Carlos De los santos - Academia.edu (original) (raw)

Papers by Carlos De los santos

Comprehensive Natural Products Chemistry, 1999

LEGICOM, 1999

Distribution électronique Cairn.info pour Victoires éditions. Distribution électronique Cairn.inf... more Distribution électronique Cairn.info pour Victoires éditions. Distribution électronique Cairn.info pour Victoires éditions. La reproduction ou représentation de cet article, notamment par photocopie, n'est autorisée que dans les limites des conditions générales d'utilisation du site ou, le cas échéant, des conditions générales de la licence souscrite par votre établissement. Toute autre reproduction ou représentation, en tout ou partie, sous quelque forme et de quelque manière que ce soit, est interdite sauf accord préalable et écrit de l'éditeur, en dehors des cas prévus par la législation en vigueur en France. Il est précisé que son stockage dans une base de données est également interdit. Article disponible en ligne à l'adresse Article disponible en ligne à l'adresse https://www.cairn.info/revue-legicom-1999-2-page-81.htm Découvrir le sommaire de ce numéro, suivre la revue par email, s'abonner... Flashez ce QR Code pour accéder à la page de ce numéro sur Cairn.info.

Journal of Natural Products, 2010

Aristolochic acids are nephrotoxic and carcinogenic natural products that have been implicated bo... more Aristolochic acids are nephrotoxic and carcinogenic natural products that have been implicated both in the endemic nephropathy in the Balkans region and ailments caused by ingestion of herbal remedies. Aristolochic acids are metabolized to active intermediates that bind to DNA. In this study, reduction of aristolochic acid I with zinc/acetic acid afforded a new product which was characterized as 9-methoxy-7-methyl-2H-1,3-oxazolo[5′,4′-10,9]phenanthro[3,4-d]1,3dioxolane-5-carboxylic acid, designated as aristoxazol, along with the expected aristolactam I. This new compound is a condensation product of aristolochic acid and acetic acid that may be related to the aristolochic acid-DNA-adducts. The proposed mechanism of formation of aristoxazol involves nucleophilic attack of acetic acid on the nitrenium ion of aristolochic acid I. On the basis of these studies a route to the metabolic activation of aristolochic acids and formation of adducts with DNA in in vitro systems is proposed and discussed. Aristolochic acids are carcinogenic and nephrotoxic nitroarenes that occur in Aristolochia species, with aristolochic acids I and II (AAI and AAII, 1 and 2, respectively; for numbering see structure 1) usually being the most abundant of these compounds. Ingestion of AAs is implicated in the type of renal fibrosis known as "Chinese herbs nephropathy" or "Balkan endemic nephropathy". 1-9 A report on the carcinogen aristolochic acid was recently issued by the U.S. Department of Health and Human Services. 10

Journal of Molecular Biology, 2001

A unique characteristic of ionizing radiation and radiomimetic anticancer drugs is the induction ... more A unique characteristic of ionizing radiation and radiomimetic anticancer drugs is the induction of clustered damage: two or more DNA lesions (oxidized bases, abasic sites, or strand breaks) occurring in the same or different strands of the DNA molecule within a single turn of the helix. In spite of arising at a lower frequency than single lesions, clustered DNA damage represents an exotic challenge to the repair systems present in the cells and, in some cases, these lesions may escape detection and/ or processing. To understand the structural properties of clustered DNA lesions we have prepared two oligodeoxynucleotide duplexes containing adjacent tetrahydrofuran residues (abasic site analogues), positioned one in each strand of the duplex in a 5 H or 3 H orientation, and determined their solution structure by NMR spectroscopy and molecular dynamics simulations. The NMR data indicate that both duplex structures are right-handed helices of high similarity outside the clustered damage site. The thermal stability of the duplexes is severely reduced by the presence of the abasic residues, especially in a 5 H orientation where the melting temperature is 5 C lower. The structures show remarkable differences at the lesion site where the extrahelical location of the tetrahydrofuran residues in the (AP) 2-5 H-staggered duplex contrasts with their smooth alignment along the sugar-phosphate backbone in the (AP) 2-3 H-staggered duplex.

HETEROCYCLES, 2003

1H NMR spectra of a series of 1-benzyl- and 1,3-dibenzylimidazolidines are analyzed and correlate... more 1H NMR spectra of a series of 1-benzyl- and 1,3-dibenzylimidazolidines are analyzed and correlated with their conformational features. The spectra are assigned on the basis of chemical shifts and proton coupling constant values, and confirmed by NOESY spectra. C 2 -Unsubstituted imidazolidines (1, 9) show a fast inversion of the nitrogen atoms. By contrast, 1,2,3-trisubstituted imidazolidines display a preferential conformation with a transoid orientation of the N 1 , N 3 , and C 2 substituents.

Biochemistry, 2010

Ionizing radiation produces a distinctive pattern of bistranded clustered lesions in DNA. A relat... more Ionizing radiation produces a distinctive pattern of bistranded clustered lesions in DNA. A relatively low number of clustered lesions may be lethal to cells when compared to a larger number of single lesions. Enzyme cleavage experiments suggest that the orientation of bistranded lesions causes differential recognition and removal of these lesions. Similar to a previous study of bistranded abasic site lesion [Hazel, R. D., Tian, K., and de los Santos, C. (2008) Biochemistry, 47, 11909-11919], the aim of this investigation was to solve the structures of two DNA duplexes each containing two synthetic apurinic/apyrimidinic (AP) residues, positioned on opposite strands and separated by two base pairs. In the first duplex the AP residues are staggered in the 3' orientation, (−3 duplex, (AP) 2 −3 duplex), while in the second duplex the AP residues are staggered in the 5' orientation (+3 duplex, (AP) 2 +3 duplex). NOESY spectra collected in 100% and 10% D 2 O buffer solutions allowed the assignment of the non-exchangeable and exchangeable protons, respectively, for each duplex. Cross peak connectivity in the non-exchangeable proton spectra indicate that the duplex is a regular right-handed helix with the AP residues and orphan bases located inside the duplexes. The exchangeable protons spectra establish the formation of Watson-Crick G•C alignment for the two base pairs between the lesion sites in both duplexes. Distance restrained molecular dynamics simulation confirmed the intra helical orientations of the AP residues. The proximity of the AP residues across the minor groove of the −3 duplex and across the major groove in the +3 duplex is similar to their locations in the case of −1 and +1 clusters. This difference in structure may be a key factor in the differential recognition of bistranded AP lesions by human AP endonuclease.

Biochemistry, 2005

Ion channels play critical roles in signaling processes and are attractive targets for treating v... more Ion channels play critical roles in signaling processes and are attractive targets for treating various diseases. Here we describe an NMR-based strategy for structural analyses of potassium channel-ligand complexes using KcsA (residues 1-132, with six mutations to impart toxin binding and to mimic the eukaryotic hERG channel). Using this approach, we determined the solution structure of KcsA in complex with the high-affinity peptide antagonist charybdotoxin. The structural data reveal how charybdotoxin binds to the closed form of KcsA and makes specific contacts with the extracellular surface of the ion channel, resulting in pore blockage. This represents the first direct structural information about an ion channel complexed to a peptide antagonist and provides an experimental framework for understanding and interpreting earlier mutational analyses. The strategy presented here overcomes many of the limitations of conventional NMR approaches to helical membrane protein structure determination and can be applied in the study of the binding of druglike molecules to this important class of proteins.

Acta Histochemica, 2013

Preservation of biomolecules is pivotal in increasingly important molecular diagnostics. Traditio... more Preservation of biomolecules is pivotal in increasingly important molecular diagnostics. Traditionally, formaldehyde is employed for such biomolecular preservation in spite of its carcinogenicity. Moreover, formaldehyde induced cross-linking during fixation is reported to alter structural and functional properties of the preserved biomolecules. Therefore, formaldehyde-free preservatives are advantageous because they are safer for laboratory personnel and they protect the structural and functional integrity of the biomolecules. Streck Cell Preservative and Cell-Free DNA BCT reagents are used as formaldehyde alternative preservatives. However, no studies have been carried out to evaluate formaldehyde concentrations in these preservatives. In this study, we evaluated the free formaldehyde concentrations of these reagents by carbon-13 (13 C) NMR spectroscopic analysis. Chemically non-invasive NMR analysis is more reliable than the traditional derivatization based techniques in formaldehyde detection. 13 C NMR technique can be used for quantitative measurement by using 13 C NMR-relaxation agents. In this manuscript, we report an optimized NMR analysis method using Gadolinium diethylenetriaminepentaacetate. Additionally the data reported herein provide spectral analyses that indicate Streck Cell Preservative and Cell-Free DNA BCT reagents do not contain detectable free formaldehyde. Therefore, these preservatives are safer alternatives than formaldehyde for laboratory use, which can protect the overall integrity of the biomolecules within preserved samples.

Acrolein is a cell metabolic product and a main component of cigarette smoke. Its reaction with D... more Acrolein is a cell metabolic product and a main component of cigarette smoke. Its reaction with DNA produces two guanine lesions c-OH-PdG, a major adduct that is nonmutagenic in mammalian cells, and the positional isomer a-OH-PdG. We describe here the solution structure of a short DNA duplex containing a single a-OH-PdG lesion, as determined by solution NMR spectroscopy and restrained molecular dynamics simulations. The spectroscopic data show a mostly regular right-handed helix, locally perturbed at its center by the presence of the lesion. All undamaged residues of the duplex are in anti orientation, forming standard Watson-Crick base-pair alignments. Duplication of proton signals near the damaged site differentiates two enantiomeric duplexes, thus establishing the exocyclic nature of the lesion. At the lesion site, a-OH-PdG rotates to a syn conformation, pairing to its counter cytosine residue that is protonated at pH 5.9. Three-dimensional models produced by restrained molecular dynamics simulations show different hydrogen-bonding patterns between the lesion and its cytosine partner and identify further stabilization of a-OH-PdG in a syn conformation by intra-residue hydrogen bonds. We compare the a-OH-PdGdC duplex structure with that of duplexes containing the analogous lesion propano-dG and discuss the implications of our findings for the mutagenic bypass of acrolein lesions.

Journal of Biomolecular Structure and Dynamics, 2015

et al., 2014). Here we present the results of the SER titration via an excess of competing oligon... more et al., 2014). Here we present the results of the SER titration via an excess of competing oligonucleotide, according to (Reynaldo, Vologodskij, Neri, & Lyamichev, 2000). The sensitivity of the HG122 promoted SER to limited heterology between the reaction substrates was also estimated. As seen from the Figure, both HG122 and Rec A protein promote SER. In both cases, SER exhibits decreased sensibility to homology violation unlike SER promoted by other agents, such as linker histone H1, or mammalian meiotic system protein Hop2. The latter exhibit the same sensitivity to heterology as that of spontaneous SER at increased temperature (Bocharova, Smirnova, & Volodin, 2012; Pezza et al., 2014). Thus, 1,3-diazaadamantane derivative HG122 acts similar to recombinase Rec A family. This ligand facilitates SER, destabilizes the DNA duplex and appreciably reduces SER sensitivity to heterology between SER substrates. Therefore, compounds of this class are worthy models for further study of different aspects of SER mechanisms.

Nucleic acids research, 2015

Formamidopyrimidine-DNA glycosylase (Fpg) excises 8-oxoguanine (oxoG) from DNA but ignores normal... more Formamidopyrimidine-DNA glycosylase (Fpg) excises 8-oxoguanine (oxoG) from DNA but ignores normal guanine. We combined molecular dynamics simulation and stopped-flow kinetics with fluorescence detection to track the events in the recognition of oxoG by Fpg and its mutants with a key phenylalanine residue, which intercalates next to the damaged base, changed to either alanine (F110A) or fluorescent reporter tryptophan (F110W). Guanine was sampled by Fpg, as evident from the F110W stopped-flow traces, but less extensively than oxoG. The wedgeless F110A enzyme could bend DNA but failed to proceed further in oxoG recognition. Modeling of the base eversion with energy decomposition suggested that the wedge destabilizes the intrahelical base primarily through buckling both surrounding base pairs. Replacement of oxoG with abasic (AP) site rescued the activity, and calculations suggested that wedge insertion is not required for AP site destabilization and eversion. Our results suggest that ...

Nucleosides, Nucleotides and Nucleic Acids, 2010

A procedure has been elaborated for stereoselective deuterium substitution of one of the diastere... more A procedure has been elaborated for stereoselective deuterium substitution of one of the diastereotopic 5-protons in 2-deoxynucleotides. The synthetic scheme uses the reduction of the 5oxosugar derivative with deuterated Alpine-Borane. The resulting deuterosugar is converted into pyrimidine nucleosides and incorporated into DNA using standard protocols. Comparison of 2D NMR spectra of the fully protonated and partially deuterated duplexes allowed us to assign diastereotopic 5 protons, increasing the number of experimental restraints used for structure determination.

Nucleic Acids Research, 2009

Nucleic Acids Research, 2011

Aristolochic acids I and II are prevalent plant toxicants found in the Aristolochiaceae plant fam... more Aristolochic acids I and II are prevalent plant toxicants found in the Aristolochiaceae plant family. Metabolic activation of the aristolochic acids leads to the formation of a cyclic N-hydroxylactam product that can react with the peripheral amino group of purine bases generating bulky DNA adducts. These lesions are mutagenic and established human carcinogens. Interestingly, although AL-dG adducts progressively disappear from the DNA of laboratory animals, AL-dA lesions has lasting persistence in the genome. We describe here NMR structural studies of an undecameric duplex damaged at its center by the presence of an ALII-dA adduct. Our data establish a locally perturbed double helical structure that accommodates the bulky adduct by displacing the counter residue into the major groove and stacking the ALII moiety between flanking bases. The presence of the ALII-dA perturbs the conformation of the 5 0-side flanking base pair, but all other pairs of the duplex adopt standard conformations. Thermodynamic studies reveal that the lesion slightly decreases the energy of duplex formation in a sequencedependent manner. We discuss our results in terms of its implications for the repair of ALII-dA adducts in mammalian cells.

Journal of the American Chemical Society, Jan 21, 2011

Base eversion is a fundamental process in the biochemistry of nucleic acids, allowing proteins en... more Base eversion is a fundamental process in the biochemistry of nucleic acids, allowing proteins engaged in DNA repair and epigenetic modifications to access target bases in DNA. Crystal structures reveal end points of these processes, but not the pathways involved in the dynamic process of base recognition. To elucidate the pathway taken by 8-oxoguanine during base excision repair by Fpg, we calculated free energy surfaces during eversion of the damaged base through the major and minor grooves. The minor groove pathway and free energy barrier (6-7 kcal/mol) are consistent with previously reported results (Qi, Y.; Spong, M. C.; Nam, K.; Banerjee, A.; Jiralerspong, S.; Karplus, M.; Verdine, G. L. Nature 2009, 462, 762.) However, eversion of 8-oxoG through the major groove encounters a significantly lower barrier (3-4 kcal/mol) more consistent with experimentally determined rates of enzymatic sliding during lesion search (Blainey, P. C.; van Oijent, A. M.; Banerjee, A.; Verdine, G. L.; ...

Journal of Computational Chemistry, 2008

FapydG is a common oxidative DNA lesion involving opening of the imidazole ring. It shares the sa... more FapydG is a common oxidative DNA lesion involving opening of the imidazole ring. It shares the same precursor as 8-oxodG and can be excised by the same enzymes as 8-oxodG. However, the loss of the aromatic imidazole in FapydG results in a reduction of the double bond character between C5 and N7, with an accompanying increase in conformational flexibility. Experimental characterization of FapydG is hampered by high reactivity, and thus it is desirable to investigate structural details through computer simulation. We show that the existing Amber force field parameters for FapydG do not reproduce X-ray structural data. We employed quantum mechanics energy profile calculations to derive new molecular mechanics parameters for the rotation of the dihedral angles in the eximidazole moiety. Using these parameters, all-atom simulations in explicit water reproduce the nonplanar conformation of cFapydG in the crystal structure of the complex with L. lactis glycosylase Fpg. We note that the nonplanar structure is stabilized by an acidic residue that is not present in most Fpg sequences. Simulations of the E->S mutant, as present in E. coli, resulted in a more planar conformation, suggesting that the highly nonplanar form observed in the crystal structure may not have direct biological relevance for FapydG.

Journal of Chemical Theory and Computation, 2009

Base flipping is a common strategy utilized by many enzymes to gain access to the functional grou... more Base flipping is a common strategy utilized by many enzymes to gain access to the functional groups of nucleic acid bases in duplex DNA which are otherwise protected by the DNA backbone and hydrogen bonding with their partner bases. Several X-ray crystallography studies have revealed flipped conformations of nucleotides bound to enzymes. However, little is known about the base flipping process itself, even less about the role of the enzymes. Computational studies have used umbrella sampling to elicit the free energy profile of the base flipping process using a pseudo-dihedral angle to represent the reaction coordinate. In this study we have used an unrestrained trajectory in which a flipped base spontaneously reinserted into the helix in order to evaluate and improve the previously defined pseudo-dihedral angle. Our modified pseudo-dihedral angles use a new atom selection to improve the numerical stability of the restraints and also provide better correlation to the extent of flipping observed in simulations. Furthermore, based on the comparison of PMFs generated using different reaction coordinates, we observed that the shape of a flipping PMF profile is strongly dependent on the definition of the reaction coordinate, even for the same data set.

Journal of Biomolecular Structure and Dynamics, 2000

ABSTRACT 2'-deoxyaristeromycin (dAr) is a nucleoside analogue that is resistant to the ac... more ABSTRACT 2'-deoxyaristeromycin (dAr) is a nucleoside analogue that is resistant to the action of DNA glycosylases. High-resolution NMR spectroscopy and molecular dynamics simulations were used to determine the three-dimensional structure of an 11-mer DNA containing a single dAr.T base pair at its center. Analysis of the spectra revealed the existence of a right-handed duplex in solution, stabilized by Watson-Crick hydrogen bonding and base-stacking interactions. The carbocyclic sugar adopted a C1'-exo conformation and sugars of the 3'-flanking base pair had puckers in the O4'-endo range. The dAr.T base pair was mildly propeller twisted, and the dAr analogue showed a positive roll with the 3'-flanking base. Our findings indicate that the observed resistance of dAr-containing oligodeoxynucleotides to the catalytic action of DNA glycosylases relates to its electronic properties rather than structure, and validate the use of dAr and related carbocyclic nucleoside analogues for biological and structure/function relationship studies.

DNA Repair, 2002

Clustered DNA damage is a hallmark of ionizing radiation. These complex lesions, composed of any ... more Clustered DNA damage is a hallmark of ionizing radiation. These complex lesions, composed of any combination of oxidized bases, abasic sites, or strand breaks within one helical turn, create a tremendous challenge for the base excision repair system, which must process the damage without generating cytotoxic double strand breaks (DSB). Clustered lesions affect the DNA incision activity of DNA glycosylases and AP endonucleases. Different levels of enzyme inhibition are dependent on lesion identity, orientation and separation. Very little is known about the simultaneous action of both classes of enzymes, which may lead to the creation of DSB. We have developed a novel substrate system of double-labeled hairpin duplexes, which allows the simultaneous determination of enzyme incision and formation of DBS. We use this system to study the processing of four clustered 8-oxoguanine/abasic site lesions by purified mouse Ogg1, human Ape1 and mouse embryonic stem cell nuclear extracts. Ape1 activity is least affected by the presence of a nearby oxidized base. In contrast, an abasic site inhibits the glycosylase and lyase activities of Ogg1 in an orientation-dependent manner. The combined action of both enzymes leads to the preferential formation of DSB with 5-overhang ends. Processing of clusters by nuclear extracts displayed similar patter of enzyme inhibition and the same preference for avoiding double strand breaks with 3-overhang ends.