Vanderbilt Chemical Biology Interface

PI: Alex Schuppe, Chemistry

The VICB mission to “harness the power of chemistry to improve human health” is best exemplified through the discovery of novel chemical methods that accelerate the synthesis of biologically active molecules and probe compounds. This can facilitate mass production of these molecules, a requirement for biological evaluation, as well as offer a way to execute structural modifications and functional optimization. I plan to uphold this mission by developing innovative “stitch-and-tailor” protocols for accessing rearranged terpenes and isotopically labeled probe compounds. A multitude of rearranged terpenes have been identified in nature, but their biological study is limited by their low natural abundance and poor synthetic accessibility. The primary challenge is posed by the highly strained, complex ring system characteristic of these terpenes. Although synthetically cumbersome, ring strain is a feature that rigidifies these molecules, suggesting that they may exhibit strong target binding and minimal off-target effects. The proposed research project overcomes this obstacle by “stitching” polyolefin precursors into a rearranged terpene scaffold through a hydroboration–carbonylation reaction (Scheme 1A). The resulting scaffolds can then be functionalized or “tailored” using late-stage diversification tactics to obtain a collection of these rearranged terpenes. Furthermore, I plan to work with the Vanderbilt High Throughput Screening facility to elucidate biological activity and explore their therapeutic potential. Incorporation of carbon isotopes into biologically active molecules has various applications in chemical biology and medicinal chemistry. This includes mechanistic probing experiments, radiopharmaceutical imaging, and metabolic profiling. Existing methods for isotopic incorporation rely on peripheral modifications, which can alter the structure and disrupt activity; or metal-catalyzed reactions, which can be costly and complicate in vivo studies. I envision leveraging hydroboration–carbonylation methodology to internally “fashion” biologically active molecules with isotopic carbons using labeled carbon monoxide (CO) (Scheme 1B). To demonstrate the utility of this application, I look forward to collaborating with the VUIIS Research Radiochemistry Core Laboratory to obtain 11CO and identify potential radiopharmaceutical targets for positron emission topography (PET).

PI: Lars Plate, CPB

Cystic fibrosis (CF) is a lethal genetic disorder affecting approximately 85,000 individuals globally [McDonald et al., 2023]. It is caused by a variety of loss-of-function mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Mutations lead predominantly to protein misfolding and degradation of the CFTR protein, an epithelial anion channel. Recent advancements have led to the development of small molecule correctors and potentiators that enhance CFTR expression and function, significantly improving treatment outcomes for about 90% of patients, particularly those carrying the ΔF508 CFTR mutation common among Caucasians. However, these therapies are less effective for approximately 10% of patients with rare and diverse CFTR mutations, creating a therapeutic gap for these individuals, particularly in non-white populations. The challenge lies in the limited throughput of existing methods to identify and characterize novel drug compounds to treat these rare mutations.

This proposal outlines a novel approach integrating computational and biochemical techniques to enhance drug discovery efforts for CF. N1303K, the third most common mutation in CF, is prone to both endoplasmic reticulum-associated autophagy and proteasomal degradation, which current correctors fail to adequately address [DeStefano et al., 2018]. Previous work from the Plate Lab identified a critical binding pocket in proximity to N1303K within the NBD2, influencing the TMD1 intercellular loop 2 (ICL2) and closely interacting with the N-terminus of the Q-loop (residues 1284-1291). This pocket is shaped by nearby residues, including R1358, a key contributor to a hydrogen bond network essential for stabilizing the interaction between N1303K and Q1291 in the NBD2 crystal structure [Vernon et al., 2017]. We first aim to conduct virtual high-throughput drug screening using Rosetta’s REvolveD (Rosetta Evolutionary Docking) program, specifically targeting the N1303K CFTR variant at this pocket. Given that the N1303K variant remains unresponsive to conventional therapeutic strategies, including temperature correction, the lysosomal cysteine protease inhibitor E-64, and the autophagy inhibitor tubacin, yet shows positive reactions to a combination of Type-I and Type-II correctors (C18 and C4 respectively), we believe it is reasonable to explore this binding pocket for potential new corrector compounds [Rapino et al., 2015].

Following a comprehensive drug screen, we will employ time-resolved proteomics in CF human bronchial epithelial (CFBE) cells treated with novel correctors to assess if these compounds can effectively rescue the N1303K variant from degradation pathways. Several time-resolved proteomic methods are under pursuit in the Plate Lab, such as an mRNA-based flash cap system, halo-tag labeling, and whole-cell unnatural amino acid labeling with a click reaction. This integrated approach will accelerate the discovery and development of effective therapies for CFTR variants currently lacking effective treatment options, thereby expanding the therapeutic landscape, and improving outcomes for a wider array of CF patients. Successful outcomes from these experiments will potentially lead to groundbreaking treatments for over 1,500 patients homozygous for this mutation worldwide.

PI: Doug Kojetin, Biochemistry

Kelly’s research project will focus on the activation mechanism of an orphan nuclear receptor (NR) called Nurr1, which regulates a gene program influences dopamine production in the brain. Activation of Nurr1 transcription by drug-like small molecules is thought to be a potential therapeutic treatment for patients with neurodegenerative disorders, including Parkinson’s and Alzheimer’s diseases.

The draggability of Nurr1 remains unclear in the field. However, an alternative way to modulate Nurr1 transcription is through ligands that target retinoid X receptor alpha (RXRα), a NR that heterodimerizes with Nurr1, which regulate Nurr1 gene programs. One aspect of Kelly’s project will build on a recent advance my lab made using molecular biophysics, structural biology, and cellular pharmacology studies, which defined a new NR drug targeting paradigm. Instead of functioning through a classical mechanism of pharmacological activation, where ligand binding increases the recruitment of coactivator complexes to target genes, we found that Nurr1-RXRα activating ligands induce heterodimer dissociation (Yu et al. eLife 2023; doi: 10.7554/eLife.85039). Kelly will develop and optimize validate a Nurr1-RXRα protein-protein interaction high-throughput screening (HTS) assay and screen up to 100K compounds with the goal of discovering new compounds that dissociate the heterodimer. She will perform downstream assays to triage compounds and the profile activity of prioritized compounds using molecular biophysics, structural biology, and cellular methods.

A second aspect of Kelly’s project will involve proteomics methods to identify cellular coregulator proteins of Nurr1, as our studies and others indicate that Nurr1-mediated transcription may not occur via recruitment of classical NR coregulator proteins. Kelly will utilize mass spectrometry proteomic methods to capture and assess the Nurr1 protein interactome in neuronal cellular models used in the field to study Nurr1-regulated gene expression and dopaminergic cellular phenotypes. Interactome targets will be validated via overexpression and knockdown coupled to transcriptional reporter assays and gene expression analysis, and a cellular NanoBiT protein-protein interaction assay.

Kelly’s project is relevant to the mission of the CBI training grant as this work lies at the interface of chemistry and biology. The screening studies will involve collaboration with the VICB HTS core and medicinal chemists to design and test new compounds. The proteomics studies will involve collaboration with the VU mass spectrometry core.

PI: Jeff Spraggins, CPB

Most of our understanding of the molecular basis of human kidney disease comes from analyzing global mRNA expression or from proteomic and metabolomic interrogation of kidney lysates. Individual cellular and molecular changes are diluted in bulk tissue analyses such that inter-individual and disease associated variation can become undetectable, and rare cell populations that may be important in disease may not be identified. Single-cell transcriptional profiling is being used to uncover cellular heterogeneity associated with disease. However, the dissociation of tissues may affect the functional properties of cells, and these approaches forgo critical spatial relationships between cellular organization and molecular distributions. The Spraggins research group has been developing integrated multimodal molecular imaging technologies, bringing together imaging mass spectrometry with various forms of microscopy and spatial transcriptomics to address these important challenges and understand the molecular drivers of important diseases such as acute kidney injury and chronic kidney disease. My research project will expand on these development efforts by enabling, for the first time, functional information to be layered onto untargeted lipid imaging data. I will develop methods for mapping membrane fluidity in tissues using specialized lipid dyes that will be integrated with highly specific imaging mass spectrometry. This will enable altered lipid species and profiles to be correlated with membrane fluidity and associated with specific membrane microdomains (e.g., lipid rafts). I am particularly excited about this project as it requires me to develop skills and establish collaborations in the areas of analytical chemistry, cell biology, bioinformatics, and renal pathology. The opportunity to develop novel technologies and apply them to problems like diabetic nephropathy offers the potential to directly impact human health and provide me with training in cutting-edge bioanalytical techniques.