Molecular Determinants for the Activation/Inhibition of Bak Protein by BH3 Peptides

Apoptosis is a key procedure for all cells. Understanding this process and its regulation has been a subject of study in the last decades. Bcl-2 family of proteins are involved in the regulation of the apoptosis through the formation of homodimers or heterodimers between anti-apoptotic and pro-apoptotic proteins. Deregulation of pro-apoptotic proteins contributes to the progression of many tumour processes. Understanding how these proteins are activated is key to find new anti-cancer treatments. As no drug capable of activating Bak has been discovered yet, studying the structural and energetic insights of the binding of the known Bak activators, BH3 peptides, is essential to design new small molecules that resemble their binding to Bak. Recently, a BH3 Bim analogue has been discovered which inactivates Bak instead of activating it. Therefore, the present work is aimed to identify how BH3 peptides activate or inactivate Bak and determine any difference between them. Determination of the structural differences between the complexes with the activator and the inhibitor has been carried out by means of the study of the fluctuations of the corresponding Principal Components. Moreover, to calculate the binding free energy of the different complexes and to determine which residues of the peptide have the largest contribution to complex formation, MMPB/GBSA approaches are used. Results obtained in this work show differences in complexes with the activator and the inhibitor in structural and energetic terms, which can be used in the design of new molecules that could activate or inactivate pro-apoptotic Bak

Insight into the binding of DFG-out allosteric inhibitors to B-Raf Kinase using molecular dynamics and free energy calculations

B-Raf mutations are identified in 40-50% of patients with melanoma and among them, the substitution of valine for glutamic acid at position 600 (V600EB-Raf) is the most frequent. Treatment of these patients with B-Raf inhibitors has been associated with a clear clinical benefit. Unfortunately, multiple resistance mechanisms have been identified and new potent and selective inhibitors are currently needed. In this work, five different type II inhibitors, which bind V600EB-Raf in its DFG-out conformation, have been studied using molecular dynamics, free energy calculations and energy decomposition analysis. The ranking of calculated MM-PB/GBSA binding affinities is in good agreement with the experimentally measured ones. The per-residue decomposition of ΔGbinding, within the MM-GBSA approach, has been used to identify the key residues governing the allosteric binding of the studied compounds to the V600EB-Raf protein kinase. Results indicate that although van der Waals interactions are key determinants for binding, hydrogen bonds also play an important role. This work also provides a better structural understanding of the binding of DFG-out inhibitors to V600EB-Raf, which can be used in a further step for rational design of a new class of B-Raf potent inhibitors

Influencia de las nuevas tecnologías en los resultados de la asignatura “Fundamentos de Química” en la Ingeniería Técnica Industrial

En la titulación de Ingeniería Técnica Industrial de la Universidad de Jaén se ha ido implantando los créditos ECTS en todas las asignaturas, empezando en 2005 con las del primer curso, para concluir en 2007 con toda la Titulación. Aquí se presenta la parte de la experiencia correspondiente a la asignatura de Fundamentos de Química.El programa es básicamente el mismo aunque hay una reducción del tiempo del profesor dedicado a las clases presenciales. Esta circunstancia llevó al profesorado que imparte Fundamentos de Química en la Ingeniería Técnica Industrial, a comenzar a usar las nuevas tecnologías enfocadas a la puesta en práctica nuevos instrumentos docentes, tratando de mejorar la calidad del proceso de enseñanza y aprendizaje.En esta comunicación se exponen los objetivos de partida para la elaboración de guías didácticas como instrumento base para el desarrollo del proceso docente, la metodología y los resultados en la evaluación de los alumnos

A multistep docking and scoring protocol for congeneric series: Implementation on kinase DFG-out type II inhibitors

AIM:Rescoring of docking-binding poses can significantly improve molecular docking results. Our aim was to evaluate postprocessing docking protocols in order to determine the most suitable methodology for the study of the binding of congeneric compounds to protein kinases. MATERIALS & METHODS:Diverse ligand-receptor poses generated after docking were submitted to different relaxation protocols. The Molecular Mechanics Poisson-Boltzmann (Generalized Born) Surface Area approach was applied for the evaluation of the binding affinity of complexes obtained. The performance of various Molecular Mechanics Poisson-Boltzmann (Generalized Born) Surface Area methodologies was compared. RESULTS:The inclusion of a postprocessing protocol after docking enhances the quality of the results, although the best methodology is system dependent. CONCLUSION:An examination of the interactions established has allowed us to suggest useful modifications for the design of new type II inhibitors.Postprint (author's final draft

Toward understanding calmodulin plasticity by molecular dynamics

Aim: Calmodulin interacts in many different ways with its ligands. We aim to shed light on its plasticity analysing the changes followed by the linker region and the relative position of the lobes using conventional Molecular Dynamics (cMD), accelerated MD (aMD) and scaled MD (sMD). Materials & Methods: Three different structures of calmodulin are compared, obtaining a total of 2.5 μs of molecular dynamics, which have been analysed using the principal component analysis and clustering methodologies Results: sMD simulations reach conformations that cMD is not able to, without compromising the stability of the protein. On the other hand, aMD requires optimization of the setup parameters to be useful. Conclusion: SMD is useful to study flexible proteins, highlighting those factors that justify its promiscuit

Fragment Dissolved molecular dynamics: A systematic and efficient method to locate binding sites.

Fragment-based drug discovery (FBDD) has been popular in the last decade, but some drawbacks, such as protein denaturation or ligand aggregation, have not yet clearly overcome in the framework of biomolecular simulations. In this work a systematic and semi-automatic method is presented as a novel proposal, named fragment dissolved Molecular Dynamics (fdMD), to improve research in future FBDD projects. Our method employs simulation boxes of solvated small fragments, adding a repulsive Lennard-Jones potential term to avoid aggregation, which can be easily used to solvate the object of interest. This method has the advantage of solvating the target with a low number of ligands, thus preventing this way denaturation of the target, while simultaneously generating a database of ligand-solvated boxes that can be used with other targets. A number of scripts are made available to analyze the results and obtain the descriptors proposed as a means of trustfully discard spurious binding sites. To test our method, four sets of different complexity have been solvated with ligand boxes and four molecular dynamics runs of 200 ns length have been run for each system, which have been extended up to 1 μs when needed. The reported results point that the selected number of replicas are enough to identify the correct binding sites irrespective of the initial structure, even in the case of proteins having several close binding sites for the same ligand. Among the proposed descriptors, average MMGBSA and average KDEEP energies emerge as the most robust ones

A Multistep docking and scoring protocol for congeneric series: Implementation on kinase DFG-out type II inhibitors

AIM: Rescoring of docking-binding poses can significantly improve molecular docking results. Our aim was to evaluate postprocessing docking protocols in order to determine the most suitable methodology for the study of the binding of congeneric compounds to protein kinases. MATERIALS & METHODS: Diverse ligand-receptor poses generated after docking were submitted to different relaxation protocols. The Molecular Mechanics Poisson-Boltzmann (Generalized Born) Surface Area approach was applied for the evaluation of the binding affinity of complexes obtained. The performance of various Molecular Mechanics Poisson-Boltzmann (Generalized Born) Surface Area methodologies was compared. RESULTS: The inclusion of a postprocessing protocol after docking enhances the quality of the results, although the best methodology is system dependent. CONCLUSION: An examination of the interactions established has allowed us to suggest useful modifications for the design of new type II inhibitors

Shedding light on the binding mechanism of kinase inhibitors BI-2536, Volasetib and Ro-3280 with their pharmacological target PLK1

In the present work, the interactions of the novel kinase inhibitors BI-2536, Volasetib (BI-6727) and Ro-3280 with the pharmacological target PLK1 have been studied by fluorescence spectroscopy and molecular dynamics calculations. High Stern-Volmer constants were found in fluorescence experiments suggesting the formation of stable protein-ligand complexes. In addition, it was observed that the binding constant between BI-2536 and PLK1 increases about 100-fold in presence of the phosphopeptide Cdc25C-p that docks to the polo box domain of the protein and releases the kinase domain. All the determined binding constants are higher for the kinase inhibitors than for their competitor for the active center (ATP) being BI-2536 and Volasertib the inhibitors that showed more affinity for PLK1. Calculated binding free energies confirmed the higher affinity of PLK1 for BI-2536 and Volasertib than for ATP. The higher affinity of the inhibitors to PLK1 compared to ATP was mainly attributed to stronger van der Waals interactions. Results may help with the challenge of designing and developing new kinase inhibitors more effective in clinical cancer therapy

Binding of the anticancer drug BI-2536 to human serum albumin. A spectroscopic and theoretical study

BI-2536 is a potent Polo-like kinase inhibitor which induces apoptosis in diverse human cancer cell lines. The binding affinity of BI-2536 for human serum albumin (HSA) protein may define its pharmacokinetic and pharmacodynamic profile. We have studied the binding of BI-2536 to HSA by means of different spectroscopic techniques and docking calculations. We have experimentally observed that the affinity of BI-2536 for HSA is higher than that of other common HSA binding drugs. Therefore, it can be postulated that the drug dose should be increased to achieve a certain concentration of free drug in plasma, although BI-2536 could also reach tumour tissues by uptaking HSA/BI-2536 complex. Only a single binding site on HSA has been observed for BI-2536 which seems to correspond to the subdomain IIA pocket. The formation of the HSA/BI-2536 complex is a spontaneous and entropy-driven process that does not cause a significant change of the secondary structure of the protein. Its endothermic character could be related to proton release. Thermodynamic analysis showed that the main protein-drug interactions are of the van der Waals type although the presence of amide and ether groups in BI-2536 could also allow H-bonding with some residues in the subdomain IIA pocket

Effect of set up protocols on the accuracy of alchemical free energy calculation over a set of ACK1 inhibitors

Hit-to-lead virtual screening frequently relies on a cascade of computational methods that starts with rapid calculations applied to a large number of compounds and ends with more expensive computations restricted to a subset of compounds that passed initial filters. This work focuses on set up protocols for alchemical free energy (AFE) scoring in the context of a Docking–MM/PBSA–AFE cascade. A dataset of 15 congeneric inhibitors of the ACK1 protein was used to evaluate the performance of AFE set up protocols that varied in the steps taken to prepare input files (using previously docked and best scored poses, manual selection of poses, manual placement of binding site water molecules). The main finding is that use of knowledge derived from X-ray structures to model binding modes, together with the manual placement of a bridging water molecule, improves the R2 from 0.45 ± 0.06 to 0.76 ± 0.02 and decreases the mean unsigned error from 2.11 ± 0.08 to 1.24 ± 0.04 kcal mol-1. By contrast a brute force automated protocol that increased the sampling time ten-fold lead to little improvements in accuracy. Besides, it is shown that for the present dataset hysteresis can be used to flag poses that need further attention even without prior knowledge of experimental binding affinitiesPeer ReviewedPostprint (published version