Evidence of destabilization of the human thymidylate synthase (hTS) dimeric structure induced by the interface mutation Q62R

In human cells, thymidylate synthase (TS) provides the only source of 2\u2019-deoxythymidyne-5\u2019-monophosphate (dTMP), which is required for DNA biosynthesis. Because of its pivotal role, human TS (hTS) represents a validated target for anticancer chemotherapy. Nonetheless, the efficacy of drugs blocking the hTS active site has limitations due to the onset of resistance in cancer cells, requiring the identification of new strategies to effectively inhibit this enzyme. Human TS works as an obligate homodimer, making the inter-subunit interface an attractive targetable area. Here, we report the design and investigation of a new hTS variant, in which Gln62, located at the dimer interface, has been replaced by arginine in order to destabilize the enzyme quaternary assembly. The hTS Q62R variant has been characterized though kinetic assay, thermal denaturation analysis and X-ray crystallography. Our results provide evidence that hTS Q62R has a reduced melting temperature. The effective destabilization of the TS quaternary structure is also confirmed by structural analysis, showing that the introduced mutation induces a slight aperture of the hTS dimer. The generation of hTS variants having a more accessible interface area can facilitate the screening of interface-targeting molecules, providing key information for the rational design of innovative hTS interface inhibitors

Optimisation of Tyrosine-based lead molecules capable of Modulation of the Peroxisome Proliferator-Activated Receptor Gamma

The peroxisome proliferator-activated receptor gamma (PPARγ) agonist rosiglitazone has recently been withdrawn from the European market and its use has been restricted in the US due to its undesirable effects which were considered to outweigh its benefits. Literature indicates that there are two agonist bound conformations of the PPARγ as exemplified by its binding to rosiglitazone (PDB ID; 1FM6) and to farglitazar (PDB ID; 1FM9). This study aims to explore these two conformations, and to evaluate whether they should be targeted separately in the context of drug design studies. Furthermore, it was aimed to design a series of molecules with the potential to act as leads in a drug design process and the capability of agonist activity at the PPARγ with an acceptable side effect profile. In silico ligand binding affinities (pKd) of rosiglitazone and farglitazar within their cognate receptors were 6.62 and 9.70 respectively. The farglitazar conformer that bound optimally within the rosiglitazone bound PPARγ ligand binding pocket was identified and its binding affinity (pKd) re-determined. An analogous conformational analysis of rosiglitazone within the farglitazar bound PPARγ ligand binding pocket was carried out. The binding affinities (pKd) for these optimum conformations were 8.12 and 6.16 respectively. De novo novel structures were generated in silico based on the tyrosine-agonist farglitazar and its cognate ligand binding pocket. Moreover, analysis of the binding modality of farglitazar indicates that this molecule accesses the PPARγ ligand binding pocket more completely than does rosiglitazone. Binding affinity studies have shown that the PPARγ ligand binding pocket adopts diverse ligand driven conformations.peer-reviewe

A structure-based proposal for the catalytic mechanism of the bacterial acid phosphatase AphA belonging to the DDDD superfamily of phosphohydrolases

The Escherichia coli gene aphA codes for a periplasmic acid phosphatase called AphA, belonging to class B bacterial phosphatases, which is part of the DDDD superfamily of phosphohydrolases. After our first report about its crystal structure, we have started a series of crystallographic studies aimed at understanding of the catalytic mechanism of the enzyme. Here, we report three crystal structures of the AphA enzyme in complex with the hydrolysis products of nucleoside monophosphate substrates and a fourth with a proposed intermediate analogue that appears to be covalently bound to the enzyme. Comparison with the native enzyme structure and with the available X-ray structures of different phosphatases provides clues about the enzyme chemistry and allows us to propose a catalytic mechanism for AphA, and to discuss it with respect to the mechanism of other bacterial and human phosphatases. (c) 2005 Elsevier Ltd. All rights reserved

Iron Binding in the Ferroxidase Site of Human Mitochondrial Ferritin

Ferritins are nanocage proteins that store iron ions in their central cavity as hydrated ferric oxide biominerals. In mammals, further the L (light) and H (heavy) chains constituting cytoplasmic maxi-ferritins, an additional type of ferritin has been identified, the mitochondrial ferritin (MTF). Human MTF (hMTF) is a functional homopolymeric H-like ferritin performing the ferroxidase activity in its ferroxidase site (FS), in which Fe(II) is oxidized to Fe(III) in the presence of dioxygen. To better investigate its ferroxidase properties, here we performed time-lapse X-ray crystallography analysis of hMTF, providing structural evidence of how iron ions interact with hMTF and of their binding to the FS. Transient iron binding sites, populating the pathway along the cage from the iron entry channel to the catalytic center, were also identified. Furthermore, our kinetic data at variable iron loads indicate that the catalytic iron oxidation reaction occurs via a diferric peroxo intermediate followed by the formation of ferric-oxo species, with significant differences with respect to human H-type ferritin

Report on the activity of the GILDA-CRG beamline 2009-2013

Index Technical description of the beamline..................................................................................................3 Introduction ....................................................................................................................................3 Optics..............................................................................................................................................3 The XAS end station........................................................................................................................6 Standard data collection setup.....................................................................................................6 Surface XAS apparata.................................................................................................................8 Recent sample environment and Instrumentation developments................................................9 The x-ray diffraction (XRD) end-station.......................................................................................13 Beamline control...........................................................................................................................14 Administrative aspects........................................................................................................................16 Organisation..................................................................................................................................16 Beamline Staff Situation................................................................................................................17 Statistical data on Users and scientific production.............................................................................21 Future perspectives and plans for upgrade.........................................................................................25 Aim of the project.........................................................................................................................25 Design...........................................................................................................................................26 Timetable......................................................................................................................................31 Overview of the overall scientific activity.........................................................................................33 Selection of five publications........................................................................................................33 Highlights of the scientific activity................................................................................................34 Local order in semiconductors..................................................................................................34 Nanotechnology.......................................................................................................................44 Cements and porous systems....................................................................................................48 Chemistry.................................................................................................................................56 Earth Science............................................................................................................................61 Environment.............................................................................................................................67 Cultural Heritage.......................................................................................................................72 Health, medicine and life science .............................................................................................77 Acknowledgements...........................................................................................................................84 References.........................................................................................................................................85 Generic References.......................................................................................................................85 GILDA 2009-2013 Publications....................................................................................................8

Repurposing the trypanosomatidic gsk kinetobox for the inhibition of parasitic pteridine and dihydrofolate reductases

Three open-source anti-kinetoplastid chemical boxes derived from a whole-cell phenotypic screening by GlaxoSmithKline (Tres Cantos Anti-Kinetoplastid Screening, TCAKS) were exploited for the discovery of a novel core structure inspiring new treatments of parasitic diseases targeting the trypansosmatidic pteridine reductase 1 (PTR1) and dihydrofolate reductase (DHFR) enzymes. In total, 592 compounds were tested through medium-throughput screening assays. A subset of 14 compounds successfully inhibited the enzyme activity in the low micromolar range of at least one of the enzymes from both Trypanosoma brucei and Lesihmania major parasites (pan-inhibitors), or from both PTR1 and DHFR-TS of the same parasite (dual inhibitors). Molecular docking studies of the protein–ligand interaction focused on new scaffolds not reproducing the well-known antifolate core clearly explaining the experimental data. TCMDC-143249, classified as a benzenesulfonamide derivative by the QikProp descriptor tool, showed selective inhibition of PTR1 and growth inhibition of the kinetoplastid parasites in the 5 µM range. In our work, we enlarged the biological profile of the GSK Kinetobox and identified new core structures inhibiting selectively PTR1, effective against the kinetoplastid infectious protozoans. In perspective, we foresee the development of selective PTR1 and DHFR inhibitors for studies of drug combinations