Scripps Research Institute, The

NIH Research Projects · FY 2026 · 2024-05

Project Summary The choroid plexus is a structure that extends from the walls of the brain’s four ventricles, floating in the cerebrospinal fluid (CSF). It is thought to be the source of CSF and thus play a role in controlling the content and volume of the fluid that bathes the brain and spinal cord. However, it is unknown whether the choroid plexus can sense CSF flow or hydrostatic pressure in the ventricles and how it might use this information to modulate CSF production. The combination of CSF, brain, and blood volumes determine intracranial pressure (ICP), and changes in one of these parameters typically leads to compensatory changes in one or both of the other two. In the absence of these compensatory mechanisms, ICP can increase past the normal range, leading to headaches, seizures, neural damage, and in extreme cases, death. Pathological ICP levels occur in several neurological injuries and diseases (traumatic brain injury, stroke, hemorrhage, tumor, hydrocephalus, and during seizures). Thus, understanding the mechanisms underlying ICP sensation and compensation could inform therapeutic strategies for handling dysregulated ICP across multiple neuropathologies. This proposal aims to investigate mechanosensation at the choroid plexus with the overarching goal of understanding ICP dynamics in health and disease. PIEZO1 is a cation channel activated by mechanical stimuli. It is expressed in choroid plexus epithelial cells (CPECs), the cell type thought to be responsible for CSF production, but its role there is entirely unknown. Remarkably, I found that conditional knockout of Piezo1 from CPECs increases seizure susceptibility in mice in the context of kainic acid-induced neuronal hyperactivity. This proposal will test what stimuli activate PIEZO1 in CPECs and how this signal might be used to regulate and stabilize ICP. More specifically, I will use electrophysiology and calcium imaging in primary cell culture and choroid plexus explants to characterize PIEZO1 activity in CPECs. I will also assess ICP dynamics after manipulation of CSF volume and neuronal activity in control mice and those lacking Piezo1 in CPECs. Finally, I will explore the downstream effects of activating PIEZO1 in CPECs, and I will test whether increasing CSF clearance at the choroid plexus might ameliorate seizure severity in the absence of choroid plexus PIEZO1. Together, the results from these experiments will contribute to our understanding of how the choroid plexus senses and regulates ICP. This knowledge could help inform how ICP dysregulation is treated in the context of neurological diseases including stroke and traumatic brain injury.

NIH Research Projects · FY 2025 · 2024-04

Project Summary/Abstract The field of cancer research and treatment has greatly advanced due to breakthroughs in DNA sequencing techniques. These developments have accelerated the identification of dependency genes and paved the way for precision medicine. Many encoded dependency proteins, however, are not known to bind endogenous small molecules and are consequently more difficult to gauge in terms of their potential for targeting by chemical probes. "Binding-first" platforms including Activity-Based Protein Profiling (ABPP) technology have revealed numerous cysteine sites amenable to small-molecule binding. However, it is unclear whether these ligandable sites are important for protein functions and could serve as starting points for small molecule drug development. To address this challenge, I have recently innovated a base editing - ABPP platform that assesses the essentiality of ligandable cysteines in cancer dependency proteins. Notably, I have uncovered several highly ligandable and functionally important sites in dependency proteins, including TOE1. In this proposal, I will utilize biochemistry, sequencing and chemical proteomic methods to 1) dissect the mechanisms of how covalent probes affect TOE1 activity and remodel RNA interactions and 2) explore the consequences of TOE1 inhibition and determinants of sensitivity in cancer. I will also describe plans for continued technology innovation to 3) identify novel functional cysteines in cancer dependency proteins using prime editing. In the long-term, I aspire to create first-in-class therapeutics targeting ligandable cysteines in Strongly Selective cancer dependencies. During the award period, I will improve my communication, writing and scientific skills and learn new lab techniques including eCLIP sequencing and prime editing. The proposed studies will be performed at the Scripps Research Institute (TSRI), a top-ranking institution renowned for its integration of state-of-the-art chemistry and biomedicine research. My mentor, Dr. Benjamin Cravatt, a leading figure in chemical biology and chemical proteomics, and co-mentors Dr. Gene Yeo, a pioneer in RNA-binding protein research, and Dr. David Liu, a leader in the field of genome editing, will provide me invaluable guidance. I will also collaborate with molecular biologist Dr. Lykke-Andersen and medical oncologist Dr. Park at UCSD. Collectively, this research and professional development plan will offer me crucial training during this transitional phase and support me in launching my own laboratory in a research university.

NIH Research Projects · FY 2026 · 2024-03

PROJECT SUMMARY Developing a vaccine that elicits HIV broadly neutralizing antibodies (bnAbs) remains a top priority in HIV vaccine design. However, typically HIV bnAbs originate from rare B-cell germline precursors (‘UCAs’) and require improbable somatic hypermutations (SHM) in HIV+ human repertoires. To reproducibly activate such B-cells, ‘Germline Targeting’ (GT) vaccine priming strategies appear to be useful for activating specific bnAb UCAs particularly in polyclonal systems including rhesus macaques, a key preclinical animal model. We recently developed a novel membrane (m)Env liposome (MEL) platform using mEnvs that contain GT mutations. One novel GT-MEL regimen targets a ‘CDRH3 dominated’ nAb response to the CD4-binding site (CD4bs) and reproducibly elicited tier 2 serum neutralization in CH103 UCA knockin (KI) mice after only two boosts. This GT-MEL regimen offers an alternative to the more commonly studied VH restricted CD4BS cluster often targeted by soluble (s)Env immunogens. Meanwhile, we have generated GT-MELs based on described GT-sEnvs that have been reported previously to elicit VH-restricted CD4BS HIV nAbs, i.e., activation of CH235 bnAb UCA in KI mice and possible CH235 homologs in macaques. The overall objective of this proposal is use novel GT-MELs to elicit HCDR3-dominated and VH restricted CD4BS responses in rhesus macaques, in addition to responses targeting HIV’s Fusion Peptide (FP). We will test a hypothesis that a CD4BS CDRH3- dominated plus VH-restricted CD4BS GT-MEL prime-boost regimen tested in macaques will elicit serum neutralization against tier 2 HIV (SA1). In SA2, we will use a fusion peptide nanoparticle (FP-NP) ‘pre-prime’ strategy followed by BG505 variant MEL boosting regimen designed to elicit tier 2 serum neutralization based in part on a prior report by others using a sEnv immunization protocol that elicited tier 2 serum neutralization and FP bnAbs. In Phase 2 of this ‘Innovation’ grant proposal, we will use the results of the above studies to integrate the CD4BS (CH505) MEL and FP (BG505) vaccine approaches into one regimen meant to elicit HIV nAbs to both the CD4BS and the FP supersites. These studies, will reveal the serum HIV nAb breadth that can be elicited by the MEL vaccine platform in rhesus macaques against multiple overlapping targets on the CD4BS and FP, and may inform the development of an HIV vaccine for humans. 1

NIH Research Projects · FY 2026 · 2024-03

Project Summary/Abstract: Alpha-1-antitrypsin (AAT) deficiency (AATD) is the most common, underdiagnosed inherited genetic condition (1:1500) and the primary modifier for chronic obstructive pulmonary disease (COPD) (300 million patients world- wide- 3 million deaths annually). AATD is an aging and genotype sensitive disease. Onset and progression results from misfolding of variant (mutant) forms of AAT in the endoplasmic reticulum (ER) of the liver leading to aggregation triggering progressive liver disease, and subsequent loss-of-function of neutrophil elastase (NE) inhibitory activity in serum and the lung leading to pulmonary failure by mid-to-late decades of life. Effective treatment of AATD is a critical unmet need. This proposal addresses an emergent challenge to understand how diverse variants in the AAT protein encoded by the genome of the world-wide population differentially contribute to both gain- and loss-of-function disease pathology and how proteostasis (the protein folding program in the cell) involving the unfolded protein response (UPR) ATF6 and IRE1/XBP1s signaling pathways, can be manipulated by small molecules to therapeutically manage disease in the individual harboring a unique variant. ATF6 and IRE/XBP1s signaling pathways adjust the capacity of the ER associated protein folding machinery including the cytosolic heat shock protein (Hsp) 70 (Hsp70) ER paralog Grp78/BIP/HSBA5 chaperone/co- chaperone system and the Hsp90 ER paralog Grp94 to restore balance in response to misfolding and ER stress. We now apply Gaussian process (GP) regression machine learning based variation spatial profiling (VSP), an innovative yet well validated tool (described in 11 publications to date for multiple proteins) that we have pioneered to advance our understanding of the spatial covariance (SCV) relationships dictating AAT fold design to develop novel therapeutic approaches for AATD. In Aims 1 and 2 we hypothesize that ATF6 and IRE1/XBP1s signaling pathways, respectively, can be adjusted to mitigate AATD using small molecule activators of these signaling pathways for nearly the entire spectrum of AAT variants in the patient population, experiments to be performed in collaboration with our Co-Investigators Drs. Kelly and Wiseman at Scripps Research. We have substantial preliminary data attesting to the effectiveness of UPR modulators across a broad spectrum of AAT variants found in the patient population (Sun et. al., (2023) Cell Chem. Biol.). We propose that these efforts will lead to effective, long term management AAT misfolding to mitigate severity of disease progression of AATD in the clinic. In Aim 3, we hypothesize that a deep understanding of the role of UPR regulated components found in the ER can explain the biology behind ATF6 and IRE1/XBP1s signaling pathway chemical modulators, providing a solid mechanistic understanding of disease to direct drug development. Through a deep understanding of GP based SCV properties of the collective of variants affecting AAT fold design driving dysfunction in the world-wide patient population, we hope to discover how precise adjustments to the proteostasis program can be used generate a precision perspective for therapeutic management of individual.

NIH Research Projects · FY 2026 · 2024-03

Summary: The sense of smell in the mouse largely occurs through the main olfactory system (MOS) which has the amazing ability to detect trace amounts of an infinite variety of volatile organic ligands. With this sensory power why does the mouse, and most terrestrial vertebrates, also have a vomeronasal organ (VNO)? Some think that since the VNO was lost during human evolution it is not important to study. First, we expect investigating the VNO will further advance our understanding of all types of chemosensation. The presence of a robust VNO across evolutionary diverse species indicates that taste and smell have functional blind spots that the VNO is filling. Understanding the VNO will enable us to know if humans are missing an animal superpower and can search for ways to augment our sensation, or if we evolved beyond its tether; perhaps losing this system partially accounts for our remarkable behavioral flexibility. Second, as an experimental model, the VNO provides unprecedented access to activate and study circuits critically important to humans. VNO mediated behavior has accelerated identification of neurons, circuits, and the brain network that underlies mouse social behavior and provides mechanistic insight to analogous circuits implicated in human social behavior. Anatomically, accessory olfaction has direct and privileged access to the social behavior brain network and genetic ablation of this system results in aberrant, dysfunctional social communication. Although humans are not thought to use olfaction to access social behavior circuits, the downstream network is critical and dysfunction leads to autism, ADHD, anxiety, or depression, but much is still unknown. Upon successful completion of this research, we will have created datasets quantitatively detailing and simultaneously bridging major unknowns about the accessory olfactory system: what it senses, metrics of neural activity encoding the environment, and properties of sensory coding in relationship to behavior. Together, our proposed work will investigate properties of VNO sensation that are expected to differ and complement the function of the MOS. Our method enables us to gain first insight about evoked sensory activity in relationship to the environment and behavior during dynamic natural behavior and is expected to generate new insight about chemosensory function.

NIH Research Projects · FY 2026 · 2024-03

Interactions between peptides and their substrates play a central role in many cellular processes and computational methods, such as automated docking, are used to gain a mechanistic understanding of these interactions. While docking small molecules works well, peptide-docking remains a challenging, relatively new, and fast evolving field with direct and high translational impact. We have been at the forefront of developing physics-based peptide-docking methods. Emerging deep learning methods (DL) such as AlphaFold are deeply impacting structural biology and have recently been shown to perform well for predicting protein-peptide interactions. We propose to further develop and enhance cutting-edge approaches for the prediction of peptide interactions with their binding partners, with a special focus on expanding coverage of the chemical space of building blocks used to synthesize therapeutic peptides. To this end, we will extend our state-of-the-art peptide docking software program AutoDock CrankPep to support non-standard amino-acids, and additional peptide cyclization modes. We will also develop novel scoring functions specifically designed for peptide docking. Furthermore, physics-based and DL-based peptide docking methods are complementary and we have evidence that they can be synergistically combined to leverage their respective strengths. These combined docking strategies will further extend the range of biological questions and biomedical targets for which peptide docking can be applied. Finally, we will extend peptide-docking methods for predicting peptide-peptide interactions. Such a method will have a significant impact on the development of peptide-based therapeutic approaches. We will continue to collaborate with experimental biologists as the biological systems they study challenge our software tools and drive their development and extension with new methods. Furthermore, these collaborations provide us with data beyond the traditional benchmark datasets for validating and refining our computational methods and predictions. These collaborations span from translational applications such as the development of therapeutics for breast cancer patients or molecules used to prevent brain damage in stroke patients, to pioneering the use of computational structural biology approaches to study small Open Reading Frame (smORFs) encoded polypeptides, an exciting emerging new frontier in biology. We have a long-standing track record of implementing best practices in software development, and producing and distributing production-grade, open-source software, widely used by large and growing community. The software tools resulting from this project will be developed and distributed using the same principles, supporting the research of many medicinal chemists and biologists and extending their use to an even wider community. These software programs will support the preclinical design of peptide-based therapeutic molecules and approaches, thereby supporting the advancement of biomedical research as well as fundamental aspects of structural biology.

NIH Research Projects · FY 2026 · 2024-02

Project Summary/Abstract The central goal of this project is to develop new sulfur fluoride exchange (SuFEx) reactions to construct small-molecule libraries containing the SVI-F motif and to explore their in vitro and in vivo functions. SuFEx is a new family of click chemistry transformations for generating diverse chemical structures bearing the SVI-F motif, such as -OSO2F (fluorosulfate), -SO2F (sulfonyl fluoride) and iminosulfur oxydifluorides. In the last seven years, we have established three versatile SulfurVI connectors: sulfuryl fluoride (SO2F2), ethenesulfonyl fluoride (ESF), and thionyl tetrafluoride (SOF4). SO2F2 selectively reacts with the – OH group of phenols to form aryl fluorosulfates, whereas SOF4 selectively reacts with primary amines to form iminosulfur oxydifluorides. Based on the aryl fluorosulfate chemistry, we invented extremely fast fluoride exchange reactions to introduce the fluorine-18 isotope into biologically active small-molecule radiotracers for positron emission tomography. By screening aryl fluorosulfate-based libraries, we discovered small-molecule covalent modifiers for Intracellular Lipid Binding Protein(s) and a platform for the late-stage drug functionalization. Building upon the above exciting discoveries, we will develop new transformations to expand the SuFEx transformation repertory and explore the feasibility of expanding fluoride exchange reactions to stable phosphorous(V) hubs. We will apply these new chemical transformations to construct screen libraries that can be used for rapid hit-to-lead optimization and for the development of covalent inhibitors and to construct stable 18F radiotracers for PET imaging.

NIH Research Projects · FY 2025 · 2024-01

PROJECT SUMMARY YME1 is an ATP-dependent protease located in the inner mitochondrial membrane, and plays a central role in executing the functional activities of the mitochondrial protein quality control network. Preserving homeostasis within the mitochondria is critical to maintaining cellular health and preventing a wide range of human diseases resulting from the dysfunction of the mitochondria, including neurodegenerative diseases, cardiovascular disorders, and cancer. YME1 is capable of proteolytically processing a wide variety of diverse mitochondrial substrates through regulatory mechanisms that remain largely unknown. One of the most intriguing aspects of YME1 proteolytic activity arises from its unique ability to perform molecular decision-making: YME1 can choose to perform 1) processive degradation of a target substrate, whereby it cleaves the substrate into small peptides, or 2) site-specific cleavage through which the substrate undergoes a single cleavage event and is subsequently released by YME1. One of the most well-characterized of these site-specific cleavage functions is the cleavage of the optical atrophy 1 (OPA1) protein, which is implicated in age-related eye disease, at an internal loop by the human YME1 protein (YME1L). This cleavage event produces two isoforms of OPA1, the ratio of which is crucial to regulating mitochondrial morphology and function. However, despite much investigation into the mechanistic bases for molecular decision-making performed by YME1 proteins, the atomic drivers that distinguish between these two degradation modes remain elusive. Yeast YME1 shares high sequence conservation with human YME1L, especially at the catalytic sites, making it an excellent molecular model for investigating this substrate processing mechanism. Interestingly, the site-specific cleavage of the yeast chaperone protein Tim10 occurs at a site that shares high sequence conservation with that of human OPA1. Thus, insights gained from studies of proteolytic processing in yeast YME1 have a strong potential to apply broadly to this mechanism in human YME1L as well as related ATP-dependent proteases. This work will focus on elucidating the mechanistic details that define how YME1 selects and distinctly processes protein substrates based on its sequence and folded state. The details of this mechanism will be investigated in two Aims. The first aim will combine structural and biochemical studies to examine how structural components of YME1 perceive and respond to specific sequences within targeted substrates. The second Aim will explore how YME1 switches between processing modalities based on a difference in the folding stability of the targeted substrate. These combined structure-function studies will define the molecular underpinnings that regulate two distinct functional activities of an ATP-dependent protease that are required for mitochondrial health. The outcomes of this study will provide a comprehensive model of the regulation of proteolytic processing by YME1, that will enhance our understanding of human YME1L function, and how perturbations of its function are associated with mitochondrial dysfunction and human disease.

NIH Research Projects · FY 2026 · 2024-01

SUMMARY Unhealthy diets and increasingly sedentary lifestyles have led to an epidemic rise in obesity and obesity-related disorders such as Type 2 diabetes (T2D) and nonalcoholic fatty liver disease (NAFLD), the latter of which can progress to the more severe condition nonalcoholic steatohepatitis (NASH). Despite their prevalence, limited options exist to treat obesity-linked diseases, particularly NAFLD/NASH. This has led to significant interest in developing new therapeutic strategies to mitigate the tissue-specific metabolic dysfunction implicated in obesity and obesity-driven pathologies. Clinical, biological, and biochemical evidence demonstrates that dysregulated signaling through the unfolded protein response (UPR), the stress-responsive signaling pathway that remodels cellular physiology to counter endoplasmic reticulum (ER) stress, is a critical determinant in dictating the metabolic imbalances associated with obesity and obesity-linked conditions. The UPR comprises three signaling pathways activated downstream of the ER stress sensing proteins IRE1, PERK, and ATF6. Signaling through the PERK arm of the UPR is generally associated with pathologic outcomes in obesity. In contrast, signaling downstream of IRE1 and ATF6, primarily mediated by the adaptive stress-responsive transcription factors XBP1s and ATF6, respectively, has been shown to protect various organs from obesity-linked alterations. This suggests that activation of protective IRE1/XBP1s and ATF6 signaling may represent a new strategy to alleviate metabolic dysfunction in obesity-linked diseases. We hypothesize that pharmacologic activation of protective IRE1/XBP1s or ATF6 signaling will foster adaptive remodeling in multiple key metabolic tissues to broadly ameliorate tissue-specific pathologies associated with obesity. We recently developed first-in- class, highly-selective IRE1/XBP1s and ATF6 activating compounds that enable us, for the first time, to determine the impact of pharmacologic activation of these protective UPR pathways in mouse models of T2D and NAFLD/NASH. Using these compounds, we have shown that pharmacologic IRE1/XBP1s or ATF6 activation stimulates adaptive remodeling that mitigates damage in multiple organs central to obesity-related conditions, including liver and pancreas. Here, we expand these findings to define how increased protective IRE1/XBP1s or ATF6 signaling corrects tissue-specific metabolic defects to enhance overall metabolic health in mouse models of T2D and NAFLD/NASH. Through these efforts, we will reveal new mechanisms whereby protective IRE1/XBP1s and ATF6 signaling remodel tissue-specific and organismal metabolism in obesity-linked disorders and establish pharmacologic IRE1/XBP1s and ATF6 activation as broadly-applicable therapeutic strategies to mitigate the systemic metabolic dysfunction seen in obesity-driven conditions such as T2D and NAFLD/NASH.

NIH Research Projects · FY 2026 · 2024-01

Summary Since the introduction of combination antiretroviral therapy (ART) the survival and quality of life of people with HIV (PWH) in the Western world has continued to improve. However, HIV infection and ART are associated with metabolic dysregulations, dyslipidemia, obesity, and increased prevalence of metabolic syndrome. Cardiovascular disease is becoming a leading cause of morbidity and mortality in PWH. Here, we propose an innovative approach to identify and characterize highly selective chemical probes to a validated therapeutic target dysregulated in HIV and chronic inflammation that has so far proved difficult to modulate selectively. To this end, using an orchestrated effort from the applicant laboratories, The Herbert Wertheim UF Scripps Institute for Biomedical Innovation & Technology (UF Scripps) in Jupiter, FL and Scripps Research in La Jolla, California, we will carry out a high-throughput screening (HTS) campaign in a 1536-well plate format in conjunction with a tiered approach to screen the ~666K UF Scripps Drug Discovery Library (UF- SDDL), which is enriched in compounds of drug likeness and chemical diversity, and the majority with high Central Nervous System Multiparameter Optimization (CNS MPO) scores. Hit-validation will be performed to identify small molecule regulators and eliminate nonspecific effectors, using parallel and orthogonal assays as well as off-target assessments using multiple counterscreens. To prioritize hit scaffold series, we will select analogs of confirmed hits from compound libraries and commercial sources. Hit scaffolds will be triaged to remove intractable molecules. We will select 3-5 scaffolds from the most promising hits, which will be profiled to verify their selectivity, potency, and lack of cytotoxicity. Leads in 2-4 series will be formulated and retested for potency/selectivity with the aim of advancing leads that can elicit the appropriate in vitro response in the aforementioned assays. This will be followed by in vivo pharmacokinetics (PK) studies to identify 1-2 top scaffolds for further investigation. Finally, the most promising 2-3 compounds with favorable drug metabolism and pharmacokinetics (DMPK) properties including high oral bioavailability, will be selected for in vivo efficacy testing. Altogether, we propose a novel strategy to establish new and more effective therapies to ameliorate HIV- associated metabolic complications, which is an important unmet clinical need and an area of high priority HIV/AIDS research within the mission of the NIDDK.

NIH Research Projects · FY 2025 · 2024-01

PROJECT SUMMARY Some autoimmune diseases, including systemic lupus erythematosus or “lupus,” are now known to occur because of the pathogenic excess of an intracellular disaccharide called Man(b1- 4)GlcNAc. The accumulation of this disaccharide in cells can lead to the erroneous activation of immune responses, leading to autoimmunity. The molecular mechanisms through which Man(b1- 4)GlcNAc regulate autoimmunity remain unknown, due to the difficulties of capturing the intracellular interactors and receptors of free oligosaccharides, like Man(b1-4)GlcNAc. We will use a chemoproteomic mass spectrometry-based approach to elucidate the interactome of Man(b1-4)GlcNAc to identify its functional receptors. The accomplishments resulting from this work will result in an enhanced understanding of aberrant glycosylation and the bioactivity of free Man(b1-4)GlcNAc, as well as pave the pathway for studying the interactomes of intracellular free oligosaccharides.

NIH Research Projects · FY 2026 · 2024-01

PROJECT SUMMARY/ABSTRACT Histone deacetylases (HAT) are zinc-dependent enzymes that catalyze the removal of acetyl groups from the epsilon amine of lysine side chains. These proteins have been widely pursued as anti-cancer drug targets, but therapeutic development has been largely unsuccessful given the essential nature of many HDAC proteins. Recently, we discovered a synthetic lethal relationship between HDAC1 and HDAC2, which is caused by recurrent chromosomal deletions that result in hemizygous deletion of HDAC1 in neuroblastoma and HDAC2 in multiple myeloma. As a result of HDAC1 deletion, neuroblastoma cells are hypersensitive to disruption of HDAC2, and vice versa in multiple myeloma. Using dTAG-mediated degradation or CRISPR/Cas9-based gene disruption, we discovered that targeting HDAC1/2 synthetic lethality (e.g. degrading HDAC2 in neuroblastoma cells with a hemizygous HDAC1 deletion) results in dissociation of the NuRD chromatin remodeler complex, of which HDAC1/2 are members. Dissociation of the complex results in degradation of NuRD subunits that are selectively required for neuroblastoma and multiple myeloma survival, suggesting that HDAC1/2 synthetic lethality can be leveraged to target subunit-specific NuRD vulnerabilities in cancer. We hypothesize that HDAC1 deletions cause the NuRD subunits, HDAC2 / MBD3 / MTA3, to be essential for neuroblastoma, whereas HDAC2 deletions cause vulnerabilities to loss of their paralogs, HDAC1 / MBD2 / MTA2, in multiple myeloma. Here, we will address this hypothesis and explore the translational potential of these vulnerabilities by developing small- molecule modulators that target NuRD structure and/or function. In Aim 1, we will (i) Determine whether MBD and MTA vulnerabilities are caused by HDAC1/2 deletions using CRISPR/Cas9, inducible RNAi, and dTAG- based approaches in vitro and in vivo, (ii) Reveal whether the loss of NuRD subunits required for cancer cell survival leads to dissociation and/or degradation of the NuRD complex using unbiased proteomics approaches, and (iii) Establish if HDAC1/MBD2/MTA2 and HDAC2/MBD3/MTA3 form distinct NuRD sub-complexes as a result of HDAC2 and HDAC1 deletions, respectively. These experiments will determine if subunit-specific NuRD vulnerabilities are caused by HDAC1/2 deletions or simply exploited by HDAC1/2 synthetic lethality. In Aim 2, we will develop small molecules targeting the NuRD complex to exploit NuRD vulnerabilities in genetically defined cancer sub-types. Specifically, we will: (i) develop paralog-selective PROTACs that distinguish between HDAC1 and HDAC2, (ii) determine the potential for covalent ligands of MTA3-Cys532 to disrupt NuRD structure and/or function in MTA3-dependent cancers, and (iii) develop MTA3-targeted PROTACs based on ligands that covalently engage MTA3-C532. Altogether, successful completion of these aims will determine the mechanisms underlying NuRD vulnerabilities in cancer and advance novel chemical tools to drug and study them.

NIH Research Projects · FY 2026 · 2024-01

Project Summary Many studies have highlighted a large incidence of hemostatic derangements in the form of hypercoagulable and hypofibrinolytic states following SARS-CoV-2 infection. These hemostatic disturbances are fueled by and a consequence of the concomitant activation of the endothelium following a severe inflammatory response, with likely contribution from many other pathways and components, such as the complement pathway and neutrophil activation. In COVID-19 patients, the resulting hyperinflammation, vascular dysfunction, and systemic hypercoagulability, collectively referred to as COVID-19-associated coagulopathy (CAC), manifests as the increased tendency of micro-thrombosis of different organs leading to organ dysfunction, venous thromboembolism, pulmonary embolism, and deep vein thrombosis. Reducing the deleterious impact of CAC during severe SARS-CoV-2 infections continues to represent a major therapeutic challenge. Despite the enormous effort exerted during the pandemic to understand the pathological mechanisms responsible for the severity of SARS-CoV-2 infections, a major knowledge gap about the drivers and mechanisms underlying CAC still exists. Furthermore, as the link between CAC and long COVID becomes more apparent, understanding CAC is more urgent than ever. Proposed studies are designed to gain knowledge on the drivers and mechanisms underlying CAC and will test the feasibility of several potential pharmacological approaches to blunt it. Levering our novel and carefully optimized mouse model of COVID-19 and CAC, relying on a human pathological SARS- CoV-2 strain and recapitulating major pathological alterations and development of CAC observed in human patients presents a unique opportunity for hypotheses-driven research in a controlled and systematic manner to gain important new insights on CAC. Mimicking worldwide observations that males are more susceptible to severe disease after SARS-CoV-2 infection compared to females, a sex-dependent bias in disease severity was also observed in our model that was accompanied by striking temporal, quantitative, and qualitative differences in the development of CAC. Aim 1 will characterize the major molecular determinants of the sex-biased disease severity and development of CAC in our mouse model of COVID-19 and CAC. Key questions that will be addressed are the extent to which the sex bias originates from hormonal differences, whether estrogens are protective and/or androgens are deleterious, and whether hormonal supplementation therapy can alter the development of CAC. These studies will help identify novel pathways and targets for CAC. Aim 2 will expand on our preliminary data to characterize von Willebrand Factor (VWF)’s function(s) and protein/protein interaction(s) that are responsible for modulating survival during SARS-CoV-2 infection and development of CAC. The results of these studies will increase our knowledge of the mechanisms involved in regulating the finely tuned interaction between the immune, endothelial, and coagulation systems upon SARS-CoV-2 infection, possibly leading to the identification of new therapeutical targets for the treatment of CAC.

NIH Research Projects · FY 2026 · 2023-12

PROJECT SUMMARY Developing a preventative HIV vaccine remains a global health priority and to be effective will most likely need to elicit broadly neutralizing antibodies (bnAbs). HIV bnAbs are derived from rare, unmutated common ancestor (UCA) B-cell precursors in elite HIV+ human repertoires. To reproducibly activate such UCA+ B-cells to affinity mature and acquire bnAb function, `Germline Targeting' (GT) vaccine protocols are being developed typically for a single bnAb lineage, and only induce sporadic and/or weak serum bnAb responses using protracted boosting schemes that are clinically impractical. Clearly, more efficient GT regimens are needed that can elicit multiple bnAb specificities, and appropriate animal models to track and follow up on promising lead neutralization signals in the serum. We recently equipped a new membrane (m)Env liposome (MEL) platform with a GT mutation in mEnv that reproducibly induces tier 2 serum neutralization using semi- polyclonal CH103 UCA knockin (KI) mice after only two boosts. This represents the first GT regimen to elicit a `CDRH3 dominated' nAb response to the CD4-binding site (CD4bs) in which UCA B-cells are also under anergy control. The overall objective of this proposal is to build on this novel GT platform, to both improve HCDR3-dominated specific responses, and to adapt it to target the relatively better-studied VH restricted- targeted CD4BS cluster and HIV's Fusion Peptide (FP) domain. We hypothesize that GT MEL sequential priming modalities (and mRNA-LNPs) tested in strategically selected CD4BS bnAb UCA knock in models (while manipulating frequencies and tracking development of multiple UCA lineages), will help us identify a vaccine regimen that will elicit HIV serum neutralization in “higher bar”, fully polyclonal human Ig pre-clinical animal models. Specifically, in Aim 1, we will optimize the existing MEL-based GT regimen, as above, in the lead CH103 UCA KI model. Then, in Aim 2, we will test the ability of mEnv modalities first to drive physiological numbers of `VRC01-class' CH31 UCA precursors towards breadth in WTàCH31 KI chimeric mice, and then evaluate CH31/CH103 UCA multi-GT mEnv regimens promoting elicitation of both CD4BS bnAb lineages (in CH31àCH103 chimeras). Finally, in Aim 3, we will test the ability of FP nanoparticle (FP-NP) prime-mEnv boosting regimens to elicit serum nAb responses in WT and UCAàWT chimeric mice, and ultimately test the best FP and CD4BS regimens combined in Omni Mice and Omni Rats, which express fully polyclonal, unrearranged human V (D)J repertoires. These studies, even if partially successful, will reveal the serum HIV nAb breadth that can be elicited by next-generation mEnv vaccine platforms against multiple overlapping targets on the CD4BS and FP, and may be advantageous in overcoming Ig repertoire holes across individuals, preventing viral escape, and requiring less overall boosting. 1

NIH Research Projects · FY 2025 · 2023-09

Abstract Opioids are highly effective at reducing pain, but their potential for addiction and overdose has led to a growing public health crisis. Researchers have attempted to develop new opioid compounds that are less likely to be abused and have fewer side effects, but these efforts have been difficult. The endogenous opioid system has multiple receptors and ligands heterogeneously expressed across different parts of the body and cell types. Tremendous work has been done to delineate the relationships between opioid receptors (ORs) and ligands. However, the specificity of ligand-receptor engagement often depends on relative affinities at predetermined targets. In general, in vivo spatial and cellular heterogeneity of the brain obscure opioid actions, making them hard to predict based on receptor affinity alone. Currently, single-cell and spatial transcriptomics are transforming our understanding of brain architecture. However, there is a significant gap in how we measure opioid actions and align them with the spatially resolved cellular atlas of the brain. Levering emerging CATCH and inverse activity marker (IAM) techniques, we propose multimodal profiling of opioid actions with spatial and single-cell resolution across the entire mouse brain. Using three pharmacologically diverse opioids, we aim to map neuronal activities and cellular binding of these drugs onto the entire mouse brain in an unbiased way and register them with cell types identified from single-cell transcriptomics. Furthermore, we will test whether a drug’s affinities across different ORs determine its in vivo cell and neural ensemble engagement. Not only would this project provide a circuit-level mechanism linking the molecular pharmacology to brain-wide opioid actions, but also lay out a roadmap for evaluating and developing new opioids, e.g., by incorporating regional and cell-type preference into the structure-activity-relationship for lead optimization or by revealing on- and off-target sites to guide further cell-type specific in vitro chemical screening and optimization.

NIH Research Projects · FY 2024 · 2023-09

Project Summary/Abstract. Electronic cigarettes are gaining popularity as alternative to traditional cigarettes, with sales increasing from $283 million in 2012 to $2.5 billion in 2018 in the US. The global e-cigarette market is estimated to reach $24.2 billion by 2024. E-cigarette solutions, also known as e-liquids, are highly variable with enticing flavors, such as tobacco, menthol, fruit, candy, and dessert. Liquid nicotine concentrations vary in these products from 0 to 100 mg/ml according to an FDA study. Although nicotine toxicity in adults is rare with an estimated lethal dose between 60 and 500 mg (0.8-6.7 mg/kg), flavored e-liquids are increasingly being ingested orally, putting children at risk for exposure to high concentrations of nicotine. Indeed, nicotine toxicity in children under 5 years of age can occur with consumption of as little as a teaspoon of liquid nicotine. The unforeseen consequences of e-cigarettes, with respect to nicotine poisoning in children, presents an unmet need to counteract the harmful and potentially fatal outcomes that may occur among this vulnerable population. It is the pediatric population we plan to address in this proposal, as a high potential of accidental ingestion of liquid nicotine from e-cigarettes exists. Despite the increase in nicotine-related poisonings reported, there is no treatment for acute nicotine toxicity. Current treatment regimens for nicotine poisoning range from supportive care, to activated charcoal, to respiratory support with mechanical ventilation. An alternative means of altering the toxicity of nicotine poisoning could come via simple sequestering of the drug. Antibodies to nicotine have been prepared as a means to block the pharmacological effects of this drug. To date, vaccines for smoking cessation have shown promise in preclinical animal models; however, in clinical studies, these vaccines failed to measure significant differences in smoking abstinence between the intervention and placebo groups. Thus, the likelihood of an antibody attenuating an acute dose of nicotine is doubtful. What is needed is a sufficient pharmacokinetic (PK) biologic with the capacity to not just sequester nicotine but also increase its metabolism. We envision a biologic able to catabolize nicotine rather than simply sequestering the drug would have the potential to treat acute nicotine poisoning. The proposal at hand details a bacterial strain, Pseudomonas putida, which has evolved to use nicotine as its sole source of carbon and nitrogen. From this bacterial strain, we will examine a first-in-class enzyme, a nicotine oxidoreductase termed NicA2, as a means to treat nicotine poisoning. Our initial characterization of the enzyme indicates that it could be an excellent candidate for altering nicotine poisoning. However, the successful demonstration of this enzyme reversing nicotine poisoning will require several experimental undertakings including: (1) Evaluating the efficacy of NicA2 to attenuate acute nicotine toxicity in rodent models including plethysmography, blood/brain distribution and lethality. (2) Directed evolution of NicA2 to increase its catalytic capacity through gene recombination and random mutagenesis.

NIH Research Projects · FY 2025 · 2023-09

Intrinsically disordered proteins (IDPs) are highly abundant in eukaryotes and play a central role in key cellular regulatory pathways and in the spatial organization of the cell. Approximately half of the proteins in the human proteome are either fully disordered or contain long disordered regions (IDRs). The cellular abundance of disordered proteins is tightly regulated and dysregulation or mutation of IDPs and IDRs is associated with devastating diseases such as cancer, diabetes, cardiovascular disease, and neurodegenerative disease. Disordered proteins are highly flexible and undergo transient and dynamic intramolecular and intermolecular interactions that are central to their regulatory functions. Molecular level characterization of the numerous human regulatory proteins that contain both structured and disordered domains represents an enormous challenge to the traditional methods of structural biology. Most studies to date have relied upon a reductionist, divide-and-conquer approach, in which the ordered and disordered regions are expressed independently and studied in isolation. However, within the cell, the folded and disordered domains of a given protein act synergistically to allow it to perform its biological function and a full understanding of the underlying molecular mechanism can only be achieved through a holistic, rather than reductionist, approach. An overarching goal of our research is to utilize a non- reductionist approach, aided by intein-based segmental isotope labeling, to characterize the structural ensemble, dynamics, and interactions of eukaryotic proteins containing both folded and disordered domains. This strategy is broadly applicable to large, dynamic proteins with disordered domains since it is relatively straightforward to identify or engineer optimal intein splice sites within disordered regions. Importantly, traceless ligation, where no cysteine or other non-native residues are introduced at the splice site, can be accomplished using the Nrdj1 intein, allowing retention of the native protein sequence and cysteine-mediated coupling of spin labels or fluorophores at desired probe sites. Initial efforts will focus on the full-length, 180 kDa tumor suppressor p53. Current structural information on p53 is largely limited to isolated domains and fails to explain how the disordered and folded regions function synergistically to control p53 activity. There is a large and growing body of evidence that the intrinsically disordered regions of p53 regulate its activity through dynamic intramolecular and intermolecular interactions that are modulated by constitutive and stress-induced post-translational modifications. This research will provide new molecular-level insights into the mechanisms by which this important tumor suppressor is regulated, as well as providing new tools for structural and dynamic characterization of large eukaryotic regulatory proteins that contain disordered regions.

NIH Research Projects · FY 2024 · 2023-09

SUMMARY Pharmacological inhibition of glucocorticoid receptor (GR) signaling can efficiently reduce alcohol intake and seeking in rodent and primate models of heavy alcohol drinking, as well as in human subjects with an alcohol use disorder (AUD). Despite the wealth of evidence supporting the therapeutic potential of GR inhibition for the treatment of AUD, the molecular mechanism mediating this effect remains unknown. Aside from acting as a transcriptional regulator, GR can bind a subset of mRNAs in the cytoplasm and elicit their rapid degradation upon ligand binding – a process called GR-mediated mRNA decay (GMD). Intriguingly, we demonstrated that the endoribonuclease RIDA, a critical component of the GMD complex, is among the most significantly upregulated proteins in the mouse medial prefrontal cortex (mPFC) during abstinence following a history of excessive alcohol drinking. The present project will test the hypothesis that hyperactive GMD contributes to alcohol intake escalation and underlies the ability of GR antagonism to reduce alcohol drinking in mice withdrawn from chronic intermittent alcohol vapor inhalation. A first aim will be to determine whether abstinence increases GMD activity in excessive alcohol drinkers. To do so, we will first determine the identity of mRNAs bound to GR in mPFC samples from alcohol-naïve mice and test whether GR activation causes their rapid degradation. We will then examine the effect of alcohol withdrawal on these potential GMD substrates and the ability of GR inhibition to prevent it. A second aim will be to determine whether blocking GMD in the mPFC via local RIDA knockdown, which will not impact GR transcriptional activity, can replicate the effect of GR antagonism on excessive alcohol consumption and cognitive impairment. Our approach capitalizes on our expertise in modeling AUD in mice and manipulating gene expression in small brain regions, combined with access to state-of-the-art core resources for RNA sequencing and bioinformatic analysis. The proposed work will enhance our understanding of the molecular mechanisms driving alcohol intake escalation and memory deficits in mice, as well as the mechanism of action of GR antagonists. It will probe for the first time the relevance of GMD in the brain and may identify a new molecular target for the treatment of AUD and other GR-related neurological and psychiatric disorders.

NIH Research Projects · FY 2025 · 2023-09

Abstract Covalent inhibitors represent some of the most successful drugs in human history, including aspirin and penicillin. Recently, targeted covalent drugs have taken center stage as a compelling approach for achieving major goals in oncology that have proven elusive for more classical reversible small molecules, including, for instance, the selective inactivation of oncogenic kinases (BTK, EGFR, FGFR, JAK3) and, most notably, the inhibition of the once-deemed undruggable KRAS protein. We are now in the midst of a resurgence of interest in covalent drugs for their demonstrated capability to engage cancer targets that have been historically considered undruggable. However, despite their proven success and inherent advantages of potency, there has been a general reluctance to develop covalent drugs due to the concern of potential irreversible off-target toxicity across different organ systems. Hence, a comprehensive understanding of both on and off-targets in vivo is critical for covalent drugs. Currently, it is impossible to determine drug binding across a whole animal with cellular and molecular resolution in mammals. Building upon a recent breakthrough in tissue imaging termed CATCH (Clearing-Assisted Tissue click Chemistry), we propose to develop a general platform for in vivo imaging of drug-target interactions with unprecedented spatial precision by integrated applications of high-resolution whole-body imaging and chemoproteomics (such as Activity-Based Proteomic Profiling, or ABPP) through the same covalent probes. This way, every cell in a living mammal targeted by the drug (both on- and off-target) can be revealed in situ and registered onto a defined protein map to screen and identify in vivo drug targets. The data stream generated by this platform could rapidly link the rich knowledge of drug affinity to the therapeutic index, therefore accelerating the translation of chemical activities into cancer therapies. Our team has well-established and complementary expertise in chemoproteomics and tissue imaging to ensure the successful execution of the project. In this IMAT R33 application, we plan to further develop CATCH to profile in vivo targets of covalent kinase inhibitors. First, we will adapt CATCH to 3D somatic tissues (Aim 1). Next, we will expand CATCH to an array of covalent BTK (Bruton’s tyrosine kinase) inhibitors (Aim 2). Finally, we will profile dose-dependent in vivo cellular targets of BTK inhibitors in the mouse cardiovascular system (Aim 3). We anticipate that these studies will establish in vivo CATCH methods for identifying targets of covalent BTK inhibitors to better understand their efficacy and toxicity. More generally, the established platform can be broadly applied to any covalent cancer drug for unbiased in vivo target identification. The pipeline, analytics, and high-resolution drug target data will be rapidly disseminated for public access and exploration, releasing an immediate, direct, and profound impact on covalent cancer drug discovery and refinement.

NIH Research Projects · FY 2025 · 2023-09

Summary The abuse of opioid drugs is associated with treatment non-compliance, greater risk of viral transmission, and more rapid clinical progression of HIV disease. The overarching hypothesis behind the present project is that the analysis of molecular profiles of neuronal and glia cells at the single cell level in drug abuse-relevant brain regions by single nucleus RNA-Seq (snRNA-Seq) will reveal key genes that are dysregulated by the interaction of HIV with opioid abuse, resulting in neurodegeneration and cognitive impairment. To test the present hypothesis, we propose to use validated systems biology strategies for the reconstruction and interrogation of a genome-scale integrated gene regulatory network in conjunction with snRNA-Seq from HIV transgenic (Tg) rats, which harbor a non-replicating HIV-1 transgene expressing chronic low-levels of multiple HIV-1 proteins in disease-relevant cell types, and wild-type rats. The occasional but limited use of a drug is clinically distinct from dependent drug use, which is characterized by the emergence of dependence and a negative emotional state when access to the drug is prevented that drives negative reinforcement, a powerful source of motivation for drug seeking. Therefore, we will use a state-of-the-art paradigm of voluntary intravenous opioid self-administration under short access (ShA) conditions, which is characterized by a non-dependent, “recreational” pattern of drug use, and long access (LgA) conditions, which leads to dependent drug intake. Escalated drug intake under LgA conditions is highly relevant to human substance use disorder (SUD) as it has been suggested that it models all 7 of the criteria for drug addiction in the Diagnostic and Statistical Manual of Mental Disorders (DSM)-IV and 7 of the 11 criteria in the DSM-V. We showed that HIV Tg rats self-administering oxycodone in this LgA paradigm of escalated self-administration display increased neural injury and cognitive impairment. The project will address the following vexing question about opioid abuse in the setting of HIV infection: what are the cell types and cell states that drive neuroinflammation, neurodegeneration, virus expression, and escalated (dependent) opioid self-administration and cognitive impairment in the setting of HIV? Overall, this collaborative interdisciplinary proposal integrating single cell level transcriptomics, state-of-the- art behavior methods in HIV Tg and wild-type rats, and computational strategies for the deconvolution of the gene regulatory network at the single cell level will elucidate key mechanisms that underlie the effects of HIV and opioid abuse and their detrimental interactions on neuroHIV progression, virus expression and persistence. The results will indicate transformative new mechanistic hypotheses that may lead to novel therapeutic concepts for opioid use disorder (OUD) in the setting of HIV and will establish key resources for the neuroHIV field to be made publicly available through the SCORCH data coordination center and other public repositories.

NIH Research Projects · FY 2026 · 2023-08

The overarching goal of this project is to obtain a deep, molecular-level understanding of P-glycoprotein (Pgp)- mediated drug transport and efflux inhibition. Pgp belongs to a very important class of ATP-binding cassette (ABC) transporters that transport substrates out of cells using the energy of ATP hydrolysis. One of the truly remarkable features of Pgp is its unusually broad polyspecificity. Pgp functions like a hydrophobic vacuum cleaner, transporting diverse molecules including many clinically useful drugs that partition into the lipid bilayer. Pgp is known to be a key determinant of the bioavailability, pharmacokinetics, and clinical efficacy of drugs in humans. Pgp also causes cellular multidrug resistance, hindering the treatment of many diseases. Because of the potential adverse effects of Pgp efflux on drugs, the US Food and Drug Administration mandates documentation of drug interactions with Pgp for approval of any new drug. Evasion or inhibition of Pgp without compromising therapeutic efficacy has been a major goal of the pharmaceutical industry, which, however, is seriously hindered by the lack in the fundamental understanding of the polyspecific Pgp-drug interactions. Our long-term goal in studying Pgp/drug interactions is to facilitate the development and discovery of new drugs that either inhibit or evade Pgp efflux. Towards this end, we propose three independent but related specific aim studies for this project, with each addressing a key question closely related to drug development. In Aim 1, we will discover bona fide Pgp antagonists for mechanistic studies. We note that many widely studied Pgp inhibitors compete with drugs for Pgp transport. We envision the discovery of novel, nonsubstrate Pgp antagonists displaying distinct properties from the known inhibitors, which is crucial to Pgp inhibition mechanistic studies and for the development of better drugs to inhibit Pgp. In Aim 2, we will interrogate how Pgp discriminates transport substrates and inhibitors. We have formulated a novel hypothesis that the two halves of Pgp may play different roles in Pgp transport and inhibition, based on several preliminary studies. We will further investigate this hypothesis by covalently attaching ligands to different binding sites in Pgp and then characterizing Pgp structure and activity to find correlations. In Aim 3, we propose to rationalize chemical strategies to modify drugs to evade Pgp transport. We will carry out systematic chemical modifications on specific ligands for Pgp structural and activity relationship studies to learn the basic chemical principles underlying Pgp-drug interactions. Our aim studies for Pgp incorporate novel chemical designs complemented by in-depth activity and structural studies. We have collected strong preliminary data to demonstrate the feasibility and novelty of each Aim study. Our approach is quite unique and will offer unprecedented insights into the complex interactions of drugs with Pgp, thus having a significant impact on drug discovery.

NIH Research Projects · FY 2025 · 2023-08

Project Summary/Abstract This proposal summarizes ongoing projects in our laboratory focused on natural product classes that have promising biological activity but are burdened with limitations that have prevented them from reaching their therapeutic potential. These classes are structurally complex and modification of their is challenging by semisynthetic and biosynthetic methods. We aim to develop fully synthetic routes to these classes from simple building blocks, enabling chemical modification to overcome their limitations. These efforts are informed by binding data (X-ray or cryo-EM) for each class. The primary goals of project outlined herein are to 1) expand structure–function relationships for each of these important classes of molecules, and 2) discover potent analogs that are suitable for hit-to-lead optimization or for use as tools to study biological systems. Additionally, development of the synthetic routes themselves is highly innovative, and is often accompanied by development of methods that are broadly applicable in chemistry. These efforts mirror our work on streptogramin and lankacidin antibiotics, which was a primary focus in our Early Stage Investigator MIRA (R35GM128656), and led to structural reassignments and to a potent hit compound with activity against resistant strains in vivo. Much of the biology for this work will be enabled by collaboration. Five of the projects summarized herein focus on the development of novel antibiotics that target the ribosome and membrane proteins. Due to our ongoing work in this area, we have several collaborations in place to evaluate the antimicrobial activity, in vivo efficacy, and target engagement of new analogs. Beyond antibiotics, we propose to synthesize and derivatize classes that target Hsp90, an anticancer target, and eEF1A, an anticancer and antiviral target. Evaluation of these compounds for inhibitory activity, isoform selectivity, and binding will be enabled by new collaborations, expanding the scope of our research. With chemical innovation paired with strong biological investigation, we anticipate that the work outlined herein will lead to exciting discoveries in chemical synthesis and to the discovery of hit compounds for the treatment of bacterial infections, cancer, and SARS-CoV-2.

NIH Research Projects · FY 2026 · 2023-08

Project Summary/Abstract: The focus of this proposal is based on our ongoing efforts to link genetic sequence variation leading to changes in the protein fold triggering human genetic disease using an unprecedented variation spatial profiling (VSP) approach we have pioneered. VSP is a Gaussian process (GP) regression machine learning approach that utilizes human variation to assign function for each residue in the protein fold responsible for the genotype to phenotype transformation driving human biology- a new technology that is universal in application to any protein. VSP is built on the general principle of spatial covariance (SCV) which describes fundamental covariant relationships between all residues dictating the protein fold and function. These spatial relationships allow us to define with assigned uncertainty the role of each residue in genetic disease to define the residue-residue interactions that drive function in protein structure using variation capture (VarC). We focus on the cystic fibrosis transmembrane conductance regulator (CFTR), the causative agent of CF, as a model protein to understand SCV/VarC relationships dictating the impact of genetic variation on folding and trafficking through the exocytic pathway. To understand how genetic variation impacting protein fold design is managed by proteostasis folding and COPII based trafficking pathways, and how we can improve function in genetic disease by promoting protein fold fitness through small molecule correctors, we propose 3 goals. In Aim 1, we will utilize SCV relationships to dissect the contribution of the Hsp70 and Hsp90 chaperone/co-chaperone proteostasis systems we hypothesize are misaligned for the proper management of naturally occurring genetic variants triggering disease- and that these components can be retuned by adjusting their activity through molecular and chemical approaches. In Aim 2, we hypothesize that the proteostasis system generates SCV-defined 'set-points'. SCV set-points are composed of select clusters of SCV defined residue-residue spatial relationships in the protein structure that serve as master regulators for presentation of CFTR to the COPII ER export machinery through a cytosolic exposed 'YKDAD' exit code. We hypothesize that COPII components differentially respond to SCV set-points impacted by genetic variation to generate disease in the individual. We will determine the impact of genetic variation for each of the steps dictating COPII assembly to understand those events responsible for pathophysiology. In Aim 3, we further hypothesize based on GP logistics that variant CFTR polypeptides will be highly responsive to novel correctors that directly interact with the fold to restore function. We will utilize an SCV- based 'triangulation' approach to identify small molecules that directly impact the stability of the YKDAD exit motif defective in F508del and other variants to identify compounds that affect a cure for CF using in silico computational screening and experimental validation. The combined efforts outlined in Aims 1-3 will allow us to define a genome based mechanistic foundation for how the fold can be reprogrammed for optimal fitness in the individual by reducing the impact of variation triggering human genetic disease.

NIH Research Projects · FY 2026 · 2023-08

Project Summary Staphylococcus bacteria are the primary cause of healthcare-associated infections in neonatal intensive care units and the leading cause of skin and soft tissue infections worldwide. In addition to causing impetigo, the most common bacterial infection in children, Staphylococcus can result in life-threatening conditions, including sepsis and Staphylococcal scalded skin syndrome. However, there are currently no effective vaccines available for Staphylococcus bacteria, preventing maternal immunization, and the emergence of antibiotic resistance impedes standard treatments. Consequently, there is a need to understand how the immune system responds to Staphylococcal infections in early life. While innate immune cells utilize germline-encoded receptors to detect conserved pathogen-associated molecular patterns, adaptive cells recombine receptor genes to generate a broad range of antigen specificities, which delays the emergence of adaptive immunity. Following development, adaptive immune cells require antigen-mediated activation within secondary lymphoid organs to express the chemokine receptors and integrins necessary for tissue homing and produce effector cytokines or antibodies. Consequently, adaptive immune cells are largely absent from most barrier tissues in early life, which instead harbor innate lymphoid cells (ILCs) and innate-like T cells. Both primarily localize to tissues and rapidly release cytokines due to their developmental acquisition of effector characteristics. While ILCs lack recombined antigen receptors, innate-like T cells express semi-invariant T cell receptors that limit their antigenic range analogously to innate immune receptors. Though these lymphocytes arise prior to the establishment of immunologic memory and are abundant in early life, their contributions to immunity during this period remain poorly understood. The primary goal of this proposal is to determine the role of innate and innate-like lymphocytes in early-life immunity, which will be accomplished by 1) developing early-life murine infection models, 2) establishing the contributions of murine innate and innate-like lymphocytes in these models, and 3) assessing the in vivo responses of human innate and innate-like lymphocytes utilizing humanized mice. Combining state-of-the-art approaches from immunology, genetics, and bioinformatics, this highly innovative project will further our understanding of early- life immunity and lead to the development of novel therapeutics.

NIH Research Projects · FY 2024 · 2023-08

PROJECT SUMMARY: Chronic inflammatory diseases afflict millions of Americans and place devasting burdens on patient’s lives. Effective treatments for these disorders are lacking, partly because the multicellular interactions between bodily systems (e.g. the peripheral nervous and immune systems) that drive inflammation are poorly understood. For example, it is known that sensory neurons play context-dependent roles in a variety of inflammatory disorders by modulating the immune response. The specific neuronal subtypes and molecular mechanisms that contribute to this remain unclear. The overarching goal of my research is to unravel the precise mechanisms by which peripheral sensory neurons influence the pathogenesis of inflammatory diseases of the skin and visceral organs and how sensory neurons are in turn influenced by the immune system. A subtype of sensory neuron that detects itch (pruriceptors), expresses a number of receptors for pro-inflammatory and immunomodulatory molecules. Thus, these pruriceptors are attractive candidates for orchestrating multicellular interactions during inflammation. However, the role of these specific sensory neurons in inflammatory disease is unstudied as, until recently, the tools to precisely target these neurons were lacking. To bridge this gap in knowledge, this proposal will elucidate the contribution of pruriceptors to distinct inflammatory states. The proposed research will interrogate pruriceptor function using genetic and viral approaches in mouse models to selectively target and ablate these neurons and measure the effects of these manipulations on mouse models of inflammatory diseases. Specifically, I will test the hypothesis that pruriceptors, as polymodal sensors of aversive chemical, mechanical, and immune stimuli, drive hypersensitivity and local inflammatory responses in a context-specific manner. During the K99 phase, I will leverage my proven track record in studying neuroimmune interactions with innovative techniques I am learning to target and manipulate specific subsets of sensory neurons and ultimately map and elucidate multicellular circuits in inflammation. Aim 1 will establish th e contributions of pruriceptors to chronic itch-evoked skin inflammation by combining viral and genetic approaches to selectively ablate pruriceptors with behavioral, immunological, electrophysiological, and transcriptomic read- outs to understand how pruriceptors contribute to skin inflammation. As molecularly similar sensory neurons of unknown function also innervate the gut, Aim 2 will translate this approach to the study of gastrointestinal tract inflammation and gut function using a model of gastritis. I will dedicate part of the K99 phase to career development activities such as mentoring, presenting at conferences, and publishing research articles. A portion of both Aims will carry over into the independent R00 phase of the award. This proposal will provide fundamental insights into the multicellular interactions underlying chronic inflammatory disease from a neuron-focused perspective. Successfully executing this training plan with the help of The Scripps Research Institute will prepare me to lead an independent research program.