DNA Binding and Cleavage Modes of Shishijimicin A

Article Cite This: J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

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DNA Binding and Cleavage Modes of Shishijimicin A Hao Zhang,† Ruofan Li,‡ Sai Ba,† Zhaoyong Lu,‡,∥ Emmanuel N. Pitsinos,*,‡,§ Tianhu Li,*,† and K. C. Nicolaou*,‡

J. Am. Chem. Soc. Downloaded from pubs.acs.org by UNIV AUTONOMA DE COAHUILA on 05/03/19. For personal use only.

†

Division of Chemistry and Biological Chemistry, School of Physical and Mathematical Sciences, Nanyang Technological University, 21 Nanyang Link, Singapore 637371, Singapore ‡ Department of Chemistry, BioScience Research Collaborative, Rice University, 6100 Main Street, Houston, Texas 77005, United States § Laboratory of Natural Products Synthesis & Bioorganic Chemistry, Institute of Nanoscience and Nanotechnology, National Centre for Scientific Research “Demokritos”, 153 10 Agia Paraskevi, Greece S Supporting Information *

ABSTRACT: Although shishijimicin A and its extreme potencies against an array of cancer cell lines have been known for more than a decade, its assumed DNA-cleaving mechanism has not been substantiated as yet. Herein we report studies that reveal binding and scission of doublestranded DNA by shishijimicin A. The results of these studies support the proposed hypothesis that DNA strand scissions are caused by 1,4-benzenoid diradicals formed by Bergman cycloaromatization of the enediyne core of shishijimicin A upon activation by thiols. In addition, double-stranded supercoiled DNA-cleavage experiments with shishijimicin A in competition with known minor groove binders, UV spectroscopic studies, and electrophoretic analysis were utilized to clarify the binding mode of the molecule to DNA. These investigations indicate that shishijimicin A binds to the minor groove of double-stranded DNA and that its β-carboline moiety plays a role in the binding through intercalation. In addition, due to the fact that naked linker regions of DNA in the interphase and metaphase of eukaryotic cells are unprotected by histone proteins during entire cell cycles and because these unprotected regions of DNA are vulnerable to attack by DNA binders, it was concluded that the observed double-strand DNA cleavage and very low sequence selectivity by shishijimicin A may account for its extraordinary cytotoxicity. Calicheamicin γI1 (4) also includes an extra carbohydrate residue (D) appended through a hexasubstituted benzene ring (C) onto the same trisaccharide chain (ABE) as in esperamicin A1 (see structure 4, Figure 1). These different structural domains determine the mode of binding and the mechanism by which these fascinating molecules cause single- and doublestrand cuts through a common DNA cleaving mechanism that involves 1,4-benzenoid diradicals generated via a Bergman cycloaromatization reaction6 in the presence of thiols. This mechanism was originally experimentally demonstrated with calicheamicin γ1I (4).7 The remarkable sequence-selective binding of calicheamicin γI1 in the minor groove of DNA through its oligosaccharide domain8 that leads to doublestrand breaks in duplex DNA was also demonstrated.9 Esperamicin A1 (3) also cleaves double-stranded DNA through the same mechanism as calicheamicin γI1 (4), although in a less sequence-selective manner.10 Its binding into the minor groove of double-stranded DNA was demonstrated to

1. INTRODUCTION Isolated in 2003 from the marine ascidian Didemnum proliferum,1 shishijimicin A (1, Figure 1) is among the most potent members of the 10-membered ring enediyne family of antitumor antibiotics, that also includes namenamicin2 (2, Figure 1), esperamicin A13 (3, Figure 1), calicheamicin γI14 (4, Figure 1), and others.5 The structures of the two marine natural products, shishijimicin A (1) and namenamicin (2), are identical with regard to their enediyne domains and their two carbohydrate residues (A and E for 1; A and C for 2, Figure 1). They differ, however, in their other structural motifs. Specifically, shishijimicin A possesses a β-carboline system (BCD; see structure 1, Figure 1), while namenamicin includes a methylthio sugar (B) and a hydroxymethyl residue, both attached through a C−C bond onto ring A (see structure 2, Figure 1). On the other hand, both terrestrial natural products, esperamicin A1 (3) and calicheamicin γI1 (4), possess rather more sophisticated molecular structures, with esperamicin A1 including an extra glycosylated hydroxyl group on its enediyne domain (W) and an anthranilate moiety (D) attached onto the extra carbohydrate moiety (C; see structure 3, Figure 1). © XXXX American Chemical Society

Received: February 15, 2019

A

DOI: 10.1021/jacs.9b01800 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

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Journal of the American Chemical Society

one of the most thoroughly investigated enediyne-containing natural products,14 calicheamicin γI1 was examined in parallel for comparison purposes (Figure 2a). Accordingly, shishijimicin A was incubated with negatively supercoiled pBR322 plasmid DNA (0.5 μg) in the presence of 1 mM dithiothreitol (DTT) at pH 7.0 and at 37 °C for 1 h in 10% (v/v) DMSO with the expectation of fragmenting DNA as depicted in Figure 2a. As shown in Figure 2b−d, increasing concentrations of shishijimicin A lead to (a) decrease of form I of pBR322 (intact DNA, supercoiled circular) with concurrent, (b) increase of form II (single-strand scissions, circular DNA) and form III (double-strand scissions, linear DNA) of pBR322. These observations prove, for the first time, that shishijimicin A is capable of causing highly efficient DNA cuts. In addition, at concentrations of 20 nM and higher (Lanes 5−8, Figure 2b), double-strand cuts become significant. At higher concentrations (≥200 nM, Lanes 8 and 9, Figure 2b), pBR322 was cleaved into low molecular weight fragments. Comparable cleavage profiles were observed by calicheamicin γI1 (Figure 2e−g). The concentration required to cleave 50% of the plasmid DNA (EC50) by shishijimicin A and calicheamicin γI1 was also assessed from plots of percentage of cleaved DNA (sum of forms II, III, and fragmented) vs concentration of enediyne. The EC50 = 14.1 ± 1.1 nM value derived for shishijimicin A (Figure 2d) is close to the EC50 = 9.8 ± 1.2 nM value acquired for calicheamicin γI1 (Figure 2g). These observations (Figure 2b−d and Figure 2e−g) indicate that shishijimicin A and calicheamicin γI1 cleave double-stranded DNA with similar efficacy. In the cases of esperamicin A1 and calicheamicin γI1, it was demonstrated that thiols play a role in triggering the Bergman cycloaromatization by reacting with the methyltrisulfide moiety of the molecule.7,9,10 Shishijimicin A is also equipped with a methyltrisulfide moiety. In order to verify the involvement of this moiety in the induction of DNA cleavage by shishijimicin A, the dependence of DNA cleavage on the concentration of the thiol present (i.e., dithiothreitol; DTT) was examined. As shown in Figure 3, shishijimicin A at 100 nM concentration (Lane 3) did not lead to cleavage of pBR322 DNA (0.5 μg) in the absence of DTT, while varying degrees of DNA cleavage occurred in the presence of increasing DTT concentrations (0.1 μM − 1.0 mM, Lanes 4−8, Figure 3). These observations, (a) verify that similar to calicheamicin γI1,7,9 thiol activation is a prerequisite for DNA strand scission caused by shishijimicin A, and (b) provide indirect evidence that Bergman cycloaromatization6 and 1,4-benzenoid diradicals are involved in the shishijimicin A-mediated DNA-cleavage. In addition to the aforementioned activation by DTT, pHdependency of DNA cleavage by shishijimicin A and calicheamicin γI1 were examined in parallel. As shown in Figure 4a,b, variation of pH from 5.5 to 8.0 did not affect the efficiency of DNA-scission by either shishijimicin A (100 nM) or calicheamicin γI1 (100 nM), a finding in agreement with the absence of base-sensitive functional groups in the molecular structures of these two natural products (see structures 1 and 4, respectively, in Figure 1). 2.2. Identity of the Reactive Species Involved in the Cleavage of DNA by Shishijimicin A. At the outset of our studies, and based on the structural similarity between shishijimicin A and calicheamicin γI1 (see structures 1 and 4, respectively, in Figure 1), we hypothesized that, as in the case of calicheamicin γI1, 1,4-benzenoid diradicals must be the

Figure 1. Molecular structures of representative 10-membered ring enediyne natural products of marine [i.e., shishijimicin A (1) and namenamicin (2)] and terrestrial [i.e., esperamicin A1 (3) and calicheamicin γI1 (4)] origins.

involve the anthranilate moiety as an intercalating structural motif.11 Namenamicin (2) was shown to be less efficient and less sequence-selective in its action as a double-stranded DNA cleaving agent than calicheamicin γI1 (4) and with a similar selectivity as that exhibited by esperamicin A1 (3).2 Interestingly, the interaction of shishijimicin A (1) with double-stranded DNA has not been studied thus far. It has, however, been suggested that it has a similar effect on DNA, and that its β-carboline structural motif plays an intercalative role in its binding mode to duplex DNA.1 Neither aspect has been experimentally verified, presumably due to the limited availability of this scarce marine natural product.1 The sufficient quantities of shishijimicin A (1) rendered available through our total synthesis,12 in combination with its potential as a payload for antibody−drug conjugates,13 has prompted the investigations described herein of the interaction of this enediyne antitumor agent with double-stranded DNA with the aim of aiding the rational design and synthesis of second-generation shishijimicin A analogues.12b Specifically, herein we report: (a) the substantiation of the ability of shishijimicin A (1) to induce double-strand scissions of duplex DNA in the presence of thiols, (b) studies of the sequence selectivity of its action on duplex DNA, (c) the nature of the reactive species involved, (d) experimental evidence supporting the interaction of shishijimicin A with the minor groove of double-stranded DNA, (e) experimental evidence for the intercalation of the β-carboline moiety of the molecule, and (f) a possible explanation of the exceptional potency of shishijimicin A (1) against cancer cells based on features of its interaction with double-stranded DNA.

2. RESULTS AND DISCUSSION 2.1. Cleavage of DNA by Shishijimicin A. To test the hypothesis that shishijimicin A, like its siblings esperamicin A1, calicheamicin γI1, and namenamicin, cleaves double-stranded DNA,1 we examined, through agarose gel electrophoresis, its reaction with pBR322 plasmid supercoiled DNA. Since it is B


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Figure 2. (a) Schematic presentation of the experiment to test the DNA-cleaving activity of calicheamicin γI1 or shishijimicin A. (b) Agarose gel electrophoretic analysis of DNA-cleaving activity by shishijimicin A. Lane 1: DNA markers; Lane 2: pBR322 alone (0.5 μg); Lane 3 to Lane 9: pBR322 (0.5 μg) was incubated with DTT (1 mM) and 5 nM (Lane 3), 10 nM (Lane 4), 20 nM (Lane 5), 50 nM (Lane 6), 100 nM (Lane 7), 200 nM (Lane 8), and 500 nM (Lane 9) of shishijimicin A, respectively. Shishijimicin A-mediated DNA-cleaving reactions were carried out in 20 mM sodium phosphate buffer (pH 7.0) with 10% (v/v) DMSO at 37 °C for 1 h. (c) Correlation between concentration of shishijimicin A and percentage of (i) uncleaved DNA (Form I, supercoiled circular), (ii) single-strand scission (Form II DNA, circular), and (iii) double-strand scission (Form III and fragmented DNA, linear). (d) Plot of cleaved DNA percentage against concentration of shishijimicin A. These results were obtained from three independent experiments, which are presented in the form of mean ± standard deviation. (e) Agarose gel electrophoretic analysis of DNA-cleaving activity by calicheamicin γI1. Lane 1: DNA markers; Lane 2: pBR322 alone (0.5 μg); Lane 3 to Lane 9: pBR322 (0.5 μg) was incubated with DTT (1 mM) and 5 nM (Lane 3), 10 nM (Lane 4), 20 nM (Lane 5), 50 nM (Lane 6), 100 nM (Lane 7), 200 nM (Lane 8), and 500 nM (Lane 9) of calicheamicin γI1, respectively. Calicheamicin γI1-mediated DNA-cleaving reactions were carried out in 20 mM sodium phosphate buffer (pH 7.0) with 10% (v/v) DMSO at 37 °C for 1 h. (f) Correlation between concentration of calicheamicin γI1 and percentage of (i) uncleaved DNA (Form I, supercoiled circular), (ii) single-strand scission (Form II DNA, circular), and (iii) double-strand scission (Form III and fragmented DNA, linear). (g) Plot of cleaved DNA percentage against concentration of calicheamicin γI1. These results were obtained from three independent experiments, which are presented in the form of mean ± standard deviation.

efficient nucleophiles, these observations suggest that, as it is the case with calicheamicin γ1I, alkylating species (i.e., electrophiles) are not involved in shishijimicin A-mediated DNA cleavage. To rule out the involvement of reactive oxygen species (ROS) in the shishijimicin A-mediated DNA-cleaving process, the effects of (a) catalase, as hydrogen peroxide (H2O2) scavenger,15 and (b) methanol, ethanol, and mannitol, as scavengers of hydroxyl radical (·OH),15 were examined. As seen in Figure 5a, the efficiency of DNA cleavage by shishijimicin A was not significantly affected in the presence

predominant culprits for the shishijimicin A-mediated DNA cleavage. The first evidence in support of this hypothesis was the observed dependency of shishijimicin A-mediated DNA cleavage on DTT (see above, Section 2.1). To rule out the involvement of alkylating species, pyridine and 4-methylaniline were added to the reaction mixtures of, (a) shishijimicin A (100 nM) and pBR322 (0.5 μg), and (b) calicheamicin γI1 (100 nM) and pBR322 (0.5 μg). No significant changes in the efficiency of DNA-cleavage by either shishijimicin A (Lanes 5 and 6 in Figure 5a) or calicheamicin γI1 (Lanes 5 and 6, Figure 5b) were noticeable. Since pyridine and 4-methylaniline are C


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Figure 3. Electrophoretic analysis of the dependence of shishijimicin A-mediated DNA cleavage on DTT concentration. Lane 1: DNA markers; Lane 2: pBR322 alone (0.5 μg); Lane 3 to Lane 8: mixtures of pBR322 (0.5 μg) and shishijimicin A (100 nM) in the absence (Lane 3) or presence of 0.1 μM (Lane 4), 1 μM (Lane 5), 10 μM (Lane 6), 100 μM (Lane 7), or 1 mM (Lane 8) of DTT. All reactions were carried out in 20 mM sodium phosphate buffer (pH 7.0) with 10% (v/v) DMSO at 37 °C for 1 h.

Figure 5. Electrophoretic analysis of the effect of nucleophiles and ROS scavengers on, (a) shishijimicin A-mediated DNA cleavage, and (b) calicheamicin γI1-mediated DNA cleavage. Lanes 1: DNA markers; Lanes 2: pBR322 alone (0.5 μg); Lanes 3 to Lanes 10: incubation of mixtures of pBR322 (0.5 μg), DTT (1 mM), and either (a) shishijimicin A (100 nM) or (b) calicheamicin γI1 (100 nM) in the (i) absence of nucleophiles or scavengers of reactive oxygen species (Lanes 3), and (ii) presence of catalase (53 μg/mL, Lanes 4), pyridine (10 mM, Lanes 5), 4-methylaniline (10 mM, Lanes 6), methanol (1 M, Lanes 7), ethanol (1 M, Lanes 8), DTT (80 mM, Lanes 9), or mannitol (100 mM, Lanes 10), respectively. All reactions were carried out in 20 mM sodium phosphate buffer (pH 7.0) with 10% (v/v) DMSO at 37 °C for 1 h.

Figure 4. Electrophoretic analysis of dependence of, (a) shishijimicin A-mediated, and (b) calicheamicin γI1-mediated DNA cleavage on variations of pH. Lanes 1: DNA markers; Lanes 2, Lanes 4, Lanes 6, Lanes 8, Lanes 10 and Lanes 12: incubations of mixtures of pBR322 (0.5 μg) and DTT (1 mM) at pH 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, respectively; Lanes 3, Lanes 5, Lanes 7, Lanes 9, Lanes 11 and Lanes 13: incubations of mixtures of pBR322 (0.5 μg), DTT (1 mM), and either (a) shishijimicin A (100 nM) or (b) calicheamicin γI1 (100 nM), and 20 mM sodium phosphate buffer with 10% (v/v) DMSO at pH 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0, respectively. All reactions were carried out at 37 °C for 1 h.

generated from the enediyne moiety through a Bergman cycloaromatization reaction.6 2.3. Sequence Selectivity of DNA-cleavage by Shishijimicin A. It has been speculated that, due to their structural differences, calicheamicin γI1 and shishijimicin A might exhibit different DNA-sequence preferences for binding and cleaving.1 Initially, to investigate whether shishijimicin A and calicheamicin γI1 exhibit similar sequence selectivity in their DNA-cleavage, two fluorophore-labeled duplex oligonucleotides containing a known calicheamicin γI1-binding site (i.e., TCCT/AGGA)9a−c and flanking oligopyrimidine-oligopurine tracts (Duplex 1 in Figure 6a and Duplex 2 in Figure 6b) were designed as probes to investigate these phenomena. When Duplex 1 (0.5 μg) was incubated with calicheamicin γI1 in the presence of DTT (1 mM), electrophoretic analysis (Lanes 3−5, Figure 6c) indicated that, (a) the amount of Duplex 1 decreased with increasing calicheamicin γ 1I concentration (Position 1, Figure 6c), and (b) simultaneous generation of a major band of shorter fragments occurred (Position 2, Figure 6c). The band of fragments at Position 2 corresponds to DNA cleavage products at the deoxycytidine site within the tracts of TCCT/AGGA, as expected.9a−c Parallel to the studies involving calicheamicin γI1, shishijimicin A was incubated with Duplex 1 (0.5 μg) in the presence of DTT (1 mM) as shown in Lanes 6−8 in Figure 6c. Similarly to the effect observed with calicheamicin γI1 (Lanes 3−5, Figure 6c), the fraction of Duplex 1 decreased with increasing concentration of shishijimicin A. However, in contrast to the observations when calicheamicin γI1 was used, only faint bands occurred in the region below Position 1 (Lanes 6−8, Figure

of catalase (Lane 4), methanol (Lane 7), ethanol (Lane 8), or mannitol (Lane 10). Similar results were obtained in the case of calicheamicin γI1 (Lanes 4, 7, 8, and 10 in Figure 5b). As already established for calicheamicin γI1,9a these observations rule out the possibility of involvement of hydrogen peroxide and hydroxyl radicals in the shishijimicin A-mediated DNAcleaving processes. However, in the presence of a commonly used scavenger of superoxide radical (O2•−), dithiothreitol (DTT),16 the efficiency of DNA-cleavage by either shishijimicin A or calicheamicin γI1 was reduced, with single-strand cuts becoming dominant (Lane 9 in Figures 5a,b). This occurs, most likely, because DTT at the concentration employed (80 mM), apart from acting as superoxide radical scavenger, can also interfere with the fate of DNA radicals either by quenching them or leading to the formation of abasic sites, rather than strand cuts.16 Considered together the above-described observations confirm beyond doubt that shishijimicin A performs its DNA cleavage through the same mechanism of action as calicheamicin γ1I ,9a namely via 1,4-benzenoid diradicals D


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Figure 6. Examination of sequence selectivity of DNA cleavage by calicheamicin γI1 and shishijimicin A. (a) Illustration of DNA sequence and calicheamicin γI1-binding site (blue box) and cleavage sites (arrows) in Duplex 1. (b) Illustration of DNA sequence and calicheamicin γI1-binding site (blue box) and cleavage sites (arrows) in Duplex 2. (c) Denaturing polyacrylamide gel electrophoretic analysis of Duplex 1 cleavage by calicheamicin γI1 and shishijimicin A. Lane 1: DNA markers; Lane 2: Duplex 1 alone (0.5 μg); Lane 3 to Lane 5: incubation of Duplex 1 (0.5 μg) and DTT (1 mM) with either 1 μM (Lane 3), 5 μM (Lane 4), or 25 μM (Lane 5) of calicheamicin γI1; Lane 6 to Lane 8: incubation of Duplex 1 (0.5 μg) and DTT (1 mM) with either 1 μM (Lane 6), 5 μM (Lane 7), or 25 μM (Lane 8) of shishijimicin A. (d) Denaturing polyacrylamide gel electrophoretic analysis of Duplex 2 cleavage by calicheamicin γI1 and shishijimicin A. Lane 1: DNA markers; Lane 2: Duplex 2 alone (0.5 μg); Lane 3 to Lane 5: incubation of Duplex 2 (0.5 μg) and DTT (1 mM) with either 1 μM (Lane 3), 5 μM (Lane 4), or 25 μM (Lane 5) of calicheamicin γI1; Lane 6 to Lane 8: incubation of Duplex 2 (0.5 μg) and DTT (1 mM) with either 1 μM (Lane 6), 5 μM (Lane 7), or 25 μM (Lane 8) of shishijimicin A. (e) Illustration of DNA sequence and calicheamicin γI1-binding (blue boxes) and cleavage sites (arrows) in Duplex 3. Gray boxes indicate estimated shishijimicin A cleavage sites. (f) Native polyacrylamide gel electrophoretic analysis of Duplex 3 cleavage by calicheamicin γI1 and shishijimicin A. Lane 1: DNA markers; Lane 2: Duplex 3 alone (0.5 μg); Lane 3 to Lane 5: incubation of Duplex 3 (0.5 μg) and DTT (1 mM) with either 1 μM (Lane 3), 5 μM (Lane 4), or 25 μM (Lane 5) of calicheamicin γI1; Lane 6 to Lane 8: incubation of Duplex 3 (0.5 μg) and DTT (1 mM) with either 1 μM (Lane 6), 5 μM (Lane 7), or 25 μM (Lane 8) of shishijimicin A. All DNA-cleaving reactions were carried out in 20 mM sodium phosphate buffer (pH 7.0) with 20% (v/v) DMSO at 37 °C for 1.5 h.

6c). Similar to the outcome of our studies with Duplex 1, a decrease in the amount of intact Duplex 2 (Position 1, Figure 6d) and very faint bands corresponding to lower molecular weight fragments were observed (Lanes 6−8, Figure 6d) upon incubation with shishijimicin A. The latter observations with shishijimicin A are indicative of a nonspecific DNA cleavage mode. More specifically, it is known that when nonspecific

DNA cleavage takes place, the probability of DNA strand scission at each individual nucleotide is nearly equal, as revealed by the absence of major bands at any particular position in the electrophoretic analysis.17 As a result of nearly random DNA cleavage by shishijimicin A, fragmentation products of Duplex 1 and Duplex 2 spread out widely in the E


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Figure 7. Examination of sequence selectivity of DNA cleavage by calicheamicin γI1 and shishijimicin A with (a) Duplex 4, (b) Duplex 5, (c) Duplex 6, and (d) Duplex 7. The DNA sequence of each Duplex is illustrated to the right of the corresponding denaturing polyacrylamide gel electrophoretic analysis. Boxes and arrows are indicating known calicheamicin γI1 binding and cleavage sites, respectively. Lanes 1a−d: DNA markers (15-bp, 30-bp, 45-bp); Lanes 2a−d: DNA alone (1 μg); Lanes 3a−d: incubation of each Duplex DNA (1 μg) with calicheamicin γI1 (50 μM) in the presence of DTT (1 mM); Lanes 4a−d: incubation of each Duplex DNA (1 μg) with shishijimicin A (50 μM) in the presence of DTT (1 mM); Lanes 5a−d: products of incubation of each Duplex with shishijimicin A (50 μM) in the presence of DTT (1 mM) followed by treatment with piperidine (0.1 M) at 90 °C for 0.5 h. All DNA-cleaving reactions were carried out in 20 mM sodium phosphate buffer (pH 7.0) with 20% (v/ v) DMSO at 37 °C for 1.5 h.

these duplexes, resulting instead in a ladder of cleavage fragments with no significant difference in their intensity upon denaturing polyacrylamide gel electrophoretic analysis (Lane 4, Figure 7a−d). In that respect, shishijimicin A resembles the nonselective behavior of calicheamicin T (a truncated calicheamicin γI1 derivative that lacks the B−C−D rings).9c Furthermore, to gain some insight into the chemical nature of the DNA fragments produced by the action of shishijimicin A on Duplexes 4−7, the initially produced fragments were treated with hot piperidine solution prior to electrophoretic analysis. If they contained abasic sites18 or base-sensitive termini (e.g., oligonucleotide 5′-aldehyde),19 then further fragmentation18 or conversion to the corresponding 3′phosphate-terminated oligonucleotides (which migrate slightly faster during denaturing polyacrylamide gel electrophoretic analysis)19 would be expected and it would result in an altered fragmentation profile (i.e., position or intensity of bands). However, the profile of the fragments remained essentially unaltered after piperidine treatment (compare Lanes 4 and 5, Figure 7a−d).

areas below the band at Position 1, resulting in indistinct individual bands (Lanes 6−8, Figure 6c,d respectively). With the aim to further clarify the issue of sequence selectivity by shishijimicin A, five additional fluorophorelabeled duplex oligonucleotides were designed and employed as probes. When Duplex 3 (Figure 6e), which contains 50 base pairs in its total length and three tracts known to be recognized by calicheamicin γI1,9a was incubated with shishijimicin A, no well-defined bands could be observed (Lanes 6−8 in Figure 6f). Instead, trailing and smearing bands occurred in this case as well (Lanes 6−8, Figure 6f) indicative of a much weaker sequence selectivity than that of calicheamicin γ1I . By interpolating the size of the cleavage fragments and overlaying them onto the sequence, a rough estimate of the possible cleavage sites was attempted (gray boxes in Figure 6e). Duplexes 4−7 (Figure 7a−d, respectively), were designed and employed as probes since their sequences, taken together, match the 244 base pair sequence of the restriction fragment that has previously been employed for the study of calicheamicin γI1 cleavage behavior.9c Once again, shishijimicin A did not exhibit any significant selectivity in its cleavage of F


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Figure 8. Examination of shishijimicin A and calicheamicin γI1-mediated alteration of pBR322 superhelical property. (a) Pictorial illustration of linking number changes that might be caused by intercalation of shishijimicin A. (b) Electrophoretic analysis of pBR322 linking number changes caused by shishijimicin A. Samples in these studies were prepared as shown in Figure 8a and described in Section 2.4. Lane 1: DNA markers; Lane 2 to Lane 8: covalently closed circular pBR322 was prepared in the absence (Lane 2) or presence of either 10 nM (Lane 3), 30 nM (Lane 4), 100 nM (Lane 5), 300 nM (Lane 6), 1 μM (Lane 7), or 3 μM (Lane 8) of shishijimicin A. (c) Diagrammatic illustration of linking number difference analysis between plasmids prepared in the absence of shishijimicin A (Lane 2) and in the presence of shishijimicin A (1 μM, Lane 7). (d) Electrophoretic analysis of pBR322 linking number changes caused by calicheamicin γI1. Samples in these studies were prepared as shown in Figure 8a and described in Section 2.4 replacing shishijimicin A by calicheamicin γI1. Lane 1: DNA markers; Lane 2 to Lane 8: covalently closed circular pBR322 was prepared in the absence (Lane 2) or presence either 10 nM (Lane 3), 30 nM (Lane 4), 100 nM (Lane 5), 300 nM (Lane 6), 1 μM (Lane 7), or 3 μM (Lane 8) of calicheamicin γI1. (e) Diagrammatic illustration of linking number differences analysis between plasmids prepared in the absence of calicheamicin γI1 (Lane 2) and in the presence of calicheamicin γI1 (1 μM, Lane 7).

expected to result in changes in its topology.20 In order to shine light on the role that the β-carboline moiety plays in shishijimicin’s action on double-stranded DNA, we investigated the linking number differences between pBR322 plasmids incubated with shishijimicin A (Product 2 of Route 2, Figure 8a) and those not exposed to shishijimicin A (Product 1 of Route 1, Figure 8a). In the first step of the experiment, pBR322 was modified using nicking endonuclease (Nt.BspQI) to relax the DNA supercoils that had existed originally in the plasmids (Step 1, Figure 8a). The resultant

In conclusion, these experiments revealed no significantly preferred DNA sequences by shishijimicin A in its cleavage of DNA. Rather, the electrophoretic fragmentation patterns observed are consistent with scissions occurring almost randomly with essentially no sequence selectivity. 2.4. Binding Mode of Shishijimicin A to DoubleStranded DNA. The possibility that the β-carboline segment of shishijimicin A confers an intercalative mode to the molecule in its interaction with double-stranded DNA has been pointed out.1 Such an intercalation of circular DNA is G


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Journal of the American Chemical Society nick-possessing plasmids were further incubated, (a) with different concentrations of shishijimicin A (Step 2 in Route 2, Figure 8a), or (b) in the absence of shishijimicin A (Step 2 in Route 1, Figure 8a). The so generated products were subjected to the action of DNA ligase in order to seal the nick sites within the circular backbones of pBR322 (Step 3 in Routes 1 and 2, Figure 8a). If the β-carboline moiety of shishijimicin A was capable of intercalating into DNA macromolecules as free carbolines do,21 then changes of linking number caused by shishijimicin A would be preserved at this stage (Step 3 in Route 2, Figure 8a). Both free and intercalated by shishijimicin A macromolecules were purified using QIAquick PCR purification kits,22 a process that allowed the removal of shishijimicin A molecules from the plasmids (Step 4 in Routes 1 and 2, Figure 8a). The linking number difference between purified Products 1 and 2 were finally determined using electrophoretic analysis in the presence of chloroquine.22,23 As shown in Figure 8b, a distribution of DNA bands emerged during the electrophoretic analysis (Area 1 in Lanes 2 to 8, Figure 8b), as it is commonly observed in determinations of linking numbers of circular plasmid DNA.23,24 In addition, we observed overall slower-migrating DNA bands in Area 1 (Figure 8b) with increasing concentrations of shishijimicin A [0 nM (Lane 2) to 3 μM (Lane 8), Figure 8b). Densitometric measurements were subsequently conducted on the bands in Lanes 2 and 7, respectively, from which graphs of distances of band migrations against intensities of the bands were plotted (Figure 8c). After Gaussian Fit24 was conducted on the obtained plot (Figure 8c), the linking number difference between plasmids in Lane 2 and those in Lane 7 in Area 1 was calculated to be −0.3. This obtained negative value (ΔLk = −0.3) indicates that shishijimicin A is indeed capable of intercalating into pBR322 in Step 2 (Route 2, Figure 8a). For comparison reasons, the above experiments were also carried out with calicheamicin γI1 (negative control) and chloroquine (positive control). As shown in Figure 8d−e, no linking number difference was detected between pBR322 alone and plasmid DNA that was treated with calicheamicin γI1. These results indicate that, although calicheamicin γ1I is reported to introduce additional writhes into supercoiled DNA,25 it does not cause significant linking number change upon its binding to nicked-circular DNA. Chloroquine, a known DNA intercalator,26 however, resulted in linking number difference (ΔLk = −1.0) between plasmids processed in its absence (Lane 2, Figure S1b, Supporting Information, SI) and in its presence (Lane 7, Figure S1b). These observations (Figure 8c and 8e; Figure S1c, SI) confirm the validity of our experimental approach and shishijimicin A’s intercalation ability into DNA. Additional evidence for the intercalation of shishijimicin A into double-stranded DNA was obtained from UV studies. It is well-known that once DNA intercalating molecules insert themselves into duplex DNA their movement is restricted. As a consequence, UV absorbance of DNA-intercalators decreases, to a certain extent, owing to quenching effects caused by duplex DNA.11b,20 Since shishijimicin A displays a maximum UV absorbance at 306 nm (ε 43000),1 UV spectra of shishijimicin A in the absence and presence of duplex forms of Calf Thymus DNA were recorded (Figures S2 and S3 in the SI) and analyzed (Figure 9). The presence of Calf Thymus DNA (0.65 mg/mL) caused ca. 20% decrease of UV absorbance by shishijimicin A at 306 nm (Figure 9), which is in accord with DNA intercalation by this enediyne antitumor

Figure 9. UV spectroscopic examination of DNA intercalation by shishijimicin A. Green Line: UV Spectrum of 0.1 mM shishijimicin A alone; Purple Line: Plot of the sum: (UV Spectrum of a mixture of 0.1 mM shishijimicin A and 0.65 mg/mL Calf Thymus DNA) minus (UV spectrum of 0.65 mg/mL Calf Thymus DNA). For UV Spectrum of 0.65 mg/mL Calf Thymus DNA and detailed description of our UV Spectroscopic studies see SI.

antibiotic. In that respect, shishijimicin A resembles esperamicin A1.11 However, as expected by its nonintercalative mode of DNA-binding,14 the presence of Calf Thymus DNA had no detectable influence on the UV absorbance by calicheamicin γI1 (Figures S2, S4, and S5 in the SI). Thus, both the results of UV absorption quenching studies (Figure 9) and the observed shishijimicin A-associated linking number change (Figure 8c) support the proposition that shishijimicin A is capable of intercalating into double-stranded DNA.1 Since (a) our studies confirm that calicheamicin γI1 does not intercalate into DNA,14 and (b) the β-carboline moiety constitutes a notable structural difference between these closely related enediynes, DNA intercalation by shishijimicin A may be attributed to its β-carboline structural motif. In order to further clarify the interaction of shishijimicin A into double-stranded DNA, we conducted cleavage of pBR322 by shishijimicin A in the presence of netropsin or distamycin A (5, 6, Figure 10a). Both these pyrrole-containing amidines are known to bind to the minor groove of DNA.27 For comparison purposes, the same experiments were carried out with calicheamicin γI1. Netropsin (5) has been reported to cause significant changes on the calicheamicin γI1 and esperamicin A1 DNA cleavage sites.9a,10b In the present study, when netropsin (Figure 10b) and distamycin A (Figure 10c) were separately preincubated with pBR322 (0.5 μg) followed by addition of calicheamicin γI1 (100 nM) no significant change in the extent of DNA cleavage was observed. These results could be attributed to two possible factors. First, the binding affinity of calicheamicin γI1 toward certain homopurine/homopyrimidine tracts is remarkably high,14 an attribute that may allow it to compete with netropsin or distamycin A for DNA minor groove binding. Second, netropsin and distamycin A preferably bind to AT-rich DNA sequences,27 a selectivity that might leave homopurine/homopyrimidine segments accessible to calicheamicin γI1.14 When pBR322 (0.5 μg) was incubated with shishijimicin A (100 nM) in the presence of netropsin or distamycin A, however, the extent of DNA cleavage decreased noticeably (Figure 10d,e, respectively). The above results indicate that, (a) interaction of shishijimicin A with DNA occurs through the minor groove, and (b) the binding affinity H


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A against a panel of cancer cell lines.1,12b Our hypothesis is based on the structural features and DNA-interaction properties of these three enediynes as determined in this and previous studies,4b,7−11,14 and the exposure of genomic DNA to their action.25,28 It is known that both interphase and metaphase DNA in eukaryotic cells is bound, for the most part, by histone proteins in the core regions of nucleosomes. Only a much smaller portion of DNA, found in the linker regions of nucleosomes, is left unprotected by histone proteins.29 Previous related studies with enediyne antibiotics revealed that the nonintercalating minor groove binder calicheamicin γI1 can damage both core and linker DNA, while the intercalating minor groove binder esperamicin A1 can only access the linker regions between histone-bound DNA.28a,b,d,e As a result of DNA packaging in nucleosomes, the binding sites (e.g., TCCT/AGGA) that calicheamicin γI1 prefers, may not be readily accessible, although present in genomic DNA. Accordingly, incorporation of DNA in nucleosomes, in general, reduces its damage by calicheamicin γI1.28b Furthermore, it is interesting to note that, (a) esperamicin A1 is highly potent against tumor cell lines28c despite the fact that its action is limited to linker nucleosomal regions,28a,b,d,e (b) it exhibits much less sequence selectivity than calicheamicin γI1,10a−c and (c) it afflicts predominantly single-strand scissions.11b Herein experimental evidence has been provided for the first time that shishijimicin A, like calicheamicin γI1, interacts with DNA through its minor groove and that this interaction leads to DNA cleavage predominantly through double-strand scissions, albeit with nearly no sequence selectivity. Furthermore, similar to the anthranilate moiety of esperamicin A1, and as indicated by linking number changes and DNA-induced quenching of shishijimicin A’s UV absorbance, the intercalation of the β-carboline domain into DNA contributes to the binding affinity of shishijimicin A to DNA. Thus, by analogy to esperamicin A1, DNA cleaving activity by shishijimicin A is expected to be focused in the more vulnerable linker regions of nucleosomes28a,b,d,e that are accessible through longer periods of the cell cycle than the histone bound core regions.29 We, therefore, propose that the combination by shishijimicin A of advantageous structural features, modes of binding, and DNA cleavage of calicheamicin γI1 (i.e., minor groove binding, double-strand cuts) and esperamicin A1 (i.e., intercalation, minor groove binding, less sequence-selective cuts) may account for its exceptionally high cytotoxicity. The results and conclusions derived from these investigations add to our understanding of structure−activity relationships of the enediyne antibiotics and should prove useful in future studies directed toward drug discovery and development efforts, particularly in the area of antibody−drug conjugates as targeted cancer therapies.13d

Figure 10. (a) Molecular structures of netropsin (5) and distamycin A (6). (b,c) Electrophoretic analysis on likely interference of calicheamicin γI1’s binding to minor grooves of DNA by netropsin (b) or distamycin A (c). Lane 1: DNA markers; Lane 2: pBR322 alone (0.5 μg); Lane 3 to Lane 6: incubation of pBR322 (0.5 μg) and either 0 μM (Lane 3), 0.1 μM (Lane 4), 1 μM (Lane 5), or 10 μM (Lane 6) of netropsin (b) or distamycin A (c) followed by addition of 100 nM calicheamicin γI1. (d,e) Electrophoretic analysis on blockages of shishijimicin A’s DNA intercalation by netropsin (d) or distamycin A (e). Lane 1: DNA markers; Lane 2: pBR322 alone (0.5 μg); Lane 3 to Lane 6: incubation of pBR322 (0.5 μg) and either 0 μM (Lane 3), 0.1 μM (Lane 4), 1 μM (Lane 5), or 10 μM (Lane 6) of netropsin (d) or distamycin A (e) followed by addition of 100 nM shishijimicin A. All of the studies shown in this figure were carried out in the presence of DTT (1 mM) in 20 mM sodium phosphate buffer (pH 7.0) with 10% (v/v) DMSO at 37 °C for 1 h.

of shishijimicin A is lower than that of calicheamicin γI1. In addition, our results reveal that netropsin displayed slightly higher inhibitory effects (Figure 10d) than distamycin A (Figure 10e). The higher inhibitory effect of netropsin vs distamycin A could be attributed to the higher binding affinity of the former toward DNA than that of the latter.27b

3. CONCLUSIONS The described investigations illuminate both the binding and the cleaving modes of shishijimicin A, and allow comparisons with the corresponding modes of calicheamicin γ1I and esperamicin A1. Thus, the suspected double-strand DNA cuts by shishijimicin A in the presence of DTT was confirmed and the intercalation of its β-carboline moiety was demonstrated, the latter feature being in line with the former. It was also shown that shishijimicin A binds to the minor groove of duplex DNA, resembling in this respect, albeit with less affinity, calicheamicin γI1. The similarities of shishijimicin A with calicheamicin γI1 also include their abilities to induce doublestrand cuts, a property not shared with esperamicin A1, whose ability to cause double-strand cuts is significantly weaker than the other two enediynes.10b,11b With regard to sequence selectivity, shishijimicin A is apparently less capable in comparison to esperamicin A1 and calicheamicin γI1, the latter being clearly the most selective of the three, targeting mostly TCCT and TTTT sites of duplex DNA.9a−c In addition to these findings, our studies suggest an explanation for the exceptionally high potency of shishijimicin

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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/jacs.9b01800.

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Experimental details, additional figures (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. I


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the aminosugar and helix binding in the thiol-induced activation of calicheamicin for DNA cleavage. J. Am. Chem. Soc. 1996, 118, 1938− 1948. (8) (a) Walker, S.; Murnick, J.; Kahne, D. Structural characterization of a calicheamicin−DNA complex by NMR. J. Am. Chem. Soc. 1993, 115, 7954−7961. (b) Paloma, L. G.; Smith, J. A.; Chazin, W. J.; Nicolaou, K. C. Interaction of calicheamicin with duplex DNA: Role of the oligosaccharide domain and identification of multiple binding modes. J. Am. Chem. Soc. 1994, 116, 3697−3708. (c) Ikemoto, N.; Kumar, R. A.; Ling, T. T.; Ellestad, G. A.; Danishefsky, S. J.; Patel, D. J. Calicheamicin−DNA complexes: Warhead alignment and saccharide recognition of the minor groove. Proc. Natl. Acad. Sci. U. S. A. 1995, 92, 10506−10510. (d) Kumar, R. A.; Ikemoto, N.; Patel, D. J. Solution structure of the calicheamicin γ1I−DNA complex. J. Mol. Biol. 1997, 265, 187−201. (e) Kalben, A.; Pal, S.; Andreotti, A. H.; Walker, S.; Gange, D.; Biswas, K.; Kahne, D. Calicheamicin−DNA recognition: An analysis of seven different drug−DNA complexes. J. Am. Chem. Soc. 2000, 122, 8403−8412. (9) (a) Zein, N.; Sinha, A. M.; McGahren, W. J.; Ellestad, G. A. Calicheamicin γ1I: An antitumor antibiotic that cleaves doublestranded DNA site specifically. Science 1988, 240, 1198−1201. (b) Zein, N.; Poncin, M.; Nilakantan, R.; Ellestad, G. A. Calicheamicin γ 1I and DNA: Molecular recognition process responsible for site-specificity. Science 1989, 244, 697−699. (c) Walker, S.; Landovitz, R.; Ding, W.; Ellestad, G. A.; Kahne, D. Cleavage behavior of calicheamicin γ1 and calicheamicin T. Proc. Natl. Acad. Sci. U. S. A. 1992, 89, 4608−4612. (d) Hangeland, J. J.; De Voss, J. J.; Heath, J. A.; Townsend, C. A.; Ding, W. D.; Ashcroft, J. S.; Ellestad, G. A. Specific abstraction of the 5′S- and 4′-deoxyribosyl hydrogen atoms from DNA by calicheamicin γ1I. J. Am. Chem. Soc. 1992, 114, 9200−9202. (e) Mah, S. C.; Townsend, C. A.; Tullius, T. D. Hydroxyl radical footprinting of calicheamicin. Relationship of DNA binding to cleavage. Biochemistry 1994, 33, 614−621. (f) Elmroth, K.; Nygren, J.; Mårtensson, S.; Ismail, I. H.; Hammarsten, O. Cleavage of cellular DNA by calicheamicin γ1. DNA Repair 2003, 2, 363−374. (10) (a) Long, B. H.; Golik, J.; Forenza, S.; Ward, B.; Rehfuss, R.; Dabrowiak, J. C.; Catino, J. J.; Musial, S. T.; Brookshire, K. W.; Doyle, T. W. Esperamicins, a class of potent antitumor antibiotics: Mechanism of action. Proc. Natl. Acad. Sci. U. S. A. 1989, 86, 2−6. (b) Sugiura, Y.; Uesawa, Y.; Takahashi, Y.; Kuwahara, J.; Golik, J.; Doyle, T. W. Nucleotide-specific cleavage and minor-groove interaction of DNA with esperamicin antitumor antibiotics. Proc. Natl. Acad. Sci. U. S. A. 1989, 86, 7672−7676. (c) Guo, Q.; Krishnan, B.; Golik, J.; Rosenberg, I. E.; Doyle, T. W.; Kallenbach, N. R.; Lu, M. Determination of DNA cleavage specificity by esperamicins. J. Biomol. Struct. Dyn. 1991, 9, 285−298. (d) Christner, D. F.; Frank, B. L.; Kozarich, J. W.; Stubbe, J.; Golik, J.; Doyle, T. W.; Rosenberg, I. E.; Krishnan, B. Unmasking the chemistry of DNA cleavage by the esperamicins: modulation of 4′-hydrogen abstraction and bistranded damage by the fucose-anthranilate moiety. J. Am. Chem. Soc. 1992, 114, 8763−8767. (11) (a) Ikemoto, N.; Kumar, R. A.; Dedon, P. C.; Danishefsky, S. J.; Patel, D. J. Esperamicin A1 Intercalates into Duplex DNA from the Minor Groove. J. Am. Chem. Soc. 1994, 116, 9387−9388. (b) Yu, L.; Golik, J.; Harrison, R.; Dedon, P. The deoxyfucose-anthranilate of esperamicin A1 confers intercalative DNA binding and causes a switch in the chemistry of bistranded DNA lesions. J. Am. Chem. Soc. 1994, 116, 9733−9738. (c) Kumar, R. A.; Ikemoto, N.; Patel, D. J. Solution structure of the esperamicin A1−DNA complex. J. Mol. Biol. 1997, 265, 173−186. (12) (a) Nicolaou, K. C.; Lu, Z.; Li, R.; Woods, J. R.; Sohn, T. I. Total synthesis of shishijimicin A. J. Am. Chem. Soc. 2015, 137, 8716− 8719. (b) Nicolaou, K. C.; Li, R.; Lu, Z.; Pitsinos, E. N.; Alemany, L. B.; Aujay, M.; Lee, C.; Sandoval, J.; Gavrilyuk, J. Streamlined total synthesis of shishijimicin A and its application to the design, synthesis, and biological evaluation of analogues thereof and practical syntheses of PhthNSSMe and related sulfenylating reagents. J. Am. Chem. Soc. 2018, 140, 12120−12136.

Hao Zhang: 0000-0002-7961-3322 Zhaoyong Lu: 0000-0002-9218-6337 Tianhu Li: 0000-0003-4749-4426 K. C. Nicolaou: 0000-0001-5332-2511 Present Address ∥

State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, 38 Tongyan Road, Tianjin 300350, China. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS T.L., H.Z, and S.B. acknowledge support by the Agency for Science, Technology and Research (A*STAR), Singapore (SERC A1883c0007). K.C.N., E.N.P., R.L., and Z.L. acknowledge support by the Cancer Prevention Research Institute of Texas (CPRIT), The Welch Foundation (grant C-1819), AbbVie Stemcentrx, and Rice University.

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DNA Binding and Cleavage Modes of Shishijimicin A

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