VTT-006, an anti-mitotic compound, binds to the Ndc80 complex and suppresses cancer cell growth in vitro

Leena J Laine,Jerry F Tien,Jenni H.E Mäki-Jouppila,Ville Härmä,Emma Kutvonen,Lila Kallio,Thomas K.M Nyholm,Pekka Tiikkainen,Charles L Asbury,Marko J Kallio,Antti Poso,Trisha N Davis,Neil T Umbreit,Gary J Gorbsky

doi:10.18632/oncoscience.549

Abstract

Hec1 (Highly expressed in cancer 1) resides in the outer kinetochore where it works to facilitate proper kinetochore-microtubule interactions during mitosis. Hec1 is overexpressed in various cancers and its expression shows correlation with high tumour grade and poor patient prognosis. Chemical perturbation of Hec1 is anticipated to impair kinetochore-microtubule binding, activate the spindle assembly checkpoint (spindle checkpoint) and thereby suppress cell proliferation. In this study, we performed high-throughput screen to identify novel small molecules that target the Hec1 calponin homology domain (CHD), which is needed for normal microtubule attachments. 4 million compounds were first virtually fitted against the CHD, and the best hit molecules were evaluated in vitro. These approaches led to the identification of VTT-006, a 1,2-disubstituted-tetrahydro-beta-carboline derivative, which showed binding to recombinant Ndc80 complex and modulated Hec1 association with microtubules in vitro. VTT-006 treatment resulted in chromosome congression defects, reduced chromosome oscillations and induced loss of inter-kinetochore tension. Cells remained arrested in mitosis with an active spindle checkpoint for several hours before undergoing cell death. VTT-006 suppressed the growth of several cancer cell lines and enhanced the sensitivity of HeLa cells to Taxol. Our findings propose that VTT-006 is a potential anti-mitotic compound that disrupts M phase, impairs kinetochore-microtubule interactions, and activates the spindle checkpoint.

Highlights

Expressed in cancer 1 (Hec1) is a conserved mitotic protein needed for faithful chromosome segregation and the maintenance of genomic balance [1, 2]
To identify small molecules that interfere with the binding of Highly expressed in cancer 1 (Hec1) to microtubules, we first performed a virtual in silico high-throughput screen (HTS) using the FRED docking software [26]
About 4 million chemical structures obtained from large-diversity compound libraries of different vendors were virtually fitted against the calponin homology domain (CHD) of Hec1 to determine their predicted binding performance

Summary

Introduction

Expressed in cancer 1 (Hec1) is a conserved mitotic protein needed for faithful chromosome segregation and the maintenance of genomic balance [1, 2]. The globular domains and N-terminal tails of the Hec1Nuf dimer face outwards towards microtubules and the Spc24-Spc dimer orients towards the centromere [2]. During the establishment of correct kinetochore-microtubule interactions, the 80 amino acid long unstructured N-terminal tail of Hec and the calponin homology domains (CHDs) of Hec and Nuf work in concert to control Ndc complex binding affinity to microtubules [9,10,11,12,13]. Aurora B phosphorylates the N-terminal tail of Hec at multiple sites and controls the establishment of correct kinetochore-microtubule attachments [14, 15] whereas Nek phosphorylates Ser165 in the CH domain and this way controls Hec mediated spindle checkpoint signalling and chromosome alignment process [16, 17]. Mps phosphorylation sites have been shown to control spindle checkpoint signalling [18]

Methods

Results

Conclusion