Theoretical-experimental study on protein-ligand interactions based on thermodynamics methods, molecular docking and perturbation models

The current doctoral thesis focuses on understanding the thermodynamic events of protein-ligand interactions which have been of paramount importance from traditional Medicinal Chemistry to Nanobiotechnology. Particular attention has been made on the application of state-of-the-art methodologies to address thermodynamic studies of the protein-ligand interactions by integrating structure-based molecular docking techniques, classical fractal approaches to solve protein-ligand complementarity problems, perturbation models to study allosteric signal propagation, predictive nano-quantitative structure-toxicity relationship models coupled with powerful experimental validation techniques. The contributions provided by this work could open an unlimited horizon to the fields of Drug-Discovery, Materials Sciences, Molecular Diagnosis, and Environmental Health Sciences

Advanced Materials Based on Nanosized Hydroxyapatite

The development of new materials based on hydroxyapatite has undergone a great evolution in recent decades due to technological advances and development of computational techniques. The focus of this review is the various attempts to improve new hydroxyapatite-based materials. First, we comment on the most used processing routes, highlighting their advantages and disadvantages. We will now focus on other routes, less common due to their specificity and/or recent development. We also include a block dedicated to the impact of computational techniques in the development of these new systems, including: QSAR, DFT, Finite Elements of Machine Learning. In the following part we focus on the most innovative applications of these materials, ranging from medicine to new disciplines such as catalysis, environment, filtration, or energy. The review concludes with an outlook for possible new research directionsThis research was funded by MINISTERIO DE CIENCIA E INNOVACIÓN (PID2019-111327GB-100).S

Conformational binding mechanism of lysozyme induced by interactions with penicillin antibiotic drugs

In this study we present an in-depth and detailed analysis of the binding process between two antibiotics (cloxacillin and dicloxacillin) and a blood serum protein (lysozyme). Our objectives have been several: to determine, at the atomic level, the structural and conformational changes that take place in both molecular structures once the complex is formed; to investigate the effect that the substitution of a hydrogen atom for a chlorine atom has on the bonding process; and to relate these local modifications with macromolecular parameters. Achieving these goals requires a multi-pronged approach and effective resource management. In our case, we have combined different experimental (isothermal titration calorimetry, UV–vis and fluorescence spectroscopy) and computational techniques (molecular docking and network models), in order to obtain comprehensive and contrasted information of the interaction process. Both approaches have showed an excellent correlation, confirming that there is a single binding site, that both penicillins are moderate binders and hydrogen bond and van der Waals forces are predominant. On the other hand, the small discrepancies between the two techniques highlighted the pressing need to approach the study of these systems from both atomic and macromolecular perspectivesThe authors acknowledge Ministerio de Ciencia e Innovación (PID2019-111327GB-100)S

Lysozyme allosteric interactions with β-blocker drugs

Effective and reliable prediction of allosteric molecular interactions involved in protein-ligand systems are essential to understand pharmacological modulation and toxicology processes that are driven by multiple factors covering from the atomistic to cellular level. Even though the interactions taking place within a defined biophysical environment are usually intricate and complex, having a preliminary knowledge of the structural determinant and biochemical function of target enzyme in the physiological or unbound state represent a step forward in the characterization of the forces involved these processes under interaction conditions as induced by drugs. In the present work, we tackle the study of relevant binding interactions between two well-recognized betablocker drugs and the lysozyme biological target from an experimental-computational perspective. In this way, molecular docking, machine learning and perturbation analysis combined with UV–vis and fluorescence measurements will allow us to determine the allosteric regulation and functional dynamics of lysozyme by binding propranolol and acebutololThe authors acknowledge Ministerio de Ciencia e Innovación (PID2019-111327GB-100)S

New Mechanistic Insights on Carbon Nanotubes’ Nanotoxicity Using Isolated Submitochondrial Particles, Molecular Docking, and Nano-QSTR Approaches

Single-walled carbon nanotubes can induce mitochondrial F0F1-ATPase nanotoxicity through inhibition. To completely characterize the mechanistic effect triggering the toxicity, we have developed a new approach based on the combination of experimental and computational study, since the use of only one or few techniques may not fully describe the phenomena. To this end, the in vitro inhibition responses in submitochondrial particles (SMP) was combined with docking, elastic network models, fractal surface analysis, and Nano-QSTR models. In vitro studies suggest that inhibition responses in SMP of F0F1-ATPase enzyme were strongly dependent on the concentration assay (from 3 to 5 µg/mL) for both pristine and COOH single-walled carbon nanotubes types (SWCNT). Besides, both SWCNTs show an interaction inhibition pattern mimicking the oligomycin A (the specific mitochondria F0F1-ATPase inhibitor blocking the c-ring F0 subunit). Performed docking studies denote the best crystallography binding pose obtained for the docking complexes based on the free energy of binding (FEB) fit well with the in vitro evidence from the thermodynamics point of view, following an affinity order such as: FEB (oligomycin A/F0-ATPase complex) = −9.8 kcal/mol > FEB (SWCNT-COOH/F0-ATPase complex) = −6.8 kcal/mol ~ FEB (SWCNT-pristine complex) = −5.9 kcal/mol, with predominance of van der Waals hydrophobic nano-interactions with key F0-ATPase binding site residues (Phe 55 and Phe 64). Elastic network models and fractal surface analysis were performed to study conformational perturbations induced by SWCNT. Our results suggest that interaction may be triggering abnormal allosteric responses and signals propagation in the inter-residue network, which could affect the substrate recognition ligand geometrical specificity of the F0F1-ATPase enzyme in order (SWCNT-pristine > SWCNT-COOH). In addition, Nano-QSTR models have been developed to predict toxicity induced by both SWCNTs, using results of in vitro and docking studies. Results show that this method may be used for the fast prediction of the nanotoxicity induced by SWCNT, avoiding time- and money-consuming techniques. Overall, the obtained results may open new avenues toward to the better understanding and prediction of new nanotoxicity mechanisms, rational drug design-based nanotechnology, and potential biomedical application in precision nanomedicineThis research was funded by FCT/MCTES through national funds (Michael González-Durruthy, Riccardo Concu, and M. Natália D.S. Cordeiro), grant UID/QUI/50006/2020, as well as by Xunta de Galicia (Juan M. Ruso), grant ED41E2018/08S

Decrypting strong and weak single-walled carbon nanotubes interactions with mitochondrial voltage-dependent anion channels using molecular docking and perturbation theory

[Abstract] The current molecular docking study provided the Free Energy of Binding (FEB) for the interaction (nanotoxicity) between VDAC mitochondrial channels of three species (VDAC1-Mus musculus, VDAC1-Homo sapiens, VDAC2-Danio rerio) with SWCNT-H, SWCNT-OH, SWCNT-COOH carbon nanotubes. The general results showed that the FEB values were statistically more negative (p  (SWCNT-VDAC1-Mus musculus) > (SWCNT-VDAC1-Homo sapiens) > (ATP-VDAC). More negative FEB values for SWCNT-COOH and OH were found in VDAC2-Danio rerio when compared with VDAC1-Mus musculus and VDAC1-Homo sapiens (p  r2 > 0.97) was observed between n-Hamada index and VDAC nanotoxicity (or FEB) for the zigzag topologies of SWCNT-COOH and SWCNT-OH. Predictive Nanoparticles-Quantitative-Structure Binding-Relationship models (nano-QSBR) for strong and weak SWCNT-VDAC docking interactions were performed using Perturbation Theory, regression and classification models. Thus, 405 SWCNT-VDAC interactions were predicted using a nano-PT-QSBR classifications model with high accuracy, specificity, and sensitivity (73–98%) in training and validation series, and a maximum AUROC value of 0.978. In addition, the best regression model was obtained with Random Forest (R2 of 0.833, RMSE of 0.0844), suggesting an excellent potential to predict SWCNT-VDAC channel nanotoxicity.Brasil. Conselho Nacional de Desenvolvimento Científico e Tecnológico; 552131/2011-3Brasil. Conselho Nacional de Desenvolvimento Científico e Tecnológico; 454332/2014-9Galicia. Consellería de Cultura, Educación e Ordenación Universitaria; R2014/03

Experimental–computational study of carbon nanotube effects on mitochondrial respiration: in silico nano-QSPR machine learning models based on new Raman spectra transform with Markov–Shannon entropy invariants

[Abstract] The study of selective toxicity of carbon nanotubes (CNTs) on mitochondria (CNT-mitotoxicity) is of major interest for future biomedical applications. In the current work, the mitochondrial oxygen consumption (E3) is measured under three experimental conditions by exposure to pristine and oxidized CNTs (hydroxylated and carboxylated). Respiratory functional assays showed that the information on the CNT Raman spectroscopy could be useful to predict structural parameters of mitotoxicity induced by CNTs. The in vitro functional assays show that the mitochondrial oxidative phosphorylation by ATP-synthase (or state V3 of respiration) was not perturbed in isolated rat-liver mitochondria. For the first time a star graph (SG) transform of the CNT Raman spectra is proposed in order to obtain the raw information for a nano-QSPR model. Box–Jenkins and perturbation theory operators are used for the SG Shannon entropies. A modified RRegrs methodology is employed to test four regression methods such as multiple linear regression (LM), partial least squares regression (PLS), neural networks regression (NN), and random forest (RF). RF provides the best models to predict the mitochondrial oxygen consumption in the presence of specific CNTs with R2 of 0.998–0.999 and RMSE of 0.0068–0.0133 (training and test subsets). This work is aimed at demonstrating that the SG transform of Raman spectra is useful to encode CNT information, similarly to the SG transform of the blood proteome spectra in cancer or electroencephalograms in epilepsy and also as a prospective chemoinformatics tool for nanorisk assessmentXunta de Galicia; GRC2014/049Xunta de Galicia; R2014/03

Targeting Beta-Blocker Drug–Drug Interactions with Fibrinogen Blood Plasma Protein: A Computational and Experimental Study

In this work, one of the most prevalent polypharmacology drug–drug interaction events that occurs between two widely used beta-blocker drugs—i.e., acebutolol and propranolol—with the most abundant blood plasma fibrinogen protein was evaluated. Towards that end, molecular docking and Density Functional Theory (DFT) calculations were used as complementary tools. A fibrinogen crystallographic validation for the three best ranked binding-sites shows 100% of conformationally favored residues with total absence of restricted flexibility. From those three sites, results on both the binding-site druggability and ligand transport analysis-based free energy trajectories pointed out the most preferred biophysical environment site for drug–drug interactions. Furthermore, the total affinity for the stabilization of the drug–drug complexes was mostly influenced by steric energy contributions, based mainly on multiple hydrophobic contacts with critical residues (THR22: P and SER50: Q) in such best-ranked site. Additionally, the DFT calculations revealed that the beta-blocker drug–drug complexes have a spontaneous thermodynamic stabilization following the same affinity order obtained in the docking simulations, without covalent-bond formation between both interacting beta-blockers in the best-ranked site. Lastly, experimental ultrasound density and velocity measurements were performed and allowed us to validate and corroborate the computational obtained resultsThis research was funded by FCT/MCTES through national funds (Michael González-Durruthy, Riccardo Concu, and M. Natália D.S. Cordeiro), grant UID/QUI/50006/2020, as well as by Xunta de Galicia (Juan M. Ruso), grant ED41E2018/08S

Carbon Nanotubes’ Effect on Mitochondrial Oxygen Flux Dynamics: Polarography Experimental Study and Machine Learning Models using Star Graph Trace Invariants of Raman Spectra

[Abstract] This study presents the impact of carbon nanotubes (CNTs) on mitochondrial oxygen mass flux (Jm) under three experimental conditions. New experimental results and a new methodology are reported for the first time and they are based on CNT Raman spectra star graph transform (spectral moments) and perturbation theory. The experimental measures of Jm showed that no tested CNT family can inhibit the oxygen consumption profiles of mitochondria. The best model for the prediction of Jm for other CNTs was provided by random forest using eight features, obtaining test R-squared (R2) of 0.863 and test root-mean-square error (RMSE) of 0.0461. The results demonstrate the capability of encoding CNT information into spectral moments of the Raman star graphs (SG) transform with a potential applicability as predictive tools in nanotechnology and material risk assessmentsInstituto de Salud Carlos III; PI13/02020Instituto de Salud Carlos III; PI13/00280Galicia. Consellería de Cultura, Educación e Ordenación Universitaria; R2014/025Galicia. Consellería de Cultura, Educación e Ordenación Universitaria; GRC2014/049Galicia. Consellería de Cultura, Educación e Ordenación Universitaria; R2014/039Ministerio de Economía y Competitividad; UNLC08-1E-002Ministerio de Economía y Competitividad ; UNLC13-13-3503Ministerio de Economía y Competitividad; CTQ2016-74881-PPaís Vasco.Gobierno; IT1045-16Brasil. Conselho Nacional de Desenvolvimento Científico e Tecnológico; 308539/2016-8Brasil. Conselho Nacional de Desenvolvimento Científico e Tecnológico; 454332/2014-

Unraveling the Compositional and Molecular Features Involved in Lysozyme-Benzothiazole Derivative Interactions

In this work we present a computational analysis together with experimental studies, focusing on the interaction between a benzothiazole (BTS) and lysozyme. Results obtained from isothermal titration calorimetry, UV-vis, and fluorescence were contrasted and complemented with molecular docking and machine learning techniques. The free energy values obtained both experimentally and theoretically showed excellent similarity. Calorimetry, UV-vis, and 3D/2D-lig-plot analysis revealed that the most relevant interactions between BTS and lysozyme are based on a predominance of aromatic, hydrophobic Van der Waals interactions, mainly aromatic edge-to-face (T-shaped) π-π stacking interactions between the benzene ring belonging to the 2-(methylthio)-benzothiazole moiety of BTS and the aromatic amino acid residue TRP108 of the lysozyme receptor. Next, conventional hydrogen bonding interactions contribute to the stability of the BTS-lysozyme coupling complex. In addition, mechanistic approaches performed using elastic network models revealed that the BTS ligand theoretically induces propagation of allosteric signals, suggesting non-physiological conformational flexing in large blocks of lysozyme affecting α-helices. Likewise, the BTS ligand interacts directly with allosteric residues, inducing perturbations in the conformational dynamics expressed as a moderate conformational softening in the α-helices H1, H2, and their corresponding β-loop in the lysozyme receptor, in contrast to the unbound state of lysozyme