Research paperThe structural requirements of histone deacetylase inhibitors: C4-modified SAHA analogs display dual HDAC6/HDAC8 selectivity
Graphical abstract
Introduction
Histone deacetylase (HDAC) proteins are key enzymes involved in epigenetic regulation of gene expression. Specifically, HDAC-mediated deacetylation of acetyllysine residues on nucleosomal histones leads to tight binding to genomic DNA, which affects accessibility and transcription [1], [2]. In addition, HDAC proteins influence protein-protein interaction, protein-DNA interaction, protein localization, and protein stability through deacetylation of non-histone substrates [3], [4]. The eighteen human HDAC proteins are divided into four classes according to their homology with yeast proteins, size, cellular localization, and number of catalytic active sites [5]. Class III (SIRT1-7) HDAC proteins are NAD+-dependent. Classes I (HDAC1, 2, 3 and 8), II (HDAC4, 5, 6, 7, 9, and 10), and IV (HDAC11) HDAC proteins are metal-dependent, and are the focus of this work [5].
HDAC proteins regulate the expression of several cancer-related proteins involved in cell signaling, transcription, and tumor suppression through the deacetylation of nucleosomal histone proteins [6], [7]. Overexpression of HDAC proteins results in unregulated transcription and aberrant protein activity and function, which is linked to several diseases, including cancer [7]. For example, HDAC1 was overexpressed in lung [8], breast [9], and colon cancers [10]. HDAC2 was overexpressed in colorectal cancer [11]. HDAC8 was highly expressed in neuroblastoma patients, leading to cancer progression and poor survival rates [12]. In addition, selective inhibition of HDAC8 induced apoptosis in leukemia and T-cell lymphoma cell lines [13]. Class II HDAC6 was overexpressed in oral squamous cell carcinoma and ovarian cancer [14], [15]. Overexpression of both HDAC6 and HDAC8 was linked to breast cancer metastasis and invasion [16].
Due to their key role in cancer, several anti-cancer agents targeting HDAC proteins have been developed [17]. HDAC inhibitors promoted apoptosis and reduced proliferation and migration through their effect on both histone and non-histone substrates [17], [18], [19]. Several HDAC inhibitors have been approved by the FDA for treatment of cancer, and several others are in clinical trials [20]. SAHA (suberoylamide hydroxamic acid, Vorinostat, Zolinza™), and Belinostat (PXD101, Belodaq™, Fig. 1) are FDA-approved for treatment of T-cell lymphoma [20], [21], [22], while Panobinostat (LBH-589, Farydak™, Fig. 1) was approved for treatment of multiple myeloma [23]. SAHA is a nonselective inhibitor that targets most of the eleven metal-dependent HDAC isoforms [24]. The nonselectivity of the FDA-approved drugs, including SAHA, might explain the side effects observed in the clinic, such as cardiac arrhythmia and thrombocytopenia [25], [26]. Moreover, the use of SAHA as a chemical tool to study the role of specific HDAC isoforms in cancer cell biology is limited due to its nonselectivity.
To overcome the limitations of nonselective drugs, several isoform selective HDAC inhibitors have been developed, with some in clinical trials. As illustrative examples, entinostat (MS-275, Fig. 1) is selective for HDAC1, 2, and 3 [24], [27], whereas tubastatin (Fig. 1) is HDAC6-selective [28], [29]. Recently, several dual HDAC6/8 selective inhibitors have been reported, including BRD-73954 and valpropylhydroxamic acid (Fig. 1) [30], [31]. HDAC inhibitors that target one or two HDAC isoforms will be valuable for development of new drugs with minimal side effects [32], [33], [34], [35]. In addition, recent reports suggested that inhibition of two HDAC isoforms is desirable by maintaining synergistic therapeutic effects in various cancers [30], [36], [37]. Related to this work, dual inhibition of HDAC6 and HDAC8 might have potential application in breast cancer angiogenesis and metastasis [13], [30]. Moreover, selective HDAC inhibitors will be useful as chemical tools to study cancer-related HDAC cell biology.
To understand the structural requirements of HDAC inhibitors, we previously synthesized SAHA analogs substituted in the linker region at carbon 2 (C2), carbon 3 (C3), carbon 5 (C5), or carbon 6 (C6) (Fig. 1) [38], [39], [40], [41]. C2-hexyl SAHA (Fig. 1) showed 49- to 300-fold HDAC6/8 dual selectivity over HDAC1, 2, and 3, with 0.6 and 2.0 μM potency against HDAC6 and HDAC8, respectively [42]. Among the C5-SAHA analogs, C5-benzyl SAHA (Fig. 1) displayed 8- to 21-fold HDAC6/8 selectivity with IC50 values of 270 and 380 nM with HDAC6 and HDAC8, respectively [41]. Some of the C3-modified SAHA analogs displayed preference for HDAC6 over HDAC1 and 3 [39], while some of the C6-modified SAHA analogs inhibited HDAC1 and 6 over HDAC3 [40]. In addition, SAHA analogs modified at the hydroxamic acid moiety had a preference for HDAC1 [43]. In this work, SAHA analogs modified at the C4 position were synthesized and screened in vitro and in cellulo for their activity and selectivity. The C4-modified SAHA analogs showed high selectivity towards HDAC6 and 8 over HDAC1, 2, and 3, with nanomolar potency against HDAC6 and HDAC8. Docking studies provided a structural rationale for the observed selectivity. These studies emphasize that modification of the SAHA linker can enhance isoform selectivity. In addition, the HDAC6/8 dual selective C4-SAHA analogs reported here have the potential to be useful pharmacological tools for biomedical research and lead compounds for anti-cancer drug development.
Section snippets
Synthesis of C4-modified SAHA analogs
Synthesis of the C4-SAHA analogs started with a cross metathesis reaction of methyl-4-pentenoate (2) with crotonaldehyde (3) using second generation Grubbs' catalyst to afford the α,β-unsaturated aldehyde (4) (Scheme 1). Different substituents were appended to 4 via 1,4-addition using organolithium cuprates, followed by Horner–Wadsworth–Emmons reaction with benzyl phosphonoacetate (5) to give the unsaturated benzyl esters (6a-f). Reduction and hydrogenolysis of 6a-f gave free acids (7a-f),
Conclusions
In conclusion, SAHA analogs modified at the C4 position were synthesized and screened for potency and selectivity. C4-SAHA analogs showed up to 1300-fold dual selectivity for HDAC6 and HDAC8 over HDAC1, HDAC2, and HDAC3. The best analogs were C4-n-butyl SAHA (1c) and C4-benzyl SAHA (1f). C4-benzyl SAHA (1f) showed the highest fold selectivity with 210- to 740-fold selectivity for HDAC6 and 8 compared to HDAC1, 2, and 3, and 140 and 57 nM IC50 with HDAC6 and HDAC8. Interestingly, the fold
Materials and instrumentation
Unless otherwise noted, chemicals were purchased from Sigma-Aldrich, Acros Organics, or Fisher Scientific. “Iron-free” glassware was prepared by rinsing glass vessels with a 5M aqueous HCl solution, followed by washing with distilled de-ionized water. “Iron-free” silica gel was prepared by washing with 5M aqueous HCl, followed by washing with distilled de-ionized water until colorless, and subsequently drying under air or in the oven at 80 °C. NMR spectra were taken on a Varian or Agilent 400
Author contribution
A. Negmeldin synthesized all analogs and performed all experiments, except for testing tubastatin and BRD-73954 in the HeLa lysate activity assay and determining the cytotoxicity of tubastatin and PCI-34051 using the MTT assay, which were performed by J. Knoff. M. Pflum conceived of the project and assisted in experimental design and interpretation. A. Negmeldin and M. Pflum wrote the manuscript.
Funding sources
We thank National Institutes of Health (GM121061) and Wayne State University for funding. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Acknowledgements
We thank Lumigen Instrument Center at Wayne State University for NMR and MS instrumentation, Y. Ge for the U937 cell line, K. Honn and Y. Ahn for use of the Alpha Innotech FluorChem imaging systems, and I. Gomes, J. Knoff, D. Nalawansha, and Y. Zhang for comments on the manuscript.
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