The Importance of Hydration Thermodynamics in Fragment‐to‐Lead Optimization

Using a computational approach to assess changes in solvation thermodynamics upon ligand binding, we investigated the effects of water molecules on the binding energetics of over 20 fragment hits and their corresponding optimized lead compounds. Binding activity and X‐ray crystallographic data of published fragment‐to‐lead optimization studies from various therapeutically relevant targets were studied. The analysis reveals a distinct difference between the thermodynamic profile of water molecules displaced by fragment hits and those displaced by the corresponding optimized lead compounds. Specifically, fragment hits tend to displace water molecules with notably unfavorable excess entropies—configurationally constrained water molecules—relative to those displaced by the newly added moieties of the lead compound during the course of fragment‐to‐lead optimization. Herein we describe the details of this analysis with the goal of providing practical guidelines for exploiting thermodynamic signatures of binding site water molecules in the context of fragment‐to‐lead optimization.     

Heterotypic Sam–Sam Association between Odin‐Sam1 and Arap3‐Sam: Binding Affinity and Structural Insights

Arap3 is a phosphatidylinositol 3 kinase effector protein that plays a role as GTPase activator (GAP) for Arf6 and RhoA. Arap3 contains a sterile alpha motif (Sam) domain that has high sequence homology with the Sam domain of the EphA2‐receptor (EphA2‐Sam). Both Arap3‐Sam and EphA2‐Sam are able to associate with the Sam domain of the lipid phosphatase Ship2 (Ship2‐Sam). Recently, we reported a novel interaction between the first Sam domain of Odin (Odin‐Sam1), a protein belonging to the ANKS (ANKyrin repeat and Sam domain containing) family, and EphA2‐Sam. In our latest work, we applied NMR spectroscopy, surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) to characterize the association between Arap3‐Sam and Odin‐Sam1. We show that these two Sam domains interact with low micromolar affinity. Moreover, by means of molecular docking techniques, supported by NMR data, we demonstrate that Odin‐Sam1 and Arap3‐Sam might bind with a topology that is common to several Sam‐Sam complexes. The revealed structural details form the basis for the design of potential peptide antagonists that could be used as chemical tools to investigate functional aspects related to heterotypic Arap3‐Sam associations.     

Bicyclic Peptide Inhibitor of Urokinase‐Type Plasminogen Activator: Mode of Action

The development of protease inhibitors for pharmacological intervention has taken a new turn with the use of peptide‐based inhibitors. Here, we report the rational design of bicyclic peptide inhibitors of the serine protease urokinase‐type plasminogen activator (uPA), based on the established monocyclic peptide, upain‐2. It was successfully converted to a bicyclic peptide, without loss of inhibitory properties. The aim was to produce a peptide cyclised by an amide bond with an additional stabilising across‐the‐ring covalent bond. We expected this bicyclic peptide to exhibit a lower entropic burden upon binding. Two bicyclic peptides were synthesised with affinities similar to that of upain‐2, and their binding energetics were evaluated by isothermal titration calorimetry. Indeed, compared to upain‐2, the bicyclic peptides showed reduced loss of entropy upon binding to uPA. We also investigated the solution structures of the bicyclic peptide by NMR spectroscopy to map possible conformations. An X‐ray structure of the bicyclic‐peptide–uPA complex confirmed an interaction similar to that for the previous upain‐1/upain‐2–uPA complexes. These physical studies of the peptide–protease interactions will aid future designs of bicyclic peptide protease inhibitors.     

Nanodisc‐Targeted STD NMR Spectroscopy Reveals Atomic Details of Ligand Binding to Lipid Environments

Saturation transfer difference (STD) NMR spectroscopy is one of the most popular ligand‐based NMR techniques for the study of protein–ligand interactions. This is due to its robustness and the fact that it is focused on the signals of the ligand, without any need for NMR information on the macromolecular target. This technique is most commonly applied to systems involving different types of ligands (e.g., small organic molecules, carbohydrates or lipids) and a protein as the target, in which the latter is selectively saturated. However, only a few examples have been reported where membrane mimetics are the macromolecular binding partners. Here, we have employed STD NMR spectroscopy to investigate the interactions of the neurotransmitter dopamine with mimetics of lipid bilayers, such as nanodiscs, by saturation of the latter. In particular, the interactions between dopamine and model lipid nanodiscs formed either from charged or zwitterionic lipids have been resolved at the atomic level. The results, in agreement with previous isothermal titration calorimetry studies, show that dopamine preferentially binds to negatively charged model membranes, but also provide detailed atomic insights into the mode of interaction of dopamine with membrane mimetics. Our findings provide relevant structural information for the design of lipid‐based drug carriers of dopamine and its structural analogues and are of general applicability to other systems.     

Residual Helicity at the Active Site of the Histidine Phosphocarrier,HPr, Modulates Binding Affinity to Its Natural Partners

The phosphoenolpyruvate-dependent phosphotransferase system (PTS) modulates the preferential use of sugars in bacteria. The first proteins in the cascade are common to all organisms (EI and HPr). The active site of HPr involves a histidine (His15) located immediately before the beginning of the first α-helix. The regulator of sigma D (Rsd) protein also binds to HPr. The region of HPr comprising residues Gly9-Ala30 (HPr^9–30), involving the first α-helix (Ala16-Thr27) and the preceding active site loop, binds to both the N-terminal region of EI and intact Rsd. HPr^9–30 is mainly disordered. We attempted to improve the affinity of HPr^9–30 to both proteins by mutating its sequence to increase its helicity. We designed peptides that led to a marginally larger population in solution of the helical structure of HPr^9–30. Molecular simulations also suggested a modest increment in the helical population of mutants, when compared to the wild-type. The mutants, however, were bound with a less favorable affinity than the wild-type to both the N-terminal of EI (EIN) or Rsd, as tested by isothermal titration calorimetry and fluorescence. Furthermore, mutants showed lower antibacterial properties against Staphylococcus aureus than the wild-type peptide. Therefore, we concluded that in HPr, a compromise between binding to its partners and residual structure at the active site must exist to carry out its function.     

Development of Fragment‐Based n‐FABS NMR Screening Applied to the Membrane Enzyme FAAH

Despite the recognized importance of membrane proteins as pharmaceutical targets, the reliable identification of fragment hits that are able to bind these proteins is still a major challenge. Among different ¹⁹F NMR spectroscopic methods, n‐fluorine atoms for biochemical screening (n‐FABS) is a highly sensitive technique that has been used efficiently for fragment screening, but its application for membrane enzymes has not been reported yet. Herein, we present the first successful application of n‐FABS to the discovery of novel fragment hits, targeting the membrane‐bound enzyme fatty acid amide hydrolase (FAAH), using a library of fluorinated fragments generated based on the different local environment of fluorine concept. The use of the recombinant fusion protein MBP‐FAAH and the design of compound 11 as a suitable novel fluorinated substrate analogue allowed n‐FABS screening to be efficiently performed using a very small amount of enzyme. Notably, we have identified 19 novel fragment hits that inhibit FAAH with a median effective concentration (IC₅₀) in the low mM –μM range. To the best of our knowledge, these results represent the first application of a ¹⁹F NMR fragment‐based functional assay to a membrane protein.     

Unraveling the Binding Mechanism of Alzheimer’s Drugs with Irisin: Spectroscopic,Calorimetric, and Computational Approaches

The prevalence of Alzheimer’s disease (AD) has been a major health concern for a long time. Despite recent progress, there is still a strong need to develop effective disease-modifying therapies. Several drugs have already been approved to retard the progression of AD-related symptoms; however, there is a need to develop an effective carrier system for the delivery of drugs to combat such diseases. In recent years, various biological macromolecules, including proteins, have been used as carriers for drug delivery. Irisin is a beneficial hormone in such diseases, including AD and related pathologies. Herein, the interaction mechanism of irisin with AD drugs such as memantine, galantamine, and fluoxetine is investigated. Fluorescence studies revealed that the above drugs bind to irisin with significant affinity, with fluoxetine having the highest binding affinity. Isothermal titration calorimetry (ITC) complemented the spontaneous binding of these drugs with irisin, delineating various associated thermodynamic and binding parameters. Molecular docking further validated the fluorescence and ITC results and unfolded the mechanism that hydrogen bonding governs the binding of fluoxetine to irisin with a significant binding score, i.e., −6.3 kcal/mol. We believe that these findings provide a promising solution to fight against AD as well as a platform for further research to utilize irisin in the drug-delivery system for an effective therapeutic strategy.     

Selective Targeting of the TPX2 Site of Importin‐α Using Fragment‐Based Ligand Design

Protein–protein interactions are difficult therapeutic targets, and inhibiting pathologically relevant interactions without disrupting other essential ones presents an additional challenge. Herein we report how this might be achieved for the potential anticancer target, the TPX2–importin‐α interaction. Importin‐α is a nuclear transport protein that regulates the spindle assembly protein TPX2. It has two binding sites—major and minor—to which partners bind. Most nuclear transport cargoes use the major site, whereas TPX2 binds principally to the minor site. Fragment‐based approaches were used to identify small molecules that bind importin‐α, and crystallographic studies identified a lead series that was observed to bind specifically to the minor site, representing the first ligands specific for this site. Structure‐guided synthesis informed the elaboration of these fragments to explore the source of ligand selectivity between the minor and major sites. These ligands are starting points for the development of inhibitors of this protein–protein interaction.     

Monitoring Binding of HIV‐1 Capsid Assembly Inhibitors Using 19F Ligand‐and 15N Protein‐Based NMR and X‐ray Crystallography: Early Hit Validation of a Benzodiazepine Series

The emergence of resistance to existing classes of antiretroviral drugs underlines the need to find novel human immunodeficiency virus (HIV)‐1 targets for drug discovery. The viral capsid protein (CA) represents one such potential target. Recently, a series of benzodiazepine inhibitors was identified via high‐throughput screening using an in vitro capsid assembly assay (CAA). Here, we demonstrate how a combination of NMR and X‐ray co‐crystallography allowed for the rapid characterization of the early hits from this inhibitor series. Ligand‐based ¹⁹F NMR was used to confirm inhibitor binding specificity and reversibility as well as to identify the N‐terminal domain of the capsid (CA_NTD) as its molecular target. Protein‐based NMR (¹H and ¹⁵N chemical shift perturbation analysis) identified key residues within the CA_NTD involved in inhibitor binding, while X‐ray co‐crystallography confirmed the inhibitor binding site and its binding mode. Based on these results, two conformationally restricted cyclic inhibitors were designed to further validate the possible binding modes. These studies were crucial to early hit confirmation and subsequent lead optimization.     

Recognition of the DNA Minor Groove by Thiazotropsin Analogues

Solution‐phase self‐association characteristics and DNA molecular‐recognition properties are reported for three close analogues of minor‐groove‐binding ligands from the thiazotropsin class of lexitropsin molecules; they incorporate isopropyl thiazole as a lipophilic building block. Thiazotropsin B (AcImPy^iPrThDp) shows similar self‐assembly characteristics to thiazotropsin A (FoPyPy^iPrThDp), although it is engineered, by incorporation of imidazole in place of N‐methyl pyrrole, to swap its DNA recognition target from 5′‐ACTA GT‐3′ to 5′‐ACGC GT‐3′. Replacement of the formamide head group in thiazotropsin A by nicotinamide in AIK‐18/51 results in a measureable difference in solution‐phase self‐assembly character and substantially enhanced DNA association characteristics. The structures and associated thermodynamic parameters of self‐assembled ligand aggregates and their complexes with their respective DNA targets are considered in the context of cluster targeting of DNA by minor‐groove complexes.     

TupA: A Tungstate Binding Protein in the Periplasm of Desulfovibrio alaskensis G20

The TupABC system is involved in the cellular uptake of tungsten and belongs to the ABC (ATP binding cassette)-type transporter systems. The TupA component is a periplasmic protein that binds tungstate anions, which are then transported through the membrane by the TupB component using ATP hydrolysis as the energy source (the reaction catalyzed by the ModC component). We report the heterologous expression, purification, determination of affinity binding constants and crystallization of the Desulfovibrio alaskensis G20 TupA. The tupA gene (locus tag Dde_0234) was cloned in the pET46 Enterokinase/Ligation-Independent Cloning (LIC) expression vector, and the construct was used to transform BL21 (DE3) cells. TupA expression and purification were optimized to a final yield of 10 mg of soluble pure protein per liter of culture medium. Native polyacrylamide gel electrophoresis was carried out showing that TupA binds both tungstate and molybdate ions and has no significant interaction with sulfate, phosphate or perchlorate. Quantitative analysis of metal binding by isothermal titration calorimetry was in agreement with these results, but in addition, shows that TupA has higher affinity to tungstate than molybdate. The protein crystallizes in the presence of 30% (w/v) polyethylene glycol 3350 using the hanging-drop vapor diffusion method. The crystals diffract X-rays beyond 1.4 Å resolution and belong to the P2₁ space group, with cell parameters a = 52.25 Å, b = 42.50 Å, c = 54.71 Å, β = 95.43°. A molecular replacement solution was found, and the structure is currently under refinement.     

NVP‐BHG712: Effects of Regioisomers on the Affinity and Selectivity toward the EPHrin Family

Erythropoietin‐producing hepatocellular (EPH) receptors are transmembrane receptor tyrosine kinases. Their extracellular domains bind specifically to ephrin A/B ligands, and this binding modulates intracellular kinase activity. EPHs are key players in bidirectional intercellular signaling, controlling cell morphology, adhesion, and migration. They are increasingly recognized as cancer drug targets. We analyzed the binding of NVP‐BHG712 (NVP) to EPHA2 and EPHB4. Unexpectedly, all tested commercially available NVP samples turned out to be a regioisomer (NVPiso) of the inhibitor, initially described in a Novartis patent application. They only differ by the localization of a single methyl group on either one of two adjacent nitrogen atoms. The two compounds of identical mass revealed different binding modes. Furthermore, both in vitro and in vivo experiments showed that the isomers differ in their kinase affinity and selectivity.     

Water makes the difference: rearrangement of water solvation layer triggers non-additivity of functional group contributions in protein-ligand binding

The binding of four congeneric peptide-like thermolysin inhibitors has been studied by high-resolution crystal structure analysis and isothermal titration calorimetry. The ligands differ only by a terminal carboxylate and/or methyl group. A surprising non-additivity of functional group contributions for the carboxylate and/or methyl groups is detected. Adding the methyl first and then the carboxylate group results in a small Gibbs free energy increase and minor enthalpy/entropy partitioning for the first modification, whereas the second involves a strong affinity increase combined with large enthalpy/entropy changes. However, first adding the carboxylate and then the methyl group yields reverse effects: the acidic group attachment now causes minor effects, whereas the added methyl group provokes large changes. As all crystal structures show virtually identical binding modes, affinity changes are related to rearrangements of the first solvation layer next to the S(2)' pocket. About 20-25 water molecules are visible next to the studied complexes. The added COO(-) groups perturb the local water network in both carboxylated complexes, and the attached methyl groups provide favorable interaction sites for water molecules. Apart from one example, a contiguously connected water network between protein and ligand functional groups is observed in all complexes. In the complex with the carboxylated ligand, which still lacks the terminal methyl group, the water network is unfavorably ruptured. This results in a surprising thermodynamic signature showing only a minor affinity increase upon COO(-) group attachment. Because the further added methyl group provides a favorable interaction site for water, the network can be reestablished, and a strong affinity increase with a large enthalpy/entropy signature is then detected.     

Radical in the Peroxide-Produced F-Type Ferryl Form of Bovine Cytochrome c Oxidase

The reduction of O₂ in respiratory cytochrome c oxidases (CcO) is associated with the generation of the transmembrane proton gradient by two mechanisms. In one of them, the proton pumping, two different types of the ferryl intermediates of the catalytic heme a₃-Cu_B center P and F forms, participate. Equivalent ferryl states can be also formed by the reaction of the oxidized CcO (O) with H₂O₂. Interestingly, in acidic solutions a single molecule of H₂O₂ can generate from the O an additional F-type ferryl form (F^•) that should contain, in contrast to the catalytic F intermediate, a free radical at the heme a₃-Cu_B center. In this work, the formation and the endogenous decay of both the ferryl iron of heme a₃ and the radical in F^• intermediate were examined by the combination of four experimental approaches, isothermal titration calorimetry, electron paramagnetic resonance, and electronic absorption spectroscopy together with the reduction of this form by the defined number of electrons. The results are consistent with the generation of radicals in F^• form. However, the radical at the catalytic center is more rapidly quenched than the accompanying ferryl state of heme a₃, very likely by the intrinsic oxidation of the enzyme itself.     

Hot‐Spot Residues in the Cytochrome P450cam–Putidaredoxin Binding Interface

Cytochrome P450cam (P450cam) is a heme‐containing monooxygenase that catalyzes the hydroxylation of D ‐camphor to produce 5‐exo‐hydroxycamphor. The catalytic cycle of P450cam requires two electrons, both of which are donated by putidaredoxin (Pdx), a ferredoxin containing a [2 Fe–2 S] cluster. Atomic‐resolution structures of the Pdx‐P450cam complex have recently been solved by X‐ray crystallography and paramagnetic NMR spectroscopy. The binding interface showed the potential electron transfer pathways and interactions between Pdx Asp38 and P450cam Arg112, as well as hydrophobic contacts between the Pdx Trp106 and P450cam residues. Several polar residues not previously recognized as relevant for binding were found in the interface. In this study, site‐directed mutagenesis, kinetic measurements, and NMR studies were employed to probe the energetic importance and role of the polar residues in the Pdx–P450cam interaction. A double mutant cycle (DMC) analysis of kinetic data shows that favorable interactions exist between Pdx Tyr33 and P450cam Asp125, as well as between Pdx Ser42 and P450cam His352. The results show that alanine substitutions of these residues and several others do not influence the rates of electron transfer. It is concluded that these polar interactions contribute to partner recognition rather than to electronic coupling of the redox centers.     

Chemical Synthesis of a Synthetic Analogue of the Sialic Acid‐Binding Lectin Siglec‐7

As a basis for the development of an artificial carbohydrate‐binding lectin, we chemically synthesized a domain of siglec‐7, a well‐characterized sialic‐acid‐binding lectin. The full polypeptide (127 amino acids) was constructed by sequential native chemical ligation (NCL) of five peptide segments. Because of poor cysteine availability for NCL, cysteine residues were introduced at suitable ligation sites; these cysteine residues were alkylated in order to mimic native glutamine or asparagine residues, or converted to an alanine residue by desulfurization after NCL. After folding the full‐length polypeptide, the sialic‐acid‐binding activity of the synthetic siglec‐7 was clearly demonstrated by STD NMR and ELISA experiments. We succeeded in the synthesis of siglec‐7 by installing three extra cysteine residues with side‐chain modifications and found that these modifications did not affect the binding activity.