Developing potent PROTACs tools for selective degradation of HDAC6 protein

Abstract

Dear Editor, Histone deacetylases (HDACs) are a family of enzymes that remove acetyl groups on histone and non-histone proteins, thereby playing a vital role in the modulation of gene expression and protein activity. Eighteen HDACs have been identified in human and subdivided into four classes including I, II (IIa, IIb), III and IV (Seto et al., 2014). Among them, HDAC6 is a unique IIb HDAC with dominant cytoplasmic localization and two functional catalytic domains. Besides the functions for deacetylation of histone, and modulation of α-tubulin, HSP90 and cortactin, HDAC6 also participates in protein trafficking and degradation, cell shape and migration (Valenzuela-Fernandez et al., 2008). The deregulation of HDAC6 is related to various diseases, such as neurodegenerative diseases, cancer and pathological autoimmune response (Batchu et al., 2016). Hence, it is especially important for directly controlling cellular HDAC6 protein levels to achieve therapeutic purposes. The traditional approaches of reducing cellular protein levels mainly rely on genetic modifications, such as RNA interference, transcription activator-like effector nucleases, recombination-based gene knockout and clustered regularly interspaced short palindromic repeats (CRISPR-Cas9) (Boettcher et al., 2015). However, these approaches have failed to a certain degree to achieve acute and reversible changes of gene function. Furthermore, the complications of potential genetic compensation and/or spontaneous mutations arising in gene-knockout models may lead to misinterpretations (Davisson et al., 2012; El-Brolosy et al., 2017). Therefore, it is urgent for developing a rapid, robust, and reversible approach to directly modulate HDAC6 protein levels. Known as a chemical based protein knockdown strategy, PROteolysis-TArgeting Chimera (PROTAC) has emerged as a novel and powerful method for the degradation of interested proteins. The PROTACs are heterobifunctional molecules, which consist of three parts: a ligand for binding target protein, a ligand for recruiting E3 ligase and a linker connecting the two ligands (Lai et al., 2017). Consequently, the PROTACs mediated interaction of the target protein and a E3 ligase caused ubiquitination and subsequent degradation of the target protein by the ubiquitin-proteasome system (UPS) (Fig. 1A). It has been proved that PROTAC technology can achieve efficient degradation of proteins with excellent selectivity in a quick and direct manner (Yang et al., 2018; Zhou et al., 2018). Moreover, the PROTAC also worked well for mutated proteins (Sun et al., 2018). Herein, we report the development of HDAC6-targeting degraders based on the PROTAC strategy. The newly designed PROTACs induced significant degradation of HDAC6 in a panel of cell lines, exhibited excellent selectivity against other HDACs, and demonstrated efficient inhibition of cell proliferation. Besides, the degradation process was well illustrated by fluorescence-based visualization. Development of selective HDAC6-degrading PROTACs. (A) The principle of PROTAC. (B) The structure of PROTAC, as shown in the upper portion. A binding mode of PROTAC (ball stick), HDAC6 (PDB 5G0J, purple) and CRL4-CRBN (PDB 2HYE and 4CI3, colored cyan and gray) was simulated by Pymol. (C) Screen for a potent HDAC6 degrader. HeLa cells were treated as indicated for 24 h. (D) Characterization of NP8-induced degradation in HeLa cells. The degradation of HDAC6 was in a dose-dependent manner. The HDAC1, HDAC2 and HDAC4 levels were not affected within 24 h. (E) NP8 caused fast degradation of HDAC6 within 2 h in HeLa cells. (F) NP8-NC1 and NP8-NC2 failed to degrade HDAC6. NP8 induced degradation was rescued by single introduction of Nexturastat A (Nex A, 300 nmol/L), Pomalidomide (Poma, 10 µmol/L) or Carfilzomib (CARF, 1 µmol/L). The concentration of NP8-NC1, NP8-NC2 and NP8 were 300 nmol/L. HeLa cells were treated with Carfilzomib for 6 h and 24 h for the rest. (G) The degradation of HDAC6 by titration of NP8 for 24 h in MM.1S cells. (H) MM.1S cells were treated with NP8 or Nexturastat A (Nex A) for 72 h. Cell viability was determined by CCK-8 assay (n = 3) To design novel HDAC6-targeting PROTACs, we chose a selective HDAC6 inhibitor Nexturastat A (Nex A) as the HDAC6 binder (Bergman et al., 2012). According to the recently released co-crystal structure of HDAC6 in complex with Nex A (Miyake et al., 2016), the aliphatic chain was oriented outside of the ligand binding pocket. Based on the PROTACs design principles, Pomalidomide (Poma, a ligand for E3 ligase CRBN) was introduced onto the end of aliphatic chain of Nex A via different linkers (Lopez-Girona et al., 2012). As shown in the simulated diagram (Fig. 1B), the PROTACs should actively bind HDAC6 and CRBN simultaneously. The synthesis of the HDAC6 degraders was shown in Supplementary Materials (Scheme 1). Next, the resulting HDAC6-targeting PROTAC molecules were tested. To evaluate the degradation capability of our PROTACs for HD