Enhancing Potency and Selectivity of a DC-SIGN Glycomimetic Ligand by Fragment-Based Design: Structural Basis

Chemical modification of pseudo-dimannoside ligands guided by fragment-based design allowed for the exploitation of an ammonium-binding region in the vicinity of the mannose-binding site of DC-SIGN, leading to the synthesis of a glycomimetic antagonist (compound 16 ) of unprecedented affinity and selectivity against the related lectin langerin. Here, the computational design of pseudo-dimannoside derivatives as DC-SIGN ligands, their synthesis, their evaluation as DC-SIGN selective antagonists, the biophysical characterization of the DC-SIGN/ 16 complex, and the structural basis for the ligand activity are presented. On the way to the characterization of this ligand, an unusual bridging interaction within the crystals shed light on the plasticity and potential secondary binding sites within the DC-SIGN carbohydrate recognition domain.     

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.     

Tetra- and Hexavalent Siglec-8 Ligands Modulate Immune Cell Activation

Carbohydrate-binding proteins are generally characterized by poor affinities for their natural glycan ligands, predominantly due to the shallow and solvent-exposed binding sites. To overcome this drawback, nature has exploited multivalency to strengthen the binding by establishing multiple interactions simultaneously. The development of oligovalent structures frequently proved to be successful, not only for proteins with multiple binding sites, but also for proteins that possess a single recognition domain. Herein we present the syntheses of a number of oligovalent ligands for Siglec-8, a monomeric I-type lectin found on eosinophils and mast cells, alongside the thermodynamic characterization of their binding. While the enthalpic contribution of each binding epitope was within a narrow range to that of the monomeric ligand, the entropy penalty increased steadily with growing valency. Additionally, we observed a successful agonistic binding of the tetra- and hexavalent and, to an even larger extent, multivalent ligands to Siglec-8 on immune cells and modulation of immune cell activation. Thus, triggering a biological effect is not restricted to multivalent ligands but could be induced by low oligovalent ligands as well, whereas a monovalent ligand, despite binding with similar affinity, showed an antagonistic effect.     

Design,Synthesis, and Biomedical Applications of Glycotripods for Targeting Trimeric Lectins

In the last decades, various efforts have been made to synthesize optimal glycotripods for targeting trimeric glycoproteins like asialoglycoprotein receptor, hemagglutinin, and langerin. All these trimeric glycoproteins have sugar binding pockets which are highly selective for a particular carbohydrate ligand. Optimized glycotripods are high affinity binders and have been used for delivering drugs or even applied as drug candidates. The selection of the tripodal base scaffold together with the length and flexibility of the linker between the scaffold and sugar residue, as important design parameters are discussed in this review.     

Quantifying Protein-Ligand Binding Constants using Electrospray Ionization Mass Spectrometry: A Systematic Binding Affinity Study of a Series of Hydrophobically Modified Trypsin Inhibitors

NanoESI-MS is used for determining binding strengths of trypsin in complex with two different series of five congeneric inhibitors, whose binding affinity in solution depends on the size of the P3 substituent. The ligands of the first series contain a 4-amidinobenzylamide as P1 residue, and form a tight complex with trypsin. The inhibitors of the second series have a 2-aminomethyl-5-chloro-benzylamide as P1 group, and represent a model system for weak binders. The five different inhibitors of each group are based on the same scaffold and differ only in the length of the hydrophobic side chain of their P3 residue, which modulates the interactions in the S3/4 binding pocket of trypsin. The dissociation constants (K_D) for high affinity ligands investigated by nanoESI-MS ranges from 15?nM to 450?nM and decreases with larger hydrophobic P3 side chains. Collision-induced dissociation (CID) experiments of five trypsin and benzamidine-based complexes show a correlation between trends in K_D and gas-phase stability. For the second inhibitor series we could show that the effect of imidazole, a small stabilizing additive, can avoid the dissociation of the complex ions and as a result increases the relative abundance of weakly bound complexes. Here the K_D values ranging from 2.9 to 17.6???M, some 1?C2 orders of magnitude lower than the first series. For both ligand series, the dissociation constants (K_D) measured via nanoESI-MS were compared with kinetic inhibition constants (K_i) in solution.     

Quantifying labile protein—Ligand interactions using electrospray ionization mass spectrometry

A new electrospray ionization mass spectrometry (ES-MS) approach for quantifying protein—ligand complexes that are prone to in-source (gas-phase) dissociation is described. The method, referred to here as the reference ligand ES-MS method, is based on the direct ES-MS assay and competitive ligand binding. A reference ligand (L_ref), which binds specifically to the protein (P), at the same binding site as the ligand (L) of interest, with known affinity and forms a stable protein—ligand complex in the gas phase, is added to the solution. The fraction of P bound to L_ref, which is determined directly from the ES mass spectrum, is sensitive to the fraction of P bound to L in solution and enables the affinity of P for L to be determined. A mathematical framework for the implementation of the method in cases where P has one or two specific ligand binding sites is given. Affinities of two carbohydrate-binding proteins, a single chain fragment of a monoclonal antibody and the lectin concanavalin A, for monosaccharide ligands are reported and the results are shown to agree with values obtained using isothermal titration calorimetry.     

Validation of an empirical RNA-ligand scoring function for fast flexible docking using RiboDock®

We report the design and validation of a fast empirical function for scoring RNA-ligand interactions, and describe its implementation within RiboDock, a virtual screening system for automated flexible docking. Building on well-known protein-ligand scoring function foundations, features were added to describe the interactions of common RNA-binding functional groups that were not handled adequately by conventional terms, to disfavour non-complementary polar contacts, and to control non-specific charged interactions. The results of validation experiments against known structures of RNA-ligand complexes compare favourably with previously reported methods. Binding modes were well predicted in most cases and good discrimination was achieved between native and non-native ligands for each binding site, and between native and non-native binding sites for each ligand. Further evidence of the ability of the method to identify true RNA binders is provided by compound selection ('enrichment factor') experiments based around a series of HIV-1 TAR RNA-binding ligands. Significant enrichment in true binders was achieved amongst high scoring docking hits, even when selection was from a library of structurally related, positively charged molecules. Coupled with a semi-automated cavity detection algorithm for identification of putative ligand binding sites, also described here, the method is suitable for the screening of very large databases of molecules against RNA and RNA-protein interfaces, such as those presented by the bacterial ribosome.     

Multiple ligand detection and affinity measurement by ultrafiltration and mass spectrometry analysis applied to fragment mixture screening

Binding affinity of a small molecule drug candidate to a therapeutically relevant biomolecular target is regarded the first determinant of the candidate's efficacy. Although the ultrafiltration-LC/MS (UF-LC/MS) assay enables efficient ligand discovery for a specific target from a mixed pool of compounds, most previous analysis allowed for relative affinity ranking of different ligands. Moreover, the reliability of affinity measurement for multiple ligands with UF-LC/MS has hardly been strictly evaluated. In this study, we examined the accuracy of K_d determination through UF-LC/MS by comparison with classical ITC measurement. A single-point K_d calculation method was found to be suitable for affinity measurement of multiple ligands bound to the same target when binding competition is minimized. A second workflow based on analysis of the unbound fraction of compounds was then developed, which simplified sample preparation as well as warranted reliable ligand discovery. The new workflow implemented in a fragment mixture screen afforded rapid and sensitive detection of low-affinity ligands selectively bound to the RNA polymerase NS5B of hepatitis C virus. More importantly, ligand identification and affinity measurement for mixture-based fragment screens by UF-LC/MS were in good accordance with single ligand evaluation by conventional SPR analysis. This new approach is expected to become a valuable addition to the arsenal of high-throughput screening techniques for fragment-based drug discovery.     

Design of Ligand Binding to an Engineered Protein Cavity Using Virtual Screening and Thermal Up-shift Evaluation

Summary Proteins could be used to carry and deliver small compounds. As a tool for designing ligand binding sites in protein cores, a three-step virtual screening method is presented that has been optimised using existing data on T4 lysozyme complexes and tested in a newly engineered cavity in flavodoxin. The method can pinpoint, in large databases, ligands of specific protein cavities. In the first step, physico-chemical filters are used to screen the library and discard a majority of compounds. In the second step, a flexible, fast docking procedure is used to score and select a smaller number of compounds as potential binders. In the third step, a finer method is used to dock promising molecules of the hit list into the protein cavity, and an optimised free energy function allows discarding the few false positives by calculating the affinity of the modelled complexes. To demonstrate the portability of the method, several cavities have been designed and engineered in the flavodoxin from Anabaena PCC 7119, and the W66F/L44A double mutant has been selected as a suitable host protein. The NCI database has then been screened for potential binders, and the binding to the engineered cavity of five promising compounds and three tentative non-binders has been experimentally tested by thermal up-shift assays and spectroscopic titrations. The five tentative binders (some apolar and some polar), unlike the three tentative non-binders, are shown to bind to the host mutant and, importantly, not to bind to the wild type protein. The three-step virtual screening method developed can thus be used to identify ligands of buried protein cavities. We anticipate that the method could also be used, in a reverse manner, to identify natural or engineerable protein cavities for the hosting of ligands of interest.     

Design of new bidentate ligands constructed of two Hoechst 33258 units for discrimination of the length of two A3T3 binding motifs

[structure: see text] The aim of this study is to develop bidentate minor-groove binders that bind the double binding motifs cooperatively. The new bidentate ligands (1) have been designed by connecting two Hoechst 33258 units with a polyether linker for cooperative binding with two remote A3T3 sites of DNA. The linker is introduced to the benzimidazole ring so that it is located at the convex side of the Hoechst unit. DNA binding affinity of the ligands was evaluated by measuring surface plasmon resonance (SPR), circular dichroism, and fluorescence spectra. Interestingly, the bidentate ligands (1) did not show affinity to DNA1 with a single A3T3 motif but showed selective affinity to DNA2 with two A3T3 motifs. The Long Bis-H (1L) having a long polyether linker showed specific binding to DNA2(6) with two A3T3 motifs separated by six nonbinding base pairs. The Long Bis-H (1L) has also shown specific binding to the three-way junction DNA4 with two A3T3 motifs. This study has demonstrated that DNA with double binding motifs can be selectively recognized by the newly designed bidentate ligands.     

On the interaction of two different types of ligands binding to the same molecule part I: basics and the transfer of the decoupled sites representation to systems with n and one binding sites

The decoupled sites representation (DSR) for one type of ligand allows to regard complex overall titration curves as sum of classical Henderson-Hasselbalch (HH) titration curves. In this work we transfer this theoretical approach to molecules with different types of interacting ligands (e.g. protons and electrons), prove the existence of decoupled systems for n ₁ and one binding sites for two different ligands, and point out some difficulties and limits of this transfer. A major difference to the DSR for one type of ligand is the loss of uniqueness of the decoupled system. However, all decoupled systems share a unique set of microstate probabilities and each decoupled system corresponds to a certain permutation of these microstate probabilities. Moreover, we show that the titration curve of a certain binding site in the original system can be regarded as linear combination of the titration curves of the individual sites of the decoupled system if the weights of the linear combination are substituted by functions in the activity of the second ligand. In the underlying model with only pairwise interaction, an important observation of our theoretical investigation is the following: Even though the binding sites of ligand L ₁ may not interact directly, they can show secondary interaction due to the interaction with the second type of ligand. This means, if the activity of the second ligand is fixed and we regard the 1-dimensional titration curve of an individual binding site for ligand L ₁ depending on its activity, we may observe a strong deviation from the classical HH shape in spite of non-interacting sites for ligand L ₁.     

Structure activity relationship by NMR and by computer: a comparative study

There has recently been considerable interest in using NMR spectroscopy to identify ligand binding sites of macromolecules. In particular, a modular approach has been put forward by Fesik et al. (Shuker, S. B.; Hajduk, P. J.; Meadows, R. P.; Fesik, S. W. Science 1996, 274, 1531-1534) in which small ligands that bind to a particular target are identified in a first round of screening and subsequently linked together to form ligands of higher affinity. Similar strategies have also been proposed for in silico drug design, where the binding sites of small chemical groups are identified, and complete ligands are subsequently assembled from different groups that have favorable interactions with the macromolecular target. In this paper, we compare experimental and computational results on a selected target (FKBP12). The binding sites of three small ligands ((2S)1-acetylprolinemethylester, 1-formylpiperidine, 1-piperidinecarboxamide) in FKBP12 were identified independently by NMR and by computational methods. The subsequent comparison of the experimental and computational data showed that the computational method identified and ranked favorably ligand positions that satisfy the experimental NOE constraints.     

Affinity Selections of DNA-Encoded Chemical Libraries on Carbonic Anhydrase IX-Expressing Tumor Cells Reveal a Dependence on Ligand Valence

DNA-encoded chemical libraries are typically screened against purified protein targets. Recently, cell-based selections with encoded chemical libraries have been described, commonly revealing suboptimal performance due to insufficient recovery of binding molecules. We used carbonic anhydrase IX (CAIX)-expressing tumor cells as a model system to optimize selection procedures with code-specific quantitative polymerase chain reaction (qPCR) as selection readout. Salt concentration and performing PCR on cell suspension had the biggest impact on selection performance, leading to 15-fold enrichment factors for high-affinity monovalent CAIX binders (acetazolamide; K_D=8.7 nM). Surprisingly, the homobivalent display of acetazolamide at the extremities of both complementary DNA strands led to a substantial improvement of both ligand recovery and enrichment factors (above 100-fold). The optimized procedures were used for selections with a DNA-encoded chemical library comprising 1 million members against tumor cell lines expressing CAIX, leading to a preferential recovery of known and new ligands against this validated tumor-associated target. This work may facilitate future affinity selections on cells against target proteins which might be difficult to express otherwise.     

On the interaction of different types of ligands binding to the same molecule Part II: systems with n to 2 and n to 3 binding sites

In the first part of this work we formulated the decoupled sites representation for two different types of ligands and highlighted special properties of the case of n binding sites for ligand L ₁ and one binding site for ligand L ₂. Moreover, for this case, we identified the microstate constants as unique components all decoupled molecules share. In the second part on hand, we investigate the cases with (n, 2) and (n, 3) binding sites. As it is difficult to solve the system of equations occurring when a molecule with more than one binding site for both ligands shall be decoupled, we present applicable calculation methods which exploit the special structure of the system of equations. Moreover, we investigate which unique properties all decoupled molecules share and show that for two different decoupled molecules with the same binding polynomial, not all microstate constants of a certain macrostate are permutations of the microstate constants of the other molecule.     

Photoswitching Affinity and Mechanism of Multivalent Lectin Ligands

Multivalent receptor–ligand binding is a key principle in a plethora of biological recognition processes. Immense binding affinities can be achieved with the correct spatial orientation of the ligands. Accordingly, the incorporation of photoswitches, which can be used to reversibly change the spatial orientation of molecules, into multivalent ligands is a means to alter the binding affinity and possibly also the binding mode of such ligands. We report a divalent ligand for the model lectin wheat germ agglutinin (WGA) containing an arylazopyrazole photoswitch. This switch, which has recently been introduced as an alternative to the more commonly used azobenzene moiety, is characterized by almost quantitative E/Z photoswitching in both directions, high quantum yields, and high thermal stability of the Z isomer. The ligand was designed in a way that only one of the isomers is able to bridge adjacent binding sites of WGA leading to a chelating binding mode. Photoswitching induces an unprecedentedly high change in lectin binding affinity as determined by isothermal titration calorimetry (ITC). Furthermore, additional dynamic light scattering (DLS) data suggest that the binding mode of the ligand changes from chelating binding of the E isomer to crosslinking binding of the Z isomer.     

Hierarchical Fluid Interface Enables Spatiotemporal Regulation of Ligand Distribution to Increase Kinetics and Thermodynamics of Interfacial Binding Reaction

In nature, regulation of the spatiotemporal distribution of interfacial receptors and ligands leads to optimum binding kinetics and thermodynamics of receptor–ligand binding reactions within interfaces. Inspired by this, we report a hie rarchical fluid interface (HieFluidFace) to regulate the spatiotemporal distribution of interfacial ligands to increase the rate and thermodynamic favorability of interfacial binding reactions. Each aptamer-functionalized gold nanoparticle, termed spherical aptamer (SAPT), is anchored on a supported lipid bilayer without fluidity, like an “island”, and is surrounded by many fluorescent aptamers (FAPTs) with free fluidity, like “rafts”. Such ligand “island-rafts” model provides a large reactive cross-section for rapid binding to cellular receptors. The synergistic multivalency of SAPTs and FAPTs improves interfacial affinity for tight capture. Moreover, FAPTs accumulate at binding sites to bind to cellular receptors with clustered fluorescence to “lighten” cells for direct identification. Thus, HieFluidFace in a microfluidic chip achieves high-performance capture and identification of circulating tumor cells from clinical samples, providing a new paradigm to optimize the kinetics and thermodynamics of interfacial binding reactions.     

人血白蛋白结合位点II的分子结合模式研究

人血白蛋白是血浆中含量最高的蛋白,是一种重要的载体蛋白,能与多种内源性和外源性物质结合。人血白蛋白主要有两个药物结合位点,位点I和位点II,其中位点II的柔性较大,对药物分子的亲和性较高。本文采用分子模拟方法,基于12种结合于位点II的小分子-人血白蛋白晶体结构,分析了相互作用能,发现12种分子的结合以疏水作用为主,静电作用为辅。进一步采用丙氨酸扫描和结合能评价,分析结合部位的关键氨基酸残基,探究结合模式的规律性,发现从位点入口到空腔内部存在静电、疏水和杂合的三层相互作用分布,共同形成了稳定的分子结合。最后采用分子对接和分子动力学模拟,预测了L-色氨酸的结合模式。研究结果有助于深入了解人血白蛋白药物位点II的小分子结合模式,为基于位点II的药物和分离配基的优化设计提供指导。     

Allosteric antagonist binding sites in class B GPCRs: corticotropin receptor 1

The 41 amino acid neuropeptide, corticotropin-releasing factor (CRF) and its associated receptors CRF₁-R and CRF₂-R have been targeted for treating stress related disorders. Both CRF₁-R and CRF₂-R belong to the class B G-protein coupled receptors for which little information is known regarding the small molecule antagonist binding characteristics. However, it has been shown recently that different non-peptide allosteric ligands stabilize different receptor conformations for CRF₁-R and hence an understanding of the ligand induced receptor conformational changes is important in the pharmacology of ligand binding. In this study, we modeled the receptor and identified the binding sites of representative small molecule allosteric antagonists for CRF₁-R. The predicted binding sites of the investigated compounds are located within the transmembrane (TM) domain encompassing TM helices 3, 5 and 6. The docked compounds show strong interactions with H228 on TM3 and M305 on TM5 that have also been implicated in the binding by site directed mutation studies. H228 forms a hydrogen bond of varied strengths with all the antagonists in this study and this is in agreement with the decreased binding affinity of several compounds with H228F mutation. Also mutating M305 to Ile showed a sharp decrease in the calculated binding energy whereas the binding energy loss on M305 to Leu was less significant. These results are in qualitative agreement with the decrease in binding affinities observed experimentally. We further predicted the conformational changes in CRF₁-R induced by the allosteric antagonist NBI-27914. Movement of TM helices 3 and 5 are dominant and generates three degenerate conformational states two of which are separated by an energy barrier from the third, when bound to NBI-27914. Binding of NBI-27914 was predicted to improve the interaction of the ligand with M305 and also enhanced the aromatic stacking between the ligand and F232 on TM3. A virtual ligand screening of ~13,000 compounds seeded with ~350 CRF₁-R specific active antagonists performed on the NBI-27914 stabilized conformation of CRF₁-R yielded a 44% increase in enrichment compared to the initially modeled receptor conformation at a 10% cutoff. The NBI-27914 stabilized conformation also shows a high enrichment for high affinity antagonists compared to the weaker ones. Thus, the conformational changes induced by NBI-27914 improved the ligand screening efficiency of the CRF₁-R model and demonstrate a generalized application of the method in drug discovery.     

Development and screening of epoxy‐spacer‐phage cryogels for affinity chromatography: Enhancing the binding capacity

Macroporous epoxy cryogels can be used as an alternative for classical matrices in affinity chromatography. Due to the structural properties of cryogels, with pores of up to 100 μm, crude samples can be processed at high speed without previous manipulations such as clarification or centrifugation. Also, we previously used a peptide‐expressing M13 bacteriophage as an affinity ligand. These ligands show high specificity toward the target to be purified. Combination of both, leads to a relative cost‐effective one‐step chromatographic set‐up delivering a high purity sample (>95%), however, so far with limited capacity. To increase the binding capacity of the affinity columns, we now inserted spacers between the chromatographic matrix and the phage ligand. Both linear spacers, di‐amino‐alkanes (C₂–C₁₀), and branched polyethyleneimine spacers with different molecular weights (800 Da–10 kDa) were analyzed. Two types of peptide expressing phage ligands, a linear 15‐mer and a cyclic 6‐mer, were used for screening. Up to a tenfold increase in binding capacity was observed depending on the combination of phage ligand and spacer type.     

EF-Tu binding peptides identified,dissected, and affinity optimized by phage display

The highly abundant GTP binding protein elongation factor Tu (EF-Tu) fulfills multiple roles in bacterial protein biosynthesis. Phage-displayed peptides with high affinity for EF-Tu were selected from a library of approximately 4.7 x 10(11) different peptides. The lack of sequence homology among the identified EF-Tu ligands demonstrates promiscuous peptide binding by EF-Tu. Homolog shotgun scanning of an EF-Tu ligand was used to dissect peptide molecular recognition by EF-Tu. All homolog shotgun scanning selectants bound to EF-Tu with higher affinity than the starting ligand. Thus, homolog shotgun scanning can simultaneously optimize binding affinity and rapidly provide detailed structure activity relationships for multiple side chains of a polypeptide ligand. The reported peptide ligands do not compete for binding to EF-Tu with various antibiotic EF-Tu inhibitors, and could identify an EF-Tu peptide binding site distinct from the antibiotic inhibitory sites.