Application of ensemble pharmacophore-based virtual screening to the discovery of novel antimitotic tubulin inhibitors

Tubulin is a well-validated target for herbicides, fungicides, anti-parasitic, and anti-tumor drugs. Many of the non-cancer tubulin drugs bind to its colchicine site but no colchicine-site anticancer drug is available. The colchicine site is composed of three interconnected sub-pockets that fit their ligands and modify others’ preference, making the design of molecular hybrids (that bind to more than one sub-pocket) a difficult task. Taking advantage of the more than eighty published X-ray structures of tubulin in complex with ligands bound to the colchicine site, we generated an ensemble of pharmacophore representations that flexibly sample the interactional space between the ligands and target. We searched the ZINC data- base for scaffolds able to fit several of the subpockets, such as tetrazoles, sulfonamides and diaryl-methanes, selected roughly ~8000 compounds with favorable predicted properties. A Flexi-pharma virtual screening, based on ensemble pharmacophore, was performed by two different methodologies. Combining the scaffolds that best fit the ensemble pharmacophore-representation, we designed a new family of ligands, resulting in a novel tubulin modulator. We synthesized tetrazole 5 and tested it as a tubulin inhibitor in vitro. In good agreement with the design principles, it demonstrated micromolar activity against in vitro tubulin polymerization and nanomolar anti-proliferative effect against human epithelioid carcinoma HeLa cells through microtubule disruption, as shown by immunofluorescence confocal microscopy. The integrative methodology succedes in the design of new scaffolds for flexible pro- teins with structural coupling between pockets, thus expanding the way in which computational methods can be used as significant tools in the drug design process.The authors acknowledge the support of the Enlighten Your Research LatinAmerica2Europe (EYR-LA2EU) programme. I.L, R.O. and P.C. were also supported by MinCiencias, University of Antioquia, Ruta N, Colombia and the Max Planck Society, Germany. L.G-Y. and R.P-L. acknowledge the support by the Consejería de Educación de la Junta de Castilla y León (SA262P18 and SA116P20) and the Spanish Ministry of Science, Innovation and Universities (RTI2018-099474-BI00), co-funded by the EU’s European Regional Development Fund-FEDER. D.R. was supported by the CONICYT-PCI grant N° REDES190074 and FONDECYT grant N° 11180604. This work has been partially carried out on the ACME cluster, which is owned by CIEMAT and funded by the Spanish Ministry of Economy and Competitiveness project CODEC2 (TIN2015-63562-R) with FEDER funds as well as supported by the CYTED co-founded RICAP Network (517RT0529)