Anti-PLK1 monoclonal antibody, clone 9D23

Polo-like kinase 1 (PLK1), a member of the Polo-like kinase family, plays an important regulatory role in mitosis and cell cycle progression. PLK1 overexpression is correlated with tumourigenesis and poor prognosis in cancer patients. Therefore, the identification of novel compounds that inhibit PLK1 would provide attractive therapeutic approaches. Although some PLK1 kinase inhibitors have been developed, their application has been limited by off-target effects. PLK1 contains a regulatory domain named the Polo-box domain (PBD), which is characteristic only for the Polo-like kinase family. This domain represents an alternative therapeutic target with higher selectivity for PLK1. In this study, we applied in silico virtual drug screening, fluorescence polarization and microscale thermophoresis to identify new scaffolds targeting the PBD of PLK1. One compound, 3-{[(1R,9S)-3-(naphthalen-2-yl)-6-oxo-7,11-diazatricyclo[7.3. 1.0(2),(7)] trideca-2,4-dien-11-yl]methyl}benzonitrile (designated compound (1)), out of a total of 30,793 natural product derivatives, inhibited the PLK1 PBD with high selectivity (IC50: 17.9 +/- 0.5 mu M). This compound inhibited the growth of cultured leukaemia cells (CCRF-CEM and CEM/ADR5000) and arrested the cell cycle in the G2/M phase, which is characteristic for PLK1 inhibitors. Immunofluorescence analyses showed that treatment with compound (1) disrupted spindle formation due to the aberrant localization of PLK1 during the mitotic process, leading to G2/M arrest and ultimately cell death. In conclusion, compound (1) is a selective PLK1 inhibitor that inhibits cancer cell growth. It represents a chemical scaffold for the future synthesis of new selective PLK1 inhibitors for cancer therapy. (C) 2018 The Authors. Published by Elsevier B.V. on behalf of Cairo University.

Signals from cell surface receptors are often relayed via adaptor proteins. NCK1 and NCK2 are Src-Homology (SH) 2 and 3 domain adaptors that regulate processes requiring a remodeling of the actin cytoskeleton. Evidence from gene inactivation in mouse suggests that NCK1 and NCK2 are functionally redundant, although recent reports support the idea of unique functions for NCK1 and NCK2. We sought to examine this question further by delineating NCK1- and NCK2-specific signaling networks. We used both affinity purification-mass spectrometry and BioID proximity labeling to identify NCK1/2 signaling networks comprised of 98 proteins. Strikingly, we found 30 proteins restricted to NCK1 and 28 proteins specifically associated with NCK2, suggesting differences in their function. We report that Nck2(-/-), but not Nck1(-/-) mouse embryo fibroblasts (MEFs) are multinucleated and display extended protrusions reminiscent of intercellular bridges, which correlate with an extended time spent in cytokinesis as well as a failure of a significant proportion of cells to complete abscission. Our data also show that the midbody of NCK2-deficient cells is not only increased in length, but also altered in composition, as judged by the mislocalization of AURKB, PLK1 and ECT2. Finally, we show that NCK2 function during cytokinesis requires its SH2 domain. Taken together, our data delineate the first high-confidence interactome for NCK1/2 adaptors and highlight several proteins specifically associated with either protein. Thus, contrary to what is generally accepted, we demonstrate that NCK1 and NCK2 are not completely redundant, and shed light on a previously uncharacterized function for the NCK2 adaptor protein in cell division.