Identification of tubulin drug binding sites and prediction of relative differences in binding affinities to tubulin isotypes using digital signal processing Ke Chen a , J. Torin Huzil b , Holly Freedman b , Parameswaran Ramachandran c , Andreas Antoniou c , Jack A. Tuszynski b , Lukasz Kurgan a, * a Department of Electrical and Computer Engineering, University of Alberta, ECEFR, 9701 116 Street, Edmonton, AB, Canada T6G 2V4 b Department of Oncology, University of Alberta, Cross Cancer Institute, 11560 University Avenue, Edmonton, AB, Canada T6G 1Z2 c Department of Electrical and Computer Engineering, University of Victoria, P.O. Box 3055 STN CSC, Victoria, BC, Canada V8W 3P6 1. Introduction Microtubules (MTs) are polymeric protein complexes con- structed from a heterodimer of two highly homologous proteins known as a- and b-tubulin. The assembly of tubulin heterodimers into a macromolecular MT complex is a tightly regulated and dynamic process [1]. MTs are involved in a broad range of cellular processes, including the maintenance of cellular morphology and active transport of cellular components throughout the cytoplasm [2,3]. One of the most critical roles MTs play, is the formation of the mitotic spindle apparatus, providing the mechanical force required for chromosome separation during mitosis [4]. During mitosis, MTs are essential for the proper assembly of the mitotic spindle apparatus, the organelle responsible for the segregation of aligned chromosomes prior to cell division. As such, MTs have become the target of numerous anti-mitotic agents, including colchicine and antitumor drugs such as the taxanes, epothilones, and Vinca alkaloids. Colchicine, a water-soluble alkaloid binds to the intradimer interface between a- and b-tubulin dimers, presum- ably disrupting the correct orientation of protofilaments within the MT [5]. Paclitaxel, which continues to be one of the most successful cancer therapeutic agents, has a unique mechanism of action as it binds to and results in the stabilization of MTs within all cells [6,7]. Derivatives of paclitaxel, such as docetaxel, have been synthesized to address the limited solubility of paclitaxel and show increased binding to a site within b-tubulin [8]. Two additional families of similar tubulin-binding drugs are the epothilones and dolastatins, which share a similar mode of action. Through structural studies, both the taxanes and epothilones were shown to bind a unique site within the b subunit of the a/b-tubulin heterodimer [9,10]. Unfortunately, neither of the structures has been able to reveal the precise mechanism of MT stabilization by these drugs. Finally, the Vinca alkaloids include vincristine, vinblastine, and vinorelbine, and are used most commonly in combination chemotherapy Journal of Molecular Graphics and Modelling 27 (2008) 497–505 ARTICLE INFO Article history: Received 8 January 2008 Received in revised form 1 September 2008 Accepted 2 September 2008 Available online 10 September 2008 Keywords: Tubulin Isotype Digital signal processing Protein hot spot Drug affinity Resonant recognition model Short-time Fourier transform Characteristic frequency ABSTRACT Microtubules are involved in numerous cellular processes including chromosome segregation during mitosis and, as a result, their constituent protein, tubulin, has become a successful target of several chemotherapeutic drugs. In general, these drugs bind indiscriminately to tubulin within both cancerous and healthy cells, resulting in unwanted side effects. However, differences between b-tubulin isotypes expressed in a wide range of cell types may aid in the development of anti-tubulin drugs having increased specificity for only certain types of cells. Here, we describe a digital signal processing (DSP) method that is capable of predicting hot spots for the tubulin family of proteins as well as determining relative differences in binding affinities to these hot spots based only on the primary sequence of 10 human tubulin isotypes. Due to the fact that several drug binding sites have already been characterized within b- tubulin, we are able to correlate hot spots with the binding sites for known chemotherapy drugs. We have also verified the accuracy of this method using the correlation between the binding affinities of characterized drugs and the tubulin isotypes. Additionally, the DSP method enables the rapid estimation of relative differences in binding affinities within the binding sites of tubulin isotypes that are yet to be experimentally determined. ß 2008 Elsevier Inc. All rights reserved. * Corresponding author. Tel.: +1 780 492 5488; fax: +1 780 492 1811. E-mail address: lkurgan@ece.ualberta.ca (L. Kurgan). Abbreviations: DSP, digital signal processing; EIIP, electron–ion interaction potential; GDP, guanosine diphosphate; GTP, guanosine triphosphate; IC 50 , half maximal inhibitory concentration; MTs, microtubules; PDB, protein data bank; RSA, relative solvent accessibility; RRM, resonant recognition model; STDFT, short-time discrete Fourier transform. Contents lists available at ScienceDirect Journal of Molecular Graphics and Modelling journal homepage: www.elsevier.com/locate/JMGM 1093-3263/$ – see front matter ß 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.jmgm.2008.09.001