RESUMEN
The new 5-substituted SN-38 derivatives, 5(R)-(N-pyrrolidinyl)methyl-7-ethyl-10-hydroxycamptothecin (1) and its diastereomer 5(S) (2), were investigated using a combination of nuclear magnetic resonance (NMR) spectroscopy and molecular modeling methods. The chemical stability, configuration stability, and propensity to aggregate as a function of concentration were determined using 1H NMR. The calculated self-association constants (Ka) were found to be 6.4 mM-1 and 2.9 mM-1 for 1 and 2, respectively. The NMR experiments were performed to elucidate the interaction of each diastereomer with a nicked decamer duplex, referred to as 3. The calculated binding constants were determined to be 76 mM-1 and 150 mM-1 for the 1-3 and 2-3 complexes, respectively. NMR studies revealed that the interaction between 1 or 2 and the nicked decamer duplex occurred at the site of the DNA strand break. To complement these findings, molecular modeling methods and calculation protocols were employed to establish the interaction mode and binding constants and to generate molecular models of the DNA/ligand complexes.
Asunto(s)
ADN , Imagen por Resonancia Magnética , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Irinotecán , ADN/químicaRESUMEN
The understanding of the mechanism of Topo I inhibition by organic ligands is a crucial source of information that has led to the design of more effective and safe pharmaceuticals in oncological chemotherapy. The vast number of inhibitors that have been studied in this respect over the last decades have enabled the creation of a concept of an 'interfacial inhibitor', thereby describing the machinery of Topo I inhibition. The central module of action of this machinery is the interface of a Topo I/DNA/inhibitor ternary complex. Most of the 'interfacial inhibitors' are primarily kinetic inhibitors that form molecular complexes with an "on-off" rate timing; therefore, all of the contacts between the inhibitor and both the enzyme and the DNA are essential to keep the complex stable and reduce the "off rate". To test this hypothesis, we designed the compound using a C-9-(N-(2'-hydroxyethyl)amino)methyl substituent in an SN38 core, with a view that a flexible substituent may bind inside the nick of a model of the DNA and stabilize the complex, leading to a reduction in the "off rate" of a ligand in a potential ternary complex in vivo. Using docking analysis and molecular dynamics, free energy calculations on the level of the MM-PBSA and MM-GBSA model, here we presented the in silico-calculated structure of a ternary complex involving the studied compound 1. This confirmed our suggestion that compound 1 is situated in a groove of the nicked DNA model in a few conformations. The number of hydrogen bonds between the components of a ternary complex was established, which strengthens the complex and supports our view. The docking analysis and free energy calculations for the receptor structures which were obtained in the MD simulations of the ternary complex 1/DNA/Topo I show that the binding constant is stronger than it was for similar complexes with TPT, CPT, and SN38, which are commonly considered as strong Topo I inhibitors. The binary complex structure 1/DNA was calculated and compared with the experimental results of a complex that was in a solution. The analysis of the cross-peaks in NOESY spectra allowed us to assign the dipolar interactions between the given protons in the calculated structures. A DOSY experiment in the solution confirmed the strong binding of a ligand in a binary complex, having a Ka of 746 mM-1, which was compared with a Ka of 3.78 mM-1 for TPT. The MALDI-ToF MS showed the presence of the biohybrid, thus evidencing the occurrence of DNA alkylation by compound 1. Because of it having a strong molecular complex, alkylation is the most efficient way to reduce the "on-off" timing as it acts as a tool that causes the cog to brake in a working gear, and this is this activity we want to highlight in our contribution. Finally, the Topo I inhibition test showed a lower IC50 of the studied compound than it did for CPT and SN38.
Asunto(s)
Camptotecina , Protones , Ligandos , Inhibidores de Topoisomerasa I/farmacología , Inhibidores de Topoisomerasa I/química , ADN-Topoisomerasas de Tipo I/metabolismo , Inhibidores de Topoisomerasa , ADN/metabolismo , Preparaciones FarmacéuticasRESUMEN
Novel nontoxic derivatives of SN38 with favorable antineoplastic properties were characterized in water solution using NMR. The phenomena observed by NMR were linked to basic pharmacological properties, such as solubility, bioavailability, chemical and stereochemical stability, and binding to natural DNA oligomers through the terminal G-C base pair, which is commonly considered a biological target of Topo I inhibitors. Compound 1, with bulky substituents at both C5(R) and C20(S) on the same side of a camptothecin core, manifests self-association, whereas diastereomers 2, with bulky C5(S) and C20(S) substituents are mostly monomeric in solution. The stereogenic center at C5 is stable in water solution at pH 5-6. The compound with an (N-azetidinyl)methyl substituent at C9 can undergo the retro Mannich reaction after a prolonged time in water solution. Both diastereomers exhibit different abilities in terms of binding to DNA oligomers: compound 1 is strongly bound, whereas the binding of compound 2 is rather weak. Molecular modeling produced results consistent with NMR experiments. These complementary data allow linking of the observed phenomena in NMR experiments to basic preliminary information on the pharmacodynamic character of compounds and are essential for planning further development research.
Asunto(s)
Antineoplásicos/química , ADN/química , Irinotecán/análogos & derivados , Simulación del Acoplamiento Molecular , Inhibidores de Topoisomerasa I/química , Antineoplásicos/toxicidad , Irinotecán/toxicidad , Inhibidores de Topoisomerasa I/toxicidadRESUMEN
The compounds 7-ethyl-9-(N-methylamino)methyl-10-hydroxycamptothecin (2) and 7-ethyl-9-(N-morpholino)methyl-10-hydroxycamptothecin (3) are potential topoisomerase I poisons. Moreover, they were shown to have favorable anti-neoplastic effects on several tumor cell lines. Due to these properties, the compounds are being considered for advancement to the preclinical development stage. To gain better insights into the molecular mechanism with the biological target, here, we conducted an investigation into their interactions with model nicked DNA (1) using different techniques. In this work, we observed the complexity of the mechanism of action of the compounds 2 and 3, in addition to their decomposition products: compound 4 and SN38. Using DOSY experiments, evidence of the formation of strongly bonded molecular complexes of SN38 derivatives with DNA duplexes was provided. The molecular modeling based on cross-peaks from the NOESY spectrum also allowed us to assign the geometry of a molecular complex of DNA with compound 2. Confirmation of the alkylation reaction of both compounds was obtained using MALDI-MS. Additionally, in the case of 3, alkylation was confirmed in the recording of cross-peaks in the 1H/13C HSQC spectrum of 13C-enriched compound 3. In this work, we showed that the studied compounds-parent compounds 2 and 3, and their potential metabolite 4 and SN38-interact inside the nick of 1, either forming the molecular complex or alkylating the DNA nitrogen bases. In order to confirm the influence of the studied compounds on the topoisomerase I relaxation activity of supercoiled DNA, the test was performed based upon the measurement of the fluorescence of DNA stain which can differentiate between supercoiled and relaxed DNA. The presented results confirmed that studied SN38 derivatives effectively block DNA relaxation mediated by Topo I, which means that they stop the machinery of Topo I activity.
Asunto(s)
Camptotecina/análogos & derivados , Camptotecina/metabolismo , Roturas del ADN de Cadena Simple , ADN-Topoisomerasas de Tipo I/química , ADN-Topoisomerasas de Tipo I/metabolismo , ADN Superhelicoidal , Inhibidores de Topoisomerasa II/farmacología , Alquilación , HumanosRESUMEN
Derivatives of SN38 were synthesized that were either monosubstituted at C-5 or C-9 or disubstituted at both C-5 and C-9. Substitution to C-5 led to the generation of pairs of diastereomers (2c-2 h) in a one-pot reaction and was readily separable by HPLC. The absolute configurations of C-5 were established by electronic circular dichroism experiments. Compounds were tested in vitro against human cancer cell lines as well as a normal cell line. The impact of compounds 2a-2j on cancer cells is significant and the IC50 values against the normal cell line are several times higher than that of SN38. Using the Mannich reaction we obtained a new innovative group of derivatives with unique biological properties that preserves the high cytotoxicity in cancer cells and eliminates the acute toxicity to non-neoplastic cells, which can be considered a breakthrough in chemotherapy with the use of topoisomerase I inhibitors from the camptothecin family.
Asunto(s)
Antineoplásicos/farmacología , Camptotecina/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Camptotecina/síntesis química , Camptotecina/química , Línea Celular , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Estructura Molecular , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
In this account we present NMR based results of the interaction of 7-ethyl-9-hydroxymethyl-10-hydroxycamptothecin (1), a derivative of SN38, with a model nicked DNA decamer mimicking the wild type DNA target of Topoisomerase I inhibitors from the camptothecin family. The title compound 1 can be considered a main metabolite of phase I in the metabolic pathway of camptothecin derivatives bearing the alkylamino substituent. Therefore, its pharmacodynamic properties are of interest. It was established by DOSY (Diffusion Ordered Spectroscopy) that compound 1 forms a fairly stable molecular complex with a model nicked DNA decamer with affinity constant Ka 3.02 mM-1. The analysis of NOESY experiments revealed intermolecular cross peaks and mutual induced shifts on both interacting components allowing the conclusion that guest molecule 1 is stacking the nitrogen bases inside the nick. MD (Molecular Dynamics) analysis of four possible inclusions of 1 inside the nick allows establishing the detailed geometry of a complex. Two conformations are suggested as the ones best representing the results of molecular modeling reconciled with experimental NOESY results. The aromatic core of both structures is stacking the nitrogen bases in a nick facing the unbroken strand with ring A. The protons in ring E interact with ribose protons of edge bases of a nick. In conclusion, it can be asserted that SN38 derivative 1 can effectively bind the molecular target of Topo I enzyme and play a role as a Topo I inhibitor.
Asunto(s)
Camptotecina/química , ADN-Topoisomerasas de Tipo I/química , ADN/química , Inhibidores de Topoisomerasa I/química , Sitios de Unión , Camptotecina/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , ADN/metabolismo , ADN-Topoisomerasas de Tipo I/metabolismo , Humanos , Enlace de Hidrógeno , Simulación de Dinámica Molecular , Resonancia Magnética Nuclear Biomolecular , Conformación de Ácido Nucleico , Inhibidores de Topoisomerasa I/metabolismoRESUMEN
PURPOSE: Identification of human insulin analogs' impurity with a mass shift +14 Da in comparison to a parent protein. METHODS: The protein sequence variant was detected and identified with the application of peptide mapping, liquid chromatography, tandem mass spectrometric analysis, nuclear magnetic resonance spectroscopy (NMR) and Edman sequencing. RESULTS: The misincorporated lysine (Lys) at asparagine (Asn) position A21 was detected in recombinant human insulin and its analogs. CONCLUSIONS: Although there are three asparagine residues in the insulin derivative, the misincorporation of lysine occurred only at position A21. The process involves G/U or A/U wobble base pairing.
Asunto(s)
Asparagina/química , Escherichia coli/metabolismo , Insulinas/metabolismo , Lisina/análisis , Cromatografía Líquida de Alta Presión/métodos , Escherichia coli/genética , Humanos , Insulinas/química , Péptidos/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrometría de Masas en Tándem/métodosRESUMEN
The NMR derived translational diffusion coefficients were performed on unlabeled and uniformly labeled 13C,15N human insulin in water, both in neat, with zinc ions only, and in pharmaceutical formulation, containing only m-cresol as phenolic ligand, glycerol and zinc ions. The results show the dominant role of the pH parameter and the concentration on aggregation. The diffusion coefficient Dav was used for monitoring the overall average state of oligomeric ensemble in solution. The analysis of the experimental data of diffusion measurements, using the direct exponential curve resolution algorithm (DECRA) allows suggesting the two main components of the oligomeric ensemble. The 3D HSQC-iDOSY, (diffusion ordered HSQC) experiments performed on 13C, 15N-fully labeled insulin at the two pH values, 4 and 7.5, allow for the first time a more detailed experimental observation of individual components in the ensemble. The discussion involves earlier static and dynamic laser light scattering experiments and recent NMR derived translational diffusion results. The results bring new informations concerning the preparation of pharmaceutical formulation and in particular a role of Zn2+ ions. They also will enable better understanding and unifying the results of studies on insulin misfolding effects performed in solution by diverse physicochemical methods at different pH and concentration.
Asunto(s)
Insulina/química , Resonancia Magnética Nuclear Biomolecular/métodos , Agregado de Proteínas , Difusión , Humanos , Ligandos , Pliegue de Proteína , Zinc/químicaRESUMEN
The synthesis of water-soluble SN38 derivatives is presented, and their stability in solutions used during drug development studies has been investigated. A preliminary study of mechanism of action of 9-aminomethyl SN38 is presented. Using NMR techniques, the interaction of the oligomer d(GCGATCGC)2 is studied, showing that the terminal GC base pairs are the main site of interaction. Using pulsed field gradient spin echo and mass spectroscopy, evidence of a spontaneous alkylation reaction of the DNA oligomer with SN38 derivatives is presented. A proposed mechanism of reaction is suggested. Copyright © 2016 John Wiley & Sons, Ltd.
Asunto(s)
Antineoplásicos/química , Camptotecina/análogos & derivados , ADN/química , Alquilación , Antineoplásicos/síntesis química , Secuencia de Bases , Camptotecina/síntesis química , Camptotecina/química , Estabilidad de Medicamentos , Irinotecán , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Estructura Molecular , Polidesoxirribonucleótidos/química , Relación Estructura-ActividadRESUMEN
A monomer structure of a novel human insulin analog A22S-B3K-B31R (SK3R) has been characterized by NMR in water/acetonitrile solution and compared with the structure of human insulin (HIS) established in the same medium. The composition of the oligomer ensemble for neat insulins in water was qualitatively assessed by monitoring, derived from NMR experiment, translational diffusion coefficient Dix10-10m2s-1, whose value is a population averaged of individual coefficients for species in oligomeric ensemble. Nanospray ESI/MS experiment was used to establish the masses of oligomers in pharmaceutical formulation of the SK3R insulin. The pharmacodynamic data were established and compared to insulin glargine characterized by the same profile of action in diabetics. The oligomerization process of insulin during development of pharmaceutical formulation with routinely used excipients has been studied using translation diffusion coefficient Dix10-10m2s-1 established in water solution. These properties were compared with those of human insulin (HIS) which is a standard reference for novel recombinant insulins.
Asunto(s)
Insulina/análisis , Insulina/química , Espectroscopía de Resonancia Magnética/métodos , Cristalografía por Rayos X/métodos , Preparaciones de Acción Retardada/análisis , Preparaciones de Acción Retardada/química , Composición de Medicamentos , Humanos , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Proteínas Recombinantes/análisis , Proteínas Recombinantes/química , Espectrometría de Masa por Ionización de Electrospray/métodosRESUMEN
Using DOSY NMR and MALDI-TOF MS techniques, we present evidence that quaternary trimethylammonium salts of topotecan, [TPT-NMe3 ](+) X(-) (X = CF3SO3, HCOO), bind covalently the natural DNA oligomer upon near UV irradiation in water under physiological conditions. It is shown that formate salt is very reactive at pH 7 and requires short irradiation time. This weak irradiation at 365 nm paves the way for a new application of TPT derivatives in clinical use, which can dramatically increase the therapeutic effects of a medicine.
Asunto(s)
ADN/química , Compuestos de Amonio Cuaternario/química , Topotecan/química , Rayos Ultravioleta , Sitios de Unión , Espectroscopía de Resonancia Magnética , Estructura Molecular , Sales (Química)/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Tris(pentafluorophenyl)corrole and its (15)N-enriched isotopomer were studied in [D8]toluene solution by 1D and 2D variable-temperature NMR techniques to establish the mechanisms of tautomerization of the NH protons inside the interior of the corrole macrocycle. Three such rate processes could be identified of which two modulate the spectral line shapes at temperatures above 205â K and the third is NMR-inaccessible as it is very fast. The latter involves the proton engaged in an unsymmetrical proton sponge unit formed by two pyrrole nitrogen atoms. Temperature and concentration dependences of the two remaining processes were determined. One of them is purely intramolecular and the other is intermolecular at low temperatures, with growing contribution of an intramolecular mechanism at elevated temperatures. The proposed microscopic mechanisms of all these processes are semi-quantitatively confirmed by quantum chemical calculations using density functional theory.
Asunto(s)
Espectroscopía de Resonancia Magnética , Porfirinas/química , Isótopos de Carbono/análisis , Flúor/análisis , Hidrógeno/análisis , Enlace de Hidrógeno , Isomerismo , Isótopos de Nitrógeno/análisis , TemperaturaRESUMEN
Twelve alkyl analogues (1-12) of the high-affinity serotonin transporter (SERT) inhibitor 6-nitroquipazine (6-NQ) were synthesized and studied using in vitro radioligand competition binding assays to determine their binding affinity (Ki ). The putative antidepressant activity of five of the binders with the highest SERT binding affinities was studied by the forced swim and locomotor activity mouse tests. The three-dimensional (3D) structures of 8 and 9 were determined using NOE NMR technique. Flexible docking of the compounds was undertaken to illustrate the binding of the compounds in the SERT model. Our results showed that several of the 6-NQ analogues are high-affinity SERT inhibitors and indicated that the octyl (8), decyl (10) and dodecyl (12) 6-NQ analogues exhibit moderate antidepressant activity.
Asunto(s)
Antidepresivos/síntesis química , Quipazina/análogos & derivados , Inhibidores Selectivos de la Recaptación de Serotonina/síntesis química , Proteínas de Transporte de Serotonina en la Membrana Plasmática/química , Animales , Antidepresivos/química , Antidepresivos/farmacología , Sitios de Unión , Masculino , Ratones , Simulación del Acoplamiento Molecular , Actividad Motora/efectos de los fármacos , Unión Proteica , Estructura Terciaria de Proteína , Quipazina/síntesis química , Quipazina/química , Quipazina/farmacología , Receptor de Serotonina 5-HT1A/química , Receptor de Serotonina 5-HT1A/metabolismo , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Inhibidores Selectivos de la Recaptación de Serotonina/química , Inhibidores Selectivos de la Recaptación de Serotonina/farmacologíaAsunto(s)
Acetonitrilos/química , Insulina Regular Humana/química , Proteínas Recombinantes/química , Agua/química , Humanos , Enlace de Hidrógeno , Insulina Regular Humana/metabolismo , Modelos Moleculares , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Ingeniería de Proteínas , Subunidades de Proteína , Proteínas Recombinantes/metabolismoAsunto(s)
Antibacterianos/aislamiento & purificación , Dihidroxifenilalanina/análogos & derivados , Inhibidores Enzimáticos/aislamiento & purificación , Streptomyces/química , Acetilación , Antibacterianos/química , Antibacterianos/farmacología , Dihidroxifenilalanina/química , Dihidroxifenilalanina/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Concentración 50 Inhibidora , Pruebas de Sensibilidad Microbiana , Resonancia Magnética Nuclear Biomolecular , Péptido Hidrolasas/metabolismo , Espectrometría de Masa por Ionización de Electrospray , Espectrofotometría Infrarroja , Espectrofotometría UltravioletaRESUMEN
New genistein derivatives were synthesized, which are fairly well soluble in water, with respect to parent genistein, and thus facilitate study of the interaction with dumbbell DNA dodecamer, mimicking the biological target for topoisomerase II inhibitors. A pulsed field gradient spin echo NMR experiment was used to check the binding and to estimate the association constants and its pH dependence of genistein with dumbbell DNA. Experimental restraints based on nuclear Overhauser spectroscopy spectra were used to calculate the NMR structure in solution in case of 6,8-disubstituted genistein with dimethylaminomethyl groups and were used in molecular modeling calculations. The structure is dynamic, and 10 molecular dynamics runs yield a family of conformations that essentially differ in a depth of inclusion of genistein into a nick. The paper experimentally shows evidence for binding, intercalation in the nick is proposed as a mode of genistein binding, and a model of the event is provided.