ACY-738 demonstrates inhibitory activity against recombinant HDAC6 (IC50: 1.7 nM), with respective average selectivity over class I HDACs being 100-fold.

HDAC6:1.7 nM

ACY-738 increases the acetylated (lysine 40) fraction of α-tubulin in RN46A-B14 cells at the concentration of 2.5?μM. The effect that ACY-738 (10 μM) induces cell death is similar to LBH589 and FK228.

ACY-738 administered at a dosage of 5 mg/kg notably increases α-tubulin acetylation in whole-brain lysates, whereas a higher dosage (50 mg/kg) fails to enhance locomotor activity in wild-type (WT) mice within a home cage setting. At a dosage of 5 mg/kg by body weight (BW), ACY-738 influences B cell differentiation in bone marrow (BM) without significantly impacting immunoglobulin G (IgG) and complement component 3 (C3) deposition in NZB/W mice. This dosage also achieves a peak plasma concentration of 1310 ng/mL approximately 0.0830 hours post-treatment. When the dosage is increased to 20 mg/kg, ACY-738 markedly reduces the severity of proteinuria in NZB/W F1 mice and significantly lowers anti-double-stranded DNA (anti-dsDNA) production as well as glomerular interleukin-6 (IL-6) and interleukin-10 (IL-10) mRNA levels by more than 50% in NZB/W mice; the latter being reduced to non-detectable levels with the 20 mg/kg treatment. Additionally, both 5 and 20 mg/kg dosages of ACY-738 decrease serum interleukin-1 beta (IL-1β) production as the NZB/W mice age.

Recombinant kinase domain of EGFR L858R and T790M-L858R mutants are incubated with EGF816 to confirm covalent modification of EGFR and site of adduction. Recombinant enzyme is incubated at room temperature with a 20-fold molar excess of compound in 40 mM Tris, pH 8, 500 mM NaCl, 1% glycerol, 5 mM TCEP for 1 h. The reaction is quenched by addition of dithiothreitol (DTT, 80-fold excess to compound) and transfer to ice. A third of the reaction (10 μL) is processed for intact MS by adding an equal volume of 6 M Guan HCl, 100 mM Tris, pH 8, 20 mM DTT, 10 mM TCEP and incubating at room temperature for 15 min. Intact MS analysis is performed on an Agilent 6520 QToF mass spectrometer equipped with a dual spray ion source (IS of 4500 V, fragmentor of 250 V, fas temp of 350°C, and skimmer of 75 V). The samples are injected onto a PLRP-S column (2.1 mm × 50 mm), heated to 60°C, and desalted for 2 min at 500 μL/min and 3% B prior to elution with a fast gradient of 3-50% B in 3 min (B, 0.1% formic acid). The data are analyzed in MassHunter for automatic peak selection, integration, and spectral deconvolution with a mass range of 15?000-75?000 Da.