University of Michigan College of Pharmacy Virtual Open House (MS & PhD Programs)

Amna Rizvi-Toner - Clinical Pharmacy Translational Science PhD Student

University of Michigan - College of Pharmacy

Dr. Daniel Hertz - Department of Clinical Pharmacy

University of Michigan - College of Pharmacy - Clinical Pharmacy

Antiviral nucleoside analogs are often developed as ester prodrugs to improve their cell permeability. Once enter cells, these ester prodrugs require several activation processes to form the active metabolite, the triphosphate nucleoside (TP-Nuc). Serine hydrolases are the major enzymes responsible for the initial step of the activation-the cleavage of the ester group, and thus playing an important role in determining the intracellular concentration of the active metabolite. In this study, we quantified the protein expression levels of serine hydrolases in the S9 fractions of different human tissues, including the lung, liver, intestine, and kidney. We evaluated the contribution of each major responsible serine hydrolase based on its activity and protein expression level in the tissues. The expression patterns of serine hydrolases in different tissues were significantly different. The activation rates of ester prodrugs are tissue-dependent. Our results suggested that intravenous or inhalation administration, by bypassing the extensive first-passing hydrolysis in the liver, may be a favorable option for some antiviral prodrugs for the treatment of COVID-19.

Jiapeng Li - Clinical Pharmacy Translational Science PhD Student

1Department of Clinical Pharmacy, College of Pharmacy, University of Michigan, 2Department of Pharmaceutical Sciences, College of Pharmacy, Northeast Ohio Medical University

Background: Sepsis induces a widespread disruption in metabolism. This is illustrated by the reported increases in acetylcarnitine (C2) and tricarboxylic acid (TCA) cycle intermediates present in sepsis non-survivors compared with survivors. C2 is thought to be produced via a shunt pathway during acetyl-CoA accumulation when entering the TCA cycle. Previous studies have suggested that sepsis non-survivors have higher levels of TCA cycle metabolites but their associations with C2 have not been studied in the context of the metabolic disruption induced by sepsis. Therefore, the purpose of this analysis was to investigate the relationship between C2 and various TCA cycle metabolites in patients with septic shock. We hypothesized there would be stronger associations between C2 and TCA metabolite levels in septic shock non-survivors than in survivors. Methods: Serum samples from a clinical trial in septic shock patients were analyzed with targeted acylcarnitine (liquid chromatography-mass spectrometry) and TCA cycle metabolite (Ion-pairing chromatography MS) assays. The current analysis was conducted using pre-treatment (T0) serum samples of the placebo group patients (n=60). The metabolite concentrations were log transformed and scaled. Multiple linear regression models were employed to identify differences in the relationships between measured C2 and TCA cycle metabolite levels (citrate/isocitrate, fumarate, malate, succinate, 𝛼-ketoglutarate) in septic shock survivors and non-survivors at 28-days and 1-year. Herein, the models were developed using C2 as the dependent variable with 3 independent variable terms; a TCA cycle metabolite (TCA), mortality, and an interaction between TCA and mortality. They were constructed so that the coefficient of the interaction term would represent a difference in the TCA-C2 relationship between the survivors and non-survivors. Hypothesis testing (t-statistic) was used to determine if a significant difference (p-value ≤ 0.05) between the groups existed. Student’s t-tests were used to corroborate the reported findings of increased C2 and TCA cycle metabolite concentrations in the non-survivors. All statistics were conducted using Rstudio. Results: A difference in the association between C2 and TCA cycle intermediates were found to be significant in 3 of the models that included 2 distinct TCA cycle metabolites (malate and succinate). The interaction term coefficient between malate and 28-day mortality was -0.528 (p= 0.05). For malate and 1-year mortality, the interaction term coefficient was -0.5461 (p= 0.03). The coefficient of the interaction between succinate and 1-year was -0.6624 (p= 0.04). Non-survivors had significantly elevated levels of malate (1-year: p= 0.02), succinate (1-year: p= 0.04), and C2 (28-days: p= 0.0009; 1-year: p= 0.002). Conclusions: Elevations in the acylcarnitines, including C2, generally reflect impaired mitochondrial fatty acid oxidation. These results suggest C2 is representative of a more broadly encompassing mitochondrial dysfunction that includes the TCA cycle. The distinction between mortality groups also indicates that the disrupted metabolic pathways may influence survival.

Marc McCann - Clinical Pharmacy Translational Science PhD Student

University of Michigan College of Pharmacy Department of Clinical Pharmacy

Dr. Gregory Amidon and MSIPS students - Nick Econom and Alysha Reichel

University of Michigan - College of Pharmacy - Master of Science in Integrated Pharmaceutical Sciences

Dr. Heather Carlson, Dr. Timothy Cernak and Dr. Roland Kersten - Department of Medicinal Chemistry

University of Michigan - College of Pharmacy - Department of Medicinal Chemistry

Sean Tanzey - Medicinal Chemistry PhD Student

University of Michigan PET Center

DICER1 is a multidomain protein that contains two RNase III domains (RNase IIIa and IIIb) that catalyze the final cleavage of a pre-microRNA to form the 3p (3’ prime end) and 5p (5’ prime end) strands of a mature microRNA (miRNA), respectively. While some initial characterization has been done to show how DICER1 binds to a pre-miRNA substrate, the exact mechanism by which the RNase III domains interact with the pre-miRNA during pre-miRNA cleavage is still largely unclear. One of the major causes behind this gap in knowledge is that no high-resolution (sub-3.5Å) structures of DICER1 are available for building a molecular model detailing how the protein engages and processes pre-miRNA substrates. Recent biochemical studies have shown that point mutations at key metal-binding residues in the RNase IIIb domain, which are common in many DICER1 Syndrome patients, cause defective miRNA processing. thus, these mutations offer a unique strategy to structurally understand how pre-miRNAs binds to the RNase III domains without interference of the full cleavage reaction. Additionally, recent work from the Garner lab has led to the discovery of distinct classes of natural products that can be used to inhibit DICER1-mediated pre-miRNA maturation. Thus, these newly discovered compounds can also be used as chemical tools to modulate DICER1 activity, and as an additional strategy by which to structurally and functionally characterize DICER1 interactions with a pre-miRNA. This research proposal aims to characterize the structure-function relationship of human DICER1 bound to pre-miRNA substrates during miRNA biogenesis. The specific objective of this proposal is to determine how alterations due to mutations in the RNase III domains can be used to lock DICER1 in different conformational states, as well as to utilize natural products to modulate pre-miRNA interactions. This work is based on the hypothesis that DICER1 must undergo key conformational changes in the RNase III domains during miRNA cleavage in order to properly orient the pre-miRNA substrate towards a favorable interaction with the critical metal binding residues found in the catalytic active site. To evaluate my hypothesis, I will combine cryo-EM structural studies as well as pre-miRNA cleavage and binding assays to study the underlying mechanism that dictates miRNA biogenesis. Furthermore, this work will shed new light on key binding pockets within DICER1 that can be targeted using small molecules and natural products as an alternative approach to therapeutically treating miRNA linked diseases.

Rachel Torrez - Medicinal Chemistry PhD Student

University of Michigan, Department of Medicinal Chemistry

Natural products have provided the framework and inspiration for numerous drug-like molecules. Here, at the University of Michigan, there has been a strong investment in continuing to investigate this fascinating field of molecules. This poster will give a brief overview of the 3 labs (Sherman, NPDC, Kersten) in the medicinal chemistry department that work to uncover the machinery required to create these complex metabolites as well as looking for novel compounds to target new areas of therapeutics.

Robert Hohlman - Medicinal Chemistry PhD Student

University of Michigan, Department of Medicinal Chemistry

Alec Desai

University of Michigan - College of Pharmacy - Department of Pharmaceutical Sciences

Jason Albert - Pharmaceutical Sciences PhD student

University of Michigan - College of Pharmacy - Department of Pharmaceutical Sciences & Medicinal Chemistry*

Dr. James Moon - Department of Pharmaceutical Sciences

University of Michigan - College of Pharmacy

Cancer is one of the most prevalent causes of death worldwide and standard treatments including surgical resection, chemotherapy, and radiation therapy have limited efficacy as indicated by tumor recurrence and metastasis. One of the reasons for this recurrence is cancer stem cells (CSCs), which are a subpopulation of cancer cells able to self-renew and sustain tumor growth after completion of conventional therapy. Due to this resistance, cancer immunotherapy delivering noted CSC biomarker (ALDH, SOX2, NANOG) antigens to deplete CSCs is a precise and sustainable approach. However, efficient delivery of antigens to immune activation sites remains a major challenge in this therapeutic approach.

We have previously developed a synthetic high-density lipoprotein (sHDL) platform for delivery of CSC antigens. sHDL’s small size (< 15 nm) and high biocompatibility make sHDL an ideal candidate for lymphatic trafficking. Here, we have identified immunogenic CSC antigens and developed antigen peptide-sHDL vaccine formulations for the treatment of CSC-containing cancers. We show that vaccinating tumor-bearing mice with sHDL nanodiscs co-loaded with ALDH, SOX2, and NANOG antigen peptides and CpG (a Toll-like receptor 9 agonist) elicits robust antigen-specific T-cell responses and delays tumor growth.

Marisa Aikins

University of Michigan, Department of Pharmaceutical Sciences

Dr. Cherie R. Dotson - College of Pharmacy / Student Services

University of Michigan - College of Pharmacy - Student Affairs