Adaptive simulations, towards interactive protein-ligand modeling

Modeling the dynamic nature of protein-ligand binding with atomistic simulations is one of the main challenges in computational biophysics, with important implications in the drug design process. Although in the past few years hardware and software advances have significantly revamped the use of molecular simulations, we still lack a fast and accurate ab initio description of the binding mechanism in complex systems, available only for up-to-date techniques and requiring several hours or days of heavy computation. Such delay is one of the main limiting factors for a larger penetration of protein dynamics modeling in the pharmaceutical industry. Here we present a game-changing technology, opening up the way for fast reliable simulations of protein dynamics by combining an adaptive reinforcement learning procedure with Monte Carlo sampling in the frame of modern multi-core computational resources. We show remarkable performance in mapping the protein-ligand energy landscape, being able to reproduce the full binding mechanism in less than half an hour, or the active site induced fit in less than 5 minutes. We exemplify our method by studying diverse complex targets, including nuclear hormone receptors and GPCRs, demonstrating the potential of using the new adaptive technique in screening and lead optimization studies.We thank Drs Anders Hogner and Christoph Grebner, from AstraZeneca, and Jorge Estrada, from BSC, for fruitful discussions and feedback on the manuscript. We acknowledge the BSC-CRG-IRB Joint Research Program in Computational Biology. This work was supported by the CTQ2016-79138-R grant from the Spanish Government. D.L. acknowledges the support of SEV-2011-00067, awarded by the Spanish Government.Peer ReviewedPostprint (published version

New rhenium complexes with ciprofloxacin as useful models for understanding the properties of [99mTc]-ciprofloxacin radiopharmaceutical

Rhenium complexes with the antibiotic ciprofloxacin have been prepared to be studied as models of technetium radiopharmaceuticals. With this aim, the new rhenium complexes 1 {[ReO(Cpf)2]Cl}, 2 {[ReO(CpfH)2]Cl3} and 3 {fac-[Re(CO)3(H2O)(Cpf)]} with ciprofloxacin (CpfH=ciprofloxacin; Cpf=conjugated base of ciprofloxacin) have been synthesised and characterised by elemental analyses, IR, NMR ((1)H, (19)F and (13)C CP-MAS) spectroscopy, as well as MS measurements. All spectroscopic data are consistent with the coordination of ciprofloxacin in all these complexes through the carbonyl and the carboxylate oxygen atoms with the formation of a six member chelate ring. The study of a Tc-ciprofloxacin solution by ESI-MS reveals the presence of [TcO(Cpf)2](+) cations, which agrees with the hypothesis that complexes 1 and 2 can be seen as model rhenium complexes of this radiopharmaceutical. Antimicrobial and DNA gyrase inhibition studies performed with complexes 2 and 3 have shown a very similar behaviour between complex 2 and the free antibiotic, whereas complex 3 exhibit a lower antimicrobial activity. Based on a joint analysis of the data reported in the literature and the chemical and biological results obtained in this study, a tentative proposal to explain some aspects of the behaviour of Tc-ciprofloxacin radiopharmaceutical has been made

Bioconjugated technetium carbonyls by transmetalation reaction with zinc derivatives

Altres ajuts: Acord transformatiu CRUE-CSICThe transmetalation reaction between zinc dithiocarbamates functionalized with organic groups and the cation fac-[Tc(HO)(CO)] has been studied as a new strategy to bind biomolecules to this radionuclide for preparing radiopharmaceuticals with high molar activity. All complexes were obtained in high yields by heating at moderate temperatures and without subsequent purification. The chemical identity was ascertained by HPLC comparison with the homologous rhenium complexes. Stability studies in cysteine solution and serum have shown a good stability of the coordination set fac-[Tc(CO)(SS)(P)]. Preliminary biological studies of the radiocomplex functionalized with D-(+)-glucosamine with carcinoma cells have been performed

A simplified implementation of the stationary liquid mass balance method for on-line OUR monitoring in animal cell cultures

This is the peer reviewed version of the following article: [Fontova, A. , Lecina, M. , López‐Repullo, J. , Martínez‐Monge, I. , Comas, P. , Bragós, R. and Cairó, J. J. (2018), A simplified implementation of the stationary liquid mass balance method for on‐line OUR monitoring in animal cell cultures. J. Chem. Technol. Biotechnol. doi:10.1002/jctb.5551], which has been published in final form at [doi:10.1002/jctb.5551]. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.BACKGROUND: Compared with other methods, the stationary liquid mass balance method for oxygen uptake rate (OUR) determination offers advantages in terms of estimation accuracy and reduction of stress. However, the need for sophisticated instrumentation, like mass flow controllers and gas analysers, has historically limited wider implementation of such a method. In this paper, a new simplified method based on inexpensive valves for the continuous estimation of OUR in animal cell cultures is evaluated. The determination of OUR values is based on accurate operation of the dissolved oxygen (DO) control loop and monitoring of its internal variables. RESULTS: The method developed was tested empirically in 2¿L bioreactor HEK293 batch cultures. OUR profiles obtained by a dynamic method, global mass balance method and the developed simplified method were monitored and compared. The results show how OUR profile obtained with the proposed method better follows the off-line cell density determination. The OUR estimation frequency was also increased, improving the method capabilities and applications. The theoretical rationale of the method was extended to the sensitivity analysis which was analytically and numerically approached. CONCLUSIONS: The results showed the proposed method to be not only cheap, but also a reliable alternative to monitor the metabolic activity in bioreactors in many biotechnological processes, being a useful tool for high cell density culture strategies implementation based on OUR monitoring.Peer ReviewedPostprint (published version

New bioconjugated rhenium carbonyls by transmetalation reaction with zinc derivatives

The transmetallation reaction between zinc dithiocarbamates and rhenium carbonyls has been used as a new strategy to link biomolecules to transition metals. The zinc(II) dithiocarbamate of isonipecotic acid (1) and the succinimidyl ester derivative (2) were prepared by straight forward procedures and were fully characterized by spectroscopic and X-ray diffraction methods, showing in both cases the presence of dinuclear complexes. Complex 2 reacted with all the primary and secondary amines studied (glycine methyl ester, β-alanine methyl ester, 1-(2-methoxyphenyl)piperazine and D-(+)-glucosamine) through the activated succinimidyl ester group, linking the metallic fragment with the biomolecule by the formation of a peptidic bond, and leading to the respective bioconjugated zinc complexes 3-6. In all cases, these zinc complexes could be isolated from the reaction medium by simple precipitation. These results evidence the potential of complex 2 to be used as a synthon to link the zinc dithiocarbamate fragment to biomolecules that contain an amine group. Complexes 3-6 were characterized by the usual spectroscopic methods and all data agree with the proposed structures, which do not contain significant interactions between the zinc fragment and the functional groups of these biomolecules. The transmetallation reaction between the zinc complexes 3-6 and the rhenium carbonyl [ReBr₃(CO)₃]²⁻ led to the expected rhenium dithiocarbamates 7-10 with no change in the organic dithiocarbamate fragments, confirming the viability of this reaction as a tool for linking biomolecules to transition elements. All complexes were characterized by spectroscopic methods and the crystal structure of 8 was studied by X-ray diffraction analysis. All data demonstrated that the biomolecule is positioned far away from the fac-{Re(CO)₃} fragment and the octahedral coordination around the metal is completed by the functionalized dithiocarbamate and a phosphine ligand. Finally, the analysis by ESI-MS spectrometry of the reaction between the zinc complex 4 and a water solution of [Re(H₂O)₃(CO)₃]+ at a very low concentration (10 ppm) showed that the transmetallation reaction took place even though the solubility of the zinc complex in water medium was as low as 0.66 ppm. This preliminary result supports the viability of this approach for the preparation of rhenium and technetium target specific radiopharmaceuticals since the preparation of these compounds are always performed in water medium

Structural changes of Arthrospira sp. after low energy sonication treatment for microalgae harvesting: elucidating key parameters to detect the rupture of gas vesicles

© . This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/The buoyancy suppression by low energy sonication (LES) treatment (0.8 W·mL-1, 20 kHz, 10 s) has recently been proposed as an initial harvesting step for Arthrospira sp. This paper aims to describe the structural changes in Arthrospira sp. after LES treatment and to present how these structural changes affect the results obtained by different analytical techniques. Transmission electron microscopy (TEM) micrographs of trichomes evidenced the gas vesicles rupture but also revealed a rearrangement of thylakoids and more visible phycobilisomes were observed. Differences between treated and untreated samples were detected by confocal microscopy, flow cytometry and optical microscopy but not by electrical impedance spectroscopy (EIS). After LES treatment, 2-fold increase in autofluorescence at 610/660 nm was measured (phycocyanin/allophycocyanin emission wavelengths) and a ten-fold decrease in side scatter light intensity (due to a reduction of trichome’s inner complexity). This was further confirmed by optical microscopy showing changes on trichomes appearance (from wrinkled to smooth).Peer ReviewedPostprint (author's final draft

Compostos de remi i tecneci pel desenvolupament de radiofàrmacs de segona generació

Aquesta tesi doctoral es troba dividida en tres parts corresponents a les tres línees d’investigació que s’ha desenvolupat: (a) Complexos de reni i Ciprofloxacina; (b) Síntesi de radiofàrmacs de 2a generació d’elevada activitat específica; i (c) Estudi de la interacció de les metal·lotioneines de mamífer amb el fragment fac-{M(CO)3} (M =Re, Tc) (a) S’ha provat de descobrir el mecanisme d’actuació del radiofàrmac experimental 99mTc-Ciprofloxacina. L’anàlisi de l’activitat antibacteriana dels compost homòleg amb reni ha demostrat que les propietats antibiòtiques del complex estan lligades al caràcter làbil del seu enllaç. Amb conseqüència, s’ha proposat un mecanisme d’actuació del radiofàrmac que està d’acord amb les dades experimentals tant de la present tesi doctoral com les publicades fins el moment.(b) Els resultats obtinguts han demostrat que és possible l’aplicació de la metodologia proposada, basada en la reacció de transmetal·lació de complexos de zinc poc solubles en aigua. Els elevats valors de puresa radioquímica dels complexos de tecneci-99m sintetitzats, tenint en compte la baixa solubilitat de la biomolècula, demostren la viabilitat del mètode ja que s’aconsegueix disminuir un ordre de magnitud la relació entre biomolècula marcada i no marcada respecte dels mètodes actuals. (c) L’estudi de la interacció de les metal·lotioneïnes MTs amb els fragments carbonil, ha demostrat ser una bona alternativa als mètodes sintètics convencionals emprats avui dia en la preparació d’agents quelants de tecneci. L’obtenció per primer cop de l’estequiometria d’un compost Tc-MTs, obre la porta tant a la preparació de radiofàrmacs mitjançant la fusió de les MTs a altres proteïnes (Tc-MT-Biomolècula) com també a la possibilitat de marcatge i estudi in vivo d’aquestes.This thesis is divided into three parts corresponding to the three lines of research that has been developed: (a) Ciprofloxacin and rhenium complexes; (b) 2nd Generation Synthesis of radiopharmaceuticals with high specific activity; and (c ) Study of the interaction of mammalian Metallothioneins with fragment fac-{M(CO)3} (M = Re, Tc) (a) It has tried to explore the action mechanism of experimental radiopharmaceutical 99mTc-Ciprofloxacina. Analysis of the antibacterial activity of the homologous rhenium compound has shown that the antibiotic properties of the complex are related to the labile nature of their bond. In consequence, it has been proposed a mechanism of action for the radiopharmaceutical that it is in agreement with the experimental data. (b) The results have shown that it is possible to apply the proposed methodology, based on transmetallation step reaction of zinc complexes poorly soluble in water. The high values of radiochemical purity of technetium-99m complexes synthesized, taking into account the low solubility of the biomolecules, it demonstrate the feasibility of the method, since the relation between labeled and unlabeled biomolecule decrease one order of magnitude compared with the currently methods. (c) The study of the interaction between Metallothioneins MTs and carbonyl fragments has proved a good alternative to the conventional synthetic methods, which are currently used in the preparation of technetium chelating agents. For the first time it has been obtained the stoichiometry of a compound Tc-MTs, opening the door for the preparation of radiopharmaceuticals through the fusion of MT to other proteins (Tc-MT-biomolecules), as well as the possibility of labeled MTs for in vivo studie

Adaptive simulations, towards interactive protein-ligand modeling

Modeling the dynamic nature of protein-ligand binding with atomistic simulations is one of the main challenges in computational biophysics, with important implications in the drug design process. Although in the past few years hardware and software advances have significantly revamped the use of molecular simulations, we still lack a fast and accurate ab initio description of the binding mechanism in complex systems, available only for up-to-date techniques and requiring several hours or days of heavy computation. Such delay is one of the main limiting factors for a larger penetration of protein dynamics modeling in the pharmaceutical industry. Here we present a game-changing technology, opening up the way for fast reliable simulations of protein dynamics by combining an adaptive reinforcement learning procedure with Monte Carlo sampling in the frame of modern multi-core computational resources. We show remarkable performance in mapping the protein-ligand energy landscape, being able to reproduce the full binding mechanism in less than half an hour, or the active site induced fit in less than 5 minutes. We exemplify our method by studying diverse complex targets, including nuclear hormone receptors and GPCRs, demonstrating the potential of using the new adaptive technique in screening and lead optimization studies.We thank Drs Anders Hogner and Christoph Grebner, from AstraZeneca, and Jorge Estrada, from BSC, for fruitful discussions and feedback on the manuscript. We acknowledge the BSC-CRG-IRB Joint Research Program in Computational Biology. This work was supported by the CTQ2016-79138-R grant from the Spanish Government. D.L. acknowledges the support of SEV-2011-00067, awarded by the Spanish Government.Peer Reviewe

Compostos de reni i tecneci pel desenvolupament de radiofàrmacs de segona generació

Aquesta tesi doctoral es troba dividida en tres parts corresponents a les tres línees d'investigació que s'ha desenvolupat: (a) Complexos de reni i Ciprofloxacina; (b) Síntesi de radiofàrmacs de 2a generació d'elevada activitat específica; i (c) Estudi de la interacció de les metal·lotioneines de mamífer amb el fragment fac-{M(CO)3} (M =Re, Tc) (a) S'ha provat de descobrir el mecanisme d'actuació del radiofàrmac experimental 99mTc-Ciprofloxacina. L'anàlisi de l'activitat antibacteriana dels compost homòleg amb reni ha demostrat que les propietats antibiòtiques del complex estan lligades al caràcter làbil del seu enllaç. Amb conseqüència, s'ha proposat un mecanisme d'actuació del radiofàrmac que està d'acord amb les dades experimentals tant de la present tesi doctoral com les publicades fins el moment.(b) Els resultats obtinguts han demostrat que és possible l'aplicació de la metodologia proposada, basada en la reacció de transmetal·lació de complexos de zinc poc solubles en aigua. Els elevats valors de puresa radioquímica dels complexos de tecneci-99m sintetitzats, tenint en compte la baixa solubilitat de la biomolècula, demostren la viabilitat del mètode ja que s'aconsegueix disminuir un ordre de magnitud la relació entre biomolècula marcada i no marcada respecte dels mètodes actuals. (c) L'estudi de la interacció de les metal·lotioneïnes MTs amb els fragments carbonil, ha demostrat ser una bona alternativa als mètodes sintètics convencionals emprats avui dia en la preparació d'agents quelants de tecneci. L'obtenció per primer cop de l'estequiometria d'un compost Tc-MTs, obre la porta tant a la preparació de radiofàrmacs mitjançant la fusió de les MTs a altres proteïnes (Tc-MT-Biomolècula) com també a la possibilitat de marcatge i estudi in vivo d'aquestes.This thesis is divided into three parts corresponding to the three lines of research that has been developed: (a) Ciprofloxacin and rhenium complexes; (b) 2nd Generation Synthesis of radiopharmaceuticals with high specific activity; and (c ) Study of the interaction of mammalian Metallothioneins with fragment fac-{M(CO)3} (M = Re, Tc) (a) It has tried to explore the action mechanism of experimental radiopharmaceutical 99mTc-Ciprofloxacina. Analysis of the antibacterial activity of the homologous rhenium compound has shown that the antibiotic properties of the complex are related to the labile nature of their bond. In consequence, it has been proposed a mechanism of action for the radiopharmaceutical that it is in agreement with the experimental data. (b) The results have shown that it is possible to apply the proposed methodology, based on transmetallation step reaction of zinc complexes poorly soluble in water. The high values of radiochemical purity of technetium-99m complexes synthesized, taking into account the low solubility of the biomolecules, it demonstrate the feasibility of the method, since the relation between labeled and unlabeled biomolecule decrease one order of magnitude compared with the currently methods. (c) The study of the interaction between Metallothioneins MTs and carbonyl fragments has proved a good alternative to the conventional synthetic methods, which are currently used in the preparation of technetium chelating agents. For the first time it has been obtained the stoichiometry of a compound Tc-MTs, opening the door for the preparation of radiopharmaceuticals through the fusion of MT to other proteins (Tc-MT-biomolecules), as well as the possibility of labeled MTs for in vivo studie

Monte Carlo Techniques for Drug Design: The Success Case of PELE

This chapter summarizes the most representative software packages that readily allow running Monte Carlo (MC) simulations in relevant scenarios for drug design. It explores in detail the Protein Energy Landscape Exploration (PELE) program, providing first the main characteristics of the technique, followed by an analysis of the different application studies in mapping protein‐ligand interactions. The ligand, formed by a rigid core and a set of rotatable side chains, is perturbed by translating and rotating it. PELE creates a list of perturbation poses, and then chooses the one with the lowest system energy. PELE was originally designed to map ligand migration pathways: its first applications aimed at finding exit pathways starting from ligand‐bound crystallographic structures. Additional applied studies have centered on modeling enzymatic mechanisms and engineering; the same techniques applied in mapping protein‐drug interactions can be used in the study of substrate recognition by enzymes.Along the development of PELE in the last years, we gratefully acknowledge financial support from the European Union (in particular from the ERC program) and from the Catalan and Spanish Governments. In addition we want to thank all present and past members from the EAPM lab. at BSC for their dedication and work.Peer Reviewe