High-Throughput Prediction of the Impact of Genetic Variability on Drug Sensitivity and Resistance Patterns for Clinically Relevant Epidermal Growth Factor Receptor Mutations from Atomistic Simulations.

Mutations in the kinase domain of the epidermal growth factor receptor (EGFR) can be drivers of cancer and also trigger drug resistance in patients receiving chemotherapy treatment based on kinase inhibitors. A priori knowledge of the impact of EGFR variants on drug sensitivity would help to optimize chemotherapy and design new drugs that are effective against resistant variants before they emerge in clinical trials. To this end, we explored a variety of in silico methods, from sequence-based to "state-of-the-art" atomistic simulations. We did not find any sequence signal that can provide clues on when a drug-related mutation appears or the impact of such mutations on drug activity. Low-level simulation methods provide limited qualitative information on regions where mutations are likely to cause alterations in drug activity, and they can predict around 70% of the impact of mutations on drug efficiency. High-level simulations based on nonequilibrium alchemical free energy calculations show predictive power. The integration of these "state-of-the-art" methods into a workflow implementing an interface for parallel distribution of the calculations allows its automatic and high-throughput use, even for researchers with moderate experience in molecular simulations.

Taste Perception: Molecular Recognition of Food Molecules.

Molecular recognition of food molecules by ion channels and G-protein coupled receptors is the basis of taste perception. We explore the chemical nature of dietary molecules, and explore how salty, sour, sweet, bitter, and umami tastes can be explained at a molecular level.

Extensive benchmark of rDock as a peptide-protein docking tool.

Peptide-protein interactions are ubiquitous in living cells and essential to a wide range of biological processes, as well as pathologies such as cancer or cardiovascular disease. Yet, obtaining reliable binding mode predictions in peptide-protein docking remains a great challenge for most computational docking programs. The main goal of this study was to assess the performance of the small molecule docking program rDock in comparison to other widely used small molecule docking programs, using 100 peptide-protein systems with peptides ranging from 2 to 12 residues. As we used two large independent benchmark sets previously published for other small-molecule docking programs (AutoDockVina benchmark and LEADSPEP), the performance of rDock could directly be compared to the performances of AutoDockVina, Surflex, GOLD, and Glide, as well as to the peptide docking protocol PIPER-FlexPepDock and the webserver HPepDock. Our benchmark reveals that rDock can dock the 100 peptides with an overall backbone RMSD below 2.5 Å in 58.5% of the cases (76% for the 47 systems of the AutoDockVina benchmark set and 43% for the 53 systems of the LEADSPEP benchmark set). More specifically, rDock docks up to 11-residue peptides with a backbone RMSD below 2.5 Å in 60.75% of the cases. rDock displays higher accuracy than most available small molecule docking programs for 6-10-residue peptides and can sometimes perform similarly to the peptide docking tool, especially at a high level of exhaustiveness (100 or 150 runs). Its performance, as is the case for many other unguided small molecule docking tools, is compromised when the peptides adopt secondary structures upon binding. However, our analyses suggest that rDock could be used for predicting how medium-sized biologically relevant peptides bind to their respective protein targets when the latter bind in an extended mode.

Binding mode prediction and MD/MMPBSA-based free energy ranking for agonists of REV-ERBα/NCoR.

The knowledge of the free energy of binding of small molecules to a macromolecular target is crucial in drug design as is the ability to predict the functional consequences of binding. We highlight how a molecular dynamics (MD)-based approach can be used to predict the free energy of small molecules, and to provide priorities for the synthesis and the validation via in vitro tests. Here, we study the dynamics and energetics of the nuclear receptor REV-ERBα with its co-repressor NCoR and 35 novel agonists. Our in silico approach combines molecular docking, molecular dynamics (MD), solvent-accessible surface area (SASA) and molecular mechanics poisson boltzmann surface area (MMPBSA) calculations. While docking yielded initial hints on the binding modes, their stability was assessed by MD. The SASA calculations revealed that the presence of the ligand led to a higher exposure of hydrophobic REV-ERB residues for NCoR recruitment. MMPBSA was very successful in ranking ligands by potency in a retrospective and prospective manner. Particularly, the prospective MMPBSA ranking-based validations for four compounds, three predicted to be active and one weakly active, were confirmed experimentally.

Virtual screening: an in silico tool for interlacing the chemical universe with the proteome.

In silico screening both in the forward (traditional virtual screening) and reverse sense (inverse virtual screening (IVS)) are helpful techniques for interlacing the chemical universe of small molecules with the proteome. The former, which is using a protein structure and a large chemical database, is well-known by the scientific community. We have chosen here to provide an overview on the latter, focusing on validation and target prioritization strategies. By comparing it to complementary or alternative wet-lab approaches, we put IVS in the broader context of chemical genomics, target discovery and drug design. By giving examples from the literature and an own example on how to validate the approach, we provide guidance on the issues related to IVS.

Confounding Factors in Targeted Degradation of Short-Lived Proteins.

Targeted protein degradation has recently emerged as a novel option in drug discovery. Natural protein half-life is expected to affect the efficacy of degrading agents, but to what extent it influences target protein degradation has not been systematically explored. Using simple mathematical modeling of protein degradation, we find that the natural half-life of a target protein has a dramatic effect on the level of protein degradation induced by a degrader agent which can pose significant hurdles to screening efforts. Moreover, we show that upon screening for degraders of short-lived proteins, agents that stall protein synthesis, such as GSPT1 degraders and generally cytotoxic compounds, deceptively appear as protein-degrading agents. This is exemplified by the disappearance of short-lived proteins such as MCL1 and MDM2 upon GSPT1 degradation and upon treatment with cytotoxic agents such as doxorubicin. These findings have implications for target selection as well as for the type of control experiments required to conclude that a novel agent works as a bona fide targeted protein degrader.

Breaking the aggregation of the monoclonal antibody bevacizumab (avastin®) by dexamethasone phosphate: insights from molecular modelling and asymmetrical flow field-flow fractionation.

PURPOSE: To investigate the mechanism behind the aggregation breaking properties of dexamethasone phosphate and related corticosteroids on the IgG1 antibody bevacizumab (Avastin®). METHODS: An in silico 3D dimer model is developed to identify the bevacizumab-bevacizumab interface, and different corticosteroids are docked onto the model to distinguish preferred binding sites. In silico predictions are validated by in vitro stability studies, where the antibody is stressed in presence or absence of each corticosteroid and formed aggregates are quantified by asymmetrical flow field-flow fractionation. RESULTS: The dimer model features one close crystal contact area: Lys445 on the Fc region interacts with one Fab arm of the second bevacizumab. Docking reveals an interaction between the phosphate group of dexamethasone phosphate and Lys445, while the rest of the molecule is hindering dimer formation. Predictions are confirmed in vitro, demonstrating that dexamethasone phosphate and betamethasone phosphate partly prevent antibody aggregation, whereas triamcinolone acetonide phosphate does not. CONCLUSIONS: Results suggest that bevacizumab monomers follow a specific mechanism to form dimers in which a protein-protein interaction hotspot can be distinguished. The dimer formation can be hindered by corticosteroids in a specific way. This approach allows a simple way to stabilize IgG1 antibodies.

Building Biological Relevance Into Integrative Modelling of Macromolecular Assemblies.

Recent advances in structural biophysics and integrative modelling methods now allow us to decipher the structures of large macromolecular assemblies. Understanding the dynamics and mechanisms involved in their biological function requires rigorous integration of all available data. We have developed a complete modelling pipeline that includes analyses to extract biologically significant information by consistently combining automated and interactive human-guided steps. We illustrate this idea with two examples. First, we describe the ryanodine receptor, an ion channel that controls ion flux across the cell membrane through transitions between open and closed states. The conformational changes associated with the transitions are small compared to the considerable system size of the receptor; it is challenging to consistently track these states with the available cryo-EM structures. The second example involves homologous recombination, in which long filaments of a recombinase protein and DNA catalyse the exchange of homologous DNA strands to reliably repair DNA double-strand breaks. The nucleoprotein filament reaction intermediates in this process are short-lived and heterogeneous, making their structures particularly elusive. The pipeline we describe, which incorporates experimental and theoretical knowledge combined with state-of-the-art interactive and immersive modelling tools, can help overcome these challenges. In both examples, we point to new insights into biological processes that arise from such interdisciplinary approaches.

A new N-methyl thymine derivative comprising a dihydroxyisobutenyl unit as ligand for thymidine kinase of herpes simplex virus type 1 (HSV-1 TK).

Molecular modeling and phosphorylation assay in vitro were employed to select a novel unsaturated 1,3-dihydroxyisobutenyl thymine derivative 6 as ligand for HSV-1 TK which may be of interest as lead for the development of an positron emission tomography (PET) imaging agent. Compound 6 was successfully prepared using modified approaches. A significant improvement over the syntheses involving pathways A and B (1% and 3% overall yield, respectively), was observed using synthetic route C (14% overall yield).

Novel Mechanism for an Old Drug: Phenazopyridine is a Kinase Inhibitor Affecting Autophagy and Cellular Differentiation.

Phenazopyridine is a widely used drug against urinary tract pain. The compound has also been shown to enhance neural differentiation of pluripotent stem cells. However, its mechanism of action is not understood. Based on its chemical structure, we hypothesized that phenazopyridine could be a kinase inhibitor. Phenazopyridine was investigated in the following experimental systems: 1) activity of kinases in pluripotent stem cells; 2) binding to recombinant kinases, and 3) functional impact on pluripotent stem cells. Upon addition to pluripotent stem cells, phenazopyridine induced changes in kinase activities, particularly involving Mitogen-Activated Protein Kinases, Cyclin-Dependent Kinases, and AKT pathway kinases. To identify the primary targets of phenazopyridine, we screened its interactions with 401 human kinases. Dose-inhibition curves showed that three of these kinases interacted with phenazopyridine with sub-micromolar binding affinities: cyclin-G-associated kinase, and the two phosphatidylinositol kinases PI4KB and PIP4K2C, the latter being known for participating in pain induction. Docking revealed that phenazopyridine forms strong H-bonds with the hinge region of the ATP-binding pocket of these kinases. As previous studies suggested increased autophagy upon inhibition of the phosphatidyl-inositol/AKT pathway, we also investigated the impact of phenazopyridine on this pathway and found an upregulation. In conclusion, our study demonstrates for the first time that phenazopyridine is a kinase inhibitor, impacting notably phosphatidylinositol kinases involved in nociception.

Synthesis, crystal structure, and in vitro biological evaluation of C-6 pyrimidine derivatives: new lead structures for monitoring gene expression in vivo.

Novel C-6 substituted pyrimidine derivatives are good substrates of herpes simplex virus type 1 thymidine kinase (HSV1-TK). Enzyme kinetic experiments showed that our lead compound, N-methyl DHBT (N-methyl-6-(1,3-dihydroxyisobutyl) thymine; N-Me DHBT), is phosphorylated at a similar rate compared to "gold standard" 9-[4-fluoro-3-(hydroxymethyl)butyl]guanine, FHBG, (K(m) = 10 ± 0.3 µM; k(cat) = 0.036 ± 0.015 sec(-1)). Additionally, it does not show cytotoxic properties on B16F1 cells up to a concentration of 10 mM. The x-ray analysis of the crystal structures of HSV1-TK with N-Me DHBT and of HSV1-TK with the fluorinated derivative N-Me FHBT confirmed the binding mode predicted by docking studies and their substrate characteristics. Moreover, the crystal structure of HSV1-TK with N-Me DHBT revealed an additional water-mediated H-bond interesting for the design of further analogues.

NADPH oxidase (NOX) isoforms are inhibited by celastrol with a dual mode of action.

BACKGROUND: Celastrol is one of several bioactive compounds extracted from the medicinal plant Tripterygium wilfordii. Celastrol is used to treat inflammatory conditions, and shows benefits in models of neurodegenerative disease, cancer and arthritis, although its mechanism of action is incompletely understood. EXPERIMENTAL APPROACH: Celastrol was tested on human NADPH oxidases (NOXs) using a panel of experiments: production of reactive oxygen species and oxygen consumption by NOX enzymes, xanthine oxidase activity, cell toxicity, phagocyte oxidase subunit translocation, and binding to cytosolic subunits of NOX enzymes. The effect of celastrol was compared with diphenyleneiodonium, an established inhibitor of flavoproteins. KEY RESULTS: Low concentrations of celastrol completely inhibited NOX1, NOX2, NOX4 and NOX5 within minutes with concentration-response curves exhibiting higher Hill coefficients and lower IC50 values for NOX1 and NOX2 compared with NOX4 and NOX5, suggesting differences in their mode of action. In a cell-free system, celastrol had an IC50 of 1.24 and 8.4 µM for NOX2 and NOX5, respectively. Cytotoxicity, oxidant scavenging, and inhibition of p47(phox) translocation could not account for NOX inhibition. Celastrol bound to a recombinant p47(phox) and disrupted the binding of the proline rich region of p22(phox) to the tandem SH3 domain of p47(phox) and NOXO1, the cytosolic subunits of NOX2 and NOX1, respectively. CONCLUSIONS AND IMPLICATIONS: These results demonstrate that celastrol is a potent inhibitor of NOX enzymes in general with increased potency against NOX1 and NOX2. Furthermore, inhibition of NOX1 and NOX2 was mediated via a novel mode of action, namely inhibition of a functional association between cytosolic subunits and the membrane flavocytochrome.