Polo-like kinase 1 (Plk1) is a key regulator of mitotic progression and cell division in eukaryotes. It is highly expressed in tumor cells and considered a potential target for cancer therapy. Here, we report the discovery and application of a novel potent small-molecule inhibitor of mammalian Plk1, ZK-Thiazolidinone (TAL). We have extensively characterized TAL in vitro and addressed TAL specificity within cells by studying Plk1 functions in sister chromatid separation, centrosome maturation, and spindle assembly. Moreover, we have used TAL for a detailed analysis of Plk1 in relation to PICH and PRC1, two prominent interaction partners implicated in spindle assembly checkpoint function and cytokinesis, respectively. Specifically, we show that Plk1, when inactivated by TAL, spreads over the arms of chromosomes, resembling the localization of its binding partner PICH, and that both proteins are mutually dependent on each other for correct localization. Finally, we show that Plk1 activity is essential for cleavage furrow formation and ingression, leading to successful cytokinesis. INTRODUCTIONThe error-free segregation of chromosomes during cell division is necessary for the maintenance of correct ploidy and genomic integrity, and errors in cell division are presumed to lead to aneuploidy and cancer (Rajagopalan and Lengauer, 2004). To ensure that daughter cells receive the correct complement of chromosomes, two key events need to be coordinated. First, chromosomes must be equally segregated, a process that depends on the mitotic spindle. Second, cytokinesis, the process dividing the cell into two, must occur between the two sets of segregated chromosomes. Both of these processes require the activity of a key cell cycle regulator, the Polo-like kinase 1 (Plk1). Plks form a conserved subfamily of serine/threonine protein kinases. The first member to be identified was Polo in Drosophila melanogaster (Llamazares et al., 1991) and, subsequently, four Plk family members have been identified in mammals Barr et al., 2004).Plk1 contains an N-terminal kinase domain and a phosphopeptide-binding C-terminal regulatory polo-box domain (PBD; Leung et al., 2002;Elia et al., 2003b). In vertebrates Plk1 has been implicated in the activation of Cdk1-cyclin B upon entry into mitosis, centrosome maturation via the recruitment of the ␥-tubulin ring complex (␥-TuRC), spindle formation, sister chromatid separation by cohesin removal from the chromosome arms, promotion of anaphase onset through direct phosphorylation of the APC/C complex as well as the inhibition of the APC/C inhibitor Emi1, and finally, mitotic exit and cytokinesis (reviewed in Barr et al., 2004). Fitting with these diverse functions, Plk1 localizes to the centrosomes, spindle poles, and kinetochores in prophase and metaphase, the central spindle in anaphase, and the midbody during cytokinesis. These localizations require the function of the PBD (Jang et al., 2002;Seong et al., 2002) and priming-kinases to generate phosphorylated docking sites that are subsequently recog...

The DNA damage response (DDR) secures the integrity of the genome of eukaryotic cells. DDR deficiencies can promote tumorigenesis but concurrently may increase dependence on alternative repair pathways. The ataxia telangiectasia and Rad3-related (ATR) kinase plays a central role in the DDR by activating essential signaling pathways of DNA damage repair. Here, we studied the effect of the novel selective ATR kinase inhibitor BAY 1895344 on tumor cell growth and viability. Potent antiproliferative activity was demonstrated in a broad spectrum of human tumor cell lines. BAY 1895344 exhibited strong monotherapy efficacy in cancer xenograft models that carry DNA damage repair deficiencies. The combination of BAY 1895344 with DNA damage-inducing chemotherapy or external beam radiotherapy (EBRT) showed synergistic antitumor activity. Combination treatment with BAY 1895344 and DDR inhibitors achieved strong synergistic antiproliferative activity in vitro, and combined inhibition of ATR and PARP signaling using olaparib demonstrated synergistic antitumor activity in vivo. Furthermore, the combination of BAY 1895344 with the novel, nonsteroidal androgen receptor antagonist darolutamide resulted in significantly improved antitumor efficacy compared with respective singleagent treatments in hormone-dependent prostate cancer, and addition of EBRT resulted in even further enhanced antitumor efficacy. Thus, the ATR inhibitor BAY 1895344 may provide new therapeutic options for the treatment of cancers with certain DDR deficiencies in monotherapy and in combination with DNA damage-inducing or DNA repair-compromising cancer therapies by improving their efficacy.

Background: BRD4 is a reader of acetylated histones.Results: Mutational analysis of BRD4 BD1 allowed the identification of three groups with different binding profiles.Conclusion: Pro-82, Leu-94, Asp-145, and Ile-146 have a differentiated role in acetyl-lysine and inhibitor interaction.Significance: Identification of residues essential for BRD4 function will guide the design of novel inhibitors.

Protein C inhibitor (PCI) is synthesized by cells throughout the male reproductive tract and is present in high concentrations (220 micrograms/ml) in seminal plasma. Seminal plasma as well as the acrosome of spermatozoa are rich in possible target proteases for PCI. We analyzed the interaction of PCI with acrosin, a serine protease stored in its zymogen form in the acrosome of spermatozoa. Purified human PCI inhibited the amidolytic activity of purified boar acrosin with an apparent second-order rate constant of 3.7 x 10(4) M-1.s-1. Inhibition was paralleled by the degradation of PCI from its 57- to its 54-kDa form. Human PCI also inhibited the amidolytic activity of activated human sperm extracts and formed complexes with acrosin as determined by an enzyme-linked immunosorbent assay. Immunocytochemistry revealed that morphologically abnormal spermatozoa stained for PCI antigen, whereas morphologically normal spermatozoa were negative. In immunoelectron microscopy, PCI was exclusively localized in the immediate vicinity of disrupted acrosomal membranes of sperm heads. These data suggest that PCI might function as a scavenger of prematurely activated acrosin, thereby protecting intact surrounding cells and seminal plasma proteins from possible proteolytic damage.

The ATR kinase plays a key role in the DNA damage response by activating essential signaling pathways of DNA damage repair, especially in response to replication stress. Because DNA damage and replication stress are major sources of genomic instability, selective ATR inhibition has been recognized as a promising new approach in cancer therapy. We now report the identification and preclinical evaluation of the novel, clinical ATR inhibitor BAY 1895344. Starting from quinoline 2 with weak ATR inhibitory activity, lead optimization efforts focusing on potency, selectivity, and oral bioavailability led to the discovery of the potent, highly selective, orally available ATR inhibitor BAY 1895344, which exhibited strong monotherapy efficacy in cancer xenograft models that carry certain DNA damage repair deficiencies. Moreover, combination treatment of BAY 1895344 with certain DNA damage inducing chemotherapy resulted in synergistic antitumor activity. BAY 1895344 is currently under clinical investigation in patients with advanced solid tumors and lymphomas (NCT03188965).