Effects of Jazz90 and Jazz167 are most likely mediated via reduction of the Erk phosphorylation, as seen in previous studies [6,38,39,40]. in PC3 and DU145 cells. There was a reduction in cyclin D1 and no significant changes in bcl-2 in DU145 and PC3 cells. Overall, the results showed that Jazz90 and Jazz167 function as cytostatic HDAC inhibitors in AR-null prostate cancer cells. 0.05. Data were analyzed using a one-way ANOVA followed by Bonferronis post-hoc test. 2.2. HDAC Inhibition and Cellular Effects on Acetylation of Histone-3 Lerociclib dihydrochloride Variant To determine if Jazz90 and Jazz167 could inhibit a range of HDACs, HDAC inhibition assays were conducted in nuclear lysates from both HeLa and PC3 cells. Both Jazz90 and Jazz167 exhibited similar HDAC inhibition to SAHA (Figure 2). Jazz90 led to HDAC inhibition of 34 and 45% at 0.1 and 0.2 M, whereas Jazz167 resulted in 38 and 53% inhibition at 0.1 and 0.2 M, respectively, in HeLa cells. SAHA led to HDAC inhibition of 44 and 59% at 0.1 and 0.2 M, respectively, in HeLa cells (Figure 2a). The positive control (trichostatin A (TSA)) led to 80% HDAC inhibition at a concentration of 0.1 M (Figure 2a). In the PC3 lysate, Jazz90 resulted in inhibition of 58 and 69% compared to 59 and 64% inhibition for Jazz167 and 57 and 63% for SAHA (Figure 2b) at 0.05 and 0.1 M, for all compounds, respectively. Open in a separate window Figure 2 HDAC inhibition elicited by Jazz90 and Jazz167. (a) HeLa nuclear lysates were treated with 0.1 and 0.2 M of Jazz90, Jazz167, and SAHA. TSA (0.1 M) was used as a positive control and a no enzyme control (No) was also used. Vehicle control (C) lysates were treated with 0.5% DMSO. (b) PC3 nuclear lysates were treated with 0.05 and 0.1 M of SAHA, Jazz90, and Jazz167. Bar graphs represent the relative fluorescence units (RFU) measured after 70 min of treatment. A two-way ANOVA was conducted followed by Bonferronis post hoc test. * indicates significant difference to control, 0.05. Images from the kinetic assays are provided in Figures S2 and S3. To validate the results from HDAC inhibition assays, molecular docking was conducted. The crystal structure of SAHA complexed with HDAC2 (PDB ID: 4LXZ) shows the hydroxamate coordinated to the zinc ion . The hydroxamate coordination is stabilized by hydrogen Lerociclib dihydrochloride bonding to histidine-146 and tyrosine-306. Hydrophobic interactions occur between the phenyl ring of phenylalanine-155 and the aliphatic chain in SAHA. An additional hydrogen bond exists between aspartic acid-102 and the amide nitrogen of SAHA (Figure 3a). Jazz90 was docked 10 times into the ligand binding site of HDAC2 and of those, only 1 1 pose showed the hydroxamate coordinating to the zinc ion (Figure 3b). Interactions between Jazz90 and HDAC2 were similar to that seen for the crystal structure of the SAHA complex with hydrophobic interactions between the alkyl chain and phenylalanine-155 as well as the phenyl ring and proline-35. Four of the remaining poses resulted in an unexpected binding mode where the pyridinecarbothioamide group coordinated to the zinc ion (Figure 3c). This form of zinc coordination was verified via the Cambridge Structural Database (CCDC) with entry CCDC 1257652 showing coordination to zinc via a pyridinecarbothioamide. The phenyl ring of Jazz90 lays between phenylalanine-155 and phenylalanine-210 forming a -stacking interaction (Figure 3c). The remaining poses Lerociclib dihydrochloride were similar to the latter orientation; however, the pyridine nitrogen did not coordinate (ring rotated by 180, not shown) and was therefore dismissed. Open in a separate window Figure 3 SAHA and Jazz90 binding to HDAC2. Jazz90 was docked Foxd1 into HDAC2 (PDB ID: 4LXZ). (a) Crystal structure of SAHA (cyan) complexed with HDAC2, (b) Jazz90 docked into HDAC2 with hydroxamate coordination to zinc, and (c) Jazz90 docked into HDAC2 with pyridinecarbothioamide coordination. Green ribbons and sticks represent the active site of HDAC2. (Blue atoms = nitrogen, yellow = sulfur, and red = oxygen). Acetylation of the histone-3 variant was assessed to understand the cellular effects.