Room 110

Room 110 | 8:45 a.m.

USING DIRECTED EVOLUTION TO ALTER THE STRUCTURE OF PROLYL-HYDROXYLASE DOMAIN TO MODULATE THE HUMAN HYPOXIA RESPONSE

Presented by: Joseph da Costa

Joseph da Costa, Prof. Ambika Bhagi-Damodaran

Dacos023@umn.edu, ambikab@umn.edu

Prolyl-Hydroxylase Domain is an enzyme in the hypoxia signaling pathway that hydrox- ylates the transcription factor HIF-1 which ultimately results in its degradation. It catalyzes this reaction by sensing and utilizing molecular oxygen as a cofactor, but this reaction does not take place under hypoxia when there is very little oxygen. Modulating the activity of PHD is of interest to researchers and has been pursued in the past by mutating specific residues with a rational design philosophy. This re- search expands the scope of the mutant screening by developing and using a directed evolutionary approach of random mutations. This method initially identified two mutants of PHD that were more active than the baseline, but further assays revealed these to be false positives as a product of experimental design. Further work is done on controlling protein concentration inconsistencies in the directed evolution methodology.

Room 110 | 9:10 a.m. 

Design, Synthesis and Evaluation of Prenylation Prosubstrates for Enhanced Cellular Uptake and In Vivo Labeling Efficiency

Presented by: Shanwen Ke

Shanwen Ke, Mark D. Distefano*

ke000051@umn.edu, diste001@umn.edu

Protein prenylation is a type of post-translational modification (PTM) that endows the pro- teins with the membrane-binding ability and signaling functions. Dysregulation of protein prenylation is commonly observed in various diseases and cancers. Designing specific lipid substrates with reporter groups allows researchers to identify and quantify the prenylated proteins by performing metabolic label- ing, enrichment and quantitative mass spectrometric analysis. However, the natural substrates for prenyl- trasferases are diphosphates, which have 3 negative charges at physiological pH that impairs their mem- brane permeability. In this work, three prosubstrates were designed and synthesized by applying and modifying ProTide chemistry (a type of prodrug strategy). Among the three prosubstrates, a monoanion- based probe showed excellent labeling in both cultured cells and mice. Excitingly, relative to previous work with diphosphate-based compounds the labeling efficiency of the new monoanionic prosubstrate increased significantly in liver, kidney, heart, lung, pancreas and brain tissue. This should facilitate more comprehensive proteomics analysis and diseases studies in the future.

Room 110 | 9:35 a.m. 

Harnessing E3 ligase scaffolding protein MAGEA11 for cancer-specific protein degradation

Presented by: Isabella E. Jacobsen

Isabella E. Jacobsen, Rui Shi, Cole R. Scholtz, William C.K. Pomerantz, and Gunda I. Georg

Jaco2013@umn,edu, georg@umn.edu

Proteolysis targeting chimeras (PROTACs) are an emerging therapeutic modality that induce protein degradation by recruiting E3 ligases. Most reported PROTACs recruit ubiquitously expressed E3 ligases, such as cereblon and von Hippel-Lindau. Of the 600+ unexploited E3 ligases, recruiting those with tissue-restricted expression is attractive for increasing the specificity of PROTACs. To this end, tissue-specific E3 ligases or E3 ligase-associated proteins that can be recruited for targeted protein degradation need to be identified.

This work describes the first reported PROTAC that recruits the tissue-specific E3 ligase scaffolding protein MAGEA11. MAGEA11 is exclusively expressed in testes and placental tissues. However, MAGEA11 is aberrantly expressed in multiple cancers, making it a viable candidate for recruitment for cancer-specific degradation. As an initial demonstration, a library of bromodomain and extra-terminal domain (BET)-targeting PROTACs that recruit MAGEA11 was synthesized, and the lead compound 105B was identified. 105B degrades BET proteins in U2OS osteosarcoma cell lines (BRD4 DC₅₀ = 133 pM, Dmax = 78%) and KYSE180 esophageal squamous cell carcinoma cells lines (BRD4 DC₅₀ = 39.8 nM, Dmax = 70%) but shows no degradation in non-cancerous HEK293T cells. Mechanistic studies confirmed 105B’s dependence on the ubiquitin-proteasome system and engagement of MAGEA11 and BRD4. Functionally, 105B decreased BET-regulated protein expression levels in both U2OS and KYSE180 cells (including C-MYC, RUNX2, and KRT14); however, improvements in affecting cell viability are still necessary. This work reports the first example of a PROTAC recruiting a tissue-specific E3 ligase for cancer-restricted BET degradation and highlights the need for further development of MAGEA11-recruiting degraders.

Room 110 | 10:00 a.m. 

PROBING PENICILLIN-BINDING PROTEIN ADAPTATIONS TO ENVIRONMENTAL STRESS

Presented by: Deborah Karunagaran

Deborah Karunagaran, Erin Carlson

karun042@umn.edu, carlsone@umn.edu

Penicillin-binding proteins (PBPs) are essential, membrane-associated enzymes involved in forming the bacterial cell wall. The cell wall acts as the first line of defense against adverse environments. PBPs are also the targets of beta-lactam antibiotics. Bacteria express multiple, functionally redundant PBPs. Recent research suggests that environmental stressors can modulate the activity of PBPs, rationalizing PBP redundancy as a survival mechanism. Bacillus is a class of bacteria that consists of extremophiles whose PBPs may play a role in survivability. Bacillus subtilis, a model bacterium of this class, shows differential PBP activity under neutral and alkaline pH. One such change is the activation of PBP1b, and the inactivation of PBP1a upon exposure to alkaline pH. This phenomenon is of particular interest due to both PBPs being products of a single gene, ponA. Moreover, PBP1b has not been fully characterized, and the adaptation that enables activity at higher pH is unknown. Existing hypotheses include proteolytic cleavage and deglycosylation of PBP1a to yield PBP1b. Our study aims to characterize PBP1b and understand the implications for protein stability under alkaline conditions. This will be achieved using an activity-based probe selective for PBP1b, followed by enrichment of PBP1b, and subsequent analysis by mass spectrometry. A workflow to enrich biotinylated proteins in the presence of B. subtilis lysate has been optimized successfully, and molecules to selectively pulldown PBP1b are being explored. Ultimately, this work contributes to the expansion of existing knowledge regarding the redundancy of PBPs, and clarifies the understanding of bacterial survival mechanisms in extreme environments.

Room 110 | 10:40 a.m.

DELETION OF RND EFFLUX PUMPS AFFECTS MOTILITY IN PSEUDOMONAS AERUGINOSA

Presented by: Umer Syed

Umer Syed, Hannah K. Lembke, Erin E. Carlson*

muham163@umn.edu; carlsone@umn.edu 

The proliferation of antibiotic resistance threatens to disrupt the efficacy of many routinely used antibiotics. Bacteria can both be intrinsically resistant and acquire genetic elements that endow resistance to antibiotics. In Gram-negative bacteria, Resistance Nodulation Division (RND) efflux pumps are major determinants of resistance and can extrude diverse classes of antibiotics even at basal levels. Resistant clinical isolates of the opportunistic, nosocomial pathogen, Pseudomonas aeruginosa, frequently feature overexpressions of four RND efflux pumps, MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY. Inhibiting these efflux pumps not only resensitizes bacteria to antibiotics but also hinders their ability to cause an infection. Despite this therapeutic promise, the role of efflux pumps in bacterial physiology and virulence has not been elucidated. To investigate the pumps’ roles in causing and sustaining infections, we constructed a library of P. aeruginosa mutants lacking one or more of its four most clinically relevant efflux pumps. We screened various virulence phenotypes of these mutants and found motility, specifically swarming, was significantly disrupted by the loss of MexAB-OprM. Further analyses into the link between efflux pump expression and virulence demonstrated that swarming motility is impaired by rendering the flagellar stator essential for swarming, MotCD, dysfunctional. This study further underscores the need to evaluate the native, antibiotic-independent function of efflux pumps as inhibiting these proteins can be leveraged as a promising therapeutic strategy to the global antibiotic resistance epidemic.

Room 110 | 11:05 a.m.

METABOLIC LABELING VERSUS IN VITRO INCORPORATION REVEALS THAT PRENYLATION STATUS IMPACTS PROTEIN STABILITY

Presented by: Anjana P. Sundaresan

Anjana P. Sundaresan, Mark D. Distefano*

paina024@umn.edu; distefano@umn.edu

Protein prenylation is an essential post-translational modification in which an isoprenoid lipid group is irreversibly attached to the C-terminus of target proteins. This modification can involve the attachment of a farnesyl chain (farnesylation) or a geranylgeranyl chain (geranylgeranylation). Protein hydrophobicity is significantly increased by this process, which results in its membrane localization, thereby facilitating signal transduction. Dysregulation of prenylated proteins is implicated in numerous diseases, making their comprehensive identification a critical research goal. To profile geranylgeranylated proteins and assess GGTase-I inhibition, AML-3 cells were treated with geranylgeranyltransferase type-I inhibitors (GGTIs) followed by metabolic labeling with C15AlkOPP, an alkyne-functionalized isoprenoid analog. The presence of inhibitor resulted in a marked reduction in metabolic labeling of geranylgeranylated proteins in proteomic studies, making them harder to detect. To address this limitation, we developed an in vitro labeling strategy, where lysates from GGTI-treated cells were incubated with excess recombinant GGTase-I and C15AlkOPP, enabling enzymatic labeling of unprenylated substrates and was expected to produce enhanced signals for GGTI-targeted proteins. Unexpectedly, proteomic analysis identified a smaller number of geranylgeranylated proteins after performing that workflow. To investigate this discrepancy, cycloheximide chase experiments were performed to assess protein stability. These studies showed that a subset of unprenylated geranylgeranylation substrates undergoes rapid degradation, whereas proteins in their unprenylated form remain comparatively stable. Together, these findings indicate that loss of prenylation compromises protein stability, linking post-translation modifications to protein turnover and impacting both cellular regulation and proteomic detection of prenylated substrates. 

Room 110 | 11:30 a.m. 

EXPANDING TARGETED PROTEIN DEGRADATION: IMPROVED DEGRADER PHYSIOCHEMICAL PROPERTIES AND NON-UBIQUITINATED DEGRADATION PATHWAYS

Presented by: Ethan Essenfeld

Ethan Essenfeld, William C.K. Pomerantz* 

essen038@umn.edu, wcp@umn.edu 

Targeted protein degradation (TPD), a therapeutic modality initially discovered in 2001, but since 2015, research efforts using this approach has exponentially increased. TPD hijacks the cell’s natural degradation system to degrade a target protein that have not been traditionally targeted by other therapeutic modalities. TPD traditionally uses E3 ligases to tag the targeted protein with polyubiquitin chains to trigger proteasomal degradation through recruitment by small molecule ligands. The most common E3 ligase utilized is Cereblon, CRBN, as it has a family of ligands that can be incorporated into degraders. The ligands, known as Immunomodulatory Drugs, IMiDs, contain multiple hydrogen bond donors that, when incorporated into full protein degraders, result in suboptimal physiochemical properties. This work is optimizing an approach to discover new CRBN ligands using Protein Observed Fluorine NMR to overcome challenges in developing a clinically viable degrader. As a new approach, recently a ligand that directly binds to the 26S proteasome, TCL1, has been used to circumvent the E3 ligase to induce degradation. This modality of degradation, known as ByeTACs, Bypassing E Ligase Targeting Chimeras, can potentially increase the scope of proteins that TPD can target. In a second direction,  new ByeTACs targeting Bromodomain PHD finger Transcription Factor, BPTF are being developed. Despite having multiple known selective inhibitors, BPTF has yet to be fully established as a viable target for TPD and as a cancer treatment. This work plans to explore the impact of degrading BPTF using ByeTACs to expand the capability of ByeTACs and BPTF as cancer treatments. 

Room 110 | 11:55 a.m.

Development Of SLO3 and CatSper Inhibitors as Non-Hormonal Male Contraceptives

Presented by: Srujana Mohanty

Srujana Mohanty, Jake Schmitt, Irina Zhilinskaya, Gunda I. Georg*

(mohan196@umn.edu and georg@umn.edu)

Male contraceptive options are limited to condoms and vasectomy, both of which have drawbacks, including inconsistent efficacy or invasiveness and limited reversibility. Therefore, a non-hormonal, on-demand, reversible, orally available male contraceptive remains a critical unmet need. CatSper and SLO3, two sperm-specific ion channels, are crucial for sperm capacitation and hyperac- tivated motility. Genetic knockout of either channel in mice causes infertility, confirming them as contraceptive targets. A high-throughput screen of ~72,000 compounds identified six CatSper-inhibitor scaffolds. Among these, compound 4a showed strong activity and minimal off-target effects, prompting further structure–activity relationship studies. These studies led to EJ 3.148, a potent dual CatSper/SLO3 inhibitor. VU0546110 and VU6032735 are known SLO3 inhibitors. Our current research focuses on synthesizing analogs of these lead compounds and exploring new scaffolds that combine key structural elements. Candidate compounds will be evaluated using in vitro assays to confirm CatSper and SLO3 inhibition. Potent and selective compounds will then undergo ADMET profiling, followed by pharmacokinetic and mating studies. The goal is to improve ion-channel selectivity, physicochemical properties, and pharmacokinetics while minimizing off-target activity. The long-term aim is to develop safe, effective, orally-available and reversible non-hormonal male contraceptives.

Room 110 | 1:35 p.m.

SCALABLE APPROACH TO RECYCLABLE CROSSLINKED POLYETHYLENE FROM HYDROXY TELECHELIC PRECURSORS

Presented by: David Santiago

David Santiago, Mara Kuenen, Brenden Hoehn, Marc Hillmyer* 

Santi299@umn.edu, hillmyer@umn.edu 

High-density polyethylene (HDPE) is a linear, semicrystalline thermoplastic used in a plethora of applications given its attractive physical properties and low cost. However, functionalization of HDPE is difficult due to the inert nature of the entirely saturated hydrocarbon backbone. Thus, the incorporation of functional groups typically requires radical chemistry, which can lead to low functionalization efficiency and deleterious side reactions. Here, we have explored a scalable, one-pot, solvent-free, atom-economic, and tunable synthesis of hydroxy-telechelic polyethylene (HO-PE-OH). The HO-PE-OH can be readily crosslinked in a condensation reaction with a common crosslinker trimethyl trimesate (TMT) to form crosslinked polyethylene (PEX), a high-performance thermoset used in diverse applications, including power cable insulation, potable water pipes, and artificial joints. While commercial PEX is composed of irreversible crosslinks, making recycling challenging, our approach incorporates labile ester linkages throughout the network, enabling chemical recycling back into the original linear HO-PE-OH and TMT. These findings establish the methodology for the scalable production of HO-PE-OH and a closed-loop chemically recyclable PEX.

Room 110 | 2:00 p.m

Investigating The Effect of Strong Dipole Incorporation in Homopolymers and Adhesive Tie Layers

Presented by: Luc G. Wetherbee

Luc G. Wetherbee, Christopher J. Kim, Jessica R. Lamb*

wethe077@umn.edu, jrlamb@umn.edu

Multilayer films are commonly employed in applications where multiple polymer properties are desired. For example, food packaging often consists of polyethylene (PE) and polar polymers to combine water and oxygen barrier properties. However, because PE is a poor adherend, adhesive tie layers are required to maintain the structural integrity of the multilayer film. Common tie layers consist of PE modified with functional groups that react directly with polar polymer; while this leads to strong adhesion, the potential recyclability of multilayer films is hindered by the formation of interfacial cross-links. To address this issue, this work is aimed at investigating the effect of strong dipole incorporation on the performance of nonreactive adhesive tie layers. The Lamb group specializes in synthesizing polyoxazolidinones (POxa), which possess remarkably strong dipoles (~5 Debye). These dipoles can be incorporated into adhesives via tandem ring-opening metathesis polymerization (ROMP)-hydrogenation of copolymers containing varying ratios of cyclooctadiene and the oxazolidinone-fused polar monomer, yielding a copolymer with a polyethylene-like backbone and strong dipoles distributed throughout. Trilayer laminates are prepared by hot pressing the adhesive between PE and nylon layers, and the adhesive strength of the multilayer film is tested using a 90° peel testing instrument. We hypothesize that this system can be leveraged to form strong nonreactive adhesive tie layers, in which there is a balance between adhesion via nonpolar and dipole–dipole interactions.

Room 110 | 2:25 p.m. 

NOVEL POLYMERIC EXCIPIENTS FOR IMPROVED ORAL DELIVERY OF CLASS IV ACTIVE PHARMACEUTICAL INGREDIENTS 

Presented by: Miles Willis

Miles Willis, Elizabeth Lopez, Theresa Reineke*

will7644@umn.edu, treineke@umn.edu

Oral delivery of many active pharmaceutical ingredients (APIs) is limited by their inherent insolubility and lack of permeability across gastrointestinal membranes. Both of these factors limit the oral bioavailability of Class IV APIs and must be overcome to effectively deliver drugs. To improve solubility many APIs are co-formulated with polymer excipients to form Amorphous Solid Dispersions (ASDs) which improve the solubility of APIs in aqueous environments through non-covalent interactions. To improve membrane permeability, APIs are often co-formulated with small molecule Permeation Enhancers (PEs) to enable membrane crossing, but these require high doses of PEs which limits drug loading. To address both issues, a novel polymer excipient library was synthesized and tested with the Class IV anticancer API, Venetoclax. This polymeric system was modeled off the commercial standard Poly (N-Vinyl-2-Pyrrolidone co Vinyl Acetate) (PVPVA) which is a known solubility enhancer with no known permeation enhancing effects. Replacing vinyl acetate with a hydrolysable PE based monomer enables the release of a food-safe PE, conferring both solubility and permeability enhancing properties to the polymer. Through systematic tuning of chemical composition, the optimal structure for solubility enhancement was determined via a non-sink dissolution assay. These novel polymeric excipients were found to improve Venetoclax solubility four-fold over the current commercial standards used in clinical formulations of Venetoclax.

Room 110 | 2:50 p.m.

BULK RING-OPENING TRANSESTERIFICATION POLYMERIZATION OF γ-METHYL ε-CAPROLACTONE USING HCl  

Presented by: Simran

Simran, Marianne S. Meyersohn, Marc A. Hillmyer*

simra005@umn.edu; hillmyer@umn.edu 

Aliphatic polyesters are a versatile class of polymers used in numerous applications ranging from biomedical implants to industrially compostable products. These are usually synthesized by controlled ring-opening transesterification polymerization (ROTEP) of lactones with tin-based catalysts, though concerns over tin toxicity have driven interest in metal-free organocatalysts. Hydrochloric acid (HCl) is a simple and effective Brønsted acid catalyst for the ROTEP of cyclic esters under solvent-free conditions at room-temperature and can be easily removed by devolatilization, further supporting cleaner and more sustainable processing. This study examines the kinetics of HCl-catalyzed bulk ROTEP of γ-methyl ε-caprolactone (γMCL), a monomer known for producing high-performance degradable thermoplastic elastomers. Kinetic analysis reveals that the polymerization rate is first-order in monomer, initiator, and catalyst concentrations. Limitations in achieving high molar mass polymers with HCl catalyst were also identified. We also discovered a one-pot method for producing crosslinked aliphatic polyester elastomers from γMCL using HCl catalysis and a bis-ε-caprolactone crosslinker. These elastomers exhibit properties on par with analogous elastomers made with tin catalyst. By varying initiator and crosslinker loading, elastomers with tunable moduli and mechanical properties were also obtained. Finally, the elastomers can be chemically recycled back to the high-purity monomer with a more environmentally benign catalyst ZnCl₂ establishing a closed-loop recycling process.