RESUMO
DB1255 is a symmetrical diamidinophenyl-dithiophene that exhibits cellular activity by binding to DNA and inhibiting binding of ERG, an ETS family transcription factor that is commonly overexpressed or translocated in leukemia and prostate cancer [Nhili, R., Peixoto, P., Depauw, S., Flajollet, S., Dezitter, X., Munde, M. M., Ismail, M. A., Kumar, A., Farahat, A. A., Stephens, C. E., Duterque-Coquillaud, M., Wilson, W. D., Boykin, D. W., and David-Cordonnier, M. H. (2013) Nucleic Acids Res. 41, 125-138]. Because transcription factor inhibition is complex but is an attractive area for anticancer and antiparasitic drug development, we have evaluated the DNA interactions of additional derivatives of DB1255 to gain an improved understanding of the biophysical chemistry of complex function and inhibition. DNase I footprinting, biosensor surface plasmon resonance, and circular dichroism experiments show that DB1255 has an unusual and strong monomer binding mode in minor groove sites that contain a single GC base pair flanked by AT base pairs, for example, 5'-ATGAT-3'. Closely related derivatives, such as compounds with the thiophene replaced with furan or selenophane, bind very weakly to GC-containing sequences and do not have biological activity. DB1255 is selective for the ATGAT site; however, a similar sequence, 5'-ATGAC-3', binds DB1255 more weakly and does not produce a footprint. Molecular docking studies show that the two thiophene sulfur atoms form strong, bifurcated hydrogen bond-type interactions with the G-N-H sequence that extends into the minor groove while the amidines form hydrogen bonds to the flanking AT base pairs. The central dithiophene unit of DB1255 thus forms an excellent, but unexpected, single-GC base pair recognition module in a monomer minor groove complex.
Assuntos
Amidinas/química , DNA/química , Tiofenos/química , Amidinas/síntese química , Amidinas/metabolismo , Sequência de Aminoácidos , Técnicas Biossensoriais , DNA/metabolismo , Pegada de DNA , Desoxirribonuclease I/metabolismo , Guanina/química , Guanina/metabolismo , Modelos Moleculares , Ressonância de Plasmônio de Superfície , Tiofenos/síntese química , Tiofenos/metabolismo , Temperatura de TransiçãoRESUMO
Heterocyclic diamidines are compounds with antiparasitic properties that target the minor groove of kinetoplast DNA. The mechanism of action of these compounds is unknown, but topological changes to DNA structures are likely to be involved. In this study, we have developed a polyacrylamide gel electrophoresis-based screening method to determine topological effects of heterocyclic diamidines on four minor groove target sequences: AAAAA, TTTAA, AAATT and ATATA. The AAAAA and AAATT sequences have the largest intrinsic bend, whereas the TTTAA and ATATA sequences are relatively straight. The changes caused by binding of the compounds are sequence dependent, but generally the topological effects on AAAAA and AAATT are similar as are the effects on TTTAA and ATATA. A total of 13 compounds with a variety of structural differences were evaluated for topological changes to DNA. All compounds decrease the mobility of the ATATA sequence that is consistent with decreased minor groove width and bending of the relatively straight DNA into the minor groove. Similar, but generally smaller, effects are seen with TTTAA. The intrinsically bent AAAAA and AAATT sequences, which have more narrow minor grooves, have smaller mobility changes on binding that are consistent with increased or decreased bending depending on compound structure.
Assuntos
Amidinas/química , Antiparasitários/química , DNA/química , Sequência de Bases , Eletroforese em Gel de Poliacrilamida , Compostos Heterocíclicos/química , Conformação de Ácido Nucleico , Ressonância de Plasmônio de SuperfícieRESUMO
Small molecule complexes with DNA that incorporate linking water molecules are rare, and the DB921-DNA complex has provided a unique and well-defined system for analysis of water-mediated binding in the context of a DNA complex. DB921 has a benzimidazole-biphenyl system with terminal amidines that results in a linear conformation that does not possess the appropriate radius of curvature to match the minor groove shape and represents a new paradigm that does not fit the classical model of minor groove interactions. To better understand the role of the bound water molecule observed in the X-ray crystal structure of the DB921 complex, synthetic modifications have been made in the DB921 structure, and the interactions of the new compounds with DNA AT sites have been evaluated with an array of methods, including DNase I footprinting, biosensor-surface plasmon resonance, isothermal titration microcalorimetry, and circular dichroism. The interaction of a key compound, which has the amidine at the phenyl shifted from the para position in DB921 to the meta position, has also been examined by X-ray crystallography. The detailed structural, thermodynamic, and kinetic results provide valuable new information for incorporation of water molecules in the design of new lead scaffolds for targeting DNA in chemical biology and therapeutic applications.
Assuntos
DNA/química , DNA/metabolismo , Conformação de Ácido Nucleico , Água/química , Amidinas/química , Amidinas/metabolismo , Sequência de Bases , Benzimidazóis/química , Benzimidazóis/metabolismo , Sítios de Ligação , DNA/genética , Desoxirribonuclease I/metabolismo , Ligação de Hidrogênio , Modelos Moleculares , Peso Molecular , Ressonância de Plasmônio de Superfície , TermodinâmicaRESUMO
A novel series of extended DAPI analogues were prepared by insertion of either a carbon-carbon triple bond (16a-d) or a phenyl group (21a,b and 24) at position-2. The new amidines were evaluated in vitro against both Trypanosoma brucei rhodesiense (T. b. r.) and Plasmodium falciparum (P. f.). Five compounds (16a, 16b, 16d, 21a, 21b) exhibited IC(50) values against T. b. r. of 9nM or less which is two to nine folds more effective than DAPI. The same five compounds exhibited IC(50) values against P. f. of 5.9nM or less which is comparable to that of DAPI. The fluorescence properties of these new molecules were recorded, however; they do not offer any advantage over those of DAPI.
Assuntos
DNA de Protozoário/efeitos dos fármacos , Fluorescência , Indóis/síntese química , Indóis/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Trypanosoma brucei rhodesiense/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Sítios de Ligação , Células Cultivadas , DNA de Protozoário/química , Avaliação Pré-Clínica de Medicamentos , Indóis/química , Estrutura Molecular , Testes de Sensibilidade Parasitária , RatosRESUMO
Most A/T specific heterocyclic diamidine derivatives need at least four A/T base pairs for tight binding to the DNA minor groove. Addition of a GC base pair to A/T sequences typically causes a large decrease in binding constant. The ability to target biologically important sequences of DNA could be significantly increased if compounds that could recognize A/T sites with an intervening GC base pair could be designed. The kinetoplast DNA sequence of parasitic microorganisms, for example, contains numerous three A/T binding sites that are separated by a single G. A series of compounds were prepared to target the AAAGTTT sequence as a model system for discovery of "G-jumpers". The new synthetic compounds have two aromatic-amidine groups for A/T recognition, and these are connected through an oxy-methylene linker to cross the GC. CD experiments indicated a minor groove binding mode, as expected, for these compounds. T(max), surface plasmon resonance, and isothermal titration calorimetry experiments revealed 1:1 binding to the AAAGTTT sequence with an affinity that depends on compound structure. Benzimidazole derivatives gave the strongest binding and had generally good solution properties. The binding affinities to the classical AATT sequence were similar to that for AAAGTTT for these extended compounds, but binding was weaker to the AAAGCTTT sequence with two intervening GC base pairs. Binding to both AAAGTTT and AATT was enthalpy driven for strong binding benzimidazole derivatives.
Assuntos
Pareamento de Bases , Ácidos Nucleicos/metabolismo , Sítios de Ligação , Calorimetria , Dicroísmo Circular , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Ressonância de Plasmônio de SuperfícieRESUMO
The DAPI structure has been modified by replacing the phenyl group with substituted phenyl or heteroaryl rings. Twelve amidines were synthesized and their DNA binding, fluorescence properties, in vitro and in vivo activities were evaluated. These compounds are shown to bind in the DNA minor groove with high affinity, and exhibit superior in vitro antitrypanosomal activity to that of DAPI. Six new diamidines (5b, 5c, 5d, 5e, 5f and 5j) exhibit superior in vivo activity to that of DAPI and four of these compounds provide 100% animal cure at a low dose of 4 × 5 mg/kg i.p. in T. b. rhodesiense infected mice. Generally, the fluorescence properties of the new analogues are inferior to that of DAPI with the exception of compound 5i which shows a moderate increase in efficacy while compound 5k is comparable to DAPI.
Assuntos
DNA/metabolismo , Fluorescência , Indóis/química , Indóis/farmacologia , Piridinas/química , Piridinas/farmacologia , Tripanossomicidas/química , Tripanossomicidas/farmacologia , Trypanosoma brucei rhodesiense/efeitos dos fármacos , Tripanossomíase Africana/tratamento farmacológico , Animais , Dicroísmo Circular , DNA/química , Modelos Animais de Doenças , Camundongos , Estrutura Molecular , Relação Estrutura-Atividade , Tripanossomíase Africana/parasitologiaRESUMO
Fifty-seven 2-phenylquinolines substituted at the phenyl group and C4 of the quinoline were synthesized and analyzed for inhibition of the immunostimulatory effect of oligodeoxynucleotides with a CpG-motif. The Fujita-Ban variant of the classical Free-Wilson analysis gave a highly significant correlation for a series of 48 relatively small molecules demonstrating that (i) the partial contributions of substituents to biological activity (EC(50)) are additive and (ii) assuming similar bioavailability for all quinolines studied, the larger molecules cannot be accommodated within a still unknown biological receptor. The results suggest interaction of a basic antagonist molecule with weakly acidic groups in the antagonist-receptor complex. N-[2-(Dimethylamino)ethyl]-2-[4-(4-methylpiperazino)phenyl]quinolin-4-amine (50) is the most effective antagonist found in this study (EC(50) = 0.76 nM).
Assuntos
Aminoquinolinas/síntese química , Imunossupressores/síntese química , Oligonucleotídeos/antagonistas & inibidores , Piperazinas/síntese química , Quinolinas/síntese química , Aminoquinolinas/química , Aminoquinolinas/farmacologia , Animais , Ilhas de CpG , Imunossupressores/química , Imunossupressores/farmacologia , Camundongos , Piperazinas/química , Piperazinas/farmacologia , Relação Quantitativa Estrutura-Atividade , Quinolinas/química , Quinolinas/farmacologia , Células Tumorais CultivadasRESUMO
One hundred seven 2-arylquinolin-4-amines were assayed in vitro for inhibition of the immunostimulatory effect of oligodeoxynucleotides containing a CpG-motif. The compounds are functionalized with various basic and non-basic groups at the aryl moiety and at the amino substituent of the quinolin-4-amine, and some of them contain an additional substituent at position 6 or 7 of the quinoline. Activities of these antagonists, expressed as EC(50) values, range from 0.2 to 200nM. A statistically significant structure-activity correlation was obtained for the Fujita-Ban variant of the classical Free-Wilson analysis. The CoMFA results derived from several models consistently indicate that electrostatic interactions of the molecules with a biological receptor contribute to biological activities to a greater extent than steric effects.
Assuntos
Adjuvantes Imunológicos/antagonistas & inibidores , Aminoquinolinas/farmacologia , Simulação por Computador , Linfoma de Células B/tratamento farmacológico , Oligodesoxirribonucleotídeos/antagonistas & inibidores , Relação Quantitativa Estrutura-Atividade , Aminoquinolinas/química , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Camundongos , Modelos Moleculares , Estrutura Molecular , Oligodesoxirribonucleotídeos/imunologia , Oligodesoxirribonucleotídeos/farmacologia , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
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 DNA's 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.