A 9 T G J3 T3t G G C

Compound per mole of hairpin) values for CCGG (open circles) and CTGGsm (black dots) versus the concentration of the unbound f-Pylmlm. The smooth lines in (c) were obtained by non-linear least-square fit of the data to a two-site binding model. studies described above and with minor groove-stacked systems that have shown cooperative binding in dimer DNA complexes 19, 21, 63 . The formamido group can stack with the other polyamide units as well as interact with DNA through H-bonds and van der...

A C C G G T

Fig. 15.5. (a) Structure of f-Imlmlm and (b) model of a 2 1 complex of f-Imlmlm with CCGG, with the triamide stacking in a staggered fashion. Fig. 15.5. (a) Structure of f-Imlmlm and (b) model of a 2 1 complex of f-Imlmlm with CCGG, with the triamide stacking in a staggered fashion. a good motif for recognition of a T-G mismatch. Unlike in a G-C base pair, the G-N2 amino group in a T-G wobble mismatched base pair is not involved in base pairing with thymine (Fig. 15.4). Therefore, the free...

A

Schematic diagrams illustrating ligand binding (a) at the end of a stack of G-quartets, and (b) between the G-quartetend and an antiparallel loop. If the initial starting model has the latter type of diagonal loop-over structure, then an intercalation site for the ligand needs to be constructed. In the case of the human Na+ solution structure, the optimal site is between the diagonal T2A loop and the G-quartet segment of the structure. It can be formed computationally by breaking...

A3

Top and side views of energy minimized models of the a and p anomers of the galactose analog of rebeccamycin. In each case, the dipole moment vector is indicated (fx 6.711 D and 6.745 D for the a and anomers, respectively). Fig. 20.10. Top and side views of energy minimized models of the a and p anomers of the galactose analog of rebeccamycin. In each case, the dipole moment vector is indicated (fx 6.711 D and 6.745 D for the a and anomers, respectively). The study of the...

AGX RT [ In CfSX Jo

The pronounced advantage of this equation is that it provides an estimate of the free energy needed to attain any degree of saturation without recourse to any specific binding model. Numerical integration of the data in Fig. 17.2b yields an estimate of AGx 7.8 kcal mol 1 for the full ligation of a daunorubicin-binding site. Free energies derived from binding constants obtained by curve fitting to specific models must agree with this model independent value if the...

Aminoglycosides

Aminoglycoside antibiotics are antibacterial agents comprised of two or more amino sugars joined to a hexose nucleus. Among these compounds neomycin, paromomycin, gentamycin, tobramycin, and sisomycin have been shown to stabilize triplex DNA 54-56 . Neomycin (Fig. 14.6b) is the most potent compound in this series and stabilizes the triplex poly(dA)-2poly(dT), while the duplex DNA poly(dA)-poly(dT) is unaffected 54, 55 . It shows greater discrimination between triplex and duplex DNA than most...

Anax Amn10pHpK

The resulting fits, which are depicted as solid lines in Fig. 21.7a,b, yield pKa values of 5.5 0.1 and 3.3 0.1 for 56MD2pMPB (Fig. 21.7a) and 5N2pMPB (Fig. 21.7b), respectively. Thus, substitution of the 5,6-methylenedioxy functionality in 56MD2pMPB with a strongly electron-withdrawing 5-nitro group to form 5N2pMPB reduces the p < a of the imidazole ring by 2.2 units. In addition to the pH-dependent profile of DNA-free 56MD2pMPB, Fig. 21.7a also shows the corresponding profile of 56MD2pMPB in...

Antigene Activity

Despite the clear demonstration that many ligands can selectively stabilize triplex DNA, there have been comparatively few studies on the use of these compounds, along with triplex-forming oligonucleotides, for inhibiting gene activity. The original work with BePI showed that this ligand enhanced the inhibition of transcription initiation produced by a triplex-forming oligonucleotide in an in vitro transcription assay 22 . BePI has also been show to promote the formation of intramolecular DNA...

Appendix Methodology for Ligand Quadruplex Modeling

Quantitative molecular modeling can be performed in order to visualize and describe in energetic terms how small molecules interact with and stabilize G-quadruplex structures. The appropriate target structure may be taken from a database (either from a crystal or a NMR structure analysis) and provides initial coordinates for the simulations. An appropriate binding site for the ligand can be chosen depending on the model being used in the study, for example within a loop in the human...

B

Base pairing schemes, (a) A G-quartet involving four guanines, (b) A C.C+ base pair found in i-DNA. (c) Base triplets. base pair is indicated by a dot, Hoogsteen or reverse-Hoogsteen pairing by * The base belonging to the third strand is indicated last. Left Hoosteen pairing. Right reverse Hoogsteen. Strand orientation is indicated by - and +, TC triplexes are composed of TA-'T and except for the G-quartet, where it may vary. C.G*C+ base triplets GA and GT triplexes are Note that the...

Bacterial top2 inhibitors

The principal gyr inhibitors are the coumarins, a family of natural products from Streptomyces species, the most relevant being novobiocin and coumermycin 119 , characterized by the structures reported in Fig. 19.10. Although they are extremely active in vitro, these compounds are not used clinically due to toxicity, low solubility, and rapid development of resistance. From a pharmacological point of view, however, they are still valuable since, unlike quinolones, a complete picture of their...

Benzopyridoindole Derivatives

The first triplex-specific ligand to be described was benzo e pyridoindole (BePI) (Fig. 14.3a) 22 . It is presumed to bind by an intercalative mechanism since vis-cometric studies showed that BePI unwinds super coiled DNA and increases the persistence length of double- and triple-stranded polynucleotides 22-24 . The ligand increases the melting temperature of triplex DNA much more than duplex DNA and it has a greater effect than the addition of polycations such as spermine. On irradiation with...

Berenil

Berenil (also known as diminazene l,3-bis(4-amidinophenyl)triazene) is a synthetic diaryldiamidine which is widely used in Africa for the treatment of try-panosomal infections in animals 7 . It is also used to treat animal babesial infections 78 . The topical use of berenil to treat human cutaneous leishmaniasis has been reported 79 . Similar to results with DAPI, berenil has been found to bind strongly to the DNA minor groove at AT sites and a second weaker interaction has been attributed to...

Binding Constants and Cooperativity

From the results obtained in SPR experiments, the DNA-binding constants for the compounds with six different hairpin oligonucleotides were determined by fitting the data as previously described 38, 39 . The square root of the product of the macroscopic association binding constants (Ki*K2) or the Ki (when the best fit was a one-site model) are shown. Because of correlation between 7< i and K2, the error in fitting individual values of J< i and K2 is larger than for the product, K K2, for...

Binding Free Energy

Binding free energies may be obtained from experimentally determined binding constants by using the standard Gibbs relationship AG RTln K, where Kis the equilibrium binding constant, R the gas constant, and T the temperature. Accurate determination of binding constants and stoichiometrics is not easy. Proper determination of complete binding isotherms demands that over a 100-fold range in free ligand concentration be covered - a daunting task 17 . Fortunately, the an-thracyclines have...

Binding of Imidazolecontaining Polyamide Analogs to TC Mismatches through a Dimeric Binding Motif Structural Studies

In 1999, Lee, Wang and co-workers demonstrated using NMR spectroscopy and molecular dynamic studies that a triimidazole analog of distamycin, AR-1-144 or f-Imlmlm (Fig. 15.5a) was able to bind to a self-complementary CCGG-containing decamer 5 '-GAACCGGTTC-3' 19 . This observation is in agreement with the polyamide pairing rules described in the previous section. According to the NMR structure, f-Imlmlm formed a 2 1 side-by-side antiparallel dimer and bound to four base pairs (Fig. 15.5B) with...

Binding of Imidazolecontaining Polyamides to TC Mismatches through a Dimeric Binding Motif Thermodynamic and Kinetic

The structural results described above indicate that a side-by-side Im Im pair could recognize T-G or G-T, thus adding to the pairing rules reported by Dervan and coworkers (Fig. 15.2) 24 . However, the molecular specificity for discrimination of T-G or G-T from Watson-Crick sequences or other mismatches by a side-by-side Im Im pair cannot be determined from the structural studies. Thermodynamics and kinetics studies can provide the basis for the definition of new recognition rules for...

Binding to Different Motifs

Most studies with triplex-binding ligands have examined their interaction with parallel (CT-containing) triplexes in contexts in which all three strands are composed of DNA (rather than RNA). BePI has been shown to stabilize triplexes containing antiparallel G-GC and T-AT triplets, but does not promote the binding of parallel GT-containing oligonucleotides 70 . Coralyne and naphthylquinoline also promote the formation of antiparallel GA- and GT-containing triplexes, though the ligands are less...

Bisamidoanthraquinones

Bis-substituted anthraquinones (Fig. 14.6a) have been shown to stabilize DNA triplexes in a manner dependent upon the position of substitution. 1,4-Disubstituted compounds bind preferentially to duplex DNA, whereas 2,6-disubstituted compounds are selective for triplex DNA 48 . Their stabilization properties are related to the position of substitution, with 2,7-disubstituted amidoanthraquinone displaying the greatest triplex affinity. The order of affinity for parallel (CT-containing) triplexes...

Bvb fay

In this equation, vB and ve are the critical saturation ratios of 56MD2pMPB and EtBr, respectively, and < j> E is the helix unwinding angle of EtBr, an angle known to be -26 67, 71 . The Affinity of Benzimidazoles for Duplex DNA is Modulated by the Structure and Electronic Properties of the Substituents on the Benzimidazole Rings We used absorption spectroscopy to detect and characterize the binding of selected BZ compounds to the poly(dA-dT)-poly(dT-dA) duplex, which hitherto will be...

C

Mechanisms of formation of the diradical intermediates by various enediynes. (a) Reductively-induced Bergman cyclization of the bicyclo 7.3.1 tridecadiynene core of calicheamicins, esperamicins, and probably namenamicin. (b) Nucleophilic attack at the CI 2 of neocarzinostatin and related enediyne chromophores results in the formation of an enyne- 3 -cumulene intermediate that undergoes cyclization between the C3 and Enyne- 3 -cumuiene Diradical intermediate C7 positions to form the...

Catalytic process

The enzymatic cycle of topi 13 can be divided into the following steps (a) binding of the enzyme to DNA, (b) cleavage of a single DNA strand, (c) passage of an intact strand through the gate, and (d) DNA religation. Once the initial protein-DNA complex is formed, a transesterfication process occurs between a key catalytic Tyr residue and a phospho-diester bond in the backbone of the single-stranded DNA segment, n type IA tops, the 3' oxygen in the backbone is displaced by a tyrosine oxygen,...

Cellular studies with DACA

An attractive property of DACA is its ability to overcome transport-generated multidrug resistance (MDR) 51 . Studies in P-glycoprotein-expressing CEM VLB100 and MRP-expressing CEM E1000 cells (sublines of the CCRF-CEM human leukemia line), and in MDR sublines of the HL60 human leukemia line, showed that DACA was much more effective than idarubicin, an anthracycline analog known for its relative ability to circumvent MDR 68 . In subcutaneous colon 38 adenocarcinomas in BDFl mice, DACA had...

Co va lent Adducts of Enediynes with Deoxyribose

In addition to DNA cleavage, enediynes have also been shown to form covalent adducts with deoxyribose in DNA. This phenomenon was first described by Goldberg and Povirk for neocarzinostatin 119, 120 . They observed the formation of a 624 22 Binding and Reaction of Calicheamicin and Other Enediyne Antibiotics with DNA Adenine Propenal Fig. 22.7. Reaction of dG with adenine propenal, derived from 4'-oxidation of deoxyribose, to form MiG, the pyrimidopuranone adductofdG 114 .

Conclusions

Carbazoles and related dibenzothiophenes and dibenzofurans proved to be an interesting series of compounds both in their DNA-binding characteristics and biological activity. The series was unique in that they were the first example of fused ring systems forming strong minor groove complexes with AT sequences. Characterization of the complexes with DNA was determined for the carbazoles since they appeared to be stronger DNA-binding agents than the corresponding isosteres. These studies...

Contributors

Institut de Recherches sur le Cancer de Lille Prof. Dr Jacqueline K. Barton Division of Chemistry and Chemical Engineering California Institute of Technology Pasadena, CA 91125 USA Dr. David W. Boykin Department of Chemistry Georgia State University Atlanta, Georgia 30303 USA Academy of Sciences of the Czech Republic Dr. Jonathan B. Chaires Department of Biochemistry University of Mississippi Medical Center 2500 North State Street Jackson. Mississippi 39216-4505 USA Division of Bioengineering...

COO a

Agarose gel analysis of the nuclease activity of Lys-Trp-Lys and compound 2. (a) pBR322 supercoiled pi asm id containing two AP sites plasmid 2 10 9 M. (b) Relaxed circular form. Lane 1 Reference lanes 2-4 Lys-Trp-Lys 10 3, 10 and 10 5 M lanes 5-8 Compound 2 lO5, 10 6, 10 7, and 10 8 M. Molecules Inducing Multiple DNA Damage Clustered lesions, or locally multiply damaged sites (LMDS), in tracks of a few base pairs appear to be very toxic as they present a challenging repair problem...

Coralyne

The berberine alkaloid coralyne (Fig. 14.5a) is a natural product containing four fused aromatic rings, which has been shown to stabilize triplexes. Initial reports showed that it bound to triplexes much better than duplexes and claimed that it had little sequence specificity, stabilizing both T-AT and C+-GC triplets 35 . However more recent studies have shown that it preferentially binds adjacent to T-AT Fig. 14.5. Chemical structures of triplex-binding ligands. (a) Coralyne 35-37 (b)...

Correlation of Cytotoxicity with Mode Strength and Kinetics of Binding

The above references 7, 12-14 demonstrate that intercalative binding is a necessary (but not sufficient) condition for useful cytotoxicity and in vivo antitumor Tab. 18.2. Alkyl-substituted amsacrines. a Drug concentration to lower the fluorescence of ethidium bound to poly(dA-dT) by 50 a measure of DNA binding strength. beAs for Table 18.1. Tab. 18.3. 2,7-Dialky I proflavines. Data from Refs 13 and 14 . a bAs for Table 18.1. c Slope of chemical shift drug-base pair ratio plot (an NMR measure...

Cr

The two hydrogen-bonding faces of a guanine base. strand directions. There is insufficient structural data at present to be able to define the rules governing quadruplex folding in general, but the limited current data set of crystal and NMR structures suggests that the major factors in defining strand polarity and loop formation are likely to be the nature of the sequence(s) intervening between the guanine repeats themselves, Fig. 13.2. The hydrogen-bonding arrangement in a...

D

A pyridine, pyrimidine, purine or aniline Fig. 8.8. Structures of platinum(ll) triamine cations (a) PtCl(dien) +, (b) PtCl(NH3)3 +, (c) c s- PtCl(NH3)2( V7-ACV) and (d) antitumor trisubstituted platinum(ll) compounds containing pyridine, pyrimidine, purine, or aniline ligand. ences between these adducts formed by JM216 and cisplatin may be deduced from the analysis by using antibodies elicited against DNA modified by cisplatin. Exploring new structural classes of platinum antitumor drugs has...

DACA and Other Acridine4carboxamides 18421 Introduction

An equally efficient way of lowering acridine p < a is to remove the resonant 9-amino group. The acridine-4-carboxamide (77, DACA) has a piQ, of only 3.54 59 and a lower binding constant (log XAt 6.12). However it was still an intercalator, and while it had lower cytotoxic potency than 67, it and many of its chromo phore-substituted analogs showed curative activity in the Lewis lung model (see examples 78-86, Tab. 18.10) 59, 60 . This and its activity against multidrug resistant cells 494 J...

Dapi

Schematic representation of the hydrogen bonding array of the complex between DAPI and d(CGCGAATTCGCG)2 based upon the crystal structure. Dashed lines represent hydrogen bonds. Fig. 16.4. Schematic representation of the hydrogen bonding array of the complex between DAPI and d(CGCGAATTCGCG)2 based upon the crystal structure. Dashed lines represent hydrogen bonds. N3 on opposite strands. Important van der Waals contacts between the walls of the groove and the aryl rings of DAPI are...

Definition

There are three generally recognized ways in which molecules can bind reversibly to double-stranded DNA. First, the recognition sequences of regulatory proteins primarily interact in the major groove, recognizing a unique pattern of H-bond donor and acceptor functions in a 10-15 base pair, usually GC-rich, cognate sequence. Secondly, cationic small molecules composed of repeated aromatic units with an annular topology similar to that of the DNA bind in the minor groove in AT-rich regions,...

Developing Molecules Capable of Recognizing Mismatches in DNA

Two recent publications show the development of molecules capable of recognizing DNA mismatches 51, 52 . One compound is a cleaving agent that recognizes destabilized regions on the DNA 51 and was able to recognize a single mismatch in a 2725-base-pair linear plasmid. However, compounds with more specificity for the different mismatches are needed to readily identify single nucleotide polymorphisms. Another new agent is capable of recognizing G-G mismatches and the compound has low affinity for...

Devising a Structural Basis for the Potent Cytotoxic Effects of Ecteinascidin 743

Hurley Introduction Ecteinascidia turbinata is a tunicate species from the Caribbean and the Mediterranean that belongs to the class Ascidiacea within the subphylum Tunicata (also called Urochordata). Ascidians, or sea squirts, are small bottom-dwelling soft-bodied marine animals that form colonies comprising many individuals, called zooids. The term tunicate derives from their characteristic protective covering, or tunic, which functions to a certain extent as an...

Dibenzodioxins

In a further attempt to develop very weakly binding tricyclic chromophores, the aromaticity of the ring system was lowered by breaking the conjugation. A survey 47 of several linear tricyclic chromophores identified the dibenzo l,4-Jdioxin (61) as potentially interesting, lacking a fully conjugated chromophore yet retaining planarity and binding by intercalation (unwinding angle 20 ) but weakly (log 7< at 5.03). While the parent compound 61 was only weakly cytotoxic (IC50 3000 nM) it showed...

DNA Sequence Recognition

The two most successful modes of recognition of specific sequences of DNA with four or more base pairs involve the two grooves of the double helix. These two modes are formation of a triple helix by oligonucleotides or analogs in the major groove and interaction of organic cations in the minor groove. A quite diverse array of DNA sequences, including both Watson-Crick and mismatched base pairs, can be recognized with current compounds that bind in the DNA minor groove and the focus of this...

DNA Topoisomerasetargeted Drugs

Sissi Introduction DNA-interacting anticancer drugs were introduced to clinical practice about half a century ago. The early interpretation of their mechanism of action essentially relied on the covalent or reversible structural changes generated in the nucleic acid upon drug binding, especially through intercalative mechanisms, that impaired the basic functions of rapidly proliferating cancer (and normal) cells. Although this in part holds true even in the light...

Drugs Targeted at Topoisomerases

Given the crucial role of tops in fundamental mechanisms of cell replication and maintenance, it can be easily understood how drugs interfering with these mechanisms could seriously impair cell functions and finally cause cell death. That tops are the biological targets for a number of leading drugs was originally shown in the early 1980s 28, 29 . The drugs were grouped into two main classes inhibitors and poisons, the latter being by far more effective. Topoisomerase inhibitors impair the...

Effect of Chromophore Crosssectional Thickness

Another important criterion for intercalation is the cross-sectional thickness of the intercalating chromophore. Although all polyaromatics have a similar cross-sectional thickness (about 3.5 A), directly attached substituents of larger cross-section also have to be considered, as first investigated by Gabbay 11 . Later work with derivatives of both amsacrine (10) 12 and proflavine (1) 13, 14 supported these findings. While amsacrine analogs with methyl, ethyl, or isopropyl substituents at the...

Effect of Chromophore Size

One important criterion determining binding mode is the size and nature of the intercalating chromophore 5 . A number of studies suggest that fused two-ring systems (naphthalene-type) are the minimum necessary for intercalative binding Small Molecule DNA and RNA Binders From Synthesis to Nucleic Acid Complexes. Volume 2. Edited by M. Demeunynck, C. Bailly, W. D. Wilson Copyright 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 6, 7 . NMR shift and line width changes show no evidence for...

Enediyne Target Recognition in Chromatin and Cells

Several studies have addressed the selection of enediyne targets in cellular and isolated chromatin, with the unsurprising result that studies with purified DNA are good predictors of target selection in cells. Beerman and co-workers demonstrated almost two decades ago that neocarzinostatin caused the release of nucleosome-sized DNA fragments from HeLa nuclei exposed to the drug 130 . Though the connection was not made at the time, the preferential cleavage of the DNA joining adjacent...

Enediyneinduced DNA Damage in Cells

To this point, we have considered enediyne target recognition and DNA damage in the context of isolated and purified DNA. However, as a logical consequence of the systematic search for antitumor natural products beginning in the 1960s and 1970s, the cellular effects of enediynes have been the subject of studies too numerous to review here. The reader is referred to the very comprehensive review of the biological and clinical aspects of the enediynes edited by Doyle and Borders 129 . Here, we...

Esperamicin Structure and Function

Esperamicin A1 has not received the attention paid to calicheamicin in spite of similar structures and cytoxotoxic potencies, possibly due to its weak sequence selectivity 27 and less efficient production of double-strand DNA lesions 22, 57, 58 . Beyond the early observaton of a minor groove binding mode for esperamicin Al, it was recognized that the deoxyfucose anthranilate side chain (Fig. 22.1) caused a reduction in the frequency of double-stranded DNA lesions 27 . The first indication of a...

Et 736

23.2 Biologjcal Activity and Characterization of the Active Compounds I 645 to the presence of an active a-carbinolamine (N-C-OH) group 6 , which is also present in naphthyridinomycins, quinocarcins, and pyrrolo(l benzodiazepine antibiotics such as anthramycin, sibiromycin, and tomaymycin. Reduction of the quinone skeleton of saframycin A to hydroquinone results in the concomitant elimination of the nitrile group and enhanced covalent binding to DNA 7 , in a reaction reminiscent of that between...

Forty Years On

Wakelin Early Experiments Prior to Molecular Modeling The quest to understand specific interactions between drugs and nucleic acids dates back a long time - more than 40 years. Even though the concept of gene targeting could not be explicitly formulated until much later, there were early realizations that DNA could provide a fine receptor for drugs. A major turning point in the history of drug binding to DNA, the publication in 1961 of the intercalation hypothesis...

Fr

3 LjQrD - -f T-O-I T-T- .3 3 LjQrD - -f T-O-I T-T- .3 Fig. 21.2. Agarose gels showing hTOPl -mediated DNA cleavage of linearized YEpG DNA induced by various benzimidazole derivatives and CPT, with the ligands and their concentrations (in units of j.iM) indicated at the tops of the lanes. Following incubation with both TOPI and the ligand, the DNA samples were treated with SDS and proteinase K and then alkali-denatured prior to loading on to a 0.8 agarose gel in 0.5X Tris-Phosphate-EDTA buffer...

H ppm

Titration of wild-type TAR RNA by increasing amounts of the ligand 1, (l) Lys-( ) Lys-(l) Asn (Upper). (Lower) Superposition of TOCSY spectra at increasing concentration of 1 (free RNA, pink and 1 1 ligand to RNA ratio, blue) shows that only resonances at bulge U23 and C24 are affected by the ligand 1 binding. Adapted from Hwang et al. 106 . Interestingly, ligand 1-TAR RNA interactions are different from TAR RNA-pep-tide or TAR-Arg complexes reported in previous NMR studies 55, 107...

H3n Ci

Structure of cisplatin, the first platinum antitumor drug used in the clinic. portant milestones in understanding DNA interactions with cisplatin are briefly addressed. Cisplatin reacts with DNA in the cell nucleus, where the concentration of chlorides is markedly lower than in extracellular fluids. The drug loses its chloride ligands in media containing low concentrations of chloride to form positively charged monoaqua and diaqua species (Fig. 8.2). It has been shown 11 that only...

Hydration Changes

Water is a key participant in biochemical reactions 45, 46 , but has received little attention in drug-DNA interaction studies. In part this is because of the lack of suitable experimental methods for unambiguously identifying hydration changes that may occur upon drug binding to DNA. The osmotic stress method alleviates the problem. In one useful version of the osmotic stress method, small neutral solutes (osmolytes) are added to macromolecule solutions to alter the water activity 45, 46 ....

Nteraction of DACA with topoisomerase

DACA stimulates the formation of cleavable complexes between topo II and DNA, inducing both DNA breakage and the formation of DNA protein crosslinks 63 . The DNA protein crosslinks formed by DACA are readily reversible, with a bell-shaped concentration dependence suggesting self-inhibition at higher drug levels, whereas induced DNA double-strand breaks continued to increase linearly with drug concentration. The results suggested that DACA interferes with the action of topo II, but by a...

I

Synthesis of neo-acridine. NHBoc TPSO v 0oAfl< rA NHBoc cg Boc di-ferf-butyldicarbonate DM F dimethylformamide TPSCI 2,4,6-triisopropyl- benzenesulfonyl chloride pyr pyridine 2.5 New RRE Ligands 31 Tab. 2.2. Approximate IC5o values ( .iM) for RevFl displacement by solid phase. Compound C50 C50 with DNA C50 with tRNA Rev acid with those measured in an excess of other nucleic acids, the RRE specificity of each compound can be evaluated. Even though both neo-acridine and the Rev...

Info

Figure 21.12 shows the LD1 spectra of the poly(dA)-poly(dT) duplex alone and in complex with either 5PTB (Fig. 21.12a), TB (Fig. 21.12b), or 5NIBB (Fig. 21.12c). Note that the LD1 spectra of the three ligand-poly(dA)-poly(dT) complexes are positive in the wavelength region (310-370 nm) where the ligands absorb, an observation consistent with each of the three TB derivatives binding in minor groove of the poly(dA)-poly(dT) duplex. Although not shown, we observed similar LD results when using...

Intercalation

Molecules that bind to DNA by intercalation represent an important class of DNA binders, which have seen significant use as drugs. The binding of this class of molecules to DNA is characterized by the insertion of planar aromatic rings in between DNA base pairs. This interaction can be quite strong despite the fact that energy is consumed to unwind the helix and unstack the base pairs to allow complex formation. Contributing to the stability of intercalation complexes are van der Waals,...

Intercalators

Ethidium bromide is well known as a duplex DNA intercalating agent and early thermal denaturation studies demonstrated that it destabilized the triplex helix formed by poly(A)-2poly(U) 71 . However, a later study showed that ethidium stabilized poly(dA)-2poly(dT) more than poly(dA)-poly(dT) 21 . This apparent selectivity for triplex DNA is partly due to the poor binding of ethidium to poly(dA)-poly(dT) because of its unusual conformation 72-74 . A separate study further demonstrated that...

Introduction

As a continuation of our search for novel dicationic antimicrobial compounds we synthesized and tested a series of 40 3.6- and 2.7-disubstituted carbazoles and related dibenzothiophenes and dibenzofurans 89-91 (Fig. 16.7). These compounds were tested for antimicrobial activity against Pneumocystis carinii pneumonia in a rat model 89-91 , a neonatal mouse model of Cryptosporidium parvum infection 18 and in vitro against a number of pathogenic fungi 13 . Since it had been previously suggested...

Isostructural is not Isoenergetic

Ideally, complete thermodynamic profiles should be obtained for the binding of all drug derivatives of interest. The labor in that task is enormous, and had been undertaken for only a few cases. For the anthracyclines, we have managed to complete a comparison of only daunorubicin and doxorubicin. The results are shown in Tab. 17.2 and Fig. 17.11. These results lead to a key conclusion. High-resolution crystal structures of daunorubicin or doxorubicin bound to DNA show essentially no differences...

J Hj h2 OJNXmN

Assembly of a metal-salen complex. designed to form neutral complexes with dicationic metals for study in organic solvents, but an anionic ligand had also been developed for aqueous application 12 . Neither of these were very appropriate for reaction with polyanionic nucleic acids under physiologically compatible conditions and, in one case, the anionic substituents changed the coordination geometry of the metal 12 . Our laboratories consequently synthesized a salen with two pendent...

Ho

Binding of a drug within a cleavage complex. The drug domains interacting with DNA and top (topi in this figure) are highlighted in black. Modern research requires the use of appropriate model systems to investigate drug effects in vivo. In this connection appropriately designed yeast systems have proved to be very useful for studying both topi and top2 poisons 31, 32 . Another key area of research is aimed at understanding the complex signaling pathways that lead to programmed cell...

Kinetic Studies

Figure 15.10 shows typical examples of the SPR sensorgrams obtained for the interaction of f-Imlmlm with three DNAs, the Watson-Crick CCGG sequence and the sequences with single and double T-G mismatches. A dramatic difference in rates of association and dissociation from the mismatch relative to the matched base pair sequence is easily visualized by eye (note the different time scales in the plots). Figure 15.11 also shows a visual comparison of the kinetic behavior for the Figure 15.10. SPR...

L

I) 3-Bromopropylphthalimide, DMF, NaH, rt, 6 days ii)12N HCI H AcOH 1 1 1 mixture, 100 C, 24h iii) para-methoxybenzylamlne, NEt3, DMF, 80 C, 2h iv) 1,3-dlamlnopropane, 140 C, 2h v) DMF, 180 C, 18h vi) BocNhHCHgfe-NI-k, 140 C, 1.5 days vii) 1N HCI in AcOH, rt, 1h. Scheme 10.3. Regioselective functionalization at position 2, 6, or 9 of the 2,6-diaminopurine 76, 77 . (i) 3-Bromopropylphthalimide, DMF, NaH, rt, 6 days, (ii) 12N HCl H20 Ac0H 1 1 1 mixture, 100 C, 24 h. (iii) para-...

Mechanism of Cytotoxicity of DNA Intercalators Topoisomerase Poisoning

The primary mechanism of the antitumor activity of DNA intercalating agents is due to their production of DNA double-strand breaks 34 , through interference with the normal functioning of the DNA enzyme topoisomerase II 35 . This discovery showed why it had not been possible to completely predict the biological activity of these compounds in terms of the nature of their binding to DNA. It also explained the dichotomous structure-activity relationships with 9-anilinoacridines, where substituents...

Mechanisms of Target Recognition

The earliest studies of enediynes pointed to a minor groove location for drug binding and DNA damage. This conclusion was based on competitive binding studies and the 3'-stagger of cleavage sites on opposing strands (27, 28). In the intervening years, however, numerous structural, biophysical, and biochemical studies have revealed a more complicated set of determinants for the recognition of DNA targets by enediynes, with the bulk of the studies focusing on espera-micin and calicheamicin.

Minor Groove Binders

In principle, one might expect that ligands which bind in the DNA minor groove will not affect the formation of triple helical nucleic acids since the ligand and the third strand are located in different DNA grooves. However, it is conceivable that minor groove ligands could enhance or inhibit triplex formation by altering the dimensions and or flexibility of the major groove, with cross-talk between the two grooves. At first sight the literature on this subject is confusing since the precise...

Minor Groove Binding

Groove binders are another major class of small molecules that bind to DNA and play important roles in drug development. In principle, molecules can bind to either the major or minor groove of DNA. Due to the great differences in dimensions of the two grooves, targeting them requires vastly different shaped molecules. The major groove, as the name implies, is much wider than the minor groove the groove width values for averaged-sequence B-form DNA are 11.6 and 6.0 A, respectively 51 ....

Mismatched Base Pair Recognition

As described in this chapter, there are now quite successful agents for the identification of T-G and G-G base pair mismatches in the context of Watson-Crick DNA base pair sequences. These agents have great potential for development of methods to identify SNPs in human populations but such development will require automation of the detection methods. The agent for G-G recognition has been successfully attached to BIACORE sensor chips and this provides a development route for rapid and automated...

Molecular Basis for Covalent Reactivity and Sequence Selectivity

As described in the previous section, the site selectivity of Et 743 is believed to be governed by a three-base-pair recognition sequence where the flanking base pairs immediately 5' and 3' to the modified guanine are involved in stabilizing the Et 743-DNA adduct. These previously reported studies provided a possible molecular basis for the recognition of, and reactivity toward, different DNA sequences by Et 743. Experiments that indicated differential stability of the high and low reactivity...

N2pMPB0 M

Results of yeast cytotoxicity assay. The yeast strain JN2-134 lacking the chromosomal copy of the TOPI gene was transformed with either the wild-type yeast TOPI gene (top row) or the wild-type human TOPI gene (bottom row). Yeast cells were serially (5-fold) diluted and grown on minimal medium containing 2 galactose and uracil as a selection marker. The medium also contained 0 (left), 1 (middle), or 10 (right) jjM 5N2pMPB. The plates were grown for 3 days at 30 C to assess the lethal...

Neocarzinostatin Recognition of Bulged DNA Structures

While most of the studies with enediynes have focused on B-DNA targets, the Goldberg group has made major inroads into defining the role of non-B-DNA sec- and causes formation of the cumulene intermediate and subsequent diradical. While the C2 radical center attacks the naphthoate moiety, the C6 radical is free to abstract a hydrogen atom from the deoxyribose in the nucleotide 3' to the bulge. Fig. 22.4. Thiol-independent base-catalyzed activation of neocarzinostatin that occurs selectively at...

Netropsin

Netropsin is a dissymmetric tripeptide isolated from Streptomyces netropsis that contains two different terminal cationic centers one an amidine and the other a guanidine. Netropsin shows experimental antiviral and antitumor activity that has been attributed to its ability to bind to DNA 64 . This tripeptide recognizes the minor groove of DNA at AT sites and occupies 5-6 base pairs 64 . The many bio- Fig. 16.2. Structures of well-studied DNA minor groove binders. Fig. 16.2. Structures of...

Oh

Neocarzinostatin adduct at the 5'-position of deoxyribose, one of the major sites of drug-induced oxidation, in native DNA with a frequency of about 5 119, 120 . While the site of adduction in the enediyne was not identified in the studies with B-DNA, a similar 5'-adduct in deoxyribose was observed in the reaction of neocarzinostatin at a DNA bulge. It was argued, on the basis of known site specificity, that the C6 radical center in neocarzinostatin is the site of adduction 121 . The proposed...

Overall Scheme for Binding and Activation

A likely scenario for a target search by calicheamicin starts with non-specific binding to DNA, possibly by superficial association in the hydrophobic minor groove, followed by tracking along the groove or along the outer surface of the helix in search of a target site. This is a reasonable model given the established one-dimensional diffusion or sliding of proteins along the DNA helix 29, 30 . The positive charge of calicheamicin might be expected to facilitate an approach to the negatively...

Oxidation of the 1Position of Deoxyribose and the Biochemistry of the Deoxyribonolactone Abasic Site

The deoxyribonolactone abasic site (Fig. 22.5) is the product of 1'-hydrogen atom abstraction by neocarzinostatin 93 , copper-phenanthroline 99 , and radiation 100, 101 . This repertoire of 1'-oxidants has now been expanded to include esperamicin Al 22 and C-1027 96 . While there is no direct chemical evidence for the formation of the deoxyribonolactone by these agents, it is highly likely given the precedent with neocarzinostatin and the production of the abasic site as a general feature of 1...

Pentamidine

Of all the minor groove-binding compounds studied to date, pentamidine (1,5-bis(4-amidinophenoxy)pentane), a synthetic molecule, has seen the most clinical use. Pentamidine has a long history of human use, starting in the 1940s when it was used to treat African trypanosomiasis and leishmaniasis 7, 9, 37, 38 . More recently, during the HFV AIDS epidemic, it has seen use as a secondary drug for treating AIDS-related P. carinii pneumonia 40-42 . Currently the drug is used to treat primary-stage...

Pharmacological Drawbacks of Tight DNA Binding the Concept of Minimal Intercalators

While tight DNA binding correlates with cytotoxicity, at least within individual drug series, it also severely limits the extravascular distributive properties of drugs, since diffusion is driven by the free drug concentration. For drugs that bind to DNA, the effective diffusion coefficient (Deff) that determines the rate of extra-vascular diffusion, is greatly lowered by DNA binding, in direct proportion with the binding constant. It is given by Deff D (l + CDNASf), where D is the diffusion...

Phenazines

Phenazine-l-carboxamides were explored as a class of linear neutral tricyclic chro-mophores that bound by intercalation (an unwinding angle of 18 for the parent 49) (Tab. 18.7) 47J. While 49 showed good cytotoxicity and modest in vivo activity 481 (Tab. 18.7), it also has significantly higher DNA binding than the corresponding 2-phenylquinoline (log I< at 6.58 compared with 5.97 for the 2-phenylquino-line). The effect of ring substituents on activity was small except at the 9-position, where...

Phenylbenzimidazoles

To explore the minimal intercalator concept further, replacement of some of the benzene rings in the 2-phenylquinolinecarboxamide chromophore with less aromatic five-ring heterocycles was studied 46 . 2-Furoyl- (39), 2-thienyl- (40), 2-pyrrolo- (41), and 2-phenylbenzimidazolecarboxamides (42) had similarly low binding constants, but only the latter showed biological activity (Tab. 18.6). Compound 42 appeared to intercalate DNA, albeit with an unwinding angle of only 11 . Structure-activity...

Phenylquinolines

Following studies 7 that the 2-phenylquinolinecarboxamide 3 was a DNA inter-calator, a study of phenyl ring substituted compounds was undertaken 45 (Tab. 18.5). The 2'-G analog 32 was the weakest DNA binder, but this did not bind by Tab. 18.5. Substituted 2-phenylquinolines. Data from Ref. 45 . aeAs for Table 18.1. f Not measured. Data from Ref. 45 . aeAs for Table 18.1. f Not measured. intercalation due to the 2'-substituent twisting the phenyl ring substantially out of coplanarity with the...

Pi

Coleman's total synthesis of azinomycin A 1 38 . The backbone was formed through reaction of the epoxide acid 51, synthesized as described above, with the phosphonate alcohol 58, formed from reaction of the glycine phosphonate 56 with racemic l-amino-2-propanol. Swern oxidation gave the backbone in the correct oxidation state and the resulting compound was reacted directly with the azabicyclic precursor prepared with the 12-OH protected as a triethylsilyl ether via a route...

PolyamideDNA Complexes

Polyamides, from natural products such as distamycin and netropsin to newer synthetic agents, are a very successful family of compounds for the sequence-specific recognition of DNA. The ability of these compounds to form stacked dim-ers in the DNA minor groove allows them to read sequence information from both strands of the double helix and greatly expands their sequence recognition capability. The emphasis to date in the development of these compounds has been on optimizing methods to link...

Potential Applications for Recognition of Mismatched Base Pairs

SNPs can be identified as mismatched base pairs by hybridization of individual genomic DNA with standard DNA sequences. Several procedures are in use to search such hybridized duplexes for mismatches by using methods such as chemical, enzymatic, or denaturation methods 52 . Automated methods that can be conducted at normal temperatures have significant advantages over methods that rely on precise selection of an elevated temperature for detection of differences in stability between Watson-Crick...

Preface

The ultimate goal of most organic-medicinal chemists is to see the small molecule that they have synthesized become a useful drug for the treatment of human diseases. Unfortunately, even with modern technology this is an extremely rare event. In most cases, the compounds designed and synthesized (generally with pain and passion) have a brief existence that does not exceed the first biological activity assay. The valley between chemistry and therapeutics is deep and difficult to cross but...

Proportions of Single and Doublestranded DNA Lesions Produced by Enediynes

One of the unique features of enediynes is their ability to produce double-stranded DNA lesions by the action of a single drug molecule 41. 87 . Like restriction en-donucleases with relatively weak sequence selectivity, the diradical form of all of the enediynes can abstract deoxyribose hydrogen atoms from both strands of DNA to produce lethal double-stranded DNA lesions. While esperamicin A1 22, 57, 58, 88 , C-1027 23, 89 , kedarcidin 90 , and neocarzinostatin 41, 87 all produce more...

PTB binding does not remove helical bends from kinetoplast DNA

Restriction fragments from Leishmania tarentolae kinetoplast DNA minicircles are known to adopt bent helical structures 118-120 . These globally bent DNA fragments arise from local bends that occur at the junctions between oligo(dA)-oligo(dT) tracts and adjacent segments of B-form DNA fill, 116, 117 . In order to yield a globally bent DNA fragment, the oligo(dA)-oligo(dT) tracts must occur in-phase with the periodicity of the DNA helix (i.e. every 10-11 base pairs) 111, 116, 117 . As noted in...

R3 pM

Inhibition of kinase activity of wild type (WT) topoisomerase I and the mutated Y723F topoisomerase I by compound R-3 125 . with the extracellular domain of the TrkA receptor so as to promote its interaction with the neurotrophin NT-3. A direct NB-506-protein interaction has also been suggested with topoisomerase I. Topoisomerase I exerts a kinase activity in addition to its primary action at the DNA level. Tazi and collaborators have demonstrated that topoisomerase I can...

References

Studies on the formation of two and three-stranded polyribonucleotides. Biochim. Biophys. Acta 1957, 26, 457-468. Sequence-specific cleavage of double-helical DNA by triple helix formation. Science 1987, 238, 645-650. 2 Felsenfeld, G., Davies, D. R., Rich, A. Formation of a three-stranded polynucleotide molecule. J. Am. Chem. Soc. 1957, 79, 2023-2024. Praseuth, d. etal. Sequence-specific recognition, photo-cross-linking and cleavage of the DNA double helix by an...

Relaxing the Specificity of Triplex Formation

Triplex-binding ligands can be used for increasing the strength of binding to sites which contain pyrimidine interruptions. By increasing the strength of binding, by up to 1000-fold, complexes can be formed at sequences for which there are no clear triplex coding rules and for which triplex formation is very weak. The naph-thylquinoline derivatives have been shown to promote the formation of triplexes at sites containing up to three consecutive base pair inversions using TCG and G-TA triplets...

Salt Dependency of Daunorubicin Binding to DNA

Daunorubicin binding to DNA is strongly dependent on the bulk salt concentration, as shown in Fig. 17.3a. The salt dependency of the binding constant shown may be interpreted by the polyelectrolyte theories of Manning 28 and Record 29 . Briefly, the salt dependence arises from a thermodynamic linkage between binding of the positively charged drug molecule and condensed sodium counterions surrounding the DNA duplex. Binding of the drug releases condensed counterions into solution, providing an...

Sequence Selectivity

Most triplex-binding ligands contain positively charged heterocycles and this positive charge is thought to prevent binding adjacent to protonated cytosine residues. These ligands therefore stabilize triplexes containing T-AT triplets better than those that are rich in C+-GC. Indeed there are very few triplex-binding ligands that do not possess positively charged chromophores the only exceptions are the bis-substituted amidoanthraquinonones, which contain positively charged side groups, and the...

Sources Biosynthesis and Structural Conservation

All of the enediynes isolated to date are produced by eubarteria of the order Acti-nomycetales (Table 22.1). One of the most intriguing features of enediynes, a feature relevant to any family of bacterially produced antibiotics, is the remarkable conservation of the antibiotic structure. In addition to the conserved core structures of the several enediyne classes (Fig. 22.1), the various appendages are also highly conserved in both structure and function. This is not surprising in light of the...

Ssc

Schematic representation of the species generated by single-strand cleavage reactions of (a) supercoiled circular dsDNA and (b) circular ssDNA. Tab. 11.3. Photocleavage of equimolar mixtures of supercoiled ds pBr322 and circular ss M13mpl9 by cyclobisintercaland 26 (DNA concentration 0.06 mM in phosphate units). Tab. 11.3. Photocleavage of equimolar mixtures of supercoiled ds pBr322 and circular ss M13mpl9 by cyclobisintercaland 26 (DNA concentration 0.06 mM in phosphate units). Both...

Stoichiometry of Complexes

The stoichiometry of binding of f-Imlmlm to CG-containing sequences was determined as 2 1 as expected for a stacked dimer 19, 22, 23, 24 . Titration of f-Imlmlm f-lmPylm X N,Y CH f-Pylmlm X CH,Y N f-lmPylm X N,Y CH f-Pylmlm X CH,Y N Py Pyrrole Im Imidazole f formamido (NHCOH) group 5-BiotinGAACCGGTC T CTTGGCCAGt C CCGG Fig. 15.7. (a) Chemical structures are shown for the polyamides f-lmlmlm, f-lmPylm, f-Pylmlm, and f-lmlm. (b) Sequences of the DNA hairpins used in this study are shown CCCC and...

Structural features

Topoisomerases I are single polypeptides with extensive secondary structure. The crystallographic structure of both prokaryotic and eukaryotic protein is now available (Fig. 19.1), and helps understanding the mode of catalytic action 15, 16 . There is a wide (25-30 A) hole in the center of the protein large enough to bind Fig. 19.1. Human reconstituted topi in covalent complex with a 22-bp DNA Fig. 19.1. Human reconstituted topi in covalent complex with a 22-bp DNA either a single- or...

Structure and Functions of DNA Topoisomerases

DNA topoisomerases are involved in the regulation of DNA topology and its degree of supercoiling 12, 13 . Two types of the enzyme are known type I tops change the degree of supercoiling of DNA by causing single-strand breaks and religation, type II tops (including bacterial gyrase) cause double-strand breaks. Topi is dispensable for a living cell whereas top2 is not. The different roles of DNA topi and top2 may reflect the need for fine regulation of DNA supercoiling efficiency. Topi and top2...

Structure of TG Mismatched Base Pairs

Because of the biological importance of the T-G mismatch, the structural and physical properties have been extensively investigated. NMR analysis of 5'-CGTGAATTCGCG-3', containing symmetrical T-G mismatches, indicates that the mismatches adopt a wobble conformation (see Fig. 15.4b), and structural perturbations are mainly in the vicinity of the mismatch and the nearest neighbor 53 . These localized perturbations in T-G mismatch containing oligonucleotides are supported by an X-ray analysis of...

Summary

Given the clinical success of the Mylotarg calicheamicin-antibody conjugate, there is now considerable impetus for biological studies aimed at understanding the mechanisms by which enediynes cause cell death. The structural and mechanistic diversity of the enediyne family suggests that, in spite of any commonality of radical-based DNA cleavage, there are likely to be many important differences in the cellular responses to individual enediynes. The success of calicheamicin has also served as...

Synthesis of Carbazoles and Related Analogs

The syntheses of 3,6-disubstituted carbazoles and 2,8-disubstituted dibenzothio-phenes were carried out in a similar manner from their corresponding unsub-stituted parent ring systems 89-91 . A typical synthetic route is shown in Scheme 16.1. For N-alkylated products the alkylation step was carried out on the dibromo or dinitrile intermediates. a) Br2) CS2 b) CuCN, DMF, reflux c) EtOH, HCI,1,4-dioxane d) RNH2, EtOH Scheme 16.1 The 2,7-disubstituted carbazoles were prepared in a similar manner...

Synthetic Approaches for Furamidine and Analogs

Our original synthesis of furamidine is outlined in Scheme 16.5 32 and is essentially the approach used by Dann 36 . The process begins with a Friedel-Crafts reaction between bromobenzene and furmaryl chloride. The reaction proceeds well when bromobenzene or carbon disulfide is used as the solvent, but gives very poor yields with other solvents such as nitrobenzene. The reduction of the dibenzoyle-thene can be achieved with Zn HOAc or with stannous chloride, however the latter is now the...

T

Illustration of the oxygenation of Co(II) + Lys-Gly-His resulting in the intermediate formation of a -peroxo dimer and final monomeric Co (111) -Lys-Gly-His metallopeptides. logical availability of molecular oxygen. Such systems could lead to agents that function in vivo 122, 123 . To examine the above, admixtures of Co(II) in the presence of one equivalent of Lys-Gly-His were allowed to react with 32 P end-labeled restriction fragment substrates in the presence or absence of ambient...