In xenograft Fingolimod studies with either the wild type or APO866 resistant Inhibitors,Modulators,Libraries variant of the HCT 116 tumour cell line mice were treated with a standard two daily i. p. injections of 15 20 mg kg APO866 in PBS with 1% hydroxypropyl b cyclodextrin 12% propylene glycol Inhibitors,Modulators,Libraries on day 0 13, starting when tumour volumes were around 100 mm3, as previously described. Statistical analyses Statistical analysis and the graphical presentation were per formed using the software GraphPad Prism v. 4. 0. Tumour doubling times were calculated using the line fitting function for exponential growth in Graph Pad Prism, and were com pared using Students t test. The outcome of xenografts in the treatment experiment was quantified as the number of days until the tumour of each individual mouse reached a size of 800 mm3, expressed as survival days.

The survival in each treatment group was compared using log rank analy sis. The level of significance was set to a p value of 0. 05. Measurement of NAMPT activity in lysates Activity of NAMPT was examined in PC 3 and PC 3 TP201565 as well as HEK293T cells and HEK NAMPTwt. Inhibitors,Modulators,Libraries NAMPT activity was measured by the con version of 14C labelled nicotinamide to 14C NMN using a method previously described using 50 ug total protein from cell lysate. Briefly, lysate was incubated with ATP, 5 phosphoribosyl 1 pyrophosphate and 14C nicotinamide in reaction buffer. The mix was transferred to glass fibre filter and washed in acetone to remove excess 14C nicotinamide. Counts per minute determined by a liquid scintillation counter.

Computer modelling of NAMPT The molecular modelling was based on the published X ray structure of NAMPT in complex with the inhibitor APO866. The structure was imported into Maestro v. 9. 0 release 111 and prepared for docking in the following way using the protein preparation Inhibitors,Modulators,Libraries wizard. Inhibitors,Modulators,Libraries First, bond orders were assigned and hydrogen atoms were added. The four selenocysteines which had been incorporated to facilitate never X ray crystallographic analysis were mutated back to cysteine. Water molecules further than 5 away from the binding site were removed, and finally the hydrogen bonding network was optimized. The Glide docking module was used for the docking of the ligands. Initially, the recep tor grid was generated. The enclosing box for the docking of TP201565 and CHS 828 was based on the position of APO866 in the X ray crystal structure. The hydroxyl groups in the receptor were allowed to rotate. For stu dies on the H191R mutation, APO866 was removed in the 2GVJ X ray structure which had already been pre pared for molecular modelling. The Prime module allowed for introduction of the mutation, which was fol lowed by sampling of Arg191 dihedral angles to obtain the most favorable orientation of the new side chain.