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Stabilized Peptide HDAC Inhibitors Derived from HDAC1 Substrate

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Stabilized Peptide HDAC Inhibitors Derived from HDAC1 Substrate Published OnlineFirst March 6, 2019; DOI: 10.1158/0008-5472.CAN-18-1421 Cancer Metabolism and Chemical Biology Research Stabilized Peptide HDAC Inhibitors Derived from HDAC1 Substrate H3K56 for the Treatment of Cancer Stem–Like Cells In Vivo Dongyuan Wang1, Wenjun Li1, Rongtong Zhao1, Longjian Chen1, Na Liu1, Yuan Tian2, Hui Zhao3, Mingsheng Xie1, Fei Lu1, Qi Fang1, Wei Liang4, Feng Yin1, and Zigang Li1 Abstract FDA-approved HDAC inhibitors exhibit dose- A DNA H3K56 H3K64 H3K79 H3K115 limiting adverse effects; thus, we sought to improve Histone 3 αN α1 α2 α3 +H N the therapeutic windows for this class of drugs. In 3 L1 L2 this report, we describe a new class of peptide- Histone tail H3K56 Cap + based HDAC inhibitors derived from the Zn2 TD: Helix stabilization linker HDAC1-specific substrate H3K56 with improved Zinc binding O NH R nonspecific toxicity compared with traditional Histone H N CH small-molecular inhibitors. We showed that our 3 O designed peptides exerted superior antiprolifera- HDACs O HN tion effects on cancer stem–like cells with minimal Acetylated OH lysine such as H3K56Ac Helix stability HDACs N-terminus for modification toxicity to normal cells compared with the small- Facile construction molecular inhibitor SAHA, which showed nonspe- HDACs inactivation in vitro and in vivo O NH O R R NH fi B NH NH ci c toxicity to normal and cancer cells. These O O O O R HN R HN peptide inhibitors also inactivated cellular HDAC1 OH OH and HDAC6 and disrupted the formation of the HDAC1, LSD1, and CoREST complex. In ovarian O Inactive R NH Active teratocarcinoma (PA-1) and testicular embryonic HDACs HDACs Ac NH O O carcinoma (NTERA-2) cell xenograft animal mod- R NH OH els (5 mice/group, 50 mg/kg, every other day, Ac Ac H3K56 Ac intraperitoneal injection), these peptides inhibited H3K56 tumor growth by 80% to 90% with negligible organ (heart, liver, spleen, lung, kidney, brain) lesions. These results represent the first attempt to design H3K56 H4K16 Ac chemically stabilized peptide inhibitors to investi- Ac H4K16 Ac H3K56 Ac – gate HDAC inhibition in cancer stem like cells. Gene repression Gene derepression These novel peptide inhibitors have significantly enhanced therapeutic window and offer promising opportunities for cancer therapy. Proliferation No proliferation Significance: Selective antiproliferative effects of stabilized peptide HDAC inhibitors toward cancer © 2019 American Association for Cancer Research stem–like cells provide a therapeutic alternative that avoids high nonspecific toxicity of current drugs. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/8/1769/F1.large.jpg. 1School of Chemical Biology and Biotechnology, Peking University Shenzhen Corresponding Authors: Zigang Li, Peking University, Shenzhen 518055 Graduate School; State Key Laboratory of Chemical Oncogenomics, Key Lab- China. Phone: 755-26033616; E-mail: [email protected]; and Feng Yin, oratory of Chemical Genomics, Peking University Shenzhen Graduate School, School of Chemical Biology and Biotechnology, Peking University Shenz- Shenzhen, China. 2School of Life Science and Engineering, Southwest Jiaotong hen Graduate School, Shenzhen University Town, Lishui Road, Xili Town, University, Chengdu, China. 3Division of Life Science, Clarivate Analytics, Beijing, Nanshan District, Shenzhen,China. Phone: 755-26033611; E-mail: China. 4Department of Radiation Oncology, The First Affiliated Hospital, Anhui [email protected] Medical University, Hefei, China. doi: 10.1158/0008-5472.CAN-18-1421 Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Ó2019 American Association for Cancer Research. www.aacrjournals.org 1769 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst March 6, 2019; DOI: 10.1158/0008-5472.CAN-18-1421 Wang et al. Introduction helical stabilization methodologies including the N-terminal Two of the most fundamental hallmarks of malignant tumors helix nucleating template (TD strategy) and the in-tether chiral are their heterogeneous nature and limitless proliferation poten- center induced peptide helicity (CIH strategy) based on the tial (1). The cancer stem cell (CSC) theory states that malignant concept that a precisely tuned in-tether chiral center could dom- proliferation can be fueled by a small subset of stem-like tumor inate the peptide's secondary structure and its biological func- cells, which possess similar properties as typical stem cells and can tions (23, 24). Peptides constructed with these methods are contribute to drug resistance and cancer relapse (2). Cancer stem– successfully utilized to target ERa–coactivator interaction and like cells have been found in many common cancer types such as p53–MDM2/MDMX interaction (25, 26). Inspired by the previ- breast cancer (3), colorectal cancer (4), ovarian cancer (5), and ous success of FK228 and our own experience in stabilized lung cancer (6). Increasing evidence has revealed that epigenetic peptides, we hypothesized that peptide substrate analogues with mediators, especially HDACs, can contribute to the emergence large binding surfaces could achieve enhanced efficiency and and maintenance of CSCs (7–9). HDACs control cancer cell reduced toxicity through effectively disrupting the interaction fortune through the removal of the acetyl group of histone and between HDACs and their substrates. The HDAC substrates nonhistone proteins. The aberrant acetylation of histones has acetylated H3K56 and H4K16 have been reported to have a been identified in oncogenesis, for example, the loss of acetylation preference for HDAC1 and were previously examined (15, 16). of H4K16 (H4K16ac) is found in various cancer types such as Thus, we chose to modify the helical region (comprising residues lymphoma, and cancers of the breast, colon, and lung (10). 45–56) of H3 and stabilized the peptide structure using our Additionally, HDACs can interact directly with some transcription developed method—the TD strategy. We envisioned that these factors, such as E2F, Stat3, p53, Rb, and NF-kB, to regulate import- stabilized peptide-based HDAC inhibitors with large interacting ant signaling pathways (11). Thus, the aberration of HDACs in interfaces could improve the molecules' selectivity for HDACs cancer cells has fueled intense interest in the development of over other nonspecific interacting proteins and ultimately reduce HDAC inhibitors for cancer therapy (12). nonspecific toxicity. In this report, we found the designed peptide HDACs are grouped into four classes based on their homology: inhibitors showed potent pan-HDAC inhibition but selective class I (HDACs 1, 2, 3, and 8), class II (HDACs 4–7, 9, and 10), and cytotoxicity toward cancer stem–like cells (PA-1 cells and þ class IV (HDAC 11) are Zn2 dependent, and class III, referred to NTERA-2 cells) along with negligible toxicity toward normal cells þ as sirtuins, are NAD dependent (13). Different HDACs are even at much higher concentrations. The superior tumor-inhibit- thought to have their own substrate specificity, including histone ing effect displayed in the PA-1 and NTERA-2 xenograft animal substrates and nonhistone substrates. However, the identification models resulted in negligible organ lesions at the high dosage of their histone substrates is difficult as different HDACs or HDAC (50 mg/kg, every other day), further supporting the efficiency and complexes may have synergistic effects (14). Nowadays, HDAC1 safety of our designed peptides. To our knowledge, this study was is reported to have preference for H3K56ac and H4K16ac, while the first to showcase the stabilized peptide as a cap group used to HDAC3 preferentially deacetylates H4K5 and H4K12 (14–16). decrease the nonspecific toxicity of HDAC inhibitors, demon- For nonhistone substrates, HDAC1 and Sirtuin 1 are reported to strating the wide potential for combined application of stabilized deacetylate P53 (17, 18), and HDAC6 is reported to deacetylate peptides with clinical approved therapeutics. lysine 40 of a-tubulin (19). þ Due to the high similarity of the Zn2 -dependent enzyme Materials and Methods pockets of HDACs, the four FDA-approved drugs are all pan- Materials HDAC inhibitors and usually share a common dose-limiting All solvents and reagents used for solid-phase peptide syn- toxicity, impeding their further clinical application (20). The thesis were purchased from Shanghai Hanhong Chemical Co., intrinsic biocompatibility and complex chemical structure J&K Co. Ltd., Shenzhen Tenglong Logistics Co., or Energy of peptides have attracted chemists to develop peptide inhibitors Chemical Co. and were used without further purification unless with improved HDAC isoform selectivity. The most famous otherwise stated. Antibodies against HDAC6, HDAC8, P21 peptide-based HDAC inhibitors are cyclic tetrapeptides equipped þ Sox2, Ac-tubulin, GAPDH, and H3 were obtained from Pro- with active Zn2 binding groups, such as the approved drug teintech; antibodies against histone H4K16ac were obtained FK228 (21). These macrocyclic peptides usually display class I from Millipore; antibodies against HDAC1, HDAC4, HDAC5, selectivity, and the selectivity is hypothesized to be attributed to SIRT6, LSD1, H3K4m1, H3K4me2, H4, and active caspase-3 were peptide complex cap groups that interact with a large area of obtained from Abcam; antibodies against HDAC2 and HDAC3 the enzyme surface (22). Despite its preference for class I HDACs, were obtained from BETHYL;
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