Discovery of Amino Alcohols as Highly Potent, Selective, and Orally Efficacious Inhibitors of Leukotriene A4 Hydrolase.

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.

Optimizing a Weakly Binding Fragment into a Potent RORγt Inverse Agonist with Efficacy in an in Vivo Inflammation Model.

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.

Feasibility and physiological relevance of designing highly potent aminopeptidase-sparing leukotriene A4 hydrolase inhibitors.

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.

Discovery of CDZ173 (Leniolisib), Representing a Structurally Novel Class of PI3K Delta-Selective Inhibitors.

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 ß 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.

Discovery of novel pyrrolidineoxy-substituted heteroaromatics as potent and selective PI3K delta inhibitors with improved physicochemical properties.

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.

Discovery and Pharmacological Characterization of Novel Quinazoline-Based PI3K Delta-Selective Inhibitors.

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.

Extracting ligands from receptors by reversed targeted molecular dynamics.

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 [Formula: see text] and of GW0072 from the peroxysome proliferator activated receptor.

MM/GBSA binding energy prediction on the PDBbind data set: successes, failures, and directions for further improvement.

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.

Molecular characterization of oxysterol binding to the Epstein-Barr virus-induced gene 2 (GPR183).

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 3ß-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.

1-Alkyl-4-phenyl-6-alkoxy-1H-quinazolin-2-ones: a novel series of potent calcium-sensing receptor antagonists.

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.

Improved model building and assessment of the Calcium-sensing receptor transmembrane domain.

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.

Receptors for protons or lipid messengers or both?

The subfamily of G protein-coupled receptors comprising GPR4, OGR1, TDAG8, and G2A was originally characterized as a group of proteins mediating biological responses to the lipid messengers sphingosylphosphorylcholine (SPC), lysophosphatidylcholine (LPC), and psychosine. We challenged this view by reporting that OGR1 and GPR4 sense acidic pH and that this process is not affected by concentrations of SPC or LPC previously reported as agonistic. The original publications describing GPR4, OGR1, and G2A as receptors for LPC or SPC have now been retracted, and the first studies exploring receptors of this family as pH sensors in physiology have appeared. Here we review the status of this field and we confirm that GPR4, OGR1, and TDAG8 should be considered as proton-sensing receptors. Negative regulation of these receptors by high micromolar concentrations of lipids appears not specific in our experiments.

Development and testing of a general amber force field.

We describe here a general Amber force field (GAFF) for organic molecules. GAFF is designed to be compatible with existing Amber force fields for proteins and nucleic acids, and has parameters for most organic and pharmaceutical molecules that are composed of H, C, N, O, S, P, and halogens. It uses a simple functional form and a limited number of atom types, but incorporates both empirical and heuristic models to estimate force constants and partial atomic charges. The performance of GAFF in test cases is encouraging. In test I, 74 crystallographic structures were compared to GAFF minimized structures, with a root-mean-square displacement of 0.26 A, which is comparable to that of the Tripos 5.2 force field (0.25 A) and better than those of MMFF 94 and CHARMm (0.47 and 0.44 A, respectively). In test II, gas phase minimizations were performed on 22 nucleic acid base pairs, and the minimized structures and intermolecular energies were compared to MP2/6-31G* results. The RMS of displacements and relative energies were 0.25 A and 1.2 kcal/mol, respectively. These data are comparable to results from Parm99/RESP (0.16 A and 1.18 kcal/mol, respectively), which were parameterized to these base pairs. Test III looked at the relative energies of 71 conformational pairs that were used in development of the Parm99 force field. The RMS error in relative energies (compared to experiment) is about 0.5 kcal/mol. GAFF can be applied to wide range of molecules in an automatic fashion, making it suitable for rational drug design and database searching.

Homology modeling of the transmembrane domain of the human calcium sensing receptor and localization of an allosteric binding site.

A homology model for the human calcium sensing receptor (hCaR) transmembrane domain utilizing bovine rhodopsin (bRho) structural information was derived and tested by docking the allosteric antagonist, NPS 2143, followed by mutagenesis of predicted contact sites. Mutation of residues Phe-668 (helix II), Arg-680, or Phe-684 (helix III) to Ala (or Val or Leu) and Glu-837 (helix VII) to Ile (or Gln) reduced the inhibitory effects of NPS 2143 on [Ca2+]i responses. The calcimimetic NPS R-568 increases the potency of Ca2+ in functional assays of CaR. Mutations at Phe-668, Phe-684, or Glu-837 attenuated the effects of this compound, but mutations at Arg-680 had no effect. In all cases, mutant CaRs responded normally to Ca2+ or phenylalanine, which act at distinct site(s). Discrimination by the Arg-680 mutant is consistent with the structural differences between NPS 2143, which contains an alkyl bridge hydroxyl group, and NPS R-568, which does not. The homology model of the CaR transmembrane domain robustly accounts for binding of both an allosteric antagonist and agonist, which share a common site, and provides a basis for the development of more specific and/or potent allosteric modulators of CaR. These studies suggest that the bRho backbone can be used as a starting point for homology modeling of even distantly related G protein-coupled receptors and provide a rational framework for investigation of the contributions of the transmembrane domain to CaR function.

Proton-sensing G-protein-coupled receptors.

Blood pH is maintained in a narrow range around pH 7.4 mainly through regulation of respiration and renal acid extrusion. The molecular mechanisms involved in pH homeostasis are not completely understood. Here we show that ovarian cancer G-protein-coupled receptor 1 (OGR1), previously described as a receptor for sphingosylphosphorylcholine, acts as a proton-sensing receptor stimulating inositol phosphate formation. The receptor is inactive at pH 7.8, and fully activated at pH 6.8-site-directed mutagenesis shows that histidines at the extracellular surface are involved in pH sensing. We find that GPR4, a close relative of OGR1, also responds to pH changes, but elicits cyclic AMP formation. It is known that the skeleton participates in pH homeostasis as a buffering organ, and that osteoblasts respond to pH changes in the physiological range, but the pH-sensing mechanism operating in these cells was hitherto not known. We detect expression of OGR1 in osteosarcoma cells and primary human osteoblast precursors, and show that these cells exhibit strong pH-dependent inositol phosphate formation. Immunohistochemistry on rat tissue sections confirms the presence of OGR1 in osteoblasts and osteocytes. We propose that OGR1 and GPR4 are proton-sensing receptors involved in pH homeostasis.

Simulation of the different biological activities of diethylstilbestrol (DES) on estrogen receptor alpha and estrogen-related receptor gamma.

We describe the application of the molecular dynamics (MD) and molecular mechanics-generalized Born/surface area (MM-GB/SA) approaches to the simulation of the different biological activity of diethylstilbestrol (DES) on two highly homologous nuclear receptors-estrogen receptor alpha (ER-alpha) and estrogen-related receptor gamma (ERR-gamma). DES exerts an agonistic effect against ER-alpha and an antagonistic effect against ERR-gamma. Using the x-ray crystal structures of ER-alpha in the canonical agonist bound form (PDB code: 3ERD) and antagonist bound form (PDB code: 3ERT), ERR-gamma homology models have been constructed for the receptor in two different conformations. MM-GB/SA binding free energy calculations of DES in the ER-alpha and ERR-gamma structures suggest that DES exhibits a greater free energy of binding in the agonist bound conformation of ER-alpha, while the antagonist bound conformation is preferred for ERR-gamma. Further dissection of the free energy contributions coupled with calculation of the ligand binding pocket volume suggests that the van der Waals interactions for DES within the smaller binding pocket volume of ERR-gamma are less favorable and this is the main factor for DES antagonism in ERR-gamma. This approach has potential general applicability to the prediction of the biological activity of nuclear receptor ligands.

Dual Recognition of Double-Stranded DNA by 2'-Aminoethoxy-Modified Oligonucleotides.

Simultaneous interaction of the 2'-aminoethoxy-modified oligonucleotides with the phosphodiester backbone (shown on the right, A) and with the bases through Hoogsteen base contacts (B) is seen at each base-pair step of the duplex DNA target. The electrostatic interaction between the protonated amino group and the negatively charged phosphate group provides for a dramatic increase in the binding affinity and the association rate constant.