Compound 7, further truncated to a simple pyrimidine, showed no activity below 5 mM. establishes the energy of the SMM approach for identifying inhibitors of E2 enzymes, focuses on with few known smallCmolecule modulators. (125 M), 10% D2O, and 4% DMSO-d6. Nuclear magnetic resonance (NMR) spectra were acquired at 298 K on a Bruker (Billerica, MA) AVANCE III 500-MHz Prkd2 spectrometer fitted having a TCI Cryoprobe with Z-gradient. Carr-Purcell-Meiboom-Gill (CPMG) data units with Presaturation Water Suppression (cpmgpr1d) were acquired using a 200-ms T2 relaxation filter (d20 = 0.001 s, L4 = 200) with an 8-s relaxation delay. O1 was arranged to 2355.14 Hz and RG to 18, and data units were acquired with 256 scans. Data were processed with the MestReNova (Santiago de Compostela, Spain) software package. The spectra of the sample with and without protein were arrayed and scaled so that the peak heights of the internal standard scores were generated for each printed compound within the array. This approach yielded 133 hits for Ubc9 having a z score greater than 4, for an overall hit rate of 0.69%. Among these, 34 of the most promising hits were selected based on high z scores, lack of binding to UbcH5b, and visual inspection of array data and chemical structures then purchased for evaluation of biochemical activity (Suppl. Fig. S1). Open in a separate window (S)-Metolachor Number 1. Small-molecule microarray screening approach for identifying compounds that bind to fluorescently tagged Ubc9. Structural points of attachment to the glass slip are indicated in reddish. The ability of each compound to inhibit sumoylation inside a reconstituted enzymatic cascade was measured at a single concentration through monitoring the conjugation of SUMO-1 to a fluorescently labeled peptide substrate by (S)-Metolachor microfluidic electrophoretic mobility shift using an assay previously developed in our laboratory (Fig. 2A and Suppl. Fig. S2).5 Compounds that caused at least a 25% decrease in sumoylation (S)-Metolachor activity (S)-Metolachor compared to controls at this sole (S)-Metolachor concentration were investigated in dose-response format to obtain full inhibitory curves (Suppl. Fig. S3). Several possible prospects exhibited either poor curves or poor solubility and were not pursued further. However, one compound with the reported structure 1 generated a complete sigmoidal inhibition curve and was consequently selected for more study. Open in a separate window Number 2. (A) Inhibition of sumoylation at 50 M by selected hits from your microarray screen (obtained from commercial sources). GA, ginkgolic acid, 30 M. Observe Supplemental Physique S2 for full graph. (B) Oxidation of compound 1 to 2 2. (C) Synthesis of inhibitors 1 and 2. The purity of the commercial sample of 1 1 was determined by liquid chromatography/mass spectrometry (LC/ MS) analysis. MS analysis revealed that the sample contained a significant quantity of an unknown molecule with of 350 mass models, 4 Daltons less than expected for 1, with very little of this expected compound observed. Given the susceptibility of tetrahydropyridines toward aromatization, we hypothesized that amine 1 could have spontaneously oxidized to the corresponding pyridine 2 upon storage, resulting in a molecule with the observed mass (Fig. 2B). We set out to confirm this hypothesis via chemical synthesis of both structures. The syntheses of 1 1 and 2 began with known aryl chloride 3 (Fig. 2C). The amine-bearing sidechain was launched by SNAr substitution, followed by acid-mediated deprotection to generate 1. After an assessment of several oxidation conditions, IBX was found suitable to furnish 2 in affordable yield. Alternatively, 2 could be produced from the known aryl chloride 4 directly by SNAr substitution. The identity of 2 as the major component of the commercial sample was confirmed by LC/MS coinjection (Suppl. Fig. S4). It is significant to note that solid 1 was also observed to oxidize partially to 2 upon standing at room heat over the course of 10 weeks (Suppl. Fig. S5), confirming that spontaneous oxidation of the tetrahydropyridine core is feasible. Compound 2 was then generated in its.