A Novel Pyrrole-Imidazole Polyamides Hoechst Conjugate

EBNA1 binds to the origin of virus plasmid replication (OriP) on EBV episome to ... Epstein-Barr virus (EBV) is one of the most understood viruses tha...
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Cite This: J. Med. Chem. 2018, 61, 6674−6684

Novel Pyrrole−Imidazole Polyamide Hoechst Conjugate Suppresses Epstein−Barr Virus Replication and Virus-Positive Tumor Growth Zhehong Cheng,†,‡,# Wei Wang,†,# Chunlei Wu,† Xiaohua Zou,§ Lijing Fang,*,† Wu Su,*,† and Pu Wang*,§

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†

Guangdong Key Laboratory of Nanomedicine, Institute of Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology, Shenzhen, Guangdong 518055, China ‡ Shenzhen College of Advanced Technology, University of Chinese Academy of Sciences, Shenzhen, Guangdong 518055, China § Shenzhen Laboratory of Antibody Engineering, Institute of Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, Guangdong 518055, China S Supporting Information *

ABSTRACT: Epstein−Barr virus (EBV) establishes latent infection and is associated with several types of lymphomas and carcinomas. EBV nuclear antigen 1 (EBNA1) is expressed in all EBV-positive tumor cells. EBNA1 binds to the origin of virus plasmid replication (OriP) on the EBV episome to initiate virus DNA replication and regulates virus gene expression as a transcriptional activator. In this study, we designed and synthesized a pyrrole−imidazole polyamide−Hoechst 33258 conjugate named EIP-2 (2), which specifically binds to the OriP region with high affinity, to interrupt EBNA1-OriP binding in vitro and in vivo. By eradicating the EBV episome in EBV-positive cells, compound 2 selectively inhibited EBV-positive cell proliferation. Moreover, the injection of 2 significantly suppressed tumor growth in the mice xenograft tumor model. These findings demonstrate that compound 2 is a potential therapeutic candidate for the treatment of EBV-associated tumors.

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INTRODUCTION Epstein−Barr virus (EBV) is one of the most-understood viruses that account for oncogenesis. EBV latent infection is associated with the pathogenesis of Burkitt’s lymphoma, Hodgkin’s disease, non-Hodgkin’s lymphoma, nasopharyngeal carcinoma, gastric adenocarcinoma, and other malignancies in immunocompromised individuals.1,2 EBV efficiently transforms resting B cells to the permanently growing lymphoblastoid cell line (LCL) in vitro, strongly supporting its association with cancer pathogenesis.1−3 During the latent infection, Epstein−Barr virus nuclear antigen 1 (EBNA1) plays a critical role in persisting viral genome and host-cell survival.4,5 The only latent viral protein expressed in all forms of viral latency, EBNA1 activates virus replication and transcription of other viral latent genes by binding to the origin of virus plasmid replication (OriP).4,5 OriP has two EBNA1recognizing regions: the family-repeat (FR) region and the dyad symmetry (DS) region. There are four conservative 18base-pair EBNA1-binding sites on DS, and the DS has been © 2018 American Chemical Society

proven both essential and sufficient for replication in the presence of EBNA1.5,6 All of the four binding sites on DS are required for efficiency of EBNA1 binding, and mutation of these binding sites strongly inhibits EBV episome replication.7 EBNA1 knockout by transcription activator-like effector nucleases caused reduced EBNA1 expression and significant cell death of EBV-positive lymphocytes.8 Disruption of EBNA1 protein expression by antisense oligonucleotides9 or RNAi10,11 inhibited EBNA1 maintenance functions for the EBV genome. Small-molecule binding to EBNA1 DNA-binding domain also eradicated EBV episomes and induced cellular apoptosis.12 Thus, the binding model of EBNA1 and OriP DS sequence is a critical portion of EBV replication and a highly potential target for selectively inhibiting virus replication and coexisting cell growth. Received: March 29, 2018 Published: July 14, 2018 6674

DOI: 10.1021/acs.jmedchem.8b00496 J. Med. Chem. 2018, 61, 6674−6684

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Figure 1. Diagram of design and structure of polyamides. (A) Schematic diagram of OriP region targeted by polyamides. EBV OriP containing family repeats (FR) and dyad symmetry (DS) is shown in the diagram. Compound 2 is expected to bind to the designated location, which is illustrated in the boxes. (B) Chemical structure and the ball−stick model of polyamides. N-Methylpyrrole rings (Py) are indicated in blue and the N-methylimidazole ring (Im) in red. Hoechst groups are indicated in green. In the ball−stick model, white balls refer to Py rings, and the black balls refer to Im rings. The curves with a tail refer to γ-aminobutyric acid. The fold lines with a cross refer to dimethylaminopropylamide. The gray ellipses refer to conjugated Hoechst.

Pyrrole−imidazole polyamides (PIPs) are a series of synthetic small molecules that bind to DNA minor groove with high affinity and specificity.13,14 The skeleton of PIPs consists of linked N-methlypyrrole (Py) and N-methlyimidazole (Im) in a hairpin structure. A face-to-face Py−Py pair selectively targets A/T or T/A base pairs, and an Im/Py pair recognizes G/C base pairs.13,15 Sequence-specific PIPs are permeable to biological membrane16 and can interfere with the binding of proteins to target DNA sequences. Therefore, PIPs can modulate gene expression, e.g., NF-κB,17 MMP-9,18 and TGF-β19 by inhibiting transcription factor−DNA complexes in designated promoters. By regulating gene expression, PIPs exhibit antitumor activity in vivo.20,21 An attempt reported by Yasuda et al. to inhibit EBNA1-OriP function by PIPs showed a visible impact on virus replication and EBNA1 function in LCLs at 40 μM concentration.22 However, the polyamides applied in their work showed preference to only one of the four EBNA1-binding sites. These indicated that PIPs were capable of disrupting EBNA1 functions, but improvement on the specificity and efficacy was required for the clinical application of PIPs to inhibit EBNA1 in EBV-associated cancers. In this study, we designed and synthesized a novel Py−Im polyamide−Hoechst 33258 (Ht) conjugate, called EIP-2 (2, Figure 1), targeting all four binding sites on the DS region of EBNA1. In addition to enhance cellular permeability and nuclear uptake, conjugating the DNA−intercalator Hoechst 33258 on the γ-turn unit of PIP improved its specificity to DS by recognizing and binding up to 10 bps of the targeted sequence. Our study demonstrated that compound 2 recognized EBNA1 binding sites with high affinity and specificity, thus remarkably inhibiting EBNA1 binding to DS both in vitro and in vivo. As a consequence, compound 2

inhibited EBV-positive cells proliferation and resulted in significant reduction of virus replication. Notably, compound 2 greatly suppressed EBV-positive tumor growth in mice xenograft models. These results suggest that compound 2 is an excellent EBNA1−OriP binding inhibitor and a promising candidate for EBV-associated cancer therapy.

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RESULTS Polyamide Design and Synthesis. The interaction between OriP and EBNA1 is the key section for initiating the DNA replication of EBV.4−7 Interruption of the EBNA1− DNA interaction is a typical strategy by which to inhibit the virus replication and thereby suppress the tumor cell growth.8−12,22 As a fluorescent cell dye for DNA, Hoechst 33258 (Ht) exhibits a strong selectivity against A- and T-rich DNA sequences.23,24 Previous researches have demonstrated that PIP−Ht conjugate was capable of recognizing around four more base pairs than PIP alone.25−28 According to our previous study in the inhibitor of Polo-like kinase 1, the γ-turn is the most ideal point for Ht conjugating.29 To design PIPs that could appropriately bind to the EBNA1-binding region on OriP DS, we analyzed the four EBNA1 binding sites (Figure 1A) and noticed that sequence 5′-AGCATAT-3′ appeared in all four binding sites, which was proven to be conservative.5,6 Regions around these conservative regions were also A- and Trich and, therefore, ideal targets for a PIP−Ht conjugate. Accordingly, we designed and synthesized 2 (Figure 1B), a novel Py−Im polyamide with Ht conjugated at the γ-turn position. Compound 2 was expected to bind to the illustrated location (Figure 1A). We also synthesized EIP-1 (1), the polyamide skeleton without Ht, and untargeted PIP (a singlepair mismatched Py−Im polyamide−Ht conjugate, 3) that was 6675

DOI: 10.1021/acs.jmedchem.8b00496 J. Med. Chem. 2018, 61, 6674−6684

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Figure 2. EBNA1-binding inhibition in vitro of polyamides monitored by surface plasmon resonance. (A) Binding model of polyamides with EBNA1-binding sites. Polyamides are expected to bind to biotin-labeled hairpin DNA sequences of EBNA1 binding site 1 and 2 and their mutant sequences at the indicated location. The mismatched base pairs were colored in red or blue for mutant sequences or 3, respectively. (B) The inhibiting level to EBNA1 binding of 1. A gradient concentration of 1 passed through the DNA-immobilized chip surface at 25 μL/min for 4 min without any disassociation, and 100 nM EBNA1 solution was injected subsequently for 3 min. The EBNA1 binding process was illustrated in the figure. (C) The inhibiting level to EBNA1 binding of 2. The assay was performed as in B. (D) The inhibiting level to EBNA1 binding of 3. The assay was performed as in B.

not supposed to bind to any of the four binding sites as a negative control. Polyamide Targeting of EBNA1-Binding Region on OriP and Blocking of EBNA1 Binding to OriP in Vitro. Polyamides were designed to bind to the EBNA1-binding region on OriP so that it would displace EBNA1 from OriP.

Therefore, we tested the binding affinity of polyamides with EBNA1 binding region on DS by surface plasmon resonance (SPR). As shown in Figures 1 and 2A, the DNA sequences and the polyamides binding model were similar between sites 1 and 4 and sites 2 and 3. Thus, sites 1 and 2 were chosen to perform the SPR assay. The calculated KD of binding affinity was shown 6676

DOI: 10.1021/acs.jmedchem.8b00496 J. Med. Chem. 2018, 61, 6674−6684

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Table 1. Polyamide Binding Affinity to DSa site 1 KD (M) −9

1.50 × 10 2.28 × 10−8 3.35 × 10−7

1 2 3

site 1 mutant specificity 1 1 0.068

KD (M)

site 2

specificity −8

2.52 × 10 7.47 × 10−7 −

0.060 0.030 −

KD (M)

site 2 mutant specificity

−9

4.52 × 10 1.29 × 10−8 2.63 × 10−7

1 1 0.049

KD (M)

specificity −8

2.06 × 10 1.87 × 10−7 −

0.219 0.069 −

a

The specificity value is calculated by dividing the KD of matched polyamide−DNA pairs by the KD of mismatched pairs (mutant DNA sequences or untargeted PIP). For the matched pairs themselves, the specificity is 1.

in Table 1. Compounds 1 and 2 had strong (KD < 10−7) binding affinity to their target sequences and had weak associations with 1-base-pair mismatched binding sites. Both 1 and 2 bound to target sequences tightly and were difficult to be disassociated (Figure S5). The compound 3 was capable to bind to the EBNA1 binding sites but was disassociated quickly afterward (Figure S5). These results confirmed the high specificity of 1 and 2 to binding sites. Compound 2 were more specific to site 2 than to site 1, probably because the estimated binding region of conjugated Ht in Site 2 had more A and T bases. In accordance with our previous study in PIPs, Ht conjugation of polyamides reduced the affinity to the target sequence.29 However, compound 2 exhibited stronger specificity to the target sequences than 1-base-pair mismatch sequences in comparison to 1. To further investigate whether polyamides binding could prevent EBNA1 interaction with DS, we performed a competitive inhibition assay. Different concentrations of polyamides were injected to pass through EBNA1 binding sites. Subsequently, an EBNA1 solution was injected without polyamide disassociation (Figure 2B−D and Table 2). The

pre-injection of 1 and 2 remarkably reduced RU rise during EBNA1 injection, indicating that EBNA1 binding was blocked by these two polyamides. Having stronger affinity to EBNA1binding sites, compound 1 blocked EBNA1 binding more efficiently than 2 at 200 nM. Although a much-higher concentration of 3 was injected, only a small RU rise reduction was observed. It was proven that compound 3 was not able to block EBNA1 binding to OriP in vitro. In conclusion, compound 1 and 2 could occupy the binding position of EBNA1 on OriP and prevent EBNA1 binding in vitro with high specificity and affinity. Polyamide Specific Inhibition of EBV-Positive Lymphoblastic Cell Growth. Confirming that compound 1 and 2 could block EBNA1 binding to OriP in vitro, we next determined if 1 and 2 could inhibit EBV-positive lymphoblastic cell growth. The typical EBV-positive Burkitt’s lymphoma cell lines Raji and Daudi and EBV-negative cell lines Jurkat and MOLT-4 were treated with 1 and 2 at 10 μM, separately. The number of living cells was counted by a trypan blue dye assay. In EBV positive cells, the living cell amount in compound 2 treatment was significantly reduced compared with 1 and the control treatment (P < 0.01) (Figure 3A, top panel). In EBV-negative cells, there was no significant difference (P > 0.05) among 1, 2, and the control treatment (Figure 3A, bottom panel). To confirm that compound 2 inhibited EBV-positive cell lines selectively, an MTT assay was performed, and the exact GI50 value was calculated in eight different cell lines, including EBV-positive cells Raji, Daudi, and B95-8 and EBV-negative cells RPMI-8226, Jurkat, MOLT4, Nalm-6, and MDA-MB-231 (Figure 3B and Table 3). The GI50 in EBV-negative cells was higher than in EBV-positive

Table 2. Polyamides Blocking EBNA1 Binding to DS in Vitro relative RU rise reduction polyamides (200 nM)

EBNA1 binding site 1

EBNA1 binding site 2

1 2 3

0.646 0.583 0.091

0.524 0.412 0.088

Figure 3. Proliferation inhibition of polyamides to EBV-positive cells. (A) Cells (5 × 105) were seeded in a 6-well plate and treated with or without 10 μM 1 or 2. Medium was refreshed every 3 days. At day 3 and day 6, living cells were counted by a 0.4% trypan blue dye assay. (B) To further test the growth inhibition to different cell lines by 2, both EBV-positive and EBV-negative cells were treated by indicated concentration of 2. Survival ratio of cells were calculated via an MTT assay. All of the statistical significances were calculated by one-way ANOVA. Double asterisks indicate P < 0.01 6677

DOI: 10.1021/acs.jmedchem.8b00496 J. Med. Chem. 2018, 61, 6674−6684

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with 10 μM 2 was significantly decreased (P < 0.01) after 120 h, while 1 and 3 had little effect on EBV copy number. Compound 2 also reduced EBV copy numbers in two additional EBV-positive cell lines (Figure 4A). To confirm whether 2 eradicated EBV episomes by blocking the binding of EBNA1 to OriP in vivo, a chromatin immunoprecipitation (ChIP) assay was performed. The results show that binding of OriP to EBNA1 in Raji cells had been significantly reduced (P < 0.01) with time prolongation, while the same concentration of 1 was not able to prevent the binding of EBNA1 and OriP (Figure 4B). These data suggest that compound 2 competed with EBNA1 binding to OriP, disrupted the interaction between EBNA1 and OriP and decreased EBV replication. The depletion of the virus finally led to cell growth inhibition. Compound 1 failing to target OriP in cells and inhibit virus replication indicated that the conjugated Ht on 2 played an important role in inhibiting EBNA1 binding and virus replication. EBNA1 binding to OriP not only induces virus replication but also activates viral latent protein transcription.5,30 To detect whether treatment of 1 or 2 modulates latent viral protein expression, a Western blotting assay was performed to determine the expression level of EBNA1 and EBNA2, another important viral protein during latent infection. In 24 h, the expression level of two proteins was decreased by the increasing dosage of 2 (Figure 4C). Treatment of 1 did not

Table 3. GI50 (in Micrometers) of PIPs to Different Cell Lines cell line Daudi (EBV+) Raji (EBV+) B95−8 (EBV+) Nalm-6 (EBV−) RPMI-8226(EBV−) MOLT-4 (EBV−) Jurkat (EBV−) MDA-MB-231 (EBV−)

1 12.88 29.44 30.20 41.88 43.89 30.27 >100 >100

± ± ± ± ± ±

2 1.58 3.56 5.42 4.77 3.24 4.01

4.02 ± 0.53 8.00 ± 2.60 9.42 ± 3.03 22.18 ± 3.29 24.50 ± 4.30 25.00 ± 3.97 >100 >100

cells. Collectively, these data suggest that compound 2 could selectively inhibited EBV-positive cells, while 1 had muchweaker inhibition. Polyamide Inhibition of EBV DNA Replication and Latent Protein Expression. Based on experiments on cell proliferation, compound 2, which did not excel in targeting DS sequences and blocking EBNA1 binding in vitro, exhibited selective inhibition to EBV-positive cell lines but not 1. We speculated that the binding of EBNA1 to OriP in vivo was blocked by 2, and the EBV episome copy numbers were therefore decreased. EBV DNA copy numbers in Raji cells treated with polyamides were detected by real-time polymerase chain reaction (PCR). The copy numbers in Raji cells treated

Figure 4. Effects of polyamides on EBNA1 functions in EBV-positive cells. (A) Raji cells were treated by 10 μM 1, 10 μM 2, or 10 μM 3. Cells were collected every day, and total DNA was harvested to perform a quantitative real-time PCR of EBV copy number. A pair of different EBV-positive cell lines, Daudi and B95-8 were also treated with 10 μM 2 to detect the effect on viral copy number. For Raji and Daudi, primers of OriP and human GAPDH gene were used to determine the amount of viral episome and the cellular DNA, respectively. For B95-8, primers for OriP and the 18s rRNA gene was used. Statistics significance was calculated by one-way ANOVA. Double asterisks indicate P < 0.01. (B) Effect of polyamides on EBNA1−OriP interaction in cells. Raji cells were treated by 10 μM 1 or 10 μM 2 for 0, 24, or 48 h, and a ChIP assay was performed with EBNA1 antibody or normal mouse IgG to detect the interaction of EBNA1 protein and OriP sequence. EBNA1-binding DNAs were immunoprecipitated and detected by quantitative real-time PCR using OriP primers. Ratio of ChIP product to 2% input sample is shown in the figure. Statistics significance was calculated by one-way ANOVA. Double asterisks indicate P < 0.01. (C) Raji cells were treated with a group of concentration of 1 or 2 for 24 h. Total proteins were extracted, and the expression level of EBNA1 and EBNA2 was detected by Western blotting. GAPDH is used as the internal control. 6678

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Figure 5. Compound 2 inhibits EBV-positive tumor and EBNA1 functions in vivo. A total of 1 ×107 Raji cells were subcutaneously injected at the left flank of every Balb/c nude mouse. When tumor size reached about 50 mm3 (1/2 × L × W2), various amounts of 2 were injected subcutaneously near the tumor every 3 days. (A) Size of the tumors were recorded every 3 days before injection of 2. (B) Mice weights were recorded before every injection. (C) After all of the injections were finished, mice were sacrificed, and the tumors were peeled. (D) The mass of each tumor was weighed and illustrated. (E) Small pieces of tumor were homogenized for total DNA extraction. DNA was later processed to perform a real-time PCR to determine the relative EBV copy number. All of the statistics significances were calculated by one-way ANOVA. A single asterisk indicates P < 0.05, and double asterisks indicate P < 0.01.

reduce the expression of EBNAs. The results suggest that compound 2 probably disrupts latent protein expression by blocking EBNA1 binding to OriP. Polyamide 2 Inhibition of Tumor Growth in Mouse Xenograft Tumor Model. Balb/c nude mice carrying Raji xenograft at left flank were treated with different dosages of 2 by subcutaneous injection every 3 days to determine its antitumor function in vivo. The size of the tumors was recorded before every injection (Figure 5A). Mice treated with 2 had significant smaller tumor (P < 0.01) than vehicle control, and a higher dosage of treatment (10 mg/kg) resulted in

significantly better (P < 0.05) antitumor effects than did the 5 mg/kg treatment. After 8 injections, the tumors in the compound 2 treated group were significantly smaller than the vehicle control group (Figure 5C,D). It showed the favorable tumor suppression effect of 2 in animals. No significant weight loss was observed in the compound 2 treated groups at two different dosages (Figure 5B), indicating the low toxicity of 2 to animals. In addition, EBV copy numbers of the xenograft tumor were determined by real-time PCR (Figure 5E). As shown in the figure, the virus copy number was significantly suppressed (P < 0.01) by 2, 6679

DOI: 10.1021/acs.jmedchem.8b00496 J. Med. Chem. 2018, 61, 6674−6684

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The mitotic segregation of EBV required both EBNA1 and OriP elements.5 During the mitotic segregation of EBV, EBV episomes were tethered to cellular chromosomes by EBNA1.34−36 Therefore, it is important for the EBNA1− OriP binding inhibitor to locate the target sequences in nucleus. Early study of inhibiting gene expression by PIPs in cell lines other than T cells met great difficulties.37 Poor nuclear uptake was presumed to be the main reason.26 In accordance with Figure 4B, 1 was not capable of binding to its target sequences in vivo. Previous research has already demonstrated that Ht conjugation on PIPs could improve its ability to penetrate cell membrane and guide the molecule to the nuclear target sequences.38,39 In this research, the cellular uptake of PIP−Hoechst was observed, and it was confirmed that 2 was successfully co-localized with chromosomes, indicating its entrance in nuclei region (Figure S6). This was also confirmed in our previous study on the Polo-like kinase 1 inhibitor.29 Better cellular and nuclear uptake would certainly help 2 interruption of EBV episome segregation. Moreover, Hoechst 33258 interacts with DNA physically and reversibly and does not cause permanent DNA damage.23 In conclusion, Ht conjugation on 2 enhanced its ability to safely inhibit EBVassociated tumors by better recognizing target sequences and improved cellular and nuclear uptake. Several works have been reported on EBV treatment by interfering EBNA1 functions. The binding structure of EBNA1 and OriP was studied and high-throughput in silico and in vitro screening was performed to select an effective inhibitor to bind to EBNA1.40 Hsp90 inhibitor down-regulated the expression of EBNA1 and prevented the EBV transformation of B lymphocytes.41 The siRNA knockdown of EBNA1 inhibited EBNA1 expression in EBV-positive epithelial and lymphoblastic cell lines,10,11 and it also suppressed DNA replication in a mini-EBV replication model.10 Roscovitine suppressed EBNA1 functions of transcription and episome persistence by inhibiting serine 393 phosphorylation.42 Most of this research focused on targeting the EBNA1 protein to inhibit EBNA1−OriP binding, but there has been little research focusing on targeting the OriP region. The attempt to inhibit EBNA1−OriP binding by PIPs22 inspired us to develop an highly specific and efficient inhibitor to prevent EBNA1-OriP binding. Compared with the polyamides already reported, compound 2 showed better efficacy in a lower dosage and specificity to all four binding sites on DS. Nude mice Xenograft tumor model demonstrated that compound 2 is safe and effective against EBV-positive tumors in vivo. EBNA1 binding to DS is necessary for DNA replication but not sufficient5,43 because viral DNA replication can initiate from other sites independently from EBNA1, such as Rep*.44 Other important viral or host genes, including LMP1,45 topoisomerase I,46 and NF-κB47 were also involved in EBVassociated tumor formation. Lytic cycle of EBV, which contribute to EBV infection, was important for EBV treatment. The polyamide developed in this study would be the core of our further study on drug combination therapy targeting EBNA1−OriP binding and other factors associated with EBVpositive tumors.

suggesting that compound 2 was able to inhibit tumor growth by preventing virus replication.

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DISCUSSION In this study, we designed and synthesized two polyamides that target OriP sequences to block EBNA1−OriP binding. Through in vitro and in vivo screening, we discovered that the Ht-conjugated polyamide 2 was capable of displacing EBNA1 from OriP and, consequently, disrupted virus replication and viral protein expression. As a result, EBVpositive cell proliferation and EBV-positive tumor growth in mice xenograft tumor model were significantly suppressed. Although DS and FR contribute to different functions of OriP, all of the binding sites on OriP are highly conserved.22 Albeit originally designed to target DS, compound 2 is quite possible to bind to FR as well (Figure S7). One of the most important functions of FR is to activate the Cp promoter and latent membrane protein (LMP) promoters, subsequently inducing the expression of EBNAs and LMPs with EBNA1 binding.31,32 The reduced expression of EBNA1 and EBNA2 by treatment with 2 indicated that compound 2 but not compound 1 inhibited the transcription function of EBNA1, most likely by interrupting the binding of EBNA1 to FR. Along with the down-regulation of the replication of the EBV genome, compound 2 inhibited both the replication and the transcription functions of EBNA1. Ht-conjugated polyamide 2 significantly inhibited EBVpositive cell proliferation and EBV-positive tumor growth, while 1 did not exhibit any significant effect in contrast. Hoechst 33 258 is a kind of synthetic minor groove-binding molecule containing bis-benzimidazole moiety. It strongly prefers but is not restricted to A- and T-rich DNA sequences, with 5 ± 1 base pairs at sites in size.23,24 It was reported that PIP−Ht conjugate was capable of recognizing around four more base pairs than PIP alone,25−28 which could improve the specificity of the polyamide toward target sequences and reduce the off-target binding. In our previous study, we synthesized a series of PIPs to target the Polo-like kinase 1 promoter sequence.29 A PIP−Hoechst conjugate in which the Ht was conjugated at the γ-turn position recognized longer sequences and was the most specific and efficient inhibitor of Polo-like kinase 1 expression among all PIP derivatives we have synthesized. Therefore, we hypothesized that Ht-conjugated 2 exhibit a better effect on EBNA1 inhibition. According to our design, compound 2 was able to cover a 6 base pair pyrrole− imidazole recognizing region and a 4−5-base-pair A- and Trich Ht-binding region in every EBNA1 binding site on DS, coming to a total of 10−11 bps. Target-specific inhibiting would certainly benefit from longer recognizing sequences. The most-efficient approach for polyamides to interrupt gene expression is to bind to key DNA elements, such as promoters, to inhibit the formation of protein−DNA complexes.13 Besides, it was revealed that if the polyamide binding region was just slightly upstream or downstream of the transcription start site of a target gene, the expression level of the target gene was not altered.33 According to Table S1, there exist a great number of highly similar sequences of EBNA1 binding sites in human genome, but only a few of them are possibly located on promoters, especially in the transcription start site. Therefore, the lack of effects on the expression level of other important genes could be expected although off-target binding of compound 2 existed.

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EXPERIMENTAL SECTION

General Information. 1-Methyl-1H-imidazole-2-carboxylic acid, dry DMF and dry THF were purchased from Sigma-Aldrich. Fmoc− hydrazinobenzoyl AM resin was purchased from Novabiochem. Boc− Py−OH, Boc−Im−OH, collidine (2,4,6-collidine), TFA, and high6680

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yl)-1H,1′H-[2,5′-bibenzo[d]imidazol]-2′-yl)phenoxy)butanamido)butanamido)-1H-pyrrole-2-carboxamido)-1H-pyrrole-2-carboxamido)-1H-imidazole-2-carboxamide (2). To the resin-bound 1 (Im−Py−Py−Py−γ(Fmoc)−Py−Py−Im−Py), 20% piperidine in DMF (3 mL) was added, and the mixture was agitated for 5 min. This operation was repeated to remove the Fmoc group. Then, the resin was washed with DMF (4 × 3 mL) and dry DMF (3 mL). Meanwhile, Hoechst 33258 acid (2 equiv) and PyBOP (2 equiv) were dissolved in dry DMF (3 mL), and DIEA (6 equiv) was added to the solution. After 2 min of stirring, the solution was transferred to the deprotected resin. The mixture was agitated for 2 h before the resin was washed with DMF (4 × 3 mL). Cupric acetate (1 equiv) and N,N-dimethyl-1,3-propane diamine (10 equiv) in DMF (1 mL) was added to the resin. The mixture was shaken overnight at room temperature. Next, the resin was removed by filtration through a disposable propylene filter and washed with MeOH (4 × 2 mL). The filtrate was dried under vacuum, dissolved in 10% MeCN/H2O (0.1% TFA), and purified by semipreparative RP-HPLC. After lyophilization, compound 2 was obtained as yellow powder (yield: 23%). HPLC analysis showed a retention time of 15.266 min and a peak area of 99.234%. HRMS (ESI) m/z [M + H]+: calcd for C84H96N27O11, 1658.7777; found, 1658.7778; [M+2H]2+, calcd for C84H97N27O11, 829.8930; found, 829.8974 (Figure S4). Synthesis of (R)-N-(5-((5-((4-((5-((5-((5-((5-((3-(Dimethylamino)propyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)carbamoyl)-1-methyl1H-pyrrol-3-yl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)carbamoyl)-1methyl-1H-pyrrol-3-yl)amino)-3-(4-(4-(5-(4-methylpiperazin-1-yl)1H,1′H-[2,5′-bibenzo[d]imidazol]-2′-yl)phenoxy)butanamido)-4oxobutyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)-1-methyl-4-(1-methyl-1H-imidazole-2-carboxamido)-1H-imidazole-2-carboxamide (3). The resin-bound polyamide sequence, Im−Im−Py−Py−γ(Fmoc)−Py−Py−Py−Py, was prepared using the identical methods with that of 1. Next, subsequent methods including removal of the Fmoc group, coupling with Hoechst 33258 acid, resin cleavage and final purification were performed according to the synthesis of 2. Compound 3 was obtained as yellow powders (yield: 21%). HPLC analysis showed a retention time of 15.713 min and a peak area of 100%. HRMS (ESI) m/z [M + H]+: calcd for C84H96N27O11, 1658.7777; found, 1658.7794; [M +2H]2+, calcd for C84H97N27O11, 829.8930; found, 829.9167; [M +3H]3+, calcd for C84H98N27O11, 553.5979; found, 553.5996 (Figure S4). Cell Line and Culture. All of the used cell lines were purchased from Core Facility of Stem Cell Research, Shanghai Institute of Biological Science CAS, and were incubated in a humidified incubator with 5% CO2 at 37 °C. Raji, B95-8, Daudi, MOLT-4, Jurkat, RPMI8226, and Nalm-6 cells were cultured in RPMI Modified (Hyclone) with 100 U/mL penicillin, 100 μg/mL streptomycin (Hyclone), and 10% FBS (Corning). MDA-MB-231 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) with high glucose (Hyclone) with 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% FBS (Biological Industrial). SPR Assay. The SPR assays were performed with a ProteOn XPR36 instrument (Bio-Rad) on a ProteOn GLH sensor chip (BioRad). Biotin-labeled short hairpin DNAs were purchased from Genewiz. The sequences are shown in Figure 3. About 16 000 RU of streptavidin (Sangon, Shanghai, China) were coated first, and 200 nM of biotin-labeled hairpin DNA was then immobilized at around 700−900 RU. The whole assay was carried out in HBS-EP buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.005% surfactant P20) at 25 °C. Polyamides samples were first diluted in ultrapure water to avoid aggregation and then diluted to a various gradient of concentrations. For EBNA1-binding inhibiting assay, 100 nM recombinant EBNA1 (Fitzgerald) was immediately injected after the injection of polyamides. Sensorgrams were analyzed by ProteOn Manager software version 3.1 to calculate association rates (ka) and dissociation rates (kd). The proper binding models (models for simultaneous ka/kd, 1:1 binding with a drifting baseline or a 1:1 binding with mass transfer; models for general fitting and steady-state affinity) were used to get better fits. Experiments were at least performed in triplicate.

performance liquid chromatography (HPLC)-grade solvents (CH3CN and MeOH) were purchased from J&K Scientific. BTC, PyBOP, cupric acetate, and N,N-dimethyl-1,3-propane diamine were purchased from Aladdin. Fmoc−D-Dab(Boc)−OH, and HOAt were purchased from GL Biochem. Hoechst 33258 acid was purchased from Beijing Fanbo Biochemicals Ltd. All commercial reagents were used as received. The purity of the compounds was determined to be >95% by analytical reverse-phase (RP)-HPLC. Analytical RP-HPLC was performed at 25 °C on the Shimadzu LC 20 using an Inertsil ODSSP column (4.6 mm × 250 mm, 5 μm, 100 Å). UV absorbance was detected using a UV detector (SPD-20A) at 254 and 310 nm. The analytical RP-HPLC gradient started at 10% of B (CH3CN) and then increased to 100% of B over 30 min (A: 0.1% TFA in water). Semipreparative RP-HPLC was performed on the ULTIMAT 3000 Instrument (DIONEX) using Thermo Scientific Hypersil GOLD column (21.2 mm × 250 mm, 5 μm). UV absorbance was measured using a photodiode array detector at 254 and 310 nm. The RP-HPLC gradient started at 10% of B (CH3CN), was held at 5 min, and then increased to 100% of B over 25 min (A: 0.1% TFA in water). Mass spectra were measured on ABI Q-star Elite. Synthesis of (R)-4-(4-(4-(2-Amino-4-(1-methyl-4-(1-methyl-4-(1methyl-4-(1-methyl-1H-imidazole-2-carboxamido)-1H-pyrrole-2carboxamido)-1H-pyrrole-2-carboxamido)-1H-pyrrole-2carboxamido)butanamido)-1-methyl-1H-pyrrole-2-carboxamido)1-methyl-1H-pyrrole-2-carboxamido)-N-(5-((3-(dimethylamino)propyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)-1-methyl-1H-imidazole-2-carboxamide (1). According to the previous methods,48,49 the synthesis of 1 was carried out using Fmoc-hydrazinobenzoyl AM NovaGel resin at a substitution level of 0.61 mmol/g. First, the resin (400 mg, 0.244 mmol, 1 equiv) was pre-swelled in DCM for 20 min in a manual solid-phase peptide synthesis vessel. Then, the Fmoc group was removed with 20% piperidine in DMF (2 × 5 min), and the resin was washed with DMF (4 × 3 mL) and dry DMF (3 mL). Meanwhile, Boc−Py−OH (234 mg, 0.976 mmol, 4 equiv) and BTC (95 mg, 0.322 mmol, 1.33 equiv) were dissolved in dry THF (2 mL), and collidine (387 μL, 2.928 mmol, 12 equiv) was added drop-wise to the THF solution. After activation for 1 min, dry DMF (1 mL) was added to the suspension followed by addition of DIEA (509 μL, 2.928 mmol, 12 equiv). When the mixture became clear, it was transferred to the deprotected resin. Coupling of the first Boc−Py−OH to the resin was taken out for 1 h to ensure sufficient reaction. The resin was washed with DMF (3 × 3 mL) and DCM (2 × 3 mL), and the Boc group was then removed using a TFA/TIS/H2O mixture (95:2.5:2.5) (2, 1, and 20 min, respectively). After washing with DCM (2 × 3 mL), DMF (3 × 3 mL), and dry DMF (3 mL), the resin was ready for the next coupling. This procedure including activation of the amino acid, coupling with the resin and deprotection of the Boc group was repeated until a polyamide sequence (Im−Py−Py−Py−γ(Fmoc)− Py−Py−Im−Py) bound to the resin was obtained. It should be noted that the addition of HOAt (4 equiv) to the activated solution of Boc− Im−OH or Fmoc−D-Dab(Boc)−OH was needed before it was transferred to the deprotected resin. All the couplings were completed in 30 min. After the resin was washed with DMF (4 × 3 mL), N,Ndimethyl-1,3-propane diamine (311 μL, 2.44 mmol, 10 equiv) in DMF (1 mL) was added to the resin. The resulting mixture was shaken for 3 h at 90 °C before it was cooled to room temperature. Then the resin was removed by filtration through a disposable propylene filter and washed with MeOH (4 × 2 mL). The filtrate was dried under vacuum, dissolved in 10% MeCN/H2O (0.1%TFA), and purified by semipreparative RP-HPLC. After lyophilization, compound 1 was obtained as fine white powders (yield: 29%). HPLC analysis showed a retention time of 15.159 min and a peak area of 95.350%. HRMS (ESI) m/z [M + H]+: calcd for C55H68N21O9, 1166.5509; found, 1166.5509; [M+2H]2+, calcd for C55H69N21O9, 583.7793; found, 583.7703 (Figure S4). Synthesis of (R)-N-(5-((3-(Dimethylamino)propyl)carbamoyl)-1methyl-1H-pyrrol-3-yl)-1-methyl-4-(1-methyl-4-(1-methyl-4-(4-(1methyl-4-(1-methyl-4-(1-methyl-4-(1-methyl-1H-imidazole-2-carboxamido)-1H-pyrrole-2-carboxamido)-1H-pyrrole-2-carboxamido)-1H-pyrrole-2-carboxamido)-2-(4-(4-(5-(4-methylpiperazin-16681

DOI: 10.1021/acs.jmedchem.8b00496 J. Med. Chem. 2018, 61, 6674−6684

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Cell Proliferation and Growth-Inhibition Assay. For cell proliferation assays, 5 × 105 EBV-positive Raji and Daudi cells and EBV-negative Jurkat and MOLT-4 cells were cultured in 2 mL of medium with 10 μM polyamides. An equal volume of fresh medium containing polyamides was added after 3 days. At days 3 and 6, living cells were counted by a trypan blue dye assay. For cell-growthinhibition assay, certain amounts of cell (for suspension cells, 7 × 103; for adherent cells, 3 × 103) were seeded into 96-well plates in quadruple with different concentration of polyamides. After 72 h of culturing, the ratio of living cells to the control were determined by a WST assay using MTT (Sigma). GI50 was calculated using sigmoidal dose−response fitting by GraphPad Prism version 5.0 software. Experiments were at least performed in triplicate. Quantitative Real-Time PCR Assay. To quantify the EBV DNA copy numbers of cell treated with polyamides, 5 × 105 Raji cells were cultured with 10 μM 1 and 2 and 10 μM 3. Cells were harvested every 24 h, and the whole-genome DNA was extracted using a genomic DNA kit (OMEGA). A total of 5 ×105 B95-8 cells and 1 × 106 Daudi cells were also cultured with 10 μM 2, and DNA was extracted. DNA was subjected to real-time PCR in triplicate with a SYBR mix (TransGen, Beijing, China). EBV OriP sequence (Genbank: NC_007605.1 8927−9060; forward primer of 5′-GGTTCACTACCCTCGTGGAA-3′ and reverse primer of 5′-TGTTACCCAAC GGGAAGCATA-3′) was used to determine the amount of EBV copy number. The human GAPDH gene (Genbank: NG_007073.2 8118− 8242; forward primer of 5′-ACCCAGAAGACTGTGGATGG-3′, reverse primer: 5′-TTCAGCTCAGGGATGACCTT-3′) for humansource Raji and Daudi cells and 18sRNA (Genbank: NT_167214.1 110426−110540, forward primer of 5′-GTGGAGCGATTTGTCTGGTT-3′ and reverse primer anf 5′-AACGCCACTTGTCCCTCTAA-3′) for B95-8 cells were used to determine the cellular DNA. DNA amplifications and fluorescence detection were performed using a CFX96 Real-Time System (Biorad, Singapore). The protocol of DNA amplifications contained an initial denaturation at 95 °C for 5 min, 40 cycles of 95 °C for 10 s, 60 °C for 30 s, and a melt curve to confirm the specificity of amplification. The amplification efficiency was calculated by standard curves. Relative amounts of EBV copies and cellular DNA were calculated by the ΔΔCt method. Each experiment was performed at least in triplicate. To determine the EBV copy number in mice tissue, pieces of tumor were homogenized in liquid nitrogen and then proceeded to DNA extraction using a genomic DNA kit. Total DNA was subjected to real-time PCR, as described above. Experiments were at least performed in triplicate. ChIP Assay. Raji cells were cultured with 10 μM 1 or 2. At 24 and 48 h, 8 × 106 cells were harvested for immunoprecipitation. Cells were fixed by 37% formaldehyde and processed with a SimpleChIP Enzymatic Chromatin IP Kit (CST). 2% volume of samples are aliquoted as input control. A total of 1 μg of anti-EBNA1 antibody (Santa Cruz) and normal mouse IgG1 (CST) were added to each group for immunoprecipitation and incubated overnight. The combination was isolated by magnetic beads. The same volume of the extracted ChIP product DNA was subjected to real-time PCR in triplicate, detecting the EBV OriP sequence with a SYBR mix. The protocol of DNA amplifications contained an initial denaturation at 95 °C for 5 min, 40 cycles of 95 °C for 15 s, 60 °C for 60 s, and a melt curve to confirm the specificity of amplification. The amplification efficiency was calculated by standard curves. Relative amounts of precipitated DNA were calculated by the ΔΔCt method. Each experiment was at least performed in triplicate. Western Blotting. Raji cells were treated with 0, 1, 2, 5, 10, or 20 μM 1 or 2 for 24 h. Cells were harvested and lysed using nondenaturing lysis buffer supplemented with a protease inhibitor cocktail. Total proteins from the supernatant were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, MA). Membranes were exposed to EBNA1 antibody (Santa Cruz), EBNA2 antibody (Millipore), and GAPDH antibody (TransGen, Beijing, China) at 4 °C overnight, followed with horseradish-peroxidaseconjugated second antibody for 1 h. Blots were detected by luminata

Classico western HRP substrate (Millipore). Experiments were at least performed in triplicate. Mouse Xenograft Tumor Experiment. Balb/c nude female mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Raji cells were collected by centrifuge and diluted to 1 × 107 in 100 μL of RPMI medium without FBS. A total of 1 ×107 Raji cells were subcutaneously injected in the left flank. An injection of 2 began when tumor size reached ∼50 mm3 (L × W × 1 /2W). Compound 2 was diluted in 100 μL of 5% DMSO/0.9% NaCl solution and subcutaneously injected around the tumor every 3 days (for vehicle, n = 6; for two experimental groups, n = 7). Tumor size and animal weight were measured before injection. After eight injections, mice were sacrificed, and the tumors were weighed and peeled for further experiments. All experiments were performed under the guidelines of the Institutional Ethical Committee of Animal Experimentation of Shenzhen Institutes of Advanced Technology, Chinese Academy of Science (approval no. SIAT-IRB-160223-YYSSUW-A0182).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.8b00496. Additional figures and tables illustrating HPLC analysis, HRMS spectra, SPR diagram of polyamide-DNA binding, in vivo cellular uptake of 2, predicted binding region on FR, and similar sequences of site 2. (PDF) Molecular formula strings. (CSV)

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AUTHOR INFORMATION

Corresponding Authors

*L.F. e-mail: [email protected]. Phone: (+86)755-86392257. *W.S. e-mail: [email protected]. Phone: (+86)755-86585203. *P.W. e-mail: [email protected]. Phone: (+86)75586585232. Fax: (+86)755-86585222. ORCID

Wu Su: 0000-0001-9958-3434 Pu Wang: 0000-0001-6555-0845 Author Contributions #

Z.C. and W.W. contributed equally.

Funding

This work was supported by the Shenzhen Sciences & Technology Innovation Council (grant nos. JCYJ20160531171744232, JCYJ20170818153538196, JCYJ20170413165916608, and JCYJ20150401150223649) and the National Natural Science Foundation of China (grant nos. 21502219, 21672254, 21778068, and 21432003). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the School of Pharmaceutical of Sun YatSen University for technical and equipment support on SPR experiments.

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ABBREVIATIONS USED MeOH, methanol; CH 3 CN, acetonitrile; BTC, bis(trichloromethyl)-carbonate; DIEA, N,N′-diisopropylethylamine; HOAt, 1-hydroxy-7-aza-benzotriazole; PyBOP, benzotriazol-1-yl-oxytripyrrolidino-phosphoniumhexafluorophosphate. 6682

DOI: 10.1021/acs.jmedchem.8b00496 J. Med. Chem. 2018, 61, 6674−6684

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DOI: 10.1021/acs.jmedchem.8b00496 J. Med. Chem. 2018, 61, 6674−6684

Journal of Medicinal Chemistry

Article

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DOI: 10.1021/acs.jmedchem.8b00496 J. Med. Chem. 2018, 61, 6674−6684