Pharmacophore-guided discovery of CDC25 inhibitors causing cell cycle arrest and cell death Zeynep Kabakci1*, Simon Käppeli1*, Giorgio Cozza2, Claudio Cantù3, Christiane König1, Janine Toggweiler3, Christian Gentili1, Giovanni Ribaudo4, Giuseppe Zagotto4, Konrad Basler3, Lorenzo A. Pinna5 and Stefano Ferrari1,6 1Institute of Molecular Cancer Research and 3Institute of Molecular Life Sciences, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland 2Department of Molecular Medicine, 4Department of Pharmacology and 5Department of Biomedical Sciences, University of Padua, Via U. Bassi 58/B, I-35131 Padua, Italy SUPPLEMENTARY FILES 1 SUPPLEMENTARY MATERIALS AND METHODS Chemistry - Commercially available chemicals were purchased from Sigma-Aldrich (Milan, Italy). For work-up and chromatographic purification, commercial grade solvents were used. Semi-preparative and preparative purifications of the synthesized compounds were carried out on Isolera One automated flash chromatography system (Biotage, Upsala, Sweden). The analytical profile of the synthesized molecules was in accordance with literature data (see below). 1H and 13C[1H] NMR spectra were recorded on a Bruker Avance III 400 MHz and a Bruker AMX 300 MHz spectrometers (Redaelli, Mucignat- Caretta et al., 2015). All spectra were recorded at room temperature. High-resolution mass spectra were recorded on an ESI-TOF Mariner from PerSeptive Biosystem (Stratford, Texas, USA), using electrospray ionization (ESI). Purity was assayed by HPLC, using a Varian Pro-Star system equipped with a Biorad 1706 UV–VIS detector and an Agilent C-18 column (5mm, 4.6mm 150mm). Water (A) and acetonitrile (B) were used as mobile phases with an overall flow rate of 1 mL/min and the following analytical method: 0 min (90% A–10% B), 15 min (10% A– 90% B), 20 min (10% A–90% B), 21 min (90% A–10% B), 255 min (90% A–10% B). Purity was over 97% (HPLC area). General procedure for the synthesis of UPD-596 (2-(2,3,4-trihydroxyphenyl)-1,4- naphthoquinone), UPD-597 (2-(2,4-dihydroxyphenyl)-1,4-naphthoquinone), UPD-786 (8-hydroxy-2-(2,3,4 trihydroxyphenyl) naphthalene-1,4-dione), UPD-787 (8-hydroxy-2- (2,4-dihydroxy-3-methylphenyl) naphthalene-1,4-dione), UPD-788 (8-hydroxy-2-(2,4,6- trihydroxyphenyl)naphthalene-1,4-dione), UPD-790 (8-hydroxy-2-(2,4- dimethoxyphenyl)naphthalene-1,4-dione), UPD-140 (2-(2',4'-dihydroxyphenyl)-8- hydroxy-1,4-naphthoquinone) and UPD-176 (5-hydroxy-2-(2,4- dihydroxyphenyl)naphthalene-1,4-dione) The preparation of the substituted naphthoquinones was performed according to previously reported procedures (Pavan, Ribaudo et al., 2017, Redaelli et al., 2015). Briefly, the opportune naphthalene-1,4-dione (2 eq) and phenol (1 eq) were reacted in a mixture of acetic acid and 2M H2SO4. After 2h of stirring at room temperature under a nitrogen atmosphere, the reaction was stopped by the addition of water and neutralized with 5% sodium bicarbonate. The mixture was extracted with ethyl acetate, which was 2 then evaporated to give the crude product. Purifications or separation of isomers were carried out by recrystallization or flash chromatography (n-hexane / ethyl acetate = 7:3 v / v). General procedure for the synthesis of UPD-793 (3-(p-tolylthio)juglone) and UPD-795 (2-(3-methoxyphenylthio)-8-hydroxynaphthalene-1,4-dione) The preparation of the sulfur-containing naphthoquinone derivatives was performed according to the previously reported procedure, with slight modifications (Redaelli et al., 2015). Juglone (2 eq) and the opportune thiophenol (1 eq) were reacted in ethanol at room temperature. After 2h of stirring at room temperature, the precipitate forming from the reaction mixture was filtered and recrystallized from ethanol to give the desired product. General procedure for the synthesis of UPD-797 (8-hydroxy-2-(piperidin-1- yl)naphthalene-1,4-dione) and UPD-798 (N-(5-hydroxy-1,4-naphthoquinon-3- yl)morpholine) The N-substituted naphthoquinones were prepared according to a previously reported procedure by reacting the precursor 3-Bromo juglone (1 eq) with a large excess of the appropriate amine (7 eq) in acetic acid. The work-up of the compounds was carried out following the reported procedure (Redaelli et al., 2015). Synthesis of UPD-724 (1,4-dihydroxy-6-anthraquinonecarboxylic acid) The compound was synthesized from trimellitic anhydride and hydroquinone by a Friedel Crafts reaction (AlCl3, NaCl, 150°C, 7 h) followed by a mild air oxidation, according to a previously reported procedure (Ambler, Broadmore et al., 1997, Zagotto, Gianoncelli et al., 2014). Synthesis of UPD-738 (5-hydroxynaphtho-1,4-quinone) 5-hydroxynaphtho-1,4-quinone (juglone) was prepared according to a literature procedure (Couladouros, Plyta et al., 1996, Zonta, Cozza et al., 2009). Synthesis of UPD-1416 (4-Hydroxy-2-methylquinoline-5,8-dione) and UPD-1419 (2- methyl-5,8-dihydro-5,8-dioxoquinoline) UPD-1416 is a commercially available compound (Ark Pharm). UPD-1419 was prepared according to a previously reported procedure, allowing the preparation of 1,4-quinone derivatives starting from 1,4-dimethoxybenzenes using NBS (Kim, Choi et al., 2001). 3 Synthesis of UPD-1382 (5,8-diaminonaphthalene-1,4-dione) The compound was prepared according to the procedure reported in patent US2399355 (Klein, 1943). In silico protein preparation - The crystal structure of human CDC25A and CDC25B were retrieved from Protein Data Bank (PDB codes: 2Z5X and 1QB0 respectively) and processed in order to remove ligands and water molecules. Hydrogen atoms were added to the protein structures using standard geometries with the Molecular Operating Environment (MOE) software (G., 2010). To minimize contacts between hydrogen atoms, the structures were subjected to Amber99 force-field minimization until the root mean square (rms) of conjugate gradient was <0.1 kcal•mol−1•Å−1 (1 Å = 0.1 nm), keeping the heavy atoms fixed at their crystallographic positions. Human CDC25A and CDC25C were built using a homology modeling approach implemented into MOE (G., 2010) with CDC25B as homologous template (PDB code: 1QB0). Sequence alignment was performed using the MOE Protein Align tool with BLOSUM62 as substitution matrix (CDC25A, 67% identity; CDC25C, 61% identity). Generation of virtual library and hit selection - An in silico virtual library was built from a proprietary database of synthetic molecules (2075 compounds) upon 2D to 3D conversion of the molecules chemical structure, optimization of compound conformers and addition of Gasteiger partial charges (MOE Suite). The strategy for hit selection is described in the Results section. In silico hit expansion/optimization - The structure of the first two hits, UPD-140 and UPD-176, provided a clue for the hit expansion/optimization phase through a Site Finder approach (MOE Suite), followed by a Molecular Docking protocol (Glide, Schrodinger Suite, (Schrödinger)). Site Finder allows the identification of pockets and surface sites by identifying regions of tight atomic packing using Alpha Shapes, a generalization of convex hulls developed in (Edelsbrunner, 1955) that has been successfully validated against several unrelated targets (Cozza, Rossetto et al., 2017, Cozza, Zanin et al., 2015). The procedure suggested two close cavities that are highly conserved in the three 4 enzymes, one of them corresponding to the phosphatase catalytic site. Based on the Site Finder indications, a molecular docking process was performed using the Glide package XP procedure (Friesner, Banks et al., 2004) (Schrödinger Suite) and focusing on compounds featuring a quinone moiety. Antibodies and chemicals - The following antibodies were used for Western blot: Rabbit monoclonal antibodies to CDK1-pThr14 (#2543), CDK1-pTyr15 (#9111), Histone H3-pSer10 (#9701) and CDC25B (#9525) were purchased from Cell Signaling Technology (Beverly, MA, USA). Mouse monoclonal antibodies to CDC25A (F-6, sc- 7389), CDC25C (C-20, sc-327) and a-tubulin (YOL 1/34, sc-53030) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The mouse monoclonal antibody MPM2 (05-368) was from Upstate (Lake Placid, NY, USA). The mouse monoclonal antibody to the HA tag (HA.11, MMS-101P) was from BioLegend (S. Diego, CA, USA). HRP-conjugated anti-mouse and anti-rabbit secondary antibodies were obtained from GE-Healthcare (Otelfingen, Switzerland). HRP-conjugated IgG-k-BP was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The following antibodies were used for immunofluorescence on a Zeiss LSM 710 confocal microscope: mouse monoclonal to beta-catenin (Cl. 14, BD transduction Lab.), rabbit polyclonal to lysozyme (A0099, Dako). Secondary antibodies were: Alexa 488 goat anti-mouse, Alexa 555 goat anti-rabbit. Nuclei were visualized with 4',6-Diamidino- 2-Phenylindole (DAPI, Sigma-Aldrich). The CDK1 inhibitor Ro-3306 (Roche, Switzerland) was dissolved in DMSO at 9 mM stock concentration and stored in aliquots at -20 °C. Thymidine was obtained from SynGen Research Inc. (Sacramento, CA, USA) and dissolved in PBS as 200 mM stock solution before use. NSC-663284 (Sigma-Aldrich, Buchs, Switzerland) as well as all UPD compounds were dissolved in DMSO at 10 mM stock concentration and stored in aliquots at -20 °C. Expression and purification of CDC25 phosphatases - CDC25A (kind gift of I. Hoffmann, Heidelberg, Germany), CDC25B and CDC25C (kind gift of B. Gabrielli, Queensland, Australia) in pGEX were expressed in E. coli BL21 cells and purified on 1 5 ml Glutathione Sepharose 4 Fast Flow columns as described (Hassepass & Hoffmann, 2004). Enzymatic assays - CDC25A, CDC25B and CDC25C were appropriately diluted in 10 mM Tris-HCl pH 8.0, 50 mM NaCl and assayed in 20 mM Tris-HCl pH 8.0, 75 mM NaCl, 0.5 mM EGTA, 1 mM DTT, 0.1 mg/ml BSA and defined concentrations of compounds. Reactions were started by addition of the substrate OMFP (0.1 mM). Assays were carried out at room temperature in duplicate or triplicate using transparent-bottom, V-shaped 96 well plates and read at defined intervals on a Molecular Devices SpectraMax microplate reader (ex: 490 nm / em: 525 nm).