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Chemical Biology of Protein Phosphorylation and Proteolytic Enzymes
- Development of Nek2 Kinase Overexpression Model in Drosophila melanogaster for Studying Metastatic Signaling, and Development of Non-toxic Drug-Like Inhibitors. Read more
About forty thousand women die each year due to breast cancer occurrences in the United States alone. Nek2 is a dimeric Ser/Thr protein kinase that localizes to centrosomes and tightly regulates centrosome organization during the cell cycle. Recently published data reveals that the expression level of Nek2 kinase in the invasive breast cancer (IBC) cell is abnormally high (3-5 fold). Despite this, it remains unknown how exactly at the molecular level Nek2 transforms normal cells to malignant tumors. It also remains unclear if presence of overabundant Nek2 plays any important role in breast cancer metastasis. In addition, suitable inhibitory agents targeting Nek2 kinase with anti-cancer activity also remain scarcely available. Until now, to probe the potential role of Nek2 kinase in cancer, and during the inhibitor development process, only artificial cell-based system has been utilized. Unfortunately, this has led to a limited clinical success so far, since the onset and progression of cancer often involves a cooperation between multiple signaling pathways. To this end, Pathak’s laboratory, in collaboration with Prof. Ross L. Cagan's laboratory (formerly at Mount Sinai School of Medicine, NY), has developed the first Nek2 overexpression model in Drosophila melanogaster, and is utilizing them for the development of non-toxic Nek2-targeting anti-cancer agents. An activity-based small molecule biosensor of Nek2 kinase is also under development. The developed inhibitor, biosensor, and the whole animal models will be utilized to dissect the undocumented function of Nek2 kinase in both normal cellular physiology and breast cancer progression. This work is currently underway in collaborations with the laboratory of and Prof. Tanaji T. Talele (St. Johns University) and Prof. Karl Fath (Queens College).
T.K. Das, D. Dana, S.S. Paroly, S.K. Perumal, S. Singh, H. Jhun, J. Pendse, R.L. Cagan, T.T. Talele, S. Kumar, “Centrosomal Kinase Nek2 Cooperates With Oncogenic Pathways To Promote Metastasis”, Oncogenesis, 2013, 2, e69; doi:10.1038/oncsis.2013.34
*Financial Support for Nek2 Kinase project is provided by National Institute of Health (NIH) to SKP, Grant # 1R15CA243109-01
Left: Nek2 Overexpression phenotype (vs wt)
Center: Tumor Cell Injection assay shows migration (metastasis) in adult flies
Right: Inhibitor screen using the developed overexpression fly model identifies two drug-like Nek2 inhibitory agents with IC50s as 282 nM (neratinib) and 673±7 nM (pelitinib).
- Development of Clickable and Tagless Inhibitory Probes of Human Cathepsin L and Cathepsin B for Functional Proteomics
Cysteine cathepsins have recently emerged as critically important group of proteolytic enzymes needed for maintenance of proper cellular function. Either gain or loss of function as a result of their misregulation has been directly associated with a variety of human pathologies, such as cancer, osteoporosis, and autoimmune and metabolic disorders. In addition to their importance in human biology, cysteine cathepsins are also implicated in many other types of diseases involving lower organisms such as viruses and parasites. Originally, cysteine cathepsins were considered proteases of endolysosomal compartment where their primary function was only to degrade the unwanted cellular proteins. The functional redundancy and the overlapping substrate preferences among the members of cysteine cathepsins were therefore considered a non-issue. However, recent decade of research work suggests to a highly specialized and context-specific function for the individual members of cysteine cathepsin enzyme family. To investigate cathepsin function, standard genetics, proteomics, and biochemical techniques have been utilized that primarily rely on the cellular ‘expression’ profile. It should, however, be noted that all newly synthesized cellular cysteine cathepsins are subject to (a) post-translational processing for activation and cellular translocation, and (b) regulation by endogenous inhibitory proteins (stefins and cystatins). For these reasons, it is imperative that technology that can directly render information about the ‘activity’ states of cellular cathepsins be developed and utilized for functional investigation. The research in Pathak's group focuses on two closely related members of cysteine cathepsins, cathepsin L and B, whose functional roles in appropriate cellular context remain poorly understood. In some cells their individual function seem redundant, and in others non-redundant and specific. The current emphasis is on development of (a) highly selective inhibitory agents, and (b) clickable and tagless (for enhanced potency) activity-based probes of cathepsin L and B that are suitable for quantitative measurement of cellular activities (not just the expression level) in living cells. The results obtained are anticipated to significantly advance our current understanding of cathepsin L and B biology, and help develop novel therapeutics.
**Financial Support for cathepsin project is provided by National Science Foundation (NSF) to SKP, Grant #1709711
Highly potent, selective, cell permeable inhibitor of human cathepsin L, KD-1 (kinact = 4.3 x 106 M 1s-1; IC50 = 3.6 ± 0.1 nM).
Highly potent, selective, cell permeable inhibitor of human cathepsin L, KD-1 (kinact = 4.3 x 106 M 1s-1; IC50 = 3.6 ± 0.1 nM).
(From Dana and Kumar et al., ChemComm (Camb.), 2014, 50, 10875-10878
[A] Chemical structure of KD-1 [B] Dose–response curves for KD-1-mediated inhibition of human liver cathepsin L. [C] Progress curve for cathepsin L-catalyzed hydrolysis of a fluorescent substrate, Z-Phe-Arg-(7-Amino-4-methylcoumarin) with various concentration of KD-1. [D] KD-1 is cell permeable and can block intracellular cathepsin L activity in live MDA-MB-231 cell line.
Development of Clickable and Tagless Activity-based Probe (catABP) of Human Cathepsin L and Cathepsin B for Functional Proteomics
Development of Clickable and Tagless Activity-based Probe (catABP) of Human Cathepsin L and Cathepsin B for Functional Proteomics
(Dana and Pathak et al., Bioorganic Chemistry, 2019, 85: 505-514
Top: Chemical structure of catABP of human cathepsin L. Bottom: [A] Imaging of active human cathepsin L, labeled with ctABP, KDP-1. [A]: Purified and activated recombinant human cathepsin L was incubated with KDP-1 probe. After reaction was quenched, the labeled enzyme was clicked with 5-TAMRA-azide, resolved using SDS-PAGE, and detected using Typhoon 9400 scanner (ex: 532 nm, em: 580 nm). The experiment indicates that as little as 1 ng (40 fmol) of active human cathepsin L can be detected. [B] Control experiments: (a) heat-denatured inactivated human cathepsin L was subjected to identical labeling and click chemistry procedures; no labeling was observed (50 ng loaded) (b) Identical labeling and detection procedures were performed as in [A], but in presence of a competitive cathepsin L inhibitor KD-1. Significantly reduced amount of human cathepsin L labeling was observed, as expected.
Other Collaborations
Other Collaborations
Development of Small Molecule Probes of Poly (ADP-ribose) polymerases (PARPs) Prof. Tanaji T. Talele (St. John's University, Queens, USA
Small Molecule Ligand for Deciphering Neuropeptide Signaling Prof. Lakshmi Devi (Icahn School of Medicine at Mount Sinai, NY, USA
Prof. Erin Bobeck (Utah State University, Logan, Utah, USA
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