A small-molecule fusion inhibitor of influenza virus is orally active in mice.

Recent characterization of broadly neutralizing antibodies (bnAbs) against influenza virus identified the conserved hemagglutinin (HA) stem as a target for development of universal vaccines and therapeutics. Although several stem bnAbs are being evaluated in clinical trials, antibodies are generally unsuited for oral delivery. Guided by structural knowledge of the interactions and mechanism of anti-stem bnAb CR6261, we selected and optimized small molecules that mimic the bnAb functionality. Our lead compound neutralizes influenza A group 1 viruses by inhibiting HA-mediated fusion in vitro, protects mice against lethal and sublethal influenza challenge after oral administration, and effectively neutralizes virus infection in reconstituted three-dimensional cell culture of fully differentiated human bronchial epithelial cells. Cocrystal structures with H1 and H5 HAs reveal that the lead compound recapitulates the bnAb hotspot interactions.

Potent peptidic fusion inhibitors of influenza virus.

Influenza therapeutics with new targets and mechanisms of action are urgently needed to combat potential pandemics, emerging viruses, and constantly mutating strains in circulation. We report here on the design and structural characterization of potent peptidic inhibitors of influenza hemagglutinin. The peptide design was based on complementarity-determining region loops of human broadly neutralizing antibodies against the hemagglutinin (FI6v3 and CR9114). The optimized peptides exhibit nanomolar affinity and neutralization against influenza A group 1 viruses, including the 2009 H1N1 pandemic and avian H5N1 strains. The peptide inhibitors bind to the highly conserved stem epitope and block the low pH-induced conformational rearrangements associated with membrane fusion. These peptidic compounds and their advantageous biological properties should accelerate the development of new small molecule- and peptide-based therapeutics against influenza virus.

Biophysical and physicochemical methods differentiate highly ligand-efficient human D-amino acid oxidase inhibitors.

Many early drug research efforts are too reductionist thereby not delivering key parameters such as kinetics and thermodynamics of target-ligand binding. A set of human D-Amino Acid Oxidase (DAAO) inhibitors 1-6 was applied to demonstrate the impact of key biophysical techniques and physicochemical methods in the differentiation of chemical entities that cannot be adequately distinguished on the basis of their normalized potency (ligand efficiency) values. The resulting biophysical and physicochemical data were related to relevant pharmacodynamic and pharmacokinetic properties. Surface Plasmon Resonance data indicated prolonged target-ligand residence times for 5 and 6 as compared to 1-4, based on the observed k(off) values. The Isothermal Titration Calorimetry-derived thermodynamic binding profiles of 1-6 to the DAAO enzyme revealed favorable contributions of both ΔH and ΔS to their ΔG values. Surprisingly, the thermodynamic binding profile of 3 elicited a substantially higher favorable contribution of ΔH to ΔG in comparison with the structurally closely related fused bicyclic acid 4. Molecular dynamics simulations and free energy calculations of 1, 3, and 4 led to novel insights into the thermodynamic properties of the binding process at an atomic level and in the different thermodynamic signatures of 3 and 4. The presented holistic approach is anticipated to facilitate the identification of compounds with best-in-class properties at an early research stage.

N'-(arylsulfonyl)pyrazoline-1-carboxamidines as novel, neutral 5-hydroxytryptamine 6 receptor (5-HT6R) antagonists with unique structural features.

The 5-HT(6) receptor (5-HT(6)R) has been in the spotlight for several years regarding CNS-related diseases. We set out to discover novel, neutral 5-HT(6)R antagonists to improve off-target selectivity compared to basic amine-containing scaffolds dominating the field. High-throughput screening identified the N'-(sulfonyl)pyrazoline-1-carboxamidine scaffold as a promising neutral core for starting hit-to-lead. Medicinal chemistry, molecular modeling, small molecule NMR and X-ray crystallography were subsequently applied to optimize the leads into antagonists (compounds 1-49) displaying high 5-HT(6)R affinity with optimal off-target selectivity. Unique structural features include a pseudoaromatic system and an internal hydrogen bond freezing the bioactive conformation. While physicochemical properties and CNS availability were generally favorable, significant efforts had to be made to improve metabolic stability. The optimized structure 42 is an extremely selective, hERG-free, high-affinity 5-HT(6)R antagonist showing good human in vitro metabolic stability. Rat pharmacokinetic data were sufficiently good to enable further in vivo profiling.

Assignment of the orphan nuclear receptor Nurr1 by NMR.

The orphan nuclear receptor Nurr1 has been implicated in a number of conditions including Parkinson's disease and Schizophrenia. As such, it is of interest to study its interactions with other proteins, possibly mediated by small molecules, considering possible use as a drug target. We produced (2)H, (15)N, (13)C labelled-Nurr1 to generate the backbone amide NH, carbonyl C', C(alpha) and C(beta) assignments. About 84.0% of residues could be assigned. Most of the 37 missing assignments fall in 3 regions of the protein. Two of these surround a putative ligand-binding region of Nurr1, suggesting that this region of the protein is flexible, despite the ligand-binding pocket being filled with hydrophobic side-chains from residues surrounding the ligand binding pocket.

Ligand-based NMR spectra demonstrate an additional phytoestrogen binding site for 17beta-hydroxysteroid dehydrogenase type 1.

The enzyme 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1) has become an important drug target for breast cancer because it catalyzes the interconversion of estrone to the biologically more potent estradiol which also plays a crucial role in the etiology of breast cancer. Patients with an increased expression of the 17beta-HSD1 gene have a significantly worse outcome than patients without. Inhibitors for 17beta-HSD1 are therefore included in therapy development. Here we have studied binding of 17beta-HSD1 to substrates and a number of inhibitors using NMR spectroscopy. Ligand observed NMR spectra show a strong pH dependence for the phytoestrogens luteolin and apigenin but not for the natural ligands estradiol and estrone. Moreover, NMR competition experiments show that the phytoestrogens do not replace the estrogens despite their similar inhibition levels in the in vitro assay. These results strongly support an additional 17beta-HSD1 binding site for phytoestrogens which is neither the substrate nor the co-factor binding site. Docking experiments suggest the dimer interface as a possible location. An additional binding site for the phytoestrogens may open new opportunities for the design of inhibitors, not only for 17beta-HSD1, but also for other family members of the short chain dehydrogenases.

Site-selective labeling strategies for screening by NMR.

NMR based screening has become an important tool in the pharmaceutical industry. Methods that provide information on the location of small molecule binding sites on the surface of a drug target (e. g. SAR-by-NMR and related techniques) are of particular interest. In order to extend the applicability of such techniques to drug targets of higher molecular weight, selective labeling strategies may be employed. Dual-amino acid selective labeling and site directed non-native amino acid replacement (SNAAR) allow for the selective detection of NMR resonances of a specific amino acid residue. This results in significantly reduced spectral complexity, which not only enables application to higher molecular weight systems, but also eliminates the need for sequential resonance assignment in order to identify the binding site. Regio-selective (or segmental) labeling of an entire protein domain of a multi domain protein may also be achieved. Labeling only a selected part of a multi domain protein (e. g. a catalytic or ligand binding domain) is an attractive way to simplify the spectral interpretation without disturbing the system under study.

Structure-based screening as applied to human FABP4: a highly efficient alternative to HTS for hit generation.

The time-limiting step in HTS often is the development of an appropriate assay. In addition, hits from HTS fairly often turn out to be false positives and generally display unfavorable properties for further development. Here we describe an alternative process for hit generation, applied to the human adipocyte fatty acid binding protein FABP4. A small molecular ligand for FABP4 that blocks the binding of endogenous ligands may be developed into a drug for the treatment of type-2 diabetes. Using NMR spectroscopy, we screened FABP4 for low-affinity binders in a diversity library consisting of small soluble scaffolds, which yielded 52 initial hits in total. The potencies of these hits were ranked, and crystal structures of FABP4 complexes for two of the hits were obtained. The structural data were subsequently used to direct similarity searches for available analogues, as well as chemical synthesis of 12 novel analogues. In this way, a series of three selective FABP4 ligands with attractive pharmacochemical profiles and potencies of 10 microM or better was obtained.

Structure-based screening and design in drug discovery.

Structure-based screening represents an integrated approach for the identification and optimization of hits by the combined use of nuclear magnetic resonance (NMR) spectroscopy, homology modeling and X-ray crystallography. A general feature of the methodology is the introduction of structure-based methods (NMR, modeling and X-ray) early in the drug discovery process to optimize hits in terms of their affinities and specificities. This approach promises to deliver leads with improved physicochemical properties as compared with leads generated from a traditional HTS program. This review presents examples of structure-based screening from published and in-house drug discovery projects.

Site-selective screening by NMR spectroscopy with labeled amino acid pairs.

A new method for site-selective screening by NMR is presented. The core of the new method is the dual amino acid sequence specific labeling technique. Amino acid X is labeled with (13)C and amino acid Y is labeled with (15)N. Provided only one XY pair occurs in the amino acid sequence, only one signal in the 1D carbonyl (13)C spectrum will display a splitting due to the (1)J(C'N) coupling. Using this labeling strategy it is possible to screen selectively for binding to a selected epitope without the need for sequence specific assignments. An HNCO spectrum (1D or 2D) can be used either directly as a screening experiment or indirectly to identify what signals to monitor in a 2D (1)H-(15)N correlation spectrum. Chemical shift perturbations upon addition of a potential ligand are easily detected even for large proteins due to the reduced spectral complexity resulting from the use of a selectively labeled sample. The new technique is demonstrated on the human adipocyte fatty acid binding protein FABP-4. Due to the reduced spectral complexity, the method should be applicable to larger proteins than are conventional methods.