BROMO-PROTOPINE, A NOVEL DERIVATIVE OF PROTOPINE WITH IMPROVED BIOAVAILABILITY AND BIOACTIVITY, DEGRADES TAU AGGREGATION THROUGH MODULATION OF HDAC6-HSP90 CHAPERONIC ACTIVITY AND IMPROVES MEMORY VIA STIMULATION OF THE RAS-GRF1/ERK PATHWAY

Abstract

Recently, we identified protopine (PRO) as a novel HDAC6 inhibitor with anti-Tau activity from the Chinese medicinal herb Corydalis yanhusuo (Durairajan et. al., 2015, Neurodegenerative disease, 15 [Suppl. 1]; p1579). In this study, we investigated the mechanism of HDAC6-Hsp90 modulation by PRO and explored whether derivatization of PRO could improve its in vivo efficacy. To optimize the anti-Alzheimer's disease (AD) activity of PRO, we synthesised 8 derivatives of PRO and carried structural activity relationship studies. The docking studies of PRO derivatives with HDAC6 was performed using Autodock program. Using HDAC overexpression, acetyl-lysine immunoprecipitation and co-immunoprecipitation assays, and Western blotting, we determined how PRO modulates the HDAC6-Hsp90 chaperonic activity and stimulated RAS-GRF1/ERK pathway in both neuronal cell lines and primary neuronal cultures. Oral and Intraperitoneal pharmacokinetic studies were conducted to estimate the relative bioavailability of compounds. For the in vivo AD study, P301S Tau and 3XTg-AD mice administered with PRO and its derivative or vehicle until the mice were 4- and 18-month old, respectively. Among synthesised PRO derivatives, only bromo-protopine (PRO-Br) showed anti-HDAC6, anti-Tau activity and memory improving activities. The binding affinity of PRO and PRO-Br with HDAC6 is 7.65 and -8.76 kcal/mol, respectively. Treatment with PRO and PRO-Br reduced both phosphorylated PHF-1 Tau and total Tau, and concomitantly increased acetylated α-tubulin and Hsp90, and Hsp70 in SHSY5Y-P301L cells. PRO-Br showed two-fold more activity in reducing HDAC6 activity and tau accumulation than PRO. In pharmacokinetic studies, PRO-Br showed enhanced brain bioavailability than PRO in both oral and intraperitoneal routes of administration. In particular, the Cmax of the PRO was 289.47 ng/gm and 76.5 ng/gm whereas PRO-Br was 425.22 ng/gm and 161.6 ng/gm in oral and intraperitoneal administration, respectively. We further investigated the anti-AD mechanism of PRO and PRO-Br and found that PRO and PRO-Br clear the insoluble Tau aggregates in P301S Tau mice by modulating HDAC6-Hsp90 chaperonic activity and improved memory of 3XTg-AD mice by stimulating RAS-GRF1/ERK/CREB pathway. PRO-Br displayed improved in vivo efficacy in terms of counteracting increased Tau aggregates, motor dysfunction, and memory dysfunction in AD mouse models. Our findings suggest that PRO-Br has potential as an anti-AD drug.