Binding site elucidation and structure guided design of macrocyclic IL-17A antagonists.

Interleukin-17A (IL-17A) is a principal driver of multiple inflammatory and immune disorders. Antibodies that neutralize IL-17A or its receptor (IL-17RA) deliver efficacy in autoimmune diseases, but no small-molecule IL-17A antagonists have yet progressed into clinical trials. Investigation of a series of linear peptide ligands to IL-17A and characterization of their binding site has enabled the design of novel macrocyclic ligands that are themselves potent IL-17A antagonists.

Target the More Druggable Protein States in a Highly Dynamic Protein--Protein Interaction System.

The proteins of the Bcl-2 family play key roles in the regulation of programmed cell death by controlling the integrity of the outer mitochondrial membrane and the initiation of the apoptosis process. We performed extensive molecular dynamics simulations to investigate the conformational flexibility of the Bcl-xL protein in both the apo and holo (with Bad peptide and ABT-737) states. The accelerated molecular dynamics method implemented in Amber 14 was used to produce broader conformational sampling of 200 ns simulations. The pocket mining method based on the variational implicit-solvent model tracks the dynamic evolution of the ligand binding site with a druggability score characterizing the maximal affinity achievable by a drug-like molecule. Major movements were observed around the α3-helical domain and the loop region connecting the α1 and α2 helices, reshaping the ligand interaction in the BH3 binding groove. Starting with the apo crystal structure, which is recognized as "closed" and undruggable, the BH3 groove transitioned between the "open" and "closed" states during equilibrium simulation. Further analysis revealed a small percentage of the trajectory frames (∼10%) with a moderate degree of druggability that mimic the ligand-bound states. The ability to attain and detect by computer simulation the most suitable conformational states for ligand binding in advance of compound synthesis and crystal structure solution is of immense value to the application and success of structure-based drug design.

Identification of protein-ligand binding sites by the level-set variational implicit-solvent approach.

Protein­ligand binding is a key biological process at the molecular level. The identification and characterization of small-molecule binding sites on therapeutically relevant proteins have tremendous implications for target evaluation and rational drug design. In this work, we used the recently developed level-set variational implicit-solvent model (VISM) with the Coulomb field approximation (CFA) to locate and characterize potential protein­small-molecule binding sites. We applied our method to a data set of 515 protein­ligand complexes and found that 96.9% of the cocrystallized ligands bind to the VISM-CFA-identified pockets and that 71.8% of the identified pockets are occupied by cocrystallized ligands. For 228 tight-binding protein­ligand complexes (i.e, complexes with experimental pKd values larger than 6), 99.1% of the cocrystallized ligands are in the VISM-CFA-identified pockets. In addition, it was found that the ligand binding orientations are consistent with the hydrophilic and hydrophobic descriptions provided by VISM. Quantitative characterization of binding pockets with topological and physicochemical parameters was used to assess the "ligandability" of the pockets. The results illustrate the key interactions between ligands and receptors and can be very informative for rational drug design.

Heterogeneous Hydration of p53/MDM2 Complex.

Water-mediated interactions play critical roles in biomolecular recognition processes. Explicit solvent molecular dynamics (MD) simulations and the variational implicit-solvent model (VISM) are used to study those hydration properties during binding for the biologically important p53/MDM2 complex. Unlike simple model solutes, in such a realistic and heterogeneous solute-solvent system with both geometrical and chemical complexity, the local water distribution sensitively depends on nearby amino acid properties and the geometric shape of the protein. We show that the VISM can accurately describe the locations of high and low density solvation shells identified by the MD simulations and can explain them by a local coupling balance of solvent-solute interaction potentials and curvature. In particular, capillary transitions between local dry and wet hydration states in the binding pocket are captured for interdomain distance between 4 to 6 Å, right at the onset of binding. The underlying physical connection between geometry and polarity is illustrated and quantified. Our study offers a microscopic and physical insight into the heterogeneous hydration behavior of the biologically highly relevant p53/MDM2 system and demonstrates the fundamental importance of hydrophobic effects for biological binding processes. We hope our study can help to establish new design rules for drugs and medical substances.

Probing the origin of structural stability of single and double stapled p53 peptide analogs bound to MDM2.

The stabilization of secondary structure is believed to play an important role in the peptide-protein binding interaction. In this study, the α-helical conformation and structural stability of single and double stapled all-hydrocarbon cross-linked p53 peptides when bound and unbound to MDM2 are investigated. We determined the effects of the peptide sequence, the stereochemistry of the cross-linker, the conformation of the double bond in the alkene bridge, and the length of the bridge, to the relative stability of the α-helix structure. The binding affinity calculations by WaterMap provided over one hundred hydration sites in the MDM2 binding pocket where water density is greater than twice that of the bulk, and the relative value of free energy released by displacing these hydration sites. In agreement with the experimental data, potentials of mean force obtained by weighted histogram analysis methods indicated the order of peptides from lowest to highest binding affinity. Our study provides a comprehensive rationalization of the relationship between peptide stapling strategy, the secondary structural stability, and the binding affinity of p53/MDM2 complex. We hope our efforts can help to further the development of a new generation p53/MDM2 inhibitors that can reactivate the function of p53 as tumor suppressor gene.

Evaluation of Hydration Free Energy by Level-Set Variational Implicit-Solvent Model with Coulomb-Field Approximation.

In this article, we systematically apply a novel implicit-solvent model, the variational implicit-solvent model (VISM) together with the Coulomb-Field Approximation (CFA), to calculate the hydration free energy of a large set of small organic molecules. Because these molecules have been studied in detail by molecular dynamics simulations and other implicit-solvent models, they provide a good benchmark for evaluating the performance of VISM-CFA. With all-atom Amber force field parameters, VISM-CFA is able to reproduce well not only the experimental and MD simulated total hydration free energy but also the polar and nonpolar contributions individually. The correlation between VISM-CFA and experiments is R2 = 0.763 for the total hydration free energy, with a root-mean-square deviation (RMSD) of 1.83 kcal/mol, and the correlation to results from TIP3P explicit water MD simulations is R2 = 0.839 with a RMSD = 1.36 kcal/mol. In addition, we demonstrate that VISM captures dewetting phenomena in the p53/MDM2 complex and hydrophobic characteristics in the system. This work demonstrates that the level-set VISM-CFA can be used to study the energetic behavior of realistic molecular systems with complicated geometries in solvation, protein-ligand binding, protein-protein association, and protein folding processes.

Role of large thermal fluctuations and magnesium ions in t-RNA selectivity of the ribosome.

The fidelity of translation selection begins with the base pairing of codon-anticodon complex between the m-RNA and tRNAs. Binding of cognate and near-cognate tRNAs induces 30S subunit of the ribosome to wrap around the ternary complex, EF-Tu(GTP)aa-tRNA. We have proposed that large thermal fluctuations play a crucial role in the selection process. To test this conjecture, we have developed a theoretical technique to determine the probability that the ternary complex, as a result of large thermal fluctuations, forms contacts leading to stabilization of the GTPase activated state. We argue that the configurational searches for such processes are in the tail end of the probability distribution and show that the probability for this event is localized around the most likely configuration. Small variations in the repositioning of cognate relative to near-cognate complexes lead to rate enhancement of the cognate complex. The binding energies of over a dozen unique site-bound magnesium structural motifs are investigated and provide insights into the nature of interaction of divalent metal ions with the ribosome.

Probing the alpha-helical structural stability of stapled p53 peptides: molecular dynamics simulations and analysis.

Reactivation of the p53 cell apoptosis pathway through inhibition of the p53-hDM2 interaction is a viable approach to suppress tumor growth in many human cancers and stabilization of the helical structure of synthetic p53 analogs via a hydrocarbon cross-link (staple) has been found to lead to increased potency and inhibition of protein-protein binding (J. Am. Chem. Soc. 129: 5298). However, details of the structure and dynamic stability of the stapled peptides are not well understood. Here, we use extensive all-atom molecular dynamics simulations to study a series of stapled alpha-helical peptides over a range of temperatures in solution. The peptides are found to exhibit substantial variations in predicted alpha-helical propensities that are in good agreement with the experimental observations. In addition, we find significant variation in local structural flexibility of the peptides with the position of the linker, which appears to be more closely related to the observed differences in activity than the absolute alpha-helical stability. These simulations provide new insights into the design of alpha-helical stapled peptides and the development of potent inhibitors of alpha-helical protein-protein interfaces.

DNA on a tube: electrostatic contribution to stiffness.

Two simple models are used to estimate the electrostatic contributions to the stiffness of short DNA fragments. The first model views DNA as two strands that are appropriately parametrized and are wrapped helically around a straight cylinder radius equal to the radius of the DNA molecule. The potential energy of the DNA due to phosphate-phosphate electrostatic interactions is evaluated assuming that the charges interact through Debye-Hückel potentials. This potential energy is compared with the potential energy as computed using our second model in which DNA is viewed as two helical strands wrapping around a curved tube whose cross-section is a disk of radius equal to the radius of the DNA. We find that the electrostatic persistence length for B-DNA molecules in the range of 105-130 bp is 125.64 angstroms (37 bp) and 76.05 angstroms (23 bp) at 5 and 10 mM monovalent salt concentration, respectively. If the condensed fraction theta is taken to be 0.715 at 10 mM, then the electrostatic persistence length is 108.28 angstroms (32 bp), while that based on taking into account end effects is 72.87 angstroms (21 bp). At 5 mM monovalent salt, the total persistence length for DNA fragments in this length range is approximately 575.64 angstroms (171 bp), using the best estimate for nonelectrostatic contribution to persistence length. Electrostatic effects thus contribute 21.8% to DNA stiffness at 5 mM for fragments between 105- to 130-bp. In contrast, electrostatics are calculated to make a negligible contribution to the DNA persistence length at physiological monovalent cation concentration. The results are compared with counterion condensation models and experimental data.

Discovering ordered phases of block copolymers: new results from a generic Fourier-space approach.

A generic Fourier-space approach to solve the self-consistent field theory of block copolymers is developed. This approach is based on the fact that, for any computational box with periodic boundary conditions, all spatially varying functions are spanned by the Fourier series determined by the size and shape of the box. The method reproduces all known diblock copolymer phases. The application of this method to a model "frustrated" triblock copolymer leads to a phase diagram with a number of new phases. Furthermore, the capability of the method to reproduce experimentally observed structures is demonstrated using the knitting pattern of triblock copolymers.