Development of Eg5 inhibitors for cancer chemotherapy
1 June 2014
Project description: The mitotic spindle is a validated target in cancer chemotherapy. Taxanes and vinca alkaloids target the MTs of the mitotic spindle and are currently used to treat a variety of tumours. However, these current drugs are prone to the development of resistance and display dose limiting toxicities.
Thus, efforts are underway to improve existing drugs and identify new protein targets in the mitotic spindle. Currently, several novel spindle targets for drug development are under investigation including members of the kinesin superfamily. Kinesins form a superfamily of proteins with key roles in intracellular transport and cell division. There may be as many as 16 different ‘mitotic’ kinesins involved in various aspects of mitotic spindle assembly and function, several of which are potential candidates for drug development in cancer chemotherapy.
Our lab’s research focuses on members of the kinesin superfamily, with an emphasis on mitotic motors and their associated proteins. To this end we apply interdisciplinary approaches to identify and/or improve inhibitors of human mitotic kinesins that may lead to new chemotherapeutics.
One prominent member is Eg5, a kinesin responsible for the formation of the bipolar spindle. Current models suggest that Eg5 separates duplicated centrosomes by pushing anti-parallel MTs apart, thus establishing the bipolar spindle. Eg5 represents an attractive chemotherapeutic target because its inhibition causes cells to arrest in mitosis (Figure 1). Prolonged mitotic arrest leads to subsequent apoptotic cell death.
Using an in vitro screening procedure, we originally identified S-trityl-L-cysteine (STLC) as a new, potent Eg5 inhibitor . We have shown that tumour cells treated with STLC are blocked in mitosis leading to apoptosis [2-3]. An initial structure-activity relationship (SAR) study identified a range of derivatives that were more potent than STLC .
To further improve the efficiency of STLC-based inhibitors and aid structure-based drug design, we determined the crystal structure of the Eg5 motor domain in complex with STLC and a range of other interesting analogues  (Figure 2). Our crystal structure revealed several important interactions in the inhibitor-binding pocket and allowed further improvement of our inhibitor analogues .
In collaboration with different chemistry groups, we tested distinct series of new STLC analogues for their inhibition of Eg5 in a variety of tumour cell lines . Based on these results, we developed a series of 4,4,4-triphenylbutan-1-amine inhibitors . This new generation systematically improved on potency: the most potent C-trityl analogues exhibit Kiapp values ≤ 10 nM and have GI50 values around 50 nM, comparable to the phase II clinical benchmarks, ispinesib and SB-743921. Crystallographic studies revealed that they adopt the same overall binding configuration as S-trityl analogues [9,10]. Evaluation of their drug-like properties revealed favourable profiles for future development. The triphenylbutanamine analogue possesses very good bioavailability (51%), and also displayed in vivo anti-tumour growth activity in nude mice xenograft studies (Figure 3) .
Our next aim is to develop STLC-based inhibitors with further improved efficacy in xenograft models of multiple myeloma alone or in combination with FDA-approved drugs, and further enhanced drug-like properties.
Researchers: Project Lead at UCL - Prof Frank Kozielski
Collaborators: The following researchers have or are still participating at this drug discovery project: Dr D Skoufias (IBS, Grenoble, France); Prof R Margolis (Sanford/Burnham Medical Research Institute, USA); Dr L Lebeau (University Strassbourg, France); Prof R Cross (Warwick Medical School, UK); Prof B Joseph (University Lyon, France); Prof B Thiede (The Biotechnology Centre of Oslo, Norway); Prof D Hackney (Carnegie Mellon University, USA); Prof J Weiss (University Heidelberg, Germany); Prof S MacKay (University Strathclyde, Scotland, UK).
1. DeBonis S, Skoufias DA, Robin G, Lebeau L, Lopez R, Margolis R, Wade RH, Kozielski F (2004) In vitro screening for inhibitors of the human mitotic kinesin, Eg5, with antimitotic and antitumor activity. Mol Cancer Ther 3: 1079-1090.
2. Skoufias, D.A., DeBonis, S., Lebeau, L., Crevel, I., Saoudi, Y., Cross, R., Wade, R.H., Hackney, D., Kozielski, F. (2006). S-Trityl-L-cysteine is a reversible, tight-binding inhibitor of the human kinesin Eg5 that specifically blocks mitotic progression. J. Biol. Chem. 281, 17559-17569.
3. Kozielski, F., Skoufias, D.A., Indorato, R.L., Saoudi, Y., Jungblut, P.R., Hustoft, H.K., Strozynski, M., and Thiede, B. (2008). Proteome analysis of apoptosis signaling by S-trityl-L-cysteine, a potent reversible inhibitor of human mitotic kinesin Eg5. Proteomics, 8, 289-300.
4. DeBonis, S., Skoufias, D.A., Indorato, R.L., Liger, F., Marquet, B., Jospeh, B., Kozielski, F. (2008). Structure-activity relationship of S-trityl-L-cysteine analogues, potent inhibitors of the human mitotic kinesin Eg5. J. Med. Chem., 51, 1115-1125.
5. Kaan, H.Y.K., Ulaganathan, V., Hackney, D.D., Kozielski, F. (2010). An allosteric transition trapped in an intermediate state of a new kinesin-inhibitor complex. Biochem. J., 425, 55-60.
6. Kaan, H.Y.K., Weiss, J., Menger, D., Ulaganathan, V., Laggner, C., Popowycz, F., Joseph, B., Kozielski, F. (2011). Structure-activity relationship and multidrug resistance study of new S-trityl-L-cysteine derivatives as inhibitors of Eg5. J. Med. Chem. 54, 1576-1586.
7. Wang, F., Good, J.A., Rath, O., Kaan, H.Y.K., Sutclife, O., MacKay, S., Kozielski, F. (2012). Triphenylbutanamines – Kinesin Spindle Protein inhibitors with in vivo anti-tumour activity. J. Med. Chem. 55, 1511-1525.
8. Abualhasan, M.N., Good, J.A., Wittayanarakul, K., Anthony, N.G., Rath, O., Kozielski, F., Sutcliffe, O.B., Mackay, S.P. (2012). Doing the methylene shuffle with STLC – further insights into the inhibition of Eg5. E. J. Med. Chem. 54, 483-498.
9. Talapatra, S.K., Schüttelkopf, A., Kozielski, F. (2012). Crystal structure of the ternary Eg5-ADP-ispinesib complex. Acta Cryst. D68, 1311-1319.
10. Talapatra, S.K., Anthony, N.G., Mackay, S.P., Kozielski, F. (2013). Mitotic kinesin Eg5 overcomes inhibition to the phase I/II clinical candidate SB743921 by an allosteric resistance mechanism. J. Med. Chem. 56, 6317-6329.
11. Good, J.A., Wang, F., Rath, O., Kaan, H.Y.K., Talapatra, S.S., Podgórski, D., MacKay, S.P., and Kozielski, F. (2013). Development of Optimized S-Trityl-L-Cysteine Based Inhibitors of Kinesin Spindle Protein with potent in Vivo Antitumor Activity in Lung Cancer Xenograft Models. J. Med. Chem. 56, 1878-1893.
Funding: Cancer Research UK
Contact: Prof Frank Kozielski