Leukotriene C4 synthase (LTC4S) is a glutathione S-transferase that mediates the biosynthesis of cysteinyl leukotriene C4 (LTC4). Cysteinyl leukotrienes (CysLTs) are lipid mediators that drive type 2 inflammation, bronchoconstriction, and itch. Thus, LTC4S represents an attractive drug target for the treatment of allergic inflammatory diseases, but to date, no LTC4S inhibitor has been tested in patients. Herein, we disclose the discovery and preclinical profiling of the highly selective, oral LTC4S inhibitor GJG057 (compound 1), which exhibits 20-fold improved potency (IC50 = 44 nM) versus clinical candidate AZD9898 (IC50 = 900 nM) in a human whole blood LTC4 release assay. GJG057 showed efficacy in a murine asthma exacerbation model as well as in a mastoparan-induced skin challenge PK/PD model and was profiled in GLP toxicology studies. Despite its promising properties, GJG057 was not progressed into clinical trials as an oral drug. Its potential as a topical drug is currently being evaluated.

The discovery of chiral amino alcohols derived from our previously disclosed clinical LTA4H inhibitor LYS006 is described. In a biochemical assay, their optical antipodes showed similar potencies, which could be rationalized by the cocrystal structures of these compounds bound to LTA4H. Despite comparable stabilities in liver microsomes, they showed distinct in vivo PK properties. Selective O-phosphorylation of the (R)-enantiomers in blood led to clearance values above the hepatic blood flow, whereas the (S)-enantiomers were unaffected and exhibited satisfactory metabolic stabilities in vivo. Introduction of two pyrazole rings led to compound (S)-2 with a more balanced distribution of polarity across the molecule, exhibiting high selectivity and excellent potency in vitro and in vivo. Furthermore, compound (S)-2 showed favorable profiles in 16-week IND-enabling toxicology studies in dogs and rats. Based on allometric scaling and potency in whole blood, compound (S)-2 has the potential for a low oral efficacious dose administered once daily.

The cytosolic metalloenzyme leukotriene A(4) hydrolase (LTA4H) is the final and rate-limiting enzyme in the biosynthesis of pro-inflammatory leukotriene B-4 (LTB4). Preclinical studies have validated this enzyme as an attractive drug target in chronic inflammatory diseases. Despite several attempts, no LTA4H inhibitor has reached the market, yet. Herein, we disclose the discovery and preclinical profile of LYS006, a highly potent and selective LTA4H inhibitor. A focused fragment screen identified hits that could be cocrystallized with LTA4H and inspired a fragment merging. Further optimization led to chiral amino acids and ultimately to LYS006, a picomolar LTA4H inhibitor with exquisite whole blood potency and long-lasting pharmacodynamic effects. Due to its high selectivity and its ability to fully suppress LTB 4 generation at low exposures in vivo, LYS006 has the potential for a best-in-class LTA4H inhibitor and is currently investigated in phase II clinical trials in inflammatory acne, hidradenitis suppurativa, ulcerative colitis, and NASH.

The transcription factor RORγt is an attractive drug-target due to its role in the differentiation of IL-17 producing Th17 cells that play a critical role in the etiopathology of several autoimmune diseases. Identification of starting points for RORγt inverse agonists with good properties has been a challenge. We report the identification of a fragment hit and its conversion into a potent inverse agonist through fragment optimization, growing and merging efforts. Further analysis of the binding mode revealed that inverse agonism was achieved by an unusual mechanism. In contrast to other reported inverse agonists, there is no direct interaction or displacement of helix 12 observed in the crystal structure. Nevertheless, compound 9 proved to be efficacious in a delayed-type hypersensitivity (DTH) inflammation model in rats.

Leukotriene A4 Hydrolase (LTA4H) is a bifunctional zinc metalloenzyme that comprises both epoxide hydrolase and aminopeptidase activity, exerted by two overlapping catalytic sites. The epoxide hydrolase function of the enzyme catalyzes the biosynthesis of the pro-inflammatory lipid mediator leukotriene (LT) B4. Recent literature suggests that the aminopeptidase function of LTA4H is responsible for degradation of the tripeptide Pro-Gly-Pro (PGP) for which neutrophil chemotactic activity has been postulated. It has been speculated that the design of epoxide hydrolase selective LTA4H inhibitors that spare the aminopeptidase pocket may therefore lead to more efficacious anti-inflammatory drugs. In this study, we conducted a high throughput screen (HTS) for LTA4H inhibitors and attempted to rationally design compounds that would spare the PGP degrading function. While we were able to identify compounds with preference for the epoxide hydrolase function, absolute selectivity was not achievable for highly potent compounds. In order to assess the relevance of designing such aminopeptidase-sparing LTA4H inhibitors, we studied the role of PGP in inducing inflammation in different settings in wild type and LTA4H deficient (LTA4H KO) animals but could not confirm its chemotactic potential. Attempting to design highly potent epoxide hydrolase selective LTA4H inhibitors, therefore seems to be neither feasible nor relevant.

The predominant expression of phosphoinositide 3-kinase δ (PI3Kδ) in leukocytes and its critical role in B and T cell functions led to the hypothesis that selective inhibitors of this isoform would have potential as therapeutics for the treatment of allergic and inflammatory disease. Targeting specifically PI3Kδ should avoid potential side effects associated with the ubiquitously expressed PI3Kα and β isoforms. We disclose how morphing the heterocyclic core of previously discovered 4,6-diaryl quinazolines to a significantly less lipophilic 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidine, followed by replacement of one of the phenyl groups with a pyrrolidine-3-amine, led to a compound series with an optimal on-target profile and good ADME properties. A final lipophilicity adjustment led to the discovery of CDZ173 (leniolisib), a potent PI3Kδ selective inhibitor with suitable properties and efficacy for clinical development as an anti-inflammatory therapeutic. In vitro, CDZ173 inhibits a large spectrum of immune cell functions, as demonstrated in B and T cells, neutrophils, monocytes, basophils, plasmocytoid dendritic cells, and mast cells. In vivo, CDZ173 inhibits B cell activation in rats and monkeys in a concentration- and time-dependent manner. After prophylactic or therapeutic dosing, CDZ173 potently inhibited antigen-specific antibody production and reduced disease symptoms in a rat collagen-induced arthritis model. Structurally, CDZ173 differs significantly from the first generation of PI3Kδ and PI3Kγδ-selective clinical compounds. Therefore, CDZ173 could differentiate by a more favorable safety profile. CDZ173 is currently in clinical studies in patients suffering from primary Sjögren's syndrome and in APDS/PASLI, a disease caused by gain-of-function mutations of PI3Kδ.

In the recent years, PI3Kδ has emerged as a promising target for the treatment of B- and T-cell mediated inflammatory diseases. We present a cellular assay activity analysis for our previously reported 4,6-diaryl quinazoline PI3Kδ inhibitor series that suggests an optimal logP range between 2 and 3. We discovered novel analogues in this lipophilicity space that feature a chiral pyrrolidineoxy-group as a replacement for the position-4 aromatic ring of 4,6-diaryl quinazolines. These Fsp3 enriched derivatives retain potency and selectivity towards PI3Kδ. Compared to 4,6-diaryl quinazolines, their permeability profile is improved and molecular weight as well as PSA are reduced. These modifications offer additional possibilities for derivative generation in a favorable physicochemical property space and thus increase the chances to identify a clinical candidate.

Inhibition of the lipid kinase PI3Kδ is a promising principle to treat B and T cell driven inflammatory diseases. Using a scaffold deconstruction-reconstruction strategy, we identified 4-aryl quinazolines that were optimized into potent PI3Kδ isoform selective analogues with good pharmacokinetic properties. With compound 11, we illustrate that biochemical PI3Kδ inhibition translates into modulation of isoform-dependent immune cell function (human, rat, and mouse). After oral administration of compound 11 to rats, proximal PD markers are inhibited, and dose-dependent efficacy in a mechanistic plaque forming cell assay could be demonstrated.

Short targeted MD trajectories are used to expel ligands from binding sites. The expulsion is governed by a linear increase of the target RMSD value, growing from zero to an arbitrary chosen final RMSD that forces the ligand to a selected distance outside of the receptor. The RMSD lag (i.e., the difference between the imposed and the actual RMSD) can be used to follow barriers encountered by the ligand during its way out of the receptor. The force constant used for the targeted MD can transform the RMSD lag into a strain energy. Integration of the (time-dependent) strain energy over time yields a value with the dimensions of “action” (i.e, energy multiplied by time) and can serve as a measure for the overall effort required to extract the ligand from its binding site. Possibilities to compare (numerically and graphically) the randomly detected exit pathways are discussed. As an example, the method is tested on the exit of bisphenol A from the human estrogen-related receptor \(\gamma\) and of GW0072 from the peroxysome proliferator activated receptor.

We validate an automated implementation of a combined Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) method (VSGB 2.0 energy model) on a large and diverse selection of protein-ligand complexes (855 complexes). Although this data set is diverse with respect to both protein families and ligands, after carefully removing flawed structures, a significant correlation (R(2) = 0.63) between calculated and experimental binding affinities is obtained. Consistent explanations for "outlier" complexes are found. Visual analysis of the crystal structures and recourse to the original literature reveal that neglect of explicit solvent, ligand strain, and entropy contribute to the under- and overestimation of computed affinities. The limits of the Molecular Mechanics/Implicit Solvent approach to accurately estimate protein-ligand binding affinities is discussed as is the influence of the quality of protein-ligand complexes on computed free energy binding values.

Oxysterols are oxygenated cholesterol derivates that are emerging as a physiologically important group of molecules. Although they regulate a range of cellular processes, only few oxysterol-binding effector proteins have been identified, and the knowledge of their binding mode is limited. Recently, the family of G protein-coupled seven transmembrane-spanning receptors (7TM receptors) was added to this group. Specifically, the Epstein-Barr virus-induced gene 2 (EBI2 or GPR183) was shown to be activated by several oxysterols, most potently by 7α,25-dihydroxycholesterol (7α,25-OHC). Nothing is known about the binding mode, however. Using mutational analysis, we identify here four key residues for 7α,25-OHC binding: Arg-87 in TM-II (position II:20/2.60), Tyr-112 and Tyr-116 (positions III:09/3.33 and III:13/3.37) in TM-III, and Tyr-260 in TM-VI (position VI:16/6.51). Substituting these residues with Ala and/or Phe results in a severe decrease in agonist binding and receptor activation. Docking simulations suggest that Tyr-116 interacts with the 3β-OH group in the agonist, Tyr-260 with the 7α-OH group, and Arg-87, either directly or indirectly, with the 25-OH group, although nearby residues likely also contribute. In addition, Tyr-112 is involved in 7α,25-OHC binding but via hydrophobic interactions. Finally, we show that II:20/2.60 constitutes an important residue for ligand binding in receptors carrying a positively charged residue at this position. This group is dominated by lipid- and nucleotide-activated receptors, here exemplified by the CysLTs, P2Y12, and P2Y14. In conclusion, we present the first molecular characterization of oxysterol binding to a 7TM receptor and identify position II:20/2.60 as a generally important residue for ligand binding in certain 7TM receptors.

Parathyroid hormone (PTH) is an effective bone anabolic agent. However, only when administered by daily sc injections exposure of short duration is achieved, a prerequisite for an anabolic response. Instead of applying exogenous PTH, mobilization of endogenous stores of the hormone can be envisaged. The secretion of PTH stored in the parathyroid glands is mediated by a calcium sensing receptor (CaSR) a GPCR localized at the cell surface. Antagonists of CaSR (calcilytics) mimic a state of hypocalcaemia and stimulate PTH release to the bloodstream. Screening of the internal compound collection for inhibition of CaSR signaling function afforded 2a. In vitro potency could be improved >1000 fold by optimization of its chemical structure. The binding mode of our compounds was predicted based on molecular modeling and confirmed by testing with mutated receptors. While the compounds readily induced PTH release after iv application a special formulation was needed for oral activity. The required profile was achieved by using microemulsions. Excellent PK/PD correlation was found in rats and dogs. High levels of PTH were reached in plasma within minutes which reverted to baseline in about 1-2 h in both species.

BACKGROUND:Mast cells are secretory immune cells whose degranulation can provoke acute allergic reactions. It is presently unclear, however, whether an individual mast cell can repeatedly degranulate or turns dysfunctional after a single antigen stimulus. This work thus aims to better define the mast cell life cycle, with particular focus on new target structures for therapeutic or diagnostic approaches in allergy.METHODS:Monoclonal antibodies were raised against degranulated cord blood-derived human mast cells. A subset of these antibodies that exclusively recognized degranulated mast cells, but did not cross-react with quiescent mast cells or other hematopoietic cell types, became key reagents in subsequent experiments.RESULTS:We identified a granular variant of tetraspanin CD63 as an exclusive molecular marker of degranulated human mast cells. Mutant analyses indicate that a cysteine cluster around residue C170 and protein glycosylation at residue N172 account for the antibody specificity. Here, we show that mast cells, which underwent an initial FcεRI-mediated degranulation, can be degranulated for at least another cycle in vitro. Repeated degranulation, however, requires an IgE/antigen stimulus that differs from the preceding one. Furthermore, the new variant-specific anti-CD63 antibodies effectively impair repeated cycles of mast cell degranulation.CONCLUSION:Our findings indicate that mast cells are stable, multiple-use cells, which are capable of surviving and delivering several consecutive hits. Surface expression of the novel CD63 variant is a distinguishing feature of such primed cells. Reagents directed against this molecular hallmark may thus become valuable diagnostic and therapeutic agents.

A three-dimensional model of the human Calcium-sensing receptor (CaSR) seven transmembrane domain was built via a novel sequence alignment method based on the conserved contacts in proteins using the crystal structure of bovine rhodopsin as the template. This model was tested by docking NPS 2143, the first identified allosteric antagonist of CaSR. In our model, Glu837 plays a critical role in anchoring the protonated nitrogen atom and hydroxy oxygen atom of NPS 2143. The phenyl moiety of the ligand contacts residues Phe668, Pro672, and Ile841. The naphthalene moiety is surrounded by several hydrophobic residues, including Phe684, Phe688, and Phe821. Our model appears to be consistent with all six residues that have been demonstrated to be critical for NPS 2143 binding, in contrast with existing homology models based on traditional sequence alignment of CaSR to rhodopsin. This provides validation of our sequence alignment method and the use of the rhodopsin backbone as the initial structure in homology modeling of other G protein-coupled receptors that are not members of the rhodopsin family.