Validation and functional characterization of a novel Kif18A small molecule inhibitor

Abstract

The accurate transmission of the genetic information from mother to daughter cells constitutes a crucial event in the life cycle of all eukaryotic cells (Morgan 2006). To fulfill this task cells rely on a specialized microtubule-based structure called the mitotic spindle (Wittmann et al. 2001). The assembly and the functions of the spindle apparatus are tightly regulated by the orchestrated interplay of dynamic microtubules and motor proteins (Wittmann et al. 2001). In addition to Cytoplasmic dynein, motor proteins of the kinesin superfamily are fundamental for the structure and function of the mitotic spindle (Goshima and Vale 2003; Zhu et al. 2005). Kif18A is a member of the kinesin-8 protein family characterized by its unique dual functionality, which couples a highly processive motor activity with the ability to destabilize microtubules by specifically depolymerizing them at their plus end (Mayr et al. 2007). Recent studies proposed Kif18A to be a key component in the process of chromosome alignment (Stumpff and Wordeman 2007). Notably, this kinesin is required to slow down the oscillatory movements of chromosomes happening in prometaphase after their binding to kinetochore-microtubules and to increase their switch rate across the spindle equatorial region, ultimately leading to the correct chromosome positioning at the metaphase plate (Stumpff et al. 2008). Other reports identified Kif18A as a regulatory partner of Cenp-E (Huang et al. 2009; Zhang and Matunis 2009), a member of the kinesin-7 family involved in chromosome alignment and checkpoint signaling pathways (Yao et al. 2000). Moreover, evidences derived from different works suggest a role of Kif18A in late mitotic events (Gatt et al. 2005; Mayr et al. 2007; Stumpff et al. 2008). The mechanisms by which Kif18A accomplishes these different functions are not fully understood, partially due to the lack of appropriate experimental tools. In this work we reported the functional characterization of the Kif18A small molecule inhibitor 4-chloro-2-nitrodiphenyl sulphone, named BTB-1. This compound originally identified in a reverse chemical genetics high-throughput screening (Grüner 2004), has been validated as a specific and potent inhibitor of the ATPase activity of Kif18A in vitro by three different unrelated approaches. BTB-1 has proven to be effective against Kif18A at low micromolar concentrations (IC50 = 1.7 μM) through a reversible inhibitory mechanism. Moreover, detailed kinetics studies revealed its inhibitory activity to be ATP competitive and microtubule uncompetitive. When tested on human cells BTB-1 induced a mitotic specific phenotype, characterized by the presence of aberrant spindle structures, misaligned chromosomes, increased mitotic index and a prolonged time to complete mitosis. Collectively, these results led to the discovery of the first small molecule inhibitor of the mitotic kinesin Kif18A, which could be employed as a powerful tool to functionally dissect and characterize the different properties of this motor protein.