Saint Louis University

NIH Research Projects · FY 2025 · 2020-12

Hepatitis B virus (HBV) is a hepatotropic DNA virus that replicates by reverse transcription. It chronically infects >250 million people worldwide and kills ~870,000 annually. Therapy primarily employs nucleos(t)ide analog drugs against viral DNA synthesis that often drive viremia below the detection limit. However, replication is not eliminated, and HBV resurges if drugs are withdrawn. Nevertheless, treatment cures up to 6% of patients, so more patients could be cured by suppressing HBV further. Reverse transcription requires the viral ribonuclease H (RNaseH) that destroys the RNA after it has been copied into DNA. Blocking the RNaseH prevents synthesis of viral genomes, including both the key nuclear cccDNA form of the genome and the DNA in virions. Drugs have not yet been designed against the RNaseH. We developed the first screening pipeline for HBV RNaseH inhibitors and found RNaseH >150 inhibitors that block HBV replication. The most effective is an N-hydroxypyridinedione (HPD) with an EC50 of 0.11 μM and a therapeutic index (TI, CC50/EC50) of 300. An HPD hit compound suppressed HBV viremia in mice with humanized livers. We also found that napthyridinones [NPTH, comprised of true napthyridinones (HNOs) and the closely related hydroxypyridopyrimidinones (HPPs)] inhibit HBV replication with EC50s as low as 0.95 μM and TIs up to 83. Achieving EC50s as low as 0.11 μM and TIs up to 350 after assessing only 51 HPDs, combined with good activity of the related NPTH chemotype, led Drs. Tavis (HBV virologist), Meyers, Zhan, and Zoidis and (medicinal chemists) to join forces to develop the HPDs and NPTHs into pre-clinical leads for novel HBV drugs. Aim 1. Lead optimization of HPD HBV inhibitors. We will synthesize up to 125 HPDs based on our existing structure-activity relationships (SAR) and evaluate their efficacy, cytotoxicity, and solubility. Aim 2. Hit-to-lead development of NPTH HBV inhibitors. We will synthesize up to 100 HNOs and 100 HPPs and evaluate their efficacy, cytotoxicity, and solubility. Aim 3. Assess specificity for HBV of the novel inhibitors. We will evaluate HPDs and NPTHs for induction of heteroduplex formation in HBV capsids and suppression of cccDNA formation. Synergy with other HBV drugs will be assessed. Selectivity will be measured against microbial pathogens and human metalloenzymes including RNaseH1. Aim 4. Evaluate pharmacological parameters for HPD and NPTH HBV inhibitors. We will assess stability, cellular permeability, plasma protein binding, pharmacokinetics, and toxicology of key inhibitors. Efficacy of the best compounds will be tested against HBV replication in HBV-infected mice carrying humanized livers. These studies will advanced HBV RNaseH inhibitors as first-in-mechanism and first-in-class HBV drug leads. The eventual anti-HBV RNaseH drugs are anticipated to be used in combination with nucleos(t)ide analogs to suppress HBV replication enough to clear HBV in many more patients than current therapies achieve.

NIH Research Projects · FY 2024 · 2020-09

Mycobacterium tuberculosis (Mtb) infects one-third of the world’s population and causes almost 1.3 million deaths per year, including 100, 000 children. Approximately 90% of infected persons have latent tuberculosis infection (LTBI), have protective immunity and remain well, but 10% develop primary tuberculosis (TB) soon after infection or reactivation TB many years later. Children are more susceptible to TB infection, due to an immature immune system. HIV infection in children markedly increases susceptibility to TB, and HIV-infected persons with LTBI have an 800-fold greater risk of developing active TB (www.cdc.gov/tb/). TB is the leading cause of death in HIV-infected persons and more than half a million coinfected people die annually. To develop adequate prophylaxis or therapy, it is important to understand immune responses to Mtb. Identification of HIV+ children with LTBI who are at greatly increased risk for development of TB would allow treating only high-risk children, facilitating completion of therapy for LTBI and preventing future development of TB. To identify these children, it is important to pinpoint the nature of the defective immune responses that permit development of active TB in HIV+LTBI+ pediatric patients. Over the past 18 years, we have published a series of articles demonstrating that human NK cells have the potential to contribute to both innate and adaptive immune responses to Mtb. Our recently published studies demonstrate that memory-like NK cells contribute to vaccine-induced protective immunity against Mtb and IL-21 is required for expansion of memory-like NK cells in both humans and mice. Based on our published studies, we hypothesize that HIV-LTBI+ children household contacts have defective memory-like NK cell expansion compared to HIV-LTBI+ adult household contacts and these defects are more severe in HIV+ LTBI+ children. The proposed studies in the current application will be performed in India as a part of RePORT-India consortium. This study will leverage the large Indo-US investment and TB/HIV research consortium of RePORT-India which has developed cohorts of TB cases and household contacts in India and has paired Indian investigators with US investigators at 6 sites. Already collected samples will be used for the proposed studies in aim 1. Our specific aims are: 1. Determine Mtb specific memory-like NK cell responses of children in a large group of household contacts of TB patients. 2.Compare the memory-like NK cell responses of HIV+ and HIV- children with LTBI. 3. Determine whether KIR haplotypes and HLA polymorphism is associated with expansion of memory-like NK cells in children.

NIH Research Projects · FY 2026 · 2019-12

Saint Louis University (SLU) seeks to expand its service to the Vaccine and Treatment Evaluation Unit (VTEU) network providing resources and expertise to help the network achieve its objectives of evaluating vaccines, preventive biologics, therapeutics and diagnostics for infectious diseases. Dr. Daniel Hoft, SLU VTEU PI, also serves as Co-PI for a VTEU Leadership Group (LG) application supported by all current VTEUs; however, regardless of who leads the LG, SLU will provide full support to promote outstanding VTEU research. Outstanding infrastructure for phase I-IV vaccine & therapeutic trials against priority pathogens: SLU has served as a VTEU for 29 years, conducted hundreds of phase I-IV trials in healthy and special populations of all ages, and can provide unique knowledge and extensive infrastructure to support VTEU network goals. Diverse knowledge scientific skills for trial design priority areas: SLU investigators include experts in vaccinology, immunology, seasonal and pandemic influenza, tuberculosis, biodefense, urgent/emergent pandemic trials, liver/enteric diseases, sexually transmitted infections, malaria/neglected tropical diseases, epidemiology and arboviral diseases. We also provide state-of-the art multi-platform omics core expertise. Urgent national preparedness trials: SLU has led urgent trials of vaccines against potential bioweapons (smallpox, anthrax, plague and tularemia), and emergent diseases (2009 H1N1 pandemic flu, avian H5 and H7 flu and Zika/Yellow Fever). In addition to streamlined institutional review and contracting, SLU can contribute containment facilities for select agent work and inpatient human challenge. Controlled Human Infection Models: SLU has developed the capacity for human challenge models to study influenza, parainfluenza, vaccinia, salmonella and tuberculosis immunity. We provide a 23-bed airborne containment facility for challenge studies with wild type GMP influenza strains, and have developed an active collaboration with SGS to standardize challenge protocols and obtain influenza challenge strains. Expertise in First-in-Human, investigator-initiated and pharmacokinetic/pharmacodynamic studies: SLU has completed dozens of first-in-human and investigator-initiated trials of vaccines against influenza, tuberculosis, HCV, enteric pathogens, potential bioweapons and emerging flaviviruses. Experimental biology studies of mucosal and systemic immunity with human samples of blood, tissue and mucosal samples have identified biomarkers and targets for iterative influenza and tuberculosis vaccine development. Sexually transmitted infection (STI) expertise: SLU's 10 years in the HIV Vaccine Trials Network provided SLU with expertise in STI. SLU also led a 44-site herpes vaccine efficacy trial (Herpevac), and two of the highest enrolling Herpevac sites have agreed to serve as Protocol-Specific Sites to expand our STI expertise. SLU ID follows ~450 HIV patients and closely collaborates with several molecular virologists. SLU looks forward to providing its expertise and capacities to meet the new ID challenges of the future.

NIH Research Projects · FY 2025 · 2019-06

Abstract Work under previous support has broadened our understanding of the factors that influence the catalytic activity of thrombin and set the stage for a structure-based characterization of how this enzyme interacts with physiological substrates. Unraveling the architecture of factors involved in blood coagulation remains a challenging task because of the difficulty of obtaining high resolution structures for proteins containing multiple domains. We have recently shown how to address this challenge by using cryo-EM, the new gold standard for the structural investigation of biological macromolecules. The proposed research project is a segue to our pioneering cryo-EM structural work on human coagulation factors V and Va free and bound to factor Xa in the prothrombinase complex and the structure of this complex bound to prothrombin. Specifically, we plan to solve the cryo-EM structures of factor V in complex with thrombin (aim 1) and its active precursor meizothrombin (aim 2) with the goal of revealing the molecular basis of a key step of the initiation phase of the coagulation response that leads to assembly of the prothrombinase complex. The proposal is supported by exciting preliminary cryo- EM maps currently refined at 6.6 Å resolution for the thrombin-factor V complex (aim 1) and 3.8 Å resolution for the meizothrombin-fV complex (aim 2). Once fully refined, these structures will reveal the distinct modes of binding of thrombin and meizothrombin at preferred sites of activation and the full architecture of meizothrombin for the first time. Underlying epitopes will be validated independently by mutagenesis of specific residues of thrombin, meizothrombin and factor V. In addition, the structures will test the hypothesis that binding of thrombin and meizothrombin to factor V rigidifies the disordered B domain and visualizes the structural determinants that keep factor V in its inactive state. Success of the proposed studies will significantly advance our basic knowledge on factor V and its activation in ways that are directly relevant to other multidomain proteins in the blood coagulation cascade and that may benefit the development of new therapeutic strategies.

NIH Research Projects · FY 2025 · 2018-06

Abstract The proposed research project continues and expands our investigation of the interaction of thrombin with the anticoagulant protein C responsible for a key feedback regulation of the coagulation response. The project addresses unresolved issues in the field using an innovative structural approach and plans to fill existing gaps in basic knowledge about protein C as a substrate of the thrombin-thrombomodulin complex and activated protein C as an enzyme that inactivates factor Va. Unraveling the architecture of multidomain factors involved in blood coagulation, complement and fibrinolysis remains a challenging task because of the difficulty of obtaining high resolution structures. This limitation is even more acute when considering complexes involving these factors and their macromolecular substrates or activators. Our approach addresses this challenge directly with cryo-EM, the new gold standard for the structural investigation of biological macromolecules. Building on our recent success in solving the structures of human coagulation factors V and Va, the proposed research project plans to revolutionize the structural enzymology of protein C in a way that is relevant to other multidomain proteins and their complexes in the blood coagulation cascade. Toward this end, we have obtained preliminary cryo-EM structures of protein C free and bound to the thrombin-thrombomodulin complex. Once fully refined as planned under aim 1, these unprecedented structures will unravel the mechanism of protein C activation and test the hypothesis that thrombomodulin promotes the interaction of thrombin with protein C by offering a scaffold that changes their conformation and alleviates electrostatic clash. In addition, we have obtained a preliminary cryo- EM structure of activated protein C free and prepared stable particles of activated protein C bound to factor Va and protein S for cryo-EM data acquisition. Progress from these studies will elucidate how the structure of activated protein C compares to that of its zymogen form and will further refine the mechanism of protein C activation. Furthermore, direct information on the epitopes of recognition of factor Va will enable a structure- based engineering of variants of activated protein C with altered specificity for potential therapeutic applications.

NIH Research Projects · FY 2026 · 2015-02

Abstract Head and neck cancer (HNC) is the sixth most prevalent cancer in the world, and oral cancer is the most common subtype. The World Health Organization estimates ~330,000 deaths per year globally. The overall survival rate (50-60%) has not improved over the past couple of decades, despite significant improvements in surgical procedure, radiotherapy, and chemotherapy. The risk factors associated with oral cancer includes tobacco use, excessive alcohol consumption, betel quid chewing, and human papillomavirus (HPV) infection. HPV infection is likely to be reduced in the future due to successful vaccination and better prognosis. There is a critical need to define the HNC disease processes, and to identify better therapeutic strategies for successful HNC patient management. Further, non-traditional therapies must be investigated as adjuncts to reduce the risk of recurrence and improve survival. In our current funding period, we made several important novel observations. We showed that bitter melon extract feeding alters c-Met signaling and prevents HNC growth in preclinical models. We subsequently showed an immunomodulatory role of bitter melon extract, although the precise mechanism is yet to be determined. Further, we found several long non-coding RNAs are altered in feeding of bitter melon extract in a carcinogen-induced model. Very recently, we identified momordicine-I, one of the active metabolites of bitter melon, which shows therapeutic anti-tumor activity in an oral cancer preclinical model. Although we gained much evidence for bitter melon in prevention of HNC from our and other studies, considerable knowledge gaps remain in understanding the mechanism of momordicine-I on tumor metabolism and immune regulation, and its therapeutic use. Based on our and others results, we hypothesize that combining momordicine-I with other current therapeutics will improve efficacy in tumor regression. We will also examine the changes in glucose and lipid metabolism and the tumor microenvironment after treatment with momordicine-I using a mouse model. Results from this study will have important translational implications and significant benefits along with current therapy. Innovation: Our study will examine for the first time the therapeutic and mechanistic effects of momordicine-I in HNC animal models for major translational impact.

NIH Research Projects · FY 2026 · 2012-09

ABSTRACT The overall goal of our proposal is to investigate how iron metabolism and erythropoietic activity are interrelated, and the mechanisms whereby dysregulation in one contributes to pathophysiology in the other. Roles for the two transferrin receptors in these processes is clear; however, little is understood regarding the signaling properties of their ligand, transferrin. Transferrin has long been recognized to deliver iron by binding to receptor, transferrin receptor 1 (TFR1), present on all cells. More recently, a homologous receptor, TFR2, has been identified almost exclusively on bone marrow and liver cells where it serves as a sensor of iron supply and a regulator of erythropoiesis. We generated mice in which one or other of the two iron binding sites (N and C) was mutated, and unable to take in iron. We observed striking differences in the regulation of iron metabolism and erythropoiesis between these mice, demonstrating that each of lobes of transferrin has distinct signaling properties. We present in this proposal new data demonstrating significant improvement in a mouse model of the human disease β-thalassemia when iron is specifically bound to the N but not C lobe. We now devote our attention to the mechanisms by which the two transferrin lobes exert their effects. We also explore the potential of increasing N lobe TF as a potential treatment approach in ineffective erythropoiesis.

NIH Research Projects · FY 2025 · 2010-08

Project Summary While many important RNA sequences have been determined, there is little definitive secondary and three-dimensional (3D) structure information about RNA. Several algorithms predict RNA secondary structure from sequence. However, they are limited by a lack of experimental parameters for non-Watson-Crick regions, the inability to incorporate non-standard nucleotides, and a lack of knowledge about how in vivo-like conditions affect stability. NMR and X-ray crystallography are powerful tools to determine RNA 3D structure but are time- and labor-intensive. With the millions of RNA sequences available, there is a need for reliable, rapid methods to predict secondary and 3D structures of RNA from sequence. Therefore, the broad, long-term objective of the PI’s laboratory is to improve RNA secondary and tertiary structure prediction from sequence. To do so, it is essential to understand RNA thermodynamics and structure. Improved nearest neighbor parameters derived from thermodynamic data can improve secondary structure prediction from sequence. To improve tertiary structure prediction, it would help to know the structural features of secondary structure motifs in solved 3D structures. Therefore, this proposal continues to investigate the thermodynamics and structures of common RNA secondary structure motifs. Specific objectives are: 1) derive improved nearest neighbor parameters to better predict RNA stability and secondary structure from sequence; 2) identify structural patterns in previously solved RNA 3D structures to improve 3D structure prediction. Design and methods include optical melting experiments and free energy parameter derivation, online tool development to annotate and compare 3D structures, and an in-depth analysis of the structural features of secondary structure motifs. This research is relevant to the NIH mission and AREA grant program objectives. An improved method to predict RNA secondary and tertiary structure from sequence is essential to move RNA research forward. Further, the work should impact researchers in any field relying on RNA structure prediction, especially those attempting to understand the structure-function relationship of RNA, understand RNA interactions with other biological molecules, target RNA with therapeutics, and utilize RNA as a therapeutic. As a result, the proposed research will advance the nation’s capacity to protect and improve health, expand knowledge in medical and associated sciences, and benefit students through exposure to and participation in research in the biomedical sciences.

NIH Research Projects · FY 2025 · 2002-05

Abstract This application is for the renewal of the Nonalcoholic Steatohepatitis Clinical Research Network (NASH CRN) clinical center at Saint Louis University and its pediatric component at Baylor University. The NASH Clinical Research Network (NASH CRN) has been sponsored by the NIDDK since 2002 with renewals in 2009, 2014 and 2019. Metabolic dysfunction associated steatotic liver disease (MASLD, previously called NAFLD) affects more than one out of three adults and one out of five children the U.S. MASLD, and especially its most severe subset, metabolic dysfunction associated steatohepatitis (MASH, previously called NASH), may lead to cirrhosis and primary liver cancer resulting in death or liver transplant which contributes to substantial health burdens and costs. The NASH CRN has been ideally and uniquely positioned to impact the growing public health significance of MASH that can only be addressed via a large research consortium. A primary objective of the NASH CRN has been to perform clinical trials of therapeutic agents for MASH in adults and children. A closely linked and high priority secondary objective is to conduct translational research in MASH and MASLD focusing on the pathogenesis that will provide the basis for understanding the natural history and developing better means of diagnosis, prevention, treatment, and clinical management. In this final phase of the NASH CRN, the adult vitamin E dosing trial (VEDS) initiated during the previous funding cycle will be completed. The longitudinal cohort Database-3 study of adults and children with MASLD will be wrapped up with closeout visits in year 1 to provide a unique repository of data and samples from a well-phenotyped cohort that can be used in future studies to prospectively define the natural history of the disease, the cardiovascular and metabolic risk factors, aid in biomarker discovery and validation to identify patients with at-risk MASH (MASH and at least stage 2 fibrosis) and identify factors affecting disease progression. The Saint Louis University Clinical Center of the NASH CRN will work collaboratively with the other clinical centers, SDCC and NIDDK to prioritize and conduct studies of existing datasets over the final three years of funding. Proposed in this application is a project for consideration that focuses on the role of the PNPLA3 I148M variant in sequestering and inactivating a protein called ABHD5 or CGI58. Human variants of this protein are associated with progressive liver disease and suppressed expression of this protein in rodents causes steatohepatitis. The underlying hypothesis is that basis for the PNPLA3 I148M variant contributing to progressive liver disease is its role of ABHD5/CGI-58 sequestration rather than its putative role in lipid droplet triglyceride turnover. Whether this project is pursued or other projects are given higher priority, the Saint Louis University clinical center will fully support the decision of the steering committee with input from the NIDDK and devote its resources to the chosen projects. The NASH CRN has had a major impact on the field and directly advances the mission of the National Institutes of Health to improve the health of the public.

NIH Research Projects · FY 2026 · 1994-12

Abstract Work under previous support has continued to advance basic knowledge in the field of hemostasis and thrombosis with significant contributions to the structural understanding of thrombin dynamics and prothrombin activation. Unraveling the architecture of coagulation factors and their complexes remains a challenging task because of the difficulty of obtaining high resolution structures for proteins containing multiple domains. We have addressed this challenge by being among the pioneers of using cryo-EM in the field of hemostasis and thrombosis. We have solved the structures of factors V, Va, V short and most notably that of the prothrombin- prothrombinase complex that has documented, for the first time, the initial step of prothrombin activation along the meizothrombin pathway. We will build upon the progress made under previous support and address the structural basis of prothrombin activation, that remains an important unresolved issue in the field. Using cryo- EM, we will dissect the entire kinetic scheme that defines how prothrombinase activates prothrombin along the meizothrombin (cleavage at R320 first) and prethrombin-2 (cleavage at R271 first) pathways. We will characterize the interaction of prothrombinase with prothrombin and meizothrombin (aim 1), and solve the structures of prothrombin, meizothrombin, and fXa in the free form (aim 2). These studies will unravel the nature of conformational changes and underlying mechanism leading to assembly of the various complexes in the kinetic scheme of activation. We will test the hypothesis that cleavage at R320 takes place with prothrombin in the collapsed closed form whilst cleavage at R271 requires meizothrombin or prothrombin to switch to the elongated open form. We also hypothesize that prothrombinase cleaves R320 and R271 in two different conformations. The proposal is supported by solid preliminary cryo-EM data of the prothrombin-prothrombinase complex on nanodiscs, the meizothrombin-prothrombinase complex, as well as of prothrombin, meizothrombin, and fXa in the free form. Success of the project will significantly advance basic knowledge on the most important step of the coagulation response and provide a strategy for the study of other interactions of biological relevance to blood physiopathology.