BTB-1

KIF18A is a kinesin family member that is involved in various cellular processes including cell division, cell transformation, and carcinogenesis. However, its possible role in the regulation of host immunity has not been examined.

The aim of this study is to investigate the functional role of KIF18A in the differentiation and activation of dendritic cells (DCs) that are the most efficient antigen-presenting cells.

A bioinformatic analysis of the KIF18A gene family was performed to understand its sequence variability and evolutionary history. To inhibit KIF18A activity, a highly specific small molecule inhibitor for KIF18A, BTB-1 was used. DCs were differentiated from mouse bone marrow (BM) cells from 6 to 7 week old C57BL/6 mice with recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF). Expression of KIF18A was measured by Western blotting. The surface expression of differentiation and activation markers on DCs were analyzed by flow cytometry.

The phylogenetic analysis revealed that the KIF18A gene family is remarkably conserved across vertebrates. Interestingly, the expression of KIF18A was increased as BM precursor cells differentiated into DCs. BTB-1 treatment strongly inhibited the differentiation of BM cells into DCs in a dose-dependent manner. Furthermore, treatment of immature DCs with BTB-1 significantly impaired the expression of activation markers on DCs including MHC class I, CD80, and CD86 upon TLR4 or TLR7 treatment.

Our results reveal that KIF18A is a critical DC differentiation and activation regulator. Therefore, KIF18A could be a potential therapeutic target for immune-mediated disorders.

The mitotic spindle, a highly dynamic structure composed of microtubules, mediates the segregation of the previously duplicated genome into the two nascent daughter cells. Errors in this process contribute to pathology including tumor formation. Key for the shape and function of the mitotic spindle are kinesins, molecular motor proteins that convert chemical energy into mechanical work. Due to their fast mode of action, small molecules are valuable tools to dissect the dynamic functions of kinesins during mitosis. In this study, we report the identification of optimized small molecule inhibitors of the mitotic kinesin Kif18A. Using BTB-1, the first identified Kif18A inhibitor, as a lead compound, we synthesized a collection of derivatives. We demonstrate that some of the synthesized derivatives potently inhibited the ATPase activity of Kif18A with a half maximal inhibitory concentration (IC50) value in the low micromolar range. In vitro analysis of a panel of Kif18A-related kinesins revealed that the two most potent compounds show improved selectivity compared to BTB-1. Structure-activity relationship studies identified substituents mediating undesired inhibitory effects on microtubule polymerization. In summary, our study provides key insights into the mechanism of action of BTB-1 and its analogs, which will have a great impact on the further development of highly selective and bioactive Kif18A inhibitors. Since Kif18A is frequently overexpressed in solid tumors, such compounds are not only of great interest for basic research but also have the potential to open up new strategies for the treatment of human diseases.