Structure-activity relationship studies of Bz amide-containing α-GalCer derivatives as natural killer T cell modulators.

CD1d is a non-polymorphic antigen-presenting glycoprotein that recognizes glycolipids as ligands. Ligands bind to the hydrophobic grooves of CD1d, and the resulting ligand-CD1d complexes activate natural killer T (NKT) cells by means of T cell receptor recognition, leading to the secretion of various cytokines. However, details of the ligand recognition mechanism of a large hydrophobic ligand binding pocket and the relationship between cytokine induction and ligand structure are unclear. We report the synthesis of α-GalCer derivatives containing a Bz amide group having various substituting groups in the ceramide moiety, and the analysis of the structure-activity relationships. The assays reveal that the Bz amide-containing CD1d ligands function as NKT cell modulators displaying Th2 cytokine biasing responses. Furthermore, molecular dynamics simulation studies suggest that the phenyl groups can interact with the aromatic amino acid residues in the lipid binding pocket of CD1d.

Water-mediated recognition of simple alkyl chains by heart-type fatty-acid-binding protein.

Long-chain fatty acids (FAs) with low water solubility require fatty-acid-binding proteins (FABPs) to transport them from cytoplasm to the mitochondria for energy production. However, the precise mechanism by which these proteins recognize the various lengths of simple alkyl chains of FAs with similar high affinity remains unknown. To address this question, we employed a newly developed calorimetric method for comprehensively evaluating the affinity of FAs, sub-Angstrom X-ray crystallography to accurately determine their 3D structure, and energy calculations of the coexisting water molecules using the computer program WaterMap. Our results clearly showed that the heart-type FABP (FABP3) preferentially incorporates a U-shaped FA of C10-C18 using a lipid-compatible water cluster, and excludes longer FAs using a chain-length-limiting water cluster. These mechanisms could help us gain a general understanding of how proteins recognize diverse lipids with different chain lengths.

General Theory of Fragment Linking in Molecular Design: Why Fragment Linking Rarely Succeeds and How to Improve Outcomes.

Linking two fragments binding in nearby subpockets together has become an important technique in fragment-based drug discovery to optimize the binding potency of fragment hits. Despite the expected favorable translational and orientational entropic contribution to the binding free energy of the linked molecule, brute force enumeration of chemical linker for linking fragments is rarely successful, and the vast majority of linked molecules do not exhibit the expected gains of binding potency. In this paper, we examine the physical factors that contribute to the change of binding free energy from fragment linking and develop a method to rigorously calculate these different physical contributions. We find from these analyses that multiple confounding factors make successful fragment linking strategies rare, including (1) possible change of the binding mode of the fragments in the linked state compared to separate binding of the fragments, (2) unfavorable intramolecular strain energy of the bioactive conformation of the linked molecule, (3) unfavorable interaction between the linker and the protein, (4) favorable interaction energies between two fragments in solution when not chemically linked that offset the expected entropy loss for the formation of fragment pair, (5) complex compensating configurational entropic effects beyond the simplistic rotational and translational analysis. We here have applied a statistically mechanically rigorous approach to compute the fragment linking coefficients of 10 pharmaceutically interesting systems and quantify the contribution of each physical component to the binding free energy of the linked molecule. Based on these studies, we have found that the change in the relative configurational entropy of the two fragments in the protein binding pocket (a term neglected to our knowledge in all previous analyses) substantially offsets the favorable expected rotational and translational entropic contributions to the binding free energy of the linked molecule. This configurational restriction of the fragments in the binding pocket of the proteins is found to be, in our analysis, the dominant reason why most fragment linking strategies do not exhibit the expected gains of binding potency. These findings have further provided rich physical insights, which we expect should facilitate more successful fragment linking strategies to be formulated in the future.

Development of self-indicating resin.

Previously, we have reported the development and application of self-indicating resins (SIR), materials which can indicate presence or absence of amines in the reaction solution by the conspicuous color change of a phenolsulfophthalein type dye immobilized on resin beads [2a]. Although the functionality necessary for attaching the dye to the resins could be readily introduced by the Suzuki-Miyaura coupling during the synthesis of the SIR 1, this approach was only applicable to the dyes containing suitable functionality for the cross-coupling reaction. In this article we describe a new approach of immobilizing the indicating dyes onto the resin support. The dyes suitable for loading onto aminomethyl polystyrene (PS) resin were prepared by Friedel-Crafts reaction of 2-sulfoterephthalic anhydride with a wide range of phenols. Using this new route, the SIR 6c was readily prepared in >100g quantities. Use of the SIR 6c in the synthesis of a 144 member urea library was demonstrated and the SIR successfully indicated the endpoint of the reaction between amines and isocyanates.

Isolated Polar Amino Acid Residues Modulate Lipid Binding in the Large Hydrophobic Cavity of CD1d.

The CD1d protein is a nonpolymorphic MHC class I-like protein that controls the activation of natural killer T (NKT) cells through the presentation of self- and foreign-lipid ligands, glycolipids, or phospholipids, leading to the secretion of various cytokines. The CD1d contains a large hydrophobic lipid binding pocket: the A' pocket of CD1d, which recognizes hydrophobic moieties of the ligands, such as long fatty acyl chains. Although lipid-protein interactions typically rely on hydrophobic interactions between lipid chains and the hydrophobic sites of proteins, we showed that the small polar regions located deep inside the hydrophobic A' pocket could be used for the modulation of the lipid binding. A series of the ligands, α-galactosyl ceramide (α-GalCer) derivatives containing polar groups in the acyl chain, was synthesized, and the structure-activity relationship studies demonstrated that simple modification from a methylene to an amide group in the long fatty acyl chain, when introduced at optimal positions, enhanced the CD1d recognition of the glycolipid ligands. Formation of hydrogen bonds between the amide group and the polar residues was supported by molecular dynamics (MD) simulations and WaterMap calculations. The computational studies suggest that localized hydrating water molecules may play an important role in the ligand recognition. Here, the results showed that confined polar residues in the large hydrophobic lipid binding pockets of the proteins could be potential targets to modulate the affinity for its ligands.

Partial reduction of pyridinium salts as a versatile route to dihydropyridones.

[reaction: see text] The addition of two electrons to a pyridinium salt turns it into a nucleophile. The intermediate generated by the reduction of such salts can be reacted successfully with a range of different electrophiles (acids, alkyl halides, and carbonyl compounds) and the intermediate hydrolyzed in situ to provide a wide range of dihydropyridones. Each position on the dihydropyridone ring is then accessible using standard synthetic manipulations.

pH indicating resins.

pH indicating resins were prepared by covalently attaching carboxylic acid derivatives of sulfthalein dyes, synthesized using a Suzuki cross-coupling, onto resin beads. The resin-bound indicators showed the expected colour changes according to pH and their behaviour was analysed using a micro UV/Vis spectrometer.

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.

GAMESS as a free quantum-mechanical platform for drug research.

Driven by a steady improvement of computational hardware and significant progress in ab initio method development, quantum-mechanical approaches can now be applied to large biochemical systems and drug design. We review the methods implemented in GAMESS, which are suitable to calculate large biochemical systems. An emphasis is put on the fragment molecular orbital method (FMO) and quantum mechanics interfaced with molecular mechanics (QM/MM). The use of FMO in the protein-ligand binding, structure-activity relationship (SAR) studies, fragment- and structure-based drug design (FBDD/SBDD) is discussed in detail.

Irreversible 4-Aminopiperidine Transglutaminase 2 Inhibitors for Huntington's Disease.

A new series of potent TG2 inhibitors are reported that employ a 4-aminopiperidine core bearing an acrylamide warhead. We establish the structure-activity relationship of this new series and report on the transglutaminase selectivity and in vitro ADME properties of selected compounds. We demonstrate that the compounds do not conjugate glutathione in an in vitro setting and have superior plasma stability over our previous series.

Discovery and structure-activity relationship of potent and selective covalent inhibitors of transglutaminase 2 for Huntington's disease.

Tissue transglutaminase 2 (TG2) is a multifunctional protein primarily known for its calcium-dependent enzymatic protein cross-linking activity via isopeptide bond formation between glutamine and lysine residues. TG2 overexpression and activity have been found to be associated with Huntington's disease (HD); specifically, TG2 is up-regulated in the brains of HD patients and in animal models of the disease. Interestingly, genetic deletion of TG2 in two different HD mouse models, R6/1 and R6/2, results in improved phenotypes including a reduction in neuronal death and prolonged survival. Starting with phenylacrylamide screening hit 7d, we describe the SAR of this series leading to potent and selective TG2 inhibitors. The suitability of the compounds as in vitro tools to elucidate the biology of TG2 was demonstrated through mode of inhibition studies, characterization of druglike properties, and inhibition profiles in a cell lysate assay.

Compound Design by Fragment-Linking.

The linking together of two fragment compounds that bind to distinct protein sub-sites can lead to a superadditivity of binding affinities, in which the binding free energy of the linked fragments exceeds the simple sum of the binding energies of individual fragments (linking coefficient E<1). However, a review of the literature shows that such events are relatively rare and, in the majority of the cases, linking coefficients are far from optimal being much greater than 1. It is critical to design a linker that does not disturb the original binding poses of each fragment in order to achieve successful linking. However, such an ideal linker is often difficult to design and even more difficult to actually synthesize. We suggest that the chance of achieving successful fragment linking can be significantly improved by choosing a fragment pair that consists of one fragment that binds by strong H-bonds (or non-classical equivalents) and a second fragment that is more tolerant of changes in binding mode (hydrophobic or vdW binders). We also propose that the fragment molecular orbital (FMO) calculations can be used to analyse the nature of the binding interactions of the fragment hits for the selection of fragments for evolution, merging and linking in order to optimize the chance of success.

Prediction of cyclin-dependent kinase 2 inhibitor potency using the fragment molecular orbital method.

BACKGROUND: The reliable and robust estimation of ligand binding affinity continues to be a challenge in drug design. Many current methods rely on molecular mechanics (MM) calculations which do not fully explain complex molecular interactions. Full quantum mechanical (QM) computation of the electronic state of protein-ligand complexes has recently become possible by the latest advances in the development of linear-scaling QM methods such as the ab initio fragment molecular orbital (FMO) method. This approximate molecular orbital method is sufficiently fast that it can be incorporated into the development cycle during structure-based drug design for the reliable estimation of ligand binding affinity. Additionally, the FMO method can be combined with approximations for entropy and solvation to make it applicable for binding affinity prediction for a broad range of target and chemotypes. RESULTS: We applied this method to examine the binding affinity for a series of published cyclin-dependent kinase 2 (CDK2) inhibitors. We calculated the binding affinity for 28 CDK2 inhibitors using the ab initio FMO method based on a number of X-ray crystal structures. The sum of the pair interaction energies (PIE) was calculated and used to explain the gas-phase enthalpic contribution to binding. The correlation of the ligand potencies to the protein-ligand interaction energies gained from FMO was examined and was seen to give a good correlation which outperformed three MM force field based scoring functions used to appoximate the free energy of binding. Although the FMO calculation allows for the enthalpic component of binding interactions to be understood at the quantum level, as it is an in vacuo single point calculation, the entropic component and solvation terms are neglected. For this reason a more accurate and predictive estimate for binding free energy was desired. Therefore, additional terms used to describe the protein-ligand interactions were then calculated to improve the correlation of the FMO derived values to experimental free energies of binding. These terms were used to account for the polar and non-polar solvation of the molecule estimated by the Poisson-Boltzmann equation and the solvent accessible surface area (SASA), respectively, as well as a correction term for ligand entropy. A quantitative structure-activity relationship (QSAR) model obtained by Partial Least Squares projection to latent structures (PLS) analysis of the ligand potencies and the calculated terms showed a strong correlation (r2 = 0.939, q2 = 0.896) for the 14 molecule test set which had a Pearson rank order correlation of 0.97. A training set of a further 14 molecules was well predicted (r2 = 0.842), and could be used to obtain meaningful estimations of the binding free energy. CONCLUSIONS: Our results show that binding energies calculated with the FMO method correlate well with published data. Analysis of the terms used to derive the FMO energies adds greater understanding to the binding interactions than can be gained by MM methods. Combining this information with additional terms and creating a scaled model to describe the data results in more accurate predictions of ligand potencies than the absolute values obtained by FMO alone.

Novel MK2 inhibitors by fragment screening.

Inhibitors of MAPKAP kinase 2 (MK2) are expected to attenuate the p38alpha signal transduction pathway in macrophages in a similar way to p38alpha inhibitors and to have a lower propensity for toxic side effects that have slowed the clinical development of the latter. Therefore, novel MK2 inhibitors may find therapeutic application in acute and chronic, TNFalpha-mediated inflammatory conditions like rheumatoid arthritis and others. Herein we have applied fragment screening, using physiologically relevant bioassays and fragment binding mode mapping by protein-observed NMR spectroscopy to the discovery of novel efficient chemical starting points for MK2.

Fragment-based identification of Hsp90 inhibitors.

Heat shock protein 90 (Hsp90) plays a key role in stress response and protection of the cell against the effects of mutation. Herein we report the identification of an Hsp90 inhibitor identified by fragment screening using a high-concentration biochemical assay, as well as its optimisation by in silico searching coupled with a structure-based drug design (SBDD) approach.

Captured and cross-linked palladium nanoparticles.

Polystyrene-poly(ethylene glycol) resin-captured cross-linked palladium nanopaticles were prepared via a straightforward route, and their heterogeneous behavior was truly confirmed by various tests. They were applied to aqueous Suzuki cross-coupling reactions with various aryl bromides and recycled up to six times without loss of activity.

The ammonia-free partial reduction of substituted pyridinium salts.

This paper reports a study into the partial reduction of N-alkylpyridinium salts together with subsequent elaboration of the intermediates thus produced. Activation of a pyridinium salt by placing an ester group at C-2, allows the addition of two electrons to give a synthetically versatile enolate intermediate which can be trapped with a variety of electrophiles. Furthermore, the presence of a 4-methoxy substituent on the pyridine nucleus enhances the stability of the enolate reaction products, and hydrolysis in situ gives stable dihydropyridone derivatives in good yields. These versatile compounds are prepared in just three steps from picolinic acid and can be derivatised at any position on the ring, including nitrogen when a p-methoxybenzyl group is used as the N-activating group on the pyridinium salt. This publication describes our exploration of the optimum reducing conditions, the most appropriate N-alkyl protecting group, as well as the best position on the ring for the methoxy group. Electrochemical techniques which mimic the synthetic reducing conditions are utilised and they give clear support for our proposed mechanism of reduction in which there is a stepwise addition of two electrons to the heterocycle, mediated by di-tert-butylbiphenyl (DBB). Moreover, there is a correlation between the viability of reduction of a given heterocycle under synthetic conditions and its electrochemical response; this offers the potential for use of electrochemistry in predicting the outcome of such reactions.

A soluble-polymer system for the asymmetric transfer hydrogenation of ketones.

The appropriate combination of methacrylate polymers permits the synthesis of a soluble polymer for use in ruthenium(II)-catalyzed asymmetric transfer hydrogenation reactions. Using a 7:3 copolymer of a poly(ethylene glycol) ester and a hydroxyethyl ester, a derived ruthenium(II)/norephedrine complex catalyses reduction of acetophenone in up to 95% yield and 81% ee.

Tetrahydrofuran-based amino acids as library scaffolds.

A furanose sugar amino acid (SAA) has been utilized as a library scaffold for the first time. Two furanose SAA scaffolds were examined to illustrate their potential for derivatization. The resulting 99-member library contained three orthogonal points of diversification that allowed easy access to ethers and carbamates from a hydroxyl moiety, a range of ureas from an azide (via an amine), and a range of amides from a methyl ester. The novel amide formation (by displacement of the methoxide from the methyl ester moiety) was achieved in good yield and purity with high structural confidence. Full characterization of several library intermediates (including a crystal structure) was obtained. The library was submitted for antibacterial screening.