Farial A. Tanious

Antiparasitic compounds that target DNA

Designed, synthetic heterocyclic diamidines have excellent activity against eukaryotic parasites that cause diseases such as sleeping sickness and leishmania and adversely affect millions of people each year. The most active compounds bind specifically and strongly in the DNA minor groove at AT sequences. The compounds enter parasite cells rapidly and appear first in the kinetoplast that contains the mitochondrial DNA of the parasite. With time the compounds are also generally seen in the cell nucleus but are not significantly observed in the cytoplasm. The kinetoplast decays over time and disappears from the mitochondria of treated cells. At this point the compounds begin to be observed in other regions of the cell, such as the acidocalcisomes. The cells typically die in 24-48h after treatment. Active compounds appear to selectively target extended AT sequences and induce changes in kinetoplast DNA minicircles that cause a synergistic destruction of the catenated kinetoplast DNA network and cell death.

Dications That Target the DNA Minor Groove: Compound Design and Preparation, DNA Interactions, Cellular Distribution and Biological Activity

Fluorescence microscopy of trypanosomes from drug treated mice shows that biologically active heterocyclic diamidines that target the DNA minor groove bind rapidly and specifically to parasite kinetoplast DNA (k-DNA). The observation that the kinetoplast is destroyed, generally within 24 hours, after drug treatment is very important for understanding the biological mechanism, and suggests that the diamidines may be inhibiting some critical opening/closing step of circular k-DNA. Given the uncertainties in the biological mechanism, we have taken an empirical approach to generating a variety of synthetic compounds and DNA minor groove interactions for development of improved and new biological activities. Furamidine, DB75, is a diphenyl-diamidine that has the curvature to match the DNA minor groove as expected in the classical groove interaction model. Surprisingly, a linear diamidine with a nitrogen rich linker has significantly stronger binding than furamidine due to favorable linker and water-mediated DNA interactions. The water interaction is very dependant on compound structure since other linear compounds do not have similar interactions. Change of one phenyl of furamidine to a benzimidazole does not significantly enhance DNA binding but additional conversion of the furan to a thiophene (DB818) yields a compound with ten times stronger binding. Structural analysis shows that DB818 has a very favorable curvature for optimizing minor groove interactions. It is clear that there are many ways for compounds to bind to k-DNA and exert specific effects on kinetoplast replication and/or transcription that are required to obtain an active compound.

The activity of diguanidino and 'reversed' diamidino 2,5-diarylfurans versus Trypanosoma cruzi and Leishmania donovani.

The in vitro activity of 20 dicationic molecules containing either diguanidino or reversed amidine cationic groups were evaluated versus Trypanosoma cruzi and Leishmania donovani. The most active compounds were in the reversed amidine series and six exhibited IC(50) values of less than 1 micro mol versus T. cruzi and five gave similar values versus L. donovani.

Antitumor Polycyclic Acridines. 20. Search for DNA Quadruplex Binding Selectivity in a Series of 8,13-Dimethylquino[4,3,2-kl]acridinium Salts: Telomere-Targeted Agents

The growth-inhibitory activities of an extensive series of quaternized quino[4,3,2- kl]acridinium salts against tumor cell lines in vitro have been measured and their biological properties interpreted in the light of differential binding to different DNA isoforms. Selectivity for quadruplex DNA binding and stabilization by compounds were explored through an array of methods: UV absorption and fluorescence emission spectroscopy, surface plasmon resonance, and competition dialysis. Quadruplex DNA interaction was further characterized through FRET and DNA polymerase arrest assays. Telomerase inhibition, inferred from the TRAP assay, is attributed to quadruplex stabilization, supported by the strong correlation (R(2) = 0.81) across the series between quadruplex DNA binding affinity and TRAP inhibition potency. Growth inhibition potency in the NCI60 human tumor cell line panel is more marked in compounds with greater DNA duplex binding affinity (R(2) = 0.82). Quantification of relative quadruplex and duplex binding affinity constants puts some of these ligands among the most selective quadruplex DNA interactive agents reported to date.

Biosensor-surface plasmon resonance methods for quantitative analysis of biomolecular interactions.

The surface plasmon resonance (SPR) biosensor method has emerged as a very flexible and powerful approach for detecting a wide diversity of biomolecular interactions. SPR monitors molecular interactions in real time and provides significant advantages over optical or calorimetric methods for systems with strong binding and low spectroscopic signals or reaction heats. The SPR method simultaneously provides kinetic and equilibrium characterization of the interactions of biomolecules. Such information is essential for development of a full understanding of molecular recognition as well as for areas such as the design of receptor-targeted therapeutics. This article presents basic, practical procedures for conducting SPR experiments. Initial preparation of the SPR instrument, sensor chips, and samples are described. This is followed by suggestions for experimental design, data analysis, and presentation. Steady-state and kinetic studies of some small molecule-DNA complexes are used to illustrate the capability of this technique. Examples of the agreement between biosensor-SPR and solution studies are presented.

Anti-Pneumocystis carinii pneumonia activity of dicationic carbazoles

Summary A series of 2,7- and 3,6-bis cationic carbazoles was synthesized and evaluated for activity against a rat model of Pneumocystis carinii pneumonia (PCP). The compounds were also tested for inhibition of topoisomerase II and binding to DNA. Several of the compounds proved to be more potent and less toxic than a standard anti-PCP drug (pentamidine). While no quantitative correlation was seen between anti-PCP activity, topoisomerase inhibition and DNA binding, a minimal level of DNA binding was found to be necessary for antimicrobial activity.

Loop and Backbone Modifications of Peptide Nucleic Acid Improve G-Quadruplex Binding Selectivity

Targeting guanine (G) quadruplex structures is an exciting new strategy with potential for controlling gene expression and designing anticancer agents. Guanine-rich peptide nucleic acid (PNA) oligomers bind to homologous DNA and RNA to form hetero-G-quadruplexes but can also bind to complementary cytosine-rich sequences to form heteroduplexes. In this study, we incorporated backbone modifications into G-rich PNAs to improve the selectivity for quadruplex versus duplex formation. Incorporation of abasic sites as well as chiral modifications to the backbone were found to be effective strategies for improving selectivity as shown by UV-melting and surface plasmon resonance measurements. The enhanced selectivity is due primarily to decreased affinity for complementary sequences, since binding to the homologous DNA to form PNA-DNA heteroquadruplexes retains high affinity. The improved selectivity of these PNAs is an important step toward using PNAs for regulating gene expression by G-quadruplex formation.

Synthesis and anti-Pneumocystis carinii pneumonia activity of novel dicationic dibenzothiophenes and orally active prodrugs.

Dicationic carbazoles have been found to be highly active against a rat model of Pneumocystis carinii pneumonia (PCP). Unfortunately, amidoxime derivatives, designed as prodrugs, were inactive against PCP even though the corresponding amidines were highly active. In the present work, a series of 2,8- and 3,7-bis cationic dibenzothiophenes was synthesized and assayed for anti-PCP activity. Three of the compounds proved to be more potent and less toxic than a standard anti-PCP drug (pentamidine) when given intravenously. Unlike the carbazoles, a dibenzothiophene amidoxime prodrug given orally reduced the parasite load by more than 99%. While no quantitative correlation was seen between anti-PCP activity and DNA binding, a strong level of DNA binding was found to be necessary for antimicrobial activity.

Different Binding Mode in AT and GC Sequences for Unfused-Aromatic Dications

We have previously synthesized a 2,5-diphenylfuranamidine dication (4) and presented evidence that this compound binds to AT sequences in DNA by a minor-groove interaction mode but binds to GC sequences by intercalation (1,2). To probe these sequence-dependent binding modes in more detail, and particularly to obtain additional evidence for the binding mode in GC rich sequences, we have synthesized and studied the DNA complexes of 1-3 which have the furan ring of 4 replaced by 2,6-substituted pyridine (1), pyrimidine (2), or triazine (3) ring systems. The three compounds with a six-membered central ring system bind to AT DNA sequences more weakly than the furan compound, but retain the minor-groove binding mode. The pyridine and pyrimidine derivatives bind to GC sequences of DNA more strongly than the furan, but the triazine derivative binds more weakly. The aromatic proton signals of 1-3, as previously observed with 4 shift upfield by approximately 0.5 ppm or greater on complex formation with polyd(G-C)2. This and other spectroscopic as well as viscosity and kinetics results indicate that 1-4 bind to GC sites in DNA by intercalation. A nonclassical intercalation model, with the twisted-unfused, aromatic ring system intercalated into an intercalation site of matching structure can explain all of our and the literature results for the GC binding mode of these unfused, aromatic compounds.

Bis-4-aminoquinolines: novel triple-helix DNA intercalators and antagonists of immunostimulatory CpG-oligodeoxynucleotides

Six dimeric 2-(2-naphthyl)quinolin-4-amines with a linker between the amino groups and eight dimeric 2-(4-anilino)quinolin-4-amines linked between the anilino groups were synthesized and evaluated for their interaction with duplex/triplex DNAs and as antagonists of immunostimulatory oligodeoxynucleotides with a CpG-motif (CpG-ODN). The most powerful triple-helix DNA intercalator known to date, with high affinity toward T.A.T triplets and triplex/duplex selectivity, was found. The potent antagonism of immunostimulatory CpG-ODN by several bis-4-aminoquinolines is not related to their DNA interactions.