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The antitumor effects of platinum(IV) complexes, considered prodrugs for cisplatin, are believed to be due to biological reduction of Pt(IV) to Pt(II), with the reduction products binding to DNA and other cellular targets. In this work we used pBR322 DNA to capture the products of reduction of oxoplatin, c,t,c-[PtCl(2)(OH)(2)(NH(3))(2)], 3, and a carboxylate-modified analog, c,t,c-[PtCl(2)(OH)(O(2)CCH(2)CH(2)CO(2)H)(NH(3))(2)], 4, by ascorbic acid (AsA) or glutathione (GSH). Since carbonate plays a significant role in the speciation of platinum complexes in solution, we also investigated the effects of carbonate on the reduction/DNA-binding process. In pH 7.4 buffer in the absence of carbonate, both 3 and 4 are reduced by AsA to cisplatin (confirmed using ((195))Pt NMR), which binds to and unwinds closed circular DNA in a manner consistent with the formation of the well-known 1, 2 intrastrand DNA crosslink. However, when GSH is used as the reducing agent for 3 and 4, ((195))Pt NMR shows that cisplatin is not produced in the reaction medium. Although the Pt(II) products bind to closed circular DNA, their effect on the mobility of Form I DNA is different from that produced by cisplatin. When physiological carbonate is present in the reduction medium, ((13))C NMR shows that Pt(II) carbonato complexes form which block or impede platinum binding to DNA. The results of the study vis-à-vis the ability of the Pt(IV) complexes to act as prodrugs for cisplatin are discussed.

For Jurkat cells in culture exposed to cisplatin (1), we measured the number of platinum adducts on DNA and showed that it is proportional to the AUC, the area under the concentration vs time curve, for cisplatin. The number of platinum-DNA adducts is measured immediately following exposure to drug. The AUC is calculated either as the product of the initial cisplatin concentration and the exposure time or as the integral under the concentration vs time curve for the unreacted dichloro species, which decreases exponentially. We also show that the number of adducts correlates with decreases in respiration, with the amount of DNA fragmentation, and with cell viability, all measured 24 h after exposure to the drug. To study the reactions of cisplatin at concentrations approaching clinical relevance (65 microM), we use two-dimensional [1H15N]HSQC NMR and the 15N-labeled form of the drug, cis-Pt(15NH3)2Cl2, 1. In the absence of cells, 1 reacts with components of the growth medium and also transforms slowly (k(h) = 0.205 h-1 at 37 degrees C) into the chloro-aquo species, cis-[Pt(15NH3)2Cl(H2O)]+ (2), which at the pH of the medium (pH 7.15), is mainly in the deprotonated chloro-hydroxy form, cis-Pt(15NH3)2Cl(OH) (4). The concentration of 2 (4), as measured by HSQC NMR, decreases due to reaction with components of the medium. In the presence of 5 million or more cells, the concentration of 1 decreases with time, but the NMR signal for 2 (4) is not seen because it is rapidly removed from solution by the cells, keeping its concentration very low. These experiments confirm that the species preferentially removed from the medium by cells is 2 (4) and not 1. Our findings are discussed in the context of a kinetic model for platination of nuclear DNA by cisplatin, which includes aquation of cisplatin outside the cell, passage of 2 (4) through the cell membrane, reaction of reactive platinum species (RPS) in the cytosol with thiols, formation of adducts between RPS and accessible sites on genomic DNA, and removal of platinum from DNA by repair. Some of the rate constants involved are measured, but others can only be estimated. Calculations with this model show that little of the platinum reacts with intracellular thiols before reaching the nuclear DNA, indicating that binding to thiols is not important in cisplatin resistance. The model also predicts the circumstances under which the amount of platination of nuclear DNA is proportional to AUC.

A Pt(IV) complex, 1-adamantanemethylamine–Pt(IV), 2, a potential prodrug of the Pt(II) anticancer agent cisplatin was synthesized by tethering 1-adamantanemethylamine to oxoplatin using a dicarboxylate linker. The host–guest inclusion complex of 2 with β-cyclodextrin, is less toxic toward neuroblastoma SK-N-SH cells than cisplatin. Reduction of the inclusion complex with ascorbic acid produces a Pt(II) product that binds to and unwinds pBR322 form I DNA in a manner that is similar to that of cisplatin.

We show how polyacrylamide gel electrophoresis of radiolabeled DNA can be used to measure the hairpin-duplex equilibrium constant for DNA in solution. As an aid to the interpretation of the experiments, the differential equations associated with diffusion, migration and chemical reaction of the DNA forms are solved and intensity patterns generated. Two kinds of experiments were performed on several DNA 12-mers: in the first, electrophoresis time was constant while DNA concentration varied; in the other, concentration was constant while time varied (a `load-and-run' gel). The observed patterns depended on the gel temperature and not the temperature at which the DNA was equilibrated before loading in the well, because reequilibration occurs before the DNA leaves the well to enter the gel proper. During this time, mixing also occurs, changing the concentration and ionic strength of the sample. A method of calculating the true DNA concentration, including the unmeasured concentration added with the radiolabel, is given. When the intensity pattern consists mainly of monomer and dimer peaks, the equilibrium constant K is easily calculated from peak intensities. However, when there is significant intensity between the peaks (which the calculations show results from monomer–dimer interconversion in the gel), K will be inaccurate. An accurate value of K may be determined from a load-and-run gel by extrapolating back to time 0. When the intensity pattern consists of a single broad peak (from rapid monomer-dimer interconversion in the gel), K cannot be calculated without additional information. The rate of interconversion increases with temperature. Estimated rates in the gel are more than an order of magnitude smaller than in bulk solution at the same temperature. Derived values of K for several DNAs are compared with literature values.

A kinetic analysis of cleavage of simian virus DNA (SV40 DNA) inside and outside green monkey BSC-1 cells by the enediyne-protein antibiotic C-1027 and its free chromophore is described. Information on rate constants was obtained by fitting populations of forms I (closed circular DNA), II (nicked circular DNA) and III (linear DNA) of SV40 DNA as a function of drug concentration to a kinetic model which includes: cutting of form I to give form II with rate constant k1, cutting of form I to give form III with rate constant k4, and cutting of form II to give form III with rate constant k2. The ratio of single-strand (ss) to double-strand (ds) cutting for the holoantibiotic and the free chromophore, k 1 k 4 , is approximately 1.8 for extracellular SV40 DNA. For intracellular DNA and extracellular DNA which has been post-treated with putrescine, ds cutting is much more probable, with k4 about four times as large as k1. This observation suggests that amine groups present in the cell are able to convert abasic sites opposite an ss break into a ds break in SV40 chromatin. The overall rate of cleavage of form-I DNA inside the cell is much larger than the rate outside, the sum k1 + k4 being about three times as large for intracellular DNA as for extracellular DNA.

The kinetics and mechanism of cleavage of DNA by the insulin-mimetic peroxo-vanadate NH4(VO(O2)2(phen)), pV, are described. In the presence of low energy UV radiation or biologically common reducing agents, pV decomposes into the monomer, dimer, and tetramer of vanadate and an uncharacterized compound of V 4+ as shown by 51 V NMR, ESR, and absorption spectra. The rate of photodecomposition of pV is reduced in the presence of calf thymus DNA, indicating that a decomposition product of the peroxo-vanadate, that is important in the destruction pathway of the complex, is interacting with DNA. This species, probably a short-lived complex of V 4+ , may also be responsible for the observed catalytic decomposition of pV in the absence of DNA by ascorbate. If closed circular pBR322 DNA is present when the peroxo-vanadate is destroyed by either UV radiation or reducing agents, the polymer may have its sugar-phosphate backbone broken. Closed circular DNA (form I) is converted into nicked circular DNA (form II) and linear DNA (form III). The amounts of the various forms produced as a function of irradiation time and peroxo-vanadate concentration were fit to a kinetic model to derive rate constants for the conversions. The kinetic analysis shows that pV is a single-strand nicking agent which exhibits some base and/or sequence preference. Furthermore, the pH dependences of the rates for conversion of form I to form II and for conversion of form II to form III are different, indicating that the nature of the chemistry at the site of cleavage on DNA influences further cutting by activated pV. Reduced amounts of DNA breakage in the presence of various salts and metal binding ligands indicate that a short-lived reactive complex of V 4+ , not the V 4+ species detected by ESR at long irradiation times, is important in the cleavage process. The susceptibility of pV to decomposition by biologically common reducing agents suggests that metabolites of the agent, and not the compound itself, are responsible for its insulin-mimetic effects.

Oligonucleotide-functionalized gold nanoparticles (AuNP) were designed and synthesized to be delivery vehicles for the clinically used anticancer drugs doxorubicin (DOX) and actinomycin D (ActD). Each vehicle contains a tailorable number of DNA duplexes, each possessing three high-affinity sequences for the intercalation of either DOX or ActD, thus allowing for control of drug loading. Drug binding was evaluated by measuring changes to DNA melting temperature, T(m), hydrodynamic diameter, D(h), and surface plasmon resonance wavelength, λ(spr), with drug loading. These studies indicate that DOX intercalates at its high-affinity sequence bound at the AuNP, and that ActD exhibits relatively weaker binding to its preferred sequence. Agarose gel electrophoresis further confirmed drug binding and revealed that particle mobilities inversely correlate with D(h). The equilibrium binding constant, K, and dissociation rate constant, β, were determined by dialysis. Results indicate that the high negative electrostatic potential within the DNA shell of the particle significantly decreases β and enhances K for DOX but has little effect on K and β for ActD. The cytotoxicity of the vehicles was studied, with IC(50) = 5.6 ± 1.1 μM and 46.4 ± 9.3 nM for DOX-DNA-AuNP and IC(50) = 0.12 ± 0.07 μM and 0.76 ± 0.46 nM for ActD-DNA-AuNP, in terms of drug and particle concentrations, respectively.

This review outlines the steps for obtaining relative binding constants for drugs from footprinting data. After correcting the autoradiographic spot intensities for differing amounts of radioactive DNA loaded into the lanes of a sequencing gel, footprinting plots, showing individual spot intensities as a function of drug concentration, are constructed. The initial relative slopes of footprinting plots are proportional to the binding constant of the drug for its DNA site. Slopes of plots outside of drug binding sites can be used to identify locations of altered DNA structure. It illustrates the power of quantitative footprinting analysis by analyzing the binding of the antiviral agent netropsin to a 139-base pair restriction fragment in the presence of the antitumor agent actinomycin D. While two netropsin binding regions are unaffected by actinomycin D a third region experiences enhanced binding in the presence of the antitumor agent.

Utilizing linear dichroism (LD), circular dichroism (CD), and fluorescence energy transfer, the binding geometries of a series of Co(3+)-porphyrins and their free ligands were examined. The compounds studied were Co-meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (CoTMPyP) and its free ligand (H2-TMPyP), Co-meso-tetrakis(N-n-butylpyridinium-4-yl)porphyrin (CoTBPyP) and its free ligand (H2TBPyP), and Co-meso-tetrakis(N-n-octylpyridinium-4-yl)porphyrin (CoTOPyP). The two non-metalloporphyrins exhibit negative LD, having angles of roughly 75 degrees relative to the DNA helix axis. They also display negative CD and a significant contact energy transfer from the DNA bases. On the other hand, the three metalloporphyrins display orientation angles of roughly 45 degrees between the porphyrin plane and the helix axis of DNA. Furthermore, they exhibit positive CD and no contact energy transfer from DNA bases. These observations show that the metalloporphyrins are not intercalated whereas non-metalloporphyrins having four freely rotating meso-aryl groups intercalate between the base pairs of DNA. In the presence of KHSO5, the cobalt porphyrins cleave closed circular PM2 DNA in a single strand manner, i.e., a single activation event on the porphyrin leads to a break in one of the DNA strands. A kinetic analysis of the cleavage data revealed that cleavage rates are in the order CoTMPyP > CoTBPyP > CoTOPyP with the difference being due to different DNA affinities rather than differences in cleavage rate-constants. Based on these and earlier observations, the metalloporphyrins appear bound to a partially melted region of DNA.

Equilibrium binding studies and viscosity experiments are described that characterize the interaction of delta- and lambda-[Ru(o-phen)3]2+ with calf thymus DNA. The mode of binding of these compounds to DNA is a matter of controversy. Both isomers of [Ru(o-phen)3]2+ were found to bind but weakly to DNA, with binding constants of 4.9 (+/- 0.3) x 10(4) M-1 and 2.8 (+/- 0.2) x 10(4) M-1 determined for the delta and lambda isomers, respectively, at 20 degrees C in a solution containing 5 mM Tris-HCl (pH 7.1) and 10 mM NaCl. We determined that the quantity delta log K/delta log [Na+] equals 1.37 and 1.24 for the delta and lambda isomers, respectively. Application of polyelectrolyte theory allows us to use these values to show quantitatively that both the delta and lambda isomers are essentially electrostatically bound to DNA. Viscosity experiments show that binding the lambda isomer does not alter the relative viscosity of DNA to any appreciable extent, while binding of the delta isomer decreases the relative viscosity of DNA. From these viscosity results, we conclude that neither isomer of [Ru(o-phen)3]2+ binds to DNA by classical intercalation.

In footprinting experiments, an increase in DNA cleavage with addition of ligand to a system may be due to a ligand-induced structural change. Ligand binding also enhances cleavage by displacing the cleavage agent from ligand-binding sites, thus increasing its concentration elsewhere. The theory and characteristics of this mass-action enhancement are given, and it is shown how it may be recognized. Results of DNase I footprinting of small oligomers, with actinomycin D as ligand, are analyzed to reveal which enhancements are due to mass action, and which can reasonably be ascribed to structural changes. Patterns in the footprinting plots from our experiments on actinomycin D binding to a 139-base-pair DNA fragment (with DNase I as a probe) are studied in the same way. The likely origins of these patterns are discussed, as are enhancements occurring with other probes commonly used in footprinting experiments.

A coupled kinetic analysis of esperamicin, calicheamicin, and DNase I cleavage of covalently closed circular PM2 DNA has been carried out. Analysis of the optical density data derived from agarose gel electrophoresis experiments shows that esperamicin A,, like the hydrolytic enzyme DNase I, produces mainly single-strand breaks in DNA. These agents cause covalently closed circular form I DNA to be initially converted to nicked circular form I1 DNA. However, the ratio of the rate constant for this process (k,') to that associated with conversion of form I1 to linear form I11 DNA ( k i ) is not consistent with completely random nicking, and some double-strand cleavage may occur. The values of k , ' / k i found for DNase I and esperamicin A, were 5.4 nd 3.9, respectively. The behavior of these agents sharply contrasts with that of esperamicin C and calicheamicin, for which double-strand cleavage of DNA is deduced from the analysis. Although the rate constant for introducing the first break in DNA for calicheamicin is lower than the corresponding rate constant for esperamicin C, the second break (in the opposing strand) is fast for calicheamicin, making it the better double-strand cleaving agent. These drugs are unique among antitumor agents in that a single activation event on the warhead portion produces a double-strand break in DNA without the need to posttreat the DNA with other agents in order to induce a cleavage. The cleavage kinetics are discussed in terms of the structural differences in these unusual anticancer drugs. The newly discovered anticancer drugs esperamicin1*2 and calicheamicin3*' (Figure 1) exhibit high potency against murine tumor lines. These compounds possess sugar groups appended to an unusual 1,5-diyn-3-ene core structure referred to as the uwarheadn. In the presence of reducing agents and D N A the warhead undergoes a rearrangement to create a phenylene diradical that causw strand breaks by attacking the sugar groups (1) Golik, J.; Clardy, J.; Dubay, G.; Groenewold, G.; Kawaguchi, H.; Konishi, M.; Krishnan, B.; Ohkuma, H.; Saitoh, K.4.; Doyle, T. W. J. Am. Chcm. Soc. 1987. 109. 3461-3462. Golik. J.: Dubav. G.: Groenwold. 0.: Kawaguchi, H.; Konishi, M.; Krishnan. B.; Ohhma, H.i Saitoh, K.4.; Doyle; *TO whom correspondence should be a d d r d . T. W. Ibid. 1987, 109, 3461-3462. 0002-7863/91/1513-5434$02.50/0 CP 1991 American Chemical Society DNA Cleavage In\ J . Am. Chem. Soc., Vol. 113. No. 14, I991 5435

The reaction of aged carboplatin (reaction of carboplatin in 24 mM NaHCO(3) for 45 h, 37 degrees, pH 8.6) with pBR322 DNA at 0 < r < 2.8, where r = [drug]/[DNA-bp], in 24 mM HEPES buffer, pH 7.4, for 24 h, followed by agarose gel electrophoresis showed DNA mobility changes consistent with unwinding closed circular DNA. However, identical experiments conducted in a two-buffer system, 24 mM HEPES plus 24 mM carbonate, showed no DNA mobility changes, indicating that carbonate blocks formation of the 1,2 intrastrand cross-link on DNA. Studies with aged carboplatin and with cisplatin carried out with ca. 4.0 < r < 10.0 in the two-buffer system show that some DNA binding and unwinding occurs for both drugs. Since carbonate inhibits the binding of aged carboplatin and cisplatin to DNA, carbonate present in the body likely modulates the reactivity of these drugs with a variety of biological targets including DNA.

The antitumor and DNA-binding properties of a group of oligomeric platinum(II) and platinum(IV) complexes are described. The compounds, having the stoichiometry [cis-Pt II (X) 2 (μ-OH] 2 (NO 3 ) 2 , where X is NH 3 , NH 2 CH 2 CH 3 , and NH 2 CH(CH 3 ) 2 , were found to be inactive or only weakly active against L-1210 leukemia. In vitro studies involving PM2-DNA show that these compounds bind to an unwind closed circular DNA in a manner similar to cis-Pt II -(NH 3 ) 2 Cl 2 . The Pt(IV) complexes produced by hydrogen peroxide oxidation of the Pt(II) dimers are inactive as antitumor agents and are incapable of unwinding PM2-DNA. The cyclotrimer [cis-Pt II (RR-DACH)(μ-OH)] 3 (NO 3 ) 3 , where RR-DACH is (R,R)-1,2 diaminocyclohexane, exhibits potent antitumor activity against L-1210 leukemia and modest activities with B-16 and M5076 tumor lines. This compound platinates DNA, causing DNA unwinding and mobility shifts

A series of tether-linked bis netropsins have been synthesized in order to assess the phasing problem, which arises because of the lack of dimensional correspondence between oligopeptides and oligonucleotides in DNA binding characteristics. The consequences of incorporating variable-length flexible and rigid tethers [poly(methylene), Z and E ethylene, m- and p-phenylene] between the two netropsin-like moieties on the DNA binding properties were assessed by DNase I footprinting. The conformational freedom associated with two netropsins linked by a flexible methylene tether allows ligand binding in both a mono- and bidentate fashion, with bidentate binding requiring a minimum linker length of (CH2)3. For compounds possessing rigid tethers, for example, cis and trans ethylene moieties, the cis geometry excludes bidentate ligation while the trans structure favors it. Bis netropsins possessing aryl linking groups have reduced DNA binding affinities. This is most plausibly due to the aryl groups, which are not coplanar with the netropsin moieties, thus blocking the ligand from penetrating deeply into the minor groove of DNA.

We report on the binding of cisplatin, carboplatin and oxaliplatin to double-stranded DNA in two different (phosphate and carbonate) buffers, using an electrochemical DNA-biosensor. The propensity of the electrophilic agent produced by hydrolysis to interact with DNA was measured as a function of the decrease of guanine oxidation signal of the metal-DNA adduct immobilized on a screen-printed electrode, by using square wave voltammetry. The results obtained confirm that carbonate reacts with platinum drugs to form activated carbonato complexes, which are able to react readily with DNA.

Carbonate in its various forms is an important component in blood and the cytosol. Since, under conditions that simulate therapy, carbonate reacts with cisplatin to form carbonato complexes, one of which is taken up and/or modified by the cell [C.R. Centerwall, J. Goodisman, D.J. Kerwood, J. Am. Chem. Soc., 127 (2005) 12768–12769], cisplatin-carbonato complexes may be important in the mechanism of action of cisplatin. In this report we study the binding of cisplatin to pBR322 DNA in two different buffers, using gel electrophoresis. In 23.8 mM HEPES, N -(2-hydroxyethyl)-piperazine- N ′-2-ethanesulfonic acid, 5 mM NaCl, pH 7.4 buffer, cisplatin produces aquated species, which react with DNA to unwind supercoiled Form I DNA, increasing its mobility, and reducing the binding of ethidium to DNA. This behavior is consistent with the formation of the well-known intrastrand crosslink on DNA. In 23.8 mM carbonate buffer, 5 mM NaCl, pH 7.4, cisplatin forms carbonato species that produce DNA-adducts which do not significantly change supercoiling but enhance binding of ethidium to DNA. This behavior is consistent with the formation of a monofunctional cisplatin adduct on DNA. These results show that aquated cisplatin and carbonato complexes of cisplatin produce different types of lesions on DNA and they underscore the importance of carrying out binding studies with cisplatin and DNA using conditions that approximate those found in the cell.

DNA platination by cisplatin (CDDP) was investigated in peripheral blood mononuclear cells and ovarian cancer cells using atomic absorption spectroscopy. Plots showing the amount of platinum (Pt) bound to DNA versus the molar concentration of cisplatin in the incubation medium ([CDDP]) were nonlinear. For [CDDP] < about 5 microM, the amount of Pt bound to DNA increased slowly with added drug. However, for larger [CDDP], the slope of the plot increased significantly. To study the role of thiols in affecting cisplatin binding to DNA, cells were treated with N-ethylmaleimide, which modifies thiol groups, rendering them incapable of binding cisplatin. Analysis using high-pressure liquid chromatography showed that approximately 99% of cellular glutathione was modified by N-ethylmaleimide. A plot of the amount of Pt bound to DNA versus [CDDP] for thiol-blocked cells is linear, with a slope similar to that of unblocked cells at high [CDDP]. Neither S-2-(3 aminopropylamino)ethanethiol (WR-1065) nor mesna, when added at clinically achievable concentrations (i.e., < approximately 300 microM), affected DNA platination. However, DNA platination was totally abolished by millimolar concentrations of the drug thiols (approximately 1.25 mM WR-1065 or approximately 5 mM mesna). Thus, the data show that endogenous thiols intercept cellular cisplatin, but this mechanism is less important at high [CDDP]. Moreover, therapeutic concentrations of drug thiols do not significantly affect DNA platination. A simple model that reproduces the experimental results of the amount of cisplatin binding to DNA as a function of [CDDP], time, and thiol content is proposed. The model takes into account passage of cisplatin and thiols through the cell membrane, binding of cisplatin to cellular thiols, and platination of DNA.

Various cleavage agents interact with circular double-stranded DNA molecules to convert the closed-circular form (form-I) to the open-circular (form-II) and linear (form-III) forms, and ultimately to small DNA fragments. The various cutting processes which take place in the DNA pool are here analyzed kinetically, and, by solving the kinetic equations, expressions are derived for the amounts of the closed-circular, open-circular, and linear forms of DNA as a function of reaction time and concentration of cleavage agent. Conversions between subspecies of forms II and III, differing in numbers of internal cuts, are taken into account. The only assumption required to solve the kinetic equations is that the concentration of cleavage agent obeys [D] = D0 f(t) where the function of time f(t) is independent of D0, the initial concentration of cleavage agent. By choosing parameters in the expressions for the calculated amounts of forms I, II and III to give the best fit to the measured amounts, one obtains information about the rates and rate constants for the conversions. The rate constants in turn give important information about the specificity and mechanism of action of the cleavage agents. The analysis is applied to the cleavage of pBR322, SV-40 and PM2 DNAs by DNase I, Fe-EDTA, and the antitumor agents calicheamicin and bleomycin. Cleavage rate constants are derived and discussed for these systems.

The fluorescence of ethidium bromide (EB) bound to equimolar amounts of supercoiled form I and unstrained linear form III pBR322, SV40 and PM2 DNA in agarose gels has been measured by scanning a photographic negative of the gel with a microdensitometer. For SV40 and PM2 DNA, commonly used staining conditions cause both forms, i.e. linear and supercoiled, to fluoresce to the same extent. This obviates the need to use a correction factor for the fluorescence of form I DNA when measuring the amount of this form relative to the amounts of unstrained forms in agarose gels. In the case of PBR322 DNA, form I was found to fluoresce approximately 20% more than form III DNA.

Chemically and photochemically induced cleavage of DNA by the insulin-mimetic compound NH4[VO(O2)2-(1,10-phenanthroline)], bpV(phen), have been studied. 51V NMR and absorption indicate that photoirradiation with low energy UV light of aqueous solutions containing bpV(phen) leads to the conversion of the compound to simple vanadates. Photoillumination of the compound in the presence of supercoiled pBR322 DNA results in cutting of the plasmid to produce nicked circular and linear DNA. Quantitative analysis of agarose gel data shows that bpV(phen) is a single strand nicking agent exhibiting sequence and/or base specificity.

The preparation and purification of PCR generated DNA fragments suitable for footprinting and classical drug binding studies is described. One of the fragments, a 214-mer derived from pBR322 DNA exhibits a biphasic melting profile. This behavior appears to be due to a non-random distribution of base pairs within the fragment causing a region rich in AT base pairs to melt prior to a segment having a high concentration of GC base pairs. The usefulness of large amounts of PCR generated DNA for footprinting and optical binding studies involving drugs is also presented and discussed.

The anticancer drug, doxorubicin (DOX), was loaded onto DNA-capped gold nanoparticles (AuNP) designed for specific DOX intercalation. Drug binding was confirmed by monitoring DNA melting temperature, AuNP plasmon resonance maximum, and hydrodynamic radius increase, as a function of [DOX]/[DNA] ratio. The capacity for drug release to target DNA was confirmed.

Absorbance and fluorescence methods, circular dichroism, UV melting experiments, viscosity, and competition dialysis were used to study the interaction of delta and lambda tris(phenanthroline)ruthenium(II) with DNA. The results of these studies indicated that both isomers bind to DNA by a single mode. The two isomers differ, however, in their effect on the hydrodynamic properties of DNA as measured by viscosity and, therefore, probably differ in their individual binding modes. The optical properties of the fully bound compounds differ from those of the free, but the perturbations of their visible absorbance and fluorescence emission spectra are modest when compared to changes observed for other DNA binding compounds. Binding of both isomers to DNA was found to be weak (in comparison to proven intercalators), with binding constants on the order of 10(4) M-1 determined for their binding to calf thymus DNA. A small, positive enthalpy was found for the binding of each isomer to DNA, suggesting that binding is entropically driven. Both isomers increased the melting temperature of DNA, with little quantitative difference between the two. A modest base specificity was found for each isomer, with the delta isomer preferentially binding to GC base pairs, and the lambda isomer preferentially binding to AT base pairs. Competition dialysis was used to examine the preference of delta and lambda Ru for right-handed B DNA and left-handed Z DNA. Neither isomer exhibits significant selectivity for these radically different DNA secondary structures.

Quantitative footprinting methods have been used to measure site-specific binding constants for the anticancer drug actinomycin D on a restriction fragment from pBR 322 DNA. Sites with the highest binding constants, 1–6 x 105M-1, possess the classical binding sequence GC, which is flanked on the 5’ side by either T or C, but not G. The sequence GGC is a weaker binding site, except for one case. A non-classical sequence, CGTC, was found to be a strong actinomycin D site. However, the value of the binding constant for this site and for other classical sites was dependent on the proximity of sites on the 139-mer and/or the nature of flanking sequences. Comparison with earlier studies indicates that the length of DNA may be important, with short oligomers yielding higher binding constants than polymeric DNA. The analysis also revealed that actinomycin D can cause anomalous rate enhancements adjacent to certain of its interaction sites when binding is probed with DNase I and the metalloporphyrin, MnT4MPyP, but not with Fe-MPE. This indicates that regions adjacent to drug binding may have altered minor groove width and/or exist in a partially melted state.

We report site-specific binding constants for the intercalating anticancer drug actinomycin D (Act-D), binding to a 139-base-pair restriction fragment from pBR 322 DNA. The binding constants are derived from analysis of footprinting experiments, in which the radiolabeled 139-mer is cleaved using DNase I, the cleavage products undergo gel electrophoresis, and, from the gel autoradiogram, spot intensities, proportional to amounts of cleaved fragments, are measured. A bound drug prevents DNase I from cleaving at approximately 7 bonds, leading to decreased amounts of corresponding fragments. With the radiolabel on the 3' end of the noncoding strand (A-label), we measured relative amounts of 54 cleavage products at 25 Act-D concentrations. For cleavage of the 139-mer with the label on the 3' end of the coding strand (G-label), relative amounts of 43 cleavage products at 11 Act-D concentrations were measured. These measurements give information about approximately 120 base pairs of the restriction fragment (approximately 12 turns of the DNA helix); in this region, 14 strong and weak Act-D binding sites were identified. The model used to interpret the footprinting plots is derived in detail. Binding constants for 14 sites on the fragment are obtained simultaneously. It is important to take into account the effect of drug binding at its various sites on the local concentration of probe elsewhere. It is also necessary to include in the model weak as well as strong Act-D sites on the carrier DNA which is present, since the carrier DNA controls the free-drug concentration. As expected, the strongest sites are those with the sequence (all sequences are 5'----3') GC, with TGCT having the highest binding constant, 6.4 x 10(6) M-1. Sites having the sequence GC preceded by G are weak binding sites, having binding constants approximately 1 order of magnitude lower than those of the strong sites. Also, the non-GC-containing sequences CCG and CCC bind Act-D with a binding constant comparable to those of the weak GGC sites. The analysis may reveal drug-induced structural changes on the DNA, which are discussed in terms of the mechanism of Act-D binding.

The theory for measuring ligand binding constants from footprinting autoradiographic data associated with a single binding site is derived. If the ligand and DNA cleavage agent compete for a common site, the spot intensities are not proportional to the amount of DNA not blocked by ligand. The analysis of a single site is experimentally illustrated by using results for the anticancer drug actinomycin D interacting with the duplex d(TAGCGCTA)2 as probed with the hydrolytic enzyme DNase I.

A system comprised of a linear scanning microdensitometer interfaced to a personal computer has been developed to facilitate analysis of ligand-DNA footprinting autoradiograms. The system, which can be used to record density and sequence information from autoradiographic films, enables the user to relate the area under an autoradiographic band to the concentration of radiolabeled molecules present in the electrophoresis gel. This report describes the computer program which performs the calculations and discusses the ability of the system to accurately determine oligonucleotide concentration, as a function of band separation, photographic response, and the computational algorithm used to calculate band areas.

Quantitative footprinting studies involving a 139-base pair restriction fragment from pBR322 DNA, a lexitropsin ligand and two different DNA cleavage agents, the enzyme DNase I and the footprinting reagent Fe(III) methidium-propyl-EDTA (Fe-MPE), are described. The autoradiographic data showed that the ligand, an analogue of netropsin possessing two N-methylimidazole groups, binds to four regions on the 139-mer which are rich in GC. Analysis of the data leading to individual binding constants for each of the four loading events on the 139-mer revealed that Fe-MPE and DNase I report the same binding constants for the lexitropsin bound to its interaction sequences. The fact that the data from both probes can be analyzed using a common model indicates that the DNA cleavage specificity of the probe and not its binding/cleavage mechanism is the important factor in reporting of site loading information in the footprinting experiment. The study also showed that under certain conditions it is possible to gain information on the density of ligand binding sites on carrier DNA by monitoring site loading events on the labeled fragment.

Raman spectra have been obtained for bleomycin A2 and selected fragments. Virtually all of the features in the bleomycin Raman spectrum can be attributed to the preresonance enhanced vibrational transitions of the 2,4′-bithiazole moiety as determined by comparison to the Raman spectra of bithiazole fragments. The Raman spectrum of bleomycin is dominated by a very intense band at 1542 cm−1 that can be approximately assigned as a bithiazole ring stretching mode. This feature may serve as a probe of bithiazole environment in bleomysin -DNA interactions.

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)

The interaction of Cu(II) with the native and heat-denatured DNA complexes of daunomycin (1) and N-(trifluoroacetyl)daunomycin (3) has been examined by using absorption and circular dichroism spectroscopies. At low rt, where rt is the input molar ratio of drug to DNA phosphate, Cu(II) interacts with the native and heat-denatured calf thymus DNA complex of daunomycin to form a ternary complex involving the aglycon portion of the antibiotic, Cu(II), and DNA. A Job plot of the titration involving Cu(II) and heat-denatured DNA shows that the Cu(II)-drug stoichiometry in the ternary complex is less than or equal to 1. Although the N-(trifluoroacetyl)daunomycin-native DNA complex does not form a ternary complex, the denatured DNA complex with the antibiotic does. Copper(II) titrations of the daunomycin-native DNA complex, at high rt, where both strongly and weakly bound antibiotic molecules are very likely present in solution result in the formation of both the ternary species as well as a binary complex involving only the metal ion and the antibiotic. The spectroscopic results indicate that in the ternary complex, the Cu(II) ion is bound to the unintercalated aglycon portion of the antibiotic and very likely also to the heterocyclic bases of DNA.

Methods for determining sequence specificities of anticancer drugs, carcinogens, and mutagens which interact with natural DNA's are presented. For drugs which nick or covalently bind to DNA and thus leave a permanent record of their residence position on the helix, the sequences important in drug action can be readily determined. For agents which interact with DNA in an equilibrium fashion, "footprinting" analysis, a technique used to investigate protein-DNA binding, has proved to be useful in studying drug-DNA interactions. The sequence specificities of a number of small ligands which interact with natural DNA's are also presented.

The DNA cleavage properties of two cationic manganese porphyrins possessing different peripheral substituents were compared. The identical nature of the strand scission patterns of the porphyrins on a 139 base pair restriction fragment of pBR-322 DNA, along with other evidence, suggests that the porphyrin is end-on bound via the minor groove in a melted or partially melted region of DNA. This unusual binding mode underscores the potential of outisde binding cationic metalloporphyrins as probes for low melting regions of DNA

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.

The results of a series of quantitative footprinting experiments of the netropsin-DNA interaction as studied using two different DNA cleaving probes, the enzyme DNase I and a cationic manganese porphyrin complex, are described. Plots of the relative change in oligonucleotide concentration as a function of drug concentration, covering approximately 110 base pairs of a DNA restriction fragment, revealed netropsin induced changes in the cleavage rates of both probes. These appeared as inhibitions for the binding sites, enhancements where no binding took place, and enhancement/inhibitions for the weak binding sites. Determination of the concentration of drug necessary to reduce the amount of a particular oligomer to half of its initial value allowed a ranking of the affinities of the various binding sites on the fragment. In addition to uncovering the location of a number of overlapping netropsin binding sites, the data allowed additional insight on the manner in which both probes alter their DNA cleavage rates in the drug-footprinting experiment.

In the footprinting experiment, an end-radiolabeled DNA restriction fragment is subjected to digest by an endonuclease in the presence and absence of a ligand which alters the endonuclease cleavage rate at sites of ligand-DNA contact. The location of these sites, and the strength of the ligand binding, are then deduced from the measured concentrations of the different oligonucleotides produced by the digest. We analyze the experiment in terms of coupled kinetic equations which take into account the cutting rates of endonuclease for sites with ligand present and absent, and the rates of binding and dissociation of the ligand to a site. As long as the ligand concentration remains essentially constant (which occurs, for example, if digest is terminated early enough to assure that all fragments result from single cuts by the endonuclease), the oligonucleotide concentrations reflect only the ligand binding equilibrium constant (ratio of rate constants) and the cutting rates in the presence and absence of ligand. We also show how the measured oligonucleotide concentrations (from, e.g. an autoradiogram) can be used to deduce the ligand equilibrium binding constants for the various sites on the polymer.

The sequence specific binding of the antibiotic (4S)-(+)-dihydrokikumycin B and its (4R)-(-) enantiomer, [(S)-1 and (R)-1, respectively] to DNA were characterized by DNase I and MPE footprinting, calorimetry, UV spectroscopy, circular dichroism, and 1H NMR studies. Footprinting analyses showed that both enantiomers [(S)-1 and (R)-1] bind to AT-rich regions of DNA. 1H NMR studies (ligand induced chemical shift changes and NOE differences) of the dihydrkikumycins with d-[CGCAATTGCG]2 show unambiguously that the N to C termini of the ligands are bound to 5'-A5T6T7-3' reading from left to right. From quantitative 1D-NOE studies, the AH2(5)-ligand H7 distance of complex A [(S)-1 plus decamer (which is bound more strongly)] and complex B [(R)-1 and decamer] are estimated to be 3.8 +/- 0.3 A and 4.9 +/- 0.4 A, respectively. This difference in binding properties is reflected in the thermodynamic profiles of the two enantiomeric ligands determined by a combination of spectroscopic and calorimetric techniques. The binding free energies (delta G degrees) of (S)-1 and (R)-1 to poly d(AT).poly d(AT) at 25 degrees C are -31.8 and -29.3 kJ mol-1, respectively while the corresponding binding enthalpies (delta H degrees) are -11.3 and -0.8 kJ mol-1. These data permit the construction of models for the binding of the enantiomeric dihydrokikumycins to DNA and account for the more efficient binding of the natural (S) isomer to DNA.

A series of metalloporphyrins linked through basic chains to certain DNA interactive groups has been synthesized. Several of these agents reproduce the characteristic properties of the antitumor glycopeptide bleomycin, including the oxygen-mediated scission of DNA in the presence of thiols, antibiobic activity under aerobic conditions, and activity against human and animal tumor models. Initial screening by scission of PM2-CCC-DNA identified six of the compounds, including those bearing acridine and acodazole intercalating groups, as the most active. The specificity of the oxygen-mediated scission of a 139 base pair HindIII/NciI restriction fragment of pBR322 by these six selected agents was then determined and compared with the action of pancreatic DNase by densitometric scans. All six of these compounds produce uniform base and sequence neutral cleavage of the restriction fragment at each base site. The six active compounds bear either of two types of intercalators, 6-chloro-2-methoxyacridine or acodazole, and with linkages to the ferric binding domain of -NH(CH2)2-, -NH(CH2)3-, -NH(CH2)4-, or -NH(CH2)3NH(CH2)3- and either porphyrin or deuteroporphyrin moieties. Comparison of the Kassoc values for binding to calf thymus DNA suggests that the enhanced binding observed with the linker -NH(CH2)3NH(CH2)3- contributes to the efficiency of sequence neutral DNA scission and may be a factor in the relative anticancer activities of these agents. The iron porphyrins give no evidence of the production of base propenals in DNA degradation, and the autoradiograms clearly indicate that a phosphate group is attached to the 5' end of the oligomer. The scission is partially suppressible by catalase and superoxide dismutase.(ABSTRACT TRUNCATED AT 250 WORDS)

The esperamicins represent a class of antitumor antibiotics characterized by an unusual chemical core structure and extremely potent cytotoxicity. The mechanism by which these drugs produce cytotoxicity was investigated and found to be related to the formation of single- and double-strand DNA breaks. Using five structurally related analogs, we defined a structure-activity relationship for cytotoxicity in various eukaryotic and DNA-repair-deficient prokaryotic cell lines, for DNA breakage in a human colon carcinoma cell line, and for DNA breakage in vitro in pBR322 DNA. Mild reducing agents such as dithiothreitol greatly increased the DNA breakage potency of these analogs in vitro. Results suggest that the pendant aromatic chromophore of esperamicin A1 may contribute to the uptake of the drug into cells but may also hinder double-strand DNA break formation. Little DNA breakage specificity was observed for the drug in a 139-base-pair fragment of pBR322 DNA. Evidence supports a previously proposed mechanism whereby esperamicins may produce the observed DNA breaks through reduction of the methyl trisulfide group to a thiolate anion followed by a Michael addition of the anion across the alpha,beta-unsaturated ketone. This addition may result in the saturation of the bridgehead double bond, thus allowing the two triple bonds to approach each other, causing cyclization of the diyn-ene to form a phenylene diradical. It is likely that this diradical is the active form of the drug responsible for single- and double-strand DNA breakage produced by this class of antitumor agents.

A series of monocationic lexitropsins, or information-reading oligopeptides, were synthesized to minimize and offset the AT bias for doubly cationic ligands bound in the minor groove of DNA. The compounds possess an N-formyl group in place of the guanidinium moiety normally present in netropsin. By systematic replacement of the N-methylpyrrole groups of the dipeptide with N-methylimidazole, a remarkably high degree of sequence specificity was obtained. One of the compounds having two N-methylimidazole residues was found to exhibit dramatically altered specificity when compared with netropsin and preferred to bind to the sequence 5'-CCGT-3' 3'-GGCA-5'. The structural elements underlying sequence recognition in terms of the model for the netropsin-DNA interaction are presented and discussed.

The ability of two platinum(IV) antitumor agents, cis,cis,trans-PtIV[(CH3)2CHNH2]2Cl2(OH)2 (2) and cis,cis,trans-PtIV(NH3)2Cl2(OH)2 (4), to interact with PM2 DNA was examined. Analysis using gel electrophoresis showed that neither compound is able to alter the electrophoretic mobilities of the three forms of PM2 DNA in the gel. However, incubation of 2 and 4 with 2 equiv of Fe(ClO4)2 X 6H2O or 1 equiv of ascorbic acid results in reduction to yield the divalent complexes cis-PtII(NH3)2Cl2 (1) and cis-PtII-[(CH3)2CHNH2]2Cl2 (3). The structures of the reduction products were characterized by using elemental analysis as well as infrared and 195Pt NMR spectroscopies. Both 1 and 3 were found to bind to and unwind supercoiled form I PM2 DNA. The aforementioned observations support the suggestion that reduction is a means of activating the antitumor properties of 2 and 4.

The ability of a group of water-soluble metalloporphyrins to cleave DNA has been investigated. Incubation of Mn3+, Fe3+, or Co3+ complexes of meso-tetrakis(N-methyl-4-pyridiniumyl)porphine (H2T4MPyP) with DNA in the presence of ascorbate, superoxide ion, or iodosobenzene results in DNA breakage. Comparisons between the rates of porphyrin autodestruction with the rates of strand scission of covalently closed circular PM2 DNA indicate that the porphyrins remain intact during the cleavage process. Analysis of the porphyrin-mediated strand scissions on a 139-base-pair restriction fragment of pBR322 DNA using gel electrophoresis/autoradiography/microdensitometry reveals that the minimum porphyrin cleavage site is (A X T)3. The cleavage pattern within a given site was found to be asymmetric, indicating that porphyrin binding and the strand scission process are highly directional in nature. In addition to an analysis of the mechanism of porphyrin-mediated strand breakage in terms of the DNA cleavage mechanism of methidium-propyl-iron-EDTA and Fe-bleomycin, the potential of the cationic metalloporphyrins as footprinting probes and as new "reporter ligands" for DNA is presented and discussed.

The synthesis, characterization, and antitumor properties of a group of platinum (IV) complexes is presented. The compounds, formed by oxidation of cis-dichlorodiammineplatinum (II) (1) or its cis-dihydroxo analogue, were characterized by elemental analysis and infrared and 195Pt NMR spectroscopies. EPR studies of aqueous solutions containing the spin trap phenyl-tert-butylnitrone and various platinum(IV) complexes revealed that the compounds are incapable of producing radical species which may in turn cause DNA breakage. It appears that the antitumor activity of the compounds is either due to Pt(IV) binding via ligand displacement to important cellular components or through the ability of the compounds to undergo in vivo reduction to platinum (II) species, which in turn exert their cytotoxic effects in a manner analogous to 1. As a group, the platinum(IV) compounds were found to be significantly less active against L-1210 leukemia than the parent platinum(II) complex, 1.

The sequence specificities of a series of cationic metalloporphyrins toward a 139 base pair restriction fragment of pBR-322 DNA have been studied by DNase I footprinting methodology. Analysis using controlled digests and quantitative autoradiography/microdensitometry revealed that the 5- and 6-coordinate complexes of meso-tetrakis(N-methyl-4-pyridiniumyl)porphine, MT4MPyP, where M is Mn, Fe, Co, and Zn, were found to bind to AT regions of DNA. Footprinting analysis involving the radiolabel on the opposing strand of restriction fragment showed site skewing in the direction of the 3' end of the fragment, indicating that the porphyrins bind in the minor groove of DNA. The significant increase in DNase I catalyzed hydrolysis observed in various regions of the fragment appeared to be primarily due to a decrease in available substrate DNA upon porphyrin binding with possible contributions from structural changes in DNA caused by ligand binding. The complexes NiT4MPyP and CuT4MPyP were found to bind to both AT and GC regions of the fragment, producing different degrees of inhibition in the two regions. Since the outside-binding porphyrins can neither intercalate or effectively hydrogen bond to DNA, they appear to read sequence by responding to steric and/or electrostatic potential effects located in the minor groove of DNA.

The DNA binding properties of the Au(I)-containing antiarthritic drug Ridaura and related Au(I) complexes have been investigated. Absorption and circular dichroism spectroscopy show that if the gold ion possesses an easily displaced ligand, e.g., Cl- or Br-, Au(I) is capable of interacting, in a nondenaturing fashion, with calf thymus DNA. Absorption studies with the four DNA nucleosides suggest that guanine and cytosine bases are important in the Au(I)DNA interaction.

Footprinting experiments for DNase I digests of a 139-base-pair segment of pBR-322 DNA in the presence of either netropsin or actinomycin D were carried out. Plots of oligonucleotide concentration as a function of drug concentration were analyzed to study the enhancement in cleavage rates at approximately 30 sites, accompanying drug binding at other sites. The pattern of enhancements is not consistent with drug-induced DNA structural changes, but agrees with a redistribution mechanism involving DNase I. Since the total number of enzyme molecules per fragment remains unchanged, drug binding at some sites increases the enzyme concentration at other sites, giving rise to increased cleavage. The consequences of the redistribution mechanism for analysis of footprinting experiments are indicated.