Synthesis and evaluation of N-aryl and N-alkenyl CBI derivatives

The preparation of a novel series of N-aryl CBI derivatives in which an aryl substituent could be used to predictably modulate the reactivity of the resulting CC-1065/duocarmycin alkylation subunit analogue is detailed and its extension to a unique series of N-alkenyl derivatives is reported. The N-aryl derivatives were found to be exceptionally stable and to exhibit well-defined relationships between structure (X-ray), reactivity, and cytotoxic potency. When combined with the results of past investigations, the studies define a fundamental parabolic relationship between reactivity and cytotoxic potency. The parabolic relationship establishes that compounds in the series should possess sufficient stability to reach their biological target (DNA), yet maintain sufficient reactivity to effectively alkylate DNA upon reaching the biological target. Just as importantly, it defined this optimal balance of stability and reactivity that may be used for future design of related analogues. Notably, the duocarmycin SA and yatakemycin alkylation subunit lies at this optimal stability/reactivity position, whereas the CC-1065 and duocarmycin A alkylation subunits lie progressively and significantly to the left of this optimal position (too reactive).     

The DNA phosphate backbone is not involved in catalysis of the duocarmycin and CC-1065 DNA alkylation reaction

The rates of DNA alkylation were established for the reaction of (+)-duocarmycin SA (1) with the native duplex d(G(1)TCAATTAGTC(11))*d(G(12)ACTAATTGAC(22)), an 11 bp deoxyoligonucleotide that contains a single high-affinity alkylation site that has been structurally characterized at exquisite resolution, and modified duplexes in which the four backbone phosphates proximal to the C4 carbonyl of bound 1 were replaced with methylphosphonates. All were found to react at comparable rates establishing that these backbone phosphates do not participate in catalysis of the DNA alkylation reaction.     

Effect of alkylation with N-methyl-N-nitrosourea and N-ethyl-N-nitrosourea on the secondary structure of DNA

We have earlier reported that alkylation of DNA by the chemical carcinogen dimethyl sulphate, which mainly alkylates N-7 of guanine and N-3 of adenine, causes the formation of partially denatured regions in double-stranded DNA (Rizvi RY, Alvi NK & Hadi SM, Biosci. Rep. 2, 315-322, 1982). It is known that the major site of alkylation in DNA by N-ethyl-N-nitrosourea (EtNu) are the phosphate groups. N-methyl-N-nitrosourea (MeNu), on the other hand, causes the alkylation of mainly guanine residues. We have therefore studied the effect of these two alkylating carcinogens on the secondary structure of DNA. DNA alkylated with increasing concentrations of EtNu and MeNu was subjected to alkaline and S1 nuclease hydrolysis. Thermal melting profiles of alkylated DNA were also determined using S1 nuclease. The results indicated that alkylation by the two alkylating agents had a differential effect on the secondary structure of DNA. EtNu-alkylated DNA was found to be more thermostable than native DNA at neutral pH. It was however more alkali-labile than MeNu-alkylated DNA. The greater stability of EtNu-alkylated DNA was considered to be due to abolition of negative charges on phosphate alkylation.     

Enantioselective affinity labelling of horse liver alcohol dehydrogenase. Correlation of inactivation kinetics with the three-dimensional structure of the enzyme.

Kinetic data for the inactivation of horse liver alcohol dehydrogenase with S-2-chloro-3-(imidazol-5-yl)propionate at pH8.2 were correlated with the three-dimensional structure of the enzyme. The R-2-chloro-3-(imidazol-5-yl)propionate enantiomer did not inactivate the enzyme, and the reaction is thus enantioselective. Inactivation follows an affinity-labelling mechanism where a reversible complex is formed before the irreversible alkylation and inactivation of the enzyme. A reversible complex is also formed with the non-inactivating enantiomer, and this shows that the selectivity occurs at the irreversible step. By using a computer-controlled display system, models of the two enantiomers of 2-chloro- and 2-bromo-3-(imidazol-5-yl)propionate were built into a model of the enzyme so that the imidazole moiety was liganded to the active-site metal, while the carboxylate group interacted with the general anion-binding site. The conformation of the imidazole derivatives and their orientation in the active site were adjusted to minimize unfavourable steric interactions. It was clear that alkylation of cysteine-46 could proceed with the S-enantiomer bound in this way, but not with the R-enantiomer. Model building thus agrees with the inactivation kinetics and indicates the structural origin of the enantioselectivity.     

(+)-CC-1065 as a probe for intrinsic and protein-induced bending of DNA

(+)-CC -1065 is biologically potent DNA-reactive antitumor antibiotic produced by Streptomyces zelensis. This antibiotic covalently modifies DNA by alkylation of N-3 of a adenine in the minor groove. As a Structural consequence of covalent modification of DNA, the helix axis id bent into the minor groove. The drug-induced bending of DNA has similarities to intrinsic. A-tract bending and the 3′ adenine of A-tracts shows a unique reactivity to alkylation by (+) -CC-1065. Upon covalent modification of A-tracts, the magnitude of bending is increased and helix is stiffened. Using high-field NMR, hydroxyl-radical footprinting and gel electrophoresis, the molecular basis for the high reactivity of the bonding sequence 5′ - AGTTA* (an asterisk indicates the covalent modification site) to (+)-CC-1065 has been shown to involve the inherent conformational flexibility of this sequence. Furthermore, these studies also demonstrate that after alkylation the drug-induced bending is focused over the TT region. By analogy with the junction bend model for A-tracts, a ‘truncated junction bend model’ is proposed for this structure. Last, the application of (+)-CC-1065 entrapped/induced bending of DNA as a probe for the Sp1-induced bending of the 21-base-pair repeat an Mu transpose bending of the att L3 sequence is described.     

Alkylation of duplex DNA in nucleosome core particles by duocarmycin SA and yatakemycin

(+)-Yatakemycin (1, Fig. 1) and (+)-duocarmycin SA (2) are exceptionally potent, naturally occurring antitumor agents that derive their biological properties through a characteristic sequence-selective DNA-alkylation reaction. Studies have shown that both the AT-rich binding selectivity (shape-selective recognition) and the alkylation catalysis (shape-dependent catalysis) that contribute to the alkylation selectivity are dependent on the DNA minor groove shape and size characteristics of an AT-rich sequence (ref. 6 and references therein; refs. 7,8). Here we report the alkylation properties of yatakemycin and duocarmycin SA on free DNA (alpha-satellite DNA) and the same sequence bound in a nucleosome core particle (NCP) modeling the state of DNA in eukaryotic cells. Both compounds showed a clear, relatively unaltered ability to alkylate DNA packaged in NCPs in terms of both alkylating efficiency and sequence selectivity, despite the steric and conformational perturbations imposed by NCP packaging. These findings highlight the dynamic nature of NCP-bound DNA and illustrate that cell- and protein-free DNA-alkylation studies of members of this class of antitumor drugs provide valuable insights into their properties.     

Inhibition of RecBCD enzyme by antineoplastic DNA alkylating agents

To understand how bulky adducts might perturb DNA helicase function, three distinct DNA-binding agents were used to determine the effects of DNA alkylation on a DNA helicase. Adozelesin, ecteinascidin 743 (Et743) and hedamycin each possess unique structures and sequence selectivity. They bind to double-stranded DNA and alkylate one strand of the duplex in cis, adding adducts that alter the structure of DNA significantly. The results show that Et743 was the most potent inhibitor of DNA unwinding, followed by adozelesin and hedamycin. Et743 significantly inhibited unwinding, enhanced degradation of DNA, and completely eliminated the ability of the translocating RecBCD enzyme to recognize and respond to the recombination hotspot chi. Unwinding of adozelesin-modified DNA was accompanied by the appearance of unwinding intermediates, consistent with enzyme entrapment or stalling. Further, adozelesin also induced "apparent" chi fragment formation. The combination of enzyme sequestering and pseudo-chi modification of RecBCD, results in biphasic time-courses of DNA unwinding. Hedamycin also reduced RecBCD activity, albeit at increased concentrations of drug relative to either adozelesin or Et743. Remarkably, the hedamycin modification resulted in constitutive activation of the bottom-strand nuclease activity of the enzyme, while leaving the ability of the translocating enzyme to recognize and respond to chi largely intact. Finally, the results show that DNA alkylation does not significantly perturb the allosteric interaction that activates the enzyme for ATP hydrolysis, as the efficiency of ATP utilization for DNA unwinding is affected only marginally. These results taken together present a unique response of RecBCD enzyme to bulky DNA adducts. We correlate these effects with the recently determined crystal structure of the RecBCD holoenzyme bound to DNA.     

DNA stretching in the nucleosome facilitates alkylation by an intercalating antitumour agent

DNA stretching in the nucleosome core can cause dramatic structural distortions, which may influence compaction and factor recognition in chromatin. We find that the base pair unstacking arising from stretching-induced extreme minor groove kinking near the nucleosome centre creates a hot spot for intercalation and alkylation by a novel anticancer compound. This may have far reaching implications for how chromatin structure can influence binding of intercalator species and indicates potential for the development of site selective DNA-binding agents that target unique conformational features of the nucleosome.     

Analysis of substrate specificity of Schizosaccharomyces pombe Mag1 alkylpurine DNA glycosylase

DNA glycosylases specialized for the repair of alkylation damage must identify, with fine specificity, a diverse array of subtle modifications within DNA. The current mechanism involves damage sensing through interrogation of the DNA duplex, followed by more specific recognition of the target base inside the active site pocket. To better understand the physical basis for alkylpurine detection, we determined the crystal structure of Schizosaccharomyces pombe Mag1 (spMag1) in complex with DNA and performed a mutational analysis of spMag1 and the close homologue from Saccharomyces cerevisiae (scMag). Despite strong homology, spMag1 and scMag differ in substrate specificity and cellular alkylation sensitivity, although the enzymological basis for their functional differences is unknown. We show that Mag preference for 1,N⁶‐ethenoadenine (εA) is influenced by a minor groove‐interrogating residue more than the composition of the nucleobase‐binding pocket. Exchanging this residue between Mag proteins swapped their εA activities, providing evidence that residues outside the extrahelical base‐binding pocket have a role in identification of a particular modification in addition to sensing damage.     

Distinct Functions of Human Cohesin-SA1 and Cohesin-SA2 in Double-Strand Break Repair

Cohesin is an essential multiprotein complex that mediates sister chromatid cohesion critical for proper segregation of chromosomes during cell division. Cohesin is also involved in DNA double-strand break (DSB) repair. In mammalian cells, cohesin is involved in both DSB repair and the damage checkpoint response, although the relationship between these two functions is unclear. Two cohesins differing by one subunit (SA1 or SA2) are present in somatic cells, but their functional specificities with regard to DNA repair remain enigmatic. We found that cohesin-SA2 is the main complex corecruited with the cohesin-loading factor NIPBL to DNA damage sites in an S/G₂-phase-specific manner. Replacing the diverged C-terminal region of SA1 with the corresponding region of SA2 confers this activity on SA1. Depletion of SA2 but not SA1 decreased sister chromatid homologous recombination repair and affected repair pathway choice, indicating that DNA repair activity is specifically associated with cohesin recruited to damage sites. In contrast, both cohesin complexes function in the intra-S checkpoint, indicating that cell cycle-specific damage site accumulation is not a prerequisite for cohesin''s intra-S checkpoint function. Our findings reveal the unique ways in which cohesin-SA1 and cohesin-SA2 participate in the DNA damage response, coordinately protecting genome integrity in human cells.     

Crystal structure of trioxacarcin A covalently bound to DNA

We report a crystal structure that shows an antibiotic that extracts a nucleobase from a DNA molecule ‘caught in the act’ after forming a covalent bond but before departing with the base. The structure of trioxacarcin A covalently bound to double-stranded d(AACCGGTT) was determined to 1.78 Å resolution by MAD phasing employing brominated oligonucleotides. The DNA–drug complex has a unique structure that combines alkylation (at the N7 position of a guanine), intercalation (on the 3′-side of the alkylated guanine), and base flip-out. An antibiotic-induced flipping-out of a single, nonterminal nucleobase from a DNA duplex was observed for the first time in a crystal structure.     

Inactivation of Cg10062, a cis-3-chloroacrylic acid dehalogenase homologue in Corynebacterium glutamicum, by (R)- and (S)-oxirane-2-carboxylate: analysis and implications

( R)- and ( S)-oxirane-2-carboxylate were determined to be active site-directed irreversible inhibitors of the cis-3-chloroacrylic acid dehalogenase ( cis-CaaD) homologue Cg10062 found in Corynebacterium glutamicum. Kinetic analysis indicates that the ( R) enantiomer binds more tightly and is the more potent inhibitor, likely reflecting more favorable interactions with active site residues. Pro-1 is the sole site of covalent modification by the ( R) and ( S) enantiomers. Pro-1, Arg-70, Arg-73, and Glu-114, previously identified as catalytic residues in Cg10062, have also been implicated in the inactivation mechanism. Pro-1, Arg-70, and Arg-73 are essential residues for the process as indicated by the observation that the enzymes with the corresponding alanine mutations are not covalently modified by either enantiomer. The E114Q mutant slows covalent modification of Cg10062 but does not prevent it. The results are comparable to those found for the irreversible inactivation of cis-CaaD by ( R)-oxirane-2-carboxylate with two important distinctions: the alkylation of cis-CaaD is stereospecific, and Glu-114 does not take part in the cis-CaaD inactivation mechanism. Cg10062 exhibits low-level cis-CaaD and trans-3-chloroacrylic acid dehalogenase (CaaD) activities, with the cis-CaaD activity predominating. Hence, the preference of Cg10062 for the cis isomer correlates with the observation that the ( R) enantiomer is the more potent inactivator. Moreover, the factors responsible for the relaxed substrate specificity of Cg10062 may account for the stereoselective inactivation by the enantiomeric epoxides. Delineation of these factors would provide a more complete picture of the substrate specificity determinants for cis-CaaD. This study represents an important step toward this goal by setting the stage for a crystallographic analysis of inactivated Cg10062.     

(+)-CC-1065 as a structural probe of Mu transposase-induced bending of DNA: overcoming limitations of hydroxyl-radical footprinting.

Phage Mu transposase (A-protein) is primarily responsible for transposition of the Mu genome. The protein binds to six att sites, three at each end of Mu DNA. At most att sites interaction of a protein monomer with DNA is seen to occur over three minor and two consecutive major grooves and to result in bending up to about 90 degrees. To probe the directionality and locus of these A-protein-induced bends, we have used the antitumor antibiotic (+)-CC-1065 as a structural probe. As a consequence of binding within the minor groove, (+)-CC-1065 is able to alkylate N3 of adenine in a sequence selective manner. This selectivity is partially determined by conformational flexibility of the DNA sequence, and the covalent adduct has a bent DNA structure in which narrowing of the minor groove has occurred. Using this drug in experiments in which either gel retardation or DNA strand breakage are used to monitor the stability of the A-protein--DNA complex or the (+)-CC-1065 alkylation sites on DNA (att site L3), we have demonstrated that of the three minor grooves implicated in the interaction with A-protein, the peripheral two are 'open' or accessible to drug bonding following protein binding. These drug-bonding sites very likely represent binding at at least two A-protein-induced bending sites. Significantly, the locus of bending at these sites is spaced approximately two helical turns apart, and the bending is proposed to occur by narrowing of the minor groove of DNA. The intervening minor groove between these two peripheral sites is protected from (+)-CC-1065 alkylation. The results are discussed in reference to a proposed model for overall DNA bending in the A-protein att L3 site complex. This study illustrates the utility of (+)-CC-1065 as a probe for protein-induced bending of DNA, as well as for interactions of minor groove DNA bending proteins with DNA which may be masked in hydroxyl radical footprinting experiments.     

Reaction of the antitumor antibiotic CC-1065 with DNA. Location of the site of thermally induced strand breakage and analysis of DNA sequence specificity

CC-1065 is a unique antitumor antibiotic produced by Streptomyces zelensis. The potent cytotoxic effects of this drug are thought to be due to its ability to form a covalent adduct with DNA through N3 of adenine. Thermal treatment of CC-1065-DNA adducts leads to DNA strand breakage. We have shown that the CC-1065 structural modification of DNA that leads to DNA strand breakage is related to the primary alkylation site on DNA. The thermally induced DNA strand breakage occurs between the deoxyribose at the adenine covalent binding site and the phosphate on the 3' side. No residual modification of DNA is detected on the opposite strand around the CC-1065 lesion. Using the early promoter element of SV40 DNA as a target, we have examined the DNA sequence specificity of CC-1065. A consensus sequence analysis of CC-1065 binding sites on DNA reveals two distinct classes of sequences for which CC-1065 is highly specific, i.e., 5'PuNTTA and 5'AAAAA. The orientation of the DNA sequence specificity relative to the covalent binding site provides a basis for predicting the polarity of drug binding in the minor groove. Stereo drawings of the CC-1065-DNA adduct are proposed that are predictive of features of the CC-1065-DNA adduct elucidated in this investigation.     

Base excision repair

Damage to DNA bases resulting from deamination, oxidation, and alkylation is mainly repaired by base-excision repair. BER is initiated by DNA glycosylases, which recognize damaged bases and excise them from DNA by hydrolyzing the N-glycosidic bond between the base and the sugar phosphate backbone of DNA to generate an abasic site. Different human and E. coli DNA glycosylases have been cloned and characterized, each one with unique substrate specificity. Some of them additionally have AP lyase activity, which enables them to cleave the bond between the sugar and phosphate 3' to the damaged site. BER consist of two repair pathways (short or long) in which one or more nucleotides are introduced respectively. In conclusion, it seems to be likely that BER pathways are essential for genomic repair and stability in living cells.     

Informational parameters of an exact DNA base sequence

A series of hierarchical chemical reactivity calculations have been performed to elucidate the alkylation properties of a methyldiazonium ion toward DNA base sites. Both MINDO/3 and CNDO/2 approximate methods have been employed. For the isolated bases the O6 of guanine is predicted to be the most reactive site. This prediction may also be relevant to single-stranded DNA chains containing guanine. For base-pairs, the N7 and O6 sites on guanine are about equally favored for alkylation. The previous study of aziridinium ion alkylation gave about the same results with N7 guanine modestly favored as the preferred site of alkylation for base-pairs. In composite we conclude that N7 guanine and/or O6 guanine will be the preferred sites for alkylation by a methyldiazonium ion but cannot distinguish between these two in terms of chemical specificity.     

DNA-binding mechanism of O6-alkylguanine-DNA alkyltransferase. Effects of protein and DNA alkylation on complex stability

The mutagenic and cytotoxic effects of many endogenous and exogenous alkylating agents are mitigated by the actions of O(6)-alkylguanine-DNA alkyltransferase (AGT). In humans this protein protects the integrity of the genome, but it also contributes to the resistance of tumors to DNA-alkylating chemotherapeutic agents. Here we report properties of the interaction between AGT and short DNA oligonucleotides. We show that although AGT sediments as a monomer in the absence of DNA, it binds cooperatively to both single-stranded and double-stranded deoxyribonucleotides. This strong cooperative interaction is only slightly perturbed by active site mutation of AGT or by alkylation of either AGT or DNA. The stoichiometry of complex formation with 16-mer oligonucleotides, assessed by analytical ultracentrifugation and electrophoretic mobility shift assays, is 4:1 on single-stranded and duplex DNA and is unchanged by several active site mutations or by protein or DNA alkylation. These results have significant implications for the mechanisms by which AGT locates and interacts with repairable alkyl lesions to effect DNA repair.     

Stereoselective covalent binding of anti-benzo(a)pyrene diol epoxide to DNA conformation of enantiomer adducts

The conformation of adducts derived from the reactions and covalent binding of the (+) and (-) enantiomers of 7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (anti-BaPDE) with double-stranded calf thymus DNA in vitro were investigated utilizing the electric linear dichroism technique. The linear dichroism and absorption spectra of the covalent DNA complexes are interpreted in terms of a superposition of two types of binding sites. One of these conformations (site I) is a complex in which the plane of the pyrene residue is close to parallel (within 30 degrees) to the planes of the DNA bases (quasi-intercalation), while the other (site II) is an external binding site; this latter type of adduct is attributed to the covalent binding of anti-BaPDE to the exocyclic amino group of deoxyguanine (N2-dG), while site I adducts are attributed to the O6-deoxyguanine and N6-deoxyadenine adducts identified in the product analysis of P. Brookes and M.R. Osborne (Carcinogenesis (1982) 3, 1223-1226). Site II adducts are dominant (approximately 90% in the covalent complexes derived from the (+) enantiomer), but account for only 50 +/- 5% of the adducts in the case of the (-)-enantiomer. The orientation of site II complexes is different by 20 +/- 10 degrees in the adducts derived from the binding of the (+) and the (-) enantiomers to DNA, the long axis of the pyrene chromophore being oriented more parallel to the axis of the DNA helix in the case of the (+) enantiomer. These findings support the proposals by Brookes and Osborne that the difference in spatial orientation of the N2-dG adducts of (-)-anti-BaPDE together with their lower abundance may account for the lower biological activity of the (-) enantiomer. The external site II adducts, rather than site I adducts, appear to be correlated with the biological activity of these compounds.     

Duocarmycin SA,a potent antitumor antibiotic,sensitizes glioblastoma cells to proton radiation

New treatment modalities for glioblastoma multiforme (GBM) are urgently needed. Proton therapy is considered one of the most effective forms of radiation therapy for GBM. DNA alkylating agents such as temozolomide (TMZ) are known to increase the radiosensitivity of GBM to photon radiation. TMZ is a fairly impotent agent, while duocarmycin SA (DSA) is an extremely potent cytotoxic agent capable of inducing a sequence-selective alkylation of duplex DNA. Here, the effects of sub-nM concentrations of DSA on the radiosensitivity of a human GBM cell line (U-138) to proton irradiation were examined. Radiation sensitivity was determined by viability, apoptosis, necrosis and clonogenic assays. DSA concentrations as low as 0.001?nM significantly sensitized U-138 cells to proton irradiation. DSA demonstrates synergistic cytotoxicity against GBM cells treated with proton radiation in vitro, which may represent a novel therapeutic alternative for the treatment of GBM.     

Chemical reactivity of protonated aziridine with nucleophilic centers of DNA bases

A series of molecular orbital calculations, using MINDO/3 and CNDO/2L methods, have been used to characterize the chemical reaction of protonated aziridine with DNA nucleophilic base sites. The N-7 atom of guanine is found to be the preferred alkylation site only when the O-6 atom of guanine is involved in base-pair hydrogen bonding. Otherwise O-6 is the predicted major site of alkylation. This indirectly suggests that protonated aziridine alkylation processes involve base-paired DNA structures, since N-7 guanine is the observed major site of alkylation. Alkylation of N-3 adenine is predicted to be more favorable than chemical attack of the N-7 adenine position. Both of these sites, however, are predicted to be less reactive than N-7 of guanine. These chemical reactivity studies resolve alkylation specifically not achieved in the DNA–alkylator physical association calculations reported in the preceding paper.