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. Author manuscript; available in PMC: 2016 Feb 19.
Published in final edited form as: Chem Biol. 2015 Feb 19;22(2):273–284. doi: 10.1016/j.chembiol.2014.12.015

Identification of HDAC Inhibitors with Benzoylhydrazide scaffold that Selectively Inhibit Class I HDACs

Yunfei Wang 1,2, Ryan L Stowe 3, Christie E Pinello 5, Guimei Tian 1, Franck Madoux 5, Dawei Li 1,7, Lisa Y Zhao 1,9, Jian-Liang Li 6, Yuren Wang 8, Yuan Wang 8, Haiching Ma 8, Peter Hodder 4,5,10, William R Roush 3, Daiqing Liao 1,*
PMCID: PMC4365786  NIHMSID: NIHMS666584  PMID: 25699604

SUMMARY

Inhibitors of histone deacetylases (HDACi) hold a considerable therapeutic promise as clinical anticancer therapies. However, currently known HDACi exhibit limited isoform specificity, off-target activity, and undesirable pharmaceutical properties. Thus, HDACi with new chemotypes are needed to overcome these limitations. Here, we identify a class of HDACi with a previously undescribed benzoylhydrazide scaffold that is selective for the class I HDACs. These compounds are competitive inhibitors with a fast-on/slow-off HDAC-binding mechanism. We show that the lead compound, UF010, inhibits cancer cell proliferation via class I HDAC inhibition. This causes global changes in protein acetylation and gene expression resulting in activation of tumor suppressor pathways and concurrent inhibition of several oncogenic pathways. The isotype selectivity coupled with interesting biological activities in suppressing tumor cell proliferation support further preclinical development of the UF010 class of compounds for potential therapeutic applications.

INTRODUCTION

Histone deacetylases (HDACs) remove the acetyl group from lysine residues of histones and other cellular proteins. HDACs are classified into four phylogenetic groups: class I (HDAC1, HDAC2, HDAC3 and HDAC8), class II (HDAC4, HDAC5, HDAC7 and HDAC9 in the class IIa subgroup, and HDAC6 and HDAC10 in the IIb subgroup), class III (Sirt1–Sirt7) and class IV (HDAC11) (Smith, et al., 2008; Yang and Seto, 2008). Classes I, IIb and IV HDACs possess bona fide Zn2+-dependent acetyl-lysine deacetylase activities. While heightened HDAC activities are implicated in several disorders including chronic neurologic, inflammatory and metabolic conditions (Christensen, et al., 2014; Fass, et al., 2013; Wagner, et al., 2013), abnormal epigenetic regulation, including globally or locally altered patterns of histone acetylation, has long been implicated in cancer etiology and progression. In particular, the roles of HDAC1, HDAC2 and HDAC3 in promoting cancer progression have been extensively documented (Muller, et al., 2013; New, et al., 2012; Wilson, et al., 2006).

Chemically diverse classes of small-molecule inhibitors of HDACs (HDACi) have been developed and characterized, and many exhibit potent anticancer properties in preclinical and clinical studies (Bolden, et al., 2006; Bradner, et al., 2010). Based on the structures of the Zn2+-chelating chemical groups, HDAC inhibitors can be divided into four major classes: hydroxamic acids, aminobenzamides, cyclic peptides and aliphatic acids. A variety of derivatives of each class have been synthesized and characterized. Three compounds, vorinostat and belinostat (hydroxamic acids) and romidepsin (a cyclic peptide), have been approved for clinical anticancer therapies (Marks, 2010; New, et al., 2012). These FDA approved drugs and a number of other HDACi have undergone clinical evaluations for treating a variety of hematological malignancies and solid tumors (New, et al., 2012).

However, there are a number of issues that may limit broad clinical utility of the currently known HDAC inhibitors. Hydroxamic acids are pan-HDACi, active against different isoforms of HDACs and feature a rather strong Zn2+-chelating group (warhead) that is also found in inhibitors of other metalloenzymes such as matrix metalloproteases and TNF-α–converting enzyme (DasGupta, et al., 2009; Lotsch, et al., 2013; Nuti, et al., 2011), although a recent study shows that metal-chelating drugs generally do not display overt off-target activities (Day and Cohen, 2013). This raises the risk of significant off-target activities and unpredictable clinical toxicity. Although several mechanisms such as the induction of apoptosis, cell cycle arrest or inhibition of DNA repair are proposed to account for the antineoplastic activities of HDACi, it remains challenging to determine precisely the importance of HDAC inhibition for anticancer effects using pan-HDACi due to off-target activities. Although yet to be proven, it is generally thought that HDACi with increased isoform-selectivity and potency would be safer agents with reduced side effects and could lead to superior clinical outcomes, because such selective compounds would only target HDAC activities that are dysregulated in a particular type of cancer without causing unnecessary toxicity stemming from inhibiting other HDAC isoforms. Thus, there have been significant efforts in drug development to identify HDACi with greater isozyme-specificity (Ononye, et al., 2012). The aminobenzamide class of HDACi is selective to class I HDACs (HDACs 1–3) and displays unique slow-on/slow-off HDAC-binding kinetics (Beconi, et al., 2012; Chou, et al., 2008; Lauffer, et al., 2013; Newbold, et al., 2013). A number of these compounds such as MS-275 (entinostat) have been tested in clinical trials to treat diverse types of human cancer (Gojo, et al., 2007; Martinet and Bertrand, 2011). However, a recent study reports that aminobenzamides seem to exhibit intrinsic liabilities including chemical instability under certain conditions, high in vivo metabolic turnover, and efficient removal by Pgp drug transporters, which may significantly hamper their potential clinical utility (Beconi, et al., 2012). Although cyclic peptides are more potent against the class I HDACs (Bradner, et al., 2010), the sulfhydryl group of romidepsin is thought to chelate zinc with little specificity (Arrowsmith, et al., 2012). Moreover, serious adverse events associated with cyclic peptides including cardiac toxicity have been reported (Martinet and Bertrand, 2011). These observations call for the development of potent and isoform-selective HDACi of novel chemotypes to overcome these limitations in order to unleash the considerable therapeutic potentials of pharmacological HDAC inhibition.

Through a high-throughput screening (HTS) effort, we discovered a lead compound that selectively inhibits HDAC1, HDAC2 and HDAC3 of the class I HDACs. This lead compound (UF010) features a previously unknown benzoylhydrazide scaffold as the HDACi pharmacophore. Initial structure-activity relationship (SAR) studies confirm the critical components of this scaffold for HDAC inhibition. Importantly, HDAC inhibition potency of UF010 and analogs correlates with their ability to impair cancer cell proliferation. In addition, UF010 alters global gene expression to activate antineoplastic pathways. Thus, this new class of HDACi can serve as powerful tools to investigate roles of HDACs in the biology of human diseases.

RESULTS

HTS identification of HDACi with novel chemotypes

To discover small-molecule HDACi with novel chemotypes, we conducted a HTS campaign of 622,360 compounds using the Scripps Drug Discovery Library. For the primary screen, we engineered a luciferase reporter under the control of the adenovirus (Ad) major late promoter (Ad-MLP-Luc) in the colon cancer HCT116 cell line. Compared to the luciferase reporter under the control of the Ad E2 early promoter (Ad-E2-Luc), the Ad-MLP-Luc reporter activity dramatically increased in a dose-dependent manner by HDACi vorinostat, or MS-275 (Fig. S1). The similar responses of the Ad-MLP-Luc reporter to different classes of HDACi indicate that HDAC inhibition is the primary mechanism for the reporter activation. Each library compound was assayed at a single point and a single dose (8.6 μM). The reporter activity was detected as luminescence readout and cell viability was monitored with the PrestoBlue dye as fluorescence intensity in a multiplex format (Fig. S2). A viability counterscreen assay was used to remove highly toxic compounds. The HTS assays were robust with Z’ of > 0.6 (Fig. S3). A specific hit-cutoff based on an average plus 3-fold standard deviation was applied, resulting in the identification of 5,868 compounds that increased the Ad-MLP-Luc activity by 9.2% of the high control (vorinostat at 28 μM). These compounds were tested in confirmation assays in triplicate at 8.6 μM, among which 1,575 compounds were confirmed to activate the Ad-MLP-Luc reporter (Fig. S3). The top 637 compounds were further tested in a 10-point dose response assays in triplicate in the Ad-MLP-Luc activation assay (Fig. S3). We then selected 315 compounds highly active in the cell-based assays for in vitro HDAC1 inhibition assays using the HDAC-Glo I/II reagents (Fig. S4). A majority (54%) of these compounds inhibited HDAC1 in vitro with a potency of <10 μM, among which there were nine hydroxamates including vorinostat and scriptaid and seven aminobenzamides (Fig. S6A). These results highlight the robustness of the cell-based Ad-MLP-Luc activation assays for identifying HDACi with significant inhibitory properties.

In subsequent studies we focused on hits with novel chemical scaffolds. A confirmed hit with a benzoylhydrazide scaffold (UF010) displayed consistent HDAC inhibition in biochemical and cell-based assays. We profiled UF010 against all Zn2+-dependent HDACs and found that UF010 is class I HDAC-selective with nanomolar potencies against HDACs 1–3 (Table 1).

Table 1.

Inhibitory Potency (IC50, μM) of Different Classes of HDAC Inhibitors against HDACs 1–11

Inhibitor Class HDAC1 HDAC2 HDAC3 HDAC8 HDAC6 HDAC10 HDAC11 HDAC4 HDAC5 HDAC7 HDAC9
UF010a Benzoyl-hydrazide 0.5 0.1 0.06 1.5 9.1 15.3 44.5 >100 >100 >100 >100
MS-275b Benzamide 0.2 0.5 0.3 >10 5.90 >10 ND >10 >10 >10 >10
Cpd60c Benzamide 0.001 0.008 0.5 >30 >30 3.4 NA >30 >30 >30 >30
SAHA hydroxamic acid 0.06 0.3 0.02 0.8 0.009 0.03 NA >10 >10 >10 >10
TSAd hydroxamic acid 0.005 0.008 0.01 0.2 0.0007 0.04 0.01 5.0 2.6 1.4 10.4
Romi-depsine Cyclic peptide 0.002 0.004 ND ND 0.8 ND ND 0.03 ND ND ND
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Note:

a

Dose response assays were done in the concentration range of 5 nM to 100 μM based on fluorescence assays;

b

HDAC6 IC50 was determined in this study; other data from (Lauffer, et al., 2013);

d

HDAC1 and HDAC6 IC50 data from literature (Lauffer, et al., 2013);

e

Data from literature (Newbold, et al., 2013). ND: not determined.

Structure-Activity Relationship (SAR) of UF010 and analogs

UF010 was resynthesized and its activities were confirmed. A total of >50 UF010 analogs with a variety of modifications of the benzoylhydrazide scaffold have been synthesized and their activities determined (data for select analogs are shown in Table 2). These SAR data indicate that a tripartite structure of this scaffold with a central −C(O)-NH-NH- unit flanked by a phenyl group and a short aliphatic chain is important for HDAC inhibition. The short 4-carbon linear aliphatic chain appears nearly optimal (Table 2). Any changes in the chain-length (increase or decrease) or structure (e.g., branched) reduced HDAC inhibition potency. As for the phenyl group, the presence of a relatively bulky substituent at the para position relative to the carbonyl group seems critical for HDAC inhibition (Table 2). Among the analogs we have examined thus far, three (SR-3208, -3302 and -3459) displayed improved potencies vs. HDACs 1–3 (Table 2).

Table 2.

Structure-Activity Relationship (SAR) of Selected UF010 Analogsa

graphic file with name nihms666584u1.jpg IC50 values (μM)b
Cpd. R1 R2 HDAC1 HDAC2 HDAC3 HDAC6 HDAC8
UF010 Br n-butyl 0.46 1.33 0.19 9.09 2.83
SR-3203 F n-butyl 13.2 15.1 1.78 nd nd
SR-3204 −OCF3 n-butyl 8.86 9.09 1.35 nd nd
SR-3205 H n-butyl >50 >50 >50 nd nd
SR-3206 −OMe n-butyl 1.91 2.52 0.43 nd nd
SR-3208 −NMe2 n-butyl 0.23 0.88 0.12 9.57 0.72
SR-3210 CI n-butyl 3.0 3.8 2.5 nd nd
SR-3302 t-butyl n-butyl 0.19 1.04 0.07 6.83 0.49
SR-3459 −CH2N3 n-butyl 0.32 0.53 0.15 10.8 0.89
SR-3212 Br n-propyl 1.70 3.88 0.22 4.63 nd
SR-3970 Br n-pentyl 1.87 1.92 0.92 5.73 4.87
SR-3367 Br n-hexyl 10.27 16.11 20.04 39.1 nd
SR-3213 Br CH2CH2Bn >50 >50 24.18 nd nd
SR-3297 Br CH2CH2(c-C5H10) 7.40 7.65 1.08 nd nd
SR-3364 Br CH2(c-C5H10) 8.81 28.45 6.98 nd nd
SR-3365 Br CH2CH2CH2(c-C5H10) >50 >50 >50 nd nd
SR-3298 −NMe2 CH2(c-C5H10) 1.76 3.25 1.67 nd nd
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Note:

a

UF010 is a high-throughput screening hit. All compounds identified with “SR” numbers are synthetic analogs.

b

HDAC activity assays were performed using the HDAC-Glo I/II reagents.

We have performed comparative molecular modeling studies of UF010 and the new analogs by using the Schrödinger modeling package. This docking study was performed without bias and in each instance the software identified the HDAC active site as the only binding site for UF010 and analogs. The butyl side chain of these compounds fills a deep hydrophobic (“foot”) pocket (Fig. S5). An immediate question was if the inhibitors are Zn binders—this modeling analysis indicated in some instances that the hydrazide carbonyl interacted in monodentate manner with the active site Zn, while in others the second (distal) hydrazide nitrogen was within coordination distance of the active site Zn. In no cases was ‘bidentate’ coordination observed; in many instances the inhibitors exhibited no interaction whatsoever with the active site Zn. Thus, it appears that the principal mode of binding of this novel class of HDACi is not due to strong interactions with the active site Zn—this distinguishes this class of inhibitors from the vast majority of other known HCAC inhibitors.

Impact of UF010 on global protein acetylation

In cell-based assays, we exposed HCT116 cells to UF010 along with other HTS hits (UF003, UF006, UF007, UF008, and UF009; the structures of these hits are shown in Fig. S6A), TSA (a hydroxamate) and MS-275 (an aminobenzamide). Data presented in Fig. 1A show that UF010 consistently induced the accumulation of acetylated histones at all sites we have examined. TSA strongly induced acetylation at some sites but weakly at other sites. In contrast, MS-275 only slightly induced acetylation at several sites but failed to induce acetylation at most sites. This is probably due to the short (1 h) exposure of the cells to MS-275, as it and other aminobenzamides are known to bind rather slowly to the active site of an HDAC (Beconi, et al., 2012; Chou, et al., 2008; Lauffer, et al., 2013). We also assessed the cellular activities of synthetic UF010 analogs. In HCT116 cells, their cellular activities are consistent with their in vitro potencies for inhibiting the deacetylation at H4K5, while for H3K18ac, some weak in vitro inhibitors (e.g., SR-3205) also notably increased acetylation (Fig. 1B), suggesting that in vitro activities do not exactly reflect cellular effects. Among these analogs, UF010 and SR-3208 consistently displayed significant inhibition of cellular HDACs (Fig. 1B).

Fig. 1. UF010 Induces the Accumulation of Protein Acetylation.

Fig. 1

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(A) HCT116 cells were exposed to TSA (0.2 μM) and other compounds at 2 μM for 1 h. UF003 and UF006–UF010 are hits identified in the HTS. Histones were extracted and subject to Western blotting with antibodies to histones with the indicated modifications or stained with colloidal blue (CBS). The antibodies against H4Kac recognize H4 acetylated at K5, 8, 12 and 16. (B) The effects of synthetic UF010 analogs on histone acetylation. HCT116 cells were exposed to TSA (0.1 μM), romidepsin (5 nM) and other compounds at 0.5 μM for 24 h. The total cell extracts were subject to Western blotting with antibodies to histones with the indicated modifications. PCNA was detected as a loading control. HCT116 (C) and A549 (D) cells were exposed to DMSO or etoposide (Etop, 10 μM) for 6 h. TSA (0.2 μM), MS-275 and UF010 (2 μM) were added 1 h before cell lysis. The total cell lysates were subject to Western blotting with antibodies to the indicated proteins. PCNA was detected as a loading control.

We then assessed the impact of UF010 on the acetylation of non-histone proteins. While TSA dramatically increased α-tubulin acetylation, UF010 had no effect (Fig. 1C and D), even at a high concentration (Fig. S6B). However, UF010 induced accumulation of acetylated p53 in both HCT116 and A549 cells after exposure to etoposide that inhibits DNA topoisomerase II, and induces double-stranded DNA breaks. As expected, etoposide activated the p53 pathway, as indicated by the accumulation of p53 and its transcriptional targets p21 encoded by CDKN1A and PUMA encoded by BBC3 (Fig. 1C and D). Both UF010 and MS-275 also notably stabilized p53 with or without etoposide treatment (Fig. 1C and D). HDAC6 (class IIb) is the major tubulin deacetylase (Hubbert, et al., 2002; Matsuyama, et al., 2002; Zhang, et al., 2003), whereas HDAC1, probably in the context of the NuRD complex, deacetylates p53 (Contreras, et al., 2013; Luo, et al., 2000). These data indicate that UF010 is a class I HDAC-selective inhibitor, in agreement with in vitro biochemical assays (Table 1).

To identify proteins whose acetylation is impacted by UF010 globally, we conducted a proteomic experiment. We found that acetylation of histones H2B, H3 and H4 at most known sites of acetylation was enriched in UF010-treated cells (Table S3). Surprisingly, no acetylated peptides of H2A were detected in our experiment, although both MS-275 and vorinostat were shown to potently induce H2A acetylation (Choudhary, et al., 2009). Acetylated peptides of several other proteins were also enriched (Table S3). Although these proteins are all known acetylated proteins (Choudhary, et al., 2009), the identified acetylation sites in nucleolin, parathymosin and PEX14 in UF010-treated cells have not been reported thus far (Table S3). Whereas it is most likely that only the most abundant acetylated peptides were detected in our limited proteomic profiling, these data suggest that UF010 exerts a distinct impact on global acetylation compared to existing HDAC inhibitors, further supporting the notion that UF010 possesses unique activities.

UF010 is a competitive HDACi with a fast-on/slow-off target binding mechanism in cells

The hydroxamic acid and aminobezamide classes of HDACi occupy the catalytic center of HDACs and display the competitive mechanism of HDAC inhibition (Chou, et al., 2008; Kral, et al., 2014; Lauffer, et al., 2013). We have conducted inhibition kinetic experiments and found that UF010 is clearly a competitive inhibitor of HDAC2 vs. its substrates (Fig. 2A), indicating that UF010 binds to the substrate pocket of the catalytic core of HDAC2.

Fig. 2. Mechanisms of HDAC Inhibition by UF010.

Fig. 2

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(A) Purified HDAC2 was incubated with various concentrations of substrate and UF010 for various lengths of time. The data were fitted based on the classical Michaelis–Menten kinetics model (Top). The Km of HDAC2 was determined to be 7.6 μM without compound UF010. The observed Km of HDAC2 was significantly increased in the presence of UF010. The Lineweaver–Burke replots of the rate data is shown in the lower panel. (B) HCT116 cells were pulse treated with the indicated HDACi for 6 h. The drugs were then washed out and regular medium was added. The cells were lysed at the indicated times after drug addition. The lysates were subject to Western blotting with the indicated antibodies. (C) HCT116 cell cultures were exposed to various doses of the indicated drugs. HDAC-Glo I/II reagent was added immediately following drug addition. Luminescence was detected 20 min after adding HDAC-Glo I/II reagent, and every 10 min thereafter up to 240 min (4 h). The HDAC inhibition IC50 was determined and plotted against time.

The hydroxamates are fast-on/fast-off HDACi, whereas the aminobenzmide HDACi display a slow-on/slow-off HDAC binding mechanism (Beconi, et al., 2012; Chou, et al., 2008; Lauffer, et al., 2013). To assess how UF010 might interact with HDACs, we exposed HCT116 cells to various HDACi for 6 h. The drugs were washed out and the cells were cultured for various lengths of time after drug washout. Consistent with published data (Lauffer, et al., 2013), vorinostat induced high levels of histone acetylation (H3K18ac) within 6 h and the acetylated histones were quickly reduced to normal levels upon the drug removal (Fig. 2B lanes 7–11). Romidepsin also quickly induced acetylation, and the acetylation levels were stable up to 24 h (18 h after drug removal) (Fig. 2A lanes 1–5), suggesting that romidepsin has a slower rate of release from HDACs than vorinostat. The accumulation of histone acetylation induced by the aminobenzamide MS-275 was apparent only at 18 h after 6 h exposure, and the acetylated histones were stable up to at least 48 h (Fig. 2B lanes 12–15), in agreement with a slow-on/slow-off mode of HDAC inhibition (Beconi, et al., 2012; Chou, et al., 2008; Lauffer, et al., 2013). For UF010, histone acetylation was induced within 6 h of exposure and the levels of induced acetylation were maintained up to 96 h (Fig. 2B lanes 17–22), suggesting that UF010 is a fast-on but slow-off inhibitor.

We assessed the HDAC inhibition activity of UF010, MS-275 and SAHA in live cell cultures. As shown in Fig. 2C, UF010 exhibited an HDAC inhibition IC50 of 1.8 μM within minutes after its addition and this inhibitory potency remained relatively constant throughout the assay period up to 4 hours. The IC50 of MS-275 reached the minimum in about two hours after drug addition and showed a moderate increase thereafter. The IC50 of SAHA reached the minimum of ~0.1 μM immediately after drug addition. These data are consistent with the binding kinetics of these inhibitors as observed in drug washout experiments. One surprising finding is that UF010 is about 5-fold more potent than MS-275 against HDACs in HCT116 (Fig. 2C) and HepG2 cells (data not shown). These experiments also indicate that UF010 quickly penetrates cell membranes and interacts with its cellular targets in cell cultures.

HDAC inhibition potency of UF010 and analogs correlates with their antiproliferative effects

To assess the effects of UF010 and analogs on cancer cell viability, we treated diverse cancer cell lines with UF010 analogs. In general, UF010 is less potent to impair the viability of cancer cells than vorinostat or MS-275. For example, the IC50 values (μM) for killing the colon cancer HCT116 cells were 11.2 for UF010, 2.1 for MS-275 and 1.2 for vorinostat. To assess potential roles of HDAC inhibition on cancer cell cytotoxicity, we exposed liver cancer cell line HepG2 to UF010 and analogs with various in vitro HDAC inhibition potencies. We found that HDAC inhibition potencies of these analogs exhibited an excellent correlation with their cytotoxicity to HepG2 cells (Fig. 3A). Indeed, SR-3208 that is 2 to 5-fold more potent than UF010 in inhibiting HDACs 1–3 is 3-fold more potent in eliciting cytotoxicity on HepG2 cells. By contrast, SR-3205 that did not inhibit HDACs in vitro exerted no growth inhibition for HepG2 cells. Similar effects were also observed in other cancer cell lines (HCT116, colon; MDA-MB-231 and HCC1957, breast cancer; Fig. 3 B–D), despite highly variable sensitivities of these cell lines to UF010 analogs. These data suggest that HDAC inhibition appears critical for the UF010 class of HDACi to halt cancer cell growth and proliferation.

Fig. 3. Suppression of Cancer Cell Viability by UF010 Analogs Correlates with Their HDAC Inhibition Potencies.

Fig. 3

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Hepatocellular carcinoma cell line HepG2 (A), colon cancer cell line HCT116 (B) and breast cancer cell lines MDA-MB-231 (C) and HCC1957 (D) were exposed to DMSO (control) or various doses of UF010 or an indicated analog. Viable cells were detected at 96 h after treatment using CellTiter-Glo assay kit. The fraction of survived cells is plotted against compound concentrations. Error bars are standard error of the mean (SEM, n = 3). The IC50 of each compound was determined using nonlinear regression curve fitting with the Prism 6 software.

UF010 was screened against the NCI-60 panel of cancer cell lines by the NCI’s Development Therapeutics Program. It inhibits proliferation of all tested cell lines (including five breast cancer and seven colon cancer cell lines) with a mean GI50 of 2.94 μM, although sensitivity to this compound varies among these cell lines (Figs. 4A and B, and Fig. S7). Cell cycle analysis using MDA-MB-231 cells exposed to UF010 or vorinostat indicate that UF010 mainly blocked G1/S transition with an increased G1 cell population and a reduced cell population in the S phase in a dose-dependent manner, while vorinostat inhibited G1/S progression at 1 μM but induced a strong G2/M block at 10 μM (Fig. 4C). The G2/M checkpoint induced by vorinostat is probably due to a strong DNA-damage response in cells treated with vorinostat (Lee, et al., 2010). To assess potential effects of UF010 on cell migration that is associated with metastatic progression, we conducted a “wound” healing assays. Monolayer MDA-MB-231 cultures were exposed to DMSO, vorinostat (SAHA) or UF010, and then scratched. Cell migration to the denuded areas was assessed. We found that UF010 at 1 μM markedly slowed migration, whereas SAHA did not significantly affect this phenotype (Fig. 4D). It is unlikely that possible cross-inhibition of matrix metalloproteases (MMPs) by UF010 contributes to the inhibition of cell migration, as SAHA containing the hydroxamic acid warhead that is known to lead to pan-MMP inhibition did not affect cell migration in our experiments. Vorinostat and other hydroxamic acid HDACi have been shown to suppress breast cancer metastases in several preclinical models (Chiu, et al., 2013; Huang, et al., 2014; Palmieri, et al., 2009), although other studies showed that HDACi of the hydroxamic acid class could augment metastatic spread of certain cancer cell lines (Lin, et al., 2012).

Fig. 4. Antiproliferation Effects of UF010.

Fig. 4

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The indicated breast (A) and colon (B) cancer cell lines in the NCI-60 panel of cancer cell lines were exposed to UF010 at various concentrations. Percent cell growth relative to the cells seeded before treatment is plotted against the UF010 concentrations in log scale. For details see Fig. S7. (C) Effects of UF010 and SAHA (vorinostat) on cell cycle progression. MDA-MB-231 cells were exposed to DMSO (control) or the indicated doses of UF010 or SAHA for 24 h. Cells were then fixed and processed for FACS analysis. Shown are the average values of two experiments along with SEM. (D) UF010 suppresses cell migration. MDA-MB-231 cells were exposed to DMSO or the indicated doses of UF010 or SAHA. At 2 h after adding a compound, the monolayer cultures were scratched and the denuded areas were photographed at the indicated time points. The uncovered areas were calculated and compared to the initial open areas. Shown are averages ± SEM (n = 3). *: P < 0.01 (vs. DMSO treatment).

UF010 activates tumor suppression pathways but inhibits oncogenic signaling

To assess cellular effects of UF010 comprehensively, we treated MDA-MB-231 cells with UF010 at 1 μM for 24 h and analyzed its impact on global gene expression in comparison to DMSO control using the Affymetrix Human Transcriptome Array 2.0, which allows for the interrogation of transcripts for splicing variants of coding and noncoding genes. The expression of a large number of transcripts was impacted due to UF010 treatment, with more downregulated transcripts than upregulated ones overall (Fig. 5A). Most of these responsive genes exhibited moderate levels of changes in their mRNA expression. Among the responsive genes that were up or downregulated by 1.5-fold, 115 were upregulated and 68 downregulated (Fig. 5A). The changes in gene expression patterns due to UF010 treatment were subject to Ingenuity Pathway Analysis. In the Biological Function analysis, the most highly affected pathways include the induction of cell death, the suppression of cell cycle progression and DNA repair (Fig. 5D). In the Upstream Regulator analysis, UF010 induced the activation of p53 and Rb tumor suppressor pathways but suppressed the MYC, MYCN, and KRAS oncogenic pathways. We showed above that UF010 induced G1 cell cycle arrest in FACS analysis (Fig. 4). Concordantly, pathway analysis of our gene expression data revealed that UF010 activated pathways that promote G1 arrest, while suppressing mechanisms that promote cell cycle progression (Fig. 5B). UF010-induced activation of CDKN1A encoding p21 was confirmed by quantitative real-time PCR (Fig. 5C). Notably, MDA-MB-231 cells express a mutant (R280K) form of p53. Thus the activation of p53 pathway by UF010 is somewhat surprising but could involve partial reactivation of p53 through increasing its acetylation, as UF010 could indeed increase the levels of acetylated p53 (Fig. 1). As expected of an HDACi, this analysis also revealed that UF010 activated overlapping downstream effectors of the known HDACi including tributyrin, butyric acid, trichostatin A (TSA), and romidepsin (Fig. 5D). The inhibition of both HDAC1 and HDAC2 was also revealed in the pathway analysis (Fig. 5D), thereby providing an independent validation of UF010 as a class I-specific HDACi. Interestingly, UF010 also seems to show overlapping functions with several other drugs especially genotoxic chemotherapeutics including cisplatin, camptothecin, doxorubicin and etoposide (Fig. 5D and Table S4). This is consistent with the ability of HDACi to elicit DNA-damage response (Lee, et al., 2010).

Fig. 5. Global Effects of UF010 on Gene Expression.

Fig. 5

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MDA-MB-231 cells were exposed to DMSO or UF010 at 1 μM for 24 h. RNAs were isolated and subject to microarray gene expression profiling. (A) Volcano plot of gene expression profiles in cell treated with UF010 in comparison to those treated with DMSO. The two vertical lines in red demark ± 0.585 of the x-axis to indicate genes that were up- or downregulated by 1.5-fold. The red horizontal line indicates -1.3 of the y-axis for P value < 0.05. (B) The cell cycle regulators that were either activated (left) or suppressed (right) based on the Ingenuity Upstream Regulator analysis. The P-value is shown in negative Log10 scale. (C) qPCR validation of UF010-induced upregulation of CDKN1A encoding p21. Shown are the average values of three experiments ± SEM. (D) Major pathways impacted by UF010 in MDA-MB-231 cells. The gene expression data were analyzed using Ingenuity pathway analysis software as in (B).

Discussion

Potent and selective HDACi serve as powerful tools for probing fundamental biological questions and for ameliorating diverse pathological conditions. Although structurally diverse classes of small-molecule HDACi have been identified, many of the known HDACi feature strong Zn-chelating warheads that could lead to unintended off-target effects. We have identified a novel group of selective class I HDAC inhibitors with UF010 as the first-in-class lead compound. This class of HDACi features a novel pharmacophore targeting class I HDACs with a benzoylhydrazide scaffold. Our preliminary SAR studies confirm a tripartite structure of this scaffold with a central −C(O)-NH-NH- unit flanked by a phenyl group and a short aliphatic chain. The central unit might provide hydrogen-bonding and weak Zn2+-chelating functionalities while the flanking hydrophobic groups interact specifically with the hydrophobic pockets in the HDAC catalytic core (Fig. S5). Of importance, weaker Zn2+-chelating warheads in HDACi might reduce off-target activities (Lobera, et al., 2013). Our data suggest that UF010 analogs display a unique HDAC inhibition pharmacology. UF010 seems more potent in suppressing the deacetylation of histones and p53 than aminobenzamides and less so than hydroxamates possessing a strong Zn2+-chelating warhead in cell-based assays. Importantly, we found that the HDAC inhibition potencies of UF010 and analogs correlate directly with their ability to suppress the survival of cancer cells (Fig. 3). Bioinformatics analysis of gene expression data also implicates the suppression of HDAC1 and HDAC2 activity by UF010 (Fig. 5D). Thus, HDAC inhibition may underlie their tumor suppressive activity. Notably, UF010 seems to exert a powerful tumor suppressive effect through activating critical tumor suppressor pathways (e.g., p53 and Rb) while inhibiting several dominant oncogenic mechanisms (e.g., MYC, MYCN and KRAS), representing novel mechanisms of action for a potential anticancer agent. Our data show that UF010 is less cytotoxic than both vorinostat and MS-275. Notably, UF010 is a more potent HDAC inhibitor than MS-275 in cell-based assays (Fig. 2C). These observations suggest that off-target activities of MS-275 might contribute significantly to its cytotoxicity. Regardless, reduced general cytotoxicity for an HDACi may be more desirable for cancer therapy, given the known clinical toxicity associated with known HDACi (Martinet and Bertrand, 2011). Furthermore, less toxic compounds might be more suitable for treating diseases such as neurologic conditions and metabolic diseases, as cell death should be avoided in these settings. In preliminary experiments, we found that UF010 has a half-life of 15.8 h in cell culture medium containing 10% fetal bovine serum (data not shown), which is similar to that of romidepsin (Furumai, et al., 2002). Additional studies will be performed in the future to characterize the pharmacokinetic properties of the UF010 class of HDACi, after more potent analogs are developed.

HDACs are assembled into several multi-subunit complexes such as the Sin3 and NuRD complexes and regulatory subunits in these complexes seem to influence the binding of existing HDAC inhibitors to HDACs. For example, whereas vorinostat exerts potent inhibition of all known HDAC complexes, aminobenzamides seem to show some selectivity against different complexes (Bantscheff, et al., 2011). Specifically, aminobenzamides seem inert vs. the Sin3 complex. However, we found that UF010 can inhibit the Sin3 complex (data not shown), further supporting the notion that UF010 has distinct HDAC inhibitory activities.

In addition to acetylation (C2), lysine residues in histones can also be modified with short-chain N-acylation such as propionylation (C3), and butyrylation (C4) (Chen, et al., 2007). Histone peptides containing such short chain acylation can bind to the bromodomains of bromodomain and extra-terminal (BET) family of proteins (e.g., BRD4), although their binding affinity is considerably weaker compared to acetylated peptides (Vollmuth and Geyer, 2010). The short aliphatic chains in the UF010 analogs share certain similarity to N-acylated lysine, which raises the question as to whether UF010 analogs might inhibit bromodomains. Although we did not directly test potential activity of UF010 analogs vs. bromodomains in vitro, we found that the bromodomain inhibitor I-BET151 synergizes with UF010 to induce cell death (data not shown), suggesting that UF010 and I-BET151 have distinct cellular targets.

Pathway analysis of gene expression data revealed that UF010 exhibits overlapping activities with a number of drugs. Not surprisingly, known HDACi including tributyrin, butyric acid, TSA and romidepsin are identified (Fig. 5D), unequivocally confirming that HDACs are the targets of UF010. Interestingly, UF010 seems to share activities with genotoxic chemotherapeutics such as cisplatin, camptothecin and etoposide. This may stem from the ability of HDACi to elicit DNA damage response (Lee, et al., 2010). This property could also be a consequence of UF010-mediated activation of the p53 pathway (Fig. 1 and Fig. 5D). Fulvestrant, a selective estrogen receptor antagonist, was also identified in our bioinformatics analysis (Fig. 5D). The class I HDAC-selective HDACi MS-275 (entinostat) has been shown to sensitize ER+ breast cancer cells to antiestrogen agents and the combination of entinostat with aromatase inhibitors, which block estrogen synthesis, exhibited improved clinical outcomes for patients with locally advanced or metastatic ER+ breast cancer in a phase II clinical trial (Sabnis, et al., 2013; Yardley, et al., 2013). Of note, the ER+ T47D breast cancer cell line is most sensitive to UF010 among the tested breast cancer cell lines (Fig. 4A). It will be of particular interest to determine the effects of UF010 on ER pathways in breast cancer.

Despite the activation of multiple tumor-suppressive pathways in UF010-treated MDA-MB-231 cells, they are relatively resistant to UF010-mediated apoptosis (IC50 of 29.1 μM, Fig. 3C). Simultaneous upregulation of stress-response or survival pathways may counteract the apoptotic mechanisms to allow UF010-treated cells to survive. The NUPR1 stress-response/chemoresistance pathway was activated in UF010-treated MDA-MB-231 cells (Supplemental Table S4), likely contributing to the survival of UF010-treated MDA-MB-231 cells. Indeed, NUPR1 seems to play a critical role in tumor initiation and progression (Cano, et al., 2014). Additionally, ERK1/2 pathway was also activated (Supplemental Table S4). These and other cell survival mechanisms are likely to confer resistance to UF010. UF010 combined with inhibitors targeting these survival pathways is predicted to enhance anticancer effects.

In summary, we have identified a new HDACi chemotype that could potentially overcome some of the noted limitations of currently known HDACi. The isotype selectivity coupled with interesting biological activities in suppressing tumor cell proliferation support further preclinical development of the UF010 class of compounds for potential therapeutic applications.

Significance

Small-molecule HDAC inhibitors (HDACi) have therapeutic potentials for treating cancer and other diseases. Three FDA-approved HDACi (vorinostat, belinostat and romidepsin) are used in clinical anticancer therapy and many other HDACi have been actively tested in clinical trials. Current challenges facing drug development of HDACi include the lack of isoform specificity, undesirable toxicity and suboptimal therapeutic efficacy against solid tumors. Most HDACi in preclinical or clinical evaluations belong to the hydroxamic acid or aminobenzamide classes. The former class lacks isoform specificity and the latter shows potentially undesirable pharmaceutical properties. Thus, the identification of HDACi with novel chemical properties and isoform-specificity may unleash the considerable therapeutic potential of targeting HDACs. Through a HTS campaign and structure-function relationship study, we have identified a class of HDACi with a previously undescribed benzoylhydrazide scaffold. These compounds are selective to the class I HDACs with nanomolar potencies. These new HDACi inhibited cancer cell proliferation through HDAC inhibition. They activated tumor suppression mechanisms while inhibiting oncogenic pathways.

Experimental Procedures

HTS Assays

HCT116 cells were stably transduced with a lentiviral vector carrying the firefly luciferase gene under the control of Ad MLP (Fig. S1). The stable HCT116-Ad-MLP-Luc cell line was used for the primary HTS assay (Fig. S2). Compounds that activated the MLP-Luc reporter were tested for in vitro assays for HDAC1 inhibition using the HDAC-Glo I//II reagents (Promega, Fig. S4). Selected hits were profiled against all zinc-dependent HDACs at the Reaction Biology Corporation.

HDAC Activity Assays

Purified HDAC1, HDAC2 and HDAC3 (in complex with the deacetylase activation domain of the human NCOR2 (amino acids 395–498)) were obtained from BPS Bioscience. The enzyme activities were initially tested in a serial dilution of each HDAC using the HDAC-Glo I/II reagents (Promega) according to manufacturer’s protocol. Luminescence was detected using the BMG POLARstar Omega microplate reader. A concentration of each HDAC within the linear response region was used for assaying inhibition of HDAC activity by UF010 and analogs. Each compound was tested in 10-point dose response assay in triplicate. IC50 values were determined through linear regression of inhibition data using the Prism 6 software.

Cell Culture, Viability Assays and Western Blotting

Cell lines were obtained from ATCC and cultured with Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% bovine calf serum, penicillin to 10 units/mL, and streptomycin to 10 μg/mL. For viability assays, 5,000 cells/well were seeded in 96-well plates. Compounds or DMSO control were added 24 h later. Viability assays were done 96 h after compound addition using the CellTiter-Glo reagents (Promega). UF010 was tested against the NCI-60 panel of cancer cell lines for a one-dose initial screening and the subsequent five-dose titration assays at the Development Therapeutics Program of the National Cancer Institute (Shoemaker, 2006). For Western blotting, cell cultures were exposed to compounds as indicated in relevant figures. Total cell lysates or isolated histones were subjected to SDS-PAGE and Western blotting essentially as described (Yang, et al., 2013). Antibodies used in this study are described in Table S1.

Cell Cycle Analysis and Migration Assay

MDA-MB-231 cells were treated with DMSO, UF010 or vorinostat for 24 h. Cells were fixed and subject to cell cycle analysis using fluorescence activated cell sorting (FACS) as described (Li, et al., 2011). For cell migration assays, a confluent monolayer culture of MDA-MB-231 cells was pretreated with DMSO, vorinostat or UF010 for 2 h. The cells were scratched with a pipette tip and the “wounded” areas were imaged at various time points. The denuded area was quantified using the TScratch software (Geback, et al., 2009), and the % areas that were not covered with cells relative to the initial denuded areas were calculated.

Gene Expression Studies

MDA-MB-231 cells were cultured in a 6-well plate. Cells were exposed in triplicate to DMSO or UF010 at 1 μM final concentration at 24 h after cell plating. Total RNAs were isolated from the treated cells using the RNeasy kit (Qiagen). The RNAs were then processed for microarray hybridization to the Affymetrix GeneChip Human Transcriptome Array 2.0. Data acquisition, processing and analysis are described in Supplemental Materials and Methods. For quantitative real-time PCR, the isolated RNAs were reverse transcribed with random hexamers using 2 μg of RNA, an RNase inhibitor and the Multiscribe reverse transcriptase (Life Technologies). The resulting cDNAs were diluted and used as input for qPCR using the SYBR green detection method. The qPCR primers are provided in Table S2. The relative levels of gene expression were determined with the ΔΔCt method. The microarray data are deposited at the NCBI GEO databank (accession # GSE56823).

Molecular Docking

Compounds for docking were generated utilizing OPLS-2005 force fields. Crystal structures against which our compounds were evaluated (denoted by the following protein databank codes HDAC1: 4BKX, HDAC2: 4LXZ, HDAC3: 4A69, HDAC8: 1W22, HDAC7: 3ZNR) were minimized by Schrödinger’s “protein preparation wizard” to 0.30 Å rmsd by adding hydrogens and adjusting bond orders where needed. For details, please see Supplemental Materials and Methods.

Synthesis of UF010 Analogs

A series of UF010 analogs were synthesized, which is described in details in the Supplemental Materials and Methods.

Supplementary Material

1
2
3

Highlights.

  • Discovery of class I HDAC-selective inhibitors with a new benzoylhydrazide scaffold

  • Nanomolar inhibition potencies against HDACs 1–3 by UF010 analogs

  • HDAC inhibition potency correlates with their cytotoxicity to cancer cells

  • UF010 activates tumor suppression mechanisms while inhibiting oncogenic pathways

Acknowledgments

We thank Pierre Baillargeon, Lina DeLuca, and Louis Scampavia for compound management and quality control, Katharine Emery for secretarial assistance, and the NCI Developmental Therapeutics Program for the NCI-60 cell line testing. The work was supported by grants from Bankhead-Coley Cancer Research Program, Florida Department of Health (09BB-11, 09BW-05 and 4BF02) (to D. Liao), and the National Institutes of Health Roadmap Initiative grant U54MH084512 (to WRR). Yunfei Wang and Dawei Li were supported in part by a scholarship from the China Scholarship Council.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

AUTHOR CONTRIBUTIONS

All authors designed and performed experiments and analyzed data. Yunfei Wang, RLS, CEP, PH, WRR, Yuren Wang and D Liao wrote the paper. HM, PH, WRR and D Liao supervised the study.

The authors declare no financial conflict of interest.

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Associated Data

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Supplementary Materials

1
NIHMS666584-supplement-1.xlsx (146.6KB, xlsx)
2
NIHMS666584-supplement-2.docx (67.3KB, docx)
3
NIHMS666584-supplement-3.pdf (3.3MB, pdf)

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