Medicinal chemistry

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Structural and Functional Characterization of Pentameric and Hexameric IgM Antibodies in Complement System Activation
(2026-04-20) Mundorff, Charles; Guttman, Miklos
Immunoglobulin M (IgM) antibodies are critical components of the immune system, characterized by their pentameric or hexameric structure and exceptional binding avidity. Understanding the structural and functional differences between these forms is essential for advancing therapeutic antibody development and furthering our understanding of immune responses. This dissertation comprises interconnected studies that advance our understanding of IgM biology through methodological innovation and biophysical characterization.Chapter 1 will introduce and provide relevant background information on the topics discussed herein including IgM biology and function, hydrogen deuterium exchange mass spectrometry (HDX-MS) as a biophysical tool to study complex systems, complement cascade activation and its role in the immune system, and the myriad other techniques employed in the studies conducted. Chapter 2 addresses a fundamental methodological challenge in hydrogen-deuterium exchange mass spectrometry: hydrogen scrambling during analysis. We systematically mapped activation thresholds for scrambling, revealing that it is a global process with peptide-specific thresholds, providing critical parameters for optimizing site-specific HDX-MS experiments. Chapter 3 presents comprehensive structural characterization of pentameric and hexameric IgM using HDX-MS, electron microscopy, and small-angle X-ray scattering. These studies reveal distinct conformational properties between the two forms, with hexameric IgM showing greater flexibility in the Fc region and more extensive conformational changes upon binding to antigen-displaying nanoparticles. Chapter 4 presents functional characterization demonstrating that hexameric IgM activates complement slightly more efficiently than pentameric IgM. Integration of structural and functional data reveals that the optimal Fc presentation in hexameric IgM facilitates more effective C1q binding and complement activation. Chapter 5 presents preliminary data on C1 complex interactions and conformational dynamics and discusses future directions for understanding the complete molecular mechanism of IgM-mediated complement activation, with implications for therapeutic antibody development.
Investigating the protective role of the hydrogen sulfide signaling pathways in cardiac and hepatic stress models
(2026-02-05) Jung, Taeyoon; Totah, Rheem A
Hydrogen sulfide (H2S) is now recognized as a pivotal endogenous gasotransmitter involved in cardiovascular and hepatic physiology and pathology. In this work, we investigate the protective role of H2S signaling pathways in cardiac and hepatic stress models, suggesting potential therapeutic applications under oxidative stress and metabolic dysfunction.Doxorubicin (Dox) is an anthracycline chemotherapy agent used to treat a wide range of malignancies. Despite its potent antitumor activity, the clinical use of Dox is limited by dose-dependent cardiotoxicity and hepatotoxicity. Evidence supports the "double-edged sword" effect of H2S in hepatocellular carcinoma cells; however, little data exists on whether, and how, H2S can protect against Dox-induced toxicity. We demonstrate that exogenously added H2S effectively protects against Dox-induced cytotoxicity in hepatic and cardiac cells, primarily through regulating WNT3 signaling and potentially altering the cell-cycle. Additionally, supplementing with H2S exhibited increased viability, decreased cell death, and reduced the production of reactive oxygen species in Dox-treated cells. Transcriptomic analyses revealed extensive modulation in gene expression following Dox-treatment that were partially mitigated by H2S, confirming its broad cytoprotective impact. Next, we evaluated the effect of H2S on phorbol 12-myristate 13-acetate-induced hypertrophy in human cardiomyocytes. H2S reversed key hypertrophic markers and induced transcriptional changes through the modulation of early-response genes, such as ARC and EGR1, highlighting its therapeutic potential in pathological cardiac hypertrophy. Furthermore, we addressed palmitic acid-induced insulin resistance in hepatic and cardiac cells. Although H2S produced only modest transcriptomic changes, it improved metabolic markers, as evidenced by RNA-sequencing analysis. Finally, we elucidated structural differences between the thiol methyltransferases (TMT) TMT1A and TMT1B that methylate hydrogen sulfide and quench its protective properties. This work highlighted critical aromatic residues that differ in the inhibitor selectivity of 2,3-dichloro-α-methylbenzylamine (DCMB). These insights will enable the development of a TMT1B selective inhibitor, advancing our understanding of enzyme specificity and providing a basis for therapeutic modulation of H2S methylation. Overall, this research underscores the multifaceted protective role of H2S, underscoring its significant therapeutic potential in reducing oxidative stress-related diseases.
Private Choices in Public Health: A Framework for Economic Epidemiological Modeling of Infectious Disease
(2025-10-02) Montano-Campos, Jose Felipe; Basu, Anirban
This dissertation investigates the role of risk compensation in infectious disease dynamics by examining how individuals adjust private preventive behaviors in response to perceived mortality risk, and how these behavioral shifts shape and are shaped by epidemic trajectories. While conventional epidemiological models assume fixed or policy-driven contact rates, real-world transmission depends critically on how individuals perceive and respond to evolving risk. Public health policies, in turn, interact with these private behaviors—amplifying or dampening their effects. The first part of this dissertation provides empirical evidence of risk compensation behavior by analyzing high-frequency mobility data across U.S. counties during the COVID-19 pandemic. Using lagged local mortality as a proxy for perceived risk, the study finds that individuals significantly reduced their mobility in response to rising deaths—particularly in high-contact, discretionary domains. These responses evolved over time and were shaped by public health interventions such as shelter-in-place orders and mask mandates, which often amplified rather than displaced private behavioral changes. The second part develops a dynamic economic-epidemiological model that endogenizes contact rates through mortality-responsive behavioral feedback. Critically, the magnitude of behavioral responsiveness estimated in the empirical analysis is used to calibrate the strength of the feedback loop—bridging observed behavior with modeled transmission. This endogenous co-evolution of behavior and epidemic severity creates a self-regulating system in which private action dynamically responds to real-time risk signals, suppressing transmission as perceived threat intensifies. The model, grounded in a delayed SEIRDS framework, demonstrates how such feedback can flatten epidemic curves, delay transmission peaks, and produce multiple waves. Introducing pandemic fatigue—modeled as declining responsiveness to risk—erodes this adaptive loop, weakening the self-limiting dynamic and enabling persistent spread. Simulations highlight the importance of accounting for behavioral adaptation in forecasting and policy design. Together, these studies offer a unified framework for understanding how private choices interact with public policy and disease dynamics through the lens of risk perception and behavioral adaptation.
Healthcare resource utilization and costs of commercial and Medicare-enrolled adult patients with Multiple Sclerosis in Urban and Rural settings
(2025-08-01) Jain, Divya; Bansal, Aasthaa
Background: Multiple sclerosis (MS) is a chronic progressive neurological disease that affects almost 1 million people in the United States, with an economic burden of over $85 billion USD. Previous studies have demonstrated that rural MS patients have reduced access to a neurologist compared to urban MS patients, but how this disparity impacted these patients was not explored. However, healthcare resource utilization and cost in MS patients in rural and urban areas has not been previously studied. Objective: The primary objective of this study was to describe the healthcare resource utilization (HCRU) and healthcare costs of commercial and Medicare-enrolled patients with MS in rural and urban settings. Methods: We conducted a retrospective cohort study using health insurance claims from the Merative MarketScanÂ® Commercial and Medicare Supplemental Databases. MS cases were identified as having ≥2 MS outpatient (OP) claims, dated ≥30 days apart, within a 1-year period or ≥1 MS inpatient (IP) claim (ICD-10: G35) during the index/case identification period. The index date was defined as the first occurrence of meeting case criteria during December 31st, 2017, to December 31st, 2022. We examined all-cause HCRU by summarizing the number of IP admissions, OP services, emergency department (ED) visits, and OP pharmacy fills in the 6 months post-index. Additionally, we assessed all-cause health care costs, including IP, OP, ED, and pharmacy, over 1 year following index using the Kaplan-Meier sample average estimator to account for censoring. We reported total healthcare costs as the sum of IP, OP, ED and OP pharmacy costs. Analyses were conducted separately for the commercial and Medicare supplemental groups; all-cause HCRU and costs were summarized overall and by rural and urban status. Results: Our cohort consisted of 47,636 commercially insured patients and 3,318 Medicare patients. During the 6 months following index date, rural commercial patients had a slightly elevated mean number of IP admissions (1.4) and length of stay (LOS) (4.4 days) than the urban patients (1.3, 4.4). This trend is not repeated in the Medicare Supplemental population. In that population, the rural cohort had a lower mean number of IP admissions (1.3) and LOS (5.00) compared to the urban cohort (1.4, 5.9). For ED visits, in both the commercial and Medicare cohorts, the rural patients had lower average ED visits than the urban patients (Commercial: 1.5 vs. 1.6 rural vs urban) (Medicare: 1.7 vs. 1.8). OP visits follow this trend with the rural cohort having fewer visits among those that did have OP visits 6 months post-index in both insurance cohorts (Commercial: 9.4 vs. 10.0 rural vs urban) (Medicare: 12.7 vs. 14.4). However, there is a difference seen in the OP pharmacy fills. For both commercial and Medicare insurance, the rural cohorts had higher mean and median OP pharmacy fills than the urban cohorts The commercial rural patients had a mean of 31.5 (SD 20.8) 30-day standardized pharmacy fills and a median of 27.3 (IQR 15.9 – 42.6) compared to the urban patients with a mean of 27.7 (SD 19.4) and median of 23.4 (IQR 13.4 – 37.3). The Medicare rural patients had a mean of 38.1 (SD 23.9) 30-day standardized pharmacy fills and a median of 35.9 (IQR 22.3 – 48.9), compared to the urban patients with a mean of 36.6 (SD 20.2) and a median of 34.2 (IQR 22.3 – 48.7). Conclusion: Rural patients tended towards lower average healthcare resource utilization (HCRU), and thus, lower costs than the urban patients, with some notable exceptions. Rural patients had higher mean and median pharmacy fills and higher costs in both insurance categories. This may point to rural MS patients relying more on prescription drugs to compensate for reduced access to medical services. MS patients require complicated care and insight into their HCRU and costs, which can aid healthcare decision-makers in optimizing care for these patients.
Healthcare Resource Utilization and Costs in Patients with Mantle Cell Lymphoma in the Third-Line Setting in the US
(2025-08-01) Weisman, Nissen; Veenstra, David; Bansal, Aasthaa
Background: Treatment of mantle cell lymphoma (MCL) is characterized by multiple lines of therapy. Previous studies have described healthcare resource utilization (HCRU) and costs of MCL in the third-line or greater (3L+) setting, but none have estimated outcomes attributable to MCL specifically. Additionally, newer therapies have recently been added to clinical guidelines.Objective: Identify the attributable HRU and cost of MCL in the year following the initiation of third-line therapy. Methods: Retrospective cohort study using MarketScan health insurance claims data. The Merative™ MarketScanÂ® Databases are U.S. claims datasets with de-identified medical and pharmacy claims linked to enrollment data for individuals with commercial or Medicare supplemental insurance. Incident third-line mantle cell lymphoma patients (MCL3L) were identified between April 1st, 2016, to December 31st, 2022, and followed up until loss of enrollment or one year after initiation of third-line therapy. Matched cohorts for the commercial and Medicare supplemental populations were selected using propensity scores based on age, sex, cancer-adjusted-CCI, region, commercial vs. Medicare supplemental payer type, and presence of non-heme cancer. Six-month HCRU use was evaluated in patients with at least 6 months of continuous enrollment post-index. Annual costs for all patients were calculated accounting for censoring using Kaplan-Meier Sample Average (KMSA). HRU were compared using a t-test for mean days of utilization and a chi-square test for the presence of utilization or not. Confidence intervals for costs were calculated using the bootstrap method for both cohort-specific outcomes and the difference between the two cohorts. Results: Third-line or later MCL patients had significantly greater HRU across all care settings compared to matched controls. Within six months post-index, a higher proportion of MCL patients had ≥1 inpatient admission (50.5% vs. 11.6%; p < 0.001), ≥1 emergency department (ED) visit (50.5% vs. 14.7%; p < 0.001), and ≥1 outpatient visit (100% vs. 50.5%; p < 0.001). Among those with ≥1 event, MCL patients had significantly higher ED visit counts (1.96 vs. 1.36; mean difference [MD] = 0.60, 95% CI: 0.07 to 1.13; p = 0.030) and outpatient visit counts (26.8 vs. 9.4; MD = 17.4, 95% CI: 13.33 to 21.47; p < 0.001). Mean number of inpatient admissions (1.81 vs. 1.36; MD = 0.45, 95% CI: –0.06 to 0.96; p = 0.098) and mean length of stay per admission (8.72 vs. 5.13 days; MD = 3.59, 95% CI: –0.24 to 7.42; p = 0.071) trended higher among MCL patients. Attributable annual costs were $6,302 (95% confidence interval [CI]: $4,907–$7,877) for pharmacy, $14,410 (95% CI: $7,482–$22,885) for inpatient services, $8,586 (95% CI: $6,414–$10,937) for outpatient services, and $204 (95% CI: $59–$353) for ED visits. The total attributable annual cost was $29,613 (95% CI: $21,585–$39,045). Discussion: We found the one-year attributable costs in third-line plus treatment of mantle cell lymphoma exceeded $32,000, driven primarily by inpatient and outpatient services. There were significant differences in the % healthcare resource utilization in the 6 months following index, but these results were only in the patients who had at least 6 months of continuous enrollment potentially under-representing sicker patients who disenroll early. As the treatment landscape continues to evolve, understanding these costs is essential for payers and policymakers evaluating the value of emerging therapies.
Cost-Effectiveness of Delaying Alzheimer’s Disease Progression with Novel Monoclonal Antibodies
(2025-08-01) Chung, Minseon Veronica; Sullivan, Sean D
Background: In the US alone, most of over 7.2 million older adults with Alzheimer’s Disease (AD) treat their AD with symptom-relieving therapies. Starting in 2021, three disease-modifying monoclonal antibodies (mAbs) entered the US market as the first disease-modifying treatments for early Alzheimer's disease (AD). Despite years of presence in the US market, the value of these mAbs remains uncertain due to high treatment-related costs and an unclear comparative value of their clinical benefit. Objectives: 1) To estimate the cost-effectiveness of adding aducanumab, lecanemab, or donanemab to the current standard of care (SOC) and 2) to evaluate the value of reducing uncertainties around the mAbs from a US health care perspective and a modified societal perspective. Methods: A Markov simulation model was developed by incorporating literature and a prior cost-impact model of delaying AD progression. The clinical benefit and cohort’s characteristics mirrored results from phase 3 clinical trials of the three agents. The price of the agents was estimated using wholesale acquisition costs published by Micromedex RED BOOK. The model used a 1-week cycle length and estimated 10-year cost impact and comparative incremental cost-effectiveness ratios (ICERs) among the SOC and the mAbs. Results were described from both a health care sector and a societal perspective. One-way sensitivity analysis was conducted to demonstrate key drivers of cost-effectiveness of the mAbs. Cost-effectiveness acceptability curve and the expected value of perfect information (EVPI) used probabilistic sensitivity analysis to demonstrate the comparative value of the mAbs and the value of resolving uncertainties. Results: The three agents were estimated to delay progression from MCI to severe AD by 6.2 - 9.2 months. The analysis showed that SOC costs $243,300 and $757,800 from the health care sector and the modified societal perspective, respectively, over 10 years. From the health care sector perspective, the mAbs incurred additional $55K, $73K, and $82K for aducanumab, lecanemab, and donanemab, respectively. From the modified societal perspectives, donanemab added $73K, and aducanumab and lecanemab added $48K and $64K to the cumulative costs, respectively. Donanemab had the higher comparative ICER at $304,200/QALY, followed by lecanemab at $175,300/QALY. Key driving factors of ICER among lecanemab and donanemab were age at the treatment initiation, patient health state utility values in earlier AD states, and the drug acquisition costs across both perspectives. SOC had more favorable cost-effectiveness at a WTP of $150K/QALY across both perspectives, and lecanemab was more favored at a WTP of $200K/QALY. The EVPI of the decisions around lecanemab versus SOC was estimated to be $143 million at WTP of $190K/QALY and $155K/QALY for the health care sector and the modified societal perspectives, respectively. The EVPI around donanemab versus lecanemab was approximately $109 million at the WTP of $300K/QALY and $275K/QALY for the healthcare sector and the societal perspectives, respectively. Conclusion: In the model simulation, the novel AD treatments incurred substantial costs with modest clinical benefits, with donanemab adding the highest cost. Assuming the treatment costs based on WAC, only lecanemab was found likely to be cost-effective under a WTP of $200K/QALY from the modified societal perspective. The cost-effectiveness of the mAbs is substantially influenced by the patient’s age at the treatment initiation, patient health state utility values at earlier AD states, and the drug acquisition costs.
Impact of Cachexia on Resource Utilization and Costs in Patients with Pancreatic, Lung, and Colorectal Cancers
(2025-08-01) Perkins, Kathryn Samantha; Carlson, Josh
Introduction: Cancer cachexia or cancer anorexia-cachexia syndrome (CACS) is a wasting disease characterized by inadequate food intake, loss of muscle mass, weight loss, inactivity, and changes in metabolism. Cachexia is estimated to affect 70-80% of pancreatic cancer patients and approximately 50% of lung and colorectal cancer patients. There is limited literature describing the impact of this condition on healthcare resource utilization and costs in the US. Objective: The primary objective of this study was to compare the healthcare resource utilization (HRU) and direct health plan costs of colorectal, lung, and pancreatic cancer patients diagnosed with cachexia to matched cancer patients without a cachexia diagnosis. Methods: We conducted a retrospective cohort study in commercial and Medicare Merative™ MarketScanÂ® claims database. Patients with cachexia were identified following a colorectal, lung, or pancreatic primary cancer diagnosis between October 1st, 2016 – December 31st, 2022. Controls with no cachexia diagnosis during the study period were 2:1 matched with cachexia cases. Mean annual healthcare resource utilization and direct health plan costs were calculated using Kaplan-Meier Sample Average (KMSA) to account for patients censored before the end of the 1-year observation period. Significant differences between groups were determined by bootstrap 95% confidence intervals. A cox proportional hazard model was conducted in the unmatched cohort to determine relative hazard of cachexia diagnosis in different cancer types. Results: After matching the sample included 34,882 cancer patients, 11,880 in the cachexia cohort and 23,002 in the control cohort. Overall healthcare costs were significantly higher in colorectal, lung, and pancreatic cancer patients with cachexia ($141,626, [95% CI: $138,004 - $144, 905]) compared to those without evidence of cachexia, ($99,104, [95% CI: $97,193 – $101,224]). Average annual hospital admissions (cachexia: 0.98 admissions, [95% CI: 0.94 – 1.02]; control: 0.60, [0.58 – 0.62]) and annual emergency room visits, (cachexia: 2.47 ER visits [95% CI: 2.38 – 2.58]; control: 1.33 [95% CI: 1.29 – 1.36]) were significantly higher among cancer patients with cachexia compared to controls. Average annual outpatient encounters (cachexia: 61.5 encounters [95% CI: 60.7 – 62.4]; control: 44.6 [95% CI: 44.0 – 45.2]) and annual prescription fills (cachexia: 37.7 fills [95% CI: 37.1 – 38.4]; control: 30.4 [95% CI: 30.0 – 30.8]) were also significantly higher in cachexia cancer patients compared to controls. Among patients who developed cachexia, pancreatic cancer patients had the shortest median time to cachexia diagnosis of 1.2 months (IQR: 0.1 - 5.1) and had a 2.9-fold greater risk of being diagnosed with cachexia compared to those with colorectal cancer, which had the lowest hazard (HR: 2.90, [95% CI: 2.79 – 3.02, p<0.0001]). Conclusion: Colorectal, lung, and pancreatic cancer patients in the US, with a concurrent diagnosis of cachexia were found to have higher healthcare resource utilization and cost of care in the year following cachexia diagnosis.
Investigating the Effects of Sequence Variation and Host-Switching Mutations on the Dynamic Activation of Influenza Hemagglutinin
(2025-01-23) Kephart, Sally; Lee, Kelly K
The process by which influenza virions enter cells involves multiple steps that are carried out by the trimeric glycoprotein hemagglutinin (HA). In addition to recognizing receptors on the cell surface, HA mediates fusion of the viral and host cell membranes following endocytosis. The multifunctionality of HA is afforded by its ability to change conformations in response to an environmental trigger (pH). Changes in pH sensitivity and acid stability are observed across divergent subtypes of HA and due to specific mutations associated with host-switching, but it remains unclear how these differences impact HA function. Recently, the changes involved in the transition from the pre- to postfusion conformation have been characterized by techniques that probe protein dynamics, providing new mechanistic insights into the process of HA-mediated fusion. Here, we apply hydrogen/deuterium exchange mass spectrometry (HDX-MS) to study how sequence variation and host-switching mutations affect HA dynamics during acid-induced activation. Using a recombinant HA construct that we show behaves similarly to the HA ectodomain cleaved from the virus (Chapter 2), we elucidate mechanistic differences that drive changes in acid stability for H5 HA with two sets of host-switching mutations (Chapter 3). Our data indicate that host-switching mutations dampen dynamics in regions that become more exposed at low pH, including the HA1-HA2 interface and fusion peptide. This result suggests that pH-responsive regions are hotspots for adaptive changes. In Chapter 4, we show that this trend persists in light of variation in activation dynamics seen across diverse subtypes. Together these data reveal a connection between activation dynamics and acid stability that is central during adaptation to a new host.
Assessing the Impact of Benzalkonium Chlorides on the Gut-Liver Axis
(2025-01-23) Lopez, Vanessa Andrea; Xu, Libin
Benzalkonium Chlorides (BACs) are highly effective disinfectants, widely used in consumer, clinical, food processing and agricultural settings. Recently, the Food and Drug Administration (FDA) called for additional safety data on BACs. Additionally, the recent COVID-19 pandemic has resulted in increased concentrations of BACs being detected in human blood samples as well as residential dust samples. Importantly, environmental toxicant exposure and the effects on the resident gut microbiome is becoming increasingly recognized as a unique mechanism of understanding human health and disease. The gut-liver axis is a unique, bidirectional link that has become recognized for the role it has in liver disease and impairment of liver function. We hypothesize is that exposure to BACs can impact the resident microbiome, which will lead to alterations in transcriptomic, sterol, lipid, and xenobiotic metabolism of the liver. In this dissertation, I describe a comprehensive animal study that elucidated BAC exposure effects on gut microbiome composition and diversity, unconjugated bile acid (BA) pools in biological extracts, and BAC and BAC metabolite profiles. Additionally, I report a thorough and comprehensive quantitation analysis of BAC and BAC metabolite distribution throughout brain, lung, blood, spleen, kidney, liver, duodenum, jejunum, ileum, large intestine, feces and urine in male and female BAC exposed mice. Next, I share a comprehensive multiomics analyses of BAC-induced changes to endogenous and exogenous metabolism in livers of BAC exposed mice including transcriptomics, sterolomics, lipidomics and cytochrome p450 activity. Finally, I conclude with overall conclusions and implications of this work, and provide insight into future studies that should be conducted. Together, this dissertation provides a comprehensive and rigorous comprehension of environmental toxicant exposure risk to human health by elucidating mechanisms by which BAC-exposure disrupts gut microbiome composition and diversity, and endogenous and exogenous metabolism in the liver.
Advancing the Development of Macrocyclic Peptide Therapeutics Using High-Throughput Mass Spectrometry Approaches
(2025-01-23) Palmer, Jonathan Thomas; Guttman, Miklos; Bhardwaj, Gaurav
Cyclic peptides are poised to revolutionize the therapeutic market as designed protein modulators. These peptides are characterized by a circular sequence of amino acids, which creates unique structural properties. Their potential as drugs is supported by existing natural products and their synthetic analogues, like vancomycin and cyclosporine2–4. The proven success of these therapeutics has stimulated interest in the discovery of new cyclic peptide drugs using advanced methods. Historically, the discovery of cyclic peptide therapeutics has relied on exploring natural product analogues and random high throughput screening2,5,6. However, these methods can be improved to test a more diverse set of cyclic peptide structures and protein targets. Recent advancements in computational protein prediction and design have made in silico screening a powerful tool for drug discovery, boosted by improvements in both physics-based and deep learning approaches. Integrating computational tools with enhanced drug discovery techniques allows for the identification of novel cyclic peptide therapeutics7,8. This strategy represents a shift from random screening to a focused approach that can uncover new structures and interactions.High-throughput screening techniques offer an efficient way to evaluate thousands of compounds for target interaction simultaneously. To match the speed of computationally identified peptide candidates, high-throughput screening is essential for cyclic peptides. One such technique, affinity selection mass spectrometry (ASMS), leverages the affinity between protein and peptide to isolate the peptide from a pool of binding candidates9. The isolated peptides are identified by characteristic fragmentation inside the mass spectrometer. However, for cyclic peptides, this characteristic fragmentation can be complex and difficult to interpret. To improve interpretation, we expanded mass spectrometry data collection with multistage MSn fragmentation and developed computational sequencing to read cyclic peptide fragmentation. This approach enables the computational assignment of unknown cyclic peptide peptide sequences, unlocking the ability to test cyclic peptides for target interaction in high throughput. Furthermore, we adapted ASMS to screen against protein targets in complex environments, including membrane-bound proteins like G-protein coupled receptors (GPCRs). Using virus-like particles to anchor GPCRs in their membranes helped preserve their native environment and allowed affinity selection against properly folded proteins. Adapting ASMS in this way opens new possibilities for discovering cyclic peptide drugs against membrane-bound proteins—a longstanding barrier in the field. Beyond discovery, high-throughput mass spectrometry approaches like HDX-MS (Hydrogen-Deuterium Exchange Mass Spectrometry) allow for the study of structural dynamics in cyclic peptides. HDX-MS is a benchtop labeling experiment that, when paired with mass spectrometry, provides insights on conformational dynamics and solvent accessibility. Applying HDX-MS to cyclic peptide pools enabled us to gain structural insights into dozens of peptides simultaneously. The data generated revealed relationships between conformational flexibility and permeability, helping identify peptide scaffolds for designing membrane-traversing peptides. Overall, these high throughput approaches significantly enhance our capability to identify and assess cyclic peptides as therapeutic candidates.
Elucidation of the Role of VraTSR and Lipid Metabolism in the Development of Resistance Phenotypes in Staphylococcus aureus
(2025-01-23) Shen, Tianwei; Xu, Libin
Staphylococcus aureus is a gram-positive bacterium, which has developed resistance to many antimicrobials. Methicillin-resistant S. aureus (MRSA) was first isolated in 1961. Since then, glycopeptides (e.g. vancomycin), lipopeptides (e.g. daptomycin), and long-acting lipoglycopeptides (e.g. dalbavancin) have been developed. MRSA, however, has developed resistance against all three types of antimicrobials over time. VraSR is one two-component system (TCS) out of the 16 prototypical TCSs in S. aureus, which is activated in response to cell-envelope-targeting antimicrobials, such as vancomycin and β-lactams. A third component, VraT, has been shown to be essential for VraSR full activation, making it a three-component regulatory system VraTSR. The molecular inducer of VraTSR has been proposed to be the inhibition of transglycosylation in the peptidoglycan layer, and hundreds of genes have been shown to be in the VraR regulon, including those related to cell wall synthesis. On the other hand, cell wall synthesis has been hypothesized to crosstalk with cell membrane metabolism via three potential ways: lipoteichoic acids (LTAs), acetyl-CoA, and lipid II. In fact, overall decreased levels of lipid abundance have been observed in multiple S. aureus strains that have developed resistance against vancomycin, daptomycin, or dalbavancin, some of which harbor mutations in VraTSR along with other mutations. None of the strains, nevertheless, have mutations only in VraTSR until we isolated S7-D2 from the parent S7 strain by serial passage against dalbavancin. S7-D2 has a non-synonymous mutation in vraT (c. 377C>T; p. P126L) compared to S7. We hypothesized this mutation to be gain-of-function. By using this strain pair and vraTSR loss-of-function mutants, we aim to elucidate the contribution of vraTSR to the remodeling of the cell envelope and the modulation of antimicrobial susceptibility. Chapter 1 provides background on S. aureus and bacterial TCSs and their relation to antimicrobial susceptibility. In Chapter 2, we applied a multi-omics methodology (transcriptomics, metabolomics, and lipidomics), along with various phenotypic characterization, to assess the role of VraTSR. We found that the loss-of-function mutations in VraTSR resulted in a general increase in antimicrobial susceptibility and that an increase in only limited lipid species was unexpectedly observed. On the contrary, the gain-of-function mutant S7-D2 (confirmed by transcriptomics) exhibited characteristic decreased levels of lipids. We also showed from the multi-omics studies several aspects that could have implications in resistance modulation, e.g., decreased membrane fluidity and upregulated arginine deiminase pathway and betaine biosynthesis pathway, and in crosstalk between cell wall and cell membrane, e.g. LTAs and acetyl-CoA. We proposed several future experiments to follow up on the observations from the multi-omics studies. From a practical standpoint, inhibiting the lipid synthesis with AFN-1252 and the VraTSR with histidine kinase inhibitors appear promising in modulating resistance to cell-envelope-targeting antimicrobials, and more studies are warranted. The agr system is another TCS in S. aureus that plays critical roles in quorum sensing and virulence. The phenol-soluble modulins (PSMs) produced from Agr and released by S. aureus have been suggested to antagonize the lipid shedding mechanism of daptomycin inactivation by binding to the released lipids. Others have shown that S. aureus survival in the presence of daptomycin is enhanced with loss of the Agr function. In Chapter 3, we examined several pairs of S. aureus strains with dysfunctional Agr (KO or mutants) for their survival with exposure to daptomycin and their lipid profile (released and membrane lipids) in static time-kill experiments and in vitro pharmacokinetic/pharmacodynamic (PK/PD) modeling. We found that the contribution of dysfunctional Agr to enhanced survival varied depending on the genetic background or the type of mutations and that the enhanced survival did not correlate with the released lipids. We proposed that PSMs might not be the only molecules released by S. aureus that contributed to the antagonizing effects. Studying other released factors might help shed light on the variations among the Agr dysfunctional mutants in terms of enhanced survival against daptomycin. Chapter 4 summarizes the overall findings and proposes future directions.
Structural Dynamic Insights into Coronavirus Spike Conformational States, Receptor Activation, and Vaccines
(2024-10-16) Chen, Chengbo; Lee, Kelly
Viruses in the Sarbecovirus subgenus have given rise to two highly transmissible coronaviruses in recent human history: severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2. These viruses enter human cells through the binding between viral spike (S) glycoprotein and a common human angiotensin-converting enzyme 2 (hACE2) receptor. However, they exhibit differences in interactions with hACE2 as well as in proteolytic processing of S that trigger the fusion machinery. Identifying the molecular basis of how these differences impact S activation as well as the effects of mutations found in novel SARS-CoV-2 variants of concern (VOCs) is key to understand S function and viral pathogenesis phenotypes. The hypothesis I am testing is that differences in structural and conformational dynamics in SARS-CoV and SARS-CoV-2 spike trimers influence their ability to bind and be activated by the hACE2 receptor. To probe the structural and dynamic differences among SARS-CoV, SARS-CoV-2 and VOCs that exhibit different transmissibility, we perform hydrogen/deuterium-exchange mass spectrometry (HDX-MS), which measures protein dynamics under native conditions. HDX-MS reveals differences in spike dynamics at various levels, which will be discussed in three chapters with specific focus. In Chapter 2, HDX-MS reveals differences in dynamics of unbound S, featuring the D614G mutation-induced S conformational switch to open states and S stability. This open conformation, involving the receptor-binding domain (RBD) in the up conformation, is impaired when its N-glycosylation at position 343 is knocked down, indicating that RBD dynamics are influenced by glycan-facilitated neighboring N-terminal domain (NTD)-RBD crosstalk. In Chapter 3, we discover that hACE2 binding leads to more prominent dynamic behaviors reflecting hACE2-induced S activation. Notable differences in transduction of allosteric changes are observed, extending from the RBD to regions proximal to proteolytic cleavage sites, suggesting that the highly dynamic fusion peptide region in SARS-CoV-2 S can confer an advantage in fusion. In Chapter 4, we investigate both S conformational dynamics and local structural ordering with a focus on mosaic spike heterotrimers mimicking possible antigenic assemblies from bivalent mRNA vaccination. Both trimer stability and antigenicity are well-conserved in the mosaic trimer formation we study. The mosaic trimer co-expressed form Omicron and Hu-1, resembling the S sequences used in mRNA vaccines, also shows prominent dynamic changes in the fusion peptide proximal region. These results provide mechanistic insights into receptor-induced S activation. In such a highly dynamic Class I fusion machine, critical variations in amino acid sequences or post-translational modifications can significantly trigger allosteric effects through dynamic motions and interactions between domains, further impacting their transmission phenotypes and viral fitness.
Elucidating the Role of Two Inotropic Agents in Cardiac Hypertrophy: the Ceramides and Apelins
(2024-10-16) Wiley, Alexandra M.; Totah, Rheem A.
The steady increase in prevalence of cardiovascular diseases (CVDs) globally, necessitatesthe urgent identification of novel druggable targets to reduce the development and progression of CVD. The inotropic agents, the ceramides and apelins, represent two potential druggable targets that influence cardiovascular function. In this dissertation, we examined the impact of silencing the ceramide synthases (CERS) responsible for the production of the putative protective very long-chain (VLC) ceramides 22:0 and 24:0 (CERS2) and detrimental long-chain (LC) 16:0 ceramide (CERS5/6) on cardiomyocyte health and induced hypertrophy. In this work, we demonstrate that the VLC and LC ceramides play contrasting roles in cardiomyocyte health. The silencing of CERS2 in healthy cardiomyocytes leads to pathway changes indicative of a decline in cardiomyocyte health, while CERS2 knockdown (KD) in cardiomyocytes subjected to hypertrophic conditions displayed an exacerbated response. Interestingly, transcriptional changes in healthy cardiomyocytes with silenced CERS5/6 resulted in pathway changes suggesting improved cardiomyocyte health, while the hypertrophic response in cardiomyocytes with CERS5/6 KD appeared tempered compared to controls. These findings suggest that VLC ceramides may be protective against CVD progression, while LC ceramides may contribute to the progression of CVD. Additionally, we probed how the different components of the apelinergic system (AS) affect both healthy cardiomyocytes and cellular hypertrophy progression. Here we show that both the apelin receptor (APJ) and apelin are necessary to elicit a protective response against cardiac hypertrophy, as well as identify many hypertrophic response changes due to the addition of the various AS components. To conclude this project, we present the development of a potential technique to enable the high-throughput screening of compounds against GPCR targets, such as APJ. We discuss the utilization of virus-like particles (VLPs) overexpressing APJ and demonstrate the ability to enrich the binding of positive control apelin, in both simple and complex mixtures containing a mock screening library with over 2500 peptides. The work in this thesis provides mechanistic insight into the impact the ceramides and apelins have on the progression of hypertrophy and proposes a unique technique that can be utilized to more efficiently generate future therapeutics targeting APJ and other GPCRs.
Temporal and Spatial Multi-omics Characterization of Smith-Lemli-Opitz Syndrome
(2024-02-12) Li, Amy; Xu, Libin
Smith-Lemli-Opitz syndrome (SLOS) is a metabolic disorder caused by genetic mutations in the DHCR7 gene, leading to defective 3β-hydroxysterol-Δ7-reductase (DHCR7), the enzyme that catalyzes the last step of cholesterol synthesis. The resulting deficiency in cholesterol and accumulation of its precursor, 7-dehydrocholesterol (7-DHC), have a profound impact on brain development, manifesting as developmental delay, cognitive impairment, moderate-to-severe intellectual disability, and behavioral deficits. Additionally, disorder severity and phenotype vary widely between individuals. Anomalies of the CNS are frequently observed in patients, with microcephaly, ventricular dilatation, and malformations of the corpus callosum and cerebellum being some of the most common. However, therapeutic interventions for the neurological aspects of SLOS are lacking. Further neurophysiological studies on SLOS mouse models are needed to understand how the loss of Dhcr7 and disruption of cholesterol biosynthesis affect the course of neurodevelopment. The objective of this dissertation is to establish the spatial and temporal lipidomic and transcriptomic changes that occur during development in a Dhcr7-KO mouse model of SLOS. Chapter 1 provides background on the history, genetic cause, and clinical phenotype of the disorder. Chapter 2 presents the results from an untargeted lipidomics analysis of developing mouse brains in wild-type (WT) and Dhcr7-KO mice. We compared relative lipid levels throughout development, from embryonic day 12.5 to postnatal day 0. The study identified differentially expressed brain lipids between WT and Dhcr7-KO mice at specific developmental time points, revealing lipid metabolic pathways that are affected in SLOS beyond the cholesterol biosynthesis pathway, such as glycerolipid, glycerophospholipid, and sphingolipid metabolism. In Chapter 3, we mapped the spatial distribution of sterols and lipids in neonatal Dhcr7-KO mouse brains using mass spectrometry imaging. We determined the sites of accumulation of sterols and major oxysterol metabolites of 7-DHC and identified differentially expressed lipids between WT and Dhcr7-KO brains. Chapter 4 presents a complementary transcriptomics analysis to Chapter 2, where we carried out RNA sequencing analysis of whole brains from WT and Dhcr7-KO mice at the same four time points spanning development. We discuss the impact of these gene expression changes and affected signaling pathways as they relate to the neurological phenotype of SLOS. In Chapter 5, we summarize the overall project findings and directions for future research.
Biochemical Characterization of Drug Metabolizing Alkyl Thiol Methyltransferase 1A and 1B
(2024-02-12) Russell, Drake Anthony; Totah, Rheem A
In humans, S-methylation of aromatic and alkyl thiols is catalyzed by thiopurine methyltransferase (TPMT), and a putative enzyme(s) thought to be responsible for alkyl thiol methyltransferase (TMT) activity. In this work, we identified METTL7A along with METTL7B as the enzymes responsible for TMT activity and renamed them thiol methyltransferase 1A and 1B (TMT1A and TMT1B), respectively. TMT1A and TMT1B exhibited many characteristics ascribed to historic TMT activity, including similar molecular weight, microsomal association, and broad substrate specificity for several alkyl thiol containing drugs, including spironolactone, captopril, and mertansine. Interestingly, TMT1A but not TMT1B is potently inhibited by a historic probe inhibitor of TMT activity, 2,3-dichloro-α-methylbenzylamine (DCMB). Utilizing DCMB to probe microsomal TMT activity suggests that TMT1A, not TMT1B, is responsible for most alkyl thiol methylation activity. To explain the difference in DCMB susceptibility, we compared the sequences of TMT1A and TMT1B and used homology models to identify key residues in the binding site. We then used site-directed-mutagenesis, to pinpoint a key tyrosine residue at position 47 (Tyr-47) in the binding site of TMT1A that was critical for DMCB inhibition. Mutating Tyr-47 to the corresponding Ser-47 in TMT1B abolished DCMB inhibition and confirmed the importance of Tyr-47 for DCMB binding and inhibition. Finally, we identified hydrogen sulfide (H2S) as a potential endogenous substrate of TMT1A and TMT1B and showed that the metabolite methyl sulfide (MeSH), similar to H2S, can efficiently adduct and sulfmethylate protein cysteines. To determine the physiological relevance of sulfmethylation, we developed a novel proteomics method to detect potential proteins adducted by MeSH. This protein adductomics method may help elucidate the endogenous role of MeSH in signaling and cell homeostasis.
Defining the Structural Features and Functional Characteristics of Complement-Active IgM
(2023-08-14) Watson, Michael Jeremy; Guttman, Miklos
Immunoglobulin M (IgM) is an extremely important, yet surprisingly under-studied glycoprotein that is shared among all vertebrates on an evolutionary timeline spanning more than 500 million years. In addition to the genetically conserved subset of ‘natural’ IgM that conveys an innate degree of protection before birth, the adaptive immune system also produces ‘induced’ IgM as the initial response to all foreign pathogens. IgM is the most potent known activator of the classical complement cascade, making it an attractive therapeutic platform for the potential treatment of a wide array of disease states; while multiple IgG molecules must bind with coordination of their Fc regions together at the membrane of a pathogenic target, a single molecule of IgM is sufficient for the full-scale activation of complement, owing to the multivalency of its secreted pentameric and hexameric isoforms. Although the relationship between IgM and complement has been appreciated for decades, the biophysical details that underlie its activation mechanism still remain largely unknown. Many past attempts have been made to uncover the structural details surrounding the IgM-mediated activation of complement, but the same physical traits that give IgM its unique properties and relationship with the immune system have also earned it a notorious reputation for being technically difficult to handle and investigate experimentally. The large size, extensive glycosylation, and high degree of flexibility has rendered IgM largely refractive to many established structural techniques, and to date no fully intact IgM structures have been resolved; all structural characterizations and protein:protein interfaces have so far been inferred from combinations of fragmented structures, homology modeling, electron microscopy, X-ray scattering, and molecular dynamics simulations. While a number of current IgM drug candidates have shown promise for the treatment of several different cancers, the biophysical details of IgM-mediated complement activation still remain poorly understood. The objective of this dissertation is to establish a baseline set of structural features and functional characteristics that define the complement-active, antigen bound form of IgM – commonly referred to as the ‘staple’ conformation – and differentiate it further from other sub-active and inactive forms. Chapter 1 provides a historical review and outlines the contextual background that currently surrounds some of the major gaps in knowledge regarding the structure and function of IgM as it relates to the activation of complement. In Chapter 2, background work is presented on the development of a low-cost, automated LC-MS system designed to improve both the throughput and consistency of decoupled hydrogen/deuterium exchange mass spectrometry (HDX-MS) samples, which is the primary structural technique used to assess the defining features of the staple conformation. In Chapter 3, the functional aspects of complement-active IgM are explored through a combination of novel ligand binding and activity assay approaches, revealing a number of key variables that directly impact the binding kinetics and activation rates of the initiating molecules of the classical cascade pathway, C1q and C1. The mechanistic role of IgM binding valency/avidity was also investigated through a prebinding approach that, to our surprise, indicated there to be no hard stoichiometric cutoff for the number of surface bound arms of IgM required to bind/activate C1q/C1. The functional characteristics determined in Chapter 3 pair directly with the structural features found in Chapter 4 (via HDX-MS) in order to complete the defining structure/function relationship that underlies the active staple conformation of IgM. Strong evidence of a mechanistic hinge, compensatory rearrangement of domains within the Fc core, and the identity of previously unreported residues that may comprise a portion of the C1q binding site (in addition to the longstanding putative location) were discovered within the multivalent surface bound form of complement-active IgM. Additionally, the existence of an unexpectedly extensive network of allosterism was observed within solution bound IgM that could add much needed clarity to the litany of confusing reports found throughout the classical literature. Finally, in Chapter 5, a brief project conclusion is provided along with a discussion of future directions. Collectively, this work establishes key structural and functional qualities that define the complement-active form of IgM, along with a number of newly developed methods, improved protocols, and a foundation for IgM structural work upon which future research can be built.
Advancing Hydrogen Deuterium Exchange Mass Spectrometry Through the development of Novel Internal Exchange Reporters
(2023-08-14) Murphree, Taylor Aiken; Guttman, Miklos; Maly, Dustin
Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful tool for protein structure analysis. The remarkable versatility and sensitivity of HDX-MS renders the technique uniquely well suited to probing challenging biological systems. Over the last two decades advancements in mass spectrometers, sample handling techniques, and software tools have brought HDX-MS into the mainstream by dramatically improving data quality. Despite this renaissance, HDX-MS measurements remain very challenging to reproduce. The strong solution dependence of the chemical process underlying amide HDX contributes significantly to irreproducibility; even small variations in temperature or pH can have a pronounced effect on the deuterium uptake by a protein. As a result, a myriad of controls have been developed to help the investigator identify and address variations in solution conditions. Among these controls are compounds which directly monitor the conditions under which amide exchange takes place. These compounds, referred to as internal exchange reporters (IERs), have been the focus of my graduate work in the Guttman Lab at the University of Washington. When my work in the area began, the concept of reporting controls had been well established. However, the reporters themselves, small unstructured peptides, were of limited utility. Under the guidance of Dr. Miklos Guttman and with the assistance of many brilliant and rather patient friends and colleagues I have been able to develop novel imidazolium based IERs. This document will describe the design, validation, and subsequent refinement of these compounds through the discussion of previously published and ongoing work.
Investigating mechanisms of neutralization of staph enterotoxin B
(2023-08-14) Vorauer, Clint; Guttman, Miklos
Staphylococcal enterotoxin B (SEB) is a small, secreted protein that causes food poisoning-like symptoms and can be lethal in microgram amounts. SEB elicits a profound immune response via the bridging of major histocompatibility complex class II (MHCII) molecules on antigen-presenting cells with T-cell receptors (TCR). Several monoclonal antibodies (mAbs) have been identified as capable of neutralizing SEB. Using structural mass spectrometry, the epitopes of several neutralizing antibodies were successfully mapped, and allosteric as well as synergistic effects that contribute to the efficacy of select mAbs were determined. Having established a robust approach for tracking mAb-SEB interactions, we examined the interactions within raw, polyclonal sera for understanding antibody responses to SEB and their resulting protective efficacy. This dissertation provides mechanistic insight into the neutralization of SEB and highlights technical advances to the Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS) method.
Bridging HIV neutralization sensitivities and Env dynamic phenotypes across genetically diverse variants
(2023-08-14) Hodge, Edgar Ashmun; Lee, Kelly K
The envelope glycoprotein (Env) is the sole target for neutralizing antibodies against HIV and the most rapidly evolving, variable part of the virus. High-resolution structures of Env trimers captured in the prefusion, closed conformation have revealed a high degree of structural similarity across diverse isolates. Biophysical data, however, indicate that Env is highly dynamic, and the level of dynamics and conformational sampling are believed to vary dramatically between HIV isolates. Dynamic differences likely influence neutralization sensitivity, receptor activation, and overall trimer stability. The primary objective of this dissertation is to understand how sequence diversity impacts dynamics, and better understand how differences in dynamics impact antibody recognition. After briefly introducing the HIV Env fusion protein, chapter 1 will review one of the primary biophysical techniques I use throughout this dissertation to characterize dynamics-- hydrogen-deuterium exchange mass spectrometry (HDX-MS) -- and highlight recent advances pertaining to its use in structural biology. In chapter 2 I use HDX-MS to map local dynamics across five native-like Env trimers from diverse isolates selected for their high yields, stability, and ability to be purified to a high degree of purity. We demonstrate that significant differences in epitope structural ordering are observed across most sites targeted by broadly neutralizing antibodies (bnAbs). We also observe isolate-dependent conformational switching that occurs over a broad range of timescales. Lastly, we report that hyper-stabilizing mutations that dampen dynamics in some isolates have little effect in others. In chapter 3 I show that variation in structural ordering in the V1/V2 apex of Env across a widely used panel of HIV-1 isolates is a major source of isolate-specific antigen variation that results in a marked effect on antibody association rates. With help from collaborators, we also report cryo-EM reconstructions of the apex-targeting PGT145 bnAb bound to two divergent Env that exhibit dramatically different degrees of structural ordering throughout much of the trimer structure. Parallel HDX-MS experiments demonstrate that the PGT145 bnAb has an exquisitely focused binding footprint at the apex and is unaffected by local and global structural fluctuations throughout the trimers. In chapter 4 we extend our characterization of Env dynamic phenotypes across trimers derived from isolates in a global panel that is representative of the vast genetic diversity of HIV-1. These data highlight the impact of extreme sequence diversity on dynamic phenotype, elucidates a structure-dynamic link between local dynamics and neutralization phenotype, and provides a mechanistic explanation for IgG recognition of Env trimers.
Lipid Peroxidation Mechanisms and Their Contribution to Ferroptosis
(2023-01-21) Do, Quynh; Xu, Libin
The attack of reactive oxygen species (ROS) on reactive lipids leads to free radical chain reactions with molecular oxygen, a process termed lipid peroxidation. Increased lipid peroxidation is associated with many human diseases, including cancer, diabetes, neurodegenerative diseases, and, more recently, cell death. The rate-determining step in lipid peroxidation reactions is usually the propagation step in which the peroxyl radical can undergo either hydrogen(H)-atom transfer or peroxyl-radical addition reaction. The rate constants of the H-atom transfer process for several reactive lipids and sterols have been measured using the peroxyl radical clock method. However, no method was available to study the peroxyl-radical addition reaction. In this dissertation, I aim to elucidate lipid peroxidation mechanisms and their relevance in ferroptosis, a form of regulated cell death driven by lipid peroxidation of polyunsaturated fatty acids (PUFAs). In this work, I modified the original linoleate-based peroxyl radical clock to enable the measurement of both the H-atom transfer and peroxyl-radical addition reactions in the propagation step. The new probe was then applied to study lipid peroxidation mechanism and kinetics for a variety of biologically important lipids, including conjugated fatty acids, sterols, coenzyme Q10, and lipophilic vitamins, such as vitamin D3 and A, for the first time. Next, I elucidated the effects of various unsaturated lipids in sensitizing ferroptosis. I then sought to elucidate the mechanism underlying the potency of the different ferroptosis-inducing conjugated and nonconjugated PUFAs. Next, I investigated the relevance of ER stress and unfolded protein response in ferroptosis execution. Finally, I summarize all the findings of this collective work and propose how research in this area can continue to expand.

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