Vanderbilt Chemical Biology Interface Training Program

2020 Trainees

Katherine Clowes (Biochemistry, PI Charles Sanders)

Long QT syndrome (LQTS) is a disorder characterized by the prolongation of the latter portion of the electrocardiogram signal (the QT interval) that increases risk of arrythmias and cardiac arrest. The most common form of LQTS, type 1 long QT syndrome (LQT1), is caused by mutations in the voltage gated potassium channel protein KCNQ1. While over 250 LQT1-associated mutations in KCNQ1 have been identified, the impacts of these mutations on the channel’s structure and function are still largely unexplored. The Sanders lab’s goal is to investigate how these mutations lead to protein dysfunction. Previous studies of mutations in the KCNQ1 voltage sensing domain found that many were destabilized and exhibited reduced cell surface trafficking. This led to the hypothesis that mistrafficking due to protein destabilization is a common cause of KCNQ1 loss of function in LQT1. I plan to determine if this theme is common to mutations in other domains of KCNQ1 by characterizing a selection of mutations in the KCNQ1 pore domain for their impact on protein expression, trafficking, stability, and function. I also plan to test the hypothesis that protein destabilization is the most common cause of mistrafficking by conducting high throughput screening for molecules that bind KCNQ1 and restore trafficking by stabilizing the protein.

Henry Schares (Chemical & Physical Biology, PI Brian Bachmann)

Modern drug discovery screening campaigns screen small molecule libraries that are usually limited to characterized chemical space and rarely include natural product (NP) scaffolds, even though each year NPs make up a large proportion of newly FDA approved drugs. In addition, these screens often focus on a single target or a simplified readout of bioactivity (eg. Cytotoxicity). This can lead to investigators overlooking compounds with novel therapeutic mechanisms and spending time and resources fruitlessly advancing leads with nonspecific activity. Our lab, in collaboration with Dr. Jonathan Irish and Dr. Brent Ferrell, previously developed the Multiplexed Activity Metabolomics (MAM) platform, a fluorescence cell barcoding and multiplexed immunoassay that allows for simultaneous assessment of multiple phenotypes such as cell type, apoptosis, cell cycle status, DNA Damage, and various cell signaling markers to paint a more nuanced picture of compound bioactivity at the single cell level. In collaboration with the NCI Natural Product Division (NCI-NPD), my project uses the MAM platform to screen a library consisting of pre-fractionated plant and marine invertebrate extracts against Acute Myeloid Leukemia (AML) cells. This library was curated by the NCI-NPD as an effort to make NP discovery compatible with high throughput screening. Because the contents of each library well are a mixture of NPs unknown in structure and number, my screening efforts are able to investigate uncharacterized chemical space, but each “hit” requires identification of the bioactive compound within the extract fraction. To do this I use the MAM platform in which the assay is preceded by fractionating and dispersing the compounds in the “hit” extract across assay wells while collecting chromatographic data to create a well-content specific chromatographic map via HPLC-MS. This aids in rapid dereplication of known compounds and rapid activity-guided isolation of unknown compounds that will go on to structure elucidation using 2D NMR techniques, target identification, assessment in primary samples, and mechanistic investigation in the context of AML therapy development.

Ruben Torres (Chemistry, PI Sandra Rosenthal)

Single Particle Tracking of Disease-linked Neuronal Signaling Membrane Proteins Using Fluorescent Nanocrystals.

The dopamine transporter (DAT) is a transmembrane protein that modulates dopamine (DA) signaling amplitude and duration in the brain by driving rapid DA reuptake into the presynaptic nerve terminal. Several lines of evidence indicate that missense mutations, in particular A559V, result in DAT dysfunction that is linked to neuropsychiatric disorders, such as bipolar disorder, attention deficit hyperactivity disorder, and autism spectrum disorder. Our goal is to link transporter surface mobility with function. What is still unknown is how protein partner interactions influence DAT A559V lateral diffusion and the degree to which DAT, and its mutant variants, diffuse and cluster in vivo. Semi-conductor nanocrystal quantum dots (QDs) offer advantageous photophysical properties such as remarkable photostability and narrow emission spectra for single particle tracking (SPT) using optical microscopy. QDs are biofunctionalized with antagonist drug derivatives for specific DAT labeling. My research project encompasses both diffusion dynamics characterization of DAT A559V with respect to agonism/antagonism of speculated protein partners, such as the DA receptor as well as probe optimization for SPT of endogenous DAT at presynaptic nerve termini in acute striatal mouse brain slices to evaluate different diffusional states. If disturbed DAT diffusion dynamics can be linked to the central cause of the previously stated diseases, then novel diffusion recovery therapeutics can be investigated, potentially supplementing classical agonist/antagonist drug targeting.

Jennifer Wurm (Quantitative & Chemical Biology, PI Lars Plate)

The CDC estimates 2.8 million people are infected each year with antibiotic-resistant bacterial infection. Without changes in current therapeutic approaches, these diseases will eventually become incurable. During my research in The Vanderbilt Laboratory for Biosynthetic Studies (VLBS), directed by Dr. Brian Bachmann, I am investigating everninomicin—an antibiotic effective against gram-positive antibiotic-resistant bacteria, produced by the organism Micromonospora carbonacea. Everninomicin inhibits bacterial protein translation by interacting with the ribosomal protein (rProtein uL16) in a binding pocket on the 50S ribosome. I analyze this structure-activity relationship by investigating how structural changes made to the everninomicin scaffold would affect binding affinity and translation inhibition. Structural analogs can be generated through use of the organism’s biosynthetic mechanisms. Of particular interest is structural biology of multiple orthoester functional groups throughout the everninomicin structure that interact with the ribosome. Additional possible engineering sites involving these orthoesters can be identified through mechanistic and biochemical studies of orthoester synthase by identifying the substrates through knockout methods and studying the enzyme-substrate interactions through metabolomics.

The major requirements of the program are Fundamentals of Chemical Biology(CPB 8320), register two semesters for Graduate Seminar in Chemical Biology(CPB 8310), one cross-disciplinary course (chemistry or one from a Basic Science department or CPB/IGP programs), Rigor and Reproducibility (PHARM 8328), and participation/membership in the Chemical Biology Association of Students (CBAS) program.