Molecular characteristics of DNA-alkylating PI polyamides targeting

ABSTRACT: The runt-related transcription factor (RUNX) family has been associated with cancer development. The binding of RUNX family members to speci...
0 downloads 0 Views 996KB Size
Subscriber access provided by University of Texas Libraries

Article

Molecular characteristics of DNA-alkylating PI polyamides targeting RUNX transcription factors Rina Maeda, Shinsuke Sato, Tomo Ohno, Gengo Kashiwazaki, Shunsuke Obata, Kaori Hashiya, Toshikazu Bando, and Hiroshi Sugiyama J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b08813 • Publication Date (Web): 02 Jan 2019 Downloaded from http://pubs.acs.org on January 3, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 7 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of the American Chemical Society

Molecular characteristics of DNA-alkylating PI polyamides targeting RUNX transcription factors Rina Maeda†, Shinsuke Sato‡, Shunsuke Obata‡, Tomo Ohno‡, Kaori Hashiya‡, Toshikazu Bando*,‡, and Hiroshi Sugiyama*,‡,§ †

Graduate School of Advanced Integrated Studies in Human Survivability, Kyoto University, Sakyo, Kyoto 606-8306, Japan



Department of Chemistry, Graduate School of Science, Kyoto University, Sakyo, Kyoto 606-8502, Japan

§

Institute for Integrated Cell-Material Science (WPI-iCeMS), Kyoto University, Sakyo, Kyoto 606-8501, Japan

Supporting Information Placeholder ABSTRACT: The runt-related transcription factor (RUNX) family has been associated with cancer development. The binding of RUNX family members to specific DNA sequences is hypothesized to promote the expression of downstream genes and cause cancer proliferation. Based on this proposed mechanism of cancer growth, we developed conjugate 1, which inhibits the binding of RUNX to its target DNA. Conjugate 1 is a DNA-alkylating pyrrole–imidazole (PI) polyamide conjugate containing chlorambucil as an anticancer agent. Conjugate 1 was reported to have a marked anticancer effect in mouse models of acute myeloid leukemia. Although the effectiveness of 1 has been demonstrated in vivo, the detailed mechanism by which it alkylates DNA is unknown. Here, we chemically elucidated the molecular characteristics of conjugate 1 to confirm its potential as a RUNX-inhibiting drug. We also generated an alternative conjugate 2, which targets the same DNA sequence, by replacing one pyrrole with b-alanine. Comparison of the characteristics of conjugates 1 and 2 suggested that reaction selectivity and binding affinity to the RUNX-binding sequence were improved by the introduction of b-alanine. These findings indicate the possibility of DNA-alkylating PI polyamides as candidates for cancer chemotherapeutics.

n

INTRODUCTION

The runt-related transcription factor (RUNX) family is an essential master regulator of cell fate in the development of cancer.1 The RUNX family consists of three members: RUNX1, RUNX2, and RUNX3, each of which has a highly conserved DNA-binding domain (Runt domain) that interacts with DNA in a specific manner.2 RUNX forms a heterodimeric complex with core-binding factor subunit-b (CBFb), which cannot bind to DNA but allosterically enhances the DNA-binding affinity of RUNX.3 RUNX1 was believed to function as an oncosuppressor of the development of leukemia.4 However, a recent study showed that RUNX1 promoted the progression of acute myeloid leukemia (AML) cells.5 Since it has been found that RUNX2 and RUNX3 are related to osteosarcoma and gastric cancer, respectively, RUNX family members are therefore suggested to be strongly associated with cancer development,6 and the RUNX family has attracted attention as a new target for cancer treatment. Our group has tried to develop a RUNX-inhibiting drug to repress the activity of the RUNX family using pyrrole–imidazole (PI) polyamides.7 PI polyamides are a class of DNA minor groove binders that specifically recognize each of the four Watson–Crick base pairs according to pairing rules.8,9 The antiparallel pairing of P/P recognizes A/T or T/A base pairs, whereas the antiparallel pairing of I/P recognizes G/C base pairs.10 By taking advantage of this unique characteristic, many types of DNA-alkylating PI polyamides have been developed by conjugating them to DNA-alkylating agents such as chlorambucil.11– 13 Chlorambucil, a nitrogen mustard derivative, which is used

clinically as an anticancer agent, alkylates DNA preferentially at N7 position of guanine and N3 position of adenine.14–16 Conjugate 1, a chlorambucil–PI polyamide conjugate, was designed to target the RUNX-binding sequence. This conjugate is proposed to suppress the expression of RUNX-target genes, such as BCL11A and TRIM24, by blocking RUNX–DNA binding. (Figure 1).

Figure 1. (A) Proposed mechanism of RUNX inhibition by conjugate 1. (B) Chemical structure of DNA–chlorambucil adduct alkylated at N3 position of adenine.

ACS Paragon Plus Environment

Journal of the American Chemical Society 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 2. (A) Chemical structures of conjugates 1-4. (B) Schematic representations of conjugates 1-2.

These genes are known to promote p53 degradation, which means that their repression induces the apoptosis of cancer cells. We reported that global regulation of the RUNX family by conjugate 1 induced a stronger antitumor effect than the suppression of individual RUNX members.7 Moreover, it was demonstrated that conjugate 1 significantly improved overall survival periods in a mouse AML model xenografted with MV4-11 cells compared with cytarabine, currently used as an anticancer drug for leukemia. This result indicated that conjugate 1 is a potential anticancer agent and preparations for its clinical application are underway. However, there is room for modification of conjugate 1 to reduce its side effects and to improve its function because the molecular basis of its activity has not been characterized fully. Therefore, in this study, we examined the DNA-alkylating mechanism of conjugate 1 to confirm the validity of its anticancer effect. The alkylating activity of this conjugate and its DNA-binding affinity for the RUNX binding site were evaluated. Furthermore, to explore the possibility of using an alternative structure, we prepared a new conjugate 2, which targets the same DNA sequence, but comprises different components. By comparing the characteristics of these conjugates, we investigated them as the candidate for RUNX-family inhibitors to treat cancer. n

The PI polyamides that were the starting materials for preparation of conjugates 1 and 2 were synthesized on oxime resin using Fmoc solid-phase synthesis as described previously.21–23 After cleavage from the resin, chlorambucil was coupled to each PI polyamide using PyBOP and DIEA. For the SPR assay, conjugates 3 and 4 were synthesized by hydrolysis of conjugates 1 and 2 to avoid an irreversible alkylation reaction. All conjugates were purified by HPLC and identified by MALDI– TOFMS before use (Figures S1–S4). We reported that conjugate 1 specifically targets the sequence 5¢-TGTGGT-3¢ in 209-bp DNA fragments.7 However, the DNA alkylation sites were poorly identified because of the low resolution of polyacrylamide gel electrophoresis (PAGE). Thus,

RESULTS AND DISCUSSION

The RUNX family has been reported to bind specifically to the consensus DNA sequence, 5¢-TGTGGT-3¢, containing a highly conserved core site, GGT.2 Based on this binding specificity, conjugate 1 was designed to recognize a 6-bp sequence consistent with the RUNX-binding site.7 To disrupt the interaction of RUNX with DNA strongly and irreversibly, chlorambucil was conjugated to the N-terminal position of the PI polyamide. We also prepared conjugate 2, in which the first pyrrole from N-terminal of conjugate 1 was replaced with b-alanine (Figure 2). It was previously reported that the introduction of b-alanine into PI polyamides significantly improved their sequence specificity because b-alanine allows the crescent-shaped ligand to fit the curvature of the DNA helix.17–19 It has also been investigated that the contiguous imidazole with higher curvature induced reducing of the association rate of PI polyamides to the target DNA.20 Hence, we focused on the first pyrrole between the continuous imidazole to ease the curvature of the conjugate by substituting b-alanine. According to the binding rule that P/b pairs recognize A/T or T/A pairs, conjugate 2 targets the same sequence as conjugate 1.

Figure 3. (A) Oligonucleotides used in PAGE analysis. (B) Thermally induced strand cleavage of 5¢-FAM-labeled DNA fragment (30 bp) by conjugates 1-2. Lane 1, ODN1; Lane 2, annealed DNA (ODN1/ODN2); Lane 3, Control; Lanes 4–7, 5 or 10 µM of 1 and 2; Lane 8, ODN3; Lane 9, ODN4. (C) Schematic representations of the alkylation sites of each conjugate. Arrows indicate alkylation sites corresponding to the sites presented in Figure 3B.

ACS Paragon Plus Environment

Page 2 of 7

Page 3 of 7 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of the American Chemical Society thorough investigation including product analysis was needed to clarify the alkylation site. To obtain detailed information about the alkylating activity of conjugates 1 and 2, we examined the alkylation of 30-bp duplex DNA using high-resolution denaturing PAGE. All the DNA oligonucleotides used in this study are listed in Figure 3A. To prepare the 5¢-FAM labeled DNA fragment, ODN1 and ODN2 were annealed at 65 °C for 10 min. Reference DNAs, ODN3 and ODN4, were prepared based on the alkylation sites suggested by the previous PAGE results. DNA alkylation was carried out at room temperature for 18 h. To visualize the alkylation sites, the alkylated DNA samples were heated at 95 °C for 10 min, then treated with piperidine to convert the modified sugar termini to 3¢-phosphate termini (Scheme S1).24,25 DNA fragments with 3¢-phosphate termini were observed as alkylated bands on the gel. Finally, the alkylation sites were determined by comparison with the reference DNAs. In this experiment, all alkylated DNA fragments migrated slightly faster than the reference DNAs because alkylated fragments with 3¢-phosphate termini shift towards the anode due to the negative charge of the phosphate group. The results of PAGE analysis are shown in Figure 3B. Conjugate 1 clearly alkylated both of the two adjacent adenines in the target sequence because two bands were observed at the same positions as ODN3 and ODN4, respectively (Lanes 4, 5). In contrast, a band with equal migration to that of ODN4 was strongly detected as a result of DNA alkylation by conjugate 2

(Lanes 6, 7), indicating that conjugate 2 selectively alkylates the A19 position of ODN1. The alkylation sites of each conjugate in accordance with the sites suggested by PAGE analysis are outlined in Figure 3C. Using the conventional method with a long DNA fragment, it was difficult to distinguish the 1-bp difference in alkylation sites. The identification of sites alkylated by chlorambucil–PI polyamides has been particularly difficult due to their flexible structures. However, the evaluation method used in this study made it possible to provide detailed information about the alkylating activity of the conjugates, and revealed that conjugate 2 had higher specificity than conjugate 1 for the alkylated adenine. As mentioned above, the flexible structure of conjugate 2 provided by b-alanine might fix the alkylating position by matching the curvature of the DNA-helix. This result suggested that the replacement of pyrrole with balanine allows the conjugate to distinguish one base difference and increases their selectivity for alkylation sites. To clarify the DNA alkylation site of conjugate 2 in more detail, we investigated the alkylation of the duplex oligonucleotide, 5¢-CATTAACCACAATTAC-3¢ (ODN5)/5¢-GTAATTG TGGTTAATG-3¢ (ODN6), which was designed based on the result of the PAGE analysis described above. HPLC analysis of the reaction mixtures containing ODN5/ODN6 and conjugate 2 revealed that ODN5 and conjugate 2 were consumed after an 18 h alkylation, with the formation of a new peak (indicated by the red arrow in Figure 4C). The new peak was determined to be

Figure 4. HPLC analysis of the alkylation of ODN5/ODN6 with conjugate 2: (A) DNA control, (B) 0 h, and (C) 18 h. 50 mM TEAA containing 9–15% acetonitrile for 15 min and 15–100% acetonitrile for 15 min over a stepwise gradient. The retention time of a new peak (indicated by the red arrow) was 19.6 min. (D) Reaction mechanism of posttreatment for characterization of ODN5–2 adduct. *The peak of injection spike presumably derived from trifluoroacetic acid (counter-ion of conjugate 2) and DMF (solvent to dissolve conjugate 2).

ACS Paragon Plus Environment

Journal of the American Chemical Society 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

derived from the ODN5–conjugate 2 adduct using a procedure reported previously.24,26,27 The collected fraction containing this adduct was heated at 90 °C for 5 min to obtain an abasic site containing ODN5. This degradation sample was subsequently reduced with 0.1 M NaBH4. The reduced product was analyzed by HPLC and its identity confirmed by comparing its retention time with that of the reference oligonucleotide prepared from uracil DNA (Figures 4D, S5). The degradation products of uracil DNA after NaBH4 reduction were identified by MALDI– TOFMS (Figure S6), suggesting that the DNA alkylation by conjugate 2 occurred specifically at the A12 position of ODN5. On the other hand, the DNA alkylation by conjugate 1 gave a complex HPLC profile that showed three new peaks after an 18 h reaction (Figure S7). Although further investigation of each peak obtained from conjugate 1 is required, the results of HPLC analysis are consistent with those of PAGE analysis. This result indicated that the introduction of b-alanine improves the reaction selectivity of the conjugate. Moreover, we confirmed that DNA-alkylation occurs at the N3 position of adenine by using the oligonucleotides containing the 3-deaza-adenine (X) and 7-deaza-adenine (Y) (Figure S8A). HPLC analysis of the reaction mixtures combining 5¢CATTAACCACAXTTAC-3¢ (ODN7)/ODN6 with conjugate 2 indicated that the consumption of ODN7 and the formation of the corresponding adduct diminished (Figure S8B-D). Whereas, the examination of alkylation of 5¢-CATTAACCACAYTTAC3¢ (ODN8)/ODN6 showed the formation of the new peak expected to correspond to ODN8-conjugate 2 adduct (Figure S8E-G). These results revealed that conjugate 2 alkylated at the N3 position of adenine.

Figure 5. (A) A biotinylated DNA oligomer used in this study. (B and C) SPR sensorgrams of conjugates 3-4 with biotinylated DNA immobilized on a sensor chip SA. (B) Conjugate 3 at concentrations of 2000, 1500, 1000, 750, 500, 375 and 250 nM (from the top). (C) Conjugate 4 at concentrations of 80, 60, 40, 30, 20, 15, 10, 7.5 and 5.0 nM (from the top).

Table 1. The values of ka, kd and KD of conjugates 3-4 calculated by curve fitting for each sensorgram

We next compared the DNA-binding affinity of conjugates 1 and 2 using an SPR assay.28,29 A biotinylated hairpin DNA including the target sequence, 5¢-TGTGGT-3¢, (shown in Figure 5A) was immobilized to a sensor chip via a biotin–streptavidin link. Solutions containing various concentrations of conjugates 3 and 4 were run over the hairpin DNA on the sensor chip. The interaction was visualized as sensorgrams (Figure 5B, C) and then the association rate (ka), dissociation rate (kd), and dissociation constant (KD) were calculated by curve fitting for each sensorgram (Table 1). The KD value of conjugate 4 was considerably lower than that of conjugate 3, which means that conjugate 4, the derivative of conjugate 2, had more than 400 times stronger binding affinity to the RUNX-binding sequence. This difference was statistically significant in triplicate experiments (Table S2). As suggested previously, the structural flexibility provided by substituting b-alanine for pyrrole improved the binding affinity and stability of the conjugate for the target sequence. Furthermore, even compared with the DNA-binding affinity of RUNX1 in the presence of CBFb, the KD value of conjugate 4 was about a quarter of that of RUNX1 (KD = 7.56 nM).30 This finding suggests that conjugate 2 has sufficient binding affinity to inhibit RUNX–DNA binding. To further examine the potential of conjugate 2 as an anticancer agent, we performed on the cytotoxicity assay against MV4-11 cells, human AML cells with wild-type p53. The cell viability and the IC50 values (the concentration required for 50% inhibition of cell growth) of each conjugate were shown in Figure 6. The IC50 values demonstrated that the cytotoxicity of conjugate 2 was nearly ten times higher than that of conjugate 1. Although there were some exceptions depending on the position of b-alanine, the replacement of pyrrole with b-alanine induced the poor nuclear localization.31 This might have caused the slight improvement of cytotoxicity compared with the significant change of DNA-binding affinity. However, it was suggested that the incorporation of b-alanine had a favorable effect also on the anticancer activity. Furthermore, we confirmed that the DNA-alkylation was essential for the cytotoxicity of conjugates 1 and 2, since conjugates 3 and 4 did not show any cytotoxicity even with high concentration. A similar result was obtained for MOLM-13, a different kind of p53 WT AML cells (Figure S9A). Additionally, we detected no cytotoxicity of conjugates 2 against CML derived K562 cells with p53 mutation (Figure S9B). This result was consistent with the previous report that conjugate 1 was not toxic against p53-mutated cancer cell lines.7 Finally, we evaluated the expression level of RUNX-target gene in MV4-11 treated by conjugates 1-4 to support the proposed mechanism. As the previous study of conjugate 1,7 conjugate 2 repressed the expression of BCL11A, one of RUNXtarget genes (Figure 7). Conjugates 3 and 4 also reduced the

ACS Paragon Plus Environment

Page 4 of 7

Page 5 of 7 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of the American Chemical Society

Figure 6. Cell viability and IC50 values of conjugates 1-4 and Chlorambucil against MV4-11 cells (n = 3). Error bars indicate SDs. *p < 0.001, by unpaired two-tailed Student’s t test. Abbreviation: ND, not determined.

relative expression level, which implies that these conjugates suppressed the gene expression regardless of DNA-alkylation. Therefore, conjugates 1-4 were suggested to promote the apoptosis of cancer cells through repressing the downstream genes of RUNX.

n

ASSOCIATED CONTENT

Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Experimental procedures, HPLC profiles and mass spectrums of conjugates 1-4, reaction mechanism of after-treatment for PAGE, HPLC profiles of product analysis, triplicate data of SPR analysis, Cell viabilities of MOLM-13 and K562 and primers for RT-qPCR (PDF).

n

AUTHOR INFORMATION

Corresponding Author *[email protected] *[email protected] Notes The authors declare no competing financial interests.

n

Figure 7. Relative expression of BCL11A in MV4-11 after 24 h treatment. Error bars indicate SEs of data from two culture wells.

n

CONCLUSION

We have investigated the validity of using chlorambucil–PI polyamides targeting the RUNX binding sequence to develop new types of anticancer agents. Conjugate 1, previously reported to have remarkable antitumor effect in vivo, showed favorable results for sequence-selectivity and binding affinity to the target sequence. We also succeeded in obtaining a potential alternative conjugate, conjugate 2, by incorporating b-alanine into the structure. The chemical properties of conjugate 2 were superior to those of conjugate 1, indicating that the substitution of b-alanine for pyrrole effectively improved the conjugate’s selectivity for alkylation sites, its binding affinity for the RUNX-binding sequence and its cytotoxicity against cancer cells. Based on its molecular characteristics, this conjugate can be predicted to have significant anticancer effects in vivo. This study contributes to chemical clarification of the possibility of conjugates 1 and 2 as candidates for cancer therapeutics. n

We thank Prof. Yasuhiko Kamikubo and Ms. Kana Furuichi for providing MOLM-13 and K562 for the cytotoxicity assay. This research was supported by AMED under Grant Number JP18am0301005 (Basic Science and Platform Technology Program for Innovative Biological Medicine), JP18am0101101 (Platform Project for Supporting Drug Discovery and Life Science Research (BINDS)), and KAKENHI Grant Number JP16H06356 by JSPS.

n

REFERENCES

(1)

Ito, Y.; Bae, S. C.; Chuang, L. S. H. The RUNX family: developmental regulators in cancer. Nat. Rev. Cancer 2015, 15, 81.

(2)

Meyers, S.; Downing, J. R.; Hiebert, S. W. Identification of AML-1 and the (8;21) Translocation Protein (AML-1/ETO) as SequenceSpecific DNA-Binding Proteins: the runt Homology Domain Is Required for DNA Binding and Protein-Protein Interactions. Mol. Cell. Biol. 1993, 13, 6336.

(3)

Bartfeld, D.; Shimon, L.; Conture, G. C.; Rabinovich, D.; Frolow, F.; Levanon, D.; Groner, Y.; Shakked, Z. DNA Recognition by the RUNX1 Transcription Factor Is Mediated by an Allosteric Transition in the RUNT Domain and by DNA Bending. Structure 2002, 10, 1395.

(4)

Liu, P.; Tarle, S. A.; Hajra, A.; Claxton, D. F.; Marlton, P.; Freedman, M.; Siciliano, M. J.; Collins, F. S. Fusion Between Transcription Factor CBFβ/PEBP2β and a Myosin Heavy Chain in Acute Myeloid Leukemia. Science 1993, 261, 1041.

(5)

Goyama, S.; Schibler, J.; Cunningham, L.; Zhang, Y.; Rao, Y.; Nishimoto, N.; Nakagawa, M.; Olsson, A.; Wunderlich, M.; Link, K. A.; Mizukawa, B.; Grimes, H. L.; Kurokawa, M.; Liu, P. P.; Huang,

EXPERIMENTAL SECTION

All experimental procedures are provided in the supporting information.

ACKNOWLEDGMENT

ACS Paragon Plus Environment

Journal of the American Chemical Society 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

G.; Mulloy, J. C. Transcription factor RUNX1 promotes survival of acute myeloid leukemia cells. J. Clin. Invest. 2013, 123, 3876. (6)

Blyth, K.; Cameron, E. R.; Neil, J. C. THE RUNX GENES: GAIN OR LOSS OF FUNCTION IN CANCER. Nat. Rev. Cancer 2005, 5, 376.

(7)

Morita, K.; Suzuki, K.; Maeda, S.; Matsuo, A.; Mitsuda, Y.; Tokushige, C.; Kashiwazaki, G.; Taniguchi, J.; Maeda, R.; Noura, M.; Hirata, M.; Kataoka, T.; Yano, A.; Yamada, Y.; Kiyose, H.; Tokumasu, M.; Matsuo, H.; Tanaka, S.; Okuno, Y.; Muto, M.; Naka, K.; Ito, K.; Kitamura, T.; Kaneda, Y.; Liu, P. P.; Bando, T.; Adachi, S.; Sugiyama, H.; Kamikubo, Y. Genetic regulation of the RUNX transcription factor family has antitumor effects. J. Clin. Invest. 2017, 127, 2815.

(8)

Tauger, J. W.; Baird, E. E.; Dervan, P. B. Recognition of DNA by designed ligands at subnanomolar concentrations. Nature 1996, 382, 559.

(9)

White, S.; Szewczyk, J. W.; Turner, J. M.; Baird, E. E.; Dervan, P. B. Recognition of the four Watson–Crick base pairs in the DNA minor groove by synthetic ligands. Nature 1998, 391, 468.

(10) Dervan, P. B.; Burli, R. W. Sequence-specific DNA recognition by polyamides. Curr. Opin. Chem. Biol. 1999, 3, 688. (11) Wurtz, N. R.; Dervan, P. B. Sequence specific alkylation of DNA by hairpin pyrrole–imidazole polyamide conjugates. Chemistry & Biology 2000, 7, 153. (12) Wang, Y. D.; Dziegielewski, J.; Wurtz, N. R.; Dziegielewska, B.; Dervan, P. B.; Beerman, T. A. DNA crosslinking and biological activity of a hairpin polyamide-chlorambucil conjugate. Nucleic Acids Res. 2003, 31, 1208. (13) Minoshima, M.; Bando, T.; Shinohara, K.; Kashiwazaki, G.; Nishijima, S.; Sugiyama, H. Comparative analysis of DNA alkylation by conjugates between pyrrole–imidazole hairpin polyamides and chlorambucil or seco-CBI. Bioorg. Med. Chem. 2010, 18, 1236. (14) Mattes, W. B.; Hartley, J. A.; Kohn, K. W. DNA sequence selectivity of guanine-N7 alkylation by nitrogen mustards. Nucleic Acids Res. 1986, 14, 2971. (15) Wang, P.; Bauer, G. B.; Bennett, R. A. O.; Povirk, L. F. Thermolabile Adenine Adducts and A・T Base Pair Substitutions Induced by Nitrogen Mustard Analogues in an SV40-Based Shuttle Plasmid. Biochemistry 1991, 30, 11515. (16) Yoon, J. H.; Lee, C. S. Sequence Specificity for DNA Interstrand Cross-linking Induced by Anticancer Drug Chiorambucil. Arch. Pham. Res. 1997, 20, 550. (17) Turner, J. M.; Swalley, S. E.; Baird, E. E.; Dervan, P. B. Aliphatic/Aromatic Amino Acid Pairings for Polyamide Recognition in the Minor Groove of DNA. J. Am. Chem. Soc. 1998, 120, 6219. (18) Bando, T.; Minoshima, M.; Kashiwazaki, G.; Shinohara, K.; Sasaki, S.; Fujimoto, J.; Ohtsuki, A.; Murakami, M.; Nakazono, S.; Sugiyama, H. Requirement of β-alanine components in sequencespecific DNA alkylation by pyrrole–imidazole conjugates with seven-base pair recognition. Bioorg. Med. Chem. 2008, 16, 2286.

Page 6 of 7

(19) Han, Y. W.; Kashiwazaki, G.; Morinaga, H.; Matsumoto, T.; Hashiya, K.; Bando, T.; Harada, Y.; Sugiyama, H. Effect of single pyrrole replacement with β-alanine on DNA binding affinity and sequence specificity of hairpin pyrrole/imidazole polyamides targeting 5’GCGC-3’. Bioorg. Med. Chem. 2013, 21, 5436. (20) Han, Y. W.; Matsumoto, T.; Yokota, H.; Kashiwazaki, G.; Morinaga, H.; Hashiya, K.; Bando, T.; Harada, Y.; Sugiyama, H. Binding of hairpin pyrrole and imidazole polyamides to DNA: relationship between torsion angle and association rate constants. Nucleic Acids Res. 2012, 40, 11510. (21) Asamitsu, S.; Kawamoto, Y.; Hashiya, F.; Hashiya, K.; Yamamoto, M.; Kizaki, S.; Bando, T.; Sugiyama, H. Sequence-specific DNA alkylation and transcriptional inhibition by long-chain hairpin pyrrole– imidazole polyamide–chlorambucil conjugates targeting CAG/CTG trinucleotide repeats. Bioorg. Med. Chem. 2014, 22, 4646. (22) Kawamoto, Y.; Sasaki, A.; Hashiya, K.; Ide, S.; Bando, T.; Maeshima, K.; Sugiyama, H. Tandem trimer pyrrole–imidazole polyamide probes targeting 18 base pairs in human telomere sequences. Chem. Sci. 2015, 6, 2307. (23) Belitsky, J. M.; Nguyen, D. H.; Wurtz, N. R.; Dervan, P. B. SolidPhase Synthesis of DNA Binding Polyamides on Oxime Resin. Bioorg. Med. Chem. 2002, 10, 2767. (24) Sugiyama, H.; Fujiwara, T.; Ura, A.; Tashiro, T.; Yamamoto, K.; Kawanishi, S.; Saito, I. Chemistry of Thermal Degradation of Abasic Sites in DNA. Mechanistic Investigation on Thermal DNA Strand Cleavage of Alkylated DNA. Chem. Res. Toxicol. 1994, 7, 673. (25) Maxam, A. M.; Gilbert, W. Sequencing End-Labeled DNA with Base-Specific Chemical Cleavages. Methods Enzymol. 1980, 65, 499. (26) Bando, T.; Narita, A.; Asada, K.; Ayame, H.; Sugiyama, H. Enantioselective DNA Alkylation by a Pyrrole-Imidazole S-CBI Conjugate. J. Am. Chem. Soc. 2004, 126, 8948. (27) Takagaki, T.; Bando, T.; Sugiyama, H. Synthesis of Pyrrole−Imidazole Polyamide seco-1-Chloromethyl-5-hydroxy-1,2-dihydro‑3H‑ benz[e]indole Conjugates with a Vinyl Linker Recognizing a 7 bp DNA Sequence. J. Am. Chem. Soc. 2012, 134, 13074. (28) Asamitsu, S.; Li, Y.; Bando, T.; Sugiyama, H. Ligand-Mediated GQuadruplex Induction in a Double-Stranded DNA Context by Cyclic Imidazole/Lysine Polyamide. ChemBioChem 2016, 17,1317. (29) Kawamoto, Y.; Sasaki, A.; Chandran, A.; Hashiya, K.; Ide, S.; Bando, T.; Maeshima, K.; Sugiyama, H. Targeting 24 bp within Telomere Repeat Sequences with Tandem Tetramer Pyrrole−Imidazole Polyamide Probes. J. Am. Chem. Soc. 2016, 138, 14100. (30) Tahirov, T. H.; Inoue-Bungo, T.; Morii, H.; Fujikawa, A.; Sasaki, M.; Kimura, K.; Shiina, M.; Sato, K.; Kumasaka, T.; Yamamoto, M.; Ishii, S.; Ogata, K. Structural Analyses of DNA Recognition by the AML1/Runx-1 Runt Domain and Its Allosteric Control by CBFβ. Cell 2001, 104, 755. (31) Edelson, B. S.; Best, T. P.; Olenyuk, B.; Nickols, N. G.; Doss, R. M.; Foister, S.; Heckel, A.; Dervan, P. B. Influence of structural variation on nuclear localization of DNA-binding polyamide-fluorophore conjugates. Nucleic Acids Res. 2004, 32, 2802.

ACS Paragon Plus Environment

6

Page 7 of 7 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of the American Chemical Society

ACS Paragon Plus Environment

7