Editorial Board of Journal of Pharmaceutical Research and Reviews
Dr. Vandna Kukshal
Department of Internal medicine
Washington University School of Medicine
Biography & Research Interest
Welcome Dr. Vandna Kukshal to Join the Editorial Board of Journal of Pharmaceutical Research and Reviews.
Post doc research, Dr. Ron Bose lab, Department of Internal medicine, Washington University School of medicine, Saint Louis, Washington University School of medicine, Saint Louis
My Postdoctoral work is focused on structural and functional characterization of epithelial growth factor Receptor (EGFR). The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that couples the binding of extracellular ligands, such as EGF and transforming growth factor-a (TGF-α), to the initiation of intracellular signaling pathways that control cell growth and proliferation. Signaling from EGFR family members underlies the onset of many cancers, and there is intense interest in understanding how these receptors are regulated. As one part of project we are trying to structurally characterize an intrinsically disordered C-terminal tail (CTT) 26 kDa domain of EGFR which undergoes phosphorylation and further recruits downstream signaling proteins. By using Small angle X –ray scattering and Mass spectroscopy techniques, we are characterizing mechanism of conformational change from unphosphorylated to phosphorylation state. In other part of the project, we are trying to develop one assay to measure dimerization of Kinase domains of EGFR using Mass spectroscopy as well as TR-FRET assay.
Post-doctoral research, Dr. Tom Ellenberger lab, Department of Biochemistry and Molecular Biophysics, Washington University School of medicine, Saint Louis
My postdoctoral work is focused on structural and functional characterization of Human DNA ligase III (LigIII), a crucial enzyme for cell survival. I characterized the role of ZnF domain of Lig III in bridging of two DNA ends together prior to intermolecular DNA ligation. I developed a TR-FRET based assay to demonstrate the bridging of two non ligatable DNA and ZnF deleted mutant was not able to do. I used Small angle X ray (SAXS) studies of different truncation mutant of ligase III to show the dynamics of two independent DNA binding modules of Lig III involved in intermolecular ligation (Kukshal V et al; NAR 2015). Ligase III expression is significantly elevated in chronic myeloid leukaemia (CML) raise the possibility of using Lig III inhibitors in combination with traditional DNA damaging cancer therapies and/or PARP-1 inhibitors to selectively kill cancerous cells. Inhibitors would also be useful tools to study the functional overlap of Lig III with other DNA ligases and its role in mitochondrial DNA replication and repair. So, to identify inhibitors of Lig III, I have developed a high throughput assay for DNA nick ligation activity and screened the Maybridge RO3 chemical fragment library to identify selective inhibitors of LigIII based on a counter screen of the library with Lig I. A group of chemical scaffolds identified by our screening experiments is undergoing further optimization using substructure and topology searching for analogs with improved potency (Kukshal V et al; Manuscript under preparation).
PhD research, R. Ravishankar lab, Central Drug Research institute Lucknow, India
My thesis work is focused on biochemical and structural characterization of eubacterial DNA ligases, which are essential for survival of eubacteria. In this context I have mainly worked on Rv3014c, a NAD+ dependent DNA ligase (LigA). I have crystallized the deletion mutant of this enzyme and solved the structure and got a unique conformation of the bound nucleotide (PDB: 3SGI). As a part of thesis work we proved the role of BRCT domain of DNA ligase A in its activity. I made site direct mutations also to probe role of different conserved residues in reaction mechanism of DNA ligation (Srivastava SK and Kukshal V; Proteins 2007). DNA ligase is an essential enzyme for different DNA transactions. So I was interested to probe how it recruits at particular pathway. For this I tried to identify the proteins which might interact with DNA ligase A and recruits it in different DNA repair pathways. I cloned, purified and crystallized and solved the structure of eubacterial DNA beta clamp and characterize its interaction with DNA ligase III (Kukshal V et al, Plos One 2012). NAD+ -dependent DNA ligase has attracted interest as a prospective broad-spectrum antibacterial target because it’s present in eubacteria and different from the eukaryotic ATP dependent DNA ligases. By using virtual screening followed by in vitro and cell based assays I identified two different classes of inhibitors specifically inhibit bacterial DNA ligase in low micromolar range (Kukshal V et al; MedChem. Comm. 2011) (Kukshal V et al; MedChem. Comm. 2012). These small molecules are in process of further optimization to increase their potency.
Structural Biology: Protein purification, Protein crystallization and X-ray diffraction Data collection and Structure determination: MR, SAD and MAD. (Program) HKL2000, CCP4, COOT Phenix (phaser, solve/resolve), CNS, molecular graphics (Pymol, Chimera, CCP4MG,), sharp, Small angle X ray scattering.
Molecular Biology and Biochemistry: Experience in many aspects of Molecular Biology and Protein Chemistry including cloning/subcloning, Gibson assembly, purification of soluble and insoluble proteins (including refolding), affinity-tagging, chromatography, electrophoresis etc., biophysical characterization with techniques like gel electrophoresis including native, SDS PAGE, CD spectroscopy, Dynamic Light Scattering, Isothermal titration calorimetry (ITC) and chromatographic methods including gel filtration, DNA –Protein interaction by EMSA and Fluorescence anisotropy assay.
Drug discovery: Biochemical assay development (fluorescence based and TR-FRET based assay) and chemical library screening, protein–ligand binding studies using Surface Plasmon Resonance (SPR), ITC and Thermal shift assays, DNA –Drug interaction, Cell based assay development, In silico docking studies using Glide and Autodock.