Trainee Research Descriptions

CBTP Trainee Research Descriptions

Timing the Kinome: Kinetic Selectivity of Kinase Inhibitors

Kinases are key modulators of cellular signaling with significant implications in oncology, making them among the most critical drug targets of the 21st century. The development of selective kinase inhibitors is a major challenge given the high structural similarity within the kinome. Most drug discovery programs select and optimize drug leads based on thermodynamic selectivity metrics such as IC50 and Kd. This approach often overlooks kinetic selectivity, which is defined as the ability of a drug to remain bound to its intended target while rapidly dissociating from off-targets. Leveraging kinetic selectivity may improve drug efficacy and reduce side effects. My project aims to explore the utility of kinetic selectivity in developing selective kinase inhibitors. The first target is Bruton's Tyrosine Kinase (BTK), which has been validated in treating B-cell malignancies with four FDA-approved BTK inhibitors and many more in clinical trials. However, selectivity issues have raised safety concerns about liver and cardiovascular damage. I am employing Promega's NanoBRET technology to investigate kinetic selectivity in live cells, offering a measure of selectivity that is more physiologically relevant than traditional biochemical methods using purified proteins, due to its ability to monitor interactions in real-time. These experiments will provide insight into drug-target interactions to assess kinetic selectivity of the inhibitors by comparing their dissociation rates for the intended target i.e., BTK versus potential off-targets, such as CSK, SYK, FRK, and LYN. Furthermore, I am developing a mechanistic model that incorporates kinetic parameters such as on- and off-rates to mathematically model the intracellular drug-target binding kinetics, predicting drug behavior in more physiologically relevant conditions. Finally, cryo-electron microscopy (CryoEM) will be used to elucidate the molecular mechanisms underlying the kinetic selectivity observed in BTK inhibitors, potentially revealing conformational changes upon inhibitor binding that drive selectivity.

A Computational Investigation of the Chaperone-Usher Pathway

Type 1 pili are an important virulence factor in uropathogenic E. coli, and a better understanding of their synthesis can open the door to new treatment options. My research focuses on investigating the chaperone-usher pathway using molecular dynamics simulations. I aim to construct accurate models of the proteins involved in the pilus synthesis pathway and to use enhanced sampling techniques to gain mechanistic insight into pili biogenesis. This work seeks to provide a granular understanding of the atomistic interactions and free energy landscapes that drive pili assembly.

Structure-Guided Design of Isoform-Specific Cyclophilin Inhibitors

Strokes are one of the leading causes of death worldwide, and ischemic strokes account for about 87% of strokes in the US and about 62% of strokes globally. Ischemic strokes are made more complicated by ischemic-reperfusion injury (IRI), in which cells begin to rapidly dysfunction and die during the restoration of blood flow to the ischemic tissue. In the heart and brain, IRI is exacerbated by dysregulation of the mitochondrial transition pore (mPTP). Cyclophilin D (CypD) is a peptidylprolyl isomerase and is a key modulator of the mPTP, where binding of CypD to the mPTP favors the open conformation of the pore. Inhibition of CypD lowers the probability of pore opening when there is substantial inorganic phosphate, Pi, present, suggesting CypD inhibition is a viable therapeutic target.

Currently, there is only one FDA-approved cyclophilin inhibitor, Cyclosporine A (CsA), which, while potent, is non-specific and binds to several cyclophilin isoforms, leading to unwanted side effects. Our group has previously shown that building compounds into the specificity pocket of the CypD active site is a promising approach to developing isoform-specific inhibitors, though these initial compounds had poor cell permeability.

To address this, we are using a fragment-based drug design approach in combination with computational docking to identify novel CypD inhibitors with more desirable small molecule chemistries. We then validate the computational results with traditional biochemical and structural biological approaches.

Role of Vacuolar-ATPase in Early and Late Endosomal Trafficking

The vacuolar-ATPase (V-ATPase) is an enzyme complex in eukaryotes that translocates protons across cell membranes. While its canonical function as a proton-pumping nanomotor has been well characterized, its noncanonical functions contributing to protein-protein interactions and cell signaling events remain less explored. Using cryo-electron microscopy and biochemical applications, our goal is to provide insight towards the V-ATPase's mechanistic roles in these processes through structural and functional analysis. Specifically, my work focuses on the V-ATPase's role in early and late endosomal trafficking.

Designing Modular Caged Cyclopropenes for Spatiotemporal Cysteine Labeling

Cysteine modifications are central regulators of protein function, redox signaling, and stress adaptation in the body, especially in redox-sensitive environments like the brain. These modifications enable dynamic and reversible cellular adaptation by altering protein structure, interactions, and activity in response to oxidative cues. Such modifications include oxidation, nitrosation, sulfinylation, sulfenylation, sulfonylation, etc. Dysregulation of these processes, specifically those involving oxidation, has been implicated in numerous neurological disorders, cancers, and autoimmune diseases that threaten human health.

Despite their importance, conventional proteomic approaches struggle to resolve cysteine modifications in living, heterogeneous systems, as they are typically performed in lysates. Additionally, most probes used in these methods are constitutively reactive and lack cell specificity, thereby sacrificing both temporal and spatial resolution and making it difficult to determine when and where these modifications occur.

To address this challenge, I am developing fast, modular caged cyclopropene probes that remain inert until activated by user-defined stimuli (light, enzymes, or both). These probes enable the detection of cysteine modifications in specific cells at defined time points in vivo (spatiotemporally), while reducing background labeling. This approach will allow us to capture transient cysteine-mediated processes and provide insight into how these modifications regulate protein activity and contribute to disease.

Chemical and Structural Determinants of Mycomembrane Fluidity and Their Correlation with Antibiotic Susceptibility in Mycobacteria

The cell envelope of mycobacteria is a formidable barrier, distinguished by multiple lipid-rich membrane layers and an outer membrane (mycomembrane) composed of exceptionally long-chain lipids (60-90 carbons) known as mycolic acids. The cell envelope is implicated in mycobacteria’s intrinsic resistance to antibiotics, and there is a prevailing hypothesis in the field that this resistance arises from the unique physical properties of the mycomembrane. One such property is membrane fluidity, which is understood as the dynamic and lateral ordering of membrane components such as lipids and proteins. The current thinking in the field is that mycomembrane fludity is largely determined by mycolic acids because (1) they are a major component of the mycomembrane, and (2) their covalent attachment to underlying arabinogalactan layers provides physical constraints. However, these assumptions about the relationship between mycolic acids, mycomembrane fluidity, and intrinsic antibiotic resistance are supported by little direct evidence. Moreover, few biophysical measurements of membrane properties have been made in live, intact cells for which the complex architecture of the cell envelope is preserved. Recently, the Seeliger group pioneered an imaging- and flow cytometry-based method for measuring membrane fluidity in live mycobacteria using the membrane-targeting and polarity-sensitive fluorophore C-laurdan. My project will leverage this approach to characterize how mycomembrane fluidity is related to 1) the chemical and structural features that govern mycolic acid conformational packing within the cell wall, and 2) antibiotic susceptibility profiles. I hypothesize that (1) mycolic acid structure and composition are major determinants of mycomembrane fluidity, and (2) cell envelope and mycomembrane fluidity correlate with antibiotic susceptibility. By untangling this relationship, this project will bridge a critical gap in our knowledge of how mycomembrane properties relate to bacterial physiology and antibiotic susceptibility, ultimately paving the way for new therapeutic strategies for treating tuberculosis and other mycobacterial infections.

Diketopiperazine Cleavable Linkers: A Novel Approach to PET Imaging

Positron emission tomography (PET) radiopharmaceuticals have rapidly advanced as powerful tools for noninvasive, functional imaging across diseases such as cancer, infection, and inflammation, yet significant challenges remain in extending their utility to complex systems like the blood–brain barrier (BBB). PET uniquely enables real-time, quantitative visualization of biological processes through radiolabeled tracers (commonly fluorine-18), whose production, radiosynthesis, and clinical deployment require highly controlled, multi-step workflows that are often inefficient and failure-prone. While established tracers such as ¹⁸F-FDG have transformed diagnostics, limitations in specificity, sensitivity, and synthesis scalability highlight the need for improved radiochemistry methods. Current radiolabeling strategies—including direct fluorination, prosthetic group chemistry, and solid-phase synthesis—often rely on harsh conditions and extensive purification, restricting their applicability to sensitive biomolecules and time-critical clinical settings. These challenges are particularly evident in BBB imaging, where existing modalities lack either sensitivity, specificity, or practicality, and PET approaches are hindered by short isotope half-lives and complex dual-tracer requirements. To address these limitations, this project proposes adapting a diketopiperazine (DKP)-based solid-support strategy to enable rapid, mild, and purification-free ¹⁸F radiolabeling in aqueous, near-physiological conditions. By integrating medicinal chemistry with streamlined radiosynthesis, this approach aims to expand the pool of viable PET tracers and develop a novel BBB imaging agent capable of selectively accumulating in regions of barrier disruption, providing a fully quantitative and biologically specific method for assessing neurodegenerative disease.

Development of Novel Atropostable Kinase Inhibitors

Andrew's research focuses on developing novel atropisomerically-stable analogues of existing kinase inhibitors, as well as chemical methodologies to synthesize such compounds asymmetrically. These compounds are designed with the hypothesis that introducing atropisomerism into kinase inhibitor scaffolds will improve their selectivity for specific kinases, addressing the problem of poor target selectivity among existing kinase inhibitors.

Regulation of the T3SS and T6SS through the NahK/HptB phosphorelay signal in Pseudomonas aeruginosa

Pseudomonas aeruginosa (P. aeruginosa) is a Gram-negative, opportunistic pathogen known for causing nosocomial infections, such as lung infections of cystic fibrosis patients, ventilator-associated pneumonia, catheter-based infections, and topical infections of burn victims and surgery patients. The pathogenicity of P. aeruginosa stems from the bacteria’s ability to form biofilms, allowing the bacteria to survive in the presence of host immune responses. Biofilms are multicellular communities formed when bacteria use extracellular appendages to attach to a surface and self-encapsulate within a thick matrix called the extracellular polymeric substance (EPS). The transition from a planktonic, motile state to a sessile state is regulated by multiple signal transduction systems that modulate intracellular levels of the signaling molecule cyclic-di-GMP. This second messenger functions as a cofactor for proteins involved in biofilm-related gene transcription. As P. aeruginosa is becoming progressively resistant to current antibiotics and treatment regimens due to biofilm formation, there is an increasing need for developing new and alternative methods of treating infections caused by this pathogen. Investigations into biofilm dispersal have shown promising results that, if characterized correctly, could improve the treatment of these infections.

P. aeruginosa secretes many virulence factors through secretion systems to cause colonization, cytotoxicity, and evasion of an innate host immune system. This pathogen utilizes Type III Secretion Systems(T3SS) and Type VI Secretion Systems(T6SS), which function antagonistically to control the switch between acute-to-chronic infections, respectively. Our lab has established phenotypic characterizations in the NahK/HptB multikinase network to support the current findings that NahK is a central regulator of biofilm formation through the HptB/RsmA system, however our data also shows that some of the phenotypes observed, such as increased intracellular c-di-GMP, EPS production, and increased transcriptional expression of T3SS, function independently of RsmA through unknown regulatory mechanisms. This project aims to investigate how the phosphorylation from NahK to HptB can regulate the production of intracellular c-di-GMP and the observed phenotypes through a RsmA-independent pathway. We also aim to probe the characterized cyclic-di-GMP regulating enzymes, known to directly modulate the HptB network, to better understand the role of cyclic-di-GMP in the transcription of secretion systems.

Novel Anionic Sphingolipids in Caulobacter crescentus

Gram-negative bacteria possess a cellular envelope consisting of an inner membrane and outer membrane. The inner membrane is structurally and functionally similar to eukaryotic plasma membranes, while the outer membrane is an asymmetric bilayer where the inner leaflet consists of phospholipids and the outer leaflet comprises mainly the lipopolysaccharide (LPS). The LPS is essential for gram-negative bacterial survival by maintaining cellular integrity and providing a protective barrier from external stressors. To complement the LPS on its outer membrane, Caulobacter crescentus produces an anionic sphingolipid, ceramide poly-phosphoglycerate (CPG2), which can permit the survival of the bacterium without the LPS. This novel anionic sphingolipid’s role in outer membrane function in Caulobacter crescentus and its putative production in other bacterial species challenge the textbook understanding of gram-negative bacterial outer membranes and sphingolipid rarity in bacteria. Therefore, it is essential to study CPG2’s biosynthetic pathway. Previous works identified a series of genes that encode proteins required for CPG2 synthesis, but only a limited subset of these proteins has been ascribed enzymatic functions. My project focuses on elucidating the biochemical functions of the remaining proteins in this novel pathway and determining their structures bound to their substrates and products. This work will aim to characterize the complete biosynthetic pathway for this novel class of sphingolipids in gram-negative bacteria and provide insight into the involved proteins’ mechanisms of action.

Mechanistic Insights and Advances in Pyridoxal-Inspired Charge Transfer Photocatalysis

Pyridoxal-5’-phosphate is an enzymatic cofactor that facilitates the transformation of canonical amino acids into other useful molecules, such as neurotransmitters, within biological systems. Harnessing the stereoelectronic features on the pyridoxal scaffold, templated with an amino acid, provides an opportunity for charge transfer photocatalysis, as demonstrated in the ambient temperature decarboxylation reported by Lipshultz and coworkers. My current project investigates the mechanism of the decarboxylation platform further; specifically, radical clock studies are being conducted to probe the nature of a key diradical intermediate. Through the design and application of photocyclization amino acid substrates, insights into the rate of hydrogen atom transfer and radical philicity (illustrated via a Hammett plot) can be deduced. These results will permit strategic development of new bioinspired synthetic transformations utilizing cofactor organocatalysis.