Small Molecule Drugs

Targeting DNA and Topoisomerase I with Indolocarbazole Antitumor Agents

Minimum Inhibitory Concentration Mic

In the cell, DNA primarily exists in a supercoiled form and therefore unwinding of DNA is required prior to using it for transcription, replication, or recombination. These functions require the services of enzymes called topoisomerases, which cleave and religate one or two strands of DNA, and hence allow the DNA to unwind 1 . For more than 15 years, these enzymes have attracted considerable attention as targets for cancer therapeutics since many drugs inhibiting them, such as topotecan,...

DNA Binding of Furamidine and Analogs

Aatt Dna Cuts

The Wilson, Neidle, and Bailly laboratories have performed extensive biophysical studies including NMR, X-ray crystallographic, and footprinting investigations which demonstrate that the primary mode of DNA binding for furamidine is at AT-rich sites of the minor groove 114-121 and that its weaker secondary binding occurs at GC sites. Similar dual binding modes have been demonstrated for the structurally related diamidines berenil and DAPI 73, 74, 88, 122-127 . The mode of binding of furamidine...

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...

Increasing Stability Small Molecule Drug

Complexation of nucleosides and nucleotides by CP 66, in aqueous solution, has been extensively studied 12 . It binds purine derivatives strongly and the com-plexation-induced NMR shift values, up to 1.7 ppm, are in favor of the inclusion of the adenine moiety into the cavity of CP66 with the sugar ring outside. NMR titration curves showed that the stoichiometry of the complexes is 1 1 for every substrate tested. Among purines, the selectivity for adenine derivatives is remarkable for example,...

Structural Studies of EcteinascidinDNA Complexes

Dna Bendability

Knowledge of the above rules and potential problems is important in order to design a suitable duplex oligonucleotide for structural studies. If the sequence contains, for example, a 5 '-AGC target triplet site on one strand followed by a cytosine, there will be an alternative binding site, 5'-GGC, on the complementary strand, and this may compromise the success of the experiment. Although inosine could be used to replace guanine at some key positions, as in the band shift experiments reported...

H r if Vn 50304 42355 16539

The experiments with alternative labels Tab. 25.3 confirmed that significant binding differentials between the NIO-protected and -deprotected PBD-resins are achievable, thus indicating the potential to develop sensitive screening procedures based on this strategy. The covalent interaction between the tethered PBD and DNA is validated by the experiment shown in Fig. 25.6 in which it can be seen that labeled DNA remains associated with the PBD-resin after four washes with buffer. The covalent...

Structural features

Torus With Small Hole

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...

The Rate of Reversal of Et 743 from Drug Modified 5AGT Is Faster Than That from Drug Modified 5AGC Sequences

Cyclization The Acetylaziridine

The first indication that the Et 743 alkylation reaction is reversible came from initial endeavors to create a 60-mer oligonucleotide containing an Et 743 site-directed adduct at either a 5'-AGTor 5'-AGC sequence. The substrates were constructed by ligating a duplex oligonucleotide modified with Et 743 at a single guanine to flanking duplex oligonucleotides on both the 5'- and 3sides. Although we succeeded at creating a site-directed adduct at a 5'-AGC sequence, attempts at generating a...

Salt Dependency of Daunorubicin Binding to DNA

Daunorubicin Binding

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...

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...

DNA Binding of Dicationic Carbazoles and Analogs

The carbazoles and related analogs were originally tested for DNA-binding affinity with calf thymus DNA by determination of changes in thermal melting ATm values 89-91 . The ATm values for selected compounds are shown Tab. 16.1. The group as a whole produced a number of high-affinity DNA-binding agents with the carbazole derivatives appearing to be stronger binders than the corresponding dibenzothiophenes and dibenzofurans. The most potent binder as measured by ATm was compound 12, Tab. 16.1 ,...

Electrochemical Detection of DNA with Small Molecules

Small molecules that can bind to nucleic acids DNA and RNA often show a characteristic change in their absorption spectra upon complexation with nucleic acids 1, 2 . Studies on the interaction of small molecules with DNA have been based on these phenomena. Since most of these DNA-binding small molecules are also electrochemically active reducible or oxidative, i.e. redox active , and their interaction with DNA can be studied electrochemically. Some examples of electrochemically active...

Neocarzinostatin Recognition of Bulged DNA Structures

Most Drugs

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...

Sources Biosynthesis and Structural Conservation

Functional Group Calicheamicin

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...

Triple Helixspecific Ligands

Triple Strand Dna

The formation of intermolecular DNA triple helices offers the possibility of designing compounds with extensive sequence recognition properties, which may be useful as antigene agents or tools in molecular biology. In these structures a third strand oligonucleotide binds in the DNA major groove, making specific contacts with substituents on the exposed faces of the base pairs. Although triplexes form with exquisite specificity they are often less stable than their duplex counterparts. One means...

References

M. S. et al Calicheamicins, a novel family of antitumor antibiotics. 1. Chemistry and partial structure of calicheamicin yll. J. Am. Chern. Soc. 1987, 109. 3464-3466. 2 Temple, R. FDA letter to WyethAyerst concerning approval of NDA 21-174. 2000. http www.fda.gov cder foi1 appletter 2000 21174ltr.pdf. 3 Dedon, P. C. The mechanism of calicheamicin cytotoxicity. Biol. Ther. Leukemia 2001, 2, 2-4. 4 Konishi, M., Ohkuma, H., Saitoh, K.-I. et al. Esperamicins, a...

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...

Introduction

The anthracycline antibiotics have been mainstays of cancer chemotherapy for over 30 years 1-6 . Daunorubicin (daunomycin) and doxorubicin (adriamycin) are the parental compounds of this class of drugs. Both are natural products, and are synthesized by particular species of Streptomyces 7, 8 . Daunorubicin is primarily used now in the treatment of leukemias. Doxorubicin has a broad spectrum of activity, and is used alone and in combination to treat a variety of cancers. Doxorubicin differs from...

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,...

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...

Parsing the Binding Free Energy

Once a detailed thermodynamic profile is obtained, it is possible to attempt parsing of the observed binding free energy for an intercalation process to reveal the underlying contributions from various molecular interactions 34, 36, 37 . The observed binding free energy (AG0bs) contains contributions from at least five component terms A G0bs AGconf + AGr+t + AGpe + AGhyd + AGmoi In this equation, AGconf is the contribution from conformational transitions in the DNA and intercalator, AGr+t is...

Top2 poisons

The number of known top2 poisons greatly exceeds that of topi poisons. Indeed different families of drugs are able to interfere with the cleavage rejoining process mediated by top2. These include epipodophyllotoxins, anthracyclines, acridines, anthraquinones, ellipticines, bisantrene, actinomycin D, terpenoids, quinolones, and flavonoids 8, 9 . The different chemotypes hardly share similar structural features. To confirm this, some are very efficient DNA binders, yet they poorly poison top2,...

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...

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...

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...

Asociation Of Molecule

SPR sensorgrams for the interaction at 25 C of f-lmlmlm with CCGG (a) CTGGsm (b), and CTGGdm (c). The concentration of the unbound polyamide varied from 7.5 x 10-7 M to 2.6 x 10 5 M in (a), from 1.0 x 10 9 M to 2.0 x 10 6 M in (b), and 4.0 x 10 6 M in (c). All the experiments were done in MES20. The right-side panel shows the models for the dimeric side-by-side binding of f-lmlmlm to CCGG, CTGGsm, and CTGGdm in the staggered (preferred) conformations. binding of f-Imlmlm to...

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...

T G G C C A3t C C A

Staggered and overlapped stacking Polyamides with a terminal formamido prefers modes of Pylmlm (a) and ImPyPy (b) are to stack in a staggered fashion, but the non- illustrated with and without a terminal formamido analogs stack in an overlapped formamido group, respectively. In the two manner, sequences the heterocycles are switched. posed that the stacking of polyamides in the formation of the dimer complexes with DNA is different when the compounds have a terminal formamido group...

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...

Structural Characterization and Synthesis of Ecteinascidins

Small Molecule Drugs

The crystal structures of N12-formyl C21-methoxy Et 729 formic acid methanol solvate dihydrate and Et 743 N12-oxide acetonitrile solvate octahydrate deposited in the Cambridge Structural Data Base under accession codes WAMBAN and WAMBER, respectively revealed two independent ecteinascidin molecules per asymmetric unit plus several solvent molecules. The molecular shape of both ecteinascidins is highly compact, with much of the surface occupied by hydrophobic methyl, methylene, and aryl-CH...

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...

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...

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 ....

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...

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...

Targeting Hiv Rna with Small Molecules

Luedtke and Yitzhak Tor Introduction The central dogma of biology states that RNA is selectively transcribed from DNA and serves as a messenger to be translated into proteins 1, 2 . Recent discoveries detailing the molecular workings of the cell have inspired new generations of chemists to search for a deeper understanding of the biophysical world. The majority of this book presents important advances in the study of DNA this chapter is one of the few to explore RNA. We have,...

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...

Amsacrlnemltoxantrone

Small Molecule Drugs

Chemical structure of selected classical top2 poisons. A further contribution to the mechanistic aspects of top2 inhibition derives from the finding that ATP-bound top2 acts as a target for antitumor drugs and discriminates between two groups of compounds ATP-insensitive top2 poisons such as amonafide, batracylin, and menadione that were poorly responsive to the ATP-bound conformation of top2, and ATP-sensitive top2 poisons including doxorubicin, etoposide, mitoxantrone, and...

Antimicrobial Activity of Furamidine and Analogs

As previously noted, furamidine has been shown to be effective against three different organisms in animal models. The effectiveness against trypanosomes was described sometime ago in both murine and simian models 32, 33 . More recently, Hall and co-workers have demonstrated the efficacy of furamidine using one intravenous dose of 2.5 pmol kg 1 against a mouse model of the disease see summary in Tab. 16.6 . In this study mice were infected with T. brucei brucei S427 clone 22 and were treated 72...

Strand IStrand II

Small Molecule Antibiotics

Irradiation time - 1h 2h 3h 1h 2h 3h - 1h 2h 3h 1h 2h 3h BisA Exo ill G A---- ---- G A Ci G2 C3 G4 Ts Ae C7 G8 C9 A10 C11X12 C13 An C15 G18 C17 A18 T19 G20 C21 G22 C23 G46 C45 G44 C43 A42 T41 G40 C39 GSS T37 G36 T35 G34 T33 G32 C31 G30 T29 A28 C27 G26C25 G24 Fig. 11.12. Cleavage pattern of a 23-mer duplex containing an apurinic site X. Lane 1 duplex incubated with exonuclease III to localize the abasic lesion lanes 2 and 1 7 C A for strand I and II lanes 3 and 10 non-irradiated strands I and II...

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...

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...

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...

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...

Synthesis of Carbazoles and Related Analogs

Small Molecule Drugs

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

Nco Acrylate

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...

Hydration Changes

Small Molecule Drugs

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 ....

Structure Activity Relationships

Small Molecule Drugs

Initial structure-activity relationships among natural members of the ecteinascidin class were based on results from in vitro cytotoxic screening assays against murine leukemia cells L1210 19 . The importance of the reactive carbinolamine for optimal activity was highlighted by the 175-fold decrease in activity in going from Et 743 to its C21-deoxy counterpart Et 745, and the 17-fold reduction in potency upon oxidation of the carbinolamine to the lactam (Et 759A). Likewise, replacement of the...

Dicationic DNA Minor Groove Binders as Antimicrobial Agents

Boy kin Introduction DNA is an attractive target for drug design due to the large amount of detailed structural information and its role as the source of genetic information 1 . The strategy of using small molecule DNA binders to alter gene expression was suggested some time ago and it has been continually refined 2-4 . The completion of numerous genomic projects now adds greater significance to this approach. Recent in vivo experiments with fruit flies...

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...

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...

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...

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...

Prodrug Approaches for Furamidine

As noted in a previous section arylamidines are quite basic and exhibit pK values of approximately 11 148 . Consequently, the low oral bioavailability of these type molecules is not unexpected. Nevertheless, very few reports have appeared describing pro-drug approaches for aryl amidines 104, 105 . Weller and co-workers have demonstrated that both amidoxime and carbamate derivatives of mono-amidines are effective pro-drugs and provide significantly improved oral bioavailability for fibrinogen...

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...

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...

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...

Triplex Cleaving Agents

Addition of an EDTA moiety to a triplex-binding ligand can convert it into a triplex-specific cleavage agent in the presence of Fe2+ and a reducing agent. EDTA conjugates, attached to the alkyamino side chain, have been prepared for BePI, BgPI 26 and BQQ 130 . The ligands cleave DNA at sites to which a triplex-forming oligonucleotide is bound, though the background cleavage in non-targeted duplex DNA is greater than that produced by EDTA-tethered oligonucleotides 3 . BgPI and BQQ conjugates...

Antimicrobial Activity of Carbazoles and Related Analogs

A number of carbazole derivatives have been shown to be highly effective in two animal models of infection and effective in vitro against several other pathogenic microbial organisms. Initial antimicrobial studies of the carbazoles against Pneumocystis carinii pneumonia in a rat model of the disease 89 and Cryptosporidium parvum in a neonatal mouse model 18 clearly showed several of the compounds to be highly active. The data for 13 compounds against the two organisms are shown in Tab. 16.3...

Substituent Contributions

Thermodynamic studies can provide quantitative information about the contribution of specific drug substituents to the binding free energy 30, 37, 50 . Rather detailed structure-affinity relationships may then be examined. The approach is as follows. For a series of drug analogs synthesized to contain the substituent changes of interest, complete binding isotherms are obtained at several salt concentrations. From these primary data, observed binding free energies are obtained, which are then...

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...

Structure of the CalicheamicinDNA Complex

A series of NMR studies over the past decade has produced a nearly complete picture of the structure of the DNA complexes of calicheamicin 32-34, 38, 45-47 and esperamicin 21, 48 . Dinshaw Patel's group has provided what are arguably Fig. 22.3. NMR structural models for the DNA complexes of calicheamicin y (left. Ref. 38 ) and esperamicin A1 (right, Ref. 48 ). The calicheamicin complex was prepared with a hairpin deoxyoligonucleotide shown beneath the image, while the esperamicin complex Fig....

Structure and Dynamics of Calicheamicin Binding Sites

Calicheamicin target selection was first proposed to involve G-C-containing tetra-purine sequences such as AGGA-TGGT 62, 63 . This led to the proposal of a critical interaction between the aryl iodide atom and the exocyclic N2 of dG 49 , which, as discussed earlier, is clearly not critical for calicheamicin target recognition. The contribution of hydrophobic interactions to the stability of the calicheamicin-DNA complex 31 suggests another mechanism for targeting of tetrapurine sequences by the...

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...

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...

Tethered Triplexbinding Ligands

Benzopyridoindole and benzopyridoquinoline triplex-binding ligands have been attached to either the 5'-end or to an internucleotide position of triplex-forming oligonucleotides 98, 99 . All these conjugates stabilize triple helical structures more than the addition of free ligand at an equivalent concentration. As expected the attached ligand had a greater effect on sequences containing longer run of contiguous thymines. Benzo h qtiinoquinoxaline was the most effective compound when attached to...

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...

Dedication of this Chapter to Professor J William Lown on the Occasion of his Retirement

William Lown was co-organized by Professors William H. Gmeiner of Wake Forest University School of Medicine and Moses Lee of Furman University and held March, 2001 on the campus of Wake Forest University School of Medicine. Both Professors Gmeiner and Lee were post-doctoral fellows in the laboratory of Professor Lown in the Chemistry Department of the University of Alberta. Among the distinguished scientists who attended the Lown Symposium were Professor Peter...

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...

Metabolism and pharmacology of DACA

The primary route of metabolism of DACA is oxidation at C-9 by aldehyde oxidase to give the acridone 87, although oxidative demethylation of the side chain dime-thylamino group has also been observed 73 . Comparative studies of the conversion of DACA to 87 were carried out using aldehyde oxidase preparations from human, guinea pig, and rat. The human enzyme had intrinsic clearance values Vmax lt m ranging from 0.27 to 0.35 mL min 1 mg 1 protein, whereas rat and guinea pig had approximately 7-...

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,...

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...

Triplex versus Quadruplexspecific Ligands and Telomerase Inhibition

Patrizia Alberti, Magali Hoarau, Lionel Guittat, Masashi Takasugi, Paola B. Arimorido, Laurent Lacroix Martin Mills, Marie-Paule Teulade-Fichou, Jeari-Pierre Vigrterort, Jeart-Marie Lehri, Patrick Mailliet, arid Jean-Lou is Mergriy A number of alternative DNA structures have been described to date. As stressed previously, DNA comes in many forms 1 . It can exist in a variety of double-, triple-, and quadruple-stranded forms. The list of alternative conformations that can be adopted by this...

Biological Activity and Characterization of the Active Compounds

Crude aqueous ethanol extracts of Ecteinascidia turbinata were shown to have powerful immunomodulating and antiproliferative properties as early as 1969 1 . Many unsuccessful attempts at isolation of the active compounds were followed by concerted efforts that led to the characterization of six alkaloids called ecteinasci-dins 729, 743, 745, 759A, 759B, and 770 (Fig. 23.1), of which Et 743 was the most Small Molecule DNA and RNA Binders From Synthesis to Nucleic Acid Complexes. Volume 2. Edited...

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...

LiNaK Rb Cs

Drug And Alcohol Side Effects

Dependency of the non-polyelectrolyte portion of the binding free energy on cation type. Triangles refer to data obtained for daunorubicin. Solid circles refer to data obtained for ethidium bromide. In order to partition the binding free energy into its enthalpic and entropic components, calorimetric studies or studies of the temperature dependence of the 17.4 Binding Enthalpy and the Temperature Dependence of Binding Fig. 17.6. Isothermal titration studies of daunorubicin binding to...

DNA Interactions of Novel Platinum Anticancer Drugs

Viktor Brabec and Jana Kasparkova Introduction Platinum compounds constitute a discrete class of anticancer agents, which are now widely used in the clinic. The interest in antitumor platinum drugs has its origin in the 1960s, with the serendipitous discovery by Rosenberg of the inhibition of division of bacterial cells by platinum complexes 1 . The first platinum anticancer drug in clinical use was cisplatin (cis-diamminedichloroplatinum(II)), which is a very simple and purely inorganic...

Type I Topoisomerases

Topi participates in the relaxation of negative super coils and has also been implicated in knotting and unknotting DNA and in linking complementary rings of single-stranded DNA into double-stranded rings 12 . The relaxation process catalysed by the enzyme essentially consists of a transient break of a segment of single-stranded DNA, followed by passing an intact strand of DNA through the gate (type I-A) or allowing rotation of the broken strand around the intact strand (type I-B), with final...

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...

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...

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...

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...

Prodrugs of Carbazoles and Related Analogs

Because aromatic amidines are strongly basic pJCa between 10 and 12 there seems very little chance that they will be absorbed from the gastrointestinal tract and therefore they do not make good candidates as oral drugs. Chemical modification of biologically active compounds to form new molecules pro-drugs , usually biologically inactive, which can be converted in vivo to the parent molecule can potentially improve physico-chemical properties such as water solubility and lipo-philicity,...

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...

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...

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...

Use of the TG Recognition Motif by Imlm Pairs to Probe the Effects of the Terminal Head Group on the Stacking of

As described above, examination of 2 1 complexes of polyamides and DNA revealed that the nature by which the heterocyclic units stack can directly impact their sequence recognition 39 . As described above, a terminal formamido group can shift stacking in the polyamide system from fully overlapped, without a formamido, to an offset or staggered motif (Fig. 15.3). This shift in stacking affects the DNA sequence recognized by polyamides and it is essential to define the stacking mechanisms in...

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...

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...

TC Mismatched DNA Base Pairs and their Biological Relevance

Mutations in DNA that go unrepaired lead to the polymorphic nature of individual genomes. Such mutations can lead to evolutionary variations and advantages, but they can also lead to disease states or tendencies. T-G mismatched base pairs in DNA (Fig. 15.4b), for example, are responsible for most of the common mutations leading to the formation of tumors in humans. Human bladder carcinoma is an example where a G-C to A T transition at the 3'-G of the GG doublet in codon 12 of the Ha-ras and...

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...

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...

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...