Fig 2: Tip60 HAT activation as a unique therapeutic strategy for restoring histone acetylation homeostasis mediated cognition in Alzheimer’s disease (AD).In the healthy brain (left), the balance between Tip60 HAT and HDAC2 enzymes maintains histone acetylation homeostasis necessary for chromatin relaxation and neural gene expression that drives dynamic neural processes. However, in the AD brain (right), loss of Tip60 HAT and/or gain of HDAC2 activity results in reduced histone acetylation levels that condenses chromatin and represses critical neuronal genes resulting in cognitive decline. Tip60 activators selectively enhance its catalytic function to restore histone acetylation levels in the AD brain that should promote expression of critical neural genes and therefore, ameliorate cognitive function in the AD brain. (Created in BioRender. Bhatnagar, A. (2025) https://BioRender.com/a43t168).

Fig 3: Pharmacophore-based virtual screening pipeline for Tip60 HAT activators.A Synthesis of compounds. CTB was used as a query ligand to screen compounds with a similar pharmacophoric model in the curated ZINC15 compound library. Top ~19,000 compounds with similar pharmacophore as CTB were then individually docked on Tip60’s HAT domain (PDB:2ou2) using GOLD protein–ligand docking software. Using a filter for Gold docking score >78, top 38 compounds with best docking scores were selected and 13 of these compounds were custom synthesized for further testing, now referred to as P1–P13. (Created in BioRender. Bhatnagar, A. (2025) https://BioRender.com/t67v064). B Pharmacophore modeling on CTB ligand. Using the Molecular Operating Environment (MOE), six pharmacophore features for CTB (gray licorice sticks) were screened as follows: F1 aromatic ring (orange); F2 aromatic ring (orange); F3 hydrogen donor (magenta); F4 hydrogen acceptor (cyan); F5 hydrophobic (green), and F6 hydrophobic (green). Different combinations of these pharmacophore features were used for virtual screening on ZINC15 compound library. Tip60 Tat-interacting protein 60 kDa, CTB N-(4-Chloro-3-triflouromethyl-phenyl)−2-ethoxy-benzamide, Hyd hydrophobic, Aro aromatic, Don donor, Acc acceptor.

Fig 4: Substructure-based virtual screening pipeline and in vivo testing for Tip60 HAT activators.A CTB was used as a query ligand to screen compounds with a similar substructure in curated ZINC15 compound library. Top 100 compounds with similar structure as CTB were then individually docked on Tip60’s HAT domain (PDB:2ou2) using GOLD protein–ligand docking software. Top 15 compounds with the best docking scores were selected and 10 out of these 15 compounds were synthesized for further testing, now referred to as C1–C10. (Created in BioRender. Bhatnagar, A. (2025) https://BioRender.com/v38v616). (B–K) In vivo locomotion testing of C1–C10 compounds using Drosophila Tip60 knockdown model under elavC155 pan-neuronal Gal4 driver to assess compound efficacy on locomotion speed. Locomotion results are for Tip60-RNAi knockdown larvae fed with vehicle only or different indicated compound concentrations prepared in vehicle, revealing high-performing compounds (B–D), low-performing compounds (E–J) and non-performing compound (K). Statistical significance was calculated using one-way ANOVA with Dunnett’s multiple comparisons. *P < 0.05; **P < 0.01, ***P < 0.001, ****P < 0.0001. For all box and whisker plots, center line represents the median. Bounds of box indicate 25th to 75th percentile. Source data are provided as a Source Data file. Precise n and P values for each experiment are included in Supplementary Data Table 7. Tip60 Tat-interacting protein 60 kDa, CTB N-(4-Chloro-3-triflouromethyl-phenyl)−2-ethoxy-benzamide, Veh vehicle, WT wild-type.

Fig 5: Oral administration of pharmacophore-based compounds prevent Tip60 knockdown-mediated locomotion deficits in vivo.A–F In vivo locomotion testing of C1–C10 compounds using Drosophila Tip60 knockdown model under elavC155 pan-neuronal Gal4 driver to assess compound efficacy on locomotion ability to cross 0.5-cm grid lines in 30 s. Locomotion results are for Tip60-RNAi knockdown larvae fed with vehicle only or different indicated concentrations of high-performing P compounds identified in in vitro HAT assay for compounds P6 (A), P10 (B), and P13 (C). Compound efficacy for third-instar larvae ability to cross entire plate for P6 (D) P10 (E), and P13 (F). Statistical significance was calculated using one-way ANOVA with Dunnett’s multiple comparisons. *P < 0.05; **P < 0.01, ***P < 0.001, ****P < 0.0001. For all box and whisker plots, center line represents the median. Bounds of box indicate 25th to 75th percentile. Source data are provided as a Source Data file. Precise n and p values for each experiment are included in Supplementary Data Table 7. Tip60 Tat-interacting protein 60 kDa, Veh vehicle, WT wild-type.