Development of First-in-Class Dual Sirt2/HDAC6 Inhibitors as Molecular Tools for Dual Inhibition of Tubulin Deacetylation

Abstract

Dysregulation of both tubulin deacetylases sirtuin 2 (Sirt2) and the histone deacetylase 6 (HDAC6) has been associated with the pathogenesis of cancer and neurodegeneration, thus making these two enzymes promising targets for pharmaceutical intervention. Herein, we report the design, synthesis, and biological characterization of the first-in-class dual Sirt2/HDAC6 inhibitors as molecular tools for dual inhibition of tubulin deacetylation. Using biochemical in vitro assays and cell-based methods for target engagement, we identified Mz325 (33) as a potent and selective inhibitor of both target enzymes. Inhibition of both targets was further confirmed by X-ray crystal structures of Sirt2 and HDAC6 in complex with building blocks of 33. In ovarian cancer cells, 33 evoked enhanced effects on cell viability compared to single or combination treatment with the unconjugated Sirt2 and HDAC6 inhibitors. Thus, our dual Sirt2/HDAC6 inhibitors are important new tools to study the consequences and the therapeutic potential of dual inhibition of tubulin deacetylation.

Structure–Activity Studies of 1,2,4-Oxadiazoles for the Inhibition of the NAD+-Dependent Lysine Deacylase Sirtuin 2

“…To remove precipitates, the solutions were centrifuged 10 min at 4 °C before pipetting. Initial crystals of the Sirt2– 16 complex were only obtained in the presence of Sirt2– Mz242 crystals as microseeds (see Figure for Mz242 structure) . These crystals formed after 3 days in wells with 0.3 μL of protein solution, 0.1 μL of Sirt2– Mz242 crystal solution, and 0.3 μL of reservoir solution in a condition of 26.5% (w/v) PEG 3350 in 0.1 M Bis-Tris at pH 6.5.…”

“…Initial crystals of the Sirt2−16 complex were only obtained in the presence of Sirt2− Mz242 crystals as microseeds (see Figure 1 for Mz242 structure). 78 These crystals formed after 3 days in wells with 0.3 μL of protein solution, 0.1 μL of Sirt2−Mz242 crystal solution, and 0.3 μL of reservoir solution in a condition of 26.5% (w/v) PEG 3350 in 0.1 M Bis-Tris at pH 6.5. Suitable crystals of the Sirt2−16 complex formed after 5 days in wells with 0.3 μL of protein solution, 0.1 μL of Sirt2− 16 crystal solution, and 0.3 μL of reservoir solution in a condition of 25% (w/v) PEG 3350 in 0.22 M Bis-Tris at pH 6.5.…”

Preferential HDAC6 inhibitors derived from HPOB exhibit synergistic antileukemia activity in combination with decitabine

“…7, 166.3, 162.6, 160.1 (d, J = 244.8 Hz), 141.2, 135.5, 133.9 (d, J = 7.3 Hz), 132.0, 131.2, 129.5, 128.5, 127.0, 125.6 (d, J = 16.2 Hz), 122.3, 113.0 (d, J = 23.4 Hz), 50.9, 47.6, 33.6, 32.4, 32.3, 25.2, 24.5, 24.4, 17.0. HRMS calculated for C24H28FN3NaO4: 464,1962, found: 464.1964 6,175.9,172.2,169.7 (d,= 244.4 Hz),150.5,150.26,144.4 (d,J = 5.6 Hz),139.1,138.9,138.4,136.5,134.4 (d,J = 15.4 Hz),131.7,122.4 (d,J = 23.4 Hz) 1, 49.9, 35.1, 33.7. 33.6, 26.6, 26.0 (2C).…”

“…5 While class I, IIa, IIb, and IV HDACs are zinc-dependent, class III isoforms, the so-called sirtuins, are nicotinamide adenine dinucleotide (NAD + )-dependent. 6 Abnormal expression of HDACs has been linked to the occurrence of different malignant tumors, making HDAC inhibitors (HDACi) a focus of intense research. 7 Currently, the FDA has approved four HDACi (Figure 1A) for the treatment of multiple myeloma and T-cell lymphoma: romidepsin, panobinostat, belinostat, and vorinostat.…”

Structure-activity studies of 1,2,4-oxadiazoles for the inhibition of the NAD+ dependent lysine deacylase Sirtuin2

“…To remove precipitates, the solutions were centrifuged 10 min at 4 °C before pipetting. Initial crystals of the Sirt2-16 complex were only obtained in presence of Sirt2-Mz242 crystals as microseeds (see Figure1for Mz242 structure) 81. These crystals formed after 3 days in wells with 0.3 µL protein solution, 0.1 µL Sirt2-Mz242 crystal solution and 0.3 µL reservoir solution in a condition of 26.5 % (w/v) PEG 3,350 in 0.1 M Bis-Tris at pH 6.5.…”