Potent and Selective Non‐hydroxamate Histone Deacetylase 8 Inhibitors

Abstract

Specific inhibition of histone deacetylase 8 (HDAC8) has been suggested as a promising option for the treatment of neuroblastoma and T-cell malignancies. A novel class of highly potent and selective HDAC8 inhibitors with a pyrimido[1,2-c][1,3]benzothiazin-6-imine scaffold was studied that is completely different from the traditional concept of HDAC inhibitors comprising a zinc binding group (ZBG), in most cases a hydroxamate group, a spacer, and a capping group that may interact with the surface of the target protein. Although lacking a ZBG, some of the new compounds were shown to have outstanding potency against HDAC8 in the single-digit nanomolar range. The pyrimido[1,2-c][1,3]benzothiazin-6-imines also inhibited the growth of solid and hematological tumor cells. The small size and beneficial physicochemical properties of the novel HDAC inhibitor class underline the high degree of drug likeness. This and the broad structure-activity relationship suggest great potential for the further development of compounds with the pyrimido[1,2-c][1,3]benzothiazin-6-imine scaffold into innovative and highly effective therapeutic drugs against cancer.

Switching the Switch: Ligand Induced Disulfide Formation in HDAC8

“…In ap revious study,w eidentified PD-404,182 as ap otent and selectiveH DAC8 inhibitor. [19] By using time resolved HPLC-MS/ MS, we also explained the exact mechanism of PD-404,182 decomposition in aqueous solution and subsequent mixed disulfide formation of various cysteines in HDAC8. [20] PD-404,182 decomposes quicklyi nto the corresponding sulfenamide, which forms mixed disulfides with cysteiner esidues for example, in HDAC8.…”

“…[12][13][14][15][16][17][18] In the effort to discover new potent and selective non-hydroxamate inhibitors of HDAC8, we identified PD-404,182 in amedium-scaled compound screeningc ampaign. [19] Further studies on the mode of protein-inhibitor interaction revealedt hat PD-404,182 is ac ovalent modifier of HDAC8, forms mixed disulfides and transformst hiol groups into thiocyanates. [20] In af urther study we found that HDAC8 is regulated by ar edox switchi nvolving cysteines Cys 102 and Cys 153 .…”

Determination of the binding mechanism of histone deacetylase inhibitors

“…A concentration series of a reference inhibitor like SAHA, trichostatin A, or SATFMK is highly recommended Add 10 μl HDAC/HDAH working solution to each well and incubate for 30 min to detect also slow binding inhibitors Add 20 μl of a mixture containing 100 μM substrate and 0.5 mg/ml trypsin Start the fluorescence measurement of continuously released AMC for a period of about 30–60 min immediately. The slope of fluorescence intensity versus time is proportional to enzyme activity ExampleFigure : Dose–response curves of selected benzothiazine‐6‐imine inhibitors of HDAC8 (Kleinschek, Meyners, Digiorgio, Brancolini, & Meyer‐Almes, )…”

“…Start the fluorescence measurement of continuously released AMC for a period of about 30-60 min immediately. The slope of fluorescence intensity versus time is proportional to enzyme activity Example Figure 6: Dose-response curves of selected benzothiazine-6-imine inhibitors of HDAC8 (Kleinschek, Meyners, Digiorgio, Brancolini, & Meyer-Almes, 2016) Figures 5 and 6.…”

“…Add 30 μl twofold concentrate of HDAC/HDAH 4. Measure FLT and ascertain that equilibrium is achieved imine inhibitors of HDAC8 (Kleinschek et al, 2016) 3.4 | Active site titration Principle Figure 11: Scheme of competitive binding assay using fluorescence polarization as readout…”