Peptide stapling by late-stage Suzuki-Miyaura cross-coupling

The development of peptide stapling techniques to stabilise alpha-helical secondary structure motifs of peptides led to the design of modulators of protein-protein interactions, which had been considered undruggable for a long time. We disclose a novel approach towards peptide stapling utilising macrocyclisation by late-stage Suzuki-Miyaura cross-coupling of bromotryptophan-containing peptides of the catenin-binding domain of axin. Optimisation of the linker length in order to find a compromise between both sufficient linker rigidity and flexibility resulted in a peptide with an increased alpha-helicity and enhanced binding affinity to its native binding partner beta-catenin. An increased proteolytic stability against proteinase K has been demonstrated

Fullerene-Based Mimics of Biocatalysts Show Remarkable Activity and Modularity.

The design of catalysts with greater control over catalytic activity and stability is a major challenge with substantial impact on fundamental chemistry and industrial applications. Due to their unparalleled diversity, selectivity, and efficiency, enzymes are promising models for next-generation catalysts, and considerable efforts have been devoted to incorporating the principles of their mechanisms of action into artificial systems. We report a heretofore undocumented catalyst design that introduces fullerenes to the field of biocatalysis, which we refer to as fullerene nanocatalysts, and that emulates enzymatic active sites through multifunctional self-assembled nanostructures. As a proof-of-concept, we mimicked the reactivity of hydrolases using fullerene nanocatalysts functionalized with the basic components of the parent enzyme with remarkable activity. Owing to the versatile amino acid-based functionalization repertoire of fullerene nanocatalysts, these next-generation carbon/biomolecule hybrids have potential to mimic the activity of other families of enzymes and, therefore, offer new perspectives for the design of biocompatible, high-efficiency artificial nanocatalysts

Why Does Asn71 Deamidate Faster Than Asn15 in the Enzyme Triosephosphate Isomerase? Answers from Microsecond Molecular Dynamics Simulation and QM/MM Free Energy Calculations

Deamidation is the uncatalyzed process by which asparagine or glutamine can be transformed into aspartic acid or glutamic acid, respectively. In its active homodimeric form, mammalian triosephosphate isomerase (TPI) contains two deamidation sites per monomer. Experimental evidence shows that the primary deamidation site (Asn71-Gly72) deamidates faster than the secondary deamidation site (Asn15-Gly16). To evaluate the factors controlling the rates of these two deamidation sites in TPI, we have performed graphics processing unit-enabled microsecond long molecular dynamics simulations of rabbit TPI. The kinetics of asparagine dipeptide and two deamidation sites in mammalian TPI are also investigated using quantum mechanical/molecular mechanical tools with the umbrella sampling technique. Analysis of the simulations has been performed using independent global and local descriptors that can influence the deamidation rates: desolvation effects, backbone acidity, and side chain conformations. Our findings show that all the descriptors add up to favor the primary deamidation site over the secondary one in mammalian TPI: Asn71 deamidates faster because it is more solvent accessible, the adjacent glycine NH backbone acidity is enhanced, and the Asn side chain has a preferential near attack conformation. The crucial impact of the backbone amide acidity of the adjacent glycine on the deamidation rate is shown by kinetic analysis. Our findings also shed light on the effect of high-order structure on deamidation: the deamidation in a small peptide is favored first because of the higher reactivity of the asparagine residue and then because of the stronger stability of the tetrahedral intermediate

The Q41R mutation in the HCV-protease enhances the reactivity towards MAVS by suppressing non-reactive pathways

Recent experimental findings pointed out a new mutation in the HCV protease, Q41R, responsible for a significant enhancement of the enzyme's reactivity towards the mitochondrial antiviral-signaling protein (MAVS). The Q41R mutation is located rather far from the active site, and its involvement in the overall reaction mechanism is thus unclear. We used classical molecular dynamics and QM/MM to study the acylation reaction of HCV NS3/4A protease variants bound to MAVS and the NS4A/4B substrate and uncovered the indirect mechanism by which the Q41R mutation plays a critical role in the efficient cleavage of the substrate. Our simulations reveal that there are two major conformations of the MAVS H1 '(p) residue for the wild type protease and only one conformation for the Q41R mutant. The conformational space of H1 '(p) is restricted by the Q41R mutation due to a pi-pi stacking between H1 '(p) and R41 as well as a strong hydrogen bond between the backbone of H57 and the side chain of R41. Further QM/MM calculations indicate that the complex with the conformation ruled out by the Q41R substitution is a non-reactive species due to its higher free energy barrier for the acylation reaction. Based on our calculations, we propose a kinetic mechanism that explains experimental data showing an increase of apparent rate constants for MAVS cleavage in Q41R mutants. Our model predicts that the non-reactive conformation of the enzyme-substrate complex modulates reaction kinetics like an uncompetitive inhibitor

Predicting the bioactive conformations of macrocycles: a molecular dynamics-based docking procedure with DynaDock

Macrocyclic compounds are of growing interest as a new class of therapeutics, especially as inhibitors binding to protein-protein interfaces. As molecular modeling is a well-established complimentary tool in modern drug design, the number of attempts to develop reliable docking strategies and algorithms to accurately predict the binding mode of macrocycles is rising continuously. Standard molecular docking approaches need to be adapted to this application, as a comprehensive yet efficient sampling of all ring conformations of the macrocycle is necessary. To overcome this issue, we designed a molecular dynamics-based docking protocol for macrocycles, in which the challenging sampling step is addressed by conventional molecular dynamics (750ns) simulations performed at moderately high temperature (370K). Consecutive flexible docking with the DynaDock approach based on multiple, pre-sampled ring conformations yields highly accurate poses with ligand RMSD values lower than 1.8 angstrom. We further investigated the value of molecular dynamics-based complex stability estimations for pose selection and discuss its applicability in combination with standard binding free energy estimations for assessing the quality of poses in future blind docking studies

Cyclization of RGD Peptides by Suzuki-Miyaura Cross-Coupling

Halogenated L- or D-tryptophan obtained by biocatalytic halogenation was incorporated into RGD peptides together with a variety of alkyl or aryl boronic acids. Suzuki-Miyaura cross-coupling either in solution or on-resin results in side chain-to-tail-cyclized RGD peptides, for example, with biaryl moieties, providing a new dimension of structure-activity relationships. An array of RGD peptides differing in macrocycle size, the presence of D-amino acid, N-methylation, or connectivity between the indole moiety and the boronic acid showed that, in particular, connectivity exhibits a major impact on affinities toward integrins, for example, a(v)beta(3). Structure-activity relationship studies yielded peptides with affinities toward a(v)beta(3) in the low nanomolar range, good selectivity, and high plasma stability. Structural characteristics of representative molecules have been investigated by molecular dynamics simulations, which allowed understanding the observed activity differences

Water interactions with hydrophobic groups: Assessment and recalibration of semiempirical molecular orbital methods

In this work, we present a study of the ability of different semiempirical methods to describe intermolecular interactions in water solution. In particular, we focus on methods based on the Neglect of Diatomic Differential Overlap approximation. Significant improvements of these methods have been reported in the literature in the past years regarding the description of non-covalent interactions. In particular, a broad range of methodologies has been developed to deal with the properties of hydrogen-bonded systems, with varying degrees of success. In contrast, the interactions between water and a molecule containing hydrophobic groups have been little analyzed. Indeed, by considering the potential energy surfaces obtained using different semiempirical Hamiltonians for the intermolecular interactions of model systems, we found that none of the available methods provides an entirely satisfactory description of both hydrophobic and hydrophilic interactions in water. In addition, a vibrational analysis carried out in a model system for these interactions, a methane clathrate cluster, showed that some recent methods cannot be used to carry out studies of vibrational properties. Following a procedure established in our group [M. I. Bernal-Uruchurtu, M. T. C. Martins-Costa, C. Millot, and M. F. Ruiz-Lopez, J. Comput. Chem. 21, 572 (2000); W. Harb, M. I. Bernal-Uruchurtu, and M. F. Ruiz-Lopez, Theor. Chem. Acc. 112, 204 (2004)], we developed new parameters for the core-core interaction terms based on fitting potential energy curves obtained at the MP2 level for our model system. We investigated the transferability of the new parameters to describe a system, having both hydrophilic and hydrophobic groups, interacting with water. We found that only by introducing two different sets of parameters for hydrophilic and hydrophobic hydrogen atom types we are able to match the features of the ab initio calculated properties. Once this assumption is made, a good agreement with the MP2 reference is achieved. The results reported in this work provide therefore a direction for future developments of semiempirical approaches that are still required to investigate chemical processes in biomolecules and in large disordered systems. (C) 2014 AIP Publishing LLC

Ca2+ binding induced sequential allosteric activation of sortase A: An example for ion-triggered conformational selection

The allosteric activation of the intrinsically disordered enzyme Staphylococcus aureus sortase A is initiated via binding of a Ca2+ ion. Although Ca2+ binding was shown to initiate structural changes inducing disorder-to-order transitions, the details of the allosteric activation mechanism remain elusive. We performed long-term molecular dynamics simulations of sortase A without (3 simulations of 1.6 mu s) and with bound Ca2+ (simulations of 1.6 mu s, 1.8 mu s, and 2.5 mu s). Our results show that Ca2+ binding causes not only ordering of the disordered beta 6/beta 7 loop of the protein, but also modulates hinge motions in the dynamic beta 7/beta 8 loop, which is important for the catalytic activity of the enzyme. Cation binding triggers signal transmission from the Ca2+ binding site to the dynamic beta 7/beta 8 loop via the repetitive folding/unfolding of short helical stretches of the disordered beta 6/beta 7 loop. These correlated structuralrearrangements lead to several distinct conformational states of the binding groove, which show optimal binding features for the sorting signal motif and feature binding energies up to 20 kcal/mol more favorable than observed for the sortase A without Ca2+. The presented results indicate a highly correlated, conformational selection-based activation mechanism of the enzyme triggered by cation binding. They also demonstrate the importance of the dynamics of intrinsically disordered regions for allosteric regulation

Tuning the Biological Activity of RGD Peptides with Halotryptophanst

An array of L- and D-halotryptophans with different substituents at the indole moiety was synthesized employing either enzymatic halogenation by halogenases or incorporation of haloindoles using tryptophan synthase. Introduction of these Trp derivatives into RGD peptides as a benchmark system was performed to investigate their influence on bioactivity. Halotryptophan-containing RGD peptides display increased affinity toward integrin alpha(nu)beta(3) and enhanced selectivity over integrin alpha(5)beta(1). In addition, bromotryptophan was exploited as a platform for latestage diversification by Suzuki-Miyaura cross-coupling (SMC), resulting in new-to-nature biaryl motifs. These peptides show enhanced affinity toward alpha(nu)beta(3), good affinity to alpha(nu)beta(8), and remarkable selectivity over alpha(5)beta(1) and alpha(IIb)beta(3) while featuring fluorogenic properties. Their feasibility as a probe was demonstrated in vitro. Extensive molecular dynamics simulations were undertaken to elucidate NMR and high-performance liquid chromatography (HPLC) data for these late-stage diversified cyclic RGD peptides and to further characterize their conformational preferences

Activity of Topotecan toward the DNA/Topoisomerase I Complex: A Theoretical Rationalization

Topotecan (TPT) is a nontoxic anticancer drug characterized by a pH-dependent lactone/carboxyl equilibrium. TPT acts on the covalently bonded DNA/topoisomerase I (DNA/TopoI) complex by intercalating between two DNA bases at the active site. This turns TopoI into a DNA-damaging agent and inhibits supercoil relaxation. Although only the lactone form of the drug is active and effectively inhibits TopoI, both forms have been co-crystallized at the same location within the DNA/TopoI complex. To gain further insights into the pH-dependent activity of TPT, the differences between two TPT:DNA/TopoI complexes presenting either the lactone (acidic pH) or the carboxyl (basic pH) form of TPT were studied by means of molecular dynamic simulations, quantum mechanical/molecular mechanical calculations, and topological analysis. We identified two specific amino acids that have a direct relationship with the activity of the drug, i.e., lysine 532 (K532) and asparagine 722 (N722). K532 forms a stable hydrogen bond bridge between TPT and DNA only when the drug is in its active lactone form. The presence of the active drug triggers the formation of an additional stable interaction between DNA and protein residues, where N722 acts as a bridge between the two fragments, thus increasing the binding affinity of DNA for Topol and further slowing the release of DNA. Overall, our results provide a clear understanding of the activity of the TPT-like class of molecules and can help in the future design of new anticancer drugs targeting topoisomerase enzymes