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. 2020 Mar 18;11(4):575–581. doi: 10.1021/acsmedchemlett.0c00046

Development of Selective Histone Deacetylase 6 (HDAC6) Degraders Recruiting Von Hippel–Lindau (VHL) E3 Ubiquitin Ligase

Ka Yang †, Hao Wu †, Zhongrui Zhang ‡, Eric D Leisten †, Xueqing Nie †, Binkai Liu †, Zhi Wen ±, Jing Zhang ±, Michael D Cunningham †, Weiping Tang †,‡,*
PMCID: PMC7153272  PMID: 32292566

Abstract

graphic file with name ml0c00046_0004.jpg

Histone deacetylase 6 (HDAC6) is involved in multiple cellular processes such as aggresome formation, protein stability, and cell motility. Numerous HDAC6-selective inhibitors have been developed as cellular chemical tools to elucidate the function of HDAC6. Since HDAC6 has multiple domains that cannot be studied by HDAC6-selective inhibitors, CRISPR-CAS9 and siRNA/shRNA have been employed to elucidate the nonenzymatic functions of HDAC6. However, these genetic methods have many limitations. Proteolysis targeting chimera (PROTAC) is an emerging technology for the development of small molecules that can quickly remove the entire protein in cells. We previously developed multifunctional HDAC6 degraders that can recruit cereblon (CRBN) E3 ubiquitin ligase. These HDAC6 degraders can degrade not only HDAC6 but also neo-substrates of CRBN. They are excellent candidates for the development of anticancer therapeutics, but the multifunctional nature of the CRBN-based HDAC6 degraders has limited their utility as specific chemical probes for the study of HDAC6-related cellular pathways. Herein we report the development of the first cell-permeable HDAC6-selective degraders employing Von Hippel–Lindau (VHL) E3 ubiquitin ligase, which does not have any known neo-substrates. The DC50’s of the most potent compound 3j are 7.1 nM and 4.3 nM in human MM1S and mouse 4935 cell lines, respectively. The Dmax’s of 3j in these two cell lines are 90% and 57%, respectively.

Keywords: HDAC6, PROTAC, degradation, VHL

As one of the major post-translational modifications (PTMs) of proteins,1 ε-acetylation of lysine residues is essential in numerous biological pathways such as cellular signaling and metabolism.2 Histone acetyltransferases (HATs) and histone deacetylases (HDACs) control the dynamic acetylation of histones and chromatin structures, thereby regulating transcriptional activation or suppression, respectively.3,4 HDAC6 is a class IIb HDAC primarily localized in the cytoplasm, while most other HDACs are localized in the nuclei.5,6 Nonhistone proteins including α-tubulin, cortactin, and HSP90 are substrates of HDAC6, which regulates cell motility and protein turnover through the removal of the ε-acetyl group in lysine residues on these substrates.7,8 HDAC6 is also known as tubulin deacetylase (TDAC) because tubulin is one of the major substrates of HDAC6.9 In addition, HDAC6 was reported to modulate PD-L1 expression via the STAT3 signaling pathway, suggesting its role in immunoregulation.10,11 Beside deacetylase activity, HDAC6 was also reported to ubiquitinate MSH2, indicating its role as an E3 ubiquitin ligase.12

HDAC6-selective inhibitors have been developed as potential therapeutics for the treatment of cancers,13 neurodegenerative diseases including Alzheimer’s, Parkinson’s, and Huntington’s diseases,14 and autoimmune and inflammatory disorders.15 These HDAC-selective inhibitors can block the enzymatic function of the C-terminal catalytic domain.16 However, HDAC6 has a N-terminal catalytic domain and a zinc-finger ubiquitin binding domain (ZnF-UBP), in addition to the C-terminal catalytic domain that can be inhibited by HDAC6-selective inhibitors.17 In order to study the functions of these domains, genetic knockdowns have been used as the primary tools.18

Knocking down or out gene(s) in biological pathways is an important method to study the cellular network. CRISPR-CAS9 and siRNA/shRNA technology have been used as the major methods in controlling protein expression at the genome and transcriptome levels.19,20 These genetic methods are powerful tools for determining the functional consequence of loss of a target gene. However, the delay between the initiation of modulation of the system to experimental observation of the loss of protein target is a significant limitation of these genetic methods, which cannot be used for assessing any acute change of a specific protein target. There are also a number of other limitations, such as challenges associated with delivery, metabolic instability, irreversible editing, and off-target effects.

The ubiquitination-proteasome system (UPS) is the major pathway for protein degradation.21 Proteolysis targeting chimera (PROTAC) can induce efficient degradation of the targeted protein via the UPS and complement the genetic tools.22 PROTAC is a bifunctional small molecule created by tethering a E3 ubiquitin ligase ligand and a ligand for a protein of interest (POI) with a linker. The chimeric molecule binds to either the POI or E3 ligase first, and then recruits one to the other to form a ternary complex.23 The proximity allows E3 ligase to induce the polyubiquitination of the POI and promotes its proteasomal degradation. The E3 ligase and the targeted protein(s) do not need to be physiologically relevant partners. Various functional proteins have been artificially targeted for ubiquitination–degradation by different E3 ligases using the PROTAC strategy.24 Beside inducing rapid degradation, PROTAC has multiple potential advantages over traditional inhibitors as chemical tools and potential therapeutics, such as substoichiometric dose, abolishing the enzyme-independent functions of a multifunctional protein, sustained downstream effects, and offering selectivity for isoforms of proteins that are otherwise difficult to differentiate.25 These features highlight the significance of targeted protein degradation (TPD) by PROTAC as a powerful alternative tool to CRSPR-CAS9 or siRNA/shRNA for the study of loss-of-function of proteins involved in various biological pathways.

We reported the first HDAC6 degrader 1 (Figure 1A) by tethering a pan-HDAC inhibitor and pomalidomide to recruit cereblon (CRBN) E3 ligase.26 Although the nonselective inhibitor can bind to all 11 isoforms of HDACs, the resulting degrader selectively induced the degradation of HDAC6. Based on this, we further developed the next generation HDAC6 degraders using Nexturastat A (Next-A), a known HDAC6-selective inhibitor, as the ligand of POI.27 Among the compounds we prepared, degrader 2 (Figure 1A) showed the highest potency for the degradation of HDAC6. It also possessed promising antimyeloma activity due to synergistic effects of HDAC6 degradation/inhibition with the degradation of Ikaros family zinc finger proteins 1 and 3 (IKZF1/3). After our work, similar HDAC6 degraders that recruit CRBN E3 ligases were reported.28 The IKZFs are not physiologically relevant substrates of CRBN, but the binding of immunomodulatory drugs (IMiDs) such as pomalidomide to CRBN can induce the degradation of IKZFs—so-called neo-substrates.29 However, the degradation of IKZFs is not always desired and the pomalidomide moiety has several other potential neo-substrates besides IKZFs.30 In addition, it has also been suggested that alternating use of PROTACs recruiting different E3 ligases may achieve durable responses for the treatment of certain types of cancers.31,32

Figure 1.

Figure 1

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Development of HDAC6 degraders. (A) Structure of CRBN-based HDAC6 degraders. (B) Structure of VHL-based degraders. (C) Screening of VHL-based degraders bearing different linker lengths. MM1S cells were treated with 100 nM degraders for 6 h and analyzed by in-cell ELISA assay. (D) Test of selected candidates with longer linkers. MM1S cells were treated with 100 nM degraders for 6 h and analyzed by Western blot.

The putative number of E3 ligases in the human genome is over 600.33 However, only inhibitor of apoptosis protein (IAP), Von Hippel–Lindau tumor suppressor protein (VHL), and CRBN, together with their ligands, are widely exploited for the development of PROTACs for the degradation of various protein targets.34 VHL is part of the VBC E3 ubiquitin complex (VHL, elongins B and C, Cul2 and Rbx1).35 Crews, Ciulli, and their co-workers adopted l-hydroxyproline (l-Hyp) as the core structure for the design and development of small-molecule VHL ligands VHL-1/VH032.36,37 Since then, VHL ligands have been applied to the development of several effective degraders that can recruit VHL E3 ligase.38−43 Herein, we describe a new class of chemical probes that can recruit VHL to degrade HDAC6 efficiently. The advantage of degraders based on VHL-ligand over those based on CRBN-ligands is the lack of any known neo-substrates.

To examine the feasibility of recruiting VHL E3 ligase to degrade HDAC6, we synthesized several series of compounds with different types and lengths of linkers between Next-A and VHL ligand VHL-1.44 Among them, the 11 compounds 3a–k (Figure 1B) showed promising activity. Within this series of compounds, the number of methylene units (n) between Next-A and the triazole ring ranged from 2 to 12. There are four methylene units between the triazole and VHL ligand. We thus termed these compounds as the “n + 4” series. During our initial attempts, we also tested the “n + 2” and “n + 3” series of compounds with various lengths of methylene units (n) between Next-A and the triazole ring. We also tested the “n + 4” series of compounds with polyethylene glycol linker for the “n” side. These compounds are generally not active degraders for the HDACs we tested (Supporting Information Figure S1). This suggests that both linker type and linker length are important parameters for the development of HDAC6 degraders recruiting VHL.

All 11 “n + 4” VHL-based potential degraders were initially tested at 100 nM and compared with CRBN degrader 2 in MM1S cells. The resulting cells were analyzed for HDAC6 protein level by in-cell ELISA (Figure 1C). We observed a clear trend of increased potency as the linker length is increased from 3a (n = 2) to 3k (n = 12). The three most potent compounds, 3i, 3j, and 3k, induced a similar level of degradation compared to degrader 2, indicating that this new class of degraders could achieve efficient depletion of HDAC6. We then used Western blot analysis to confirm the ELISA results (Figure 1D). Degraders 3g–k downregulated HDAC6 following a linker-length-dependent manner at 100 nM. Meanwhile, we observed that degraders 3h–j increased acetylated tubulin more than others.

To further examine the efficiency of degraders, we performed a dose response study of 3j, 3k, and 2 at concentrations ranging from 1 nM to 3 μM for 6 h in MM1S cells by ELISA (Figure 2A). The DC50 obtained for 2 was 2.2 nM, which was close to our previously reported data (DC50 = 1.6 nM).27 Degraders 3j and 3k were slightly less potent in terms of DC50. However, both of them showed similar Dmax (∼90%) to 2 (∼86%). It requires more degraders based on VHL-ligands to achieve similar degradation level of HDAC6 compared to degraders derived from CRBN ligands. However, degraders containing VHL-ligand showed similar maximal HDAC6 depletion to degraders derived from CRBN ligands.

Figure 2.

Figure 2

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Profiling the efficiency and mechanism of VHL-based degraders by in-cell ELISA or Western blot. (A and B) Dose response of degraders from 1 nM to 300 nM or to 30 μM for 4 h in MM1S. (C) Selectivity for increasing acetylated tubulin over acetylated histone. MM1S cells were treated with HDAC6 degraders or HDAC inhibitors for 6 h. (D) Time-course study of degraders. MM1S cells were treated with 100 nM 3j or 2 for from 0 to 6 h. (E and F) Mechanistic study of degrader 3j. MM1S cells were pretreated with pathway inhibitors or competitive ligands for 1 h and then treated with 100 nM degrader 3j. Concentration used: MG132 (1 μM), Bortezomib (Bortz, 1 μM), MLN4924 (5 μM), and Next-A (5 μM).

Degrader 3j was chosen for our further studies on its dose response, mechanism, and selectivity. We treated MM1S cells with this degrader at concentrations ranging from 1 nM to 30 μM for 4 h and analyzed them by Western blot (Figure 2B). HDAC6 was degraded strongly at as low as 10 nM of the degrader and the maximal effect was reached around 100 nM, which was consistent with in-cell ELISA results. PROTACs often become less effective at higher concentrations because the formation of more binary complexes (E3-PROTAC and POI–PROTAC) than ternary complex (E3-PROTAC-POI), which is often referred as “hook effect”.45 The “hook effect” for HDAC6 degrader 3j was observed at 3 μM or higher concentrations. The acetylation level of tubulin was dose-dependent and reached the maximal effect around 1 μM of concentration. Significant increase of acetylated tubulin was only observed when the concentration of degrader 3j was 300 nM or higher. In addition to the HDAC6 level, the amount of acetylated tubulin also depends on other factors, such as the efficiency of acetyltransferase-catalyzed acetylation of tubulin and the efficiency of HDAC6-catalyzed deacetylation of tubulin. Overall, the HDAC6 level is correlated with the amount of acetylated tubulin. The effect on the protein level of other HDACs was minimal. At least, no obvious degradation of other HDACs was observed when 3j reached its maximal effect for the degradation of HDAC6 at around 100 nM concentration. In addition, we did not observe obvious degradation of IKZFs, which were often degraded when thalidomide analogues were used as the E3 ubiquitin ligase recruiting ligands (Supporting Information Figure S2). When HDAC6-selective degraders (3j and 2) and HDAC6-selective inhibitor Next-A were compared with pan-HDAC inhibitor SAHA at different concentrations, selective increase of acetylated tubulin over acetylated histone was confirmed for HDAC6-selective degraders (Figure 2C). Our results collectively suggested that degrader 3j is a selective chemical probe for the degradation of HDAC6.

To further probe the efficiency of degrading HDAC6, a time-course study was performed (Figure 2D). Both 3j and 2 started to reduce HDAC6 protein level at 30 min and reached maximal degradation around 4 h. Both 3j and 2 also induced the increase of tubulin acetylation as expected; but the latter was slightly more potent. All of our data indicate that degrader 3j based on a VHL ligand can effectively degrade HDAC6 selectively in cellular assays.

To gain more evidence to support the hypothesis that the VHL-based degraders suppress HDAC6 protein level via the ubiquitination–proteasome pathway, we conducted a series of experiments by cotreating the HDAC6 degraders with competitive pathway inhibitors (Figure 2E). MM1S cells pretreated with proteasome inhibitor (MG132 and Bortezomib) showed no obvious HDAC6 degradation by 3j, supporting that the proteolysis of HDAC6 occurred in proteasome. Next-A abolished the effect of HDAC6 degradation by 3j by competitively binding to HDAC6. MLN 4924, an inhibitor of NEDD8-activating enzyme (NAE), can deactivate the E3 ligase activity and abolish the 3j-induced degradation, indicating that the neddylation and E3 were required for VHL-based PROTACs.46,47 VHL-1 completely recovered the HDAC6 level at 5 μM or above (Figure 2F), demonstrating that VHL E3 was required for efficient degradation by the VHL-ligand based degraders.

To explore the generality of 3j for the degradation of HDAC6, we tested its effect on HDAC6 in other human cell lines including HEK293T, U87MG, A549, and MCF-7 (Supporting Information Figure S3). Across all cell lines, HDAC6 was depleted in a dose-dependent manner, though the potency varies in these cell lines, which may be due to either the different expression levels of VHL E3 ligase or different resynthesis rates of HDAC6 in these cell lines. Furthermore, we compared the degradation efficiency of HDAC6 induced by 2 and 3j in mouse immortalized cell line 4935 by Western blot and in-cell ELISA. In concentrations ranging from 10 nM to 10 μM, VHL-based degrader 3j induced more HDAC6 degradation and tubulin acetylation than CRBN-based degrader 2 (Figure 3A). The estimated DC50’s are about 12 and 360 nM for 3j and 2, respectively, based on the band intensity in Figure 3A. ELISA results also showed that 3j was more potent than 2 and their calculated DC50’s were about 4.3 nM and 18 nM, respectively (Figure 3B). Western blot and ELISA do not always give the same results. The difference between these two assays in mice cells (Figures 3A/3B) appeared to be bigger than that in human cells (Figures 2A/2B). The background of ELISA in mice cells was higher than that in human cell line, which might contribute to the bigger difference. The maximal degradation percentage for HDAC6 induced by 3j is 57%, which is better than the Dmax of 45% induced by compound 2. Our data indicated that the relative potency of different PROTACs may vary among different species. The VHL E3 ligase-based degrader 3j could effectively knockdown HDAC6 in mice cell line. It is worth mentioning that the selectivity of HDAC inhibitors is less documented for mouse HDACs than human HDACs.

Figure 3.

Figure 3

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Effect of VHL-based degrader 3j on the degradation of HDAC6 in mouse cell line. VHL-based degrader 3j is less dependent on species than CRBN-based degrader 2. Mouse 4935 cells were treated for 6 h and analyzed by Western blot (A) and in-cell ELISA (B).

In summary, we have developed a new class of HDAC6 degraders by recruiting VHL instead of CRBN as the E3 ubiquitin ligase. The linker length required for VHL-ligand based degraders is much longer than that of CRBN-ligand based degraders. Compound 3j was identified as the most potent candidate among this class of HDAC6 degraders. Mechanistic studies demonstrated that 3j targeted HDAC6 for proteasomal degradation. Compound 3j did not induce the degradation of IKZF1/3, which were often the targets of previous CRBN-ligand based degraders. The potency, selectivity, and activity in a broad scope of cell lines underscore the utility of 3j as a specific chemical probe for the degradation of HDAC6 to study HDAC6-related biological pathways. The investigation of HDAC6 associated biological pathways by comparing the effect of its degraders and inhibitors is ongoing and will be reported in due course.

Methods

A detailed description of the methods and materials used is provided in the Supporting Information.

Acknowledgments

The authors would like to thank the University of Wisconsin Carbone Cancer Center’s (UWCCC) Consultation Panel for support of this project. This work is also supported in part by NIH/NCI P30 CA014520-UW Comprehensive Cancer Center Support (CCSG). Jing Zhang thanks NIH (R01 CA152108) and pilot funding from UWCCC Developmental Therapeutics Program for financial support.

Glossary

Abbreviations

PTM

post-translational modification

HAT

histone acetyltransferase

HDAC

histone deacetylase

TDAC

tubulin deacetylase

ZnF-UBP

zinc-finger ubiquitin binding domain

UPS

ubiquitination–proteasome system

PROTAC

proteolysis targeting chimera

POI

protein of interest

TPD

targeted protein degradation

CRBN

cereblon

Next-A

Nexturastat A

IKZF

Ikaros family zinc finger protein

IMiD

immunomodulatory drug

IAP

inhibitor of apoptosis protein

VHL

Von Hippel–Lindau tumor suppressor protein

L-hyp

L-hydroxyproline

ELISA

enzyme-linked immunosorbent assay

DC50

half maximal degradation concentration

Dmax

maximal degradation

NAE

NEDD8-activating enzyme.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.0c00046.

  • Supplemental figures, detailed biology methods, synthetic procedures, and compound characterizations (PDF)

Author Contributions

∥ K.Y. and H.W. contributed equally. K.Y. designed and completed most biochemical and cell-based assays. H.W. designed and synthesized all compounds. All authors contributed to discussions.

The authors declare the following competing financial interest(s): A provisional patent on compounds in this manuscript has been filed.

Supplementary Material

ml0c00046_si_001.pdf (5.1MB, pdf)

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