These ligands were docked onto the binding pocket of the PDB file 3C45 with eHiTS v2009 (SimBioSys Inc., Toronto, Canada; http://www.simbiosys.ca/ehits) [63] using default docking conditions, with the exception of the length of the sides of the cubic box encompassing the DPP-IV binding site, which was ??increased from 10 A to 15 A. Next, the resulting docked poses were again filtered with Phase through the common pharmacophore using the same filtering conditions as in the first Phase run (with the exception that no reorientation of the docked poses was allowed during the search). Thus, only docking poses compatible with the pharmacophore survived this filter. Finally, in the last step of the VS protocol, the poses that were hits in this second pharmacophore screen were submitted to a shape and electrostatic potential comparison with the DPP-IV inhibitor present in the 3C45 PDB file to rescore the hits. This comparison was completed with EON v2.0.1 (OpenEye Scientific Software, Inc., Santa Fe, New Mexico, USA; http://www.eyesopen.com) and used the Electrostatic Tanimoto combo (ET_combo) score as the similarity criterion. The ET_combo score is the sum of two calculations: (a) the Shape Tanimoto (ST) score, which is a quantitative measure of three-dimensional overlap (where 1 corresponds to a perfect overlap; i.e. the same shape), and (b) the Poisson-Boltzmann Electrostatic Tanimoto (ET_pb) score, which compares the electrostatic potential of two small molecules and ranges from 1 (identical potential) to a negative value that results from the overlap of positive and negative charges.
Structural Similarity Analysis
The molecules that survived the electrostatics/shape similarity filter were merged with 2,342 known DPP-IV inhibitors obtained from the BindingDB database [64] and then clustered using Canvas v1.2 (Schrodinger LLC., Portland, USA; http://www. ?schrodinger.com). MOLPRINT2D fingerprints [65], using a fingerprint precision of 32 bits, were calculated for each molecule, and hierarchical clustering, based on Tanimoto similarities, was subsequently obtained. The number of clusters obtained was defined using the Kelley criterion [66], corresponding to a Tanimoto coefficient of 0.775 in this case.
Behavioural Assessment of UWA-101 in the MPTPlesioned Common Marmoset
Ethics statement. Five female common marmosets (Callithrix jacchus, 300?00 g; Harlan, Madison, USA) were kept under conditions of controlled temperature (2562uC) and lighting (12 h light/ dark cycle, 08:00 lights on). Animals were cared for in accordance with an IACUC approved by University Health Network Animal Care Committee protocol and with the regulations defined by the Canadian Council on Animal Care. Animals were housed in groups of 2? and had unrestricted access to food, fresh fruit supplements and water, and their home cage was enriched with primate toys, perches and auditory stimuli. Efforts were made to reduce to a minimum the number of animals required for statistically valid analyses and to minimise their suffering. Prior to the start of studies, animals were acclimatised to handling, administration of subcutaneous (s.c.) treatments, and transfer to observation cages.
Induction of Parkinsonism and Dyskinesia in the Common Marmoset
Animals were rendered parkinsonian by administration of MPTP hydrochloride (2 mg/kg s.c. daily, for 5 d; Sigma, St Louis, USA). Following MPTP treatment, marmosets entered a 12 week recovery period to allow parkinsonian symptoms to develop and stabilise. Treatment-related complications, including dyskinesia and psychosis-like behaviours, were induced by administration of oral ProlopaH (L-DOPA/ benserazide 15/3.75 mg/kg twice daily; Hoffmann-La Roche Limited, Mississauga, Canada) for a minimum of 30 d. This treatment regimen has been demonstrated to produce a stable model of L-DOPA-induced motor and nonmotor complications [16,17]. The animals used in the present experiment had not been used in previous studies and were drug?naive, with the exception of L-DOPA.
Administration of UWA-101 in Combination with L-DOPA to the Parkinsonian Marmoset
On days of behavioural assessment, marmosets were administered L-DOPA/ benserazide 25/6.25 mg/kg s.c. (Sigma-Aldrich, St Louis, USA) in combination with either vehicle (NaCl 0.9%) or UWA-101 (1, 3, 6, 10 mg/kg of drug free base) s.c. at 09:00. The drug administration schedule was fully randomised between and within animals, according to a Latin square design (Experimental Design Generator And Randomiser (EDGAR), http://www. edgarweb.org.uk/), in which all animals received all treatments. Immediately after treatment administration, marmosets were placed individually into observation cages (0.8 6 0.8 6 0.7 m) containing food, water and a wooden perch, and were left undisturbed for the 6 h duration of the experiment. Behaviour was recorded via DVD footage for post hoc analysis by a neurologist specialised in movement disorders blinded to the treatment. As in the previous experiment [13], at least 48 h were left between each treatment.
Materials and Methods UWA-101 Synthesis
UWA-101 (Figure 1) was synthesised by reductive amination of piperonyl cyclopropyl ketone with methylamine as described Behavioural Analysis
Behavioural analysis was performed according to previously published methods [16,18,19]. Parkinsonian disability scores were rated for 5 min every 10 min. The following items were rated: range of movement (0?), bradykinesia (0?), posture (0?), and attention/ alertness (0?). For each of the aforementioned items, the higher the score, the greater the disability. A global parkinsonian disability score was calculated as a combination of the aforementioned behaviours according to the following formula: (range of movement 6 1) + (bradykinesia 6 3) + (posture 6 9) + (alertness 6 9). The maximal parkinsonian disability score per 5 min observation period was 36. L-DOPA-induced dyskinesia and psychosis-like behaviours were assessed concomitantly with parkinsonian disability. Dyskinesia were rated from 0?. Choreiform and dystonic dyskinesia were rated separately and the score given reflected the most disabling dyskinesis observed, either chorea or dystonia, for every 5 min period of evaluation.