Structure‐Based Discovery of A Small Molecule Inhibitor of Histone Deacetylase 6 (HDAC6) that Significantly Reduces Alzheimer's Disease Neuropathology

Abstract Histone deacetylase 6 (HDAC6) is one of the key histone deacetylases (HDACs) that regulates various cellular functions including clearance of misfolded protein and immunological responses. Considerable evidence suggests that HDAC6 is closely related to amyloid and tau pathology, the two primary hallmarks of Alzheimer's disease (AD). It is still unclear whether HDAC6 expression changes with amyloid deposition in AD during disease progression or HDAC6 may be regulating amyloid phagocytosis or neuroinflammation or other neuropathological changes in AD. In this work, the pathological accumulation of HDAC6 in AD brains over age as well as the relationship of its regulatory activity ‐ with amyloid pathogenesis and pathophysiological alterations is aimed to be enlightened using the newly developed HDAC6 inhibitor (HDAC6i) PB118 in microglia BV2 cell and 3D‐AD human neural culture model. Results suggest that the structure‐based rational design led to biologically compelling HDAC6i PB118 with multiple mechanisms that clear Aβ deposits by upregulating phagocytosis, improve tubulin/microtubule network by enhancing acetyl α‐tubulin levels, regulate different cytokines and chemokines responsible for inflammation, and significantly reduce phospho‐tau (p‐tau) levels associated with AD. These findings indicate that HDAC6 plays key roles in the pathophysiology of AD and potentially serves as a suitable pharmacological target through chemical biology‐based drug discovery in AD.


Figure S2 :
Figure S2: Robust stability of PB118 in human serum; (a) Stability of PB118 compound in human serum was monitored for 24 h and measured using reverse phase High-Pressure Liquid Chromatography (HPLC).(b) Half-life (t1/2) of PB118 was calculated using this equation, which is ~30.2 h.

Figure S3 :
Figure S3: ADME/PK studies with PB118; HD Biosciences Co., Ltd performed the ADME studies and in vivo PK profiling.C57BL/6 mice were administered with 1mg/kg PB118 (left).For brain /plasma study, it was followed by the collection of blood and brain samples at 30 min, 1 hr, and 4 hr time points.Samples were processed using acetonitrile precipitation and analyzed by LC-MS/MS (right).(Reprinted with permission from Bai et al, Acta Pharm.Sin.B. 2022, 12(10), 3891, Ref 15, Copyright with Elsevier)

Figure S5 :
Figure S5: Stereomicroscopic images of mouse microglia BV2 cells before and after treatment with PB118 and cell viability assay by LDH assay.Representative stereo microscopic images before (a) and after (b) treatment with PB118.(c) Cell viability was analyzed by the LDH assay for cells treated with LPS +/-PB118 (500 nM and 1000 nM).Mean ± SEM; n = 3; No significant difference was observed after analyzing Student's t-test (p=0.768).Scale bar corresponded to 100 µm.

Figure S6 :
Figure S6: LDH assay to measure the 3D cell viability after treating with PB118.3D cell viability was analyzed by the LDH assay after cells were treated with PB118 at various concentrations (0, 1000 nM and 5000 nM).Mean ± SEM; n = 3; significance was measured after analyzing two-tailed Student's t-test (ns= non-significant).

Figure S7 :
Figure S7: LDH assay to measure the effect of PB118 on HMC3 cells.Cell viability was analyzed by the LDH assay after HMC3 cells were treated with PB118 at various concentrations (0, 500 nM and 1000 nM).Mean ± SEM; n = 3; statistical significance was measured after analyzing two-tailed student's t-test (ns=non-significant). Scale bar corresponded to 100 µm.

Figure S8 :
Figure S8: Negative staining confocal images of BV2 cells stained with only secondary antibody (in the absence of primary antibody); Donkey Anti-mouse Alexa 488, Donkey Antirabbit Alexa 594, and Hoechst (Alexa 405) imaged with a Nikon C2 confocal microscope using 40x objective in (a) bright field (b) 488 channel, (c) 405 channel, (d) 594 channel with (e) merged image.Scale bar corresponded to 10 µm.