Spectroscopy and NMR Unit

Madrid, Spain

Spectroscopy and NMR Unit

Madrid, Spain
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Campos-Olivas R.,Spectroscopy and NMR Unit
Current Topics in Medicinal Chemistry | Year: 2011

Over the past three decades nuclear magnetic resonance spectroscopy has been developed into a mature technique for the characterization of interactions of small molecule ligands with their corresponding protein and nucleic acid receptors. In fact, a significant number of industrial and academic laboratories employ NMR for screening small molecule compound collections for binding to defined macromolecular targets, thus potentially providing initial, low affinity hits for a fragment-based approach in the drug discovery process. NMR is also applied to interrogate hits obtained by high throughput screening using biochemical assays and by virtual screening methods, for their ability to physically interact with the target receptor. In favorable cases a variety of NMR-based methods can also provide essential information to validate the hit, rank the different hits according to affinity, and to structurally analyze the ligand-target complex, thus providing essential information for structure-based optimization and medicinal chemistry. In this review a comprehensive overview of the large variety of NMR methods to study interactions between small molecule ligands and macromolecular receptors is provided, summarizing the physico-chemical bases of the different receptor- and ligand-observed experiments. The application of these methods for compound library screening and hit validation, with special emphasis on their contribution to fragment-based drug discovery strategies, is illustrated by recent examples selected from the literature and work in my laboratory. © 2011 Bentham Science Publishers Ltd.


Carranza G.,University of Cantabria | Castano R.,University of Cantabria | Fanarraga M.L.,University of Cantabria | Villegas J.C.,University of Cantabria | And 10 more authors.
Cellular and Molecular Life Sciences | Year: 2013

Tubulin cofactors (TBCs) participate in the folding, dimerization, and dissociation pathways of the tubulin dimer. Among them, TBCB and TBCE are two CAP-Gly domain-containing proteins that together efficiently interact with and dissociate the tubulin dimer. In the study reported here we showed that TBCB localizes at spindle and midzone microtubules during mitosis. Furthermore, the motif DEI/M-COO- present in TBCB, which is similar to the EEY/F-COO- element characteristic of EB proteins, CLIP-170, and α-tubulin, is required for TBCE-TBCB heterodimer formation and thus for tubulin dimer dissociation. This motif is responsible for TBCB autoinhibition, and our analysis suggests that TBCB is a monomer in solution. Mutants of TBCB lacking this motif are derepressed and induce microtubule depolymerization through an interaction with EB1 associated with microtubule tips. TBCB is also able to bind to the chaperonin complex CCT containing α-tubulin, suggesting that it could escort tubulin to facilitate its folding and dimerization, recycling or degradation. © 2012 Springer Basel AG.


Garavis M.,CSIC - Institute of Physical Chemistry "Rocasolano" | Garavis M.,Autonomous University of Madrid | Lopez-Mendez B.,Spectroscopy and NMR Unit | Somoza A.,CSIC - National Center for Biotechnology | And 5 more authors.
ACS Chemical Biology | Year: 2014

Telomeric repeat-containing RNA (TERRA) is a novel and very attractive antitumoral target. Here, we report the first successful application of 19F-NMR fragment-based screening to identify chemically diverse compounds that bind to an RNA molecule such as TERRA. We have built a library of 355 fluorinated fragments, and checked their interaction with a long telomeric RNA as a target molecule. The screening resulted in the identification of 20 hits (hit rate of 5.6%). For a number of binders, their interaction with TERRA was confirmed by 19F- and 1H NMR as well as by CD melting experiments. We have also explored the selectivity of the ligands for RNA G-quadruplexes and found that some of the hits do not interact with other nucleic acids such as tRNA and duplex DNA and, most importantly, favor the propeller-like parallel conformation in telomeric DNA G-quadruplexes. This suggests a selective recognition of this particular quadruplex topology and that different ligands may recognize specific sites in propeller-like parallel G-quadruplexes. Such features make some of the resulting binders promising lead compounds for fragment based drug discovery. © 2014 American Chemical Society.


Martin-Pintado N.,CSIC - Institute of Physical Chemistry "Rocasolano" | Deleavey G.F.,McGill University | Portella G.,University of Barcelona | Campos-Olivas R.,Spectroscopy and NMR Unit | And 3 more authors.
Angewandte Chemie - International Edition | Year: 2013

Polarizing C-H⋯O hydrogen bonds: The structure of oligonucleotides containing alternating and contiguous tracts of 2′F-RNA and 2′F-ANA nucleotides reveals that nonconventional FC-H⋯O hydrogen bonds have a strong stabilizing effect on 2′-fluorinated duplexes. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


Martin-Pintado N.,CSIC - Institute of Physical Chemistry "Rocasolano" | Yahyaee-Anzahaee M.,McGill University | Campos-Olivas R.,Spectroscopy and NMR Unit | Noronha A.M.,McGill University | And 4 more authors.
Nucleic Acids Research | Year: 2012

We report here the first structure of double helical arabino nucleic acid (ANA), the C2'-stereoisomer of RNA, and the 2'-fluoro-ANA analogue (2'F-ANA). A chimeric dodecamer based on the Dickerson sequence, containing a contiguous central segment of arabino nucleotides, flanked by two 2'-deoxy- 2'F-ANA wings was studied. Our data show that this chimeric oligonucleotide can adopt two different structures of comparable thermal stabilities. One structure is a monomeric hairpin in which the stem is formed by base paired 2'F-ANA nucleotides and the loop by unpaired ANA nucleotides. The second structure is a bimolecular duplex, with all the nucleotides (2'F-ANA and ANA) forming Watson-Crick base pairs. The duplex structure is canonical B-form, with all arabinoses adopting a pure C2'-endo conformation. In the ANA:ANA segment, steric interactions involving the 2'-OH substituent provoke slight changes in the glycosidic angles and, therefore, in the ANA:ANA base pair geometry. These distortions are not present in the 2'F-ANA:2'F-ANA regions of the duplex, where the -OH substituent is replaced by a smaller fluorine atom. 2'F-ANA nucleotides adopt the C2'-endo sugar pucker and fit very well into the geometry of B-form duplex, allowing for favourable 2'F⋯⋯⋯H8 interactions. This interaction shares many features of pseudo-hydrogen bonds previously observed in 2'F-ANA:RNA hybrids and in single 2'F-ANA nucleotides. © 2012 The Author(s).


Stella S.,Macromolecular Crystallography Group | Stella S.,Copenhagen University | Molina R.,Macromolecular Crystallography Group | Lopez-Mendez B.,Spectroscopy and NMR Unit | And 7 more authors.
Acta Crystallographica Section D: Biological Crystallography | Year: 2014

DNA editing offers new possibilities in synthetic biology and biomedicine for modulation or modification of cellular functions to organisms. However, inaccuracy in this process may lead to genome damage. To address this important problem, a strategy allowing specific gene modification has been achieved through the addition, removal or exchange of DNA sequences using customized proteins and the endogenous DNA-repair machinery. Therefore, the engineering of specific protein-DNA interactions in protein scaffolds is key to providing 'toolkits' for precise genome modification or regulation of gene expression. In a search for putative DNA-binding domains, BurrH, a protein that recognizes a 19 bp DNA target, was identified. Here, its apo and DNA-bound crystal structures are reported, revealing a central region containing 19 repeats of a helix-loop-helix modular domain (BurrH domain; BuD), which identifies the DNA target by a single residue-to-nucleotide code, thus facilitating its redesign for gene targeting. New DNA-binding specificities have been engineered in this template, showing that BuD-derived nucleases (BuDNs) induce high levels of gene targeting in a locus of the human haemoglobin β (HBB) gene close to mutations responsible for sickle-cell anaemia. Hence, the unique combination of high efficiency and specificity of the BuD arrays can push forward diverse genome-modification approaches for cell or organism redesign, opening new avenues for gene editing. © 2014 International Union of Crystallography.


PubMed | University of Florence, Spectroscopy and NMR Unit, University College London and Spanish National Cancer Research Center
Type: | Journal: Scientific reports | Year: 2016

Understanding the conformational changes associated with the binding of small ligands to their biological targets is a fascinating and meaningful question in chemistry, biology and drug discovery. One of the most studied and important is the so-called DFG-flip of tyrosine kinases. The conserved three amino-acid DFG motif undergoes an in to out movement resulting in a particular inactive conformation to which type II kinase inhibitors, such as the anti-cancer drug Imatinib, bind. Despite many studies, the details of this prototypical conformational change are still debated. Here we combine various NMR experiments and surface plasmon resonance with enhanced sampling molecular dynamics simulations to shed light into the conformational dynamics associated with the binding of Imatinib to the proto-oncogene c-Src. We find that both conformational selection and induced fit play a role in the binding mechanism, reconciling opposing views held in the literature. Moreover, an external binding pose and local unfolding (cracking) of the aG helix are observed.


Campos-Olivas R.,Spectroscopy and NMR Unit
Biomolecular NMR assignments | Year: 2011

The Src tyrosine kinase is the paradigm of an oncogenic kinase, and of regulation by intramolecular inhibitory interactions, as well as an important anticancer target due to its roles in cell proliferation and metastasis. The assignment of backbone (1)H(N), (13)C(α), (13)CO, and (15)N, and sidechain (13)C(β) resonances of the catalytic domain of Src (283 residues) in complex with the anticancer drug Imatinib is reported here. Consistent with previous X-ray studies of the same complex, most signals from the activation loop are not detected, indicating that, even in the presence of the drug, it probably adopts highly heterogeneous conformations in intermediate exchange. For the rest of the polypeptide backbone, assignments have been completed for ~88% of residues, with only a few solvent-exposed amides remaining unassigned.


Sancho P.,Queen Mary, University of London | Sancho P.,Stem Cells and Cancer Group | Burgos-Ramos E.,Stem Cells and Cancer Group | Tavera A.,Stem Cells and Cancer Group | And 12 more authors.
Cell Metabolism | Year: 2015

The anti-diabetic drug metformin targets pancreatic cancer stem cells (CSCs), but not their differentiated progenies (non-CSCs), which may be related to distinct metabolic phenotypes. Here we conclusively demonstrate that while non-CSCs were highly glycolytic, CSCs were dependent on oxidative metabolism (OXPHOS) with very limited metabolic plasticity. Thus, mitochondrial inhibition, e.g., by metformin, translated into energy crisis and apoptosis. However, resistant CSC clones eventually emerged during treatment with metformin due to their intermediate glycolytic/respiratory phenotype. Mechanistically, suppression of MYC and subsequent increase of PGC-1α were identified as key determinants for the OXPHOS dependency of CSCs, which was abolished in resistant CSC clones. Intriguingly, no resistance was observed for the mitochondrial ROS inducer menadione and resistance could also be prevented/reversed for metformin by genetic/pharmacological inhibition of MYC. Thus, the specific metabolic features of pancreatic CSCs are amendable to therapeutic intervention and could provide the basis for developing more effective therapies to combat this lethal cancer. © 2015 Elsevier Inc.


PubMed | Bioinformatics Unit and Structural Biology and Biocomputing Programme, The Francis Crick Institute, Stem Cells & Cancer Group, Queen Mary, University of London and Spectroscopy and NMR Unit
Type: Journal Article | Journal: Cell metabolism | Year: 2015

The anti-diabetic drug metformin targets pancreatic cancer stem cells (CSCs), but not their differentiated progenies (non-CSCs), which may be related to distinct metabolic phenotypes. Here we conclusively demonstrate that while non-CSCs were highly glycolytic, CSCs were dependent on oxidative metabolism (OXPHOS) with very limited metabolic plasticity. Thus, mitochondrial inhibition, e.g., by metformin, translated into energy crisis and apoptosis. However, resistant CSC clones eventually emerged during treatment with metformin due to their intermediate glycolytic/respiratory phenotype. Mechanistically, suppression of MYC and subsequent increase of PGC-1 were identified as key determinants for the OXPHOS dependency of CSCs, which was abolished in resistant CSC clones. Intriguingly, no resistance was observed for the mitochondrial ROS inducer menadione and resistance could also be prevented/reversed for metformin by genetic/pharmacological inhibition of MYC. Thus, the specific metabolic features of pancreatic CSCs are amendable to therapeutic intervention and could provide the basis for developing more effective therapies to combat this lethal cancer.

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