Sunday, April 28, 2024
4/28/2024
PROJECT SUMMARY/ABSTRACT Nek2, a ser/thr kinase, localizes to centrosomes and plays a crucial role in mitosis during centrosome disjunction. Aberrant Nek2 function has been linked to a wide variety of human diseases, including highly invasive cancers. Despite this, many of its cellular function remain poorly understood. This is primarily due to the lack of selective small molecule probes needed for functional studies. For example, temporal and spatial windows of Nek2 activation with regards to mitotic entry and exit are not well defined. Although it has been shown that a kinetochore-localizing protein Mad2 is a physiological substrate of Nek2 kinase, no Nek2 `activity' at the kinetochore (and microtubule) has ever been detected. Whether Nek2 activity is subject of regulation by other mitotic kinases, such as Aurora A and Cdc1, also remains a matter of speculation. In Aim 1 of this application, we plan to develop a Nek2-specific, activity-based, small molecule biosensor probe and demonstrate its utilizations in investigating the undocumented function of this kinase with exquisite temporal and spatial control. Specifically, we will (i) Develop efficient and selective fluorescence-based Nek2 biosensors (kcat/Km = 106-107 M- 1s-1); our working hypothesis is that upon phosphorylation by Nek2 kinase, biosensor probes will exhibit robust fluorescence enhancement (~300-800%) (ii) Construct photoactivatable analogues of developed Nek2 biosensor and demonstrate its utilization (as proof-of-principle) in monitoring Nek2 kinase activity at the kinetochore in live cells. The proposed study is innovative because it will result in development of FIRST highly selective and efficient small molecule biosensor of Nek2 kinase activity with a robust fluorescence readout. Furthermore, Nek2 kinase is notably overabundant in Triple Negative Breast Cancer (TNBC) – a highly metastatic cancer for which currently no targeted therapeutics exists. In our laboratory using a whole animal in-vivo model, we have shown that Nek2 kinase can cooperate with other oncogenes to promote metastasis. In this study, we also discovered a new Nek2 pharmacophore derived from EGFR-targeting drug candidates. Since the expression of both Nek2 and EGFR is strongly elevated in 38 percent of TNBC tumors, we hypothesize that an inhibitory agent capable of suppressing both Nek2 and EGFR kinase activities simultaneously will be an effective strategy for TNBC intervention. To test this hypothesis, in Aim 2 we propose to develop a new class of lead chemical agents that inhibit the function of both of these target oncogenes in TNBC. Once the lead compounds are identified, we will evaluate their efficacy in TNBC cell lines. By simultaneously inhibiting two complementary targets Nek2 and EGFR, our goal is to develop a lead compound that addresses the tumors' complexity and plasticity. Finally, the project proposed herein will provide our undergraduate students a direct hands-on research experience in area of medicinal chemistry, computational modeling, enzymology, and cell biology. Additionally, it will significantly enhance the research environment of Queens College, a primarily undergraduate institution.
HHS’ Tracking Accountability in Government Grants System (TAGGS) website provides access to detailed descriptions of grants, loans, aggregated direct payments and other types of financial assistance awarded by the HHS Operating Divisions (OPDIVs) and the Office of the Secretary Staff Divisions (STAFFDIVs).
