Temperature artifacts in protein structures bias ligand-binding predictions

X-ray crystallography is the gold standard to resolve conformational ensembles that are significant for protein function, ligand discovery, and computational methods development. However, relevant conformational states may be missed at common cryogenic (cryo) data-collection temperatures but can be populated at room temperature. To assess the impact of temperature on making structural and computational discoveries, we systematically investigated protein conformational changes in response to temperature and ligand binding in a structural and computational workhorse, the T4 lysozyme L99A cavity. Despite decades of work on this protein, shifting to RT reveals new global and local structural changes. These include uncovering an apo helix conformation that is hidden at cryo but relevant for ligand binding, and altered side chain and ligand conformations. To evaluate the impact of temperature-induced protein and ligand changes on the utility of structural information in computation, we evaluated how temperature can mislead computational methods that employ cryo structures for validation. We find that when comparing simulated structures just to experimental cryo structures, hidden successes and failures often go unnoticed. When using structural information in ligand binding predictions, both coarse docking and rigorous binding free energy calculations are influenced by temperature effects. The trend that cryo artifacts limit the utility of structures for computation holds across five distinct protein classes. Our results suggest caution when consulting cryogenic structural data alone, as temperature artifacts can conceal errors and prevent successful computational predictions, which can mislead the development and application of computational methods in discovering bioactive molecules.

Temperature artifacts in protein structures impact the utility of structural information in computation by misleading validation and application of computational methods in discovering bioactive molecules.     

Relationship among ligand conformations in solution, in the solid state, and at the Hsp90 binding site: geldanamycin and radicicol

The unknown effects of a receptor's environment on a ligand's conformation presents a difficult challenge in predicting feasible bioactive conformations, particularly if the receptor is ill-defined. The primary hypothesis of this work is that a structure's conformational ensemble in solution presents viable candidates for protein binding. The experimental solution profile can be achieved with the NAMFIS (NMR analysis of molecular flexibility in solution) method, which deconvolutes the average NMR spectrum of small flexible molecules into individual contributing conformations with varying populations. Geldanamycin and radicicol are structurally different macrocycles determined by X-ray crystallography to bind to a common site on the cellular chaperone heat shock protein 90 (Hsp90). Without benefit of a receptor structure, NAMFIS has identified the bioactive conformers of geldanamycin and radicicol in CDCl3 solution with populations of 4% and 21%, respectively. Conversely, docking the set of NAMFIS conformers into the unliganded proteins with GLIDE followed by MM-GBSA scoring reproduces the experimental crystallographic binding poses.     

Short and diverse route toward complex natural product-like macrocycles

[reaction: see text] A general strategy toward macrocyclic compounds using multicomponent reaction (MCR) chemistry, e.g., Passerini and Ugi variants, and ring-closing metathesis (RCM) is introduced. The corresponding bifunctional isocyanides carboxylic acids bearing a terminal olefin are easy to prepare from the corresponding commercially available starting materials. Advantageously, this strategy allows fast access to a diverse conformational space of natural product-like macrocycles and could thus be of interest in the discovery of novel bioactive agents.     

A robust force field based method for calculating conformational energies of charged drug-like molecules

The binding affinity of a drug-like molecule depends among other things on the availability of the bioactive conformation. If the bioactive conformation has a significantly higher energy than the global minimum energy conformation, then the molecule is unlikely to bind to its target. Determination of the global minimum energy conformation and calculation of conformational penalties of binding is a prerequisite for prediction of reliable binding affinities. Here, we present a simple and computationally efficient procedure to estimate the global energy minimum for a wide variety of structurally diverse molecules, including polar and charged compounds. Identifying global energy minimum conformations of such compounds with force field methods is problematic due to the exaggeration of intramolecular electrostatic interactions. We demonstrate that the global energy minimum conformations of zwitterionic compounds generated by conformational analysis with modified electrostatics are good approximations of the conformational distributions predicted by experimental data and with molecular dynamics performed in explicit solvent. Finally the method is used to calculate conformational penalties for zwitterionic GluA2 agonists and to filter false positives from a docking study.     

Flexible ligand docking without parameter adjustment across four ligand–receptor complexes

Understanding molecular recognition is one of the fundamental problems in molecular biology. Computationally, molecular recognition is formulated as a docking problem. Ideally, a molecular docking algorithm should be computationally efficient, provide reasonably thorough search of conformational space, obtain solutions with reasonable consistency, and not require parameter adjustments. With these goals in mind, we developed DIVALI (Docking wIth eVolutionary AlgorIthms), a program which efficiently and reliably searches for the possible binding modes of a ligand within a fixed receptor. We use an AMBER-type potential function and search for good ligand conformations using a genetic algorithm (GA). We apply our system to study the docking of both rigid and flexible ligands in four different complexes. Our results indicate that it is possible to find diverse binding modes, including structures like the crystal structure, all with comparable potential function values. To achieve this, certain modifications to the standard GA recipe are essential. © 1995 John Wiley & Sons, Inc.     

Macrocycles All Aflutter: Substitution at an Allylic Center Reveals the Conformational Dynamics of [13]‐Macrodilactones

The shapes adopted by large‐ring macrocyclic compounds play a role in their reactivity and their ability to be bound by biomolecules. We investigated the synthesis, conformational analysis, and properties of a specific family of [13]‐macrodilactones as models of natural‐product macrocycles. The features of our macrodilactones enabled us to study the relationship between stereogenic centers and planar chirality through the modular synthesis of new members of this family of macrocycles. Here we report on insights gained from a new [13]‐macrodilactone that is substituted at a position adjacent to the alkene in the molecule. Analysis of the compound, in comparison to an α‐substituted regioisomer, by using X‐ray crystallography, NMR coupling constants, and reaction‐product characterization in concert with computational chemistry, revealed that the alkene unit is dynamic. That is, the data support a model in which the alkene in our [13]‐macrodilactones oscillates between two conformations. A difference in reactivity of one conformation compared to the other leads to manifestation of this dynamic behavior. The results underscore the local conformational dynamics observed in some natural‐product macrocycles, which could have implications for biomolecule binding.     

Conformational and docking studies of acyl homoserine lactones as a robust method to investigate bioactive conformations

A method aiming at investigating possible bioactive conformations of acyl homoserine lactone (AHL) quorum sensing (QS) modulators is established. The method relies on the exhaustive conformational analysis of AHLs by varying torsion angles around the amide group then on the selection of the closest conformation to those known from co-crystallized XRD data of AHL-receptor complexes. These latter are then docked as rigid ligand within the receptor binding site, leading to interactions with binding site residues which are highly consistent as compared with the data arising from XRD studies. The method is first validated using AHLs for which XRD data of their complexes with their cognate receptor are available, then extended to examples for which the binding mode is still unknown.Three compounds were used to validate the method: hexanoyl homoserine lactone (HHL) as an example of autoinducer, 3-oxo-butanoyl homoserine lactone (OBHL), as a representative model of 3-oxo-AHLs, and 4-(4-chlorophenoxy)butanoyl homoserine lactone (CPOBHL) as an example of a QS inhibitor. The conformational analysis of these three compounds to their cognate protein (TraR, SdiA, LasR and CviR) provides the data which enable the next rigid docking step. Further rigid docking of the closest conformations compared to the known bioactive ones within the binding sites allows to recover the expected binding mode with high precision (atomic RMSD < 2 Å). This “conformational analysis/torsion angle filter/rigid ligand docking” method was then used for investigating three non-natural AHL-type QS inhibitors without known co-crystallized XRD structures, namely was 2-hexenoyl homoserine lactone (HenHL), 3-oxo-4-phenylbutanoyl homoserine lactone (OPBHL) and 3-(4-bromophenyl)propanoyl homoserine lactone (BPPHL). Results provide insights into their possible binding mode by identifying specific interactions with some key residues within the receptor binding site, allowing discussion of their biological activity.     

Flexible protein‐protein docking based on Best‐First search algorithm

We developed a new high resolution protein‐protein docking method based on Best‐First search algorithm that loosely imitates protein‐protein associations. The method operates in two stages: first, we perform a rigid search on the unbound proteins. Second, we search alternately on rigid and flexible degrees of freedom starting from multiple configurations from the rigid search. Both stages use heuristics added to the energy function, which causes the proteins to rapidly approach each other and remain adjacent, while optimizing on the energy. The method deals with backbone flexibility explicitly by searching over ensembles of conformations generated before docking. We ran the rigid docking stage on 66 complexes and grouped the results into four classes according to evaluation criteria used in Critical Assessment of Predicted Interactions (CAPRI; “high,” “medium,” “acceptable,” and “incorrect”). Our method found medium binding conformations for 26% of the complexes and acceptable for additional 44% among the top 10 configurations. Considering all the configurations, we found medium binding conformations for 55% of the complexes and acceptable for additional 39% of the complexes. Introducing side‐chains flexibility in the second stage improves the best found binding conformation but harms the ranking. However, introducing side‐chains and backbone flexibility improve both the best found binding conformation and the best found conformation in the top 10. Our approach is a basis for incorporating multiple flexible motions into protein‐protein docking and is of interest even with the current use of a simple energy function. © 2010 Wiley Periodicals, Inc. J Comput Chem, 2010     

Large scale free energy calculations for blind predictions of protein–ligand binding: the D3R Grand Challenge 2015

We describe binding free energy calculations in the D3R Grand Challenge 2015 for blind prediction of the binding affinities of 180 ligands to Hsp90. The present D3R challenge was built around experimental datasets involving Heat shock protein (Hsp) 90, an ATP-dependent molecular chaperone which is an important anticancer drug target. The Hsp90 ATP binding site is known to be a challenging target for accurate calculations of ligand binding affinities because of the ligand-dependent conformational changes in the binding site, the presence of ordered waters and the broad chemical diversity of ligands that can bind at this site. Our primary focus here is to distinguish binders from nonbinders. Large scale absolute binding free energy calculations that cover over 3000 protein–ligand complexes were performed using the BEDAM method starting from docked structures generated by Glide docking. Although the ligand dataset in this study resembles an intermediate to late stage lead optimization project while the BEDAM method is mainly developed for early stage virtual screening of hit molecules, the BEDAM binding free energy scoring has resulted in a moderate enrichment of ligand screening against this challenging drug target. Results show that, using a statistical mechanics based free energy method like BEDAM starting from docked poses offers better enrichment than classical docking scoring functions and rescoring methods like Prime MM-GBSA for the Hsp90 data set in this blind challenge. Importantly, among the three methods tested here, only the mean value of the BEDAM binding free energy scores is able to separate the large group of binders from the small group of nonbinders with a gap of 2.4 kcal/mol. None of the three methods that we have tested provided accurate ranking of the affinities of the 147 active compounds. We discuss the possible sources of errors in the binding free energy calculations. The study suggests that BEDAM can be used strategically to discriminate binders from nonbinders in virtual screening and to more accurately predict the ligand binding modes prior to the more computationally expensive FEP calculations of binding affinity.     

Triptycene-derived calixarenes,heterocalixarenes and analogues

With unique three-dimensional triptycene derivatives as the building blocks, several kinds of novel macrocyclic compounds including triptycene-derived calixarenes, heterocalixarenes, N(H)-bridged azacalixarenes, homooxacalixarene analogues, and tetralactam macrocycles could be conveniently synthesized with satisfactory yields by one-pot method or two-step fragment coupling reactions. With rigid triptycene moiety, these macrocyclic hosts not only have large enough cavities, but also show specific fixed conformations. These structural features made them exhibit well molecular recognition toward small organic molecules, fullerenes and organic dyes. Moreover, these macrocycles could also show interest self-assembly abilities in both solution and solid state, which will make them be broad application prospects.     

Exploring ensembles of bioactive or virtual analogs of X-ray ligands for shape similarity searching

Shape similarity searching is a popular approach for ligand-based virtual screening on the basis of three-dimensional reference compounds. It is generally thought that well-defined experimentally determined binding modes of active reference compounds provide the best possible basis for shape searching. Herein, we show that experimental binding modes are not essential for successful shape similarity searching. Furthermore, we show that ensembles of analogs of X-ray ligands—in the absence of these ligands—further improve the search performance of single crystallographic reference compounds. This is even the case if ensembles of virtually generated analogs are used whose activity status is unknown. Taken together, the results of our study indicate that analog ensembles representing fuzzy reference states are effective starting points for shape similarity searching.     

New Chiral Macrocyclic Compounds from D-Mannitol

We have recently reported the synthesis of new chiral macrocyclic polyhydroxyethers by reduction of cyclodextrins 1. These compounds display appreciable conformational freedom in solution as it occurs with the ionophores. Our chiral macrocycles may be considered as built by units of alditol (1 → 4) alditols. Such units, conveniently substituted, prepared by us by reduction of disaccharide derivatives2, are possible synthons for the synthesis of other macrocyclic polyhydroxyethers in which the nature and number of alditol (1 → n) alditol components can be varied at will. We are interested in the preparation and the structural studies of these type of receptors since the synthesis of new chiral macrocycles is a topic of interest and the building of chiral cavities may be of importance in the study of host-guest interactions. We now report on the preparation, from D-mannitol, a readily available starting material with C₂symmetry, and tetraethylene glycol, of the chiral macrocycles 1, 2, and 3, as model compounds in exploring the synthesis of more complex macrocyclic polyhydroxyethers derived from alditol (1 → n) alditol. Other macrocyclic compounds from D mannitol have been previously synthesised.     

The unique properties observed for the unsymmetrical macrocyclic compounds with the highly distorted structure

A new class of aza-macrocycles with the highly distorted structure was found to exhibit unique properties. These macrocycles react with various lithium salts to form lithium complexes and their lithium complexation reactions depend on a substituent on the macrocyclic ring; slower rates and larger equilibrium constants were observed for the macrocycle with a bulkier substituent. The irradiation of these macrocycles by UV light was found to lead to the isomerization, and the photoisomerization rate of macrocycle with the bulky substituent was much faster. The highly distorted structure of these macrocycles makes it much easier to change the conformation of macrocyclic skeleton and these macrocycles have a variety of conformations. The factors to govern this conformational change were therefore explored. The solvent effect was examined by ¹H NMR spectroscopy, because these macrocycles have a strong intramolecular hydrogen bond in the ring. As a result, the solvent was found to have a big effect on the ¹H NMR spectra of macrocycles that could be explained in terms of the conformational change of macrocycle. This finding suggests the solvent to be an important way of controlling the conformation.     

Conformational selection of protein kinase A revealed by flexible-ligand flexible-protein docking

Protein kinases have high structural plasticity: their structure can change significantly, depending on what ligands are bound to them. Rigid-protein docking methods are not capable of describing such effects. Here, we present a new flexible-ligand flexible-protein docking model in which the protein can adopt conformations between two extremes observed experimentally. The model utilized a molecular dynamics-based simulated annealing cycling protocol and a distance-dependent dielectric model to perform docking. By testing this model on docking four diverse ligands to protein kinase A, we found that the ligands were able to dock successfully to the protein with the proper conformations of the protein induced. By imposing relatively soft conformational restraints to the protein during docking, this model reduced computational costs yet permitted essential conformational changes that were essential for these inhibitors to dock properly to the protein. For example, without adequate movement of the glycine-rich loop, it was difficult for the ligands to move from the surface of the protein to the binding site. In addition, these simulations called for better ways to compare simulation results with experiment other than using the popular root-mean-square deviation between the structure of a ligand in a docking pose and that in experiment because the structure of the protein also changed. In this work, we also calculated the correlation coefficient between protein-ligand/protein-protein distances in the docking structure and those in the crystal structure to check how well a ligand docked into the binding site of the protein and whether the proper conformation of the protein was induced.     

Naringenin and quercetin – potential anti-HCV agents for NS2 protease targets

Nonstructural proteins of hepatitis C virus had drawn much attention for the scientific fraternity in drug discovery due to its important role in the disease. 3D structure of the protein was predicted using molecular modelling protocol. Docking studies of 10 medicinal plant compounds and three drugs available in the market (control) with NS2 protease were employed by using rigid docking approach of AutoDock 4.2. Among the molecules tested for docking study, naringenin and quercetin revealed minimum binding energy of ? 7.97 and ? 7.95 kcal/mol with NS2 protease. All the ligands were docked deeply within the binding pocket region of the protein. The docking study results showed that these compounds are potential inhibitors of the target; and also all these docked compounds have good inhibition constant, vdW+Hbond+desolv energy with best RMSD value.     

Novel inhibitor discovery through virtual screening against multiple protein conformations generated via ligand-directed modeling: a maternal embryonic leucine zipper kinase example

Kinase targets have been demonstrated to undergo major conformational reorganization upon ligand binding. Such protein conformational plasticity remains a significant challenge in structure-based virtual screening methodology and may be approximated by screening against an ensemble of diverse protein conformations. Maternal embryonic leucine zipper kinase (MELK), a member of serine-threonine kinase family, has been recently found to be involved in the tumerogenic state of glioblastoma, breast, ovarian, and colon cancers. We therefore modeled several conformers of MELK utilizing the available chemogenomic and crystallographic data of homologous kinases. We carried out docking pose prediction and virtual screening enrichment studies with these conformers. The performances of the ensembles were evaluated by their ability to reproduce known inhibitor bioactive conformations and to efficiently recover known active compounds early in the virtual screen when seeded with decoy sets. A few of the individual MELK conformers performed satisfactorily in reproducing the native protein-ligand pharmacophoric interactions up to 50% of the cases. By selecting an ensemble of a few representative conformational states, most of the known inhibitor binding poses could be rationalized. For example, a four conformer ensemble is able to recover 95% of the studied actives, especially with imperfect scoring function(s). The virtual screening enrichment varied considerably among different MELK conformers. Enrichment appears to improve by selection of a proper protein conformation. For example, several holo and unliganded active conformations are better to accommodate diverse chemotypes than ATP-bound conformer. These results prove that using an ensemble of diverse conformations could give a better performance. Applying this approach, we were able to screen a commercially available library of half a million compounds against three conformers to discover three novel inhibitors of MELK, one from each template. Among the three compounds validated via experimental enzyme inhibition assays, one is relatively potent (15; K(d) = 0.37 μM), one moderately active (12; K(d) = 3.2 μM), and one weak but very selective (9; K(d) = 18 μM). These novel hits may be utilized to assist in the development of small molecule therapeutic agents useful in diseases caused by deregulated MELK, and perhaps more importantly, the approach demonstrates the advantages of choosing an appropriate ensemble of a few conformers in pursuing compound potency, selectivity, and novel chemotypes over using single target conformation for structure-based drug design in general.     

Conformational energy penalties of protein-bound ligands

The conformational energies required for ligands to adopt their bioactive conformations were calculated for 33 ligand–protein complexes including 28 different ligands. In order to monitor the force field dependence of the results, two force fields, MM3 and AMBER, were employed for the calculations. Conformational analyses were performed in vacuo and in aqueous solution by using the generalized Born/solvent accessible surface (GB/SA) solvation model. The protein-bound conformations were relaxed by using flat-bottomed Cartesian constraints. For about 70% of the ligand–protein complexes studied, the conformational energies of the bioactive conformations were calculated to be 3 kcal/mol. It is demonstrated that the aqueous conformational ensemble for the unbound ligand must be used as a reference state in this type of calculations. The calculations for the ligand–protein complexes with conformational energy penalties of the ligand calculated to be larger than 3 kcal/mol suffer from uncertainties in the interpretation of the experimental data or limitations of the computational methods. For example, in the case of long-chain flexible ligands (e.g. fatty acids), it is demonstrated that several conformations may be found which are very similar to the conformation determined by X-ray crystallography and which display significantly lower conformational energy penalties for binding than obtained by using the experimental conformation. For strongly polar molecules, e.g. amino acids, the results indicate that further developments of the force fields and of the dielectric continuum solvation model are required for reliable calculations on the conformational properties of this type of compounds.