RESUMEN
Orthogonally positioned diamino/dicationic polyamides (PAs) have good water solubility and enhanced binding affinity, whilst retaining DNA minor groove and sequence specificity compared to their monoamino/monocationic counterparts. The synthesis and DNA binding properties of the following diamino PAs: f-IPI (3a), f-IPP (4), f-PIP (5), and f-PPP (6) are described. P denotes the site where a 1-propylamino group is attached to the N1-position of the heterocycle. Binding of the diamino PAs to DNA was assessed by DNase I footprinting, thermal denaturation, circular dichroism titration, biosensor surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC) studies. According to SPR studies, f-IPI (3a) bound more strongly (K(eq)=2.4×10(8) M(-1)) and with comparable sequence selectivity to its cognate sequence 5'-ACGCGT-3' when compared to its monoamino analog f-IPI (1). The binding of f-IPI (3a) to 5'-ACGCGT-3' via the stacked dimer motif was balanced between enthalpy and entropy, and that was quite different from the enthalpy-driven binding of its monoamino parent f-IPI (1). f-IPP (4) also bound more strongly to its cognate sequence 5'-ATGCAT-3' (K(eq)=7.4×10(6) M(-1)) via the side-by-side stacked motif than its monoamino analog f-IPP (2a). Although f-PPP (6) bound via a 1:1 motif, it bound strongly to its cognate sequence 5'-AAATTT-3' (K(eq)=4.8×10(7) M(-1)), 15-times higher than the binding of its monoamino analog f-PPP (2c), albeit f-PPP bound via the stacked motif. Finally, f-PIP (5) bound to its target sequence 5'-ATCGAT-3' as a stacked dimer and it has the lowest affinity among the diamino PAs tested (Keq <1×10(5) M(-1)). This was about two times lower in affinity than the binding of its monoamino analog f-PIP (2b). The results further demonstrated that the 'core rules' of DNA recognition by monoamino PAs also apply to their diamino analogs. Specifically, PAs that contain a stacked IP core structure bind most strongly (highest binding constants) to their cognate GC doublet, followed by the binding of PAs with a stacked PP structure to two degenerate AT base pairs, and finally the binding of PAs with a PI core to their cognate CG doublet.
Asunto(s)
ADN/metabolismo , Imidazoles/química , Imidazoles/farmacología , Nylons/química , Nylons/farmacología , Secuencia de Bases , Sitios de Unión , Dicroismo Circular , ADN/química , Huella de ADN , Diseño de Fármacos , Formamidas/química , Formamidas/farmacología , Conformación de Ácido Nucleico , Pirroles/química , Pirroles/farmacología , Resonancia por Plasmón de Superficie , TermodinámicaRESUMEN
With the aim of incorporating a recognition element that acts as a fluorescent probe upon binding to DNA, three novel pyrrole (P) and imidazole (I)-containing polyamides were synthesized. The compounds contain a p-anisylbenzimidazolecarboxamido (Hx) moiety attached to a PP, IP, or PI unit, giving compounds HxPP (2), HxIP (3), and HxPI (4), respectively. These fluorescent hybrids were tested against their complementary nonfluorescent, non-formamido tetraamide counterparts, namely, PPPP (5), PPIP (6), and PPPI (7) (cognate sequences 5'-AAATTT-3', 5'-ATCGAT-3', and 5'-ACATGT-3', respectively). The binding affinities for both series of polyamides for their cognate and noncognate sequences were ascertained by surface plasmon resonance (SPR) studies, which revealed that the Hx-containing polyamides gave binding constants in the 10(6) M(-1) range while little binding was observed for the noncognates. The binding data were further compared to the corresponding and previously reported formamido-triamides f-PPP (8), f-PIP (9), and f-PPI (10). DNase I footprinting studies provided additional evidence that the Hx moiety behaved similarly to two consecutive pyrroles (PP found in 5-7), which also behaved like a formamido-pyrrole (f-P) unit found in distamycin and many formamido-triamides, including 8-10. The biophysical characterization of polyamides 2-7 on their binding to the abovementioned DNA sequences was determined using thermal melts (ΔT(M)), circular dichroism (CD), and isothermal titration calorimetry (ITC) studies. Density functional calculations (B3LYP) provided a theoretical framework that explains the similarity between PP and Hx on the basis of molecular electrostatic surfaces and dipole moments. Furthermore, emission studies on polyamides 2 and 3 showed that upon excitation at 322 nm binding to their respective cognate sequences resulted in an increase in fluorescence at 370 nm. These low molecular weight polyamides show promise for use as probes for monitoring DNA recognition processes in cells.
Asunto(s)
ADN/metabolismo , Diseño de Fármacos , Imidazoles/química , Conformación de Ácido Nucleico , Nylons/química , Nylons/metabolismo , Pirroles/química , Secuencia de Bases , Calorimetría , Dicroismo Circular , ADN/química , ADN/genética , Desoxirribonucleasa I/metabolismo , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/química , Colorantes Fluorescentes/metabolismo , Nucleasa Microcócica/metabolismo , Modelos Moleculares , Nylons/síntesis química , Espectrometría de Fluorescencia , Especificidad por Sustrato , Resonancia por Plasmón de SuperficieRESUMEN
An orthogonally positioned diamino/dicationic polyamide f-IPI 2 was synthesized. It has enhanced binding affinity, and it showed comparable sequence specificity to its monoamino/monocationic counterpart f-IPI 1. Results from CD and DNase I footprinting studies confirmed the minor groove binding and selectivity of polyamides 1 and 2 for the cognate sequence 5'-ACGCGT-3'. SPR studies provided their binding constants: 2.4 × 10(8)M(-1) for diamino 2, which is â¼4 times higher than 5.4 × 10(7)M(-1) for its monoamino analogue 1.
Asunto(s)
ADN/química , Imidazoles/química , Imidazoles/metabolismo , Nylons/química , Pirroles/química , Pirroles/metabolismo , Dicroismo Circular , Huella de ADN , Desoxirribonucleasa I/química , Imidazoles/síntesis química , Nylons/síntesis química , Nylons/metabolismo , Pirroles/síntesis química , Resonancia por Plasmón de SuperficieRESUMEN
N-Methyl imidazole (Im) and N-methyl pyrrole (Py)-containing polyamides that can form stacked dimers can be programmed to target specific DNA sequences in the minor groove of DNA and control gene expression. Polyamides are being investigated as potential medicinal agents for treating diseases including cancer. The naturally occurring polyamide distamycin binds as a dimer in the minor groove of DNA and recognizes sequences rich in A/T and T/A base pairs indiscriminately. Synthetic analogs of distamycin that incorporate N-methylimidazole into the heterocyclic core have been shown to bind to G/C rich sequences with a high degree of specificity. The purpose of this study is to investigate the behavior of polyamides containing the 2,5-linked N-methylpyrrole-2-carboxamide or pyrrole(H) [Py(H)] moiety upon binding to DNA. The synthesis and biophysical characteristics of two polyamides PyPyPyPy(H) 2 and ImPyPyPy(H) 3 designed to test the binding preference of a Py/Pyrrole(H) pairing [Py/Py(H)] and a [Im/Py(H)] is described. Studies utilizing circular dichroism, thermal denaturation (ΔT(M)), biosensor-surface plasmon resonance and DNase I footprinting show that an [Im/Py(H), 3] pairing prefers a G/C or C/G pairing whilst a [Py/Py(H), 2] pairing tolerates A/T or T/A base pairs and avoids a G/C base pair.
Asunto(s)
Composición de Base , ADN/química , Nylons/química , Pirroles/química , Secuencia Rica en At , Sitios de Unión , Dicroismo Circular/métodos , Secuencia Rica en GC , Imidazoles/química , Modelos Moleculares , Estructura Molecular , Resonancia por Plasmón de Superficie/métodosRESUMEN
The synthesis, DNA binding characteristics and biological activity of an N-formamido pyrrole- and imidazole-containing H-pin polyamide (f-PIP H-pin, 2) designed to selectively target the ICB2 site on the topoIIalpha promoter, is reported herein. Thermal denaturation, circular dichroism, isothermal titration calorimetry, surface plasmon resonance and DNase I footprinting studies demonstrated that 2 maintained the selectivity of the unlinked parent monomer f-PIP (1) and with a slight enhancement in binding affinity (K(eq)=5 x 10(5)M(-1)) to the cognate site (5'-TACGAT-3'). H-pin 2 also exhibited comparable ability to inhibit NF-Y binding to 1, as demonstrated by gel shift studies. However, in stark contrast to monomer 1, the H-pin did not affect the up-regulation of topoisomerase IIalpha (topoIIalpha) in cells (Western blot), suggesting that the H-pin does not enter the nucleus. This study is the first to the authors' knowledge that reports such a markedly different cellular response between two compounds of almost identical binding characteristics.
Asunto(s)
Antígenos de Neoplasias/genética , ADN-Topoisomerasas de Tipo II/genética , Proteínas de Unión al ADN/genética , Formamidas/química , Imidazoles/química , Nylons/química , Pirroles/química , Animales , Sitios de Unión , Calorimetría , Línea Celular , Dicroismo Circular , Ratones , Nylons/síntesis química , Nylons/farmacología , Regiones Promotoras Genéticas , Desnaturalización Proteica , Resonancia por Plasmón de Superficie , Temperatura de TransiciónRESUMEN
A total of 24 novel 2,5-diaryl-1,3,4-oxadiazoline analogs of combretastatin A-4 (CA-4, 1) were designed, synthesized, and evaluated for biological activities. The compounds represent two structural classes; the Type I class has three methoxy groups on the A ring and the Type II class has a single methoxy group on the A ring. Biological evaluations demonstrate that multiple structural features control the biological potency. Four of the compounds, 2-(3'-bromophenyl)-5-(3'',4'',5''-trimethoxyphenyl)-2-acetyl-2,3-dihydro-1,3,4-oxadiazoline (9l), 2-(2',5'-dimethoxyphenyl)-5-(3''-methoxyphenyl)-2-acetyl-2,3-dihydro-1,3,4-oxadiazoline (10h), 2-(3',4',5'-trimethoxyphenyl)-5-(3''-methoxyphenyl)-2-acetyl-2,3-dihydro-1,3,4-oxadiazoline (10i), and 2-(3',5'-dimethoxyphenyl)-5-(3''-methoxyphenyl)-2-acetyl-2,3-dihydro-1,3,4-oxadiazoline (10j), have potent antiproliferative activities against multiple cancer cell lines. Mechanistic studies indicate that they retain the microtubule disrupting effects of compound 1, including microtubule loss, the formation of aberrant mitotic spindles, and mitotic arrest. Compound 10i inhibits purified tubulin polymerization and circumvents drug resistance mediated by P-glycoprotein and betaIII tubulin expression. The oxadiazoline analog 10i is a promising lead candidate worthy of further investigation.
Asunto(s)
Diseño de Fármacos , Oxadiazoles/química , Estilbenos/síntesis química , Estilbenos/farmacología , Animales , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Células HeLa , Humanos , Ratones , Estructura Molecular , Estereoisomerismo , Estilbenos/química , Relación Estructura-ActividadRESUMEN
Imidazole and pyrrole-containing polyamides belong to an important class of compounds that can be designed to target specific DNA sequences, and they are potentially useful in applications of controlling gene expression. The extent of polyamide curvature is an important consideration when studying the ability of such compounds to bind in the minor groove of DNA. The current study investigates the importance of curvature using polyamides of the form f-Im-Phenyl-Im, in which the imidazole heterocycles are placed in ortho-, meta-, and para-configurations of the phenyl moiety. The synthesis and biophysical evaluation of each compound binding to its cognate DNA sequence (5'-ACGCGT-3') and a negative control sequence (5'-AAATTT-3') is reported, along with their comparison to the parent binder, f-Im-Py-Im (3). ACGCGT is a medicinally significant sequence present in the MluI cell-cycle box (MCB) transcriptional element found in the promoter of a gene associated with cell division. The results demonstrated that the para-derivative has the greatest affinity for its cognate sequence, as indicated via thermal denaturation, CD, ITC, SPR analyses, and DNase I footprinting. ITC studies showed that binding of the para-isomer (2c) to ACGCGT was significantly more exothermic than binding to AAATTT. In contrast, no heat change was observed for binding of the meta- (2b) and ortho- (2a) isomers to both DNAs, due to low binding affinities. This is consistent with results from SPR studies, which indicate that the para-derivative binds in a 2:1 fashion to ACGCGT and binds weakly to ACCGGT (K = 1.8 x 10(6) and 4.0 x 10(4) M(-1), respectively). Interestingly, it binds in a 1:1 fashion to AAATTT (K = 5.4 x 10(5) M(-1)). The meta-compound does not bind to any sequence. The para-derivative also was the only compound to show an induced peak via CD at 330 nm, indicative of minor groove binding, and produced a DeltaT(m) value of 5.8 degrees C. Molecular modeling experiments have been performed to determine the shape differences between the three compounds, and the results indicate that the para-derivative 2c has a closest curvature to previously synthesized polyamides. DNase I footprinting studies confirmed earlier observations that only the para-derivative 2c produced a footprint with ACGCGT (1 microM) and no significant footprint was observed at any sites examined for meta-2b and ortho-2a analogs up to 40 microM. The results of these studies suggest that the shape of the ortho- and meta- derivatives is too curved to match the curvature of the DNA minor groove to facilitate binding. The para-derivative gives the highest binding affinity in the series and the results illustrate that 4-aminobenzamide is a reasonable substitute for 4-aminopyrrole-2-carboxylate.
Asunto(s)
ADN/genética , ADN/metabolismo , Nylons/química , Nylons/metabolismo , para-Aminobenzoatos , Ácido 4-Aminobenzoico/química , Secuencia de Bases , Benzamidas , Calorimetría , Dicroismo Circular , ADN/análisis , ADN/química , Desoxirribonucleasa I/metabolismo , Modelos Moleculares , Conformación Molecular , Desnaturalización de Ácido Nucleico , Nylons/farmacología , Especificidad por Sustrato , Resonancia por Plasmón de Superficie , Temperatura de TransiciónRESUMEN
The N-terminal formamido group on imidazole- and pyrrole-containing polyamides causes stacked polyamides to bind in the minor groove of DNA in the staggered motif, and it also increases the binding affinity compared to those of non-formamido compounds. To further investigate the role of the N-terminal acylamido in affecting sequence specificity and binding affinity, six polyamide analogues containing the core triheterocyclic structure IPI were designed and synthesized, and the acylamido moiety reported herein includes the following: formamido (f-IPI, 1), acetamido (Ac-IPI, 2), trifluoroacetamido (Tf-IPI, 3), N-methylureido (Mu-IPI, 4), N-methylpyrrole-2-carboxamido (PIPI, 5), and the (13)C-labeled formamido-IPI compound ((13)C-f-IPI, 6). In addition, two nonacylated IPI compounds were also synthesized and examined, namely, the amino-containing (NH(2)-IPI, 7) and non-formamido (nf-IPI, 8) compounds. The binding characteristics of compounds 1-8 were investigated using methods of molecular biology and biochemistry, which included biophysical techniques, such as DNA melts, circular dichroism, isothermal titration calorimety, and surface plasmon resonance and DNase I footprinting. With the exception of nf-IPI and NH(2)-IPI, all other compounds preferentially interacted with the cognate sequence, 5'-ACGCGT-3'. The biophysical results suggest that all six compounds bind within the minor groove at their cognate DNA sequence as stacked, staggered, antiparallel dimers. The order from highest to lowest binding affinities is as follows: f-IPI > P-IPI > Ac-IPI > Mu-IPI > Tf-IPI >> NH(2) and nf-IPI. Hence, having an acylamido moiety at the N-terminus is important for the binding of polyamides to DNA in a stacked and staggered motif. According to footprinting analysis, P-IPI (5), Ac-IPI (2), Mu-IPI (4), and Tf-IPI (3) exhibited some enhancement in sequence preference for their cognate 5'-ACGCGT-3' over f-IPI (1). NMR analysis of the [(13)C]f-IPI-CGCGnmr complex showed a slight downfield shift in the formamido (13)C signal indicating that the moiety remained intact. The trend in binding affinity suggests that steric factors play a role, in which small and planar aromatic acylamido units such as f, Ac, and P are preferred. Polar groups, such as in Mu-IPI (4) and Tf-IPI (3), afforded negative effects on binding affinity, compared to that of Ac-IPI (2).
Asunto(s)
ADN/química , Imidazoles/química , Nylons/química , Pirroles/química , Secuencia de Bases , Sitios de Unión , Dicroismo Circular , ADN/metabolismo , Huella de ADN , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Nylons/metabolismo , Sensibilidad y Especificidad , Resonancia por Plasmón de Superficie , Termodinámica , VolumetríaRESUMEN
Imidazole (Im) and Pyrrole (Py)-containing polyamides that can form stacked dimers can be programmed to target specific sequences in the minor groove of DNA and control gene expression. Even though various designs of polyamides have been thoroughly investigated for DNA sequence recognition, the use of H-pin polyamides (covalently cross-linked polyamides) has not received as much attention. Therefore, experiments were designed to systematically investigate the DNA recognition properties of two symmetrical H-pin polyamides composed of PyImPyIm (5) or f-ImPyIm (3e, f=formamido) tethered with an ethylene glycol linker. These compounds were created to recognize the cognate 5'-ACGCGT-3' through an overlapped and staggered binding motif, respectively. Results from DNaseI footprinting, thermal denaturation, circular dichroism, surface plasmon resonance and isothermal titration microcalorimetry studies demonstrated that both H-pin polyamides bound with higher affinity than their respective monomers. The binding affinity of formamido-containing H-pin 3e was more than a hundred times greater than that for the tetraamide H-pin 5, demonstrating the importance of having a formamido group and the staggered motif in enhancing affinity. However, compared to H-pin 3e, tetraamide H-pin 5 demonstrated superior binding preference for the cognate sequence over its non-cognates, ACCGGT and AAATTT. Data from SPR experiments yielded binding constants of 1.6x10(8)M(-1) and 2.0x10(10)M(-1) for PyImPyIm H-pin 5 and f-ImPyIm H-pin 3e, respectively. Both H-pins bound with significantly higher affinity (ca. 100-fold) than their corresponding unlinked PyImPyIm 4 and f-ImPyIm 2 counterparts. ITC analyses revealed modest enthalpies of reactions at 298 K (DeltaH of -3.3 and -1.0 kcal mol(-1) for 5 and 3e, respectively), indicating these were entropic-driven interactions. The heat capacities (DeltaC(p)) were determined to be -116 and -499 cal mol(-1)K(-1), respectively. These results are in general agreement with DeltaC(p) values determined from changes in the solvent accessible surface areas using complexes of the H-pins bound to (5'-CCACGCGTGG)(2). According to the models, the H-pins fit snugly in the minor groove and the linker comfortably holds both polyamide portions in place, with the oxygen atoms pointing into the solvent. In summary, the H-pin polyamide provides an important molecular design motif for the discovery of future generations of programmable small molecules capable of binding to target DNA sequences with high affinity and selectivity.
Asunto(s)
Imidazoles/química , Nylons/síntesis química , Pirroles/química , Secuencia de Bases , Calorimetría , Dicroismo Circular , Simulación por Computador , Modelos Moleculares , Desnaturalización de Ácido Nucleico , Nylons/química , Resonancia por Plasmón de Superficie , Termodinámica , VolumetríaRESUMEN
Fifteen curcumin analogs were synthesized and tested for in-vitro cytotoxicity towards B16 and L1210 murine cancer cell lines using an MTT assay. Significant activity was discovered for two analogs: 8 (B16 IC(50) = 1.6 microM; L1210 IC(50) = 0.35 microM) and 9 (B16 IC(50) = 0.51 microM; L1210 IC(50 )= 1.2 microM). Several other analogs exhibited notable cytotoxicity. The data from quantitative structure-activity relationships suggest that large electron-withdrawing substituents placed in the meta-position of the arylidene aryl rings enhance potencies. Compounds 8 and 9 were found using a cell-based assay to have virtually no effects on microtubules at concentrations up to 40 microM. These results suggest that tubulin inhibition is not the principal mechanism by which the curcumin analogs act.
Asunto(s)
Antineoplásicos/síntesis química , Curcumina/análogos & derivados , Ciclohexanonas/síntesis química , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Línea Celular Tumoral , Ciclohexanonas/administración & dosificación , Ciclohexanonas/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Concentración 50 Inhibidora , Ratones , Microtúbulos/efectos de los fármacos , Relación Estructura-Actividad Cuantitativa , Tubulina (Proteína)/efectos de los fármacosRESUMEN
Seven N-terminus modified derivatives of a previously published minor-groove binding polyamide (f-ImPyIm, 1) were synthesized and the biochemical and biophysical chemistry evaluated. These compounds were synthesized with the aim of attaining a higher level of sequence selectivity over f-ImPyIm (1), a previously published strong minor-groove binder. Two compounds possessing a furan or a benzofuran moiety at the N-terminus showed a footprint of 0.5microM at the cognate ACGCGT site (determined by DNase I footprinting); however, the specificity of these compounds was not improved. In contrast, PyImPyIm (4) produced a footprint of 0.5microM but showed a superior specificity using the same technique. When evaluated by thermal melting experiments and circular dichroism using ACGCGT and the non-cognate AAATTT sequence, all compounds were shown to bind in the minor-groove of DNA and stabilize the cognate sequence much better than the non-cognate (except for the non-amido-compound that did not bind either sequence, as expected). PyImPyIm (4) was interesting as the DeltaT(m) for this compound was only 4 degrees C but the footprint was very selective. No binding was observed for this compound with a third DNA (non-cognate, ACCGGT). ITC studies on compound 4 showed exothermic binding with ACGCGT and no heat change was observed for titrating the compound to the other two DNA sequences. The heat capacity (DeltaC(p)) of the PIPI/ACGCGT complex calculated from the hydrophobic interactions and SASA calculations was comparable to the experimental value obtained from ITC (-146calmol(-1)K(-1)). SPR results provided confirmation of the sequence specificity of PyImPyIm (4), with a K(eq) value determined to be 7.1x10(6) M(-1) for the cognate sequence and no observable binding to AAATTT and ACCGGT. Molecular dynamic simulations affirmed that PyImPyIm (4) binds as a dimer in an overlapped conformation, and it fits snugly in the minor-groove of the ACGCGT oligonucleotide. PyImPyIm (4) is an especially interesting molecule, because although the binding affinity is slightly reduced, the specificity with respect to f-ImPyIm (1) is significantly improved.
Asunto(s)
Furanos/química , Imidazoles/química , Nylons/química , Análisis de Secuencia de ADN/métodos , Secuencia de Bases , Huella de ADN , Desoxirribonucleasa I/química , Modelos Moleculares , Estructura Molecular , Nylons/síntesis química , Sensibilidad y Especificidad , Resonancia por Plasmón de Superficie , Factores de TiempoRESUMEN
Studies on the binding of a triamide f-IPI (1) to its cognate sequence labeled with a 2-aminopurine (2AP or G( *)) group are described. ITC studies showed that f-IPI (1) bound to the cognate site (ACG( *)CGT) with only 3.5-fold lower affinity than binding to the unlabeled DNA (ACGCGT) (K(eq)=2 x 10(7) and 7 x 10(7)M(-1), respectively). Titration of f-IPI (1) to both sequences gave strong induced bands at 330 nm via circular dichroism studies. The compound also gave comparable DeltaT(m) values of 5.0 and 7.8 degrees C, respectively. These techniques also proved that the sequence selectivity of f-IPI (1) was uncompromised, as only limited binding to the non-cognate sequence ACCG( *)GT was observed. Fluorescence studies demonstrated a 2:1 ligand:DNA binding motif as anticipated, and indicated that the limit of detection for this technique was 20muM DNA concentration. The results demonstrate that 2-aminopurine is a sufficient substitute for guanine in a G.C base pair useful in DNA binding studies.
Asunto(s)
2-Aminopurina/química , Citosina/química , ADN/química , Nylons/química , Oligonucleótidos/química , Espectrometría de Fluorescencia , Emparejamiento Base , Sitios de UniónRESUMEN
Eleven 1,2,3,4-tetrahydro-2-thioxopyrimidine analogs of combretastatin-A4 (CA-4) were synthesized and their cytotoxicity against the growth of two murine cancer cell lines (B16 melanoma and L1210 leukemia) in culture was determined using an MTT assay. Two 2-thioxopyrimidine analogs 8f and 9a exhibited significant activity (IC50<1 microM for L1210 and <10 microM for B16 cells). Exposure of A-10 cells to 8f and 9a produced a significant reduction in cellular microtubules in interphase cells, with an EC50 value of 4.4 and 2.9 microM, respectively, for microtubule loss. Molecular modeling studies using MacSpartan indicated that the two active 2-thioxopyrimidine analogs preferably adopt a twisted conformation, similar to CA-4, affirming that conformation and structure are connected to activity.
Asunto(s)
Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Pirimidinas/química , Estilbenos/síntesis química , Estilbenos/farmacología , Animales , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ratones , Estilbenos/químicaRESUMEN
The synthesis and DNA binding characteristics of a polyamide-intercalator conjugate, designed to inhibit NF-Y binding to the ICB-2 site of the topoisomerase IIalpha promoter and up-regulate the expression of the enzyme in confluent cells, are reported. Thermal denaturation and CD titration studies demonstrated binding to the cognate sequence (5'-AAGCTA-3'). Formation of ligand-induced CD bands at approximately 330 nm provided indication that the molecule interacts selectively in the minor groove of DNA. Intercalation was evidenced by a fivefold increase in emission of the intercalator moiety upon binding to the ICB-2 hairpin oligonucleotide. An increase in viscosity of a solution of calf-thymus DNA on addition of the conjugate provided further evidence. The binding affinity of the conjugate was ascertained using SPR (5.6x10(6) M(-1)), which according to a gel shift assay was capable of inhibiting the binding of NF-Y at a concentration of 50 microM. DNaseI footprinting, using the topoIIalpha promoter sequence, highlighted the specificity of the conjugate for the cognate site (5'-AAGCTA-3'). Finally, through Western blot analysis, confluent murine NIH 3T3 cells treated with conjugate were found to have enhanced expression of topoIIalpha. These results suggest that the conjugate can enter the nucleus, bind to its target site, presumably as a stacked dimer, and up-regulate the expression of topoIIalpha by blocking the binding of NF-Y.
Asunto(s)
Antígenos de Neoplasias/genética , Factor de Unión a CCAAT/antagonistas & inhibidores , ADN-Topoisomerasas de Tipo II/genética , Proteínas de Unión al ADN/genética , Sustancias Intercalantes/farmacología , Factores de Transcripción/antagonistas & inhibidores , Células 3T3 , Animales , Secuencia de Bases , Sitios de Unión , ADN , Dimerización , Sustancias Intercalantes/química , Ratones , Nylons/química , Nylons/farmacología , Regiones Promotoras Genéticas , Unión Proteica/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The polyamide N-formamido imidazole-pyrrole-imidazole (f-ImPyIm) binds with an exceptionally high affinity for its cognate site 5'-ACGCGT-3' as a stacked, staggered, and noncovalent cooperative dimer. Investigations are presented into its sequence specificity and binding affinity when linked covalently as an H-pin "dimer". Five f-ImPyIm cross-linked analogues with six to nine methylene linkers and an eight-linked ethylene glycol linker were examined to investigate the effect of linkage and linker length on DNA binding. Thermal denaturation studies on short DNA hairpins showed preferential binding by both f-ImPyIm (DeltaTm = 7.8 degrees C) and its cross-linked derivatives (DeltaTm > 30 degrees C) at 5'-ACGCGT-3', indicating sequence specificity was retained on linkage. DNase I footprinting confirmed strict cognate site selectivity and demonstrated that affinity increased with linker length (f-ImPyIm-9 = f-ImPyIm-8 = f-ImPyIm-EG-8 > f-ImPyIm-7 > f-ImPyIm-6). The eight- and nine-linked derivatives bound at 100-fold lower concentrations at the cognate site relative to f-ImPyIm-6, and with 10-fold higher affinity than unlinked f-ImPyIm. Use of an ethylene glycol linkage in f-ImPyIm-EG-8 to improve solubility slightly increased the cognate site affinity relative to those of f-ImPyIm-8 and f-ImPyIm-9, although some selectivity was lost at high ligand concentration. CD demonstrated that cognate site binding by eight and nine-linked compounds occurred in the minor groove. SPR analysis gave a binding affinity (K) for f-ImPyIm-EG-8 at the cognate site of 2 x 10(10) M-1, representing a 100-fold increase relative to that of f-ImPyIm. This study demonstrates that the high-affinity cooperative binding of f-ImPyIm can be enhanced significantly by suitable covalent linkage, while maintaining its strict cognate site selectivity.
Asunto(s)
ADN/química , Imidazoles/química , Pirroles/química , Antivirales/química , Secuencia de Bases , Sitios de Unión , Dicroismo Circular , Huella de ADN , Desoxirribonucleasa I/farmacología , Dimerización , Distamicinas/química , Imidazoles/síntesis química , Imidazoles/metabolismo , Cinética , Modelos Químicos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Desnaturalización de Ácido Nucleico , Pirroles/síntesis química , Pirroles/metabolismo , Sensibilidad y Especificidad , Resonancia por Plasmón de Superficie , Temperatura , VolumetríaRESUMEN
Fourteen N-acetylated and non-acetylated 3,4,5-tri- or 2,5-dimethoxypyrazoline analogs of combretastatin-A4 (1) were synthesized. A non-acetylated derivative (5a) with the same substituents as CA-4 (1) was the most active compound in the series, with IC(50) values of 2.1 and 0.5 microM in B16 and L1210 cell lines, respectively. In contrast, a similar compound with an acetyl group at N1 of the pyrazoline ring (6g) showed poor activity in the cell lines studied. A cell-based assay indicated that compound 5a caused extensive microtubule depolymerization with an EC(50) value of 7.1 microM in A-10 cells while no activity was seen with the acetylated compound. Molecular modeling studies showed that these compounds possess a twisted conformation similar to CA-4 (1).
Asunto(s)
Diseño de Fármacos , Pirazoles/química , Estilbenos/química , Estilbenos/farmacología , Animales , Línea Celular , Ratones , Modelos Moleculares , Estilbenos/síntesis químicaRESUMEN
Five polyamide derivatives with rationally modified C-terminus moieties were synthesized and their DNA binding specificity and affinity determined. A convergent approach was employed to synthesize polyamides containing an alkylaminopiperazine (4 and 5), a truncated piperazine (6), or an alkyldiamino-C-terminus moiety (7 and 8) with two specific objectives: to investigate the effects of number of potential cationic centers and steric bulk at the C-terminus. CD studies confirmed that compounds 4, 5, 7, and 8 bind in the minor groove of DNA. The alkylpiperazine containing compounds (4 and 5) showed only moderate binding to DNA with DeltaT(m) values of 2.8 and 8.3 degrees C with their cognate sequence, respectively. The alkyldiamino compounds (7 and 8) were more impressive producing a DeltaT(m) of >17 and >22 degrees C, respectively. Compound 6 (truncated piperazine) did not stabilize its cognate DNA sequence. Footprints were observed for all compounds (except compound 6) with their cognate DNA sequence using DNase I footprinting, with compound 7 producing a footprint of 0.1 microM at the expected 5'-ACGCGT-3' site. SPR analysis of compound 7 binding to 5'-ACGCGT-3', 5'-ACCGGT-3', and 5'-AAATTT-3' produced binding affinities of 2.2x10(6), 3.3x10(5), and 1x10(5)M(-1), respectively, indicating a preference for its cognate sequence of 5'-ACGCGT-3'. These results are in good agreement with the footprinting data. The results indicate that steric crowding at the C-terminus is important with respect to binding. However, the number of cationic centers within the molecule may also play a role. The alkyldiamino-containing compounds (7 and 8) warrant further investigation in the field of polyamide research.
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Amidas/química , ADN/efectos de los fármacos , Distamicinas/síntesis química , Distamicinas/farmacología , Imidazoles/química , Pirroles/química , Secuencia de Bases , Sitios de Unión , Unión Competitiva , Dicroismo Circular/métodos , ADN/química , Huella de ADN/métodos , Distamicinas/química , Datos de Secuencia Molecular , Estructura Molecular , Nylons/síntesis química , Nylons/química , Nylons/farmacología , Sensibilidad y Especificidad , Estereoisomerismo , Relación Estructura-Actividad , Especificidad por Sustrato , Resonancia por Plasmón de Superficie/métodos , Temperatura , Factores de TiempoRESUMEN
An N-formamido pyrrole- and imidazole-containing triamide (f-PIP) has been shown by DNase I footprinting, SPR, and CD studies to bind as a stacked dimer to its cognate sequences: 5'-TACGAT-3' (5'-flank of the inverted CCAAT box-2 of the human topoisomerase IIalpha promoter) and 5'-ATCGAT-3'. A gel shift experiment provided evidence for f-PIP to inhibit protein-DNA interaction at the ICB2 site. Western blot studies showed that expression of the topoisomerase IIalpha gene in confluent NIH 3T3 cells was induced by treatment with f-PIP. The results suggested that the triamide was able to enter the nucleus, interacted with the target site within ICB2, inhibited NF-Y binding, and activated gene expression.
Asunto(s)
ADN-Topoisomerasas de Tipo II/genética , Distamicinas/farmacocinética , Regulación Neoplásica de la Expresión Génica , Imidazoles/farmacocinética , Regiones Promotoras Genéticas , Pirroles/farmacocinética , Células 3T3 , Animales , Secuencia de Bases , Dicroismo Circular , Huella de ADN , Desoxirribonucleasa I , RatonesRESUMEN
The synthesis and DNA-binding properties of a novel naphthalimide-polyamide hairpin (3) designed to target the inverted CCAAT box 2 (ICB2) site on the topoisomerase IIalpha (topoIIalpha) promoter are described. The polyamide component of 3 was derived from the minor-groove binder, 2, and tailored to bind to the 5'-TTGGT sequence found in and flanking ICB2. The propensity of mitonafide 4 to intercalate between G-C base pairs was exploited by the incorporation of a naphthalimide moiety at the N terminus of 2. Hybrid 3 targeted 5'-CGATTGGT and covered eight contiguous base pairs, which included the underlined ICB2 site. DNase I footprinting analysis with the topoIIalpha promoter sequence demonstrated that 3 bound selectively to the ICB2 and ICB3 sites. Thermal-denaturation studies confirmed these results, and the highest degree of stabilization was found for ICB2 and -3 in preference to ICB1 (4.1, 4.6, and 0.6 degrees C, respectively). CD studies confirmed minor-groove binding and suggested a 1:1 binding stoichiometry. Emission-titration experiments established intercalative binding. Surface plasmon resonance results showed strong binding to ICB2 (2.5x10(7) M(-1)) with no observable binding to ICB1. Furthermore, the binding constant of 3 to ICB2 was larger than that of the parent polyamide 2. The increased binding affinity was primarily due to a reduction in the dissociation-rate constant of the polyamide-DNA complex, which can be attributed to the N-terminal naphthalimide moiety. In addition, the binding site of 3 was larger than that of 2, which innately improved sequence selectivity. We conclude that the polyamide-naphthalimide 3 selectively binds to the ICB2 site by simultaneous intercalation and minor-groove binding, and warrants further investigation as a model compound for the regulation of topoIIalpha gene expression.