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The use of DNA equilibrium binding molecules to transfer alkyl groups to specific positions on DNA is an approach to generating cytotoxic DNA damage while avoiding the formation of promutagenic lesions that increase the risk for the development of secondary cancer. We have previously reported that in vitro a neutral DNA equilibrium binding agent based on an N-methylpyrrolecarboxamide dipeptide (lex) and modified with an O-methyl sulfonate ester functionality (Me-lex) selectively affords N3-methyladenine lesions in >90% yield relative to the formation of other adducts. While in vitro interactions between the lex dipeptide and DNA have been thoroughly studied, in vivo interactions are more difficult to elucidate. We report herein the relationship between the in vivo formation of N3-methyladenine and toxicity in wild-type and base excision repair defective mutant Escherichia coli. In addition, it is demonstrated that both N3-methyladenine adduction and cytotoxicity can be inhibited in vivo with netropsin, a potent competitive inhibitor of binding of lex to DNA. The results show a clear relationship between the levels of N3-methyladenine and toxicity in an alkA/tag glycosylase mutant that cannot remove the adduct from its genome. For methyl methanesulfonate, which does not sequence selectively methylate DNA, a relationship between the formation of N3-methyladenine and toxicity is also observed. However, netropsin affects neither the level of N3-methyladenine nor the toxicity of methyl methanesulfonate in E. coli.

The Human Genome Project as well as sequencing of the genomes of other organisms offers a wealth of DNA targets for both therapeutic and diagnostic applications, and it is important to develop additional DNA binding motifs to fully exploit the potential of this new information. We have recently found that an aromatic dication, DB293, with an amidine-phenyl-furan-benzimidazole-amidine structure can recognize specific sequences of DNA by binding in the minor groove as a dimer [Wang, L., Bailly, C., Kumar, A., Ding, D., Bajic, M., Boykin, D. W., and Wilson, W. D. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 12-16]. The dimer binding is strong, highly cooperative and, in contrast to many closely related heterocyclic dications, has both GC and AT base pairs in the minor groove binding site. The aromatic heterocycle stacked dimer is quite different in structure from the polyamide-lexitropsin type compounds, and it is a dication while all lexitropsin dimers are monocations. The heterocyclic dimer represents only the second small molecule class that can recognize mixed sequences of DNA. To test the structural limits on the new type of complex, it is important to probe the influence of compound charge, chemical groups, and structural features. The effects of these compound molecular variations on DNA complex formation with several DNA sequences were evaluated by DNase I footprinting, CD and UV spectroscopy, thermal melting, and quantitative analysis with surface plasmon resonance biosensor methods. Conversion of the amidines to guanidinium groups does permit the cooperative dimer to form but removal of one amidine or addition of an alkyl group to the amidine strongly inhibited dimer formation. Changing the phenyl of DB293 to a benzimidazole or the benzimidazole to a phenyl or benzofuran also inhibited dimer formation. The results show that formation of the minor groove stacked-dimer complex is very sensitive to compound structure. The discovery of the aromatic dimer mode offers new opportunities to enhance the specificity and expand the range of applications of the compounds that target DNA.

Circular dichroism (CD), isothermal calorimetric titrations (ITC), and temperature-dependent UV spectroscopy were used to investigate binding of the minor groove-directed ligands distamycin A (Dst) and netropsin (Net) to the following duplexes: d(GTTAGTATTTGG). d(CCAAATACTAAC), d(GTTAGTATATGG).d(CCATATACTAAC), d(GTTAGTACTTGG). d(CCAAGTACTAAC), and d(GTTAGTAGTTGG).d(CCAACTACTAAC). Our results reveal that Dst binds within the minor grooves of these dodecamers that contain five-AT and/or four-AT.GC binding sites exclusively in a dimeric high-affinity 2:1 binding mode (K approximately 10(16) M(-)(2)). By contrast, Net exhibits high-affinity binding only when it binds in a 1:1 mode (K(1) approximately 10(9) M(-)(1)) to the two duplexes that contain five-AT sites (5'-TATTT-3' and 5'-TATAT-3'). Its further binding to these two duplexes occurs in a low-affinity mode (K(2) approximately 10(6) M(-)(1)) and results in the formation of 2:1 Net-DNA complexes. To the other two duplexes that contain sequences with at most three AT consecutive base pairs Net binds in two distinctive low-affinity 1:1 binding modes (K(1) approximately 10(7) M(-)(1), K(2) approximately 10(6) M(-)(1)). Competition experiments (CD and ITC titrations) reveal that Dst entirely displaces Net from its 1:1 and 2:1 complexes with any of the four duplexes. We discuss and interpret our optical and calorimetric results in the context of the available structural information about the complexes between DNA and the sequence-specific minor groove binders Dst and Net.

The crystal structure of netropsin bound to the decamer d(CGCAATTGCG) has been determined at 2.4 A resolution. This is the first example of a crystal structure of netropsin bound to decamer DNA. The central eight bases of each DNA single-strand base pair with a self-complementary strand to form an octamer B-DNA duplex. These duplexes lie end to end within the unit cell. The terminal 5'-C and G-3' bases are unpaired and interact with the neighboring duplexes via interactions within both the major and minor groove to form base triplet interactions of the type C(+)-G x C and G*(G x C), respectively. The triplet interaction of the type C(+)-G x C is known to exist within triplex DNA with the C+ base oriented parallel with the Watson-Crick guanine base to which it hydrogen bonds. The netropsin molecule lies within the minor groove of the octamer duplex and assumes a class I type position, with bifurcated hydrogen-bonding interactions from the amide groups of the netropsin to the A x T base pairs of the minor groove. The netropsin molecule fits within a five base pair long minor groove site by bending of the flexible amidinium group at one end of the drug.

The interaction of the minor groove binding drug distamycin with the T-A-T triple helix and the A-T double helix was studied using circular dichroism spectroscopy and thermal denaturation. The triple helix was made by the oligonucleotide (dA)12-x-(dT)12-x-(dT)12, where x is a hexaethylene glycol chain bridged between the 3'-phosphate of one strand and the 5'-phosphate of the following strand. This oligonucleotide is able to fold back on itself to form a very stable triplex. Changing the conditions allows the same oligonucleotide to be in a duplex form with a dangling arm. Circular dichroism spectroscopy demonstrates that the distamycin A molecule can bind to the triple-stranded form of this oligonucleotide. Spectral analysis shows that the bound distamycin exhibits a conformation and an environment slightly different from those which are observed when the drug is bound to the corresponding double-stranded structure. Furthermore, a second type of complex which is observed in the double-strand binding (two stacked distamycins in the minor groove) is not observed with the triple-stranded host. When distamycin is added to the triplex made of unbridged chains (dA)12 + 2(dT)12, the triplex dissociates to give a double-stranded structure. Thermal denaturation experiments demonstrate that distamycin binding destabilizes the triplex whereas it stabilizes the duplex. These results are compared with those obtained by the same experimental approaches on other minor groove binding drugs.

Combilexins are a group of DNA ligands having a sequence-specific minor groove binding element combined with an intercalating chromophore which stabilizes the DNA complex and can interfere with topoisomerases. In this study, complementary methods of spectroscopy (absorption, circular dichroism, electric linear dichroism) and biochemistry (viscometry, footprinting) have been applied to explore the nature of the complex formed between a new amsacrine-4-carboxamide-netropsin combilexin and DNA or chromatin. Collectively, the structural and kinetic data concur that the conjugate threads through the DNA double helix so as to intercalate its acridine chromophore, leaving the netropsin moiety and the methanesulfonanilino group positioned within the minor and major grooves of the double helix, respectively. The hybrid retains the AT selectivity conferred by the netropsin moiety. The threading-type intercalation process, evidenced by stopped-flow measurements, is affected when the DNA is wrapped around histones. The composite drug can bind to both the DNA linker segments and the nucleosomal cores in chromatin though, unlike its constituents, it antagonizes the salt-induced condensation of chromatin. As far as its mode of binding to DNA is concerned, the netropsin-amsacrine hybrid molecule exhibits structural features reminiscent of the antitumor antibiotics nogalamycin and pluramycin. The design of DNA-threading combilexins provides an original route for the development of sequence-specific ligands capable of forming stable complexes with DNA.

Five classic DNA minor groove-binding drugs and a series of bis-linked lexitropsins based on netropsin and distamycin have been screened for their effectiveness in inhibiting transcription by HIV-1 reverse transcriptase (RT) on a poly(rA).oligo(dT) template-primer (TP). The two most effective drugs, 3,5 m-pyridyl-linked bisdistamycin (MPyr) and trans-vinyl-linked bisdistamycin (TVin), show (1) enhanced inhibition in reactions initiated with pre-incubated enzyme template-primer (ETP) and (2) reduced affinity for a "free" TP analog, when compared with the parent drug distamycin. All three drugs lack the ability to inhibit processive incorporation of nucleotide, suggesting drug intervention instead at initiation or termination of processive cycles. The two bis-linked drugs exhibit different kinetic behavior with reverse transcriptase's two substrates: template-primer and nucleotide. When primer is the variable substrate, TVin is partially noncompetitive and MPyr is dead-end competitive (Ki = 6.5 microM). With nucleotide as substrate, TVin is noncompetitive at low drug concentrations and MPyr is uncompetitive. Gel band mobility shift assays with MPyr indicate that the drug inhibits via entrapment of TP on the enzyme rather than displacement of TP from the enzyme surface. The conformation of nucleic acid is most likely altered upon MPyr binding, enhancing the induced fit of enzyme to hybrid duplex. The relevance of this novel mode of inhibition is considered in relation to enzyme association/dissociation with TP that occurs prior to (-)-DNA strand transfer, and to the structural implications of an enzyme-bound hybrid RNA/DNA nucleic acid.

An X-ray crystal structure has been solved of the complex of a dicationic lexitropsin with a B-DNA duplex of sequence CGCGAATTCGCG. The lexitropsin is identical to netropsin except for replacement of the first methylpyrrole ring by methylimidazole, converting a =CH- to =N-. Crystals are isomorphous with those of the DNA dodecamer in the absence of drug. Although the =N- for =CH- substitution was intended to make that locus on the drug molecule compatible with a G.C base pair, electrostatic attraction for the two cationic ends of the drug predominates, and this lexitropsin binds to the same central AATT site as does the parent netropsin. But unlike netropsin, this lexitropsin exhibits end-for-end disorder in the crystal. Both orientations were refined separately to completion. Final residual errors at 2.25 A resolution for the 2358 reflections above 2 sigma in F are R = 0.165 for one orientation (LexA) with 37 water molecules and 0.164 for the inverted drug orientation (LexB) with 40 water molecules. This molecular disorder is probably attributable to a weakening of binding to the AATT site occasioned by the imidazole-for-pyrrole substitution.

A 2.3-A X-ray crystal structure analysis has been carried out on the antitumor drug anthramycin, covalently bound to a ten base pair DNA double helix of sequence C-C-A-A-C-G-T-T-G-G. One drug molecule sits within the minor groove at each end of the helix, covalently bound through its C11 position to the N2 amine of the penultimate guanine of the chain. The stereochemical conformation is C11S, C11aS. The natural twist of the anthramycin molecule in the C11aS conformation matches the twist of the minor groove as it winds along the helix; a C11aR drug would only fit into a left-handed helix. The C11S attachment is roughly equatorial to the overall plane of the molecule, whereas a C11R attachment would be axial and would obstruct the fitting of the drug into the groove. The six-membered ring of anthramycin points toward the 3' end of the chain to which it is covalently attached or toward the end of the helix. The acrylamide tail attached to the five-membered ring extends back along the minor groove toward the center of the helix, binding in a manner reminiscent of netropsin or distamycin. The drug-DNA complex is stabilized by hydrogen bonds from C9-OH, N10, and the end of the acrylamide tail to base pair edges on the floor of the minor groove. The origin of anthramycin specificity for three successive purines arises not from specific hydrogen bonds but from the low twist angles adopted by purine-purine steps in a B-DNA helix. Binding of anthramycin induces a low twist at T-G in the T-G-G sequence of this DNA-drug complex, by comparison with the structure of the free DNA. The origin of anthramycin's preference for adenines flanking the alkylated guanine arises from a netropsin-like fitting of the acrylamide tail into the minor groove.

We use high-precision acoustic and densimetric techniques to determine, at 25 degrees C, the changes in volume, delta V, and adiabatic compressibility, delta Ks, that accompany the binding of netropsin to the poly(dAdT).poly(dAdT) and poly(dA).poly(dT) duplexes, as well as to the poly(dT).poly(dA).poly(dT) triplex. We find that netropsin binding to the heteropolymeric poly(dAdT).poly(dAdT) duplex is accompanied by negative changes in volume, delta V, and small positive changes in compressibility, delta Ks. By contrast, netropsin binding to the homopolymeric poly(dA).poly(dT) duplex is accompanied by large positive changes in both volume, delta V, and compressibility, delta Ks. Furthermore, netropsin binding to the poly(dT).poly(dA).poly(dT) triplex causes changes in both volume and compressibility that are nearly twice as large as those observed when netropsin binds to the poly(dA).poly(dT) duplex. We interpret these macroscopic data in terms of binding-induced microscopic changes in the hydration of the DNA structures and the drug. Specifically, we find that netropsin binding induces the release of approximately 22 waters from the hydration shell of the poly(dAdT).poly(dAdT) heteropolymeric duplex, approximately 40 waters from the hydration shell of the poly(dA).poly(dT) homopolymeric duplex, and about 53 waters from the hydration shell of the poly(dA).poly(dT), induces the release of 18 more water molecules than netropsin binding to the heteropolymeric duplex, poly(dAdT).poly(dAdT). On the basis of apparent molar volume, phi V, and apparent molar adiabatic compressibility, phi Ks, values for the initial drug-free and final drug-bound states of the two all-AT duplexes, we propose that the larger dehydration of the poly(dA).poly(dT) duplex reflects, in part, the formation of a less hydrated poly(dA).poly(dT)-netropsin complex compared with the corresponding poly(dAdT).poly(dAdT)-netropsin complex. In conjunction with our previously published entropy data [Marky, L. A., & Breslauer, K. J. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 4359-4363], we calculate that each water of hydration released to the bulk solvent by ligand binding contributes 1.6 cal K-1 mol-1 to the entropy of binding. This value corresponds to the average difference between the partial molar entropy of water in the bulk state and water in the hydration shells of the two all-AT duplexes. When netropsin binds to the poly(dT).poly(dA).poly(dT) triplex, the changes in both volume and compressibility suggest that the binding event induces more dehydration of the triplex than of the duplex state. Specifically, we calculate that netropsin binding to the poly(dT).poly(dA).poly(dT) triplex causes the release of 13 more waters than netropsin binding to the poly(dA).poly(dT) duplex.(ABSTRACT TRUNCATED AT 400 WORDS)

TBP (TATA box binding protein), a general transcription factor required for proper initiation of gene expression by RNA polymerase II, and minor groove binding drugs (MGBs) both interact with DNA within the minor groove at AT sites. This study has evaluated MGBs as inhibitors of DNA/TBP complex formation by gel mobility shift assays. Our results demonstrate that reversible MGBs (DAPI, distamycin A, Hoechst 33258, and netropsin) are effective inhibitors of the formation of DNA/TBP complex and that distamycin A is the most potent (0.16 microM inhibits TBP complex formation by 50%). CC-1065, a drug that covalently binds to DNA in the minor groove, is even more active than distamycin A (0.00085 microM inhibits TBP complex formation by 50%). Significantly more CC-1065 (0.009 microM) is required to break up preformed DNA/TBP complex compared to the drug concentration needed to prevent complex formation. In comparison, the order of drug addition has little influence on the ability of reversible MGBs to disrupt DNA/TBP complex. In the presence of TFIIA, a factor that enhances TBP association with DNA, greater drug concentrations (distamycin A and CC-1065, respectively) are needed to disrupt a preformed complex of DNA/TBP/TFIIA. In comparison to MGBs, drugs capable of binding to DNA by intercalation are generally weaker at blocking TBP complex formation except for hedamycin, which can intercalate and irreversibly bind to DNA and is as effective as reversible MGBs.

In order to further characterize the binding of the hybrid molecule NetOPC [bis(pyrrolecarboxamide)-oxazolopyridocarbazole conjugate] to double-stranded DNA, we have performed a molecular modeling study to investigate the binding modes of the complexes possibly formed between NetOPC and synthetic polynucleotides poly [(dA-dT)]2, poly [(dA).d(T)], and poly [d(G-C)]2 and interpreted the results in the light of the experimentally determined binding parameters. In agreement with experimental data, the modeling study suggests that whatever was the binding mode of the complex formed, the complexation energy is markedly lower (thus favorable) for AT-containing polynucleotides than for poly d[(G-C)]2. With both poly [d(A).d(T)] and poly [d(A-T)]2, the most energetically favored complex has netropsin and OPC moieties bound simultaneously in the minor groove of DNA. The second favored complex exhibits the bimodal binding, i.e., intercalation of OPC and minor groove binding of the netropsin moiety. For both types of complex, the energy of complex formation is slightly lower with poly [d(A).d(T)]. The binding site sizes of the modeled complexes are about seven and four base pairs to the full groove and bimodal binding, respectively.

The chromophore of the anticancer drug neocarzinostatin (NCS-Chrom) oxidatively cleaves single-stranded or duplex DNA site-specifically in the absence of activating thiol provided that the DNA contains a bulged structure. Point mutations, deletions, and insertions in the DNA analogue and its complement of the 3'-terminus of yeast tRNA(Phe) show that for a single-stranded DNA to be cleaved by NCS-Chrom the DNA must generate a hairpin structure with an apical loop and at least a two-base-pair stem hinged to a region of duplex structure via a bulge containing a target nucleotide at its 3' side. The size of the loop is not critical so long as it contains at least three nucleotides; the bulge requires a minimum of two nucleotides but must have fewer than five. With a notable exception involving base-pair changes immediately 3' to the bulge, base changes in the bulge and base-pair changes immediately 5' to the bulge retain substrate activity for NCS-Chrom. Maintenance of the bulged structure requires stable duplex regions on each side of the bulge. A similar bulged structure, but lacking a loop, formed by the annealing of a linear 8-mer and a 6-mer is an excellent target for cleavage in the thiol-independent reaction. Drugs such as netropsin, which sequester the DNA into nonbulge containing structures inhibit the reaction. In the absence of O2 strand cleavage is blocked and quantitatively replaced by a presumed drug-DNA covalent adduct.(ABSTRACT TRUNCATED AT 250 WORDS)

The molecular structure of the complex between the minor groove binding drug netropsin and the dodecamer d(CGCAAATTTGCG) has been solved and refined by X-ray diffraction analysis to an R-factor of 19.8% and 2.2-A resolution. The drug lies in the narrow minor groove of the B-DNA fragment, covering five of the six A.T base pairs (from A5.T20 to T9.A16). The long six A.T base pair tract allows the drug to bind in a position that optimizes its contacts with the DNA, establishing hydrogen bonds with O2 of thymines and N3 of adenines. The DNA molecule shows a high propeller twist only at the A6.T19 step of the A-tract. Two three-centered hydrogen bonds are observed in the major groove at half of the A-tract

A series of sulfonate esters that are attached to a noncationic minor-groove-binding N-methylpyrrole dipeptide (Lex) related to netrospin have been synthesized. The compounds prepared differ in two respects: (1) the length [(CH2)2 vs (CH2)8] of the tether between the DNA affinity binding portion of the molecule and the sulfonate ester and (2) whether a methyl group [MeOSO2(CH2)n-Lex] or the dipeptide including the aliphatic tether [MeSO2O(CH2)n-Lex] is covalently transferred to the DNA. The DNA-cleavage patterns of these bimolecular alkylating compounds have been mapped in 32P-end-labeled restriction fragments using neutral thermal hydrolysis and alkali treatment to expose single-strand breaks at bases with thermally labile modifications. In contrast to the alkylation of DNA by simple alkyl alkanesulfonate esters, that predominantly yield major-groove alkylation at N7-guanine, the modification of DNA by MeOSO2(CH2)n-Lex and MeSO2O(CH2)n-Lex occurs primarily at N3-adenine residues associated with previously footprinted Lex DNA affinity binding regions. The ratio for the formation of N3-methyladenine (minor groove) to N7-methylguanine (major groove) in calf thymus DNA is 1:7 for dimethyl sulfate, while only the former adenine product is observed with MeSO2O(CH2)n-Lex indicating the change in groove specificity. DNA cleavage by MeOSO2(CH2)n-Lex and MeSO2O(CH2)n-Lex is efficiently inhibited by the coaddition of distamycin; however, only the DNA damage generated by the latter is blocked by NaCl. As expected, increasing the length of the (CH2)n tether from n = 2 to n = 8 moves the alkylation site by 1-2 base pairs further from the affinity binding domain. Finally, a comparison of the methylation patterns of MeOSO2(CH2)n-Lex as a function of tether length provides an insight into Lex sequence and orientational preferences.

The DNA binding properties of a series of imidazole-containing and C-terminus-modified analogues 4-7 of distamycin are described. These analogues contain one to four imidazole units, respectively. Data from the ethidium displacement assay showed that these compounds bind in the minor groove of DNA, with the relative order of binding constants of 6 (Im3) > 7 (Im4) > 5 (Im2) > 4 (Im1). The reduced binding constants of these compounds for poly(dA-dT) relative to distamycin, while they still interact strongly with poly(dG-dC), provided evidence of GC sequence acceptance. The preferences for GC-rich sequences by these compounds were established from a combination of circular dichroism (CD) titration, proton nuclear magnetic resonance (1H-NMR), and methidiumpropylethylenediaminetetraacetate-iron(II) [MPE.Fe-(II)] footprinting studies. In the CD studies, these compounds produced significantly larger DNA-induced ligand bands with poly(dG-dC) than poly(dA-dT) at comparable ligand concentrations. 1H-NMR studies of the binding of 5 to d-[CATGGCCATG]2 provided further evidence of the recognition of GC sequences by these compounds, and suggested that the ligand was located on the underlined sequence in the minor groove with the C-terminus oriented over the T residue. MPE footprinting studies on a GC-rich BamHI/SalI fragment of pBR322 provided unambiguous evidence for the GC sequence selectivity for some of these compounds. Compounds 4 and 7 produced poor footprints on the gels; however, analogues 5 and 6 gave strong footprints.(ABSTRACT TRUNCATED AT 250 WORDS)

Exposure of cells to alkylating agents produces DNA lesions, most of which are repaired. However some alkyl lesions persist and play a role in inducing point mutations and the subsequent carcinogenic conversion. O6-Ethylguanine (e6G) is a relatively persistent alkylation lesion caused by the exposure of DNA to N-ethyl-N-nitrosourea. We study the consequence of the e6G incorporation in DNA by X-ray crystallography. We have obtained crystals of the modified DNA dodecamer d(CGC[e6G]AATTCGCG) and the unmodified d(CGCGAATTCGCG), complexed to the minor groove binding drug netropsin. The space group of both crystals is P2(1)2(1)2(1), isomorphous to other related dodecamer DNA crystals. The structures have been solved by the molecular replacement method and refined by the constrained least-squares procedure to R-factors of approximately 16% at resolution of approximately 2.5 A. The two independent e6G-C base pairs in the DNA duplex adopt different base-pairing schemes. The e6G4-C21 base pair has a configuration similar to a normal Watson-Crick base pair, except with one three-centered hydrogen bond pair and one direct hydrogen bond between e6G4 and C21. In contrast, the e6G16-C9 base pair adopts a wobble configuration. The ethyl group is in the proximal orientation (to N7) in both base pairs. These observations enrich and support those found in the crystal structure of d(CGC[e6G]AATTCGCG), complexed to minor groove binding drugs Hoechst 33258 and Hoechst 33342 [Sriram et al. (1992) EMBO J. 11, 225-232]. We suggest that a dynamic equilibrium between these two configurations for the e6G-C base pair is likely and would present an ambiguous signal to the cellular transcription, replication, or repair mechanisms. In contrast, thymine can pair with e6G in only one way, albeit imperfect, mimicking a Watson-Crick base pair. This may be a plausible explanation of why thymine is found preferentially incorporated across the e6G during replication. In addition, we analyze the influence of the alkylation lesion on DNA and the molecular details of netropsin-DNA interaction. In the present two new netropsin complexes, the netropsin spans across five base pairs (starting halfway between C3-G22 and e6G4-C21 base pairs and ending at T8-A17 base pair) in the narrow minor groove. This is in contrast to the earlier crystal structure of netropsin complexed with another DNA dodecamer having the same AATT central core sequence, d(CGCGAATT[br5C]GCG) [Kopka et al. (1985) J. Mol. Biol. 272, 390-395]. In the latter structure, the netropsin lies between G4-br5C21 and br5C9-G16 base pairs.(ABSTRACT TRUNCATED AT 400 WORDS)

We report the DNA binding properties of two hybrid molecules which result from the combination of the DNA sequence-specific minor groove ligand netropsin with the bithiazole moiety of the antitumor drug bleomycin. The drug-DNA interaction has been investigated by means of electric linear dichroism (ELD) spectroscopy and DNase I footprinting. In compound 1 the two moieties are linked by a flexible aliphatic tether while in compound 2 the two aromatic ring systems are directly coupled by a rigid peptide bond. The results are consistent with a model in which the netropsin moiety of compound 1 resides in the minor groove of DNA and where the appended bithiazole moiety is projected away from the DNA groove. This monocationic hybrid compound has a weak affinity for DNA and shows a strict preference for A and T stretches. ELD measurements indicate that in the presence of DNA compound 2 has an orientation typical of a minor groove binder. Similar orientation angles were measured for netropsin and compound 2. This ligand which has a biscationic nature tightly binds to DNA (Ka = 6.3 x 10(5) M-1) and is mainly an AT-specific groove binder. But, depending on the nature of the sequence flanking the AT site first targeted by its netropsin moiety, the bithiazole moiety of 2 can accommodate various types of nucleotide motifs with the exception of homooligomeric sequences. As evidenced by footprinting data, the bithiazole group of bleomycin acts as a DNA recognition element, offering opportunities to recognize GC bp-containing DNA sequences with apparently a preference (although not absolute) for a pyrimidine-G-pyrimidine motif. Thus, the bithiazole unit of bleomycin provides an additional anchor for DNA binding and is also capable of specifically recognizing particular DNA sequences when it is appended to a strongly sequence selective groove binding entity. Finally, a model which schematizes the binding of compound 2 to the sequence 5'-TATGC is proposed. This model readily explains the experimentally observed specificity of this netropsin-bithiazole conjugate.

The crystal structure of the DNA dodecamer C-G-T-G-A-A-T-T-C-A-C-G has been determined at a resolution of 2.5 A, with a final R factor of 15.8% for 1475 nonzero reflections measured at 0 degrees C. The structure is isomorphous with that of the Drew dodecamer, with the space group P2(1)2(1)2(1) and cell dimensions of a = 24.94 A, b = 40.78 A, and c = 66.13 A. The asymmetric unit contains all 12 base pairs of the B-DNA double helix and 36 water molecules. The structure of C-G-T-G-A-A-T-T-C-A-C-G is very similar to that of C-G-C-G-A-A-T-T-C-G-C-G, with no major alterations in helix parameters. Water peaks in the refined structure appear to represent a selection of peaks that were observed in the Drew dodecamer. The minor-groove spine of hydration at 2.5 A is fragmentary, but as Narendra et al. (1991) [Biochemistry (following paper in this issue)] have observed, lowering the temperature leads to a more complete representation of the spine.

We have investigated some properties related to interaction with DNA and recognition of AT-rich sequences of netropsin-oxazolopyridocarbazole (Net-OPC) (Mrani et al., 1990), which is a hybrid groove-binder-intercalator. The hybrid molecule Net-OPC binds to poly[d(A-T)] at two different sites with Kapp values close to 7 x 10(6) and 6 x 10(8) M-1 (100 mM NaCl, pH 7.0). Data obtained from melting experiments are in agreement with these values and indicate that Net-OPC displays a higher binding constant to poly[d(A-T)] than does netropsin. On the basis of viscometric and energy transfer data, the binding of Net-OPC to poly[d(A-T)] is suggested to involve both intercalation and external binding of the OPC chromophore. In contrast, on poly[d(G-C)], Net-OPC binds to a single type of site composed of two base pairs in which the OPC chromophore appears to be mainly intercalated. The binding constant of Net-OPC to poly[d(G-C)] was found to be about 350-fold lower than that of the high-affinity binding site in poly[d(A-T)]. As evidenced by footprinting data, Net-OPC selectively recognizes TTAA and CTT sequences and strongly protects the 10-bp AT-rich DNA region 3'-TTAAGAACTT-5' containing the EcoRI site. The binding of Net-OPC to this sequence results in a strong and selective inhibition of the activity of the restriction endonuclease EcoRI on the plasmid pBR322 as substrate. The extent of inhibition of the rate constant of the first strand break catalyzed by the enzyme is about 100-fold higher than the one observed in the presence of netropsin under similar experimental conditions.

The influence of cosolutes and DNA sequence on the interaction of netropsin with three duplexes has been studied by isothermal titration calorimetry. In buffer, netropsin forms two complexes with a net stoichiometry of 1:1 in the minor groove of the oligonucleotide (GCGCGAATTCGCGC)2. One complex has a weaker affinity and is more enthalpically favored relative to the other one, consistent with previous studies [Freyer, M. W., et al. (2006) Biophys. Chem. 126, 186-196]. With the cosolutes betaine and 2-methyl-2,4-pentanediol, the enthalpy and heat capacity changes indicate that the complex with weaker affinity is disfavored relative to the complex with higher affinity. With (CGCGCAATTGCGCG)2, netropsin has one binding mode in buffer, and complex formation is not influenced by the cosolutes. The similarities of the enthalpy and heat capacity changes suggest that netropsin interacts similarly with these two oligonucleotides in the presence of cosolutes. The oligonucleotide (GCGCAAATTTGCGC)2 also forms two complexes with netropsin, and the complex with weaker affinity is again disfavored by the cosolutes. Thus, the interaction of netropsin with these A/T binding sites is influenced both by the bases adjacent to the binding site and by cosolutes. We suggest that these two factors influence the conformation of the minor-groove binding site of DNA.

Minor groove specific DNA equilibrium binding peptides (lex) based on N-methylpyrrole-carboxamide and/or N-methylimidazolecarboxamide subunits have been modified with an O-methyl sulfonate ester functionality to target DNA methylation in the minor groove at Ade/Thy- and/or Gua/Cyt-rich sequences. HPLC and sequencing gel analyses show that the Me-lex compounds all selectively react with DNA to afford N3-alkyladenine as a major adduct. The formation of the N3-alkyladenine lesions is sequence-dependent based on the equilibrium binding preferences of the different lex peptides. In addition to the reaction at adenine, the molecules designed to target Gua/Cyt sequences also generate lesions at guanine; however, the methylation is not sequence dependent and takes places in the major groove at the N7-position. To determine if and how the level of the different DNA adducts and the sequence selectivity for their formation affects cytotoxicity, the Me-lex analogues were tested in wild type Escherichia coli and in mutant strains defective in base excision repair (tag and/or alkA or apn). The results demonstrate the importance of 3-methyladenine, and in some cases 3-methylguanine, lesions in cellular toxicity, and the dominant protective role of the DNA glycosylases. There is no evidence that the sequence specificity is related to toxicity.

The herpes simplex virus type 1 origin binding protein (UL9) is a sequence-specific DNA binding protein. Several studies have demonstrated that UL9 binds to the 11-base pair sequence 5'-CGTTCGCACTT-3' primarily, or solely, through interaction with the major groove. Minor groove binding ligands, such as distamycin, netropsin, and GLX, an indole-linked dimer of netropsin, can effectively disrupt the UL9-DNA complex only when their DNA binding sites are coincident with the right side of the DNA binding site of the protein and overlap with the protein binding site by two (TT) base pairs. These results suggest that the right side of the UL9-DNA complex has a unique structure that is sensitive to minor groove ligand binding. In addition, a biphasic displacement curve was observed with GLX, which suggests two modes of ligand binding which have different effects on UL9-DNA complexes. Using a fluorescence-based hybridization stabilization assay, we determined that GLX can bind to its binding site as an overlapping dimer (i.e., 2:1 stoichiometry). Footprinting of UL9-DNA complexes with the minor groove directed chemical nuclease 1,10-phenanthroline copper confirms that the DNA conformation at the position of the right-side ligand binding site of GLX is altered and has a widened minor groove. In contrast, it is well established that at 1:1 stoichiometries, AT sequence specific ligands, such as netropsin, distamycin, and GLX, prefer uniform, narrow minor grooves. The opposing conformational requirements of UL9 and lower concentrations of GLX at the ligand binding A-tract overlapping the right side of the protein binding site indicate that allosteric inhibition, rather than direct steric competition, contributes to ligand-induced protein displacement. At higher GLX concentrations, giving 2:1 binding in a widened minor groove, co-binding with UL9 is allowed. A model is presented that is consistent with these observations, and implications for targeted regulation of gene transcription are discussed.

The effects of a wide range of DNA binding drugs on peptide nucleic acid (PNA) binding to double-stranded DNA by strand displacement have been investigated using a gel retardation assay. The bis-PNA [H-(Lys)-TTJTTJTTTT-(eg)(3)-TTTTCTTCTT-Lys-NH(2)] was used together with a 248 bp DNA fragment containing an appropriate target for the PNA. Most of the ligands that were studied, including DNA minor groove binders as well as intercalators and bis-intercalators, either have no effect or strongly inhibit PNA binding to DNA. By contrast, quinoxaline antibiotics facilitate PNA-DNA complex formation. The "PNA-helper" effect of echinomycin was studied in more detail using time and temperature dependence experiments to elucidate the mechanism. PNA binding to DNA follows pseudo-first-order kinetics, but the initial rate of binding is accelerated more than 10-fold in the presence of 10 microM echinomycin. The activation energy for PNA binding to dsDNA is lowered 2-fold by the antibiotic (45 vs 90 kJ/mol in the control). The reasons why quinoxalines promote the binding of PNA to DNA are not entirely clear but may well include distortions (opening) of the double helix that facilitate PNA invasion. This study establishes that the efficacy of DNA-targeted PNA antigene molecules could potentially be enhanced by judiciously adding certain DNA-interactive ligands.

The sequence and structural selectivity of 15 different DNA binding agents was explored using a novel, thermodynamically rigorous, competition dialysis procedure. In the competition dialysis method, 13 different nucleic acid structures were dialyzed against a common ligand solution. More ligand accumulated in the dialysis tube containing the structural form with the highest ligand binding affinity. DNA structural forms included in the assay ranged from single-stranded forms, through a variety of duplex forms, to multistranded triplex and tetraplex forms. Left-handed Z-DNA, RNA, and a DNA-RNA hybrid were also represented. Standard intercalators (ethidium, daunorubicin, and actinomycin D) served as control compounds and were found to show structural binding preferences fully consistent with their previously published behavior. Standard groove binding agents (DAPI, distamycin, and netropsin) showed a strong preference for AT-rich duplex DNA forms, along with apparently strong binding to the poly(dA)-[poly(dT)](2) triplex. Thermal denaturation studies revealed the apparent triplex binding to be complex, and perhaps to result from displacement of the third strand. Putative triplex (BePI, coralyne, and berberine) and tetraplex [H(2)TmPyP, 5,10,15, 20-tetrakis[4-(trimethylammonio)phenyl]-21H,23H-porphine, and N-methyl mesoporphyrin IX] selective agents showed in many cases less dramatic binding selectivity than anticipated from published reports that compared their binding to only a few structural forms. Coralyne was found to bind strongly to single-stranded poly(dA), a novel and previously unreported interaction. Finally, three compounds (berenil, chromomycin A, and pyrenemethylamine) whose structural preferences are largely unknown were examined. Pyrenemethylamine exhibited an unexpected and unprecedented preference for duplex poly(dAdT).

In an attempt to obtain sequence specific DNA-cleaving molecules, we have synthesized a series of hybrid minor groove binders composed of a photoactiveable isoalloxazine (flavin) chromophore linked through a polymethylenic chain to a bis-pyrrolecarboxamide moiety related to netropsin. Like netropsin, the hybrid derivatives preferentially bind to A+T-rich sequences. Activation of the flavin chromophore by visible light results in the appearance of single strand breaks in the vicinity of the DNA binding site. We have further investigated the cleavage affinity properties of one of these compounds referred to as netropsin-flavin (Net-Fla) and considered as representative of the series. Net-Fla cleaves only one strand at a specific locus downstream of 5'-AAAT-3', upstream of 5'-TAAA-3' and on either side of a 5'-AAAA-3' sequence. Net-Fla cleaves both strands downstream to 5'-AATT-3'. This makes the properties of Net-Fla similar to that of a restriction endonuclease and provides additional insight into establishing the rules for the readout of B-DNA helix by non-nucleotidic compounds. Using molecular modeling, we show that Net-Fla binds to an asymmetric site in one orientation. The values of the energetic minima lie in the same order as expected from the cleavage patterns, which suggests that the oriented cleavage is a consequence of a sequence-oriented binding of Net-Fla in the DNA minor groove.

The X-ray crystal structure of the complex of the B-DNA dodecamer CGCGAATTCGCG with the antitumor drug netropsin has been reexamined to locate the drug accurately for computer-based drug design. The optimum solution is with the drug centered in the AATT region of the minor groove, making three good bifurcated hydrogen bonds with adenine N3 and thymine O2 atoms along the floor of the groove. Pyrrole rings of netropsin are packed against the C2 positions of adenines, leaving no room for the amine group of guanine and, hence, providing a structural rationale for the A.T specificity of netropsin. An alternative positioning in which the drug is shifted along the minor groove by ca. one-half base pair step is rejected on the basis of free R factor calculations and the appearance of the original drug-free difference maps. Final omit maps, although of more pleasing appearance, are not a dependable means of discriminating between right and wrong structures. The shifted alternative drug position ignores potential hydrogen bonding along the floor of the groove, provides no explanation for netropsin's observed A.T specificity, and is contradicted by NMR results [Patel, D. J. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 6424].

We use high-precision acoustic and densimetric techniques to determine, at 25 degrees C, the changes in volume, delta V, and adiabatic compressibility, delta Ks, that accompany the binding of netropsin to the poly(dAdT).poly(dAdT) and poly(dA).poly(dT) duplexes, as well as to the poly(dT).poly(dA).poly(dT) triplex. We find that netropsin binding to the heteropolymeric poly(dAdT).poly(dAdT) duplex is accompanied by negative changes in volume, delta V, and small positive changes in compressibility, delta Ks. By contrast, netropsin binding to the homopolymeric poly(dA).poly(dT) duplex is accompanied by large positive changes in both volume, delta V, and compressibility, delta Ks. Furthermore, netropsin binding to the poly(dT).poly(dA).poly(dT) triplex causes changes in both volume and compressibility that are nearly twice as large as those observed when netropsin binds to the poly(dA).poly(dT) duplex. We interpret these macroscopic data in terms of binding-induced microscopic changes in the hydration of the DNA structures and the drug. Specifically, we find that netropsin binding induces the release of approximately 22 waters from the hydration shell of the poly(dAdT).poly(dAdT) heteropolymeric duplex, approximately 40 waters from the hydration shell of the poly(dA).poly(dT) homopolymeric duplex, and about 53 waters from the hydration shell of the poly(dA).poly(dT), induces the release of 18 more water molecules than netropsin binding to the heteropolymeric duplex, poly(dAdT).poly(dAdT). On the basis of apparent molar volume, phi V, and apparent molar adiabatic compressibility, phi Ks, values for the initial drug-free and final drug-bound states of the two all-AT duplexes, we propose that the larger dehydration of the poly(dA).poly(dT) duplex reflects, in part, the formation of a less hydrated poly(dA).poly(dT)-netropsin complex compared with the corresponding poly(dAdT).poly(dAdT)-netropsin complex. In conjunction with our previously published entropy data [Marky, L. A.,Breslauer, K. J. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 4359-4363], we calculate that each water of hydration released to the bulk solvent by ligand binding contributes 1.6 cal K-1 mol-1 to the entropy of binding. This value corresponds to the average difference between the partial molar entropy of water in the bulk state and water in the hydration shells of the two all-AT duplexes. When netropsin binds to the poly(dT).poly(dA).poly(dT) triplex, the changes in both volume and compressibility suggest that the binding event induces more dehydration of the triplex than of the duplex state. Specifically, we calculate that netropsin binding to the poly(dT).poly(dA).poly(dT) triplex causes the release of 13 more waters than netropsin binding to the poly(dA).poly(dT) duplex.(ABSTRACT TRUNCATED AT 400 WORDS)

Short peptides derived from chromosomal proteins have previously been proposed to bind specifically to the minor groove of A,T-rich DNA [for a review, see M. E. A. Churchill and A. A. Travers (1991) Trends Biochem. Sci. 16, 92-97]. Using NMR spectroscopy, we investigated the DNA binding of SPRKSPRK, which is one such A,T-specific motif. Under the conditions studied SPRKSPRK interacts only nonspecifically with d(CGCAAAAAAGGC).d(GCCTTTTTTGCG). The peptides TPKRPRGRPKK, PRGRPKK, and PRGRP derived from the non-histone chromosomal protein HMG-I/Y, however, bind specifically to the central A,T sites of d(CGCAAATTTGCG)2 and d(CGCGAATTCGCG)2. 2D NOE measurements show that the RGR segment of each peptide is in contact with the minor groove. The arginine side chains and the peptide backbone are buried deep in the minor groove, in a fashion generally similar to the antibiotic netropsin. Under the same conditions the peptide PKGKP does not interact with the same oligonucleotide duplexes, indicating that the arginine guanidinium groups are major determinants of the A,T specificity.

NMR spectroscopy combined with molecular modeling was used to characterize a heterodimeric complex with Dst and 2-ImN bound in the minor groove of d(GCCTAACAAGG)•d(CCTTGTTAGGC) (1:1:1 2-ImN•Dst•DNA complex). The imidazole-pyrrole-pyrrole ligand 2-ImN spans 5'-GTTA-3' of the TAACA•TGTTA binding site with the imidazole nitrogen specifically recognizing the guanine amino group. The Dst ligand lies along the 5'-AACA-3' sequence and complements the 2-ImN ligand in the formation of the antiparallel side-by-side heterodimeric complex. Titrations of the same site with Dst or 2-ImN alone yield homodimeric complexes (2:1 ligand.DNA) of lower stability than the 1:1:1 2-ImN•Dst•DNA complex. Dst and 2-ImN binding to d(CGCAAACTGGC)•d(GCCAGTTTGCG) was also investigated. The 1:1:1 2-ImN•Dst•DNA complex is again the most stable complex with the AAACT•AGTTT site and is similar to the TAACA•TGTTA complex. No monomeric binding of either 2-ImN or Dst was observed to either site.

The designed peptides pyridine-2-carboxamidonetropsin (2-PyN) and 1-methylimidazole-2-carboxamidonetropsin (2-ImN) are crescent-shaped analogs of the natural products netropsin and distamycin A. 2-PyN and 2-ImN bind the 5'-TGTCA-3' sequence as antiparallel side-by-side dimers in the minor groove of DNA. The binding affinities of 2-PyN and 2-ImN to four different 5-bp sites on DNA were determined by quantitative MPE-Fe(II) footprint titration and compared with the tripeptide D from distamycin. The binding affinities of D to the sites 5'-TTTTT-3' and 5'-TGTCA-3' are 2.6 x 10(7) and < 1 x 10(5) M-1, respectively (pH 7.0, 100 mM NaCl). 2-PyN binds these sites with similar affinities, 2.3 x 10(5) and 2.7 x 10(5) M-1, respectively. The affinities of 2-ImN to the same two sites are < 5 x 10(4) and 1.4 x 10(5) M-1, respectively. Substitution of an N-methylpyrrole-2-carboxamide of the distamycin tripeptide by 1-methylimidazole-2-carboxamide has changed the specificities for the two binding sites by a factor of 10(3). The data for 2-PyN and 2-ImN binding the 5'-TGTCA-3' site are best fit by a cooperative binding curve consistent with 2:1 peptide-DNA complexes.

Exposure of cells to alkylating agents produces DNA lesions, most of which are repaired. However some alkyl lesions persist and play a role in inducing point mutations and the subsequent carcinogenic conversion. O6-Ethylguanine (e6G) is a relatively persistent alkylation lesion caused by the exposure of DNA to N-ethyl-N-nitrosourea. We study the consequence of the e6G incorporation in DNA by X-ray crystallography. We have obtained crystals of the modified DNA dodecamer d(CGC[e6G]AATTCGCG) and the unmodified d(CGCGAATTCGCG), complexed to the minor groove binding drug netropsin. The space group of both crystals is P2(1)2(1)2(1), isomorphous to other related dodecamer DNA crystals. The structures have been solved by the molecular replacement method and refined by the constrained least-squares procedure to R-factors of approximately 16% at resolution of approximately 2.5 A. The two independent e6G-C base pairs in the DNA duplex adopt different base-pairing schemes. The e6G4-C21 base pair has a configuration similar to a normal Watson-Crick base pair, except with one three-centered hydrogen bond pair and one direct hydrogen bond between e6G4 and C21. In contrast, the e6G16-C9 base pair adopts a wobble configuration. The ethyl group is in the proximal orientation (to N7) in both base pairs. These observations enrich and support those found in the crystal structure of d(CGC[e6G]AATTCGCG), complexed to minor groove binding drugs Hoechst 33258 and Hoechst 33342 [Sriram et al. (1992) EMBO J. 11, 225-232]. We suggest that a dynamic equilibrium between these two configurations for the e6G-C base pair is likely and would present an ambiguous signal to the cellular transcription, replication, or repair mechanisms. In contrast, thymine can pair with e6G in only one way, albeit imperfect, mimicking a Watson-Crick base pair. This may be a plausible explanation of why thymine is found preferentially incorporated across the e6G during replication. In addition, we analyze the influence of the alkylation lesion on DNA and the molecular details of netropsin-DNA interaction. In the present two new netropsin complexes, the netropsin spans across five base pairs (starting halfway between C3-G22 and e6G4-C21 base pairs and ending at T8-A17 base pair) in the narrow minor groove. This is in contrast to the earlier crystal structure of netropsin complexed with another DNA dodecamer having the same AATT central core sequence, d(CGCGAATT[br5C]GCG) [Kopka et al. (1985) J. Mol. Biol. 272, 390-395]. In the latter structure, the netropsin lies between G4-br5C21 and br5C9-G16 base pairs.(ABSTRACT TRUNCATED AT 400 WORDS)

Affinity cleaving proteins have been synthesized based on the DNA-binding domain of the yeast transcriptional activator GCN4 with the DNA cleaving moiety Fe·EDTA attached at the NH_2 terminus [Oakley, M. G., & Dervan, P. B. (1990) Science 248, 847]. Cleavage patterns generated by Fe·EDTA-GCN4(226-281) bound to the DNA sites 5'-CTGACTAAT-3' and 5'-ATGACTCTT-3' reveal that the NH_2 termini of the GCN4 DNA-binding domain are located in the major groove of DNA, 9-10 base pairs apart, consistent with a Y-shaped dimeric structure. 1-Methylimidazole-2-carboxamide netropsin (2-ImN) is a designed synthetic peptide which binds in the minor groove of DNA at 5'-TGACT-3' sites as an anti parallel, side-by-side dimer [Mrksich, M., Wade, W. S., Dwyer, T. J ., Geierstanger, B. H., Wemmer, D. E., & Dervan, P. B. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 7586]. Through the use of Fe·EDTAGCN4( 226-281) as a sequence-specific footprinting agent, it is shown that the dimeric protein GCN4-(226-281) and the dimeric peptide 2-ImN can simultaneously occupy their common binding site in the major and minor grooves of DNA, respectively. The association constants for 2-ImN in the presence and in the absence of Fe·EDTA-GCN4(226-281) are found to be similar, suggesting that the binding of the two dimers is not cooperative.

The crystal structure of the complex formed between the dodecanucleotide d(CGCGAATTCGCG)2 and the drug pentamidine, which is active against the Pneumocystis carinii pathogen in AIDS patients, has been determined to a resolution of 2.1 A and an R-factor of 19.4%. Analysis of the structure has shown the drug to be bound in the 5'-AATT minor groove region of the duplex, with the amidinium groups H-bonded to adenine N3 atoms in an interstrand manner. The drug molecule adopts an extended conformation, and the immediate binding site spans four base pairs. Structural details of the drug-DNA interactions are discussed, and comparison is made with the dodecamer complex of the structurally similar berenil ligand.

The interaction of thioformyldistamycin, an amide isostere of the naturally occurring antibiotic distamycin A, with a self-complementary decadeoxynucleotide duplex, d(CGCAATTGCG)2, has been examined using a variety of high-field 1H-NMR techniques. The ligand exhibits two forms in solution arising from geometric isomerism due to restricted rotation around the thioformamide bond. Only the thermodynamically more stable Z-form is shown to bind to the oligonucleotide along its minor groove at the central 5'-AATT segment with the end groups of the ligand extending into the flanking GC regions but without any close contact at the amidinium terminus. Cross-peaks involving characteristic intra- and interresidue proton connectivities in the 2D experiments (COSY and NOESY) were employed to assign individual resonances of both strands in the asymmetric DNA-drug complex. The solution structure of the complex was constructed by molecular mechanics calculations based upon initial estimates of drug-DNA NOE contacts and further refined through energy minimization. These results complement previous structural studies on distamycin and other lexitropsins with oligonucleotides. The exchange of the ligand between two equivalent binding sites on the DNA sequence was estimated to occur at 40 s-1 with a free energy of activation of 16.5 kcal.mol-1 at 321-326 K. There was no evidence of formation of a 2:1 drug-oligomer complex, in contrast to the case of the natural product, which is attributed to steric demands of the larger sulfur atom.

The synthesis and characterization of a series of compounds that contain an N-alkyl-N-nitrosourea functionality linked to DNA minor groove binding bi- and tripeptides (lexitropsins or information-reading peptides) based on methylpyrrole-2-carboxamide subunits are described. The lexitropsins (lex) synthesized have either a 3-(dimethylamino)propyl or propyl substituent on the carboxyl terminus. The preferred DNA affinity binding sequences of these compounds were footprinted in 32P-end-labeled restriction fragments with methidiumpropyl-EDTA.Fe(II), and in common with other structural analogues, e.g., distamycin and netropsin, these nitrosoureas recognize A-T-rich runs. The affinity binding of the compound with the dimethylamino terminus, which is ionized at near-neutral pH, appeared stronger than that observed for the neutral dipeptide. The sequence specificity for DNA alkylation by (2-chloroethyl)nitrosourea-lex dipeptides (Cl-ENU-lex), with neutral and charged carboxyl termini, using 32P-end-labeled restriction fragments, was determined by the conversion of the adducted sites into single-strand breaks by sequential heating at neutral pH and exposure to base. The DNA cleavage sites were visualized by polyacrylamide gel electrophoresis and autoradiography. The alkylation of DNA by Cl-ENU-lex was compared to that by N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea (CCNU), which has no DNA affinity binding properties. While all the Cl-ENU compounds generate DNA breaks as a consequence of the formation of N7-alkyl-guanine, the Cl-ENU-lex compounds induced, in a time- and dose-dependent fashion, intense DNA cleavage bands at adenine, cytosine, and thymine residues associated with affinity binding sites. These non-G cleavages induced by Cl-ENU-lex were inhibited by the coaddition of distamycin at concentrations that did not affect G alkylation break sites. CCNU, even at much higher concentrations, does not generate any similar detectable lesions at non-G sites. Therefore, linking the Cl-ENU moiety to minor groove binders is a viable strategy to qualitatively and quantitatively control the delivery and release of the ultimate DNA alkylating agent in a sequence-dependent fashion.

Large variations in alkylation intensities exist among guanines in a DNA sequence following treatment with chemotherapeutic alkylating agents such as nitrogen mustards, and the substituent attached to the reactive group can impose a distinct sequence preference for reaction. In order to understand further the structural and electrostatic factors which determine the sequence selectivity of alkylation reactions, the effect of increased ionic strength, the intercalator ethidium bromide, AT-specific minor groove binders distamycin A and netropsin, and the polyamine spermine on guanine N7-alkylation by L-phenylalanine mustard (L-Pam), uracil mustard (UM), and quinacrine mustard (QM) was investigated with a modification of the guanine-specific chemical cleavage technique for DNA sequencing. For L-Pam and UM, increased ionic strength and the cationic DNA affinity binders dose dependently inhibited the alkylation. QM alkylation was less inhibited by salt (100 mM NaCl), ethidium (10 microM), and spermine (10 microM). Distamycin A and netropsin (100 microM) gave an enhancement of overall QM alkylation. More interestingly, the pattern of guanine N7-alkylation was qualitatively altered by ethidium bromide, distamycin A, and netropsin. The result differed with both the nitrogen mustard (L-Pam less than UM less than QM) and the cationic agent used. The effect, which resulted in both enhancement and suppression of alkylation sites, was most striking in the case of netropsin and distamycin A, which differed from each other. DNA footprinting indicated that selective binding to AT sequences in the minor groove of DNA can have long-range effects on the alkylation pattern of DNA in the major groove.

It has been previously reported that a neutral DNA equilibrium binding agent based on an N-methylpyrrolecarboxamide dipeptide (lex) and modified with an O-methyl sulfonate ester functionality (MeOSO(2)-lex) selectively affords N3-methyladenine lesions. To study the interaction of the neutral lex dipeptide with calf thymus DNA, we have prepared stable, nonmethylating sulfone analogues of MeOSO(2)-lex that are neutral and cationic. Thermodynamic studies show that both the neutral and monocationic sulfone compounds bind to DNA with K(b)'s of 10(5) in primarily entropy-driven reactions. To determine how the cytotoxic N3-methyladenine adduct generated from MeOSO(2)-lex is repaired in E. coli, MeOSO(2)-lex was tested for toxicity in wild-type E. coli and in mutant strains defective in base excision repair (tag and/or alkA glycosylases or apn endonuclease), nucleotide excision repair (uvrA), and both base and nucleotide excision repair (tag/alkA/uvrA). The results clearly demonstrate the cellular toxicity of the N3-methyladenine lesion, and the protective role of base excision glycosylase proteins. A novel finding is that in the absence of functional base excision glycosylases, nucleotide excision repair can also protect cells from this cytotoxic minor groove lesion. Interaction between base and nucleotide excision repair systems is also seen in the protection of cells treated with cis-diamminedichloroplatinum(II) but not with anti-(+/-)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene.

Two general approaches have been taken to understand the mechanism of the reversible binding of the nonprotein chromophore of neocarzinostatin to DNA: (1) measurement of the relative affinity of the chromophore for various DNAs that have one or both grooves blocked by bulky groups and (2) studies on the influence of adenine-thymine residue-specific, minor groove binding agents such as the antibiotics netropsin and distamycin on the chromophore-DNA interaction. Experiments using synthetic DNAs containing halogen group (Br, I) substituents in the major groove or natural DNAs with glucosyl moieties projecting into the major groove show that obstruction of the major groove does not decrease the binding stoichiometry or the binding constant for the DNA-chromophore interaction. Chemical methylation of bases in both grooves of calf thymus DNA, resulting in 13% methylation of N-7 of guanine in the major groove and 7% methylation of N-3 of adenine in the minor groove, decreases the binding affinity and increases the size of the binding site for neocarzinostatin chromophore. Similar results were obtained whether binding parameters were determined directly by spectroscopic measurements or indirectly by measuring the ability of the DNA to protect the chromophore against degradation. On the other hand, netropsin and distamycin compete with neocarzinostatin chromophore for binding to the minor groove of DNA, as shown by their decrease in the ability of poly(dA-dT) to protect the chromophore against degradation and their reduction in chromophore-induced DNA damage as measured by thymine release.(ABSTRACT TRUNCATED AT 250 WORDS)

A series of promoters with nine base-pair substitutions in the spacer DNA separating the -10 and -35 regions was used to demonstrate that Escherichia coli RNA polymerase is sensitive to events affecting the spacer DNA--a region not directly contacted by the enzyme. The drugs distamycin A and netropsin specifically enhanced the rate of functional complex formation at a promoter bearing a substitution of nonalternating A-T base pairs. The effect is exerted at an early step in the RNA polymerase-promoter interaction. We hypothesize that a drug-induced structural alteration in the spacer DNA occurs, similar to that normally resulting from RNA polymerase binding. These findings are relevant to an understanding of potential mechanisms of transcription activation.

We report dichroism and equilibrium binding studies of netropsin (Net) and distamycin A3 (Dist) binding to deoxyribonucleic acid (DNA). We show that at low degrees of binding (r) to calf thymus DNA, Net induces a considerable increase in the apparent DNA length (14 A/drug molecule bound), closely analogous to the results reported earlier for Dist. In addition, we show that chicken erythrocyte DNA shows length changes similar to those of calf thymus DNA upon distamycin binding. DNA length reaches a maximum at 1 bound drug/20-30 base pairs and then decreases to its initial value by r = 0.1. This effect is not seen for two other DNAs with nearly identical A + T base pair content and may therefore arise from the details of base sequence or base modification in eukaryotic DNA. We also show that Dist binding to calf thymus DNA at low r values is positively cooperative and shows a DNA affinity which is primarily nonionic. We demonstrate that independent of the DNA to which they are bound, the Net and Dist transition moments are inclined by 43 +/- 3 degrees from the helix axis, consistent with the idea that both drugs bind inside and parallel to the DNA small groove. From dichroism measurements, we show that the conformational change induced in calf thymus DNA by Dist does not kink or bend the helix and does not substantially alter the average inclination of the bases. Finally, we outline a statistical mechanical theory for calculation of binding isotherms when binding is coupled to a DNA structural change.

The results of quantitative footprinting studies involving the antiviral agent netropsin and a DNA-cleaving cationic metalloporphyrin complex are presented. An analysis of the footprinting autoradiographic spot intensities using a model previously applied to footprinting studies involving the enzyme DNase I [Ward, B., Rehfuss, R., Goodisman, J., & Dabrowiak, J. C. (1988) Biochemistry 27, 1198-1205] led to very low values for netropsin binding constants on a restriction fragment from pBR-322 DNA. In this work, we show that, because the porphyrin binds with high specificity to DNA, it does not report site loading information in the same manner as does DNase I. We elucidate a model involving binding equilibria for individual sites and include competitive binding of drug and porphyrin for the same site. The free porphyrin and free drug concentrations are determined by binding equilibria with the carrier (calf thymus DNA) which is present in excess and acts as a buffer for both. Given free porphyrin and free netropsin concentrations for each total drug concentration in a series of footprinting experiments, one can calculate autoradiographic spot intensities in terms of the binding constants of netropsin to the various sites on the 139 base pair restriction fragment. The best values of these binding constants are determined by minimizing the sum of the squared differences between calculated and experimental footprinting autoradiographic spot intensities. Although the determined netropsin binding constants are insensitive to the value assumed for the porphyrin binding constant toward its highest affinity sites, the best mean-square deviation between observed and calculated values, D, depends on the choice of (average) drug binding constant to carrier DNA, Kd.(ABSTRACT TRUNCATED AT 250 WORDS)

Viscometric titrations of bacteriophage PM2 closed circular DNA, in addition to spectrophotometric and fluorometric methods, were used to investigate the mode of DNA binding of a number of antitrypanosomal and antitumor compounds. Several classes of compounds were identified which failed to unwind PM2 DNA, which appeared to have a large DNA binding site of at least 4 base pairs and which often showed considerable selectivity of binding to poly[d(AT)] as opposed to poly[d(GC)]. The classes included the antiviral antibiotics distamycin and netropsin, bisamidines such as the trypanocidal drug berenil, phthalanilide bisamidines, aromatic bis(guanylhydrazones), and the bisquaternary ammonium heterocycles. It is proposed that the compounds all bind in the minor groove of the DNA double helix.

/sup 1/H and /sup 31/P NMR spectral analysis of a chromomycin/d(ATGCAT)/sub 2/ complex provides strong evidence for a nonintercalative mode of drug binding. Investigation of the imino proton region of the duplex suggests a protection of one of the two guanine imino protons from fast exchange with the bulk water up to at least 45 /sup 0/C by the drug. Subsequent one-dimensional nuclear Overhauser enhancement experiments place the exchangeable chromomycin chromophoric hydroxyl proton

Comparative DNA equilibrium binding studies with mithramycin (MTR) and ethidium bromide in the presence and in the absence of second drugs were investigated by spectral titrations. Unusual curvatures (in contrast to those due to neighbor exclusion or anticooperativity) are found in the Scatchard plots of MTR-DNA titrations in the presence of netropsin, a minor-groove binder. Parallel studies with ethidium bromide indicate that although the presence of netropsin significantly reduces the binding ability of ethidium, no unusually curved Scatchard plots are obtained. The unusual curvature exhibited by the Scatchard plots of MTR titrations in the presence of netropsin indicates that the binding of netropsin greatly affects the MTR binding to DNA and can be simulated by an explicit incorporation of the second drug-DNA interaction in the binding formalism. Since netropsin is a minor-groove binder, its interference with the binding of MTR is in accord with the notion that MTR also binds at this groove. The observation of negligible effects on the DNA binding ability of MTR in the presence of either a major-groove or a phosphate group binder lends further support to this conclusion. Consistent with its guanine specificity, studies with synthetic polynucleotides suggest that MTR exhibits negligible affinity for poly(dA-dT).poly(dA-dT) or poly(dA).poly(dT).(ABSTRACT TRUNCATED AT 250 WORDS)

Distamycin and netropsin, a class of minor groove binding nonintercalating agents, are characterized by their B-DNA and A-T basespecific interactions. To understand the CQI I ~OIT~ ~ I ~ ~aOnMd ~c hemical basis of the above specificities, the DNA-binding characteristics of a novel synthetic analogue of distamycin have been studied. The analogue, mPD derivative, has the requisite charged end groups and a number of potential hydrogen-bonding loci equal to those of distamycin. The difference in the backbone curvatures of the ligands, distamycin, the mPD derivative, and NSC 101327 (another structurally analogous compound),is a major difference between these ligands. UV and CD spectrosoopic studies reported here show the following salient features: The mPD derivative recognizes only B-DNA, to which it binds via the minor groove. On the other hand, unlike distamycin, it binds with comparable affinities to A-T and G-C base pairs in a natural DNA. These DNA-binding properties are compared with those reported earlier for distamycin and NSC 101327 [Zimmer, Ch., & Wahnert, U. (1986) Prog. Biophys. Mol. Biol. 47, 31-1121. The backbone structures of these three ligands were compared to show the progressive decrease in curvatures in the order distamycin, mPD derivative, and NSC 101327. The plausible significance of the backbone curvature vis-&vis the characteristic B-DNA and AT-specific binding of distamycin is discussed. To our knowledge, this is the first attempt (with a model synthetic analogue) to probe the possible influence of backbone curvature upon the specificity of interactions of the distamycin class of groove-binding ligands with DNA.

The molecular structure of the complex between a minor groove binding drug (netropsin) and the DNA dodecamer d(CGCGATATCGCG) has been solved and refined by single-crystal X-ray diffraction analysis to a final R factor of 20.0% to 2.4-A resolution. The crystal is similar to that of the other related dodecamers with unit cell dimensions of a = 25.48 A, b = 41.26 A, and c = 66.88 A in the space group P2(1)2(1)2(1). In the complex, netropsin binds to the central ATAT tetranucleotide segment in the narrow minor groove of the dodecamer B-DNA double helix as expected. However, in the structural refinement the drug is found to fit the electron density in two orientations equally well, suggesting the disordered model. This agrees with the results from solution studies (chemical footprinting and NMR) of the interactions between minor groove binding drugs (e.g., netropsin and distamycin A) and DNA. The stabilizing forces between drug and DNA are provided by a combination of ionic, van der Waals, and hydrogen-bonding interactions. No bifurcated hydrogen bond is found between netropsin and DNA in this complex due to the unique dispositions of the hydrogen-bond acceptors (N3 of adenine and O2 of thymine) on the floor of the DNA minor groove. Two of the four AT base pairs in the ATAT stretch have low propeller twist angles, even though the DNA has a narrow minor groove. Alternating helical twist angles are observed in the ATAT stretch with lower twist in the ApT steps than in the TpA step.

A theory for deriving drug-DNA site binding constants from footprinting data is presented. Plots of oligonucleotide concentration, as a function of drug concentration, for various cutting positions on DNA are required. It is assumed that the rate of cleavage at each nucleotide position is proportional to the concentration of enzyme at that nucleotide and to the probability that the nucleotide is not blocked by drug. The probability of a nucleotide position not being blocked is calculated by assuming a conventional binding equilibrium for each binding site with exclusions for overlapping sites. The theory has been used to evaluate individual site binding constants for the antiviral agent netropsin toward a 139 base pair restriction fragment of pBR-322 DNA. Drug binding constants, evaluated from footprinting data in the presence of calf thymus DNA and poly(dGdC) as carrier and in the absence of carrier DNA, were determined by obtaining the best fit between calculated and experimental footprinting data. Although the strong sites on the fragment were all of the type (T.A)4, the value of the binding constant was strongly sequence dependent. Sites containing the dinucleotide sequence 5'-TA-3' were found to have significantly lower binding constants than those without this sequence, suggesting that an adenine-adenine clash produces a DNA structural alteration in the minor groove which discourages netropsin binding to DNA. The errors, scope, and limitations associated with the method are presented and discussed.

We report the first measurement of the magnetic circular dichroism (MCD) of the basic polypeptide antibiotic netropsin (Nt). The MCD shows that the longest wavelength absorption band of Nt is the sum of more than one component and permits a radically new interpretation of the circular dichroism of the complex which Nt forms with DNA. We conclude that Nt has no major effect on the CD and thus the helical structure of the bases of the DNA to which it is bound. Thus the ability of Nt to inhibit the function of DNA polymerase, RNA polymerase, and the photoreactivating enzyme must be mediated by factors other than a distortion of the helical structure of the bases.

The lifetimes for exchange of the imino protons in the dodecanucleotide d(C-G-C-G-A-A-T-T-C-G-C-G) upon binding of netropsin and/or actinomycin have been measured by proton nuclear magnetic resonance experiments. At high temperature these lifetimes were found to measure the lifetimes for opening of the base pairs in the double helix. Comparison of the opening rates in the dodecamer with those in the complex with netropsin (which binds at the -A-A-T-T- sequence) shows that there is not only a large kinetic stabilization of the A-T base pairs at the binding site but also a significant stabilization of the G-C base pairs adjacent to the netropsin binding site. For the complex with actinomycin, which intercalates at the G-C sites in the double strand, the lifetimes of the base pairs at the binding site increase upon binding of actinomycin, and the A-T base pairs in the central core are slightly kinetically destabilized by the actinomycin binding. The activation energies for exchange of the imino protons were also measured in the complexes and indicate that the mechanism for exchange of the imino protons is individual base-pair opening, where one base pair opens independently of the others. The effects of drug binding on the dynamics of individualmore » base pairs in a double-stranded helix are discussed.« less