High-mobility group box 1 protein (HMGB1) is a nuclear component, but extracellularly it serves as a signaling molecule involved in acute and chronic inflammation, for example in sepsis and arthritis. The identification of HMGB1 inhibitors is therefore of significant experimental and clinical interest. We show that glycyrrhizin, a natural anti-inflammatory and antiviral triterpene in clinical use, inhibits HMGB1 chemoattractant and mitogenic activities, and has a weak inhibitory effect on its intranuclear DNA-binding function. NMR and fluorescence studies indicate that glycyrrhizin binds directly to HMGB1 (K(d) approximately 150 microM), interacting with two shallow concave surfaces formed by the two arms of both HMG boxes. Our results explain in part the anti-inflammatory properties of glycyrrhizin, and might direct the design of new derivatives with improved HMGB1-binding properties.

CONSPECTUS: This Account highlights recent advances and discusses major challenges in the field of drug-target recognition, binding, and unbinding studied using metadynamics-based approaches, with particular emphasis on their role in structure-based design. Computational chemistry has significantly contributed to drug design and optimization in an extremely broad range of areas, including prediction of target druggability and drug likeness, de novo design, fragment screening, ligand docking, estimation of binding affinity, and modulation of ADMET (absorption, distribution, metabolism, excretion, toxicity) properties. Computationally driven drug discovery must continuously adapt to keep pace with the evolving knowledge of the factors that modulate the pharmacological action of drugs. There is thus an urgent need for novel computational approaches that integrate the vast amount of complex information currently available for small (bio)organic compounds, biologically relevant targets and their complexes, while also accounting accurately for the thermodynamics and kinetics of drug-target association, the intrinsic dynamical behavior of biomolecular systems, and the complexity of protein-protein networks. Understanding the mechanism of drug binding to and unbinding from biological targets is fundamental for optimizing lead compounds and designing novel biologically active ones. One major challenge is the accurate description of the conformational complexity prior to and upon formation of drug-target complexes. Recently, enhanced sampling methods, including metadynamics and related approaches, have been successfully applied to investigate complex mechanisms of drugs binding to flexible targets. Metadynamics is a family of enhanced sampling techniques aimed at enhancing the rare events and reconstructing the underlying free energy landscape as a function of a set of order parameters, usually referred to as collective variables. Studies of drug binding mechanisms have predicted the most probable association and dissociation pathways and the related binding free energy profile. In addition, the availability of an efficient open-source implementation, running on cost-effective GPU (i.e., graphical processor unit) architectures, has considerably decreased the learning curve and the computational costs of the methods, and increased their adoption by the community. Here, we review the recent contributions of metadynamics and other enhanced sampling methods to the field of drug-target recognition and binding. We discuss how metadynamics has been used to search for transition states, to predict binding and unbinding paths, to treat conformational flexibility, and to compute free energy profiles. We highlight the importance of such predictions in drug discovery. Major challenges in the field and possible solutions will finally be discussed.

Plant homeodomain (PHD) fingers are often present in chromatin-binding proteins and have been shown to bind histone H3 N-terminal tails. Mutations in the autoimmune regulator (AIRE) protein, which harbours two PHD fingers, cause a rare monogenic disease, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). AIRE activates the expression of tissue-specific antigens by directly binding through its first PHD finger (AIRE-PHD1) to histone H3 tails non-methylated at K4 (H3K4me0). Here, we present the solution structure of AIRE-PHD1 in complex with H3K4me0 peptide and show that AIRE-PHD1 is a highly specialized non-modified histone H3 tail reader, as post-translational modifications of the first 10 histone H3 residues reduce binding affinity. In particular, H3R2 dimethylation abrogates AIRE-PHD1 binding in vitro and reduces the in vivo activation of AIRE target genes in HEK293 cells. The observed antagonism by R2 methylation on AIRE-PHD1 binding is unique among the H3K4me0 histone readers and represents the first case of epigenetic negative cross-talk between non-methylated H3K4 and methylated H3R2. Collectively, our results point to a very specific histone code responsible for non-modified H3 tail recognition by AIRE-PHD1 and describe at atomic level one crucial step in the molecular mechanism responsible for antigen expression in the thymus.

Asparagine deamidation at the NGR sequence in the 5th type I repeat of fibronectin (FN-I 5 ) generates isoDGR, an ␣v␤3 integrin-binding motif regulating endothelial cell adhesion and proliferation. By NMR and molecular dynamics studies, we analyzed the structure of CisoDGRC (isoDGR-2C), a cyclic ␤-peptide mimicking the FN-I 5 site, and compared it with NGR, RGD, or DGR-containing cyclopeptides. Docking experiments show that isoDGR, exploiting an inverted orientation as compared with RGD, favorably interacts with the RGD-binding site of ␣v␤3, both recapitulating canonical RGD-␣v␤3 contacts and establishing additional polar interactions. Conversely, NGR and DGR motifs lack the fundamental pharmacophoric requirements for high receptor affinity. Therefore, unlike NGR and DGR, isoDGR is a new natural recognition motif of the RGDbinding pocket of ␣v␤3. These findings contribute to explain the different functional properties of FN-I 5 before and after deamidation, and provide support for the hypothesis that NGR 3 isoDGR transition can work as a molecular timer for activating latent integrin-binding sites in proteins, thus regulating protein function.

Solution NMR data can provide information about the interactions of molecules in the first stages of crystallisation, and 1 H NMR complexation-induced changes in chemical shift can be used to determine high resolution three-dimensional structures of complexes in solution. The concentration-dependent changes in 1 H NMR chemical shift of two different compounds have been used to make predictions about how these compounds pack in the crystalline state. The structures of dimer motifs characterised in solution agree very well with the structures of dimers found in the corresponding X-ray crystal structures. Solution NMR experiments therefore provide an experimental tool for probing the organisation of molecules in the solid state.

Background: Asparagine deamidation at Asn-Gly-Arg (NGR) sites leads to the isoAsp-Gly-Arg (isoDGR) integrin-binding motif formation. Results: Ceruloplasmin (Cp), which contains two NGR sites and is oxidized in cerebrospinal fluid (CSF) in neurodegenerative diseases, can, undergo oxidation-induced structural changes fostering NGR deamidation with gain of integrin binding and signaling properties, in vitro and ex vivo in pathological CSF. Conclusion: Cp NGR motifs can deamidate acquiring integrin-binding functions. Significance: Cp structural changes favor NGR deamidation.

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In this paper, we introduce the BiKi Life Sciences suite. This software makes it easy for computational medicinal chemists to run ad hoc molecular dynamics protocols in a novel and task-oriented environment; as a notebook, BiKi (acronym of Binding Kinetics) keeps memory of any activity together with dependencies among them. It offers unique accelerated protein-ligand binding/unbinding methods and other useful tools to gain actionable knowledge from molecular dynamics simulations and to simplify the drug discovery process.