Drug Discovery Day: 2018

Student Poster Session

Abstract Book

November 8, 2018

Hahn Hall South Atrium

Organized by

The Virginia Tech Center for

Drug Discovery

www.vtcdd.science.vt.edu

Sponsored by

The Fralin Life Science Institute

www.fralin.vt.edu

VTCDD Poster Session 11/8/18 A01(Drug Discovery)

ANTIBODY-SPECIFIC BIOSIMILAR DESIGN FOR ORPHAN BIOLOGICS: A CASE STUDY OF IN SILICO METHODS APPLIED TO ECULIZUMAB

Yueying Wang 1 , David Barto 1 , Gavin Vess 2 , Sean Reiter 3 , Eryney Marrogi 1 , and Anne Brown 1, * 1 Department of Biochemistry, Virginia Tech, Blacksburg, Virginia, (US) 2 Academy of Integrated Science, Virginia Tech, Blacksburg, Virginia, (US) 3 Department of Mathematics, Virginia Tech, Blacksburg, Virginia, (US) wqiao97@vt.edu Over 25 million Americans suffer from a currently-known rare disease, defined by the National Institute of Health (NIH) as a disease affecting fewer than 200,000 patients in the United States.1 Treatment of rare disease has become increasingly dependent on orphan drugs, agents developed specifically to treat patients afflicted by orphan diseases. However, these treatments are expensive, with an average cost of $111,820 per year of treatment - far exceeding the average $23,300 per year of treatment for mainstream drugs.2 Without competition, pharmaceutical companies may charge what the market will bear - hence, the average cost of orphan drugs will continue to rise for an already-vulnerable patient population. The development of a succinct drug design process by analyzing the efficacy of different in silico methods is integral to creating competition to arrest rising drug costs. In this study, we sought to identify the best available computational tools for biologic drug design to allow a more streamlined production of generics, or biosimilars, using eculizumab as a model monoclonal antibody. A series of eculizumab homology models were replicated with five different tools and underwent QMEAN model quality assessment, protein-protein docking and interaction analysis with complement component 5 (CC5), and network analysis of protein-protein interaction networks. QMEAN model quality assessment data showed SWISS-MODEL homology models were on average more similar to the original eculizumab crystal structure scores than models generated with other software. However, Verify 3D results showed models generated in MOE were more similar to the original eculizumab structure than models generated in SWISS-MODEL. From these results, SWISS-MODEL and MOE were determined to generate the most accurate homology models of the tools studied; the two also generated similar quality results. Biologic drug design utilizing these software should be aware of the limitations presented in this study moving forward. Further studies to develop and demonstrate viable homology models to eculizumab which may act as treatment options are desired long-term goals. References

1. Health, N. I. o., Rare Diseases. Services, D. o. H. a. H., Ed. nih.gov: 2017. 2. Joseph A. DiMasi, H. G. G., Ronald W. Hansen, Innovation in the pharmaceutical industry: New

estimates of R&D costs. Journal of Health Economics. We would like to thank Dr. Anne Brown, Dr. David Bevan, and Bevan-Brown Lab researchers for their continued assistance with this project. Additionally, we would like to extend thanks to the Bioactivity Design Team at Virginia Tech for their unwavering support.

VTCDD Poster Session 11/8/18 A02 (Drug Discovery)

EXPLORING NEW CHEMICAL MOIETIES ON SPHK2 INHIBITORS: EXPLOITING THE HYDROPHOBIC CORE OF THE SPHINGOSINE BINDING CAVITY

Ben A. Levy 1 , David R. Bevan 1 , Webster Santos 2 and Anne M. Brown 2, * 1 Department of Biochemistry, Virginia Tech, Blacksburg, Virginia, (United States) 2 Department of Chemistry, Virginia Tech, Blacksburg, Virginia, (United States) BenL2@vt.edu Abstract Multiple diseases, such as cancer, chronic inflammation, and Alzheimer’s, have been linked to the overexpression of sphingosine-1-phosphate (S1P). S1P is a secondary messenger molecule involved in multiple cellular processes such as cell differentiation and proliferation. Sphingosine kinases (SphKs) have been shown to be the sole enzymes to synthesize S1P from sphingosine and are found as two isoforms: sphingosine kinase 1 (SphK1) and sphingosine kinase 2 (SphK2). Given the role of SphKs in synthesizing S1P and in cell proliferation pathways, SphKs are an attractive drug target. In silico approaches, such as molecular docking, are appropriate to assess the exploitability of the binding cavities of these isoforms and to determine rationale for new chemical characteristics needed in isoform-selective inhibitors. This research assesses a set of SphK2-selective inhibitors to explore the role of certain functional groups for influencing isoform selectivity. Specifically, the role and placement of a trifluoromethyl, tert-butyl, and isopropyl group on a phenyl moiety was explored and the predicted position demonstrated high SphK2 selectivity, in agreement with in vitro results. Additionally, it was observed that the position of a hydrophobic moiety on the central phenyl ring influenced slight, but specific placement and interaction types in the hydrophobic core of the binding pocket of SphK2. Ultimately, this work aids in proposing rationale to predict and justify inhibitor selectivity and affinity in SphKs, as well as predict new chemical characteristics to add to enhance inhibitor design based on binding cavity features. This work is supported by: NIH | National Institute of General Medical Sciences (NIGMS) - NIH RO1 GM104366

VTCDD Poster Session 11/8/18 A03 (Drug Discovery)

INFLUENCE OF ELECTRONIC POLARIZATION ON THE STRUCTURE AND DYNAMICS OF G-QUADRUPLEXES WITH DISTINCT FOLDED TOPOLOGIES Alexa Salsbury 1 , Brian Ratnasinghe 2 , Jesse Pinkman 2 , Danielle Porier 2 , and Justin Lemkul * 1 Department of Biochemistry, Virginia Tech, Blacksburg, VA, (United States) asalsbur@vt.edu Abstract Guanine-quadruplexes (GQs) are guanine-rich, noncanonical nucleic acid structures that play fundamental roles in genomic stability and the regulation of gene expression. GQs are enriched in promoter sequences of growth regulatory genes and proto-oncogenes; as such, they feature in various cancers and neurodegenerative diseases, and are novel targets for drug design. GQs exhibit diverse topologies influenced by atomistic interactions and while they have become popular subjects of experimental and computational research, forces governing their dynamics are not fully understood, leaving several promising drug targets unexplored. Towards drug design and to advance our understanding of nucleic acid dynamics, we performed molecular dynamics simulations of three GQs with distinct conformations ( c-kit1 , c-kit2 , and bcl-2 ). The c-kit proto-oncogene encodes for a receptor tyrosine kinase linked to gastrointestinal stromal tumors, mast cell disease, and leukemia and forms c-kit1 and c-kit2 GQs. Similarly, the bcl-2 oncogene contains a GQ-forming sequence and is implicated in a variety of cancers. Simulations of these structures were performed with the CHARMM36 (C36) additive force field and the Drude-2017 polarizable force field, which assigns negatively charged particles to all heavy atoms in the system instead of approximating polarization. By modeling GQs with and without the inclusion of electronic polarization, we can contextualize multi-body effects influencing ion interaction and stability. Polarizable simulations revealed large differences between base dipole moments of GQs and B-DNA and distinct ion binding pathways. Such descriptions of GQ-ion dynamics help expand fundamental knowledge on noncanonical nucleic acids that can be leveraged for drug design.

Support was provided by the Virginia Tech Office of the Provost, College of Agriculture and Life Sciences, and Department of Biochemistry. Computing time and resources were provided by Virginia Tech Advanced Research Computing.

VTCDD Poster Session 11/8/18 A04 (Drug Discovery)

ANTIBIOTIC DISCOVERY: NEW APPROACHES TO REINVIGORATE ESTABLISHED MEDICINES

Andrew N. Lowell 1, * 1 Department of Chemistry, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061 alowell@vt.edu

Loss of effectiveness in clinical antibiotics is deeply concerning, especially when viewed historically. Prior to the development of antibiotics, sepsis and other bacterial infections were often a swift death sentence. Today, unchecked infections remain a pressing medical need because delayed or incorrect treatment will still result in significant loss of life, even in developed countries. 1 The ability of pathogenic bacteria to overcome an antibiotic depends on a resistance mechanism, a specific protein or mutation that prevents a formerly efficacious drug from effectively inhibiting cell growth. While these can develop spontaneously, most are promulgated via horizontal gene transfer: a susceptible species receives the protective gene from a resistant species, becoming itself resistant. To counter the problem of growing resistance, our group is pursuing new avenues beyond the identification of additional antibiotics. Our bidentate antibiotic approach (Figure 1) improves upon hybrid antibiotics 2-3 by utilizing biochemical knowledge of where and how a drug binds to its biological target and what resistance mechanism prevents drug action. By coupling drug two established drugs in such a manner that they can both engage their pharmacophore simultaneously , we are testing the hypotheses that 1) linked daughter antibiotics will have better efficacy than co-administration of their parent compounds, and 2) daughter antibiotics will be

more effective against resistant pathogens because of an anchoring effect provided by covalent linkage to another antibiotic. Another intriguing augmentation of the existing antibiotic arsenal is the identification and development of adjuvants 4 that rescue the activity of a particular drug against resistant pathogens (Figure 2). We have developed an assay 5 and adapted it to detect antibiotic adjuvants from a variety of resources, including small molecule and natural product libraries. Natural product sources are particularly desirable it is likely evolution has already created small molecules that function as antibiotic adjuvants. Successful identification and development of antibiotic/adjuvant combination therapies is especially advantageous as these drugs would be effective against both resistant and susceptible pathogens, thus reducing the prevalence of drug-resistant bacteria. Figure 2: Identification of Antibiotic Adjuvants 1. CDC. Antibiotic Resistance Threats in the United States, 2013. 2. Tevyashova, A. N.; Olsufyeva, E. N.; Preobrazhenskaya, M. N., Design of dual action antibiotics as an approach to search for new promising drugs. Russ. Chem. Rev. 2015, 84 (1), 61-97. 3. Parkes, A. L.; Yule, I. A., Hybrid antibiotics - clinical progress and novel designs. Expert Opin. Drug Discovery 2016, 11 (7), 665-680. 4. Bernal, P.; Molina-Santiago, C.; Daddaoua, A.; Llamas, M. A., Antibiotic adjuvants: identification and clinical use. Microb. Biotechnol. 2013, 6 (5), 445-449, 5 pp. 5. Lowell, A. N.; Santoro, N.; Swaney, S. M.; McQuade, T. J.; Schultz, P. J.; Larsen, M. J.; Sherman, D. H., Microscale Adaptation of In Vitro Transcription/Translation for High-Throughput Screening of Natural Product Extract Libraries. Chem. Biol. Drug Des. 2015, 86 (6), 1331-1338.

VTCDD Poster Session 11/8/18 A05 (Drug Discovery)

ELENOLIC ACID MOIETY ALLEVIATES HYPERGLYCEMIA THROUGH STIMULATING GLP-1 SECRETION IN DIET-INDUCED OBESE MICE AND INSULIN-DEFICIENT DIABETIC MICE

Yao Wang 1 , Jing Luo 1 , Hana Alkhalidy 1 , Bin Xu 2 and Dongmin Liu 1, * 1 Department of Human Nutrition, Foods and Exercise. Blacksburg, VA 2 Department of Biochemistry, Blacksburg, VA Email: yaow@vt.edu Abstract Objectives: Type 2 diabetes (T2D) is characterized by insulin resistance and β-cell dysfunction [1]. The incretin hormone glucagon-like peptide-1 (GLP-1), which is released from gut L-cells, plays a critical role in regulating glucose homeostasis via potentiating glucose-stimulated insulin secretion (GSIS) and promoting pancreatic β-cell proliferation and survival [2]. Therefore, targeting GLP-1 secretion from intestinal L-cells could be an effective strategy for treating and preventing T2D. The present study was aimed at identifying bioactive compounds targeting intestinal GLP-1 secretion to prevent and treat T2D in mouse model. Here, we report that a new olive-derived elenolic acid moiety (EA) is a potent agonist for GLP-1 secretion in vitro and in vivo, which exhibited an antidiabetic potential in diet-induced obese mice and insulin-deficient diabetic mice. Methods: Mouse GLUTag L-cells and primary mouse ileum crypts were incubated with various concentrations of EA for GLP-1 secretion measurement. The concentration of active GLP-1 was determined using an ELISA kit. Intracellular [Ca 2+ ] response to different treatments was determined using Fluo-4AM indicator. The acute effect of EA on GLP-1 level in vivo was evaluated by measuring plasma total GLP-1 concentrations before and after a single dose of vehicle or EA gavage. Chronic effects of EA on glycaemia in vivo were investigated on diet-induced obese mice and insulin-deficient diabetic mice. Male mice were maintained in a high-fat diet (HFD) for 5 months to fully develop obesity before oral administration of vehicle or 50mg/kg/day EA for 8-week. Insulin-deficient mice were induced by 5 injections of 40mg/kg streptozotocin (STZ) as previous reported [3] and orally treated with vehicle or 50mg/kg EA for 2 weeks. Body weight, food intake and blood glucose were measured weekly. Glucose tolerance test and insulin tolerance test were performed at the end of the study. Results: In vitro, EA dose dependently induced GLP-1 secretion in both clonal L-cells and primary ileum crypts, with 10 µM inducing maximal effect (2-fold of control). In addition, EA stimulated fast and transient intracellular [Ca 2+ ] increase. Inhibition of phospholipase C (PLC) activity by U73122 ablated EA-stimulated intracellular Ca 2+ release and GLP-1 secretion in L-cells. Consistently, intracellular inositol trisphosphate was elevated 2.5-fold after EA addition, suggesting that EA activates PLC. Moreover, EA-triggered GLP-1 secretion from L-cells was eliminated by YM-254890, a specific Gαq antagonist. In vivo, a single dose of EA significantly elevated plasma tGLP-1 concentration (Control=7.72±0.54 pM vs. EA= 13.61±2.2 pM, p<0.05). Further, daily EA administration mitigated HFD-induced fasting hyperglycemia and glucose intolerance, which was associated with improved insulin sensitivity in diet-induced obese mice. In addition, EA treatment alleviated STZ-induced hyperglycemia and glucose intolerance. Conclusions: EA effectively increased GLP-1 level in vitro and in vivo. In addition, EA exhibited antidiabetic effects in diet-induced obese mice and insulin-deficient mice. References 1. Abdul-Ghani MA, Tripathy D, DeFronzo RA: Contributions of β-cell dysfunction and insulin resistance

to the pathogenesis of impaired glucose tolerance and impaired fasting glucose . Diabetes care 2006, 29 (5):1130-1139.

2. Kjems LL, Holst JJ, Vølund A, Madsbad S: The influence of GLP-1 on glucose-stimulated insulin secretion: effects on β-cell sensitivity in type 2 and nondiabetic subjects . Diabetes 2003, 52 (2):380-386.

3. Wang Z, Gleichmann H: GLUT2 in pancreatic islets: crucial target molecule in diabetes induced with multiple low doses of streptozotocin in mice . Diabetes 1998, 47 (1):50-56.

This work was supported by grants from the NCCIH of NIH (R01AT007077 and 1R01AT007566-01).

VTCDD Poster Session 11/8/18 A06 (Drug Discovery)

EXAMINING SPECIFICITY FOR ERK8 OF TRYPANOSOMA BRUCEI THROUGH IN SILICO TECHNIQUES

Amanda K. Sharp 1 , Anne M. Brown 1,2 , Brittney L. Worrell 1 , David R. Bevan 1 , and Zachary B. Mackey 1 * 1 Department of Biochemistry, Virginia Tech, Blacksburg, VA, (Montgomery) 2 Department of University Libraries, Virginia Tech, Blacksburg, VA, (Montgomery) aksharp@vt.edu

Human African Trypanosomiasis (HAT), commonly referred to as African sleeping sickness, affects thousands of East Africans every year. Trypanosoma brucei is the parasitic organism responsible for HAT and is transmitted by the tsetse fly. The serotype, T. brucei rhodesiense , causes the acute form of HAT and can lead to death within a matter of months if left untreated. Current therapeutics cause severe side effects, even death, which demonstrates need for both insight into potential drug target mechanisms and novel therapeutic design. Extracellular-signal regulated kinase 8 (ERK8) has been discovered as an essential kinase for survival and proliferation of T. brucei , while expressed only in select human tissue cells. Exploiting potential binding cavity differences in these orthologs is a route to new therapeutic design. In silico techniques were utilized in this study to probe the ATP binding pocket of T. brucei ERK8 (TbERK8) against human ERK8 (HsERK8) to identify key residue and potential chemical feature differences. Molecular docking of experimentally determined, ortholog-specific inhibitors revealed different modes of binding and a shift in position of residues within the active site between kinases. The TbERK8 ATP binding cavity was found to be a smaller and more hydrophobic pocket as compared to HsERK8. Collectively, molecular docking of orthologue-specific inhibitors demonstrates that TbERK8 contains exploitable residue and binding pocket features that can be targeted for selectivity over HsERK8, leading to the potential for design of a new class of inhibitors. This information provides a buildable scaffold in targeting ortholog-specific kinases to further potential therapeutic drug design for the infectious organism, T. brucei .

VTCDD Poster Session 11/8/18 A07 (Drug Discovery)

COLLAGEN MODEL PEPTIDES

Paul Arcoria , Jiajia Li, and Felicia A. Etzkorn,* Department of Chemistry, Virginia Tech, Blacksburg, VA. Correspondence: fetzkorn@vt.edu Parc17@vt.edu We explore the structure and stability of the collagen triple helix by synthesizing peptide mimetics. Suitable mimics will be incorporated into biomaterials for potential use in joint replacement, the cellular matrix, or drug delivery applications. Through molecular modeling and chemical intuition, isosteres were designed to further increase the stability of the triple helix conformation that is necessary for the unique structural properties of collagen. After our initial discovery that simple alkene isosteres do not stabilize collagen, 1-2 ab initio calculations revealed that fluoroalkene and chloroalkene isosteres may be more stabilizing (Etzkorn, F.A.; Ware, R.I.; Pester, A.M.; Troya, D. unpublished results). The theoretical stability of haloalkene mimics is due to the presence of n-to-π* interactions that are missing in the alkene isostere. We have performed asymmetric α-hydroxymethylation on cyclopentanone with L-threonine as a chiral catalyst and achieved some degree of enantiomeric excess (Scheme 1). 3 Both stereoisomers of 3 were obtained by a Horner-Wadsworth-Emmons reaction, which will be separated via chromatography.

1. Dai, N.; Etzkorn, F. A., J. Am. Chem. Soc. 2009, 131 , 13728-13732. 2. Dai, N.; Wang, X. J.; Etzkorn, F. A., J. Am. Chem. Soc. 2008, 130 , 5396-5397. 3. Chen, A.; Xu, J.; Chiang, W.; Chai, C. L. L., Tetrahedron 2010, 66 (7), 1489-1495.

VTCDD Poster Session 11/8/18 A08 (Drug Discovery)

SYNTHESIS OF PENTENOYL-SER-PRO DIPEPTIDE AND PCPDA TO MAKE FULL-LENGTH BORA PROTEIN BY IN VITRO TRANSCRIPTION-TRANSLATION

Leanne M. Aakjar 1 , Nick Jones 1 , Felicia A. Etzkorn 1

Department of Chemistry, Virginia Polytechnic Institute and State University, Blacksburg, VA 24060 leanne16@vt.edu , jnicho7@vt.edu , fetzkorn@vt.edu

Proline isomerization has been found to be an important part of cell cycle control. 1 Aurora Kinase A, a Ser/Thr kinase, and its partner protein Bora are part of cell cycle regulation at the G2/M interphase. 2 In this work we report the protection of a dipeptide with compatible protecting groups for in vitro transcription translation and its activation with methylene nitrile. Activation of 4-pentenoic acid was carried out with Cl-HOBt which was found to protect both the amine group and alcohol side chain of the serine residue. A model compound has been activated and coupled to TBS-protected adenosine and a purification method is currently being worked out. Synthesis of the dinucleotide for transfer of the

dipeptide and the mimics has also begun. The adenosine piece has been protected and coupled to the cytosine piece. Once both of the pieces have been synthesized they will be coupled, ligated to tRNA, and used to insert our compounds into full-length Bora protein.

1. Ping Lu, K.; Hanes, S. D.; Hunter, T., A human peptidyl-prolyl isomerase essential for regulation of mitosis. Nature 1996, 380 (6574), 544-547. 2. Satinover, D. L.; Leach, C. A.; Stukenberg, P. T.; Brautigan, D. L., Activation of Aurora-A kinase by protein phosphatase inhibitor-2, a bifunctional signaling protein. P Natl Acad Sci USA 2004, 101 (23), 8625-8630.

VTCDD Poster Session 11/8/18 A09 (Drug Discovery)

HIGHLY EFFICIENT SYNTHESIS OF NOVEL CARBOLINE DERIVATIVES

Sezgin Kiren, 1, * Ayanna Brooks, Jada Graves 1 Department of Chemistry, Winston Salem State University, Winston Salem, NC 27110 kirens@wssu.edu Abstract γ-Carbolines are indole and pyridine containing alkaloids, which have been reported to posses many biological properties such as antiplasmodial and anticancer activities. We present herein the efficient synthesis of novel derivatives of these heterocycles, which host fused indole, pyridine and pyrrole rings. These molecules will be accessed from commercially available and inexpensive starting materials in a couple of steps using the very-well established reactions.

References 1) Chen, J.; Chen, W.; Hu, Y. Synlett, 2008, 1, 77 2) (a) Grellier, P.; Ramiaramanana, L.; Millerioux, V.; Deharo, E.; Schrével, J.; Frappier, F.; Trigalo, F.; Bodo, B.; Pousset, J.-L. Phytotherapy Res. 1996, 10, 317. (b) Kirby, G. C.; Paine, A.; Warhurst, D. C.; Noamese, B. K.; Phillipson, J. D. Phytotherapy Res. 1995, 9, 359. (c) Cimanga, K.; De Bruyne, T.; Pieters, L.; Vlietinck, A.; Turger, C. A. J. Nat. Prod. 1997, 60, 688.

VTCDD Poster Session 11/8/18 A10 (Drug Discovery)

C1-METHYL, SPIRO- AND RING-EXPANDED ANALOGS OF THE ANTIMALARIAL LEAD COMPOUND MMV008138 Sha Ding 1 , Maryam Ghavami 1 , Joshua H. Butler 2 , Emilio F. Merino 2 , Carla Slebodnick 1 , Maria B. Cassera 2 , and Paul R. Carlier 1, * 1 Department of Chemistry, Virginia Tech, Blacksburg, VA, 24060 (U.S.A) 2 Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, (USA)  sding@vt.edu Isopentenyl pyrophosphate (IPP) biosynthesis is an attractive target for antimalarial drug discovery. Not only is IPP essential for parasite development, but malaria parasites synthesize it via the MEP pathway, which is absent in humans. A phenotypic screen of the 400 compounds of the “Malaria Box” against Plasmodium falciparum -infected red blood cells identified MMV008138 ( 1a ) as an inhibitor of IPP biosynthesis. 1 Subsequent exploration of numerous analogs determined that i) antimalarial potency requires at least one halogen substituent at 2’- or 4’- of the D-ring, 2,3 and ii) that inhibition of P. falciparum growth was well-correlated to inhibition of the MEP pathway enzyme Pf IspD. 3

Current lead optimization work involves improving blood exposure of 1a and its analogs. To block potential oxidative metabolism of the C1-H bond, analogs 2a - d featuring C1-Me substitution, and spiro-analogs 3b and 3d appeared attractive. These compounds were prepared by Pictet-Spengler reaction of Trp-O i -Pr with substituted acetophenones and indanones, followed by hydrolysis. Interestingly, in the course of preparing 2 , C-ring-expanded analogs 4 were isolated. Further investigation revealed important conditions in controlling the selectivity for 2 and 4 . References (1) Bowman, J. D.; Merino, E. F.; Brooks, C. F.; Striepen, B.; Carlier, P. R.; Cassera, M. B. Antimicrob. Agents.

Chemother. 2014 , 58 , 811-819. (2) Yao, Z.-K.; Krai, P. M.; Merino, E. F.; Simpson, M. E.; Slebodnick, C.; Cassera, M. B.; Carlier, P. R. Bioorg. Med.

Chem. Lett. 2015 , 25 , 1515-1519. (3) Ghavami, M.; Merino, E. F.; Yao, Z.-K.; Elahi, R.; Simpson, M. E.; Fernández-Murga, M. L.; Butler, J. H.;

Casasanta, M. A.; Krai, P. M.; Totrov, M. M.; Slade, D. J.; Carlier, P. R.; Cassera, M. B. ACS Infect. Dis. 2018 , 4 , 549-559.

Acknowledgement We thank the NIH (AI128362) for financial support.

VTCDD Poster Session 11/8/18 A11 (Drug Discovery)

HETEROCYCLIC D-RING ANALOGS OF THE ANTIMALARIAL LEAD COMPOUND MMV008138 Haibo Li 1 , Maryam Ghavami 1 , Emilio F. Merino 2 , Zhongke Yao 1 , Daniel J. Slade 3 , Joshua H. Butler 2 Maria B. Cassera 2 , and Paul R. Carlier 1, * 1 Department of Chemistry, Virginia Tech, Blacksburg, VA, 24060 (U.S.A) 2 Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, (USA) 3 Department of Biochemistry, Virginia Tech, Blacksburg, VA, 24060 (U.S.A) lhaibo91@vt.edu The malaria parasite Plasmodium falciparum utilizes the methylerythritol phosphate (MEP) pathway to synthesize the essential isoprenoid precursors IPP and DMAPP. Phenotypic screening of the “Malaria

Box” identified (1 R ,3 S ) configured MMV008138 ( 1a ) as a MEP pathway inhibitor. 1 Subsequently numerous D-ring analogs were synthesized by Pictet-Spengler reaction of (S)-tryptophan methyl ester 2 with aldehydes 3 followed by hydrolysis. The potency of these compounds ( 1a - g ) to inhibit parasite growth (Dd2 strain P. falciparum ) and the activity of the target enzyme ( Pf IspD) were then assessed. As we will show, compounds featuring 2ʹ,4ʹ-dihalogen substitution of the D-ring ( 1a - h ) possess good potency in both assays. 2.3 In this poster we will describe the synthesis and evaluation of 7 heterocylic D-ring analogs 1h-n , including synthesis of the requisite dihalogenated thiophene aldehydes 3l - n .

1. Bowman, J. D.; Merino, E. F.; Brooks, C. F.; Striepen, B.; Carlier, P. R.; Cassera, M. B., Antimicrob Agents Chemother 2014 , 58 (2), 811-819.

2. Yao, Z.-K.; Krai, P. M.; Merino, E. F.; Simpson, M. E.; Slebodnick, C.; Cassera, M. B.; Carlier, P. R., Bioorg Med Chem Lett 2015 , 25 (7), 1515-1519.

3. Ghavami, M.; Merino, E. F.; Yao, Z.-K.; Elahi, R.; Simpson, M. E.; Carlier, P. R.; Cassera, M. B., ACS Infectious Diseases 2018, 4 (4), 549-559.

Acknowledgement We thank the NIH (AI128362) for financial support.

VTCDD Poster Session 11/8/18 A12 (Drug Discovery)

A NEW METHOD FOR STEREOCHEMICAL ASSIGNMENT OF 1,3-DISUBSTITUTED TETRAHYDRO- β -CARBOLINES Kristýna Cagašová , Maryam Ghavami, Zhong-Ke Yao and Paul Carlier* Department of Chemistry, Virginia Tech, Blacksburg, VA, 24061 (U.S.A.) pokludok@vt.edu 1,3-Disubsubstituted tetrahydro-β-carbolines are prominent in medicinal chemistry. Representative examples include the erectile dysfunction drug tadalafil ( 1 ), 1 the anticancer candidate AZD9496 ( 2 , currently in Phase I trials), 2 and preclinical antimalarial agents 3 3 and 4a . 4-5

The tetrahydro-β-carboline scaffold is conveniently prepared via Pictet-Spengler reaction of tryptophan esters and aldehydes, which typically gives a mixture of cis - and trans -isomers (e.g. 6a , 7a ). Historically, the assignment of cis - and trans - relative stereochemistry has relied upon the empirical rule based on 13 C NMR chemical shifts developed by Cook and co-workers. 6 In this poster we present a stereochemical assignment method that relies on 1 H NMR coupling constants, which we believe has a more secure theoretical foundation than the aforementioned 13 C NMR method. The conformational preferences of 6a and 7a (and their unsubstituted phenyl analogs 6b , 7b ) predicted by 1 H NMR coupling constants and NOE measurements are confirmed by density functional theory (DFT) calculations. In turn we show that the 1 H NMR coupling constant-based stereochemical assignment of 26 aromatic and 5 aliphatic analogs of tetrahydro-β-carboline methyl esters 6 and 7 matches those made by the 13 C NMR empirical rule. Finally, we used DFT to calculate 13 C NMR chemical shifts for 6a / 7a and 6b / 7b . We find that DFT accurately reproduces the upfield shift of C1 and C3 in trans -configured 6a / 6b relative to cis -configured 7a / 7b , which the 13 C NMR empirical rule predicts. However, our examination of the calculated 13 C NMR chemical shifts for individual conformers of 6a / 6b demonstrates that the observed upfield shifts are not the simple consequence of steric crowding, contrary to the proposed “γ-gauche effect” basis of the 13 C NMR empirical rule. References [1] Laine, A. E.; Lood, C.; Koskinen, A. M. Molecules 2014, 19 (2), 1544-1567. [2] Weir, H. M.; Bradbury, R. H.; Lawson, M.; Rabow, A. A.; Buttar, D.; Callis, R. J.; Curwen, J. O.; de Almeida, C.; Ballard, P.; Hulse, M.; Donald, C. S.; Feron, L. J. L.; Karoutchi, G.; MacFaul, P.; Moss, T.; Norman, R. A.; Pearson, S. E.; Tonge, M.; Davies, G.; Walker, G. E.; Wilson, Z.; Rowlinson, R.; Powell, S.; Sadler, C.; Richmond, G.; Ladd, B.; Pazolli, E.; Mazzola, A. M.; Cruz, C.; De Savi, C. Cancer Res. 2016, 76 (11), 3307-3318. [3] Gupta, L.; Srivastava, K.; Singh, S.; Puri, S. K.; Chauhan, P. M. S. Bioorg. Med. Chem. Lett. 2008, 18 (11), 3306-3309. [4] Yao, Z.-K.; Krai, P. M.; Merino, E. F.; Simpson, M. E.; Slebodnick, C.; Cassera, M. B.; Carlier, P. R. Bioorg. Med. Chem. Lett. 2015, 25 (7), 1515-1519. [5] Ghavami, M.; Merino, E. F.; Yao, Z.-K.; Elahi, R.; Simpson, M. E.; Fernández-Murga, M. L.; Butler, J. H.; Casasanta, M. A.; Krai, P. M.; Totrov, M. M.; Slade, D. J.; Carlier, P. R.; Cassera, M. B. ACS Infect. Dis. 2018, 4 (4), 549-559. [6] Ungemach, F.; Soerens, D.; Weber, R.; DiPierro, M.; Campos, O.; Mokry, P.; Cook, J. M.; Silverton, J. V. J. Am. Chem. Soc. 1980, 102 (23), 6976-6984. We thank the NIH for financial support (AI128362).

VTCDD Poster Session 11/8/18 A13 (Drug Discovery)

TETRAPEPTIDE PRESERVES BIOENERGETIC STRUCTURE FUNCTION BY AGGREGATING THE MITOCHONDRIAL LIPID CARDIOLIPIN

Mitchell Allen 1 , Edward Ross Pennington 2 , Saame Raza Shaikh 2 , Xianlin Han 3 , and David Brown* 1

1 Virginia Tech, Blacksburg, VA 2 University of North Carolina, Chapel Hill, NC 3 University of Texas Health Science Center, San Antonio, Texas Mallen9@vt.edu Mitochondrial structure and function are inextricably linked, with decrements in structure-function noted across pathologies. Elamipretide (Elam) is a cell-permeable tetrapeptide currently being utilized in clinical trials for genetic and age-related mitochondrial diseases. We determined if Elam restored mitochondrial structure-function in pathological mitochondria, and complemented these studies with synthetic models of mitochondrial membranes to test Elam-cardiolipin interactions. Myocardial uptake and mitochondrial localization of fluorescently-tagged Elam was confirmed using multi-photon imaging of the intact rat heart and in cells. Hearts were subjected to injury using acute ischemia-reperfusion, with a subset of hearts receiving Elam at reperfusion. Mitochondrial respiratory activities were determined at each complex using high-resolution respirometry. Decrements across complexes were noted with injury, with Elam improving respiration across all complexes. Serial block face scanning electron microscopy was employed for high-resolution imaging and three-dimensional reconstructions of mitochondrial cristae. There was a robust decrease in the number of cristae junctions, cristae density, and mitochondrial networking in injured hearts. These morphological defects were improved with Elam. The loss of cardiolipin content after injury (obtained using shotgun lipidomics) was not abrogated by Elam. Synthetic lipid membranes were constructed to model the mitochondrial membrane changes in the experimental groups. As expected, the spatial clustering of membrane lipids was lowered by decreasing the cardiolipin content in the disease model. Elam treatment acutely restored the biophysical structure of the diseased model membrane toward the healthy control. These data indicate that Elam can act by aggregating mitochondrial membranes, a property that is lost if vesicles did not contain cardiolipin. Elam represents a promising approach to improve bioenergetics in diseases by rescuing aberrant mitochondrial structure. Acknowledgements: Grants from Stealth Biotherapeutics and the NIH R01 HL123647 (all to DAB) made these studies possible.

VTCDD Poster Session 11/8/18 A14 (Drug Discovery)

PROTECTIVE MITOCHONDRIAL MECHANISMS IN PEROXIDE STRESSED MYOBLASTS

FOSTERED BY CAT-4001

Authors: Alexander H. Thomson 1 , Mitchell E. Allen 1 , Justin B. Perry 1 , Andrew J. Nichols 2 , John F. Reilly 2 , Pradeep

Bista 2 , Diana Lee 2 , Chi B. Vu 2 , David A. Brown 1

Afiliations: 1 Virginia Tech, Department of Human Nutrition, Foods, and Exercise and the Virginia Tech Center for

Drug Discovery, Blacksburg, VA, 2 Catabasis Pharmaceuticals, One Kendall Square, Suite B14202, Cambridge, MA

E-mail: alext17@vt.edu , brownda@vt.edu

Mitochondrial dysfunction is noted across disease states and is a primary target for emerging treatments. In these studies, we determined the bioenergetic efficacy of a novel cell-permeable compound, CAT-4001. CAT-4001 is a conjugate of monomethyl fumarate (activates Nrf2) and docosahexaenoic acid (inhibits NF-kB). Once inside cells, the conjugate was hydrolyzed by intracellular enzymes to synergistically decrease inflammation (by inhibiting NF-kB) and stimulate cellular resistance to oxidative stress (by activating Nrf2). Oxygen consumption rates (OCR) were assessed in undifferentiated C2C12 myoblasts exposed to a 500µM hydrogen peroxide insult. The effects of CAT-4001 were compared to positive controls N-acetylcysteine (NAC) and catalase, using a 24-hour treatment paradigm. Peroxide treatment significantly decreased maximal OCR ( 90±19 pmol/min ) compared to untreated control ( 184±34 pmol/min, P<0.05). Treatment with 1µM CAT-4001 significantly improved maximal OCR (206±19 pmol/min) to levels comparable to positive controls (192±25 pmol/min for 5mM NAC and 194±19 pmol/min for catalase). 10µM of CAT-4001 resulted in substantially higher maximal OCR (348±20 pmol/min). ATP-dependent respiration was also significantly reduced after peroxide insult, decreasing ATP-dependent OCR from 51±8 pmol/min in untreated cells to 32±5 pmol/min after hydrogen peroxide. NAC and catalase treatment led to modest improvements in ATP-dependent respiration that did not reach statistical significance (47±5 pmol/min and 49±4 pmol/min, respectively). Both 1µM and 10µM CAT-4001 evoked significantly higher ATP-dependent respiration (69±5 pmol/min and 113±5 pmol/min, respectively; P<0.05 compared to all other peroxide-treated groups). These data highlight the efficacy of CAT-4001 as a treatment to improve mitochondrial function by improving cellular tolerance to oxidative insults.

VTCDD Poster Session 11/8/18 A15 (Drug Discovery)

MENSACARCIN IMPAIRS METABOLISM IN HUMAN MELANOMA CELL LINES.

Grace N. Davis 1 , Mitchell E. Allen 1 , Justin B. Perry 1 , Alexander H. Thomson 1 , Kalyn S. Specht 1 , Elizabeth N. Kaweesa 2 , David A. Brown 1 *, Sandra Loesgen 2 1 Department of Human Nutrition, Foods, and Exercise, Virginia Tech, Blacksburg, VA (USA) 2 Department of Chemistry, Oregon State University, Corvallis, OR, (USA) Graced96@vt.edu It is estimated that over 91,000 people in the US will be diagnosed with melanoma in 2018 alone. 1 FDA approved therapies, such as vemurafenib, are available for individuals with melanoma but progressive resistance to these drugs often complicates treatment. Mensacarcin, a polyketide found in soil bacteria, has been shown to potently induce cell death in human melanoma cell lines yet its mechanism of action is relatively unknown. This study employed a Seahorse XF96 Analyzer to determine a metabolic mechanism in which mensacarcin may induce cell death in human melanoma cells (SK-Mel-5EK, SK-Mel-28). In both melanoma cell lines, 1uM mensacarcin treatment significantly decreased mitochondrial function as assessed by maximal oxygen consumption (SK-Mel-5EK 37.03±5.09 vs control 72.73±2.84 fmol/min, and the SK-Mel-28 52.06±1.71 fmol/min vs control 64.56 ± 2.04 fmol/min). When mensacarcin was combined with the melanoma standard of care, vemurafenib (2uM), the maximal oxygen consumption significantly decreased when compared to vemurafenib treatment alone (1.60±0.48 fmol/min vs 33.49±1.75 fmol/min, vemurafenib + mensacarcin vs vemurafenib). In studies examining glycolytic metabolism, mensacarcin (2uM) significantly decreased the glycolytic capacity in SK-Mel-5EK cells (19.33± 2.22 fmol/min vs 27.36±1.44 fmol/min, treatment vs control; P<0.05). These data highlight the ability of mensacarcin to favorably alter melanoma cell metabolism and support its development as both a primary and adjunctive therapy for the treatment of melanoma.

1. Key Statistics for Melanoma Skin Cancer. Retrieved from https://www.cancer.org/cancer/melanoma-skin-cancer/about/key-statistics.html on 10/28/18.

VTCDD Poster Session 11/8/18 A16 (Drug Discovery)

STRUCTURE-ACTIVITY RELATIONSHIP STUDY OF ANILINOPYRAZINES AS MITOCHONDRIAL

UNCOUPLERS

Jacob H. Murray 1 , Stefan Hargett 2 2 , Kyle Hoehn, 3 2,3 * and Webster L. Santos 4 1 * 1 Department of Chemistry and Center for Drug Discovery, Virginia Tech, Blacksburg, VA, USA 2 Department of Pharmacology, University of Virginia, Charlottesville, VA, USA 3 Department of Biotechnology and Biomolecular Sciences, University of New South Wales, Kensington, Australia Jmurray7@vt.edu Mitochondrial Uncouplers dissipate the proton gradient of the electron transport chain resulting in an increase in metabolic rate. Moderate uncoupling of the mitochondria has potential medicinal benefits in treating numerous diseases including: obesity, diabetes, parkinsons disease, and aging. 1 In previous work, BAM15, a mitochondrial uncoupler with specificity for the mitochondria, was discovered. 2 A structure-activity relationship (SAR) study showed the importance of all 3 substituent groups of the molecule. Removal of the furazan moiety resulted in total loss of uncoupling activity. 3 In this work, we will discuss the structure-activity relationship study of analinopyrazines as mitochondrial uncouplers. Modification of the aniline tail groups of the molecule regained activity. Evaluation of 22 bis-aniline pyrazine derivatives demonstrated the importance of placement, substitution, and proximity of the aniline groups to it’s efficacy, potency, and toxicity. (1) Childress, E. S.; Alexopoulos, S. J.; Hoehn, K. L.; Santos, W. L. Small Molecule Mitochondrial Uncouplers

and Their Therapeutic Potential. J. Med. Chem. 2018 , 61 (11), 4641–4655. (2) Kenwood, B. M.; Weaver, J. L.; Bajwa, A.; Poon, I. K.; Byrne, F. L.; Murrow, B. A.; Calderone, J. A.;

Huang, L.; Divakaruni, A. S.; Tomsig, J. L.; Okabe, K.; Lo, R. H.; Cameron Coleman, G.; Columbus, L.; Yan, Z.; Saucerman, J. J.; Smith, J. S.; Holmes, J. W.; Lynch, K. R.; Ravichandran, K. S.; Uchiyama, S.; Santos, W. L.; Rogers, G. W.; Okusa, M. D.; Bayliss, D. A.; Hoehn, K. L. Identification of a Novel Mitochondrial Uncoupler That Does Not Depolarize the Plasma Membrane. Mol. Metab. 2014 , 3 (2), 114–123.

(3) Kenwood, B. M.; Calderone, J. A.; Taddeo, E. P.; Hoehn, K. L.; Santos, W. L. Structure-Activity Relationships of Furazano[3,4-b]Pyrazines as Mitochondrial Uncouplers. Bioorganic Med. Chem. Lett. 2015 , 25 (21), 4858–4861.

We thank Continuum Biosciences, Inc., Virginia Center for Aging, and Virginia Catalyst for funding our work.

VTCDD Poster Session 11/8/18 A17 (Drug Discovery)

DISCOVERY OF A BRANCHED PEPTIDE THAT RECOGNIZES THE REV RESPONSE ELEMENT (RRE) RNA AND BLOCKS HIV ‐ 1 REPLICATION

Ashley N. Peralta , 1 Yumin Dai , 1 Jessica E. Wynn, 1 Chringma Sherpa, 2 Bhargavi Jayaraman, 3 Hao Li, 1 Astha Verma, 1

Alan D. Frankel, 2 Stuart F. Le Grice, 2 and Webster L. Santos 1,*

1 Department of Chemistry and Center for Drug Discovery, Virginia Tech, Blacksburg, Virginia 24060, United States 2 Basic Research Laboratory, National Cancer Institute, Frederick, Maryland 21702, United States 3 Department of Biochemistry and Biophysics, University of California, San Francisco, California 94158, United States reu13ap@vt.edu daiym@vt.edu We synthesized and screened a unique 46,656- member library composed of unnatural amino acids that revealed several hits against RRE IIB RNA. Among the hit peptides identified, peptide 4A5 was found to be selective against competitor RNAs and inhibited HIV-1 Rev-RRE RNA interaction in cell culture in a p24 ELISA assay. Biophysical characterization in a ribonuclease protection assay suggested that 4A5 bound to the stem-loop region in RRE IIB while SHAPE MaP probing with 234 nt RRE RNA indicated additional interaction with secondary Rev binding sites. Taken together, our investigation suggests that HIV replication is inhibited by 4A5 blocking binding of Rev and subsequent multimerization.

This work was supported by the National Institutes of Health (Grant RO1 GM093834).

VTCDD Poster Session 11/8/18 A18 (Drug Discovery)

DEVELOPMENT OF SELECTIVE SPHINGOSINE KINASE INHIBITORS WITH PF-543 AS LEAD COMPOUND

Hao Li 1 , Laura G. Wonilowicz 2 , Yugesh Kharel 3 , Anne M. Brown 2 , David R. Bevan 2 , Kevin R. Lynch 3 and Webster L. Santos 1, * 1 Department of Chemistry and Virginia Tech Center for Drug Discovery, Virginia Tech, Blacksburg, VA 24061, (USA) 2 Department of Biochemistry, Virginia Tech, Blacksburg, VA 24061, (USA) 3 Department of Pharmacology, University of Virginia, Charlottesville, VA 22908, (USA) lhao1@vt.edu Sphingosine kinase 1 (SphK1) is the key enzyme catalyzing the formation of sphingosine-1-phosphate (S1P), which is an important signaling molecule regulates multiple biological process including inflammatory responses. 1 Elevated SphK1 activity as well as upregulated S1P level is linked to various diseases, such as cancer, fibrosis and sickle cell disease. Therefore, there is a growing interest in studying on SphK1 as a potential target for aforementioned diseases. Through high throughput screening, various SphK1 inhibitors have been discovered, among which PF-543 is the most potent and selective inhibitor reported to date (K i =3.6 nM, >100 folds’ selectivity for SphK1). Previous research indicated that SphK1 inhibitor PF-543 are effective in reducing S1P levels and slowing down the development of sickle cell disease in vivo. 2 However, the lack of in vivo stability of PF-543 still makes it necessary to develop inhibitors with improved pharmacokinetic profile. In this study, PF-543 was employed as the lead compound, and the influence of different tails groups and head groups upon binding affinity and in vivo stability were investigated. In brief, ( R )-prolinol based derivatives with various tail groups including alkyl, alkoxy and biphenyl groups were synthesized. Their inhibition potency was tested by broken-cell assay, and hit compounds were further evaluated on yeast cell assay for EC 50 values. U937 cell line and mice model were utilized for hit compounds to quantify S1P reduction in vitro and in vivo . Our preliminary results indicated compounds 2.14d was best hit discovered, with 88% SphK1 inhibition at 1 μ M. In addition, compound 2.14d , with K i of 0.68 μ M and EC 50 of 0.15 μ M, reduced 90% S1P of U937 cells (1 μ M) and 15% plasma S1P in mice model (10 mg/kg, 3 h). Further modification of head group produced compound 3.14c , with EC 50 of 0.39 μ M and similar in vivo activity (14% plasma S1P reduction, 10 mg/kg, 6h), indicating potential improvement of in vivo stability.

References

1. Lima, Santiago, Sheldon Milstien, and Sarah Spiegel. "Sphingosine and sphingosine kinase 1 involvement in endocytic membrane trafficking." Journal of Biological Chemistry 292.8 (2017): 3074-3088.

2. Zhang, Yujin, et al. "Elevated sphingosine-1-phosphate promotes sickling and sickle cell disease progression." The Journal of clinical investigation 124.6 (2014): 2750-2761.

Acknowledgement We thank the NIH grants R01 GM104366 and R01 GM067958 for funding.

VTCDD Poster Session 11/8/18 A19 (Drug Discovery)

TRANSITION METAL-FREE TRANS HYDROBORATION OF ALKYNOIC ACID DERIVATIVES

Ashley Gates 1 , Russell Fritzemeier 1 , Xueying Guo 2 , Zhenyang Lin 2 , and Webster L. Santos 1, * 1 Department of Chemistry, Virginia Tech, Blacksburg, VA, (USA) 2 Department of Chemistry, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR (China) agates15@vt.edu Boronic esters are excellent functional handles in the development of novel drugs and drug derivatives. The installation of a boronic ester allows for many types of structural modifications, including the Suzuki-Miyaura cross-coupling for the formation of new C–C bonds. 1 This work provides a phosphine-catalyzed method to install boronic esters on alkynoate esters and amides in an E -selective fashion. The transition metal-free hydroboration affords β-borylacrylates and β-borylacrylamides (Figure 1a), which are obtained in good to excellent yields. This method was employed in the synthesis of a novel boron-containing scaffold (Figure 1b), as well as in the derivatization of pargyline, an MAO inhibitor (Figure 1c). 2

1. Chem. Soc. Rev. 2014, 43 , 412-443. 2. J. Org. Chem. , 2018 , 83 (17), pp 10436–10444.

We acknowledge financial support by the National Science Foundation (CHE -1414458) and the Research Grants Council of Hong Kong (16304017).

VTCDD Poster Session 11/8/18 A20 (Drug Discovery)

BRØNSTED BASE MEDIATED TRANSITION METAL-FREE TRANS -HYDROBORATION OF PROPARGYLAMIDES TO 2-VINYLBORONATES: SUBSTRATE SCOPE AND MEDICINAL APPLICATIONS R. Justin Grams , Russell Fritzemeier, Carla Slebodnick, and Webster L. Santos*

Department of Chemistry, Virginia Tech, Blacksburg, VA, U.S.A. justing4@vt.edu Vinylboronic acid derivates provide unique access to polysubstituted alkenes, a common structural motif in pharmaceuticals. Until recently, transition metal catalysts, such as Cu, were employed to borylate alkynes to generate cis-borylalkenes. 1 However, methods developed within the past decade no longer require transition metal catalysts. Instead, phosphine catalysts activate alkynoates via an allenyl intermediate to produce an oxyanion that regio- and stereoselectivly mono- or diborylates the alkyne. 2-5 The resulting trans -1- or 1,2-vinylboronates can then undergo selective α- and/or β-substitution. For example, analogues of tamoxifen, the most prescribed breast cancer therapy, can be readily synthesized by borylation of aryl alkynoates or alkynamides followed by Suzuki-Miyaura cross-coupling to yield densely functionalized tri- or tetra-substituted alkenes. Novel tamoxifen analogues are highly desired as new therapies for breast cancer but also to combat resistance against tamoxifen. While diboration of alkynoates has been studied extensively, literature on regio- and stereospecific monoboration of alkynamides is scarce. Thus, we set out to explore the conditions necessary to borylate primary and secondary alkynamides. As seen in the scheme below, a brønsted base initiates the formation of an oxyanion, followed by rapid borylation of the alkyne at the β-carbon, producting the trans -acrylamide due to coordination of the Lewis acidic boron with the amide oxygen. The trans -2-alkynamides generated from this reaction can then be converted to oxaboroles via sodium borohydride reduction 2 or substitution by the Suzuki-Miyaura cross-coupling. Furthermore, these compounds have the potential to provide access to novel oxaboroles, a novel pharmacophore, and polysubstituted alkenes.

(1) Chem. Soc. Rev. 2018, 47 , 7477–7494. (2) J. Org. Chem. 2018, 83 , 10436–10444. (3) Org. Lett. 2018, 20 , 1861-1865. (4) Chem. Eur. J. 2017, 23 , 15534–15537. (5) Org. Lett. 2015, 17 , 1304–1307. We thank the Virginia Tech and the NSF (CHE-1414458) for funding this research.

VTCDD Poster Session 11/8/18 A21 (Drug Discovery)

FACILE SYNTHESIS OF AIR-STABLE ORGANODIFLUOROBORANES FOR THE FORMATION OF MONO-SUBSTITUTED OXABOROLES.

Eric Medici 1 , Connor Szwetkowski 1 , Laura Wonilowicz 1 , Russell Fritzemeier 1 and Webster Santos 1, * 1 Department of Chemistry, Virginia Tech, 900 West Campus Drive, Blacksburg, VA 24061. ermedici@vt.edu and santosw@vt.edu

Organoborate salts are useful intermediates in many synthetic transformations. [1] While the synthesis and utility of trifluoroborate salts is well-documented, examples of the synthesis and transformation of neutral sp 2 hybridized difluoroborate derivatives are limited. [2,3]

We report the synthesis and transformation of air-stable organodifluoroboranes. Surprisingly, the reaction of ( E )-β-borylacrylamides and KHF 2 results in pinacol deprotection and formation of difluoroborane product, in contrast to formation of the trifluoroborate salt. [4] The corresponding organodifluoroboranes are air, moisture, as well as silica gel stable. Substrate scope and limitations are presented as well as transformations demonstrating the unique reactivity of these substrates.

We have shown that our organodifluoroboranes can be reduced under mild conditions to form

mono-substituted oxaboroles at the 3-position. These molecules resemble benzoxaboroles, which have been shown to have biological activity and are present in FDA approved pharmaceuticals, such as Tavaborole and Crisaborole. [5,

6]

[1] Vo, C. V. T.; Mitchell, A.; and Bode, J. W. JACS . 2011 , 133 , 14082-14089. [2] Bachollet, S. P. J. T.; Vivat, J. F.; Cocker, D. C.; Adams, H.; and Harrity, J. P. A. Chem. Eur. J . 2014 , 20 , 12889-12893. [3] Osmialowski, B.; Zakrzewska, A.; Jedrzejewska, B.; Grabarz, A.; Zalesny, R.; Bartkowiak, W.; and Kolehmainen, E. J. Org. Chem . 2015 , 80 , 2072-2080. [4] Audi, H.; Remond, E.; Eymin, M. J.; Tessier, A.; Malacea-Kabbara, R.; and Juge, S. Eur. J. Org. Chem. 2013 , 7960-7972. [5] Sharma, N. and Sharma D. J. Pharmacol. Pharmacother. 2015 , 6 (4), 236-239. [6] Paller, A. S.; Hebert, A. A.; et al. J. Am. Acad. Dermatol. 2016 , 75 (3), 494-503. Thanks to NSF Funding (CHE-1414458) for financial support.

VTCDD Poster Session 11/8/18 B01 (Target Validation)

DISTINCT CONTROL OF PERIOD2 DEGRADATION AND CIRCADIAN RHYTHMS BY THE ONCOPROTEIN AND UBIQUITIN LIGASE MDM2 Xianlin Zou 1, ‡ , JingJing Liu 1,2,‡ , Tetsuya Gotoh 1,5 , Anne M. Brown 3 , Liang Jiang 1 , Esther L. Wisdom 1 , Jae Kyoung Kim 4 , & Carla V. Finkielstein 1,* 1 Integrated Cellular Responses Laboratory, Department of Biological Sciences, Biocomplexity Institute, Virginia Tech, Blacksburg, VA, United States 2 Current address: Department of Computational Biology, St Jude Children’s Research Hospital, Memphis, TN, United States 3 Research and Informatics, University Libraries, Virginia Tech, Blacksburg, VA, United States 4 Department of Mathematical Sciences, Korea Advanced Institute of Science and Technology, Daejeon, South Korea 5 Current address: Laboratory of Cell Systems, Institute for Protein Research, Osaka University, Osaka, Japan ‡ These authors contributed equally to this work. * Corresponding author. Email: finkielc@vt.edu xlzou@vt.edu Abstract The circadian clock relies on posttranslational modifications to set the timing for degradation of core regulatory components, which drives clock progression. Ubiquitin-modifying enzymes that target clock components for degradation mainly recognize phosphorylated substrates 1 . Degradation of the circadian clock component PERIOD 2 (PER2) is mediated by its phospho-specific recognition by β-transducin repeat–containing proteins (β-TrCPs), which are F-box–containing proteins that function as substrate recognition subunits of the SCF β-TRCP ubiquitin ligase complex 2 . However, this mode of regulating PER2 stability falls short of explaining the persistent oscillatory phenotypes reported in biological systems lacking functional elements of the phospho-dependent PER2 degradation machinery 2,3,4,5 . We identified PER2 as a previously uncharacterized substrate for the ubiquitin ligase mouse double-minute 2 homolog (MDM2) and found that MDM2 targeted PER2 for degradation in a manner that did not depend on PER2 phosphorylation. Deregulation of MDM2 plays a major role in oncogenesis by contributing to the accumulation of genomic and epigenomic alterations that favor tumor development. MDM2-mediated PER2 turnover was important for defining the circadian period length in mammalian cells, a finding that emphasizes the connection between the circadian clock and cancer. Our results not only expand MDM2’s suite of specific substrates beyond the cell cycle to include circadian components, but they also uncover previously unknown regulatory players that likely impact our view of how other mechanisms crosstalk and modulate the clock itself, a previously unknown druggable node that is often found to be deregulated during tumorigenesis. References 1. K. Stojkovic, S. S. Wing, N. Cermakian, A central role for ubiquitination within a circadian clock protein modification code. Front Mol Neurosci 7, 69 (2014). 2. K. Ohsaki, K. Oishi, Y. Kozono, K. Nakayama, K. I. Nakayama, N. Ishida, The role of β-TrCP1 and β-TrCP2 in circadian rhythm generation by mediating degradation of clock protein PER2. J Biochem 144, 609-618 (2008). 3. H. Lee, R. Chen, Y. Lee, S. Yoo, C. Lee, Essential roles of CKIdelta and CKIepsilon in the mammalian circadian clock. Proceedings of the National Academy of Sciences of the United States of America 106, 21359-21364 (2009). 4. K. M. Walton, K. Fisher, D. Rubitski, M. Marconi, Q. J. Meng, M. Sladek, J. Adams, M. Bass, R. Chandrasekaran, T. Butler, M. Griffor, F. Rajamohan, M. Serpa, Y. Chen, M. Claffey, M. Hastings, A. Loudon, E. Maywood, J. Ohren, A. Doran, T. T. Wager, Selective inhibition of casein kinase 1 epsilon minimally alters circadian clock period. J Pharmacol Exp Ther 330, 430-439 (2009). 5. M. D'Alessandro, S. Beesley, J. K. Kim, Z. Jones, R. Chen, J. Wi, K. Kyle, D. Vera, M. Pagano, R. Nowakowski, C. Lee, Stability of Wake-Sleep Cycles Requires Robust Degradation of the PERIOD Protein. Curr Biol, (2017). Funding acknowledgement: National Science Foundation MCB division (MCB-1517298)

VTCDD Poster Session 11/8/18 B02 (Target Validation)

CIRCADIAN REGULATION OF THE P53 RESPONSE IN CANCER THERAPEUTICS Esther L. Wisdom 1 , Philip Stauffer 1 , Sarah K. Jachim 1 , Xianlin Zou 1 , Abbey Workmeister 1 , Amanda M. Locker 1 , Kedi Cao 1 and Carla V. Finkielstein 1, * 1 Department of Biological Sciences, Virginia Tech, City, Virginia, (United States of America) Presenter email: elwisdom@vt.edu Abstract The p53 tumor suppressor transcriptionally regulates cell cycle progression and triggers death processes in response to genotoxic stress and thus, it prevents the accumulation of mutations in daughter cells and the development of cancer. One option for cancer treatment for mutants of p53 that are transcriptionally inactive as result of structural rearrangements is to use a drug that rescues the mutant’s native confirmation and thereby restores p53’s regulatory functions. One mutant that shows promise for this approach contains a Tyr to Cys mutation (named p53YC hereafter). Mutant p53YC has a destabilizing crevice due to the amino acid substitution and is more than 80% unfolded at body temperature [1, 2]. Due to p53YC’s largely unfolded state, it doesn’t trigger cell cycle arrest or apoptotic pathways that control cell proliferation and prevent cancer [3]. Aminobenzothiazole derivatives are able to bind to the crevice formed by the YC mutation in p53. The crevice sits at the interface of binding with PER2 a circadian protein that regulates p53 stability, activity, and shuttling between cellular compartments [4-6]. This led us to hypothesize that treatment with aminobenzothiazole derivatives would be more effective if delivered at a time-of-day when PER2 expression in the tumor is low. First, we looked at the effect of different mutations in p53 and PER2 on their binding especially mutants in the region near the YC crevice. The YC mutant showed increased binding to PER2 and mutants near the YC crevice showed a variety of binding affinities. We monitored the effect of drugs on p53 and PER2 binding using immunoprecipitation. Higher concentrations of aminobenzothiazole reduced PER2 and p53 binding. We also monitored the circadian expression and stability of PER2 and p53 in the presence of aminobenzothiazole. Docking modeling was used to visualize theses interactions. Our findings place PER2 as a direct regulator of p53 and provide insights on PER2’s relevance in cancer therapeutics. References 1. Joerger, A.C., H.C. Ang, and A.R. Fersht, Structural basis for understanding oncogenic p53 mutations and

designing rescue drugs. Proceedings of the National Academy of Sciences of the United States of America, 2006. 103 (41): p. 15056-15061.

2. Liu, X., et al., Small molecule induced reactivation of mutant p53 in cancer cells. Nucleic Acids Res, 2013. 41 (12): p. 6034-44.

3. Joerger, A.C. and A.R. Fersht, The Tumor Suppressor p53: From Structures to Drug Discovery. Cold Spring Harbor Perspectives in Biology, 2010. 2 (6): p. a000919.

4. Gotoh, T., et al., The circadian factor Period 2 modulates p53 stability and transcriptional activity in unstressed cells. Mol Biol Cell, 2014. 25 (19): p. 3081-93.

5. Gotoh, T., et al., Association of the circadian factor Period 2 to p53 influences p53's function in DNA-damage signaling. Mol Biol Cell, 2015. 26 (2): p. 359-372.

6. Gotoh, T., et al., Model-driven experimental approach reveals the complex regulatory distribution of p53 by the circadian factor Period 2. Proceedings of the National Academy of Sciences of the United States of America, 2016. 113 (47): p. 13516-13521.

Funding Funding for this project was provided by National Science Foundation, MCB Division to CVF and Virginia Tech Department of Biological Sciences, Stacey Smith Biology Research Excellence Award to ELW.

VTCDD Poster Session 11/8/18 B03 (Target Validation)

INSIGHTS INTO MITOCHONDRIAL FUNCTION FROM A NOVEL MODEL OF LEBER’S HEREDITARY OPTIC NEUROPATHY

Justin B. Perry 1 * , Aloka B. Bandara 1 , Paloma Ruiz 2 , Dain Ruiz 2 , Zefeng Wang 2 , Joseph Ruiz 2 , David A. Brown 1 1 Department of Human Nutrition, Foods, and Exercise, Virginia Tech, Blacksburg, VA, (USA) 2 Enzerna Biosciences, Raleigh NC, (USA) perryj16@vt.edu Few viable treatment options for Leber’s hereditary optic neuropathy (LHON) patients exist, due in part to the difficulty in creating experimental models of LHON. Targeted disruption of proteins encoded by mitochondrial DNA has been bolstered in recent years by several promising approaches, yet each of these has their limitations. In this study, we utilized Artificial Site-specific RNA Endonucleases (ASREs), a novel mitochondrial RNA engineering platform, to create a cell based model of LHON by combining ASRE ablation of a target gene with the expression of corresponding human disease allele integrated into the nuclear genome as a transgene. Human embryonic kidney (HEK293) cells and C2C12 cells were transfected with a drug-inducible ASRE designed to decrease the expression of complex I subunit ND1 (ASRE-ND1). In a separate cohort, the ASRE-engineered cells were transfected with piggyBac (PB) transposon vectors that carried a cumate-inducible wild type ND1 (PB-ND1) or an ND1 gene associated with LHON [PB-ND1(G3640A)]. High-resolution respirometry in permeabilized cells revealed lower complex I-dependent respiration after ASRE-ND1 activation (13.2±0.7 pmol/sec*million cells vs 11.0±0.4 pmol/sec*million cells). Expressing wild type PB-ND1 rescued complex I-dependent respiration to 14.5±0.4 pmol/sec*million cells. Interestingly, expression of the PB-ND1(G3460A) LHON mutant in ASRE-ND1 cells did not restore complex I-dependent respiration. Respiration in ASRE-ND1 cells expressing PB-ND1(G3460A) was acutely improved with idebenone. These data highlight the feasibility of creating cell models of mitochondrial disease using the ASRE platform. We anticipate this technology will lead to the development of new screening platforms for emerging therapeutic interventions.

VTCDD Poster Session 11/8/18 B04 (Target Validation)

AGGREGATION OF HUMAN TAU ISOFORM PROTEINS IN ALZHEIMER’S DISEASE Darren Dougharty 1,^ , Sid Madhavan 1,2,^ , Christopher Tan 1 , Alisa Bondar 1 , Shradha Lad 1 , Madeleine Marcus 1 , Frances Henderson 1 , Jianyong Li 1 , George Bloom 5 , Ling Wu 1 , and Bin Xu 1,2,3,4 * 1 Department of Biochemistry, Virginia Tech, Blacksburg, VA 24061 2 School of Neuroscience, Virginia Tech, Blacksburg, VA 24061 3 Center for Drug Discovery, Virginia Tech, Blacksburg, VA 24061 4 Center for Transformative Research on Health Behaviors, Virginia Tech, Blacksburg, VA 24061 Departments of Biology, Cell Biology, and Neuroscience, University of Virginia, Charlottesville, VA 22903 ^ Equally Contributed thomas69@vt.edu , ghrelin@vt.edu , and binxu@vt.edu (correspondance) Abstract Alzheimer’s Disease (AD) is the sixth leading cause of death in the United States, the most common cause of dementia, and is also one of the most expensive diseases in the U.S., due to the intensive care needed for the patients and the lack of therapeutic drugs for treatment. A trademark of AD is neurofibrillary tangles made of tau protein that damage neurons as hyperphosphorylation causes tau proteins to become insoluble and form highly ordered aggregates. Tau has six alternatively spliced isoforms in human brain, containing 0, 1, or 2 N-terminal inserts, and 3 or 4 C-terminal microtubule binding repeats referred as 0N3R, 1N3R, 2N3R, 0N4R, 1N4R, and 2N4R tau. To better understand the mechanisms and identify key sequences in tau that drive the formation of these tangles, we characterized the six tau isoforms and two series (total 13 proteins) of amino-terminal and carboxy-terminal truncation mutants of the largest isoform, 2N4R. Using recombinant technology, biochemical and biophysical techniques, we expressed and purified all six human tau isoforms in excellent quality that are aggregation-competent and are also capable to form filaments as those in AD. We also purified 13 truncation mutants. From detailed aggregation analysis, we discovered the critical role of the second and the third repeats (R2 and R3) in tau aggregation formation and kinetics. Presence of R2 (4R isoforms) significantly delays aggregation in comparison with their 3R counterparts (absence of R2 sequence). Deletion of R2 and R3 repeats abrogates protein aggregation. But such deletion mutants regained aggregation competency when a hexameric peptide sequence from either R2 or R3 was added. Moving forward, we will correlate tau aggregation with their cytotoxicity in neuronal cells, Neuro2A. We will also perform structural studies (such as cryo-EM) through collaboration of various tau isoforms to further understand the mechanisms of tau aggregation and to discover tau aggregation inhibitors in the future. References: Goedert M, Eisenberg DS, Crowther RA. (2017) Propagation of Tau Aggregates and Neurodegeneration. Annu Rev Neurosci. 40:189-210. Fitzpatrick AWP, Falcon B, He S, Murzin AG, Murshudov G, Garringer HJ, Crowther RA, Ghetti B, Goedert M , Scheres SHW. (2017) Cryo-EM Structures of tau filaments from Alzheimer’s disease. Nature. 547:185-90. Acknowledgements: This work is in part supported by grants from the Alzheimer’s and Related Diseases Research Award Fund (ARDRAF) from Virginia Center on Aging (to L.W. and B.X.), the Commonwealth Health Research Board (CHRB), Diabetes Action Research and Education Foundation (DAREF) to B.X., Fralin Summer Research Fellowship (to C.T.), Translational Obesity Undergraduate Research (TOUR) Fellowship (to D.D.). We thank VTCDD Screening Lab for instrument access.

VTCDD Poster Session 11/8/18 B05 (Target Validation)

BIOENERGETIC CAPACITY OF SPINDLE-SHAPED C2C12 MIGRATION AND INSIDE-OUT FORCE PRODUCTION Kalyn Specht 1 , Abinash Padhi 2 , Alexander H. Thomson 1 , Amrinder S. Nain 2 , and David A. Brown 1 1 Deparment of Human Nutrition, Foods, and Exercise and the Virginia Tech Center for Drug Discover, Virginia Tech, Blacksburg, Virginia, USA 2 Department of Mechanical Engineering, Virginia Tech, Blacksburg, Virginia, USA kspecht@vt.edu Cell migration plays a key role in many cellular processes such as morphogenesis and wound healing. Cellular migration starts with cells sensing their environment through formation of finger-like protrusions which can lead to whole cell-body migration. Furthermore, cells generate force (inside out, IO) and also respond to forces that originate outside the extracellular matrix (ECM) (outside in, OI) . In this study, we engineered fiber networks of 1

controlled diameter (curvature) and alignment, with STEP (spinneret-based tunable engineered parameters) to evaluate the bioenergetics of C2C12 cell migration and IO force production. To ensure cell adhesion, the scaffolds were coated with fibronectin, a glycoprotein within the extracellular matrix. To determine the contribution of different metabolic pathways in migrating cells, we introduced different mitochondrial inhibitors, including antimycin a (AMA) [2 µM] and oligomycin [2.5 µM] and varying concentrations of a glycolysis inhibitor, 2-deoxy-d-glucose (2DG). Despite immediate effects on oxygen consumption rate (OCR) (pre: 94.4±1.2 pmol/min to post: 30.7±6.9 pmol/min, n=11; P<0.05) cell migration velocity did not significantly change until hour 3 (34.8±2.3 µm/hr and 23.3±2.9 µm/hr for pre-inhibition and post-inhibition respectively, n=21; P<0.05). Next, we treated cells with oligomycin, and again found cell migration to decrease significantly at hour 3 (pre: 30.1±3.8 µm/hr and post: 27.3±2.8 µm/hr, n=17; P<0.05). Inhibition of glycolysis with 2DG [12 mM], had immediate effects on extracellular acidification rate (ECAR, pre: 116.6±2.8 mpH/min to post: 97.1±3.9, n=12; P<0.05) and significantly decreased cell migration velocity within one hour and remained decreased for the subsequent 6-hour treatment (pre: 32.9±3.8 µm/hr to post: 20.9±1.4 µm/hr, n=25; P<0.05). Furthermore, addition of 2DG [50mM], even further decreased ECAR (pre: 116.6±2.8 mpH/min to post: 57.4±3.7, n=12; P<0.05) and cell velocity, suggesting a dose-dependent effect (pre: 31.4±1.4 µm/hr to post: 4.9±0.7 µm/hr, n=22; P<0.05). By altering the substrates present in cell culture media with the addition of galactose for 14 days, cell migration velocity decreased with AMA after 1 hour (pre: 33.0±3.3 µm/hr to 1 hour post: 3.9±0.3 µm/hr, n=17; P<0.05). In addition to observing the energetics of C2C12 cell migration, we set out to further evaluate the effects of bioenergetics inhibition on IO forces. C2C12 cells were adhered to suspended fiber networks, and deflection of these fibers can be converted to a force measurement . 2

Mitochondrial inhibition with AMA [2 µM] did not significantly change C2C12 force production over the course of the six-hour experiment (pre: 107.3±9.3 nN to post: 82.2±12.8 nN, n=12; P=NS). Similarly, inhibition of glycolysis with 2DG did not significantly alter C2C12 force production (pre: 107.3±9.3 nN to post: 117.4±12.1 nN, n=15; P=NS). Altogether our data suggest that myoblast migration is moderately dependent on mitochondrial respiration, but heavily reliant on glycolysis in conventional (glucose) cell culture conditions. However, forcing cells to rely on mitochondrial bioenergetics (by the removal of glucose and addition of galactose) augmented the contribution of mitochondrial energetics for cell migration. Interestingly, inhibition of glycolysis or mitochondrial OXPHOS did not significantly change C2C12 force production.

1 Goldfinger, L.E. 2013. Integrin Signaling, 2 nd Ed. Elsevier, Dordrecht, The Netherlands. 2 Kamm, R., J. Lammerding, and M. Mofrad. 2010. Cellular nanomechanics. In Springer Handbook of Nanotechnology. B. Bhushan, editor. Springer, Berlin, Germany, pp. 1171-1200.

VTCDD Poster Session 11/8/18 B06 (Target Validation)

A NON-PHARMACOLOGICAL THERAPY IMPROVES SPATIAL MEMORY, WASTE CLEARANCE AND NEUROTRANSMISSION IN NATURALLY AGED RAT MODEL OF ALZHEIMER'S DISEASE Tyler Lucas

2 , Hope Tobey

1 , Tyler Lucas

2 , Douglas Bledsoe

1 , Mike Mykins

1 , Caroline Campbell

1 , Stuart S. Berr

3 , Todd Sasser

4 , Cesar Molinos

5 , Richard

Helm 6 , Liang Shan

1 , Per Gunnar Brolinson

1 , Bradley G. Klein

2&7 , Blaise M. Costa

1,2,6&7* 1 Edward Via Virginia College of Osteopathic Medicine,

2 Virginia–Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, VA, USA.

3 Department

of Radiology and Medical Imaging, University of Virginia School of Medicine, Charlottesville, VA, USA. 4 Bruker BioSpin Preclinical Imaging, MA, USA.

5 Bruker Española, Spain.

6 Department of Biochemistry,

7 School of Neuroscience, Virginia Tech, Blacksburg, VA, USA.

Tylucas@vt.edu Abstract: In the aging brain, reduction in the pulsation of cerebral vasculature and reduced fluid circulation cause impairment in fluid exchange between different compartments that pave a foundation for neuroinflammation that results in Alzheimer’s disease (AD). The role of CNS lymphatic vessels in clearance of brain derived metabolic waste products, open an unprecedented capability to increase the clearance of macromolecules such as amyloid beta (Aβ) proteins. However, currently there is no physiological or pharmacological mechanism available to increase fluid circulation in the aging brain. In the present study, we demonstrate a significant improvement in spatial memory, after seven days of cranial osteopathic manipulation (COM) therapy, naturally aged rat model of AD. Immunoassay analysis and live animal PET imaging reveal that COM treatment reduces Aβ levels, activates astrocytes and improves excitatory neurotransmission in aged rat brain. These findings for the first time demonstrate a mechanism of COM treatment that has been clinically practiced for decades. This study and further investigations in this direction will help clinicians promote COM as an evidence based adjunct treatment strategy for AD. 1. Iliff, J. J.; Wang, M.; Zeppenfeld, D. M.; Venkataraman, A.; Plog, B. A.; Liao, Y.; Deane, R.; Nedergaard, M., Cerebral arterial pulsation drives paravascular CSF-interstitial fluid exchange in the murine brain. J Neurosci 2013, 33 (46), 18190-9. 2. Aspelund, A.; Antila, S.; Proulx, S. T.; Karlsen, T. V.; Karaman, S.; Detmar, M.; Wiig, H.; Alitalo, K., A dural lymphatic vascular system that drains brain interstitial fluid and macromolecules. The Journal of experimental medicine 2015, 212 (7), 991-9. 3. Louveau, A.; Smirnov, I.; Keyes, T. J.; Eccles, J. D.; Rouhani, S. J.; Peske, J. D.; Derecki, N. C.; Castle, D.; Mandell, J. W.; Lee, K. S.; Harris, T. H.; Kipnis, J., Structural and functional features of central nervous system lymphatic vessels. Nature 2015, 523 (7560), 337-41. This work was funded by Research Eureka Accelerator Program (REAP), Edward Via Virginia College of Osteopathic Medicine, Fund # 10295 to BC, HT, PGB and BK.

VTCDD Poster Session 11/8/18 C01 (Drug Delivery)

POLYMER DESIGN FOR DRUG DELIVERY SYSTEMS: ACID AND HYDROLYTICALLY CLEAVABLE NETWORK SCAFFOLDS FOR PERSONALIZED DOSAGE PHARMACEUTICALS

Emily M. Wilts 1 , James R. Brown 1 , Jana Herzberger 1 , Nicholas Chartrain 2 , Keyton Feller 2 , Christopher B. Williams 2 , and Timothy E. Long 1* 1 Department of Chemistry, Macromolecules Innovation Institute, Blacksburg, VA, USA 2 Department of Mechanical Engineering, Macromolecules Innovation Institute, Blacksburg, VA, USA emilyw16@vt.edu , brownjr2@vt.edu Water-soluble polymers are the most common materials applied for drug delivery systems, enabling drug encapsulation and slow release on-demand. The advent of 3D printing allows to customize drug carriers for patient’s needs; for example, 3D printed oral drug delivery tablets enable personal dosage, which will help minimize drug side effects. In addition, one can imagine designing degradable medical devices or cell scaffolds, which exhibit a complex geometry and tailored dissolution to improve drug release. Here, we present two different degradable, polymeric structures suitable for 3D printing. Each 3D printing technique requires tailored polymer properties to maximize success. We developed polyethylene glycol-based polymers to generate degradable drug delivery vehicles. These polymers are suitable for 3D printing techniques utilizing UV-light, e.g. stereolithography. UV-crosslinking leads to hydrogels, which are stable at physiological conditions and degrade at slightly acidic pH. These structures are promising to implant in tumor tissue, providing slow release of anti-tumor drugs. Further, we developed poly(lactic-co-glycolic) acid-based hydrolytically cleavable photo-active precursors with a range of degradation rates. Characterization through thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), and 1H NMR spectroscopy elucidated the chemical and thermal properties of the synthetic tri-block copolymers. Preliminary photo-kinetic experiments and cell studies revealed the potential for stereolithography 3D printing and low toxicity. References:

1. R. Schultz, P. M. Lambert, N. A. Chartrain, D. M. Ruohoniemi, Z. Zhang, C. Jangu, M. Zhang, C. B. Williams, T. E. Long. 3D Printing Phosphonium Ionic Liquid Networks with Mask Projection Microstereolithography. ACS Macro Lett. 2014, 3, 1205-1209.

2. J. Herzberger, J. R. Brown, N. A. Chartrain, C. B. Williams, T. E. Long, 2018, in preparation. 3. Yu, D. G.; Branford-White, C.; Yang, Y-C.; Zhu, L-M.; Welbeck, E. W.; Yang, X-L. A novel fast

disintegrating tablet fabricated by three-dimensional printing. Drug. Dev. Ind. Pharm. 2009, 35, 1530-6. 4. Moon, N. G.; Mazzini, F.; Pekkanen, A. M.; Wilts, E. M.; Long, T. E. Macromol. Chem. Phys. 2018, 219,

1800177.

VTCDD Poster Session 11/8/18 C02 (Drug Delivery)

SELF-IMMOLATIVE PERSULFIDE-RELEASING PRODRUGS

Kearsley Dillon 1 , Chad Powell 1 , Yin Wang 1 , Ryan Carrazone 1 , Veronica Ringel 2 , Kristen Eden 2 , McAlister Council-Troche 2 , Coy Allen 2 , and John Matson 1 * 1 Department of Chemistry, Virginia Tech, Blacksburg, Virginia 2 Virginia-Maryland College of Veterinary Medicine, Blacksburg, Virginia kman56@vt.edu Abstract Small molecule persulfides (R–SSH) have recently been recognized as key players in redox balance and cell signaling, but the extent of their role remains unclear. Early research into small molecule persulfides (R–SSH) has been hampered by a lack of donor molecules, as the few examples persulfide donors in the literature suffer from limited benchtop stability, poorly understood release mechanisms, and/or ill-defined persulfide species. To mitigate the issues of persulfide stability and ill-defined persulfide species being released, we report the synthesis of a family of protected N -acetyl cysteine (NAC) persulfide prodrugs. Communicated here is the synthesis and characterization of three persulfide-releasing prodrugs, termed BDP-NAC, NDP-NAC, and EDP-NAC, which are responsive to reactive oxygen species, nitroreductase, and esterases, respectively. The prodrugs display excellent benchtop stability and release the desired NAC persulfide with half-lives ranging from 1.5 ± 0.3 h – 7.5 ± 0.3 h, as determined by NMR spectroscopy and LCMS. Additionally, the persulfide prodrugs were found to be biocompatible in the presence of H9C2 cardiomyocytes and in the case of BDP-NAC were able to rescue cells under oxidative condition to a greater degree than common H 2 S donors. NDP-NAC, the nitroreductase-responsive persulfide prodrug, exhibited pronounced effects on the microbiome in mice, causing a 10-fold upregulation in probiotic bacteria relative to controls. This work was supported by the National Science Foundation (DMR-1454754) and the National Institutes of Health (R01GM123508). We thank Dr. Tijana Grove and Dr. Webster Santos and their students for experimental assistance as was as Dr. Medhi Ashraf-Khorassani and McAlister Council-Troche for help with LCMS.

VTCDD Poster Session 11/8/18 C03 (Drug Delivery)

DESIGN AND FABRICATION OF CHITOSAN NANOPARTICLES CONTAINING THE NUCLEOSIDE METABOLIC INHIBITOR, GEMCITABINE

Austin Fergusson 1 , Richey M. Davis 2, 3, 4, * (etc) 1 Graduate Program in Translational Biology, Medicine, and Health, Virginia Tech, Blacksburg, VA, (USA) 2 Department of Chemical Engineering, Virginia Tech, Blacksburg, VA, (USA) 3 Macromolecules Innovation Institute, Virginia Tech, Blacksburg, VA, (USA) 4 Faculty of Health Sciences, Virginia Tech, Blacksburg, VA, (USA) austinf9@vt.edu , rmdavis@vt.edu Gemcitabine is approved for the treatment of advanced pancreatic cancer in patients where surgical resection is impossible and treatment with other chemotherapies has been unsuccessful. Following phosphorylation, gemcitabine leads to cell death by inhibiting DNA synthesis once it is incorporated into DNA. Successful intravenous delivery of gemcitabine to the target site is complicated by its notoriously short half-life due to rapid enzymatic degradation. In order to protect the drug from enzymatic degradation and prolong its release, we have encapsulated it within polymeric nanoparticles made from chitosan using the flash nanoprecipitation technique. Flash nanoprecipitation causes rapid, turbulent mixing with mixing times on the order of tens of milliseconds, and produces nanoparticles with controlled sizes and drug loadings. We have characterized the nanoparticle synthesis parameter space by using a factorial design in order to determine an optimal formulation. Our manufacturing process produces chitosan nanoparticles with high gemcitabine loadings.

Support from the Translational Biology, Medicine, and Health Graduate Program for A.F. is gratefully acknowledged along with additional support from the Institute for Critical Technology and Applied Science.

Author Poster Number

Aakjar Leanne A08

Alkhalidy Hana A05

Allen Mitchel A13, A14, A15

Allen Coy C02

Arcoria Paul A07

Bandara Aloka B03

Barto David A01

Berr Stuart B06

Bevan David A02, A06, A18

Bista Pradeep A14

Bledsoe Douglas B06

Bloom George B04

Bondar Alisa B04

Brolinson Per G. B06

Brooks Ayanna A09

Brown Anne A01, A02, A06, A18, B01

Brown David A13, A14, A15, B03, B05

Brown James C01

Butler Joshua A10, A11

Cagasova Kristyna A12

Campbell Caroline B06

Cao Kedi B02

Carlier Paul A10, A11, A12

Carrazone Ryan C02

Cassera Maria A10, A11

Chartrain Nicholas C01

Costa Blaise B06

Council-Troche McAlister C02

Dai Yumin A17

Davis Grace A15

Davis Richey C03

Dillon Kearsley C02

Ding Sha A10

Dougharty Darren B04

Eden Kristen C02

Etzkorn Felicia A07, A08

Feller Keyton C01

Fergusson Austin C03

Finkielstein Carla B01, B02

Frankel Alan A17

Fritzemeier Russell A19, A20, A21

Gates Ashley A19

Ghavami Maryam A10, A11, A12

Gotoh Tetsuya B01

Grams Justin A20

Graves Jada A09

Guo Xueying A19

Han Xianlin A13

Hargett Stefan A16

Helm Richard B06

Henderson Frances B04

Herzberger Jana C01

Hoehn Kyle A16

Jachim Sarah B02

Jayaraman Bhargavi A17

Jiang Liang B01

Jones Nick A08

Kaweesa Elizabeth A15

Kharel Yugesh A18

Kim Jae K. B01

Kiren Sezgin A09

Klein Bradley B06

Lad Shradha B04

Le Grice Stuart A17

Lee Diana A14

Lemkul Justin A03

Levy Ben A02

Li Jiajia A07

Li Haibo A11

Li Hao A17, A18

Li Jianyong B04

Lin Zhenyang A19

Liu Dongmin A05

Liu JingJing B01

Locker Amanda B02

Loesgen Sandra A15

Long Timothy E. C01

Lowell Andrew A04

Lucas Tyler B06

Luo Jing A05

Lynch Kevin A18

Mackey Zachary A06

Marcus Madeleine B04

Marrogi Eryney A01

Matson John C02

Medici Eric A21

Merino Emilio A10, A11

Midhavan Sid B04

Molinos Cesar B06

Murray Jacob A16

Mykins Mike B06

Nain Amrinder B05

Nichols Andrew A14

Padhi Abinash B05

Pennington Edward A13

Peralta Ashley A17

Perry Justin A14, A15, B03

Pinkman Jesse A03

Porier Danielle A03

Powell Chad C02

Ratnasinghe Brian A03

Reilly John A14

Reiter Sean A01

Ringel Veronica C02

Ruiz Paloma B03

Ruiz Dain B03

Ruiz Joseph B03

Salsbury Alexa A03

Santos Webster A02, A16, A17, A18, A19, A20, A21

Sasser Todd B06

Shaikh Saame A13

Shan Liang B06

Sharp Amanda A06

Sherpa Chringma A17

Slade Daniel A11

Slebodnick Carla A10, A20

Specht Kalyn A15, B05

Stauffer Philip B02

Szwetkowski Connor A21

Tan Christopher B04

Thompson Alexander A14, A15, B05

Tobey Hope B06

Verma Astha A17

Vess Gavin A01

Vu Chi A14

Wang Yueying A01

Wang Yao A05

Wang Zefeng B03

Wang Yin C02

Williams Christopher C01

Wilts Emily C01

Wisdom Esther B01, B02

Wonilowicz Laura A18, A21

Workmeister Abbey B02

Worrell Brittney A06

Wu Ling B04

Wynn Jessica A17

Xu Bin A05, B04

Yao Zhongke A11, A12

Zou Xianlin B01, B02