Boosting Affinity by Correct Ligand Preorganization for the S2 Pocket of Thrombin: A Study by Isothermal Titration Calorimetry,Molecular Dynamics,and High‐Resolution Crystal Structures

Structural preorganization to fix bioactive conformations at protein binding sites is a popular strategy to enhance binding affinity during late‐stage optimization. The rationale for this enhancement relates to entropic advantages assigned to rigidified versus flexible ligands. We analyzed a narrow series of peptidomimetics binding to thrombin. The individual ligands exhibit at P2 a conformationally flexible glycine, more restricted alanine, N‐methylglycine, N‐methylhomoalanine, and largely rigidified proline moiety. Overall, affinity was found to increase by a factor of 1000, explained partly by an entropic advantage. All ligands adopt the same binding mode with small deviations. The residual mobility of the bound ligands is decreased across the series, and a protein side chain differs in its order/disorder behavior along with changes in the surface‐water network pattern established across the newly generated protein–ligand surfaces. The enthalpy/entropy inventory displays a rather complex picture and emphasizes that thermodynamics can only be compared in terms of relative differences within a structurally similar ligand series.     

NMR‐Guided Molecular Docking of a Protein–Peptide Complex Based on Ant Colony Optimization

Standard docking approaches used for the prediction of protein–ligand complexes in the drug development process have problems identifying the correct binding mode of large flexible ligands. Herein we show how additional experimental data from NMR experiments can be used to predict the binding mode of a mucin 1 (MUC‐1) pentapeptide recognized by the breast‐cancer‐selective monoclonal antibody SM3. Distance constraints derived from trNOE and saturation transfer difference NMR experiments are combined with the docking approach PLANTS. The resulting complex structures show excellent agreement with the NMR data and with a published X‐ray crystal structure. The method was then further tested on two complexes in order to demonstrate its more general applicability: T‐antigen disaccharide bound to Maclura pomifera agglutinin, and the inhibitor SBi279 bound to S100B protein. Our new approach has the advantages of being fully automatic, rapid, and unbiased; moreover, it is based on relatively easily obtainable experimental data and can greatly increase the reliability of the generated structures.     

On the Implication of Water on Fragment‐to‐Ligand Growth in Kinase Binding Thermodynamics

A ligand‐binding study is presented focusing on thermodynamics of fragment expansion. The binding of four compounds with increasing molecular weight to protein kinase A (PKA) was analyzed. The ligands display affinities between low‐micromolar to nanomolar potency despite their low molecular weight. Binding free energies were measured by isothermal titration calorimetry, revealing a trend toward more entropic and less enthalpic binding with increase in molecular weight. All protein–ligand complexes were analyzed by crystallography and solution NMR spectroscopy. Crystal structures and solution NMR data are highly consistent, and no major differences in complex dynamics across the series are observed that would explain the differences in the thermodynamic profiles. Instead, the thermodynamic trends result either from differences in the solvation patterns of the conformationally more flexible ligand in aqueous solution prior to protein binding as molecular dynamics simulations suggest, or from local shifts of the water structure in the ligand‐bound state. Our data thus provide evidence that changes in the solvation pattern constitute an important parameter for the understanding of thermodynamic data in protein–ligand complex formation.     

Carbohydrate–PNA and Aptamer–PNA Conjugates for the Spatial Screening of Lectins and Lectin Assemblies

Nucleic acid architectures offer intriguing opportunities for the interrogation of structural properties of protein receptors. In this study, we performed a DNA‐programmed spatial screening to characterize two functionally distinct receptor systems: 1) structurally well‐defined Ricinus communis agglutinin (RCA₁₂₀), and 2) rather ill‐defined assemblies of L‐selectin on nanoparticles and leukocytes. A robust synthesis route that allowed the attachment both of carbohydrate ligands—such as N‐acetyllactosamine (LacNAc), sialyl‐Lewis‐X (sLe^X), and mannose—and of a DNA aptamer to PNAs was developed. A systematically assembled series of different PNA–DNA complexes served as multivalent scaffolds to control the spatial alignments of appended lectin ligands. The spatial screening of the binding sites of RCA₁₂₀ was in agreement with the crystal structure analysis. The study revealed that two appropriately presented LacNAc ligands suffice to provide unprecedented RCA₁₂₀ affinity (K_D=4 μM ). In addition, a potential secondary binding site was identified. Less dramatic binding enhancements were obtained when the more flexible L‐selectin assemblies were probed. This study involved the bivalent display both of the weak‐affinity sLe^X ligand and of a high‐affinity DNA aptamer. Bivalent presentation led to rather modest (sixfold or less) enhancements of binding when the self‐assemblies were targeted against L‐selectin on gold nanoparticles. Spatial screening of L‐selectin on the surfaces of leukocytes showed higher affinity enhancements (25‐fold). This and the distance–activity relationships indicated that leukocytes permit dense clustering of L‐selectin.     

Drug Screening Boosted by Hyperpolarized Long‐Lived States in NMR

Transverse and longitudinal relaxation times (T_1ρ and T₁) have been widely exploited in NMR to probe the binding of ligands and putative drugs to target proteins. We have shown recently that long‐lived states (LLS) can be more sensitive to ligand binding. LLS can be excited if the ligand comprises at least two coupled spins. Herein we broaden the scope of ligand screening by LLS to arbitrary ligands by covalent attachment of a functional group, which comprises a pair of coupled protons that are isolated from neighboring magnetic nuclei. The resulting functionalized ligands have longitudinal relaxation times T₁(¹H) that are sufficiently long to allow the powerful combination of LLS with dissolution dynamic nuclear polarization (D‐DNP). Hyperpolarized weak “spy ligands” can be displaced by high‐affinity competitors. Hyperpolarized LLS allow one to decrease both protein and ligand concentrations to micromolar levels and to significantly increase sample throughput.     

The Role of Flexibility in the Rational Design of Modularly Assembled Ligands Targeting the RNAs that Cause the Myotonic Dystrophies

Modularly assembled ligands were designed to target the RNAs that cause two currently untreatable neuromuscular disorders, myotonic dystrophy types 1 (DM1) and 2 (DM2). DM1 is caused by an expanded repeating sequence of CUG, and DM2 is caused by expanded CCUG repeats. Both are present in noncoding regions and fold into hairpins with either repeating 1×1 nucleotide UU (DM1) or 2×2 nucleotide 5′‐CU/3′‐UC (DM2) internal loops separated by two GC pairs. The repeats are toxic because they sequester the RNA splicing regulator muscleblind‐like 1 protein (MBNL1). Rational design of ligands targeting these RNAs was enabled by a database of RNA motif–ligand partners compiled by using two‐dimensional combinatorial screening (2DCS). One 2DCS study found that the 6′′‐azido‐kanamycin A module binds internal loops similar to those found in DM1 and DM2. In order to further enhance affinity and specificity, the ligand was assembled on a peptoid backbone to precisely control valency and the distance between ligand modules. Designed compounds are more potent and specific binders to the toxic RNAs than MBNL1 and inhibit the formation of the RNA–protein complexes with nanomolar IC₅₀ values. This study shows that three important factors govern potent inhibition: 1) the surface area sequestered by the assembled ligands; 2) the spacing between ligand modules since a longer distance is required to target DM2 RNAs than DM1 RNAs; and 3) flexibility in the modular assembly scaffold used to display the RNA‐binding module. These results have impacts on the general design of assembled ligands targeting RNAs present in genomic sequence.     

Chiral Graphs: Reduced Representations of Ligand Scaffolds for Stereoselective Biomolecular Recognition,Drug Design,and Enhanced Exploration of Chemical Structure Space

Rational structure-based drug design relies on a detailed, atomic-level understanding of protein–ligand interactions. The chiral nature of drug binding sites in proteins has led to the discovery of predominantly chiral drugs. A mechanistic understanding of stereoselectivity (which governs how one stereoisomer of a drug might bind stronger than the others to a protein) depends on the topology of stereocenters in the chiral molecule. Chiral graphs and reduced chiral graphs, introduced here, are new topological representations of chiral ligands using graph theory, to facilitate a detailed understanding of chiral recognition of ligands/drugs by proteins. These representations are demonstrated by application to all ≈14 000+ chiral ligands in the Protein Data Bank (PDB), which will facilitate an understanding of protein–ligand stereoselectivity mechanisms. Ligand modifications during drug development can be easily incorporated into these chiral graphs. In addition, these chiral graphs present an efficient tool for a deep dive into the enormous chemical structure space to enable sampling of unexplored structural scaffolds.     

Halogen bonding at the active sites of human cathepsin L and MEK1 kinase: efficient interactions in different environments

In two series of small‐molecule ligands, one inhibiting human cathepsin L (hcatL) and the other MEK1 kinase, biological affinities were found to strongly increase when an aryl ring of the inhibitors is substituted with the larger halogens Cl, Br, and I, but to decrease upon F substitution. X‐ray co‐crystal structure analyses revealed that the higher halides engage in halogen bonding (XB) with a backbone C?O in the S3 pocket of hcatL and in a back pocket of MEK1. While the S3 pocket is located at the surface of the enzyme, which provides a polar environment, the back pocket in MEK1 is deeply buried in the protein and is of pronounced apolar character. This study analyzes environmental effects on XB in protein–ligand complexes. It is hypothesized that energetic gains by XB are predominantly not due to water replacements but originate from direct interactions between the XB donor (C_aryl? X) and the XB acceptor (C?O) in the correct geometry. New X‐ray co‐crystal structures in the same crystal form (space group P2₁2₁2₁) were obtained for aryl chloride, bromide, and iodide ligands bound to hcatL. These high‐resolution structures reveal that the backbone C?O group of Gly61 in most hcatL co‐crystal structures maintains water solvation while engaging in XB. An aryl? CF₃‐substituted ligand of hcatL with an unexpectedly high affinity was found to adopt the same binding geometry as the aryl halides, with the CF₃ group pointing to the C?O group of Gly61 in the S3 pocket. In this case, a repulsive F₂C? F???O?C contact apparently is energetically overcompensated by other favorable protein–ligand contacts established by the CF₃ group.     

Potent Fluoro‐oligosaccharide Probes of Adhesion in Toxoplasmosis

Unnatural, NMR‐ and MRI‐active fluorinated sugar probes, designed and synthesised to bind to the pathogenic protein TgMIC1 from Toxoplasma gondii, were found to display binding potency equal to and above that of the natural ligand. Dissection of the binding mechanism and modes, including the first X‐ray crystal structures of a fluoro‐oligosaccharide bound to a lectin, demonstrate that it is possible to create effective fluorinated probe ligands for the study of, and perhaps intervention in, sugar–protein binding events.     

Methyl,Ethyl, Propyl,Butyl: Futile But Not for Water,as the Correlation of Structure and Thermodynamic Signature Shows in a Congeneric Series of Thermolysin Inhibitors

Water is ubiquitously present in any biological system and has therefore to be regarded as an additional binding partner in the protein–ligand binding process. Upon complex formation, a new solvent‐exposed surface is generated and water molecules from the first solvation layer will arrange around this newly formed surface. So far, the influence of such water arrangements on the ligand binding properties is unknown. In this study, the binding modes of nine congeneric phosphonamidate‐type inhibitors with systematically varied, size‐increasing hydrophobic P₂′ substituents (from methyl to phenylethyl) addressing the hydrophobic, solvent‐exposed S₂′ pocket of thermolysin were analyzed by high‐resolution crystal structures and correlated with their thermodynamic binding profiles as measured by isothermal titration calorimetry. Overall, ΔΔG spreads over 7.0 kJ mol^?1, ΔΔH varies by 15.8 kJ mol^?1, and ?TΔΔS by 12.1 kJ mol^?1. Throughout the series, these changes correlate remarkably well with the geometric differences of water molecules arranged adjacent to the P₂′ substituents. Ligands with medium‐sized P₂′ substituents exhibit highest affinities, presumably because of their optimal solvation patterns around these complexes. The addition, removal, or rearrangement of even a single methyl group can result in a strong modulation of the adjacent water network pattern shifting from enthalpy to entropy‐driven binding. In conclusion, the quality of a water network assembled around a protein–ligand complex influences the enthalpy/entropy signature and can even modulate affinity to a surprising extent.     

Exploring the Structural Space of the Galectin‐1–Ligand Interaction

Galectin‐1 is a tumor‐associated protein recognizing the Galβ1‐4GlcNAc motif of cell‐surface glycoconjugates. Herein, we report the stepwise expansion of a multifunctional natural scaffold based on N‐acetyllactosamine (LacNAc). We obtained a LacNAc mimetic equipped with an alkynyl function on the 3′‐hydroxy group of the disaccharide facing towards a binding pocket adjacent to the carbohydrate‐recognition domain. It served as an anchor motif for further expansion by the Sharpless–Huisgen–Meldal reaction, which resulted in ligands with a binding mode almost identical to that of the natural carbohydrate template. X‐ray crystallography provided a structural understanding of the galectin‐1–ligand interactions. The results of this study enable the development of bespoke ligands for members of the galectin target family.     

Evaluation of solvent accessibility epitopes for different dehydrogenase inhibitors

Knowledge about the orientation of ligands or inhibitors bound to a protein is vital for the development of new drugs. It was recently shown that solvent accessibility epitopes for protein ligands can be mapped by transferring magnetization from water molecules to the ligand to derive the ligand orientation. This is based on the fact that NMR signals of ligands arising from magnetization transferred from solvent molecules via the protein have a different sign from those arising from direct magnetization transfer from bulk water. Herein we critically evaluate the applicability of solvent accessibility mapping to derive binding orientations for ligands of two dehydrogenases (AKR1C3 and HSD17beta1) with very different binding pockets, including complexes in which the ligand is buried more deeply inside the protein. We also evaluate the possibility of using co-solvents, such as DMSO, for magnetization transfer.     

Catalyst Stability Determines the Catalytic Activity of Non‐Heme Iron Catalysts in the Oxidation of Alkanes

A series of iron(II) bis(triflate) complexes [Fe(L)(OTf)₂] containing linear tetradentate bis(pyridylmethyl)diamine ligands with a range of ligand backbones has been prepared. The backbone of the ligand series has been varied from a two‐carbon linkage [ethylene ( 1 ), 4,5‐dichlorophenylene ( 2 ) and cyclohexyl ( 3 )] to a three‐carbon [propyl ( 4 )) and a four‐carbon linkage (butyl ( 5 )]. The coordination geometries of these complexes have been investigated in the solid state by X‐ray crystallography and in solution by ¹H and ¹⁹F NMR spectroscopy. Due to the labile nature of high‐spin iron(II) complexes in solution, dynamic equilibria of complexes with different coordination geometries (cis‐α, cis‐β and trans) are observed with ligands 2 – 5 . In these cases, the geometry observed in the solid state does not necessarily represent the only or even the major geometry present in solution. The ligand field strength in the various complexes has been investigated by variable temperature magnetic moment measurements and UV‐vis spectroscopy. The strongest ligand field is observed with the most rigid ligands 1 and 2 , which generate complexes [Fe(L)(OTf)₂] with a cis‐α coordination geometry and the corresponding complexes [Fe(L)(CH₃CN)₂]²⁺ display spin crossover behaviour. The catalytic properties of the complexes for the oxidation of cyclohexane, using hydrogen peroxide as the oxidant, have been investigated. An increased flexibility in the ligand results in a weaker ligand field, which increases the lability of the complexes. The activity and selectivity of the catalysts appear to be related to the strength of the ligand field and the stability of the catalyst in the oxidising environment.     

Multipose Binding in Molecular Docking

Molecular docking has been extensively applied in virtual screening of small molecule libraries for lead identification and optimization. A necessary prerequisite for successful differentiation between active and non-active ligands is the accurate prediction of their binding affinities in the complex by use of docking scoring functions. However, many studies have shown rather poor correlations between docking scores and experimental binding affinities. Our work aimed to improve this correlation by implementing a multipose binding concept in the docking scoring scheme. Multipose binding, i.e., the property of certain protein-ligand complexes to exhibit different ligand binding modes, has been shown to occur in nature for a variety of molecules. We conducted a high-throughput docking study and implemented multipose binding in the scoring procedure by considering multiple docking solutions in binding affinity prediction. In general, improvement of the agreement between docking scores and experimental data was observed, and this was most pronounced in complexes with large and flexible ligands and high binding affinities. Further developments of the selection criteria for docking solutions for each individual complex are still necessary for a general utilization of the multipose binding concept for accurate binding affinity prediction by molecular docking.     

Influences of Ligand Structure and pH on the Adsorption with Hydrophobic Charge Induction Adsorbents: A Case Study of Antibody IgY

The adsorption behaviors of hydrophobic charge induction chromatography (HCIC) adsorbents with different functional ligands were investigated with immunoglobulin of egg yolk (IgY) as a model antibody. The adsorption isotherm and retention behavior in the column were studied, and the influences of the ligand structure and the pH on the adsorption were discussed. The results indicated that the pI of the target protein and pKa of HCIC ligand are the important parameter to determine the maximum adsorption pH of HCIC adsorbent, and high adsorption of IgY was found at pH 5 for all five adsorbents tested. Some differences could be found for different HCIC adsorbents, and the ligand structure influenced pH effect on the binding/elution of target protein. 2-mercapto-1-methyl-imidazole (MMI) ligand with a sulfone group showed a high adsorption capacity and strong pH-sensitivity, which would be more suitable for antibody purification. Moreover, the retention experiments indicated that IgY could be efficiently eluted from the adsorbents with 4-mercapto-ethyl-pyridine (MEP) or MMI as the ligand at acid conditions, while 2-mercapto-benzimidazole (MBI) ligand showed some difficulties on the elution. The retention study would help in defining not only the effective pH of elution for a given protein but also the elution efficiency of a given adsorbent.     

A Comparison of the Binding of the Ligand Trimethoprim to Bacterial and Vertebrate Dihydrofolate Reductases

In this paper, we report on studies of ligand binding to the enzyme dihydrofolate reductase (DHFR). Energy minimizations of four complexes of DHFR with the inhibitor trimethoprim, an antibiotic, and the cofactor NADPH have been carried out in order to investigate the energetics responsible for the 100,000-fold increase in binding affinity of trimethoprim to E. coli DHFR compared with chicken liver DHFR. Several factors suggested to be responsible for the enhanced binding in bacterial DHFR's were investigated in terms of intermolecular and intramolecular energetics. The strain energies of trimethoprim in the four complexes were calculated and found to be about 6 kcal mol⁻¹ in all complexes of the two species. In the binary complex of chicken liver DHFR, where the largest variation was observed, 2 kcal mol⁻¹ higher than in the other complexes, it was found that this increase was compensated for by the slightly more favorable intermolecular interaction of the trimethoxyphenyl moiety with the protein. Comparison of the minimized binary and ternary complexes of E. coli allowed us to investigate the cooperativity in the binding of trimethoprim and NADPH in the bacterial enzyme in terms of the underlying intermolecular forces. This cooperativity was found to be due to a direct trimethoprim - NADPH interaction in the E. coli enzyme rather than enhanced protein-inhibitor interactions induced upon binding of the cofactor. These interactions are not as favorable in the vertebrate enzyme, consistent with the significantly diminished cooperativity observed in this enzyme.     

Experimental and computational active site mapping as a starting point to fragment-based lead discovery

Small highly soluble probe molecules such as aniline, urea, N‐methylurea, 2‐bromoacetate, 1,2‐propanediol, nitrous oxide, benzamidine, and phenol were soaked into crystals of various proteins to map their binding pockets and to detect hot spots of binding with respect to hydrophobic and hydrophilic properties. The selected probe molecules were first tested at the zinc protease thermolysin. They were then applied to a wider range of proteins such as protein kinase A, D ‐xylose isomerase, 4‐diphosphocytidyl‐2C‐methyl‐D ‐erythritol synthase, endothiapepsin, and secreted aspartic protease 2. The crystal structures obtained clearly show that the probe molecules populate the protein binding pockets in an ordered fashion. The thus characterized, experimentally observed hot spots of binding were subjected to computational active site mapping using HotspotsX. This approach uses knowledge‐based pair potentials to detect favorable binding positions for various atom types. Good agreement between the in silico hot spot predictions and the experimentally observed positions of the polar hydrogen bond forming functional groups and hydrophobic portions was obtained. Finally, we compared the observed poses of the small‐molecule probes with those of much larger structurally related ligands. They coincide remarkably well with the larger ligands, considering their spatial orientation and the experienced interaction patterns. This observation confirms the fundamental hypothesis of fragment‐based lead discovery: that binding poses, even of very small molecular probes, do not significantly deviate or move once a ligand is grown further into the binding site. This underscores the fact that these probes populate given hot spots and can be regarded as relevant seeds for further design.     

Acetylcholine Promotes Binding of α‐Conotoxin MII at α3β2 Nicotinic Acetylcholine Receptors

α‐Conotoxin MII (α‐CTxMII) is a 16‐residue peptide with the sequence GCCSNPVCHLEHSNLC, containing Cys2–Cys8 and Cys3–Cys16 disulfide bonds. This peptide, isolated from the venom of the marine cone snail Conus magus, is a potent and selective antagonist of neuronal nicotinic acetylcholine receptors (nAChRs). To evaluate the impact of channel–ligand interactions on ligand‐binding affinity, homology models of the heteropentameric α₃β₂‐nAChR were constructed. The models were created in MODELLER with the aid of experimentally characterized structures of the Torpedo marmorata‐nAChR (Tm‐nAChR, PDB ID: 2BG9) and the Aplysia californica‐acetylcholine binding protein (Ac‐AChBP, PDB ID: 2BR8) as templates for the α₃‐ and β₂‐subunit isoforms derived from rat neuronal nAChR primary amino acid sequences. Molecular docking calculations were performed with AutoDock to evaluate interactions of the heteropentameric nAChR homology models with the ligands acetylcholine (ACh) and α‐CTxMII. The nAChR homology models described here bind ACh with binding energies commensurate with those of previously reported systems, and identify critical interactions that facilitate both ACh and α‐CTxMII ligand binding. The docking calculations revealed an increased binding affinity of the α₃β₂‐nAChR for α‐CTxMII with ACh bound to the receptor, and this was confirmed through two‐electrode voltage clamp experiments on oocytes from Xenopus laevis. These findings provide insights into the inhibition and mechanism of electrostatically driven antagonist properties of the α‐CTxMIIs on nAChRs.     

Tumor-Suppressor p53TAD1–60 Forms a Fuzzy Complex with Metastasis-Associated S100A4: Structural Insights and Dynamics by an NMR/MD Approach

Conformationally flexible protein complexes represent a major challenge for structural and dynamical studies. We present herein a method based on a hybrid NMR/MD approach to characterize the complex formed between the disordered p53TAD^1–60 and the metastasis-associated S100A4. Disorder-to-order transitions of both TAD1 and TAD2 subdomains upon interaction is detected. Still, p53TAD^1–60 remains highly flexible in the bound form, with residues L26, M40, and W53 being anchored to identical hydrophobic pockets of the S100A4 monomer chains. In the resulting “fuzzy” complex, the clamp-like binding of p53TAD^1–60 relies on specific hydrophobic anchors and on the existence of extended flexible segments. Our results demonstrate that structural and dynamical NMR parameters (cumulative Δδ, SSP, temperature coefficients, relaxation time, hetNOE) combined with MD simulations can be used to build a structural model even if, due to high flexibility, the classical solution structure calculation is not possible.