Intuitive, But Not Simple: Including Explicit Water Molecules in Protein-Protein Docking Simulations Improves Model Quality

Characterizing the nature of interaction between proteins that have not been experimentally co-crystallized requires a computational docking approach that can successfully predict the spatial conformation adopted in the complex. In this work, the Hydropathic INTeractions (HINT) force field model was used for scoring docked models in a data set of 30 high-resolution crystallographically characterized “dry” protein-protein complexes, and was shown to reliably identify native-like models. However, most current protein-protein docking algorithms fail to explicitly account for water molecules involved in bridging interactions that mediate and stabilize the association of the protein partners, so we used HINT to illuminate the physical and chemical properties of bridging waters and account for their energetic stabilizing contributions. The HINT water Relevance metric identified the ‘truly’ bridging waters at the 30 protein-protein interfaces and we utilized them in “solvated” docking by manually inserting them into the input files for the rigid body ZDOCK program. By accounting for these interfacial waters, a statistically significant improvement of ~24% in the average hit-count within the top-10 predictions the protein-protein dataset was seen, compared to standard “dry” docking. The results also show scoring improvement, with medium and high accuracy models ranking much better than incorrect ones. These improvements can be attributed to the physical presence of water molecules that alter surface properties and better represent native shape and hydropathic complementarity between interacting partners, with concomitantly more accurate native-like structure predictions

A Hydrophobic Gate in an Ion Channel: The Closed State of the Nicotinic Acetylcholine Receptor

The nicotinic acetylcholine receptor (nAChR) is the prototypic member of the `Cys-loop' superfamily of ligand-gated ion channels which mediate synaptic neurotransmission, and whose other members include receptors for glycine, gamma-aminobutyric acid, and serotonin. Cryo-electron microscopy has yielded a three dimensional structure of the nAChR in its closed state. However, the exact nature and location of the channel gate remains uncertain. Although the transmembrane pore is constricted close to its center, it is not completely occluded. Rather, the pore has a central hydrophobic zone of radius about 3 A. Model calculations suggest that such a constriction may form a hydrophobic gate, preventing movement of ions through a channel. We present a detailed and quantitative simulation study of the hydrophobic gating model of the nicotinic receptor, in order to fully evaluate this hypothesis. We demonstrate that the hydrophobic constriction of the nAChR pore indeed forms a closed gate. Potential of mean force (PMF) calculations reveal that the constriction presents a barrier of height ca. 10 kT to the permeation of sodium ions, placing an upper bound on the closed channel conductance of 0.3 pS. Thus, a 3 A radius hydrophobic pore can form a functional barrier to the permeation of a 1 A radius Na+ ion. Using a united atom force field for the protein instead of an all atom one retains the qualitative features but results in differing conductances, showing that the PMF is sensitive to the detailed molecular interactions.Comment: Accepted by Physical Biology; includes a supplement and a supplementary mpeg movie can be found at http://sbcb.bioch.ox.ac.uk/oliver/download/Movies/watergate.mp

Understanding Molecular Interactions: Application of HINT-based Tools in the Structural Modeling of Novel Anticancer and Antiviral Targets, and in Protein-Protein Docking

Computationally driven drug design/discovery efforts generally rely on accurate assessment of the forces that guide the molecular recognition process. HINT (Hydropathic INTeraction) is a natural force field, derived from experimentally determined partition coefficients that quantifies all non-bonded interactions in the biological environment, including hydrogen bonding, electrostatic and hydrophobic interactions, and the energy of desolvation. The overall goal of this work is to apply the HINT-based atomic level description of molecular systems to biologically important proteins, to better understand their biochemistry – a key step in exploiting them for therapeutic purposes. This dissertation discusses the results of three diverse projects: i) structural modeling of human sphingosine kinase 2 (SphK2, a novel anticancer target) and binding mode determination of an isoform selective thiazolidine-2,4-dione (TZD) analog; ii) structural modeling of human cytomegalorvirus (HCMV) alkaline nuclease (AN) UL98 (a novel antiviral target) and subsequent virtual screening of its active site; and iii) explicit treatment of interfacial waters during protein-protein docking process using HINT-based computational tools. SphK2 is a key regulator of the sphingosine-rheostat, and its upregulation /overexpression has been associated with cancer development. We report structural modeling studies of a novel TZD-analog that selectively inhibits SphK2, in a HINT analysis that identifies the key structural features of ligand and protein binding site responsible for isoform selectivity. The second aim was to build a three-dimensional structure of a novel HCMV target – AN UL98, to identify its catalytically important residues. HINT analysis of the interaction of 5’ DNA end at its active site is reported. A parallel aim to perform in silico screening with a site-based pharmacophore model, identified several novel hits with potentially desirable chemical features for interaction with UL98 AN. The majority of current protein-protein docking algorithms fail to account for water molecules involved in bridging interactions between partners, mediating and stabilizing their association. HINT is capable of reproducing the physical and chemical properties of such waters, while accounting for their energetic stabilizing contributions. We have designed a solvated protein-protein docking protocol that explicitly models the Relevant bridging waters, and demonstrate that more accurate results are obtained when water is not ignored

Computational ligand design and analysis in protein complexes using inverse methods, combinatorial search, and accurate solvation modeling

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2006.Vita.Includes bibliographical references (p. 207-230).This thesis presents the development and application of several computational techniques to aid in the design and analysis of small molecules and peptides that bind to protein targets. First, an inverse small-molecule design algorithm is presented that can explore the space of ligands compatible with binding to a target protein using fast combinatorial search methods. The inverse design method was applied to design inhibitors of HIV-1 protease that should be less likely to induce resistance mutations because they fit inside a consensus substrate envelope. Fifteen designed inhibitors were chemically synthesized, and four of the tightest binding compounds to the wild-type protease exhibited broad specificity against a panel of drug resistance mutant proteases in experimental tests. Inverse protein design methods and charge optimization were also applied to improve the binding affinity of a substrate peptide for an inactivated mutant of HIV-1 protease, in an effort to learn more about the thermodynamics and mechanisms of peptide binding. A single mutant peptide calculated to have improved binding electrostatics exhibited greater than 10-fold improved affinity experimentally.(cont.) The second half of this thesis presents an accurate method for evaluating the electrostatic component of solvation and binding in molecular systems, based on curved boundary-element method solutions of the linearized Poisson-Boltzmann equation. Using the presented FFTSVD matrix compression algorithm and other techniques, a full linearized Poisson-Boltzmann equation solver is described that is capable of solving multi-region problems in molecular continuum electrostatics to high precision.Michael Darren Altman.Ph.D

Inhibitor Synthesis and Biophysical Characterization of Protein–Ligand–Solvent Interactions An Analysis of the Thermodynamics and Kinetics of Ligand Binding to Thermolysin

In the pre-clinical development stages of most drug design campaigns, the equilibrium binding affinity of a prospective lead candidate, in the form of an IC50, Kd or ΔG° value, is the most commonly employed benchmark parameter for its effectiveness as a putative drug. Hydrogen bonding, van der Waals and electrostatic interactions, as well as hydrophobic effects are among the most prominent factors that contribute to binding. In structure based design approaches, these interactions can routinely be linked to a structural motif of a drug molecule, which can greatly assist in the construction of compounds with a desired set of properties. Equilibrium binding affinity can also be expressed in terms of kinetics, were the steady-state constant Kd is defined as the ratio of the rate constants of dissociation (kd) and association (ka). The thermodynamic expression ΔG° can be subdivided into an enthalpic (ΔH°) and an entropic (–TΔS°) term. In either case, the molecular mechanisms that define the kinetics of binding or the compensation of enthalpic and entropic contributions are not fully understood. The goal of this dissertation is the in-depth investigation of the molecular processes that drive protein–ligand interactions. A special focus is set on the partitioning of thermodynamic and kinetic parameters into their respective microscopic elements. For this, the metalloprotease thermolysin (TLN) is used as a model system. This protein is well characterized and represents a robust system with excellent crystallographic properties and a thoroughly documented inhibitor class. The first publication (Chapter 2) presents an improved strategy for the synthesis and purification of phosphonamidate peptides that are known as potent inhibitors of TLN. Due to the inherent instability of the phosphorous–nitrogen bond, the introduction of polar functional groups into the inhibitor scaffold is quite challenging. Here, a synthetic strategy is presented that minimizes the amount of hydrolysis during peptide coupling, deprotection and purification through the use of an allyl-based protection system and a solid-phase extraction (SPE) protocol for the final purification step. This allows the synthesis of highly pure TLN inhibitors incorporating a variety of functional groups for use in biophysical experiments. In the second publication (Chapter 3), a strategy for the design of inhibitors is highlighted, which relies on the targeted design of water networks that are formed around a protein–ligand complex. Based on information from a previous study, the shape of a hydrophobic portion of a TLN ligand is altered in a way that allows a beneficial stabilization of water molecules in the first solvation layer of the complex. Supported by molecular dynamics simulations, a series of diastereomeric inhibitors is synthesized and the binding process is characterized by X-ray crystallography, isothermal titration calorimetry (ITC) and surface plasmon resonance spectroscopy (SPR). The optimization of the hydrophobic P2’ moiety results in a 50-fold affinity enhancement compared to the original methyl substituted ligand. This improvement is mainly driven by a favorable enthalpic term that originates from the stabilization of water polygons in the solvation shell. In the follow-up study in Chapter 4, the binding signature of a series of inhibitors that place a charged and polar moiety in the solvent exposed S2’ pocket of TLN is investigated. Here, a partially hydrated ammonium group is gradually retracted deeper into the hydrophobic protein environment. From the crystal structures it is evident that the polar ligands do not recruit an increased amount of water molecules into their solvation layer when compared to related analogues that feature a purely aliphatic residue at the solvent interface. The penalty for the partial desolvation of the charged functional group, in combination with the lack of a strongly ordered water network, results in a severe affinity decrease that is driven by an unfavorable enthalpic term. The deep, hydrophobic S1’ pocket of TLN determines the substrate specificity of the protease and is commonly addressed by high affinity inhibitors. Experimental evidence from previous studies suggests, however, that this apolar crevice is only poorly solvated in the absence of an interaction partner. With the study in Chapter 5, an attempt for the experimental analysis of the hydration state of the S1’ pocket is presented. For this, a special inhibitor is designed that transforms the protein pocket into a cavity, while simultaneously providing enough empty space for the accommodation of several water molecules. A detailed analysis of an experimentally phased electron density map reveals that the cavity remains completely unsolvated and thus, vacuous. As an intriguing prospect for the exploitation of such poorly hydrated protein pockets in drug design, the placement of an iso-pentyl moiety in the ligand’s P1’ position results in a dramatic, enthalpically driven gain in affinity by a factor of 41 000. With a detailed structural analysis of a series of chemically diverse TLN inhibitors, the kinetics of the protein–ligand binding process are investigated in Chapter 6. From the SPR derived kinetic information, it becomes apparent that the nature of the functional group in the P2’ position of a thermolysin inhibitor has a significant impact on its dissociation kinetics. This property can be linked to the interaction between the respective functionality of a ligand and Asn112, a residue that lines the active site of the protease and is commonly believed to align a substrate for proteolytic cleavage. This residue undergoes a significant conformational change when the protein transitions from its closed state to its open form, from which a ligand is released. Interference with this retrograde induced-fit mechanism through strong hydrogen-bonding interactions to an inhibitor results in a pronounced deceleration of the dissociation process. The case of the known inhibitor ZFPLA demonstrates that a further restriction of the rotation of Asn112 by a steric barrier in the P1 position of a ligand, can reduce the rate constant of dissociation by a factor of 74 000. Fragment-based lead discovery has become a popular method for the generation of prospective drug molecules. The weak affinity of fragments and the necessity for high concentrations, however, can result in false-positive signals from the initial binding assays that routinely plague fragment-based screening. The pursuit of such a “red herring” can lead to a significant loss of time and resources. In Chapter 7, a molecule that emerged as one of the most potent binders from an elaborate fragment screen against the aspartic protease endothiapepsin is identified as a false-positive. Detailed crystallographic, HPLC and MS experiments reveal that the affinity detected in multiple assays can in fact be attributed to another compound. This entity is formed from the initially employed molecule in a reaction cascade that results in a major rearrangement of its heterocyclic core structure. Supported by quantum chemical calculations and NMR experiments, a mechanism for the formation of the elusive compound is proposed and its binding mode analyzed by X-ray crystallography

Systematic Correlation of Structural, Thermodynamic and Residual Solvation Properties of Hydrophobic Substituents in Hydrophobic Pockets Using Thermolysin as a Case Study

Water molecules participate besides protein and ligand as an additional binding partner in every in vivo protein–ligand binding process. The displacement of water molecules from apolar surfaces of solutes is considered the driving force of the hydrophobic effect. It is generally assumed that the mobility of the water molecules increases through the displacement, and, as a consequence, entropy increases. This explanation, which is based on experiments with simple model systems, is, however, insufficient to describe the hydrophobic effect as part of the highly complex protein–ligand complex formation process. For instance, the displacement of water molecules from apolar surfaces that already exhibit an increased mobility before their displacement can result in an enthalpic advantage. Furthermore, it has to be considered that by the formation of the protein–ligand complex a new solvent-exposed surface is created, around which water molecules have to rearrange. The present thesis focuses on the impact of the latter effect on the thermodynamic and kinetic binding properties of a given ligand. A congeneric ligand series comprised of nine ligands binding to the model protein thermolysin (TLN) was analyzed to determine the impact of the rearrangement of water molecules around the surface of a newly formed protein–ligand complex on the thermodynamic binding properties of a ligand. The protein–ligand complexes were characterized structurally by X-ray crystallography and thermodynamically by isothermal titration calorimetry (ITC). The only structural difference between the ligands was their strictly apolar P2’ substituent, which changed in size from a methyl to a phenylethyl group. The P2’ group interacts with the flat, apolar, and well-solvated S2’ pocket of TLN. Depending on the bound ligand, the solvent-exposed surface of the protein–ligand complex changes. The ITC measurements revealed strong thermodynamic differences between the different ligands. The structural analysis showed ligand-coating water networks pronounced to varying degrees. A pronounced water network clearly correlated with a favorable enthalpic and less favorable entropic term, and overall resulted in an affinity gain. Based on these results, new P2’ substituents were rationally designed with the aim to achieve stronger stabilization of the adjacent water networks and thereby further increase ligand affinity. First, the quality of the putative water networks was validated using molecular dynamics (MD) simulations. Subsequently, the proposed ligands were synthesized, crystallized in complex with TLN, and analyzed thermodynamically. Additionally, a kinetic characterization using surface plasmon resonance (SPR) was performed. The crystallographically determined water networks adjacent to the P2’ substituents were in line with their predictions conducted by MD simulations. The ligands showed increasingly pronounced water networks as well as a slight enthalpy-drive affinity increase compared to the ligands from the initial study. The ligand with the highest affinity showed an almost perfect water network as well as a significantly reduced dissociation constant. To analyze the influence of the ligand-coating water networks on the kinetic binding properties of a ligand, seventeen congeneric TLN ligands exhibiting different P2’ groups were kinetically (by SPR) and crystallographically characterized. The different degree of the water network stabilization showed only a minor influence on the binding kinetic properties. By contrast, the strength of the interaction between the ligand and Asn112 proved crucial for the magnitude of the dissociation rate constant. A strong interaction resulted in a considerably prolonged residence time of the ligand by hindering TLN to undergo a conformational transition that is necessary for ligand release. In the last study, the reason for the exceptionally high affinity gain for addressing the deep, apolar S1’ pocket of TLN with apolar ligand portions was investigated. Therefore, a congeneric TLN ligand series substituted with differently large apolar P1’ substituents (ranging from a single hydrogen atom to an iso-butyl group) was analyzed. The exchange of the hydrogen atom at the P1’ position with a single methyl group already results in a 100-fold affinity increase of the ligand. To elucidate the molecular mechanism behind this considerable affinity gain, the solvation state of the S1’ pocket was carefully analyzed. The results strongly indicate that the S1’ pocket is completely free of the presence of any water molecules. Thus, the huge affinity gain was attributed to the absence of an energetically costly desolvation step. The data presented in this thesis show that to describe the thermodynamic signature of the hydrophobic effect it is necessary to explicitly consider the change of the thermodynamic properties of every involved water molecule. Solely considering the buried apolar surface area and assigning an entropic term to it is not sufficient. The increasing stabilization of the water network adjacent to the protein-bound ligand represents a promising approach — quite independent of specific properties of the target protein — to optimize the thermodynamic profile of a given ligand. This approach also allows fine-tuning of the kinetic binding parameters

T-cell epitope prediction and immune complex simulation using molecular dynamics: state of the art and persisting challenges

Atomistic Molecular Dynamics provides powerful and flexible tools for the prediction and analysis of molecular and macromolecular systems. Specifically, it provides a means by which we can measure theoretically that which cannot be measured experimentally: the dynamic time-evolution of complex systems comprising atoms and molecules. It is particularly suitable for the simulation and analysis of the otherwise inaccessible details of MHC-peptide interaction and, on a larger scale, the simulation of the immune synapse. Progress has been relatively tentative yet the emergence of truly high-performance computing and the development of coarse-grained simulation now offers us the hope of accurately predicting thermodynamic parameters and of simulating not merely a handful of proteins but larger, longer simulations comprising thousands of protein molecules and the cellular scale structures they form. We exemplify this within the context of immunoinformatics

In silico tumor-targeting technologies for the evasion of acidity-induced multidrug resistance

The physiology of tumors is tied to MDR mechanisms that hamper chemotherapeutic effects, particularly passive membrane crossing compounds, like hydrophobic Lewis base drugs. Although the lysosomal entrapment phenomena remains to be fully understood, this pH-dependent MDR mechanism induces drug sequestration in the acidic lysosomal lumen. Overcoming the MDR requires multi-pronged therapies, which often overlook an ubiquitous tumor trait: the extracellular acidity of the tumor microenvironment (TME). To address this, pHLIP peptides have emerged as an acidity-selective technology for tumor-targeting drug delivery. We focused on refining our protocols with enhanced sampling techniques and tumor-like features to improve the predictive abilities of the CpHMD-L methodology and augment the realism of these biomolecular models, thus bridging the gap to in vivo and cellular conditions. The optimized protocol coupled the CpHMD-L method with a pHRE scheme, providing a robust baseline. Then, we applied the protocol to study the diverging therapeutic efficiency of the wt and an over-performing Var3 peptide. A novel implementation of a pH gradient CpHMD-L method successfully reproduced experimental performances, thus elucidating pivotal residues electrostatic networks that dictate peptides thermodynamic stability in TME conditions. A multi-peptide study highlighted the remarkable effects of permuting arginines in modulating the local vicinity of key aspartates. These findings heavily correlate with their tumor-targeting performance, supporting more rational and in silico-based approaches to peptide design. Finally, the pH-dependent mechanism of lysosomal entrapment was modelled, hinting at the important role of acidity in Lewis base drugs membrane intercalation. Additional pH-dependent permeability calculations, using a novel US-CpHMD method, identified the TME acidity as an additional MDR defense mechanism that impairs clinical efficiency. It also revealed an intrinsic flaw of these compounds, since they preferably target healthy cells. These findings have important implications in rational drug design, especially of conjugated therapies with pHLIP-like drug delivery systems to overcome these challenges

Biophysical studies of protein-ligand interactions and the discovery of FKBP12 inhibitors

The principal aim of this study was to discover, through virtual screening, new nonimmunosuppressive inhibitors for the human immunophilin FKBP12, a target of the immunosuppressant drugs rapamycin and FK506. The enzyme acts as peptidyl-prolyl isomerase catalysing protein folding in the cell. Structurally similar isomerase domains are important for molecular recognition in multi-domain chaperone proteins. FKBP inhibitors have been shown to have protective effects against nerve damage and are therefore interesting targets for the treatment of neurodegenerative diseases. Virtual screening has been used to discover novel inhibitors for protein drug targets. Recent advances in computational power and the availability of large virtual libraries, such as the EDULISS database at Edinburgh University, have enhanced the appeal of this approach. X-ray structures of known protein-ligand complexes were examined to obtain an understanding of the key non-covalent interactions in the FKBP12 binding pocket. Virtual screening hits were selected using macromolecular docking and programs that employed a ligand-based approach. The bulk of the virtual screening in this study used Edinburgh University’s in-house program LIDAEUS. In the course of this study nearly three hundred compounds were screened in the laboratory using biophysical and biochemical binding assays. Thirty four compounds were found to have an affinity for FKBP12 of less than one hundred micromolar. To test virtual hits, it was necessary to select the most appropriate medium-throughput biophysical assay. The aim was to employ methods with sufficient sensitivity to detect compounds with affinity in the order of one hundred micromolar, coupled with the capacity to screen hundreds of compounds in a week. This study used a wide variety of biophysical techniques, these including: electrospray ionisation mass spectrometry, surface plasmon resonance and isothermal titration calorimetry. There was a particular emphasis on the quality of data from electrospray ionisation mass spectrometry. A correlation was found between the cone voltages that gave 50 % dissociation of the complex with the enthalpic contribution to the free energy of binding. From the careful examination of the differences in charge-state distributions between a pure protein and a protein-ligand mixture, it was possible to determine if a protein-ligand complex had been present in solution prior to dissociation during the electrospray process. This observation provides the basis for an assay that could be of general utility in detecting very weak inhibitors