Crystal Structure of Antagonist Bound Human Lysophosphatidic Acid Receptor 1.

Lipid biology continues to emerge as an area of significant therapeutic interest, particularly as the result of an enhanced understanding of the wealth of signaling molecules with diverse physiological properties. This growth in knowledge is epitomized by lysophosphatidic acid (LPA), which functions through interactions with at least six cognate G protein-coupled receptors. Herein, we present three crystal structures of LPA1 in complex with antagonist tool compounds selected and designed through structural and stability analyses. Structural analysis combined with molecular dynamics identified a basis for ligand access to the LPA1 binding pocket from the extracellular space contrasting with the proposed access for the sphingosine 1-phosphate receptor. Characteristics of the LPA1 binding pocket raise the possibility of promiscuous ligand recognition of phosphorylated endocannabinoids. Cell-based assays confirmed this hypothesis, linking the distinct receptor systems through metabolically related ligands with potential functional and therapeutic implications for treatment of disease.

Preventive and Therapeutic Efficacy of ONO-4641, a Selective Agonist for Sphingosine 1-Phosphate Receptor 1 and 5, in Preclinical Models of Type 1 Diabetes Mellitus.

ONO-4641, 1-({6-[(2-methoxy-4-propylbenzyl)oxy]-1-methyl-3,4-dihydronaphthalen-2-yl}methyl)azetidine-3-carboxylic acid (ceralifimod), is a second-generation sphingosine 1-phosphate receptor agonist selective for sphingosine 1-phosphate receptors 1 and 5, and has clinical effects in multiple sclerosis. The objective of the present study was to explore other potential indications for ONO-4641 based on its immunomodulatory effects. ONO-4641 was tested in non-obese diabetic (NOD) mice, an animal model of spontaneous type 1 diabetes mellitus, an autoimmune disease with unmet medical needs. ONO-4641 at a dose of 0.1 mg/kg prevented the onset of diabetes mellitus in NOD mice. Furthermore, ONO-4641 at doses of 0.03 and 0.1 mg/kg decreased diabetic prevalence in NOD mice after the onset of diabetes mellitus in a dose-dependent manner. Histopathological analysis demonstrated that insulin-positive areas in the islets of mice administered 0.03 and 0.1 mg/kg ONO-4641 showed a tendency of high values although they were not significantly different from the Control group, which was treated with vehicle. These observations suggest ONO-4641 may delay the onset and progression of type 1 diabetes mellitus.

Towards a TREK-1/2 (TWIK-Related K+ Channel 1 and 2) dual activator tool compound: Multi-dimensional optimization of BL-1249.

This letter describes a focused, multi-dimensional optimization campaign around BL-1249, a fenamate class non-steroidal anti-inflammatory and a known activator of the K2P potassium channels TREK-1 (K2P2.1) and TREK-2 (K2P10.1). While BL-1249 has been widely profiled in vitro as a dual TREK-1/2 activator, poor physicochemical and DMPK properties have precluded a deeper understanding of the therapeutic potential of these key K2P channels across a broad spectrum of peripheral and central human disease. Here, we report multi-dimensional SAR that led to a novel TREK-1/2 dual activator chemotype, exemplified by ONO-2960632/VU6011992, with improved DMPK properties, representing a new lead for further optimization towards robust in vivo tool compounds.

Discovery, Characterization, and Effects on Renal Fluid and Electrolyte Excretion of the Kir4.1 Potassium Channel Pore Blocker, VU0134992.

The inward rectifier potassium (Kir) channel Kir4.1 (KCNJ10) carries out important physiologic roles in epithelial cells of the kidney, astrocytes in the central nervous system, and stria vascularis of the inner ear. Loss-of-function mutations in KCNJ10 lead to EAST/SeSAME syndrome, which is characterized by epilepsy, ataxia, renal salt wasting, and sensorineural deafness. Although genetic approaches have been indispensable for establishing the importance of Kir4.1 in the normal function of these tissues, the availability of pharmacological tools for acutely manipulating the activity of Kir4.1 in genetically normal animals has been lacking. We therefore carried out a high-throughput screen of 76,575 compounds from the Vanderbilt Institute of Chemical Biology library for small-molecule modulators of Kir4.1. The most potent inhibitor identified was 2-(2-bromo-4-isopropylphenoxy)-N-(2,2,6,6-tetramethylpiperidin-4-yl)acetamide (VU0134992). In whole-cell patch-clamp electrophysiology experiments, VU0134992 inhibits Kir4.1 with an IC50 value of 0.97 µM and is 9-fold selective for homomeric Kir4.1 over Kir4.1/5.1 concatemeric channels (IC50 = 9 µM) at -120 mV. In thallium (Tl+) flux assays, VU0134992 is greater than 30-fold selective for Kir4.1 over Kir1.1, Kir2.1, and Kir2.2; is weakly active toward Kir2.3, Kir6.2/SUR1, and Kir7.1; and is equally active toward Kir3.1/3.2, Kir3.1/3.4, and Kir4.2. This potency and selectivity profile is superior to Kir4.1 inhibitors amitriptyline, nortriptyline, and fluoxetine. Medicinal chemistry identified components of VU0134992 that are critical for inhibiting Kir4.1. Patch-clamp electrophysiology, molecular modeling, and site-directed mutagenesis identified pore-lining glutamate 158 and isoleucine 159 as critical residues for block of the channel. VU0134992 displayed a large free unbound fraction (fu) in rat plasma (fu = 0.213). Consistent with the known role of Kir4.1 in renal function, oral dosing of VU0134992 led to a dose-dependent diuresis, natriuresis, and kaliuresis in rats. Thus, VU0134992 represents the first in vivo active tool compound for probing the therapeutic potential of Kir4.1 as a novel diuretic target for the treatment of hypertension.

Discovery of novel S1P2 antagonists, part 3: Improving the oral bioavailability of a series of 1,3-bis(aryloxy)benzene derivatives.

The structure of the S1P2 antagonist 1 has been modified with the aim of improving its oral bioavailability. The chemical modification of the alkyl chain and carboxylic acid moieties of 1 led to significant improvements in the oral exposure of compounds belonging to this series. The optimization of the ring size of the urea portion of these molecules also led to remarkable improvements in the oral exposure. Based on these changes, the pyrrolidine derivative 16 was identified as a suitable candidate compound and showed excellent pharmacokinetic profiles in rat and dog, while maintaining high levels of potency and selective antagonistic activity toward S1P2.

Discovery of novel S1P2 antagonists. Part 2: Improving the profile of a series of 1,3-bis(aryloxy)benzene derivatives.

Our initial lead compound 2 was modified to improve its metabolic stability. The resulting compound 5 showed excellent metabolic stability in rat and human liver microsomes. We subsequently designed and synthesized a hybrid compound of 5 and the 1,3-bis(aryloxy) benzene derivative 1, which was previously reported by our group to be an S1P2 antagonist. This hybridization reaction gave compound 9, which showed improved S1P2 antagonist activity and good metabolic stability. The subsequent introduction of a carboxylic acid moiety into 9 resulted in 14, which showed potent antagonist activity towards S1P2 with a much smaller species difference between human S1P2 and rat S1P2. Compound 14 also showed good metabolic stability and an improved safety profile compared with compound 9.

Discovery of novel S1P2 antagonists. Part 1: discovery of 1,3-bis(aryloxy)benzene derivatives.

The structure-activity relationships of a novel series of sphingosine-1-phosphate receptor antagonists have been examined in detail. The initial hit compound 1 was modified through synthesis to improve its S1P2 activity. The synthesis of a series of analogs revealed that 1,3-bis(aryloxy)benzene derivatives, as represented by 22, are potent and selective S1P2 antagonists.

Further optimization of the M5 NAM MLPCN probe ML375: tactics and challenges.

This Letter describes the continued optimization of the MLPCN probe ML375, a highly selective M5 negative allosteric modulator (NAM), through a combination of matrix libraries and iterative parallel synthesis. True to certain allosteric ligands, SAR was shallow, and the matrix library approach highlighted the challenges with M5 NAM SAR within in this chemotype. Once again, enantiospecific activity was noted, and potency at rat and human M5 were improved over ML375, along with slight enhancement in physiochemical properties, certain in vitro DMPK parameters and CNS distribution. Attempts to further enhance pharmacokinetics with deuterium incorporation afforded mixed results, but pretreatment with a pan-P450 inhibitor (1-aminobenzotriazole; ABT) provided increased plasma exposure.

Structure-activity relationship studies of S1P agonists with a dihydronaphthalene scaffold.

Structure-activity relationship (SAR) of sphingosine-1-phosphate receptor agonists with a dihydronaphthalene scaffold was investigated. Compound 1 was modified to improve S1P(1) agonistic activity and in vivo peripheral lymphocyte lowering (PLL) activity without impairing selectivity over S1P(3) agonistic activity. A detailed SAR study of the terminal lipophilic part revealed that the introduction of substituents on the propylene linker and the terminal benzene ring influences in vitro and PLL activities. Compound 6n bearing a (S)-methyl group at the 2-position on the propylene linker and chlorine at the para-position on the terminal benzene ring showed potent hS1P(1) agonistic activity with excellent selectivity over hS1P(3) and in vivo PLL activity in mice.

Structure-activity relationship studies of sphingosine-1-phosphate receptor agonists with N-cinnamyl-ß-alanine moiety.

Structure-activity relationship of sphingosine-1-phosphate receptor agonist was examined. In terms of reducing the flexibility of molecule, hit compound 1 was modified to improve S1P(1) agonistic activity as well as selectivity over S1P(3) agonistic activity. Novel S1P agonists with cinnamyl scaffold or 1,2,5,6-tetrahydropyridine scaffold were identified.

Discovery of S1P agonists with a dihydronaphthalene scaffold.

Structure-activity relationship of sphingosine-1-phosphate receptor agonists was examined. Cinnamyl derivative 1 was modified to improve S1P(1) agonistic activity as well as selectivity over S1P(3) agonistic activity. Dihydronaphthalene derivative 10d was identified as a potent S1P(1) receptor agonist with high selectivity against S1P(3) and enhanced efficacy in lowering peripheral lymphocyte counts in mice.

Discovery of a 1-Methyl-3,4-dihydronaphthalene-Based Sphingosine-1-Phosphate (S1P) Receptor Agonist Ceralifimod (ONO-4641). A S1P1 and S1P5 Selective Agonist for the Treatment of Autoimmune Diseases.

The discovery of 1-({6-[(2-methoxy-4-propylbenzyl)oxy]-1-methyl-3,4-dihydronaphthalen-2-yl}methyl)azetidine-3-carboxylic acid 13n (ceralifimod, ONO-4641), a sphingosine-1-phosphate (S1P) receptor agonist selective for S1P1 and S1P5, is described. While it has been revealed that the modulation of the S1P1 receptor is an effective way to treat autoimmune diseases such as relapsing-remitting multiple sclerosis (RRMS), it was also reported that activation of the S1P3 receptor is implicated in some undesirable effects. We carried out a structure-activity relationship (SAR) study of hit compound 6 with an amino acid moiety in the hydrophilic head region. Following identification of a lead compound with a dihydronaphthalene central core by inducing conformational constraint, optimization of the lipophilic tail region led to the discovery of 13n as a clinical candidate that exhibited >30 000-fold selectivity for S1P1 over S1P3 and was potent in a peripheral lymphocyte lowering (PLL) test in mice (ED50 = 0.029 mg/kg, 24 h after oral dosing).

ML418: The First Selective, Sub-Micromolar Pore Blocker of Kir7.1 Potassium Channels.

The inward rectifier potassium (Kir) channel Kir7.1 (KCNJ13) has recently emerged as a key regulator of melanocortin signaling in the brain, electrolyte homeostasis in the eye, and uterine muscle contractility during pregnancy. The pharmacological tools available for exploring the physiology and therapeutic potential of Kir7.1 have been limited to relatively weak and nonselective small-molecule inhibitors. Here, we report the discovery in a fluorescence-based high-throughput screen of a novel Kir7.1 channel inhibitor, VU714. Site-directed mutagenesis of pore-lining amino acid residues identified glutamate 149 and alanine 150 as essential determinants of VU714 activity. Lead optimization with medicinal chemistry generated ML418, which exhibits sub-micromolar activity (IC50 = 310 nM) and superior selectivity over other Kir channels (at least 17-fold selective over Kir1.1, Kir2.1, Kir2.2, Kir2.3, Kir3.1/3.2, and Kir4.1) except for Kir6.2/SUR1 (equally potent). Evaluation in the EuroFins Lead Profiling panel of 64 GPCRs, ion-channels, and transporters for off-target activity of ML418 revealed a relatively clean ancillary pharmacology. While ML418 exhibited low CLHEP in human microsomes which could be modulated with lipophilicity adjustments, it showed high CLHEP in rat microsomes regardless of lipophilicity. A subsequent in vivo PK study of ML418 by intraperitoneal (IP) administration (30 mg/kg dosage) revealed a suitable PK profile (Cmax = 0.20 µM and Tmax = 3 h) and favorable CNS distribution (mouse brain/plasma Kp of 10.9 to support in vivo studies. ML418, which represents the current state-of-the-art in Kir7.1 inhibitors, should be useful for exploring the physiology of Kir7.1 in vitro and in vivo.

Prevention of GVHD and graft rejection by a new S1P receptor agonist, W-061, in rat small bowel transplantation.

BACKGROUND: In small bowel transplantation (SBTx), inhibition of both graft-versus-host disease (GVHD) and allograft rejection is necessary. METHODS: We investigated the potency of a new sphingosine-1-phosphate receptor agonist, W-061, for these two immune responses in SBTx. W-061 has a completely different molecular structure from FTY720. Heterotopic SBTx was performed from Wistar-Furth (WF) into (WF×ACI) F1 rats as a GVHD model or F1 to WF rats as a rejection model. Recipients were orally given 3 mg/kg/day W-061 for 14 days after SBTx. Recipient survival, body weight, histopathology, lymphocyte subpopulations, and the cytokine profile were evaluated. RESULTS: W-061 treatment significantly prolonged graft survival over 100 days in four out of six recipients in the GVHD group and over 60 days in three out of six recipients in the rejection group. W-061 strongly inhibited GVHD and rejection as seen histopathologically in comparison with untreated control rats. W-061 caused a significant reduction in donor-derived T cells in target organs and infiltrating T cells in allografts by promoting these cells to home into the secondary lymphoid tissues and sequestrating those cells there. W-061 significantly decreased production of interferon-γ in target organs and allografts. CONCLUSION: Therefore, these data suggest that W-061 has considerable potential as a new therapeutic immunosuppressant in patients with SBTx.

Therapeutic effects of novel sphingosine-1-phosphate receptor agonist W-061 in murine DSS colitis.

Although IL-17 is a pro-inflammatory cytokine reportedly involved in various autoimmune inflammatory disorders, its role remains unclear in murine models of colitis. Acute colitis was induced by 2.5% dextran sodium sulfate (DSS) treatment for 5 days. A novel sphingosine-1-phosphate receptor agonist W-061, a prototype of ONO-4641, was orally administered daily, and histopathological analysis was performed on the colon. The number of lymphocytes and their cytokine production were also evaluated in spleen, mesenteric lymph node, Peyer's patch and lamina propria of the colon. Daily administration of W-061 resulted in improvement of DSS-induced colitis, and significantly reduced the number of CD4+ T cells in the colonic lamina propria. Numbers of both Th17 and Th1 cells were reduced by W-061 treatment. W-061, however, had no influence on the number of Treg cells in lamina propria. Thus, Th17 and Th1 cells in lamina propria were thought to be the key subsets in the pathogenesis of DSS-induced colitis. In conclusion, W-061 may be a novel therapeutic strategy to ameliorate acute aggravation of inflammatory bowel diseases.