the N7-position of guanine are hydrolytically labile and excreted in urine, leading to their potential use as biomarkers of BD exposure. Our laboratory has developed ultra-sensitive nanoLC/ESI+-HRMS3 based methods for quantitative analysis of N-7-(1’-hydroxy-3’-buten-2’-yl) guanine (EB-GII) adducts in human urine. We evaluated urinary EB- GII as a biomarker of exposure to butadiene in smokers by investigating its stability and association with smoking and quantified EB-GII levels in smokers belonging to three different ethnic groups. We found that levels of urinary EB-GII are the highest in Japanese Americans, who have the lowest risk of lung cancer compared to Whites and Native Hawaiians. Furthermore, we investigated BD metabolism, DNA adduct formation, and biological effects in HapMap cells, human-derived lymphoblastoid cells that have been extensively genotyped and can be used to further investigate inter-individual and ethnic differences in susceptibility to BD in a cell culture model.

Keywords: DNA adducts, mass spectrometry, lung cancer

Poster #11 Engineering anti-EpCAM Fibronectin Based Nanorings for Immunotherapy Ellie Mews and Carston R Wagner University of Minnesota Engineering cell-cell interactions has proven to be quite valuable due to the vast number of therapeutic applications that benefit from this technology. Although genetically engineering artificial receptors onto a patient’s cells has shown some success, preparation is costly, current applications are limited, and modifications are permanent. To address some of these concerns, our lab has developed a non-genetic approach to facilitate selective cell-cell interactions using various targeted protein scaffolds. One such scaffold is based on the human tenth type III fibronectin domain (Fn3), which can be engineered to target specific cell antigens. Our lab has generated a range of Fn3’s that bind to the Epithelial Cell Adhesion Molecule (EpCAM), a cancer antigen overexpressed on multiple solid tumors1. We are able to fuse these Fn3’s to our multivalent chemically self- assembling nanorings (CSANs) to form an antigen-specific construct. The diversity of the CSAN construct can be expanded by fusing additional scaffolds that target other cellular antigens, creating a bispecific nanoring. Our lab has shown that functionalizing the CSAN construct with a cancer antigen-targeting scaffold and a T-cell targeting single chain antibody fragment (anti-CD3 scfv) forms a mixture of bispecific nanorings with the ability to traffic T-cells to the tumor2. This previous work suggests that an EpCAM Fn-CD3 bispecific CSAN can direct T-cell recognition and activation against EpCAM overexpressing cancer cells. However, forming a bispecific EpCAM Fn-CD3 CSAN has revealed a potential dependence on the targeting scaffold, the kinetics of ring formation, and characteristics of T-cell interactions with the target.

Keywords: EpCAM, immunotherapy, protein engineering

Poster #12 Interrogating Environmental Contaminate Activity Against the Druggable GPCR-ome via PRESTO- TANGO Joshua C Wilkinson and David Roman University of Iowa Exposure to environmental contaminants can result in myriad conditions ranging from developmental disorders to pulmonary conditions to neurological pathologies. Literature precedent and new demonstrates specific GPCRs can be activated by some classes of environmental contaminants, including PCBs, pesticides, chloropropanols, and others. When interrogating GPCR’s with small-molecule based screens, the traditional paradigm is to test hundreds of thousands of compounds against a single target. In recent literature, an open-source method “PRESTO-TANGO” (Parallel Receptor-ome Expression and Screening via Transcriptional Output – TANGO) has been described as a feasible approach to simultaneously screen the entire druggable human GPCR-ome against a smaller collection of compounds via a G protein-independent β-arrestin recruitment assay. We plan to utilize PRESTO-TANGO to generate a matrix showing activity of a small library of known environmental contaminants against the known druggable GPCR-ome of 315 GPCRs. Upon identification of novel contaminant- GPCR interactions, further investigation into the specific effects of the GPCR and cognate contaminant ligand will then be pursued. This work intends to identify unique receptor-ligand interactions that would serve valuable for further studies probing the physiological effects elicited by environmental contaminants. Furthermore, the application of PRESTO-TANGO in this manner could present a novel platform for initial screens of environmental contaminants.

Keywords: GPCRs Screen Contaminants

Poster #12 PEPTIDE AGONISTS OF THE DELTA OPIOID RECPTOR (DOR) WITH HIGH G PROTEIN SIGNALING BIAS AND IMPROVED DRUG-LIKE CHARACTERISTICS Krishna K. Sharma, Robert J. Cassell, Richard M. van Rijn and Ryan A. Altman University of Kansas Endogenous opioid peptides regulate activity within the central nervous system (CNS), and are particularly interesting for treating pain, depression, and anxiety. Unfortunately, clinical use of peptide-based agents is restricted by poor physicochemical and biophysical properties, which limit penetration into the CNS. Therefore, many peptide-based probes cannot be employed clinically for treating many disease states. To address this problem, the Altman group explores the use of fluorinated peptidomimetics (FPMs) and peripheral derivatization to improve the drug-like properties of peptides, and to deliver peptides into the CNS. In this poster, we describe the design, synthesis, opioid activity and signaling bias of fluoroalkene {Tyr1- ψ[(Z)CF=CH]-Gly2} analogues of the endogenous opioid neuropeptide, Leu-enkephalin, with the fluoroalkene analogue emerging as an orally-active, stable and CNS-distributed in vivo probe. Further optimization using C- terminus derivatization improved opioid potency, selectivity, and signaling bias, and this series of compounds displayed high G protein signaling bias. To further improve potency, receptor selectivity and signaling bias, we also investigated C-3 Phe4- substituted analogues of Leu-Enkephalin, and the halogenated analogues demonstrated very high DOR potency and selectivity compared with Leu-Enkephalin. Further work will aim to combine favorable structural features and deliver analogues with improved opioid potency, selectivity, and signaling bias, and physicochemical and biophysical drug-like characteristics.

Keywords: Pain, opioid peptides, Leu-enkephalin, fluorinated peptidomimetics, signaling bias

Poster #13 SYNTHESIZING AN 18F PROBE OF PET IMAGING FOR ESTROGEN RECEPTOR POSITIVE (ER+) BREAST CANCER Shuvra Debnath and Stephen G. DiMagno University of Illinois The positron emission tomography (PET) is a non-invasive imaging technique which is used for staging of cancer metastasis, assessing treatment strategies, and monitoring the effects of therapy with appropriate radiotracers. Because of the increased glucose metabolism in most types of tumors, 18F-fluorodeoxyglucose PET imaging (FDG-PET) is the most common imaging technique. But this method is dependent on the higher energy requirement of the cancer cells, and does not provide further information on the type of cancer cells to inform treatment. Metastatic estrogen receptor (ER ) positive breast cancer causes death of more women than any other types of breast cancers annually. Almost two-thirds of the breast cancers are estrogen receptor positive (ER+) and 50 % of these cancers developed resistance to ER targeted endocrine therapy. 18F-fluoroestradiol (18F-FES) has been used for PET imaging to detect ER+ breast cancer. Off-target uptake and extensive metabolism associated with 18F-FES results in poor localization of metastases in the abdominal region. 18F-FES is appropriate for imaging late stage breast cancer that already spread to bone, but imaging of ER receptors in soft tissue tumours like in breast is extremely difficult. Hence, a new high affinity ER+ ligand labelled with radioactive fluorine which would localize only in the tissue of interest and provide a parallel and potentially superior probe for ER+ breast cancer imaging.

Keywords: Positron Emission Tomography (PET), Estrogen Recept0or (ER), Metastatic breast cancer. Poster #14 Tertiary Fragment Screen into Glutamate Racemase Grant Cooling and Ashley Spies University of Iowa The rising number of multi-drug resistant bacteria has increased the need for novel antimicrobial agent development. A potentially powerful target is the enzyme glutamate racemase (GR), which is absent in humans but present in all bacterial cells. This enzyme is responsible for the interconversion of L-glutamate and D- glutamate, which is incorporated into bacterial cell walls. GR is able to catalyze this stereoinversion without the use of cofactors, such as metals or pyrodoxial phosphate, for the catalytic acidification of the Cα-H bond. Studies into inhibition of this enzyme are being done with GR expressed with a Helicobacter pylori (Hp) plasmid. GR expressed in this system is being used in a tertiary fragment screening campaign to determine fragments able to bind to allosteric pockets. Differential Scanning Fluorimetry is being conducted as the primary screen to identify potential hits. Primary hits will be compared to a compound already found to bind allosteric pockets as a positive control. Surface Plasmon Resonance and Crystallography will be conducted as the secondary and tertiary screens. High resolution crystal structures have already be solved to establish a system to investigate identified hits.

Keywords: Glutamate Racemase

Poster #15 Development of CatSper Inhibitors as Potential Male Contraceptives Brian Gabet and Gunda I. Georg University of Minnesota To date, there is an unmet need for selective, non-hormonal male contraceptive agents. The cation channel of sperm (CatSper) is a sperm-specific, voltage-gated ion channel which regulates the internal calcium concentration in mature spermatozoa. These channels are activated by the alkaline environment of the female reproductive tract, resulting in an influx of Ca2+ and hyperactivated motility that is necessary for fertilization. Mutations within CatSper, as confirmed by mouse knockout studies, lead to complete infertility in males with no other observable phenotypes. A 35,000 compound HTS campaign, using human sperm and a developed FLIPR assay to measure Ca2+ influx yielded seven hits with acceptable potency. Two of these hits, GPHR-00032750 and GPHR- 00213869, were selected for additional SAR studies. Analogs of these hits are being designed and tested in a variety of assays, including computer-assisted sperm analysis (CASA), FLIPR, and off-target glycerol assays to measure the ability of compounds to reduce hyperactivation, Ca2+ influx, and measure selectivity, respectively. The design and development of CatSper-specific inhibitors which reduce hyperactive motility may provide a reversible, selective, and safe approach to male contraceptive.

Keywords: CatSper, male contraceptive Poster #16 Discovery of Allosteric Inhibitors of NF-κB Inducing Kinase (NIK) Jian Tang, Özlem Demir, Garrett Chan, Alex M Ayoub, Rommie E Amaro and Daniel A. Harki University of Minnesota Dysregulated activation of non-canonical NF-κB (ncNF-κB) signaling contributes to the pathogenesis of various autoimmune and inflammatory diseases and human cancers. In the ncNF-κB pathway, NF-κB-inducing kinase (NIK) is a central regulatory component and its activity is essential for ncNF-κB activation. Consequently, selective small molecule inhibitors of NIK are highly desired as mechanistic chemical probes and potential therapeutics. Although active in mouse disease models, current ATP-competitive NIK inhibitors suffer from either off-target effects or poor drug-like properties. Allosteric kinase inhibitors are a validated approach towards achieving high selectivity for a desired kinase target, thereby overcoming limitations of ATP-competitive inhibitors. Therefore, in this project, we are developing allosteric NIK inhibitors. Through molecular dynamics (MD) simulations, we identified two putative allosteric sites on NIK that are amenable to small molecule binding. A virtual screen against these sites was performed that yielded 120 high-scoring small molecules that were subsequently screened for NIK enzymatic inhibition. Biochemical assays revealed 13 compounds that inhibit NIK enzymatic activity at or below 100 µM. Ongoing work is focused on further characterization and optimization of these hit compounds. Our novel NIK inhibitors, once fully optimized, will represent the first-in-class NIK allosteric modulators with anticipated high selectivity for NIK and the ability to regulate aberrant ncNF-κB signaling in disease models.

Keywords: Kinase, allosteric inhibitor, NF-κB, NIK

Poster #17 Overcoming the Cell Membrane Barrier in Non-Viral Gene Delivery with Pore-Forming Polyacridine Peptide Conjugates Nathan Delvaux and Kevin Rice University of Iowa Although significant advancements in the field of non-viral gene therapy have been made towards the design of polymers that can effectively stabilize DNA in biological systems, bypassing the cell membrane remains a challenge for many vectors. Active targeting of DNA nanoparticles with ligands directed towards specific endocytic cell surface receptors can be an effective approach to this obstacle; however, receptor-mediated uptake faces its own set of problems. Receptor endocytosis is saturable, limiting the maximal rate of migration across the cell membrane, as well as generally dependent on nanoparticle size and surface charge, potentially requiring optimization of the nanoparticle for each receptor. As non-specific cell entry alternatives, cell-penetrating peptides have found use in non-viral gene delivery. Melittin is a peptide derived from bee venom that forms pores in lipid bilayers. Several melittin analogues have been designed with increased pore-forming potency and decreased overall charge, facilitating control over nanoparticle surface charge. To capitalize on the pore-forming characteristics of these analogues, we conjugated them to a polyacridine DNA binding peptide with a variety of bioconjugate linkers to form DNA nanoparticles with inherent cell-penetrating properties. These conjugates were tested in vitro for the ability to transfect HepG2 cells using luciferase expression as a readout. Direct conjugation of the melittin analogues to the polyacridine peptide resulted in luciferase expression almost equivalent to polyethylenimine, the gold standard for in vitro gene transfection. Thus, these pore-forming peptides can be used in non-viral gene delivery to facilitate cell entry and transfection.

Keywords: Non-viral gene delivery, melittin

Poster #18 Nitric Oxide is a regulator of RNA methylation by controlling the expression of FTO. Hannah Petraitis and Douglas D. Thomas University of Illinois Dysregulation of gene expression sits at the core of many disorders. At the RNA level, reversible post- transcriptional modifications (PTMs) influence RNA metabolism and localization in the cell. N6- methyladenosine (m6A) is the most prevalent mammalian mRNA modification, accounting for more than 80% of all RNA methylation. Fat Mass And Obesity-Associated Protein (FTO) mediates reversible demethylation of m6A and N6, 2-O-dimethyladenosine (m6Am), making FTO a powerful regulator of mRNA stability. Like other members of the alpha-ketoglutarate dependent dioxygenase family of enzymes, FTO is potentially subject to catalytic inhibition by the free radical signaling molecule, nitric oxide (NO). The purpose of this study is to determine the role of NO in the expression and activity of FTO through observation and quantification of m6A, adding to our understanding of NO as a global epigenetic regulator of gene expression. We observed the effect of an exogenous NO donor, DETA/NO, on mRNA and protein levels of FTO. This was done in a dose and time- dependent manner. mRNA levels and protein expression of FTO indicated an increase following exposure to NO. To confirm NO’s ability to inhibit catalytic activity, we will observe m6A through LC-MS/MS in the future, predicting to see absolute increase in m6A methyl modifications. Our preliminary analysis suggests that NO acts as an important regulator of FTO to significantly alter global m6A RNA levels. These findings further our understanding of endogenous regulators of RNA methylation status, which is vital toward examining the role(s) of NO in a wide range of human disorders.

Keywords: nitric oxide, RNA, epigenetics

Poster #19 S-Adamantyl Group Directed Site-Selective Acylation and Its Applications in the Streamlined Assembly of Oligosaccharides Stephanie Blaszczyk and Weiping Tang University of Wisconsin Carbohydrates are structurally diverse and synthetically challenging molecules that play indispensable roles in biological processes, including growth, development, inflammation, infection and cell signaling. Chemists' inability to efficiently synthesize complex carbohydrates hinders biologists' ability to probe how their structural differences affect biological function. As such, the site-selective functionalization of carbohydrates is an active area of research. We previously reported that a cation-lone pair interaction can direct site-selective acylation in O-glycoside trans-1,2-diols using a chiral catalyst. We speculated that we could selectively functionalize S- glycosides via a similar interaction. Surprisingly, the sterically bulky adamantyl substituent of the S-glycoside directed site-selective acylation at the C2 position of S-glycosides through dispersion interactions between the adamantyl C-H bonds and the π-system of the cationic acylated catalyst. This interaction may have broad implications in many other chemical reactions. Because of their stability, chemical orthogonality, and ease of activation for glycosylation, the site-selective acylation of S-glycosides significantly streamlines oligosaccharide synthesis. This methodology will have broad applications in the synthesis of complex carbohydrates for basic research and drug discovery.

Keywords: carbohydrates, site-selective functionalization, glycosylation Poster #20 Modular synthesis and reactivity profiling studies of diverse and stereochemically enriched C-, S-, and P- based electrophilic macrocycles. Gihan C. Dissanayake, Salim Javed, Dimuthu A. Vithanage, Arghya Ganguly and Paul R. Hanson The University of Kansas A modular, pot-economical and library amenable strategy for the facile synthesis of diverse C-, S-, and P-containing -unsaturated 12- to 22-membered macrocycles is reported. The key reactions involved in the synthesis of these electrophilic macrocycles are iterative one-pot sequential RCM/CM/H2, iterative regio- and diastereoselective cuprate addition, protecting group manipulation and a final one-pot sequential RCM/deprotection. These electrophilic macrocyclic probes have been evaluated for reactivity against a range of nucleophiles to elucidate structural reactivity relationships. and will further be subjected to Activity-Based Protein Profiling (ABPP) and Cysteine Quantitation (Cys-Q) studies. Collectively, these studies will contribute to further our understanding to identify “hot spot residues” in the proteome. Keywords: electrophilic macrocycles, reactivity profiling, pot-economic synthesis

Poster #21 Covalent Inhibitors Target O-GlcNAc Transferase in Cells Matthew Worth, Arielis Estevez Davila, Chia-Wei Hu, Dacheng Fan, Hao Li, Dongsheng Zhu, and Jiaoyang Jiang University of Wisconsin O-GlcNAc transferase (OGT) catalyzes the attachment of O-linked β-N-acetylglucosamine (O-GlcNAc) to over 1,000 nucleocytoplasmic proteins. This modification, termed O-GlcNAcylation, regulates proteins involved in a variety of cellular processes including transcription, translation, and signal transduction. Elevated levels of OGT and O-GlcNAcylation have been detected in many diseases including most types of cancer, and O-GlcNAc dysregulation has been linked to tumor progression. However, the paucity of suitable OGT inhibitors has impeded the dissection of OGT function in cells. A number of OGT inhibitors have previously been published, but each suffers from limitations such as toxicity, poor potency, or lack of specificity. This poster presents our design, synthesis and characterization of a new class of OGT covalent inhibitors that target a unique cysteine residue in OGT active site. Our top analogs irreversibly inhibit OGT in vitro, and X-ray crystal structures confirmed the covalent mechanism of inhibition. Cell permeable analogs of these compounds reduced cellular O-GlcNAc levels more effectively than one of the best reported OGT inhibitors. Furthermore, click chemistry and in-gel fluorescence assay have verified OGT as the primary target of our inhibitor in living cells. These inhibitors are expected to be powerful tools to study the biological functions of OGT and O-GlcNAcylation.

Keywords: O-GlcNAc, carbohydrate, covalent inhibitor

Poster #22 Rearrangement of 2-Chloroquinazolin-4(3H)-ones to Afford Cyclic Guanidines with Potential Axial Chirality Gang Yan, Bereket Zekarias, Victor Jaffett, Xiaoyu Li and Jennifer E. Golden University of Wisonsin The conversion of quinazolinones to (E)-arylamidines is a cornerstone of the Golden laboratory as part of a medicinal chemistry program focused on the development of antiviral agents. In the exploration of structure- activity relationships, we discovered that 2-chloroquinazolin-4(3H)-ones formed novel cyclic guanidines when treated with N,N’-dialkylethylenediamine or N,N’-dialkyl-1,3-propanediamine. After optimization, the transformation was used with various diamines and substituted 2-chloroquinazolinones to afford more than 25 novel guanidines in up to 95% yield. Interestingly, preliminary studies suggest that the reaction with symmetrical N,N’-dimethylethylenediamine may generate products bearing an axis of symmetry along the C-N double bond of the guanidine. This was supported by the equivalency of methylene-associated proton and carbon nuclei of the cyclic guanidine moiety in 1H and 13C NMR, respectively, and the presence of one proton NMR singlet representative of all six protons of the two N-methyl groups. Asymmetrically substituted diamines appeared to afford enantiomeric products with a chiral axis based on NOE correlations. An analysis of the data leading to this hypothesis will be shown, and the discovery and scope of the reaction leading to these compounds will be discussed.

Keywords: Reaeeangement, Guanidines, Axial Chirality

Poster #23 Discovery of Novel N-(4-hydroxybenzyl)valine Hemoglobin Adducts in Human Blood. Amanda Degner, Henrik Carlsson, Isabella Karlsson, Johan Eriksson, Suresh Pujari, Natalia Tretyakova and Margareta Tӧrnqvist University of Minnesota Electrophilic compounds are common endogenous and exogenous exposures in humans and can form adducts with nucleophilic sites of proteins and DNA. However, the identities and sources of many of these adducts are unknown. Our previous adductomics study detected 19 unknown adducts to N-terminal valine in hemoglobin. The third most abundant of these unknown adducts has a m/z of 595.153, which corresponds to an added mass of 106.042 Da to valine, and had yet to be identified. From the elemental composition of C7H6O, we hypothesized that the precursor of this adduct was an unsubstituted quinone methide. Standards for 2- and 4-quinone methide valine adducts were synthesized, spiked into human blood, derivatized, and analyzed by LC-HRMS/MS using an Orbitrap Q-Exactive mass spectrometer. Retention time, accurate mass, and fragmentation was compared between the standards and the unknown adduct. The accurate mass of both 2- and 4-OHBn-Val-FTH matched the unknown. However, the retention time (18.58 min) and fragmentation pattern of 2-OHBn-Val-FTH did not match the unknown adduct, but instead matched another previously unidentified adduct that was present at a lower abundance. The retention time (17.58 min) and fragmentation pattern for 4-OHBn-Val-FTH matched that of the unknown adduct, with major fragments of m/z 390, 445, and 373. Thus, the adduct was identified as N-(4- hydroxybenzyl)valine. Valine adducts formed from 2- and 4-hydroxybenzaldehyde were found to have identical structures to the adducts formed from 2- and 4-quinone methide, respectively, suggesting that these benzaldehydes could also be possible precursors to the identified adducts in addition to quinone methides.

Keywords: Adductomics, quinone methide, hemoglobin adduct

Poster #24 Development of Novel Small Molecule PCSK9 Modulators Haibo Xie and Weiping Tang University of Wisconsin Cardiovascular diseases (CVDs) are the leading cause of death globally. Hypercholesterolemia is a well-known risk factor for CVDs. The important role of proprotein convertase subtilisin/kexin type 9 (PCSK9) in cholesterol homeostasis has been confirmed by gain- and loss-of-function PCSK9 variations in human populations and two recent FDA approvals of monoclonal antibody (mAb) drugs against PCSK9. Considering the cost and other complications associated with mAbs, small molecules that can reduce the PCSK9 protein level emerge as a promising strategy. We recently identified a series of small molecule PCSK9 modulators that can reduce the PCSK9 protein levels in cell-based phenotypic screening.

Keywords: PCSK9, LDLR, Diindolylmethane

Poster #25 Anthrax Antitoxin Lead Optimization via Bioisosteric Replacement and Other In Silico Strategies McDermott, C. M. and Ambrose, E. A. University of Minnesota Bacillus anthracis, the causative agent of the deadly bacterial infection anthrax, is a well-known bioterrorism agent in need of effective countermeasures. Current treatment options include antibiotics and antibody-based therapeutics, but neither directly targets the primary cause of the lethality of these infections: the lethal factor (LF), a zinc-metalloprotease and component of the tripartite exotoxin that the bacteria secrete. LF, which interferes with cellular immune defense mechanisms and induces endothelial cell apoptosis, has therefore become a popular target for the development of new anthrax therapeutics; however, no LF inhibitors have yet been approved to treat anthrax. Recently, we performed a large-scale experimental high-throughput screen and identified two promising small molecules active against LF. Here we report the structures of these hits as well as efforts to increase their solubility for structural biology studies while retaining their inhibitory activity towards LF, primarily via targeted bioisosteric replacement and a variety of virtual screening techniques. We also employed biophysical fragment-based screening to identify functional groups that increase inhibitory activity against LF, and these results are presented herein. Finally, we report new structural biology data crucial in elucidating the binding modes of our novel compounds as well as key structural features that contribute to strong and specific LF inhibition.

Keywords: Anthrax, Biological Warfare, Computational Chemistry

Poster #26 Incorporation of Agouti-Related Protein (AgRP) Human Single Nucleotide Polymorphisms in the AgRP- Derived Macrocyclic Scaffold c[Pro-Arg-Phe-Phe-Asn-Ala-Phe-DPro] Decreases Melanocortin-4 Receptor Antagonist Potency Zoe M. Koerperich, Mark D. Ericson, Katie T. Freeman and Carrie Haskell-Luevano University of Minnesota The melanocortin receptors belong to the family of G protein-coupled receptors (GPCRs). Five melanocortin receptors have been discovered to date, which respond to both endogenous agonists and antagonists.1 The peptide antagonists Agouti Signaling Protein (ASP) and Agouti-Related Protein (AgRP) both contain the putative sequence Arg-Phe-Phe in the carboxyl terminal region, which is hypothesized to be responsible for both receptor affinity and antagonist activity.2 This sequence is located on an exposed ß-hairpin loop, which can be mimicked in a smaller amenable scaffold. This study focused on exploring four SNPs that were deposited into the NIH Variation Viewer3, which result in missense mutations in the proposed active loop of AgRP.4 It was hypothesized that the SNPs would alter AgRP signaling in the MC4R cascade, which may have physiological consequences in humans. The SNPs were incorporated into macrocyclic peptide scaffolds c[Pro-Arg-Phe-Phe-Asn-Ala-Phe-DPro] and c[Pro-Arg-Phe-Phe- Dap-Ala-Phe-DPro] which were previously reported to have nanomolar potency at the MC4R.5 References : 1. Ericson, M.D. et al. Biochim. Biophys. Acta. Mol. Basis. Dis. 2017, 1863, 2414-2435. 2. Bolin, K.A. et al. FEBS Lett. 1999, 451, 125-131. 3. https://www.ncbi.nlm.nih.gov/variation/view/ 4. Ericson, M.D. et al. ACS Chem. Neurosci. 2018, 9, 1235-1246. 5. Ericson, M.D. et al. J. Med. Chem. 2015, 58, 4638-4647.

Keywords: Melanocortin Receptors; Agouti-Related Protein; Macrocycles

Poster #27 Intramolecular C–H Functionalization to Electrophilic Probes: Generation of Novel Triazole-Containing Sultams Viena U. Thomas, Andie Jo Cassity, Maria A. Khan, Nicole Marie Windmon, Naeem Asad, Kyu Ok Jeon, Anna J. Diepenbrock, Qin Zang, Jung Ho Jun, Paul R. Hanson The University of Kansas The development of chemically unique electrophilic probes capable of modulating biological nucleophiles is the focal point of this study. Rapid and robust synthetic methods capable of generating structurally, electronically and stereochemically attenuable scaffolds are necessary for generating a broad spectrum of molecular probes. The design and synthesis of small libraries of triazole-containing sultam derivatives employing intramolecular C– H functionalization of an appendant triazole moiety are reported. This efficient method has enabled the synthesis of small libraries of diverse electrophilic triazole-fused sultams. The electrophilic character of these novel sultams are being investigated for their reactivity patterns with a variety of small nucleophiles.

Keywords: Electrophilic probes, C–H functionalization, triazole-fused sultams

Poster #28 Chemical Tools to Elucidate Mechanisms of Dopamine Toxicity in Parkinson’s Disease Alexander K. Hurben and Todd Doran University of Minnesota Parkinson’s disease (PD) is characterized by abnormal motor symptoms due to selective loss of dopaminergic neurons, yet the mechanisms that trigger neurodegeneration are unclear, confounding the design of therapeutics and diagnostics that modify disease at pre-symptomatic stages. The selective loss of nigrostriatal neurons suggests that dopamine (DA) may play a key role in this oxidative damage. Indeed, cytosolic and interstitial dopamine readily auto-oxidizes to form reactive dopaquinone electrophiles (DQEs), which post-translationally modify proteins leading to tissue damage. Dopamine-adducted proteins (DAPs) form via conjugate addition at the o- quinone moiety. We hypothesized that the accumulation of DAPs contributes to initial nigrostriatal death in PD. To better understand how DAPs lead to toxicity, we sought to profile the DAP proteome. While current attempts to profile DAPs in PD provide compelling evidence for their involvement in PD pathophysiology, they are unlikely to be comprehensive. To define the relationship between DAPs and PD progression, we developed DQE- like chemical probes with attenuated reactivity that react like DQEs. Based on these results, we probed lysates and found that such adducts also form when DQEs are added to cell lysates. Finally, we demonstrated that proteome-wide profiling of DAPs is possible with our chemoproteomic enrichment probes. Collectively, the probes and methods described herein provide new tools to profile and validate novel physiologically-relevant DAPs missed in previous proteomic studies. These future studies will thus lead to a more complete understanding of how discrete dopamine-protein adducts elicit their toxic behavior throughout early stages of PD.

Keywords: Parkinson's disease, Dopamine, Oxidation

Poster #29 Baicalein is a phytohormone that signals through the progesterone and glucocorticoid receptors Julia R. Austin, Brenna Kirkpatrick and Joanna E. Burdette University of Illinois Progestin therapy is used to treat many gynecological diseases that result from dysregulated progesterone. Cardiovascular disease is a side effect that is associated with progestin therapy. Alternative progestins that are selective for the progesterone receptor (PR) are necessary. Herbal supplements are becoming popular, although it is unclear what they contain. Previous studies have shown that some herbal supplements contain compounds that modify steroid signaling and our lab has identified apigenin and kaempferol as molecules in herbal supplements that act similarly to progesterone, termed phytoprogestins. Using structure activity relationships and literature reviews, we selected three flavones, tangeretin, wogonin, and baicalein, to determine if they had progestin activity. Of the three, only baicalein demonstrated progestin antagonist activity in the progesterone response element luciferase assay. To further confirm that baicalein signaled through PR, its ability to trigger PR degradation and to block breast cancer cell proliferation were measured. Baicalein did not degrade PR nor did it inhibit proliferation of breast cancer cells. Progestins are known to bind to GR, so we investigated if baicalein activates the glucocorticoid receptor (GR). Baicalein was found to be a GR agonist. Baicalein did not induce GR degradation but inhibited proliferation of an ovarian cancer cell line in a dose dependent manner. In summary, baicalein is a phytohormone that blocks progesterone receptors but acts as an agonist on glucocorticoid receptors. If further experiments can confirm that baicalein signals through GR this will connect a widely used compound with a well-accepted cellular target for anti-inflammation and anti-tumor action, GR.

Keywords: Progestins, glucocorticoids, herbal supplements

Poster #30 Development of Potent HDAC6 Selective Degraders Ka Yang, Hao Wu, Zhongrui Zhang, Haibo Xie, Eric D. Leisten, Ziyuan Li and Weiping Tang University of Wisconsin Most histone deacetylases (HDACs) are epigenetic regulators that control the acetylation level of histones. As class IIb HDACs, HDAC6 modulates the acetylation of cytoplasmatic proteins such as α-Tubulin and HSP90. HDAC6 is involved in multiple disease-relevant pathways. We designed and developed the first degrader for zinc-dependent HDACs by using proteolysis targeting chimera (PROTAC) strategy. Through screening the expression level of HDAC proteins after the treatment of PROTACs, we found that PROTACs with a non- selective HDAC inhibitor linked to E3 ligand Pomalidomide was able to degrade HDAC6 selectively. Mechanistic investigation illustrated the critical role of CRBN E3 ligases and the ubiquitination-proteasome system in HDAC6-targeting degradation. We subsequently developed the second generation of HDAC6 degraders by using selective HDAC6 inhibitor as the “warhead”. The new generation of degraders exhibited improved potency and selectivity. The most potent degrader has a 1.6 nM of DC50 for HDAC6 and inhibits the proliferation of multiple myeloma (MM) cells at low nM concentrations as well. We are currently exploring the therapeutic potential of HDAC6 degraders in various cancers and other diseases.

Keywords: HDAC6, PROTAC, Cancer Treatment

Poster #31 Site- and Stereoselective Alkylation of Carbohydrates by OH Insertion Xiaolei Li and Weiping Tang University of Wisconsin Selective functionalization of hydroxyl (O-H) groups in carbohydrates represents a long-standing challenge in chemistry. We recently discovered that metal carbenes could selectively insert into hydroxyl groups of carbohydrates, representing a mild and powerful tool for addressing the site-selectivity issue during the modification of carbohydrates. We observed excellent regioselectivity and good stereoselectivity for a broad range of substrates. Density functional theory (DFT) calculations have been carried out to understand the regio- and stereochemistry.

Keywords: Siteselective, Regioselective, O-H insertion

Poster #32 Development of Gamma-Functionalized Hydrocarbon Stapled Peptides as Inhibitors of the Estrogen Receptor/Coactivator Interaction Kornelia J. Skowron, Thomas Speltz, Zamia Siddiqui, Jeanne Danes, Sean Fanning, Colin Fowler, Chris Mayne, Emad Tajkhorshid, Geoffrey L. Greene, Jonna Frasor and Terry W. Moore University of Illinois Resistance to endocrine therapy has become a limitation in the treatment of estrogen receptor positive breast cancer. Mutations in the estrogen receptor ligand-binding domain, specifically Y537S and D538G, have been reported to play a significant role in the mechanism of resistance by decreasing the efficacy of tamoxifen, the standard of care for estrogen receptor-positive breast cancer patients. Coactivator binding inhibitors have been reported to be effective in inhibiting proliferation facilitated by the estrogen receptor as an alternative method to endocrine therapy. Work previously completed in our lab indicated that steroid receptor coactivator-mimicking hydrocarbon-stapled peptides have high affinity for the wild-type receptor and higher affinity against Y537S and D538G. Molecular dynamics and structure-based rational design indicated the possibility of developing high affinity functionalized hydrocarbon stapled peptide inhibitors of the estrogen receptor – steroid receptor coactivator interaction. The development of new amino acids λR and λS for stapling indicated that the incorporation of a methyl in the γ- position of stapling amino acid S5 improves the potency in comparison to the unsubstituted amino acid. Interestingly, the stapled peptide with the γ-methyl binds within 4 Å of D538. Our current work includes the development of functionalized stapled peptides for selectively inhibiting ER-D538G. TR-FRET is used to validate the efficacy and positioning of the staple. In particular, we have examined the selectivity of stapled peptides for estrogen receptor alpha over other nuclear receptors.

Keywords: protein-protein interactions, peptide

Poster #33 Targeting KRAS with small molecules at a novel site Saad A Alqarni, Kiira M Ratia, Priyanka Gajwani, Laura Bloem, Hyun Lee, Laura M Kim, John O'Brien and Gregory R Thatcher University of Illinois Mutations in RAS genes are involved in almost 30% of all human cancers, thus they are a promising avenue to explore for cancer therapeutics. RAS is a GTPase and controls deferent cell functions including proliferation. The RAS gene family contains (KRAS, NRAS and HRAS). In some types of cancers, K-RAS mutations occur in much higher percentages such as pancreatic cancer in which 90% of cases harbor K-RAS mutations. There have been no clinical drugs that directly inhibit KRAS proteins, thus providing an exciting novel area for research. We hypothesize that a small molecule inhibitor of KRAS signaling can provide a therapeutic remedy for cancer. A high throughput screening identified novel small molecules that bind to KRAS at a novel site with good potency. Furthermore, we have demonstrated a preliminary structure-activity relationship (SAR) data. This work provides an exciting area for identifying chemical probes that inhibit KRAS downstream signaling.

Keywords: cancer research

Poster #34 Further Exploration of the SAR of Imidazoquinolines; Identification of Potent C7 Substituted Imidazoquinolines Jordan Hunt and Laird Forrest Toll-like receptors 7 and 8 (TLR7/8) are targets for immunostimulatory agents to induce cell-mediated immune responses in vaccines or host immune response in cancer treatments. Small molecule agonists of TLR7/8, such as imidazoquinolines, are of great interest to medicinal chemists. Imidazoquinolines have been extensively modified to understand the structure activity relationship (SAR) at the N1- and C2-positions resulting in the clinical drug imiquimod, resiquimod, and several other highly potent analogues in preclinical and early clinical trials for cancer treatment and as vaccine adjuvants. However, the phenyl ring’s SAR has not been fully elucidated in the literature. This study examines the SAR of varying N1-, C2- and C7-substituted imidazoquinolines to show that TLR7/8 can tolerate changes at the C7 position and produce new functional groups to change the physical chemical properties of the molecule. These compounds not only demonstrated that TLR7/8 tolerate changes at the C7 position but can increase potency.

Keywords: TLR7/8, imidazoquinolines, SAR

Poster #35 The Mechanistic Role of Metal Ions, Ca2+ and Mg2+, in RGS: G-Protein Interactions Joe O'Brien and David Roman University of Iowa Regulator of G protein signaling (RGS) proteins are negative regulators of G protein-coupled receptor (GPCR) signaling through their ability to act as GTPase activating proteins (GAPs) for some Ga subunits. The RZ subfamily, of which RGS17 is a member, binds to activated Gao, Gaz, and Gail-3 proteins to modulate downstream pathways, including those involved in formation of cyclic AMP. In contrast to other RGS proteins, less is known about the regulation of RZ family members. Both Crystallization and 1H-15N 2D HSQC NMR experiments revealed an interaction of the metal ion Ca2+ with RGS17 at a defined binding site. Subsequent protein-protein interaction experiments, using AlphaScreen were used to assess the impact of the ions Ca2+ and Mg2+ on the RGS17 interaction with activated Gao. The results indicate that both Ca2+ and Mg2+ have an effect of promoting the RGS17-Gao interaction. These studies will extend to examining the selectivity and affinity of RGS17 for other physiologically relevant divalent metal cations, such as Zn2+, Cu2+, and Mn2+. In addition, the residues of RGS17 that bind Ca2+ are conserved in multiple RGS proteins. The functional impact of metal ion binding is likely not limited to RGS17 and a more in-depth evaluation of these proteins for metal binding deserves further attention.

Keywords: Metal, Binding, activity

Poster #36 Characterizing Calpain-1 Inhibition as a Therapeutic Approach to Neurodegeneration Rachel C Knopp, Ammar Jastaniah, Oleksii Dubrovskyi, Sue Lee, Leon Tai and Gregory R.J. Thatcher University of Illinois Calpain-1 (CAPN1) is calcium-sensitive cysteine protease found hyper-activated in the early pathogenesis of many neurodegenerative diseases including Alzheimer’s disease (AD), traumatic brain injury (TBI) and ischemic stroke (IS). This over-activation leads to the aberrant release of other cysteine proteases, unregulated activity of many neuroprotective and neurodegenerative signaling cascades, and eventually results in cell death. Research reports have long suggested that inhibition of calpain, both selectively and nonselectively, is an effective therapeutic approach to neurodegenerative disease. This research explores CAPN1 as a drug target, specifically seeking to determine how inhibition selectivity affects therapeutic efficacy. We hypothesized that given the multifaceted nature of neurodegenerative diseases, nonselective targeting would have superior advantages over selective targeting. To test this theory, we looked at neuroprotection in an in vitro assay simulating ischemic stroke, neuroinflammation in a mild TBI animal study, and attenuation of blood brain barrier (BBB) disruption in brain endothelial cells (BECs). Enzyme inhibition profiles of small molecule inhibitors were characterized through kinetic assays and validated by monitoring breakdown products of known CAPN1 substrates. All inhibitors were differentially neuroprotective against oxygen glucose deprivation-induced cell death at different treatment paradigms (pretreatment, ischemia, and reperfusion). Dual and selective inhibition were equally protective against scopolamine-induced cognitive impairment and reduced neuroinflammation 24 hours post- mTBI. Future studies will focus on the blood brain barrier, specifically characterizing the effect of inhibition strategies on tight junction and adhesion proteins, along with angiogenesis.

Keywords: neurodegeneration, cysteine protease inhibition, selectivity

Poster #37 Evaluating the effect of oregano essential oil and rosemary extract on Sestrin 2 expression in HCT 116 colon cells Jacob Veenstra and Jeremy Johnson University of Illinois The purpose of this study was to determine the effect of oregano essential oil (OEO) and rosemary extract (RE) on Sestrin 2 expression and regulation of mTORC1. Sestrin 2 is a multi-functional protein regulated by Nrf2 transcriptional activity. Sestrin 2 has been shown to regulate the activity of mTORC1, a determining factor in cell proliferation, survival, protein synthesis, and autophagy. Methods: Phytochemicals found in Mediterranean herbs were evaluated for promoting Sestrin 2 protein expression in HCT 116 colon cells. Carnosic acid, carnosol, and carvacrol were identified as promising inducers of Sestrin 2 expression. Carnosic acid and carnosol are primarily found in RE, while carvacrol is the primary component of OEO. This study used OEO and two types of RE, a water soluble extract and an oil soluble extract. Western blot analysis was performed to determine the effect of upregulation of Sestrin 2 by OEO and RE treatment on mTOR phosphorylation and effect on its downstream constituents. Results: Western blot analysis on HCT 116 cells treated with OEO and RE for 24 hours showed that OEO and oil soluble RE significantly increased Sestrin 2 expression at concentrations of 50 μg/mL. Immunoblot with OEO also showed that increasing Sestrin 2 expression decreased phosphorylated p70S6K1, suggesting an inhibitory effect on mTOR activity. Conclusion: This study suggests that OEO and RE, along with its principal components, are effective inducers of Sestrin 2 and autophagy modulation. Sestrin 2 induction could be a possible mechanism for preventing or treating certain gastrointestinal diseases associated with inflammation.

Keywords: phytochemicals, Sestrin 2

Poster #38 Identification of a Fluorinated Pyronin Fluorophore that Enables Visible Blue Light to Rapidly Depolarize Mitochondria Zhe Gao and Blake Peterson University of Kansas Small molecules that covalently modify biomolecules represent important therapeutics and molecular probes. To provide novel chemical tools that modify amines in living cells, we synthesized analogues of the fluorophore pyronin B. This cell-permeant cationic fluorophore has been extensively used to study mitochondria, where it accumulates due to the strongly negative potential that exists across mitochondrial inner membranes. Because this negative membrane potential is essential for critical functions of mitochondria, including the biosynthesis of ATP and calcium homeostasis, depolarization of mitochondria is associated with a wide range of pathologies. Consequently, small molecules that affect the mitochondrial membrane potential are of interest for studies of neurodegenerative disease, obesity, cardiomyopathy, cancer and aging. To increase the chemical reactivity of pyronin B, we installed fluorine atoms to enhance its electrophilicity. In 2,7-difluoropyronin B, this fluorination enabled reversible attack by nucleophilic amines, typically with some additional oxidation to form fluorescent 9- aminopyronins. Irradiation with visible blue light was found to enhance this oxidation step, enabling optical control over amino modification. In living HeLa cells, 2,7-difluoropyronin B localized in mitochondria, similar to the parent fluorophore, and was converted at least partially to fluorescent aminopyronins in these organelles, as evidenced by spectral profiling confocal microscopy. Irradiation of these cells with blue light rapidly depolarized mitochondria, presumably by enhancing oxidative modification of mitochondrial amines. The unique reactivity of 2,7-difluoropyronin B with amines offers a novel method to control mitochondrial function with visible light.

Keywords: Mitochondria, Fluorophore, Depolarization

Poster #39 Human Hepatic Microsomal Sulfatase Catalyzes the Hydrolysis of Polychlorinated Biphenyl Sulfates Kris Tuttle and Michael W. Duffel University of Iowa Polychlorinated biphenyls (PCBs) are environmentally persistent toxicants both from legacy sources such as materials often found in older buildings and from new sources such as inadvertent byproducts of manufacturing. PCBs and their metabolites have adverse health effects ranging from cardiovascular disease and diabetes to endocrine disruption and cancer. Some PCBs accumulate in tissues unchanged, while those with lower numbers of chlorine atoms readily undergo biotransformation into hydroxylated PCB metabolites (OH-PCBs). Among other potential metabolic fates, the sulfation of OH-PCBs, catalyzed by cytosolic sulfotransferases, results either in excretion or in transport and re-uptake of the resulting PCB sulfates. Following transport of a PCB sulfate into tissues, steroid sulfatase (STS) might catalyze hydrolysis back to the corresponding OH-PCB, which is usually more toxic than the PCB sulfate. STS is important in the hydrolysis of sulfate esters of endogenous steroids as well as xenobiotic sulfates. We hypothesized that the STS in human hepatic microsomes would catalyze the hydrolysis of PCB sulfates. Our results indicate that human hepatic microsomes (a pooled preparation from 50 male and female donors) catalyze this reaction. We examined twelve PCB sulfates, and all served as substrates for the microsomal sulfatase. Initial velocities for the sulfatase-reaction ranged from 2-19-fold greater than the rate observed with dehydroepiandrosterone sulfate, a physiologically important substrate used for comparison. Thus, the microsomal sulfatase-catalyzed conversion of PCB sulfates to their corresponding OH-PCBs indicates a dynamic interchange between these PCB metabolites that may be an important component of their transport and toxicity. [Supported by NIH P42 ES013661].

Keywords: PCB sulfates and sulfatase

Poster #40 Probing substrate recognition and proteolytic site selection by γ-secretase with designed helical peptides Anija mol T Philip, Kathleen Meneely, Todd Williams and Michael S. Wolfe University of Kansas Alzheimer’s disease (AD) is the primary cause of dementia worldwide. Although recognized as a disease for over a century, the molecular etiology of AD remains unclear. The primary component of the characteristic cerebral plaques of AD is the amyloid β-peptide (Aβ), and cleavage within the transmembrane domain (TMD) of the amyloid precursor protein (APP) by γ-secretase is the final step in Aβ production. γ-Secretase is a membrane- embedded aspartyl protease, with its active site within the boundaries of the lipid bilayer. The enzyme initially cleaves at the ε site to produce 48- or 49-residue Aβ, and this is followed by carboxypeptidase trimming every 3 residues to shorter, secreted forms of Aβ. Substrate-based helical peptides are potent inhibitors of the enzyme, suggesting a helical conformation of the TMD is important for substrate recognition. We hypothesize that the face of the helix of TMD substrate that initially interacts with the protease dictates the initial site of proteolysis. To test this hypothesis, we have designed helical peptide substrates based on the APP TMD sequence. Judicious installation of the helix-inducing residue aminoisobutyric acid (Aib) leads to Aib residues along one face of the helix and APP TMD residues along the remaining face. Moreover, Aib installation facilitates the synthesis and solubility of these highly hydrophobic peptides. Three different Aib-containing peptides were synthesized, to present three different faces of the APP TMD helix. Incubation of these designed substrates with γ-secretase followed by mass spectrometric analysis of the proteolytic fragments revealed subtle but consistent changes in the ratio of cleavage site selection. These results suggest that while the helical face of APP TMD substrate can influence cleavage site selection, other factors are likely involved. Variants of these designed substrates that incorporation AD-causing APP mutations are currently being investigated and will be discussed.

Keywords: Gamma Secretase, Alzheimer’s disease

Poster #41 Vaccine-driven pharmacodynamic dissection of ketamine psychoactivity Zhen Zheng and Cody Wenthur University of Wisconsin Developing small-molecule vaccination to define the mechanism of action for complex psychoactive mixtures is a new chemical neuroscience approach. Recently, the method for Determining the Identity of Species Supporting Expression of CNS-activity Through Incremental Vaccination (DISSECTIV) was reported and utilized for deconvolution of the actively contributing molecules to the psychoactivity of the chimeric stimulant fenethylline. With DISSECTIV, mixtures of psychoactive substances can be pharmacologically dissected in vivo by designing haptens that promote the in vivo generation of small-molecule binding antibodies to either unambiguously assign a given behavioral outcome to a single species within the mixture or identify such behavior as an effect only arising from the concerted action of multiple chemical species. Ketamine, a prescription general anesthetic, has attracted broad attention recently due to its rapid and sustained antidepressant effects. However, its mechanism of action remains controversial. It has been proposed that hydroxynorketamine (HNK), a major metabolite of ketamine, is the relevant active species, acting via an alternative mechanism from ketamine. In this study, we aim to resolve this potential polypharmacology of ketamine using the DISSECTIV method. We synthesized several haptens for ketamine and HNK, conjugated the haptens to keyhole limpet hemocyanin to generate the immunogenic species for vaccination, and intraperitoneally administrated the vaccine to Swiss Webster mice. Antibody titers generated in the serum were assessed using enzyme-linked immunosorbent assay (ELISA) analysis. Moreover, antibody-mediated alterations in the pharmacokinetics of ketamine and HNK were also evaluated using liquid chromatography - mass spectrometry (LC-MS) analysis.

Keywords: ketamine psychoactivity, small-molecule vaccination

Poster #42 Structural Exploration of the HA-Binding Groove in CD44 using Disaccharide Mimetics William McCue and Dr. Barry Finzel University of Minnesota CD44 is a cell surface hyaluronan (HA) binding protein implicated in a variety of different cancers because it can modulate tumor cell adhesion, growth, and therapeutic resistance. High molecular weight-HA (HMW-HA; 107 Da) stimulates cancer progression by binding to CD44 and activating oncogenic signals which can be inhibited using very small (<104 Da) HA fragments to disrupt HMW-HA/CD44 interactions. The goal of this project is to develop small molecules that selectively block binding of HMW-HA by CD44 to evaluate their potential to limit cancer progression and improve response to existing anticancer therapies. Our previous work has confirmed that analogs containing an 8-amino tetrahydroisoquinoline (THIQ) pharmacophore bind CD44 with moderate affinity. Crystallography has shown that THIQ analogs bind near the CD44 HA binding groove, leading to the design of a series of THIQ-linked oligosaccharides predicted to extend into the binding site of HA at subsites Glc-5 and NaG-6. The THIQ should impart selectivity for CD44 over other HA-binding proteins, while computational modeling predicts increasing affinity with the addition of each additional saccharide unit to the pharmacophore. However, when a THIQ-glucuronate conjugate was synthesized and tested with our immobilized HMW-HA SPR assay, it produced no effect on CD44 binding at 10 mM. Currently, we are employing computational methods to design alternatives to the glucoronate portion so extended analogs may span the Glc-5 subsite without strain and reach into NaG-6 where additional strong interactions with CD44 are possible. A summary of recent modeling studies will be presented.

Keywords: CD44 Structure Biology

Poster #43 Identification of inhibitors of MSI2-RNA interaction through NMR fragment screening Minli Xing, Lan Lan, Justin T. Douglas, Philip Gao, Robert P. Hanzlik, Jeff Aubé and Liang Xu University of Kansas Musahi-2 (MSI2) is an RNA-binding protein (RBP) that plays a key regulator role in cellular functions, especially in post-transcriptional regulations. MSI2 can bind to target mRNA and regulate translation of proteins that involve in oncogenic pathway, and participate in cancer stem cell populations maintenance, cancer invasion regulation, and metastasis and development of more aggressive cancer phenotype. Thus, MSI2 is a promising therapeutic target for cancer. MSI2 contains two N-terminal RRMs (RNA-recognition Motifs) RRM1 and RRM2, which mediate the binding to their target mRNAs. Compounds that can disrupt MSI2-RNA interaction represent promising drug candidates for cancer. In this study, we used saturation transfer difference (STD) NMR based fragment screening to search for fragments that bind to MSI2-RRM1, and 2D 1H-15N heteronuclear single quantum coherence (HSQC) titration experiments to identify ligand binding sites. The chemical shift perturbations analysis revealed that the fragment hits bind to the RNA binding site of MSI2-RRM1. The fragments identified in this study are starting points for developing more potent inhibitors of MSI2-RNA interaction.

Keywords: Musashi2, fragment screening, nuclear magnetic resonance spectroscopy

Poster #44 Identification of Selective APOBEC3 Ligands by Mass Spectrometry Fragment Screening Katherine Jones, Daniel Harki University of Minnesota Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3 (APOBEC3) is a family of seven single- stranded (ss)DNA cytosine-to-uracil deaminase enzymes (A3A/B/C/D/F/G/H) that function as part of an overlapping immune defense system against foreign DNA. APOBEC3-catalyzed deamination has also been associated with genomic mutation and the evolution of drug resistance mutations in HIV and various cancers, signifying an important therapeutic target. Previous work by the Harki lab and collaborators have identified inhibitors of A3G that are modestly potent and cross-reactive. The goal of this project is to perform a mass spectrometry-based binding screen to identify new A3G ligands, as well as ligands for the cancer-associated deaminase A3B. The ligands that are identified from screening will be characterized and optimized leveraging medicinal chemistry, structural biology, computational chemistry, and biochemical assays to develop novel A3- specific inhibitors. A whole protein mass spectrometry-based binding screen to identify ligands of A3 proteins has been developed. This assay allows for the identification of covalently-bound fragments identified by a shift in mass and relatively quantified as the percent of ligand bound. Early work on this project has confirmed binding of iodoacetamide to A3G C-terminal domain (ctd), and an initial pilot screen has been performed against A3Gctd using a library (N=35) of fragments containing reversible electrophiles. These results and the future directions of the project will be presented.

Keywords: mass spectrometry, fragment tethering, HTS

Poster #45 Nanodisc technology for studies of small molecule-membrane interactions Sahishna Phaniraj, Blake R. Peterson University of Kansas Nanodiscs are engineered lipoparticles composed of a phospholipid bilayer surrounded by two membrane scaffold proteins. These particles can be used to mimic biomembranes, and they can be considered a state-of-the-art chemically-defined lipid bilayer. Nanodiscs are under investigation as tools for targeted drug delivery, for functional studies of membrane proteins by surface plasmon resonance and other biophysical techniques, and for structural studies of membrane proteins by nuclear magnetic resonance, among other methods. The ability to engineer the membrane scaffold protein to create structures of well-defined molecular size and shape, in addition to the interchangeability and accessibility of the lipid and scaffold components, makes nanodiscs a versatile platform for diverse studies of model membranes. Standard nanodiscs made from the membrane scaffold protein MSP1D1 exhibit some variability in size, structure, and stability. Some of this variability results from non-covalent self-assembly of this protein. In our research, we are working to create novel covalently-linked membrane scaffold proteins that might provide better-defined and more stable nanodiscs. By modifying MSP1D1, we created a cyclic disulfide membrane scaffold protein that we termed MSPSS_M6. These nanodiscs are more thermally stable and more uniform than those derived from MSP1D1. We are currently using these nanodiscs to study interactions of membrane-disruptive peptides and small fluorescent molecules with lipid bilayers. We demonstrate that nanodiscs derived from MSPSS_M6 uniquely enable visualization of pore formation by short synthetic peptides using transmission electron microscopy. This approach is under investigation as a tool for elucidating mechanisms of action of biologically active peptides and small molecules.

Keywords: Nanodiscs, Membrane-disruptive peptides

Poster #46 DIPTOINDONESIN G AND ITS ANALOGUES AS SELECTIVE MODULATORS OF ESTROGEN RECEPTORS Dan Yin and Weiping Tang University of Wisconsin Estrogen receptors play a very important role in breast cancers. Over 50% of breast cancers overexpress ERα and about 70% of them respond to selective estrogen receptor modulators (SERMs). However, ERβ is regarded as a ‘‘tumor suppressor’’ in many cancer types due to its anti-proliferative effects. Recently, we identified Diptoindonesin G (Dip G), which significantly increases ERβ protein stability while decreasing ERα protein levels. In consideration of its novel bioactivity, we developed a practical and versatile synthetic strategy for the synthesis of Diptoindonesin G and its analogues. Preliminary structure–activity relationship (SAR) studies uncovered the critical and dispensable phenolic hydroxyl groups in the natural product.

Keywords: Diptoindonesin G, SERMs, total synthesis

Poster #47 Bacterial Histidine Kinase: Development of Novel Catalytic Domain Inhibitors Conrad Fihn and Erin Carlson University of Minnesota Two-component systems (TCSs) are the main signal transduction pathways used by bacteria to regulate a variety of processes including bacterial development, metabolism, virulence mechanisms, and antibiotic resistance. TCSs consist of a homodimeric membrane-bound sensor enzyme, a histidine kinase (HK), and a cognate effector, the response regulator (RR). The high degree of sequence conservation in the catalytic and ATP-binding (CA) domain of HKs and their essential role in bacterial signal transduction make them an attractive target because inhibitors of this site may possess broad-spectrum antibacterial activity. Severe infections cause by ESKAPE pathogens, such as Pseudomonas aeruginosa, have multiple mechanisms for virulence that are associated with TCSs. Inhibitors that prevent bacterial signal transduction could lead to a new mechanism for the treatment of infectious disease. In my initial work, I have performed structure-activity relationship (SAR) studies with 3- aminobenzothiazole based compounds that target the CA domain that our group identified through a small molecule, high-throughput screening (HTS) campaign against HK853 (Thermotoga maritima). The most potent compounds discovered in these studies possess IC50 values in the low μM range, while also exhibiting activity against two additional HKs. Recently, we have shown that these compounds are effective in whole cells with anti- virulence activity against P. aeruginosa, reducing biofilm production, virulence-associated motility, and toxin production. Through docking studies and protein mutational analysis, we postulate the S-NH-N triad in these 3- aminobenzothiazole compounds that enable hydrogen bonding to three conserved residues in the active site is required for inhibitory activity. SAR studies seem to confirm this hypothesis.

Keywords: Anti-virulence, bacterial signaling, Pseudomonas

Poster #48 Phosphate Tether-Mediated Studies Towards the Synthesis of Leustroducsin B and 13-desmethyl lyngbouilloside Ganguly, A.; Bodugam, M.; Javed, S.; Jayasinghe, S., McParland, J. P.; Whitehead, A.; Hanson, P. R. The University of Kansas Phosphate tether-mediated synthetic studies towards the bioactive natural product Lestroducsin B, a potent colony-stimulating factor inducer and 13-desmethyl-lyngbouilloside, an unnatural analog of lyngbouilloside are reported. The synthetic strategy to the novel phosphorylated polyene-, polyol- and pyranone-containing natural product Leustroducsin B involves a ring-closing metathesis (RCM), oxymercuration/Jones oxidation, cross metathesis (CM) with a lactol-olefinic subunit, and 1,2 addition of the Li-enolate of EtOAc as the key steps for the installation of the “western segment” of this molecule. This tether-mediated approach was also explored for the efficient, pot-economical and library amenable synthesis of macrolactone core of 13-desmethyl- lyngbouilloside. The key reactions involved in the synthesis are iterative one-pot sequential RCM/CM/chemoselective hydrogenation, regio- and diastereoselective cuprate addition, a one-pot sequential Pd- catalyzed reductive allylic transposition/methylation/phosphate tether removal, Roskamp homologation and Yamaguchi macrolactonization.

Keywords: Phosphate tether, Leustroducsin B, 13-desmethyl lyngbouilloside, Macrolactonization

Poster #49 Investigation of β-Lactones as Selective Activity-Based Probes for Penicllin-Binding Proteins Josh Shirley and. Erin E. Carlson University of Minnesota With the introduction of penicillin in the 1940s, β-lactam antibiotics became, and have remained, the most widely used class of antibacterial agents. The targets of the β-lactam antibiotics are penicillin-binding proteins (PBPs), which function to synthesize the peptidoglycan layer of the bacterial cell wall. PBPs synthesize peptidoglycan through transglycosylase activity to generate glycan strands, as well as transpeptidation activity to cross-link stem peptides of the newly synthesized glycan strands. All PBPs possess a catalytic serine residue in their peptidase domain that is essential for substrate turnover and is the site of inhibition by the β-lactams. Although these proteins have been exploited for decades, the individual function and regulation of each homolog in various organisms is not well understood. A key issue in addressing this is a lack of chemical tools that specifically target individual homologs to promote their study. To overcome this, we have employed β-lactones in the design of PBP-selective activity-based probes and have identified a scaffold that possesses a unique PBP selectivity profile. This is a highly significant result, as β-lactones were not previously known to inhibit PBPs. Through the investigation of an initial β-lactone library, it was hypothesized that these compounds may be interacting with the PBP active site in a manner that differs from β-lactams. Our current work aims to develop new β-lactone analogues to target additional PBPs and understand structure-activity relationships between selective probes, as well as the spatial and temporal regulation of PBPs in various species.

Keywords: Bacteria, PBP, ABPP

Poster #50 Next Generation Pyrimidine-Based Photoactivable Catch and Release DNA Decoys Samantha A. Kennelly, Ruber E. Eckermann, Ramkumar Moorthy and Daniel A. Harki University of Minnesota Transcription factors (TFs) are one of the largest classes of human proteins, stemming from over 2000 protein coding genes. Extracellular signals activate or repress TF pathways, which ultimately results in changes to TF- DNA occupancy and concomitant gene expression. Consequently, chemical probes that can regulate TF-DNA binding in a spatial and temporal manner can yield powerful tools for characterizing gene expression. The NF- κB family of DNA-binding proteins are master-regulators of the cellular inflammatory response. The Harki lab has recently reported a new class of TF-targeting DNA decoys for the study of NF-κB-DNA binding (Struntz and Harki, ACS Chem. Biol. 2017). These NF-κB-targeted Catch and Release DNA Decoys (CRDDs) utilize single- photon UV-irradiation to cleave the anomeric bond, leading to depurination between the sugar and nucleobase to achieve dissociation of the NF-κB-CRDD complex. CRDDs are advantageous to standard DNA decoys as they offer precise temporal control over transcription. Here the toolbox of CRDD nucleotides was expanded with the development of novel nitro-pyrimidine nucleoside analogues that can photochemically depurinate similarly to indole predecessors. This poster will report nitro-pyrimidine nucleoside synthesis, mechanistic studies of depurination, and the stabilities of CRDDs containing next-generation nitro-pyrimidine nucleotides.

Keywords: Chemical Biology, Nucleosides, Photochemistry

Poster #51 Connecting Remote C–H Bond Functionalization and Decarboxylative Coupling Using Simple Amines Manikandan Selvaraju, Francisco de Azambuja, Ming-Hsiu Yang, Alexander C. Brueckner, Paul Ha-Yeon Cheong and Ryan A. Altman University of Kansas Transition metal-catalyzed C–H functionalization and decarboxylative coupling are two of the most notable advances in synthetic strategies developed in the last 30 years. To connect these two reaction pathways, we exploit simple amine bases and a Pd-based catalyst system to promote a para-selective C–H functionalization reaction from benzylic electrophiles. Experimental and computational mechanistic studies suggest a pathway involving an uncommon Pd-catalyzed dearomatization of the benzyl moiety followed by a base-enabled rearomatization through a formal 1,5-hydrogen migration. This reaction complements “C–H activation” strategies that convert inert C–H bonds into C–metal bonds prior to C–C bond formation by inverting the sequence and promoting C–C bond formation prior to deprotonation. These studies provide an opportunity to develop general para-selective C– H functionalization reactions from benzylic electrophiles and show how new reactive modalities may be accessed with careful control of reaction conditions.

Keywords: Decarboxylation, para C-H functionalization

Poster #52 Modulation of estrogen biosynthesis in estrogen receptor positive breast cancer cells by Humulus lupulus and its bioactive compounds Amanda C. Maldonado, Atieh Hajirahimkhan, Shao-Nong Chen, Guido F. Pauli, Birgit M. Dietz and Judy L. Bolton University of Illinois Obese menopausal women have a substantially higher risk of developing estrogen receptor positive breast cancer. Higher estrogen levels in the fat tissue surrounding their mammary epithelium and systemic inflammation due to obesity may work in concert to enhance breast cancer risk. Many obese menopausal women have turned to botanical supplements, such as Humulus lupulus (hops), to seek relief from menopausal symptoms. Little is known about the biological effects that these botanicals may have in modulating key network pathways responsible for the production of estradiol/estrone. Aromatase (CYP19A1) and aldo-keto reductase (AKR1C3) are two enzymes responsible for the key steps in the biosynthesis of 17- β-estradiol (E2) in the breast tissue. Downregulation of CYP19A1 and AKR1C3 may decrease E2 biosynthesis and thus the risk for breast carcinogenesis. This study expands on the in vitro effects of hops and its bioactive compounds XH, 8-PN, 6-PN in modulating these enzymes in MCF-7:WS8 cells and MCF-7 cells overexpressing AKR1C3 (MCF-7:1C3). qRT-PCR was used to quantify changes in CYP19A1 and AKR1C3 mRNA expression. Hops and its bioactive compounds 6-PN and XH significantly upregulated CYP19A1 and AKR1C3. In-cell Western analysis was used to confirm relative protein expression of AKR1C3 and CYP19A1. The mechanism of action for the increase in CYP19A1 and AKR1C3 expression by hops will be analyzed through luciferase activity. This study highlights the importance of elucidating the relative risk/benefit ratio of botanical supplements for women’s health. Supported by NIH grant P50 AT00155 provided by ODS and NCCIH; and American Cancer Society grant PF- 18-049-01-NEC.

Keywords: botanicals, phytoestrogens, menopause

Poster #53 Targeting fluorescent sensors to endoplasmic reticulum membranes enables detection of peroxynitrite during phagocytosis Kelsey Knewtson and Blake Peterson University of Kansas Peroxynitrite is a highly reactive oxidant derived from superoxide and nitric oxide. In normal vertebrate physiology, some phagocytes deploy this oxidant as a cytotoxin against foreign pathogens. To provide a new approach for detection of endogenous cellular peroxynitrite, we synthesized fluorescent sensors targeted to membranes of the endoplasmic reticulum (ER). The very high surface area of these membranes was envisioned as a vast intracellular platform for the display of sensors to transient reactive species. By linking an ER-targeted profluorophore to reactive phenols, sensors were designed to be cleaved by peroxynitrite and release a highly fluorescent ER-associated rhodol. Studies of kinetics in aqueous buffer revealed a linear free energy relationship where electron-donating substituents accelerate this reaction. However, in living cells, the efficiency of detection of endogenous cellular peroxynitrite was directly proportional to association with ER membranes. By incorporating a 2,6-dimethylphenol to accelerate the reaction and enhance this subcellular targeting, endogenous peroxynitrite in living RAW 264.7 macrophage cells could be readily detected after addition of antibody- opsonized tentagel microspheres, without additional stimulation, a process undetectable with other known fluorescent sensors. This approach provides uniquely sensitive tools for studies of transient reactive species in living mammalian cells.

Keywords: ROS detection

Poster #54 Discovery of CDK2 Allosteric Inhibitors for Male Contraception and Cancer Therapy Nan Wang and Gunda I. Georg University of Minnesota Cyclin-dependent kinase 2 (CDK2) is a druggable target for male contraception due to the fact that CDK2 knock- out mice are sterile and have a normal life span. It is also an anticancer target because overexpression of cyclin E causes abnormal regulation of the cell-cycle in various cancer cells. Via developing novel allosteric inhibitors targeting the previously unrecognized allosteric site of CDK2, we aim to achieve a higher degree of kinase selectivity. 8-Anilino-1-naphthalene sulfonic acid (ANS) was confirmed crystallographically to bind to CDK2 in an allosteric manner.1 We first used ANS as a lead to develop analogs to achieve better affinity for the ANS allosteric site of CDK2 and developed a novel effective Ullmann coupling reaction to synthesis ANS analogs. Seventeen analogs were synthesized and tested in a radioactive kinase activity assay. Five analogs showed ~50% inhibition of CDK2 activity. ANS has been used frequently as fluorescent probe to study a variety of biological systems. Therefore, we investigated the fluorescent properties of the ANS analogs. Four of them possess a higher quantum yield than ANS in ethylene glycol. Particularly, analog WN-2-7 displayed a higher enhancing factor (φethylene glycol/φH2O) which indicates a larger increase in quantum yield of the fluorophore upon change from an aqueous to a nonpolar environment. Reference: 1. Betzi, S.; Alam, R.; Martin, M.; Lubbers, D. J.; Han, H.; Jakkaraj, S. R.; Georg, G. I.; Schonbrunn, E. Discovery of a potential allosteric ligand binding site in CDK2. ACS Chem. Biol. 2011, 6 (5), 492-501.

Keywords: Cyclin-dependent kinase 2, allosteric inhibition, ANS

Poster #55 NMR SAMPLE DEGASSING CAN AIDS IN THE FULL SPIN ANALYSIS OF NATURAL PRODUCTS Daniel Zagal and Guido F. Pauli University of Illinois Atmospheric paramagnetic oxygen plays an important role on the relaxation properties (T1 and T2) of NMR samples. The aim of this work was to investigate the 1H NMR spectra of degassed and non-degassed samples of strychnine, a natural product widely used for establishing NMR experiments to observe any changes or improvements in signal intensity and lineshape. A stock solution of known concentration was divided in two aliquots to prepare degassed and non-degassed samples of identical concentrations contained in 5mm NMR tubes. The oxygen-free sample was degassed using argon and prepared under an argon atmosphere. Interestingly, sample degassing produced a separation of otherwise overlapping spin multiplets for hydrogens near nitrogen atoms. The least affected signals were the aromatic ring hydrogens, which were not in the vicinity of heteroatoms, and were only shifted by 0.006 – 0.029 ppm. The most affected signals corresponded to hydrogens geminal to heteroatoms and were shifted by as much as 0.162 ppm. Additionally, there was an increase in relaxation times T1 and T2 relative to non-degassed samples from 2.73 to 3.38 s and 0.96 to 1.66 s, respectively. All coupling constants remained unchanged for both degassed and non-degassed samples. We conclude that, even though the effects of sample degassing are known to be negligible in most cases, except for its expected effect on T1 and T2, it can aid in simplifying the analysis of multiplicities in highly complex spectra. This effect appears to occur most notably in compounds with hydrogens near N- and/or O- containing functional groups.

Keywords: sample degassing, natural products, NMR

Poster #56 Design and Synthesis of Bivalent Inhibitors for Bromodomain and Extra-Terminal （BET）family proteins Xianghong Guan and Gunda I. Georg University of Minnesota Extra-Terminal (BET) family proteins (BRD2, BRD3, BRD4 and BRDT) are transcriptional coactivators that interact with acetylated lysine residues of histones. Each BET protein has two bromodomains. BET proteins are involved in multiple cell functions and processes. Among them, BRD4 was identified as a promising target for cancer therapy1 and BRDT emerged as a potential target for male contraception.2 Therefore, targeting BET family proteins is of interest, however high sequence homology between different BET family proteins is a significant challenge for the discovery of selective inhibitor for a specific family member. Recent research revealed that bivalent inhibitors can either induce the dimerization of proteins intermolecularly or bind to both bromodomains intramolecularly.3,4 In light of the new mechanism of BET bivalent inhibitors, we prepared bivalent inhibitors based on a potent BET inhibitor SG3-179.5 Symmetric inhibitors with a PEG1 linker maintained potency and selectivity similar to the parent compound with activity against all BET proteins. Through iterative optimization of attachment and linker chemistry, a series of symmetric bivalent inhibitor with PEG- linkers (PEGn, n=2–5) exhibited up to 70-fold higher selectivity for BRDT-1 over BRD4-1 in the AlphaScreen assay. Compared to the parent compound, these compounds also showed improvements in potency (~10 fold) for BRDT-1. The selectivity profile of one of the compounds, GXH-II-052, was validated in a BROMOscan assay, which confirmed selectivity for BRDT-1. These compounds were about two to six-fold more selective for tandem BRDT compared to BRD4. A following multiple myeloma (MM1.S) cell-based assay indicated these bivalent agents penetrated through cellular membrane and down regulated c-Myc level. The obtained crystal structure BRDT-1 in complex with GXH-II-052 disclosed how small molecule induced the dimerization of BRDT-1, which is the first crystallographic evidence for BRDT dimerization. Notably, the dimerization mode of BRD4-1 was very different from BRDT-1, suggesting a new selectivity improvement strategy for BET family proteins. Further dimerization mode exploration and selectivity optimization are underway.

Keywords: BET, Bivalent, Selectivity, male contraception

Poster #57 Chronochemical Informatics: Small-molecule Libraries at KU 1947-2017 Zachary Pearson and Zarko Boskovic University of Kansas The composition of small-molecule collections used in biological assays are a key determinant of screening outcomes. Therefore, it is important to develop efficient metrics to measure the structural diversity of such collections and to quantify the evolution of the structures populating them over periods of significant developments in the chemical methods used for their synthesis. Here, we present the Joseph H. Burckhalter collection of small molecules -- a library of 5,000 compounds synthesized between 1947 and circa 1980 by Dr. Burckhalter, his team, and collaborators who shared their samples with him. Compounds populating this collection are a reflection of Dr. Burckhalter's research on malaria and cancer therapies as well as fluorescent imaging. The library has been physically curated, and used as a platform to develop a process to measure structural diversity. We contrasted structural features of the molecules comprising this collection to a subset of the collection synthesized more recently (2007-2017) at the University of Kansas as part of the Chemical Methodology and Library Development initiative of the National Institutes of Health. We used cheminformatic methods from RDKit suite of Python modules to define a framework that can be used to evaluate chemical libraries in terms of intra-library diversity of its members and inter-library similarity between entire collections. Metrics that quantify complexity and three-dimensionality are presented, as are the attempts to infer unknown biological activities by clustering compounds from the Burckhalter collection to structurally similar known bioactives.

Keywords: cheminformatics, compound library

Poster #58 Cross-Reactive Antibodies as Theranostic Tools against Synthetic Cannabinoid Intoxication Adam Worob and Cody Wenthur University of Wisconsin Synthetic cannabinoids (SCBs) are compounds designed to mimic the pharmacology of psychoactive components in cannabis through agonism of the CB1 receptor. These compounds are inexpensive, readily available, and cryptic to most methods of forensic identification, making them appealing drugs of abuse (DoAs) for younger populations and individuals motivated to avoid a positive drug screen. The diverse nature of SCB structures is thought to contribute to their unique toxicological profile compared to recreational cannabis. Clinical presentations of suspected SCB toxicity are currently addressed through symptom treatment followed by retroactive confirmatory screening after patient discharge. The dual need for improved diagnostic and therapeutic intervention in SCB intoxication can be addressed with antibodies generated in the presence of SCB-resembling small molecule haptens conjugated to immune-activating proteins. Similar antibody strategies for nicotine, heroin, and other DoAs have shown potential in both detecting these small molecules and sequestering them peripherally in vivo. To account for the ever-increasing number of structurally similar SCBs, we are generating antibodies against generalized haptens comprised of known SCB motifs. Such haptens will be achieved through combinatorial parallel syntheses with different core, linker, head and tail groups, mimicking the structures of the prototypical naphthylalkylindole (JWH) series of SCBs. Antibodies produced against these haptens are hypothesized to possess higher promiscuity in SCB binding compared to those produced against single, high fidelity haptens. We have begun vaccination of Swiss-Webster mice with initial SCB haptens and suitable adjuvants, and the diagnostic capabilities of these antibodies are being evaluated through in vitro ELISA methods.

Keywords: Haptens, Synthetic Cannabinoids

Poster #59 Development of a Broadly Applicable Method for Identification and Analysis of Polychlorinated Biphenyl Sulfates in Human Serum Duo Zhang, Panithi Saktrakulkla, Kris Tuttle, Rachel Marek, Hans-Joachim Lehmler, Keri Hornbuckle and Michael Duffel University of Iowa Polychlorinated biphenyls (PCBs) are environmentally persistent toxic industrial chemicals derived from intentional legacy uses and from unintended byproducts in consumer products. PCB sulfates are metabolic products derived from hydroxylated metabolites of PCBs (OH-PCBs). Both OH-PCBs and PCB sulfates may exert multiple toxicological effects on human health such as disrupting thyroid hormone transport and inhibiting steroid sulfotransferases. Although PCB 11 sulfate has been previously detected in human serum samples, the lack of a generally applicable method for a broad range of PCB sulfates in human serum has limited our understanding of their prevalence and importance. We propose a method that employs acetonitrile extraction of the PCB sulfates from serum followed by differential analysis with, and without, hydrolysis to OH-PCBs catalyzed by a sulfatase from Helix pomatia. The specificity of a crude preparation of this enzyme for PCB sulfates indicates the feasibility of its use for their quantitative hydrolysis. OH-PCBs are quantitated by GC/MS/MS after derivatization to their corresponding methoxy PCBs. Using 13C-OH-PCB 12 as surrogate standard, we were able to recover 94 ± 5 % of 10 ng of 4-PCB 11 sulfate added to 4 ml of serum. We are now finalizing the method by performing L-tyrosine-ethyl-ester-Sepharose affinity column to completely remove glucuronidase from crude Helix pomatia juice instead of adding the glucuronidase inhibitor, D-saccharic acid 1,4-lactone. The purified sulfatase has a broad specificity for PCB sulfates with no contaminant glucuronidase activity. Evaluation of the purified enzyme in the analytical procedure is in progress. [Supported by NIH P42 ES013661]

Keywords: PCB Sulfate Identification

Poster #60 Design and Synthesis of Dihydropyridine Analogs as BRDT Selective Inhibitors for Male Contraception Jiewei Jiang and Gunda I. Georg University of Minnesota Bromodomains are essential protein recognition domains that bind to N-ε-acetylated lysine side chains of histones and recruit other transcription factors during posttranscriptional regulation processes. The BET (bromodomain and extra-terminal) proteins are a sub-family of bromodomain-containing proteins consisting of BRD2, 3, 4, and T, which have attracted attention due to their essential roles in the various diseases. Although the most extensively studied BRD4 protein has been recognized as a promising anti-cancer target, BRDT is selectively expressed in the testis and plays a crucial role in spermatogenesis. Knock-out study in mouse model suggested that BRDT-selective inhibitors could become a male contraceptive agent. A tricyclic, dihydropyridine scaffold was identified from virtual high throughput screening and demonstrated promising activities against BET family proteins. The co-crystal structure with BRD4-1 revealed that modifications at the lactone ring could direct functional groups into the ZA channel and interact with surrounding residues, which might improve BET affinity as well as selectivity. Therefore, the lactam, acyclic ester and acyclic amide subsets were designed, synthesized, and tested. Three lactam analogs with submicromolar affinities towards BRD4 and BRDT showed promising activities in cell-based assays. Whereas, BRDT selectivity was not observed presumably due to the orientation of the lactam side chain not optimal for engaging Arg54 in BRDT. Meanwhile, the crystallographic data indicated the macrocyclization strategy to the acyclic ester scaffold for the potency improvement. Further structural derivatizations focus on strengthening the interactions between the lactam side chain and unique so as to optimize selectivity

Keywords: Male contraception, BRDT

Poster #61 Development of an Activity-Based Probe for PLP-Dependent Enzymes in Mtb Scott I. Brody and Courtney C. Aldrich University of Minnesota Pyridoxal-5-phosphate dependent enzymes (PLP-DEs) catalyze an extraordinary diversity of chemical reactions in both primary and secondary metabolic pathways. The human genome encodes more than 400 PLP-DEs while many pathogenic microorganisms contain dozens that are essential for growth and survival. A number of FDA- approved drugs are known that covalently modify PLP-DEs exemplified by vigabatrin, carbidopa, D-cycloserine, and eflornithine used to treat epilepsy, Parkinson’s, tuberculosis, and African sleeping sickness, respectively. Moreover, drug discovery efforts are ongoing for numerous PLP-DEs in oncology, infectious disease, and neuroscience. In general, many of these aforementioned drugs are characterized by substantial side effects that are incompletely understood, but likely due to inhibition of other functionally related enzymes. Activity-based protein profiling (ABPP) is a powerful technique that has become popular in the field of chemical biology for characterizing enzyme selectivity and target identification of tool compounds; however, there is remarkably no described probe for PLP-DEs, one of the most important enzyme classes in biology and human medicine. We have successfully synthesized an probe capable of covalently labeling PLP-DEs. Preliminary data using purified IlvE from Mycobacterium tuberculosis, a PLP-DE aminotransferase responsible for biosynthesis of the branched- chain amino acids, shows our probe is fully capable of labeling. The native substrate for IlvE bears no structural resemblance to this probe suggesting promiscuous labeling of other PLP-DEs. This work will allow for profiling PLP-DEs and the many FDA-approved drugs that target them to determine the mechanistic basis of their toxicity and identify other unknown mechanisms of action of these multi-targeting inhibitors.

Keywords: activity-based profiling, enzyme inhibition, Mycobacterium tuberculosis

Poster #62 Histone deacetylases upregulate hepatitis B virus biosynthesis by stabilizing the viral core protein Taha Y. Taha, Alan McLachlan and Pavel A. Petukhov University of Illinois Hepatitis B virus (HBV) infection is a worldwide health problem and is endemic in many regions of Asia and Africa. Infection can be acute or chronic and clinical consequences range from subclinical to fatal. Although HBV Infection can be prevented by vaccination with hepatitis B surface antigen (HBsAg), chronic infection remains a major clinical problem (200-500 million worldwide and 1 million deaths annually). Current treatments include interferon therapy, which is associated with significant adverse events, and nucleos(t)ide analogs, which target the viral polymerase and have been associated with increasing resistance. Therefore, understanding of the regulation of HBV biosynthesis by host factors is critical to the development of novel therapeutics. Histone deacetylases (HDACs) have been shown to modestly inhibit HBV transcription, likely via deacetylation of histones assembled with the viral genome. Most of these studies were based on HDAC inhibitor treatment without a clear mechanistic understanding of the effect of HDACs on HBV biosynthesis. We aimed to elucidate this effect directly by overexpressing HDACs 1-8 in hepatoma and non-hepatoma cells and evaluating viral RNA, DNA, and protein levels. Surprisingly, overexpression of HDACs, led to upregulation of HBV biosynthesis at the post- transcriptional level. The HBV core protein (HBcAg) was significantly upregulated upon overexpression of HDACs, in particular HDAC5, without any effect on viral RNA levels. Elucidation of the post-transcriptional mechanism of HDAC-induced upregulation of HBcAg and HBV replication will pave the way for the development of novel HBV therapeutics.

Keywords: Hepatitis B virus, histone deacetylases

Poster #63 The biogenic aldehyde 3,4-dihydroxyphenylacetaldehyde is scavenged by carnosine and L-cysteine Rachel Crawford and Dr. Jonathan Doorn University of Iowa Parkinson’s disease, a neurodegenerative disease, is associated with cell death of the dopaminergic cells of the substantia nigra. The disease state is accompanied by motor issues such as tremors and bradykinesia. The neurotransmitter dopamine is metabolized by monoamine oxidase to 3,4-dihydroxyphenylacetaldehyde (DOPAL), a biogenic aldehyde that is known for its toxicity. In healthy individuals, DOPAL is further metabolized to non-reactive compounds. It is hypothesized that the pathogenesis of Parkinson’s could occur due to aberrant dopamine levels, and consequently aberrant levels of biogenic aldehydes such as DOPAL (this is known as the “catecholaldehyde hypothesis”). The reactivity and toxicity of DOPAL is therefore of great interest, as well as the mechanisms by which the body scavenges this aldehyde. Two potential scavengers of DOPAL include L-cysteine and carnosine. L-cysteine is a non-essential amino acid whose thiol group is important in metabolism and detoxification. Carnosine is a dipeptide that is present in millimolar concentrations in the heart and brain. Research through HPLC-ECD analysis has indicated that both peptides form conjugates with DOPAL. Cell-based assays imply that carnosine and L-cysteine are protective in the case of assault through excess DOPAL or the pesticide rotenone.

Keywords: aldehydes, Parkinson's Disease, carnosine

Poster #64 Studies to improve cellular penetration of quinazolinediones Eden E. D. Maack and Robert J. Kerns University of Iowa Topoisomerases are enzymes responsible for managing DNA topology, supercoiling, and catenation. Catalytic inhibitors of topoisomerases and topoisomerase ‘poisons’ are of pharmacological interest because of their cytotoxic potential as antibiotics or anticancer agents. Fluoroquinolones are clinically used antibiotic topoisomerase poisons, meaning that they bind bacterial type II topoisomerases to form a complex with the topoisomerase and nicked DNA. The binding of topoisomerase poisons prevents the religation of nicked DNA, ultimately leading to DNA damage and cell death. Fluoroquinolones have also been developed to poison human type II topoisomerases, making anticancer fluoroquinolones. However, anticancer topoisomerase poisons like etoposide or anticancer fluoroquinolones can lead to treatment-related malignancies. As such, for anticancer purposes, catalytic inhibitors that do not poison topoisomerases are desirable. Our lab has discovered that N1- biphenyl fluoroquinolones catalytically inhibit both human type I and type II topoisomerases without poisoning. Fluoroquinolones face target-mediated antibiotic resistance in the clinic, where amino acid substitutions in bacterial topoisomerases prevent fluoroquinolone binding. Quinazoline-2,4-diones (diones) share a similar core structure with fluoroquinolones and are capable of binding and poisoning topoisomerases in the same manner as fluoroquinolones but are equipotent with wild-type and fluoroquinolone resistant topoisomerases. N1-biphenyl diones have also been found to inhibit human topoisomerases as well as or better than N1-biphenyl fluoroquinolones. However, diones have poor human or bacterial cellular penetration and are highly susceptible to bacterial efflux pumps. Four approaches to improving the cellular penetration characteristics of diones will be discussed, with a focus on the synthesis of antibiotic quinazolinedione dimers.

Keywords: Synthesis, Antibiotic, Anticancer

Poster #65 Nitric Oxide Regulates DNA Methylation in Cancer Marianne Palczewski and Douglas Thomas University of Illinois Epigenetic regulation by nitric oxide (NO) has a documented and specific role in cancer and various other disease states. We show here a unique relationship between nitric oxide and DNA-demethylating enzymes, namely alpha- ketoglutarate dependent dioxygenase (ABH2) and ten-eleven translocation enzyme (TET). ABH2 is a DNA repair enzyme that uses iron as a cofactor to catalyze the repair of alkylated 1-methyladenine and 3-methylcytosine on dsDNA by removing methyl marks. We describe the mechanism by which NO directly inhibits ABH2 activity using a fluorescent enzymatic assay, western blot, PCR, and northern blot. As DNA methylating agents are an important class of chemotherapeutic drugs, our results suggest that NO-associated tumors may be especially sensitive to these agents by virtue of NO-mediated inhibition of DNA repair processes (demethylation). Using MDA-MB-231 triple-negative breast cancer cells, we demonstrate that a methylating agent (MMS) is significantly more cytotoxic when combined with an NO-donor (DETA/NO) and that this has a stronger effect than NO alone. Similarly, TET enzymes catalyze the successive demethylation of 5-methylcytosine (5mC) to unmodified cytosine by way of several oxidative products (the first of which is 5-hydroxymethylcytosine (5hmC)). Using western blot, PCR, and immunoprecipitation to measure protein and 5hmC levels we provide direct evidence that NO inhibits TET activity and regulates its expression level. Because changes in DNA methylation patterns significantly alter downstream protein expression and cell viability, this study elucidates a novel and targetable pathway for NO in cancer progression and treatment.

Keywords: epigenetics, nitric oxide, cancer

Poster #66 Electrophilic probes for deciphering substrate recognition by O-GlcNAc transferase Dacheng Fan and Jiaoyang Jiang University of Wisconsin O-linked b-N-acetylglucosamine (O-GlcNAc) transferase (OGT) is an essential human glycosyltransferase that adds O-GlcNAc modifications to numerous proteins. However, little is known about the mechanism with which OGT recognizes various protein substrates. Here we report on GlcNAc electrophilic probes (GEPs) to expedite the characterization of OGT–substrate recognition. Data from mass spectrometry, X-ray crystallization, and biochemical and radiolabeled kinetic assays support the application of GEPs to rapidly report the impacts of OGT mutations on protein substrate or sugar binding and to discover OGT residues crucial for protein recognition. Interestingly, we found that the same residues on the inner surface of the N-terminal domain contribute to OGT interactions with different protein substrates. By tuning reaction conditions, a GEP enables crosslinking of OGT with acceptor substrates in situ, affording a unique method to discover genuine substrates that weakly or transiently interact with OGT. Hence, GEPs provide new strategies to dissect OGT–substrate binding and recognition.

Keywords: O-GlcNAc transferase, substrate recognition, electrophilic probes

Poster #67 Synthesis of Novel, FASN-Specific PET Tracers Desire M. Ortiz Torres and Dr. Stephen DiMagno University of Illinois Globally, more than 1.3 million new cases of prostate cancer were diagnosed in 2018 and more than 307,000 men died from the disease. Although early clinical detection of prostate cancer has improved, it is largely based on a risk stratification system comprised of clinical T-stage, PSA count, and Gleason score. Unfortunately, these parameters are neither sensitive to nor specific for aggressive disease. Thus, it remains difficult to identify patients with aggressive or indolent forms of the disease. New, selective biomarkers for the identification of prostate tumors with poor prognosis and for monitoring tumor response to therapy are a clinical necessity. Fatty acid synthase (E.C.2.3.1.85; FASN), a lipogenic enzyme, is a potentially valuable clinical biomarker whose expression and activity could be imaged using radiolabeled small molecules to identify tumor phenotype and inform treatment strategy. Positron emission tomography (PET) imaging allows for non-invasive study of biological systems in vivo. The aim of this study is to develop small molecule PET tracers to image FASN expression in vivo with a view toward predictive and prognostic characterization of prostate cancer. To assess FASN expression, a series of novel tracers labeled with 11C or 18F will be synthesized based on the most potent FASN inhibitors reported to date.

Keywords: Synthesis, novel pet probes, prostate cancer

Poster #68 Discovery of APOBEC3B DNA Cytosine Deaminase Ligands by Protein Observed Fluorine NMR Screening Michael Grillo and Daniel Harki University of Minnesota Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (A3) catalyzes C-to-U deamination in single- stranded (ss)DNA as a function of the innate immune defense against pathogenic DNA. A3s cause hypermutation leading to genomic instability and clearance by the host immune system. One A3 enzyme in particular, A3B, is overexpressed in various cancer types and is a source of genomic mutations that result in tumor evolution and the development of drug resistance. A3B is nonessential in humans making it an exciting new target for cancer therapy. High-throughput screening campaigns have identified covalent inhibitors of A3G, but no A3B small molecule inhibitors have yet been reported. Recently, a co-crystal structure of A3B with a fragment-sized molecule has been solved indicating that A3B can be targeted by fragments. Consequently, we have initiated a solution-phase fragment screening campaign to identify A3B-binding molecules. Our approach utilizes Protein Observed Fluorine (PrOF) NMR to identify A3B-binding ligands. PrOF NMR involves incorporating fluorinated amino acids into the target protein and observing changes in the 19F-NMR resonance shifts upon titration of ligands. These changes in resonance shifts not only indicates that a molecule is binding, but also gives information on where the molecule is binding, which yields a powerful tool for the discovery of enzyme ligands. This poster will highlight recent efforts to develop a PrOF-based assay for A3B ligand screening and preliminary chemical matter discovered through our efforts.

Keywords: PrOF, APOBEC3, screening

Poster #69 Investigation into the Mechanism of Action of Selective Estrogen Receptor Modulators in Ebola Virus Disease Laura Cooper, Rui Xiong, Adam Schafer, Han Cheng, Yangfeng Li, Lijun Rong and Gregory R.J. Thatcher University of Illinois Filoviruses (Ebola and Marburg virus) cause severe hemorrhagic fever in humans and nonhuman primates. The recent ongoing outbreak of Ebola virus in the Democratic Republic of the Congo has 773 confirmed cases. In West Africa, from 2014-16, the deadliest outbreak of Ebola virus resulted in 11,310 deaths out of 28,616 confirmed cases. Currently, there are no FDA-approved therapeutics or vaccines to treat filovirus infections. One ideal target for combating filovirus infection and disease is at the entry step. The host cell attachment, endosomal entry and membrane fusion of filoviruses is mediated by a single glycoprotein (GP, cleaved by furin to form GP1 and GP2 subunits). Toremifene (SERM, Selective Estrogen Receptor Modulator) binds in the cavity between GP1 and GP2 leading to potent inhibition of Ebola virus entry and to a lesser extent, Marbury virus entry. Residues lining this binding pocket are highly conserved with the exception of Marburg virus, suggesting these compounds in theory should not bind well to the Marburg GP. A thorough analysis of this large binding pocket was characterized using single point mutation to determine key residues and to assess the binding of toremifene to the Ebola GP. In addition, insight gained from the mutational analysis aided in the rational drug design of a series of novel toremifene derivatives. In this presentation, we show that toremifene has anti-Marburg activity and introduces a series of novel derivatives of toremifene with increased potency. Furthermore, we provide the first evidence of a possible dual mechanism of action of these molecules in filovirus entry inhibition.

Keywords: Drug Discovery, Mechanism of Action, Viral Entry

Poster #70 A Farnesyltransfersase Mutant with Dual Orthogonality for Enzymatic Labeling Garrett Schey and Mark D. Distefano University of Minnesota Protein prenylation is a post-translational modification where a 15 carbon farnesyl or 20 carbon geranylgeranyl isoprenoids is appended to the C terminal end of a protein by either farnesyltransferase or geranylgeranyl transferase type 1, respectively. The isoprenoids are attached to the Cysteine residue of a four amino acid CaaX box sequence. Protein based conjugates have a wide variety of applications, including protein-fluorophore constructs that are frequently used to study cellular localization and protein-protein interactions. Strategies to selectively label proteins have previously relied on the genetic insertion of non-natural amino acids with biorthogonal functional groups. This can result in difficult protein expression on a large scale. Recently, using enzymes to selectively label proteins has been of interest due to their high efficacy under mild conditions. There are several features of farnesyltransferase that make it a candidate for enzymatic labeling applications. First, nearly any protein can become a substrate for farnesyltransferase by genetically inserting a CaaX box at the C terminus of the protein. This is a small modification that is unlikely to affect protein function. In addition, the active site of farnesyltransferase is promiscuous and can transfer a variety of isoprenoid probes with biorthogonal functional groups such as an alkyne or azide. These probes can then undergo conventional biorthogonal reactions for further study. We have designed an enzyme that combines two previous rationally designed farnesyltransferase mutants that will simultaneously have a high affinity for a large coumarin-containing isoprenoid probe and specifically recognize a non-native CaaX box, allowing for highly selective labeling.

Keywords: enzymatic labeling, rational enzyme design, protein prenylation

Poster #71 Nucleophilic Reactivity of 2-Alkyl Quinazolinones in the Synthesis of (E)-Benzamidines Jhewelle N. Fitz-Henley, Soren D. Rozema and Jennifer E. Golden University of Wisconsin The Golden group has discovered and pioneered the chemical conversion of electrophilic chloroalkylquinazolinones to benzamidines to generate useful anti-infective agents. To incorporate chiral elements into this scaffold and explore new structure-activity relationships, we studied the use of the chloroalkylquinazolinone core as a nucleophilic species with various chiral electrophiles. Our results indicated that a lithiated methylquinazolinone was more productive in the reaction with cyclic sulfamidates. Reaction optimization on a simplified substrate revealed a successful conversion of 2-methylquinazolinone to N-Boc-γ- aminoquinazolinone under mild conditions in 59% yield when 3-Boc-1,2,3-oxathiazolidine-2,2-dioxide was employed as the electrophile. Progress towards the generation of chiral (E)-benzamidines will be addressed.

Keywords: Quinazolinone Chemistry, Amidine Synthesis, Methodology Development

Poster #72 PCB-52 and Metabolites: Toxicity to the Dopaminergic System Brianna Cagle, Hans Lehmler and Jonathan Doorn University of Iowa Polychlorinated biphenyls (PCBs) are man-made dielectrics that were banned worldwide in 2001. Humans are exposed to PCBs through inhalation or the consumption of contaminated food and water. PCBs are risk factors for neurotoxicity and learning abilities in both the developing and adult brain. A current public health concern is the presence of PCBs in schools. PCBs accumulate in brain tissue and alter the dopaminergic system. For example, certain PCBs lower extra-cellular dopamine concentrations, inhibit tyrosine hydroxylase, and are associated with free radical production. Furthermore, they impair dopamine packaging and re-uptake into synaptosomes. PCB 52 is a lower-chlorinated biphenyl which shows toxicity in neuronal cell lines and is a major component of indoor air. PCBs are often hydroxylated via P450 metabolism in the body to potentially more toxic hydroxylated metabolites. Preliminary results show that 4-OH PCB 52 is toxic to PC12 cells – a dopaminergic cell line – after 24 hours at 5µM, while PCB-52 did not show toxicity until 25µM. Preliminary studies also show decreased levels of dopamine in the medium of PCB-52 treated dopaminergic cells. Both PCB-52 and 4-OH PCB-52 were found to increase reactive oxygen species in dopaminergic N27 cells. Future studies include analyzing changes in gene expression to explore alterations in dopamine cell trafficking. PCB 52 and 4-OH PCB 52 have previously shown toxicity in neuronal cell lines, yet little is known about how PCB-52 and 4-OH PCB-52 modulate the dopaminergic system.

Keywords: dopamine, neurodevelopmental disorders, polychlorinated biphenyls

Poster #73 Evaluating Histidine Triad Nucleotide Binding Protein 1 (Hint1) as a Target for Pain Therapy Max Dillenburg and Carston R. Wagner University of Minnesota Histidine triad nucleotide binding protein 1 (Hint1) has emerged as a key protein involved in the regulation of pain, opioid tolerance, and addiction properties. Specifically, Hint1 plays an important role in mediating the regulatory activity of N-methyl-D-aspartate receptor (NMDAR) on the mu-opioid receptor (MOR) following MOR activation. Hint1 is a part of the histidine triad (HIT) superfamily and possesses nucleoside phosphoramidase and acyl-AMP hydrolase activity. Inhibition of Hint1 with small molecule inhibitors developed by our lab, including tryptamine guanosine carbamate (TrpGc), has been shown to reduce the ability of NMDAR to regulate MOR activity, resulting in increased morphine antinociception and decreased development of tolerance in mice. Additionally, inhibition of Hint1 has been shown to antagonize the suppressive effects of morphine on NMDA evoked behavior. Recently, we have shown that TrpGc is able to attenuate neuropathic pain in mice with cisplatin induced peripheral neuropathy, a condition that affects many patients receiving chemotherapy.

Keywords: Hint1, Pain

Poster #74 Translesion Synthesis past DNA-Peptide Crosslinks is Dependent on Local DNA Sequence Context Jenna Thomforde and Natalia Tretyakova University of Minnesota DNA protein crosslinks (DPCs) can form when cellular proteins are covalently trapped on DNA following exposure to chemotherapeutic agents, UV radiation, reactive oxygen species, transition metals, and aldehydes. The bulky lesions that form as a result can inhibit DNA repair, replication, and transcription. 5-Formyl cytosine (5fC) is an epigenetic modification to DNA containing a reactive aldehyde group that reacts with H2A and H4 histone proteins, amongst others. These DPCs can undergo proteolytic cleavage to produce DNA-peptide conjugates. The Tretyakova lab has shown that while DNA-protein crosslinks block replication, DNA-peptide crosslinks can be bypassed by human translesion synthesis (TLS) polymerases ƞ and κ in an error-prone manner. However, the role of the DNA sequence context in DNA replication of the DNA-peptide crosslinks has not been studied. In the present study, 5fC-containing DNA of varying sequences were crosslinked to an 11-mer peptide via an oxime ligation method using oxo-lysine. The bypass of DNA-peptide crosslinks by TLS human polymerase ƞ in vitro was investigated, including the steady state kinetics of incorporation of correct and incorrect base addition opposite the DPC lesion. We observed significant differences in the efficiency of nucleotide incorporation. In an additional study, lesion bypass by replicative human polymerases δ and ε was investigated, which showed that these replicative polymerases can bypass the DNA peptide lesions in an error-free manner. Taken together, these results indicate that the effects of DNA-peptide cross-links of DNA replication are strongly dependent on DNA sequence context and the identity of DNA polymerase.

Keywords: DNA-protein crosslinks, DNA polymerase

Poster #75 Rationally Designed Inhibitors of TIlivalline Biosynthesis in Whole Cell Klebsiella oxytoca Evan Alexander and Courtney Aldrich University of Minnesota The microbiota is a collective term used to describe all bacteria, fungi, viruses and archaea that reside in and on a human host. Most of these upwards of 5,000 different species of microorganisms work symbiotically with the host. Klebsiella oxytoca is an opportunistic bacteria species found in ~10% of healthy individuals’ microbiota. Upon treatment with penicillin based antibiotics, constitutive beta-lactamase enzymes allow K. oxytoca to not only survive, but thrive. This leads to dysbiosis and severe colon damage through the secondary metabolite tilivalline. With rationally designed inhibitors we sought to circumvent this colon damage by blocking the biosynthetic pathway using potent adenylate bioisosteres.

Keywords: Natural Products, NRPS, Drug Development

Poster #76 Novel Aminoquinoline Inhibitors of Antibacterial Resistant Gram+ Pathogens John Schultz and Courtney Aldrich University of Minnesota Infections with drug resistant Gram+ bacteria pose a serious public health hazard that cause a broad spectrum of disease in normal and immunocompromise hosts. Among Gram+ bacteria, methicillin-resistant Staphylococcus aureus (MRSA), Enterococci, S. pneumoniae are the most problematic. New anti-bacterial scaffolds are needed to target these resistant bacteria. Through a phenotypic high throughput screen (HTS) we found two hits with similar 4-aminoquinoline scaffolds that showed bacteriostatic effects against MRSA strains (4-8 µg/mL MIC). A synthetic campaign was carried through to further understand the SAR around the compounds’ bacteriostatic properties, reduce the cLogP, and increase aqueous solubility of the 4-aminoquinolines. A three-step synthetic scheme gave quick access to various substituted 4-aminoquinoles with late stage diversification by Buchwald- Hartwig amination led to compounds that had drastically increased potency (0.063 µg/mL MIC) compared to the initial HTS hits. Synthetic efforts to lower the cLogP values of the 4-aminoquinolines was successfully enacted through modification of the quinoline core to a quinazoline while retaining similar bacteriostatic activity (0.5 µg/mL MIC). Preliminary in vitro cell toxicity studies have shown no eukaryotic cell toxicity at values near the MIC of the most active compounds. Our current lead compounds have been shown to act as membrane disruptors in MRSA in vitro via fluorescent microscopy that lead to MRSA cell lysis in only 15 min.

Poster #77 Development of Selective Covalent Estrogen Receptor Degraders (SCERDs) as Novel Therapeutics for the Treatment of Resistant Breast Cancers Carlo I. Rosales, Rui Xiong and Gregory R. J. Thatcher University of Illinois Breast cancer is the second leading cause of cancer related deaths in American women. Activation of estrogen receptor alpha (ERα) is the primary proliferative mechanism of breast cancer cells, making it a logical target for therapy. ER ligands with antiestrogenic activity, such as the selective estrogen receptor modulators and selective estrogen receptor degraders have proven clinically successful as treatments for breast cancer; however, resistance in up to 50% of patients is a therapeutic challenge. Once resistant, breast cancers become endocrine-independent. Thus, there is an unmet, urgent need for novel therapy. Irreversible inhibition is an attractive approach because inhibition is reliant on the rate of protein turnover rather than competition at the binding site. However, there exists an aversion to covalent inhibitors in drug design due to perceived safety concerns regarding irreversible modification of off-target proteins. This can be attenuated by reducing the reactivity of the covalent warhead; a relatively poor electrophile decreases the intrinsic promiscuity. Only within the binding pocket and with sufficient propinquity to the electrophilic residue is covalent attachment achievable. C530 of ER is a non-catalytic cysteine found between helixes 11 and 12. Our lab has also synthesized a series of benzothiophene-containing, high affinity ER ligands. By attaching a covalent warhead to these compounds, the reactive group can be anchored and correctly positioned relative to C530. Using this combination of high affinity cores and finely tuned electrophilic moieties, selective covalent estrogen receptor degraders (SCERDs) can be designed and have the potential to overcome resistance in breast cancer.

Keywords: Breast Cancer, Endocrine Resistance, Covalent Inhibitors

Poster #78 Humulus lupulus and 6-PN Activate AhR to Promote Breast Cancer Chemoprevention through Epigenetic Regulation of CYP1A1 Ryan T Hitzman, Tareisha L Dunlap, Shao-Nong Chen, Guido F Pauli, Birgit M Dietz and Judy L Bolton University of Illinois Many postmenopausal women frequently use Botanical Dietary Supplements (BDS), such as hops. Compounds found in hops showed chemopreventive activities with often unknown mechanisms, and importance. This study expands on the in vitro effects of hops and its bioactives. Estrogen carcinogenesis is in part mediated by the oxidative estrogen (E2) P450 1B1 mediated metabolite, E2-3,4-quinone (genotoxic pathway). P450 1A1 (CYP1A1) converts E2 to a benign 2-hydroxylated product (estrogen detoxification pathway), yet E2 enhances the genotoxic pathway through epigenetic repression of CYP1A1. Transcription of CYP1A1/1B1 are dependent upon the aryl hydrocarbon receptor (AhR). In-Cell Western, used to quantify relative protein expression of ER, showed AhR dependent degradation of ER when MCF-7 cells were treated with 6-PN. MCF-7 breast cancer cells transiently transfected with XRE-luciferase exhibited AhR dependent luciferase activity for treatments with hops and 6-PN. qRT-PCR was used to quantify changes in CYP1A1 and CYP1B1 transcription. Hops and 6-PN significantly upregulated CYP1A1 (estrogen detoxification pathway) in the presence of E2. The mechanism of action for the increase in CYP1A1 expression was analyzed. A chromatin immunoprecipitation (ChIP) assay for DNA methyltransferase 1 (DNMT1) in the presence of E2 alone increases DNMT1 accumulation at the CYP1A1 promoter, eluding to increased methylation and CYP1A1 downregulation. When the same ChIP assay was performed with E2 and hops or 6-PN there was significant reversal of DNMT1 association with the CYP1A1 promoter, correlating with the preferential increase in CYP1A1 transcription observed with hops and 6-PN. The current studies describe a new chemoprevention pathway for hops. This highlights the importance of elucidating bioactivities for individual phytochemicals and extract standardization to these compounds for women’s health BDS. Supported by NCCIH/NIH grants: 1F31AT010090-01; P50 AT000155.

Keywords: breast cancer, hops, metabolism, estrogen, epigenetics

Poster #79 Identification of xanthones from the mangosteen fruit that promote androgen receptor degradation. Mirielle Nauman and Jeremy Johnson University of Illinois The purpose of this study is to determine how isoprenylated xanthones disrupt androgen receptor function in prostate cancer cells. Xanthones are a class of chemical compounds isolated from the purple mangosteen fruit (Garcinia mangostana) native to Southeast Asia. α-Mangostin is the most common xanthone. Androgen receptor (AR) degradation represents an important strategy to overcome drug resistance to FDA approved anti-androgens used in prostate cancer. Methods: Two different prostate cancer cell lines, 22Rν1 and LNCaP, are used through- out these experiments. The cytotoxicity of α-mangostin after 24, 48, and 72 hours is evaluated through MTT assays. Prostate cancer cells are treated with α-mangostin for 24 hours and are lysed, and then the total protein content is isolated. Total AR, phosphorylation of the AR at Serine 81, and BiP proteins are analyzed through immunoblotting. Results: α-Mangostin has also been shown to interact with AR through a fluorescence polarization assay, suggesting there is a direct interaction between α-mangostin and the ligand binding domain of AR. Immunoblot data reveals a dose and time dependent decrease in AR, coupled with an increase in the chaperone protein BiP. Further analysis of post-translational modifications identified a reduction in phosphorylation of AR at S81. Together, upregulation of BiP and inhibition of AR phosphorylation inhibit the nuclear translocation of AR. This leads to an inhibition of transcription of downstream genes that are necessary for cell growth and proliferation Both upregulating BiP and inhibiting AR post-translational modifications inhibit the nuclear translocation of AR, thereby inhibiting the transcription of downstream genes that are necessary for cell growth and proliferation. Conclusion: These results suggest that α-mangostin promotes AR degradation by inhibiting nuclear translocation, which could be effective in drug resistant prostate cancer cases.

Keywords: prostate cancer, androgen receptor, degradation, xanthones, mangosteen