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Introduction: MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1) is a key mediator of the NF-κB signaling pathway, the main driver of a subset of B-cell lymphomas and functions by forming a complex with CARMA1 and BCL10 to mediate antigen receptor-induced lymphocyte activation. MALT1 is considered a potential therapeutic target for several subtypes of non-Hodgkin B-cell lymphomas and chronic lymphocytic leukemia (CLL). Previously, we described the discovery of novel and potent MALT1 inhibitors with anti-proliferative effects in non-Hodgkin B-cell lymphoma cells. Here, we highlight the strong anti-tumor activity of our MALT1 inhibitors across multiple tumor models and the combination potential with agents including standard-of-care. Results: Novel small molecule MALT1 inhibitors were identified using Schrodinger's proprietary physics-based free energy perturbation (FEP+) modeling technology. These molecules demonstrate strong MALT1 protein binding affinity, potent inhibition of MALT1 enzymatic activity and anti-proliferative activity in the activated B-cell (ABC) subtype of diffuse large B cell lymphoma (DLBCL) cell lines such as OCI-LY3 and OCI-LY10. In combination with approved agents, these inhibitors demonstrate strong combination potential with Bruton's tyrosine kinase (BTK) inhibitors such as ibrutinib in ABC-DLBCL cell lines. In ABC-DLBCL CDX models, our representative MALT1 inhibitor induces tumor regression as a single agent and complete tumor regression in combination with ibrutinib. Our representative MALT1 inhibitor, when tested in LY2298 PDX models, demonstrates similar results. In addition, our representative MALT1 inhibitor was explored in a CDX model derived from a Mantle cell lymphoma REC-1 cell line, and demonstrates strong anti-tumor activity of ~78% tumor growth inhibition (TGI) as a single agent. Conclusions: Schrodinger's novel, potent MALT1 protease small molecule inhibitors are efficacious in in vitro B-cell lymphoma cell proliferation assays and in in vivo B-cell lymphoma xenograft models. These data suggest that targeting MALT1 may expand therapeutic options for patients with selected B-cell lymphomas, such as ABC-DLBCL, with the possibility of expanding into other B-cell lymphomas such as MCL. Furthermore, these small molecule MALT1 inhibitors demonstrate potential in combination with BTKi to overcome drug-induced resistance in patients with relapsed/refractory B-cell lymphomas. Taken together, the data presented here strongly underscore the therapeutic potential of our MALT1 inhibitor and support further evaluation in clinical trials. Disclosures Weiss: Schrodinger: Current Employment; ARTham Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Introduction: Ubiquitin-specific protease 7 (USP7) is a deubiquitinase that regulates several proteins involved in cell cycle, DNA repair, genomic stability, and epigenetics and has been implicated in cancer progression. A key substrate of USP7 is MDM2, the oncogenic E3 ubiquitin ligase that promotes degradation of the tumor suppressor p53. USP7-mediated stabilization of MDM2 leads to the degradation of p53, preventing cell cycle arrest and the induction of apoptosis and promoting tumor cell growth. In addition to the MDM2-p53 pathway, USP7 regulates a number of other substrates involved in cancer, including PIM2 kinase, MYCN, the DNA methyltransferase DNMT1, and the PTEN tumor suppressor. Consistent with its function promoting oncogenic signaling pathways, genetic and pharmacological inhibition of USP7 has been shown to inhibit growth of a number of tumor cell lines in vitro and in vivo, and this anti-tumor activity is significantly enriched among cells expressing wild type p53. Thus, inhibition of USP7 is a promising therapeutic strategy, especially in cancers carrying wild type p53 that can be reactivated by suppression of MDM2. Results: We have discovered a new class of potent and selective USP7 inhibitors. These compounds bind to USP7 and prevent deubiquitinase activity in biochemical activity assays with picomolar potency. Our compounds induce accumulation of p53 and exhibit IC50s below 50 nM in cell viability assays in Multiple Myeloma and Acute Myeloid Leukemia (AML) cell lines. In AML cell lines, our USP7 inhibitors strongly synergize with the approved Bcl-2 inhibitor venetoclax. Unlike MDM2 antagonists currently undergoing clinical trials in AML, these USP7 inhibitors do not lead to a significant increase in MDM2 levels, and our compounds result in a more modest induction of p53, both of which could provide significant safety benefits. We have demonstrated differential sensitivity to MDM2 antagonists and our USP7 inhibitors in select cell lines and show that this effect is dependent upon p53 induction. Our compounds are orally bioavailable with a desirable PK profile in mice and induce p53 in tumor cells in CDX mouse models of Multiple Myeloma. Conclusions: We have identified novel, potent, orally bioavailable USP7 inhibitors that lead to p53 accumulation and cytotoxicity in cancer cells. We demonstrate mechanistic differences between USP7 and MDM2-p53 antagonists, which may lead to a safety advantage. We observe strong synergy between USP7 inhibitors and an approved treatment. Our lead compounds have favorable drug-like properties, promising mouse PK profiles, and can induce p53 accumulation in mouse CDX tumor models. These data demonstrate the therapeutic potential of our USP7 inhibitors and reveal specific opportunities for their use in the treatment of Multiple Myeloma and AML. Citation Format: Alan Futran, Tao Lu, Katherine Amberg-Johnson, Xiaoxiao Yang, Saidi He, Jeffrey Bell, Sarah Boyce, Markus Dahlgren, Karen Dingley, Liping Fang, Heidi Koldsoe, Zef Konst, Fang-Yu Lin, Robert Pelleltier, Heng Qian, Mats Svensson, Michael Trzoss, Jie Xu, Shuping Xu, Zhaowu Xu, Engin Yapici, Yan Zhang, Li Xing, Takao Suzuki, Xianhai Huang, Jiayi Xu, Hamish Wright, Kristian Jensen, Wayne Tang, Tao Guo, Karen Akinsanya, David Madge. Discovery of novel, potent USP7 inhibitors that upregulate p53 leading to anti-proliferative effects in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1338.

Introduction: MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1), was identified as a translocation protein fused with cIAP2 in mucosa-associated lymphoid tissue (MALT) B cell lymphomas. MALT1, a key mediator of NF-κB signaling and the main driver of a subset of B-cell lymphomas, functions via formation of a complex with CARMA1 and BCL10 to mediate antigen receptor-induced lymphocyte activation. MALT1 has been considered as a potential therapeutic target for several non-Hodgkin B cell lymphomas as well as chronic lymphocytic leukemia (CLL). Here, we describe the discovery of novel, potent MALT1 inhibitors that result in antiproliferative effects in non-Hodgkin B-cell lymphoma cells. Results: We have identified novel small molecule MALT1 inhibitors using our proprietary physics-based Free Energy Perturbation (FEP+) modeling technology. Our compounds show potent (sub nM) inhibition of MALT1 enzymatic activity, as well as high binding affinity (sub nM) to MALT1 protein measured by Surface Plasmon Resonance (SPR). BCL10 is a binding partner of MALT1 that is cleaved by MALT1 at the C-terminus. Our inhibitors were efficacious in a target engagement assay showing prevention of BCL10 cleavage in Activated B-cell (ABC) subtype of diffuse large B cell lymphoma (DLBCL) cell lines OCI-LY3 and OCI-LY10, which are Bruton tyrosine kinase (BTK) inhibitor ibrutinib-resistant and -responsive respectively. Our compounds are potent inhibitors of IL10 secretion in both OCI-LY3 and OCI-LY10 cells, which is consistent with the inhibition of NF-κB signaling. We also examined the effect of our MALT1 inhibitors on ABC-DLBCL cell proliferation. Our inhibitors demonstrated potent anti-proliferative effects in both OCI-LY3 and OCI-LY10 cell lines, as well as synergistic effects with ibrutinib in a BTKi sensitive ABC-DLBCL cell panel. Examinations of a protease panel and off-target safety screening panel, as well as in vivo high dose tolerability study showed our compound had excellent selectivity and significant safety margin. Plasma IL10 and tumor BCL10 have been identified as robust PD markers in PK/PD studies in both OCI-LY3 and OCI-LY10 tumor bearing mice. Dose-dependent tumor growth inhibition was observed after 3 weeks of treatment in OCI-LY3 xenograft model, with efficacy also observed in combination with venetoclax. Ongoing work: We are continuing to explore the synergistic effects of our compounds with BTK inhibitors in B-cell lymphoma mouse models. Preliminary data showed potent inhibition of IL-2 secretion in Jurkat cells from our compound treatment. Additional studies are ongoing to elucidate the role of MALT1 inhibition in Treg as well as Teffector cells in vitro and in vivo. Refinement of the current inhibitor series, using co-crystal structures, is in progress in preparation for further development of optimized molecules. Conclusion and Future Plans: We have identified novel potent MALT1 protease small molecule inhibitors that are efficacious in the in vitro B-cell lymphoma cell proliferation assays and in the in vivo B-cell lymphoma xenograft model. Our data suggest that targeting MALT1 may expand therapy options for patients with selected B-cell lymphomas, such as ABC-DLBCL. Our work provided insight into the anti-tumor efficacy of our inhibitors in B-cell lymphomas as single agent, and ongoing work will continue to assess the potential combination with BTKi to overcome drug-induced resistance in patients with relapsed/refractory B-cell lymphoma. Disclosures Yin: Schrodinger: Current Employment, Current equity holder in publicly-traded company. Zhe:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Placzek:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Trzoss:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Krilov:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Feng:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Lawrenz:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Pelletier:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Lai:Triplet Therapeutics: Current Employment, Current equity holder in private company. Bell:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Calkins:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Grimes:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Tang:Schrodinger: Current Employment, Current equity holder in publicly-traded company. McRobb:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Gerasyuto:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Feher:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Mondal:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Jensen:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Wright:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Akinsanya:Schrodinger: Current Employment, Current equity holder in publicly-traded company.

Aims To characterize pharmacokinetic parameters of MK-0916 and its safety and tolerability in lean, healthy male subjects following single and multiple oral doses. To assess (by stable-isotope labelling) the in vivo inhibition of cortisone-to-cortisol conversion following oral MK-0916. Methods Data are presented from two randomized, controlled, double-blind, rising-dose phase I studies. In the first study, subjects received single oral doses of 0.4–100 mg MK-0916 (n = 16). In the second study, subjects received 0.2–225 mg MK-0916 followed by daily doses of 0.2–100 mg for 13 days beginning on day 2 or day 15 (n = 80). Plasma and urine drug concentrations were measured for pharmacokinetic analysis. For pharmacodynamic analysis, concentrations of plasma [13C4]cortisol were measured by high-pressure liquid chromatography and tandem mass spectrometry following a single oral dose of 5 mg [13C4]cortisone. Results Doses ≥3 mg were rapidly absorbed (time at which maximal concentration was achieved in plasma, 1.1–1.8 h). Exposure (measured as the area under the concentration–time curve from 0 to 168 h) increased approximately in proportion to dose. Values for the maximal plasma concentration and the plasma concentration at 24 h increased in excess of dose proportionality at doses 6 mg. In subjects dosed with 6 mg MK-0916 once daily for 14 days, the mean trough plasma concentration was 240 nm and in vivo cortisone-to-cortisol conversion was inhibited by 84%. The relationship between plasma MK-0916 and hepatic 11β-hydroxysteroid dehydrogenase type 1 inhibition was well represented by a simple Emax model with an IC50 of 70.4 nm. Exposure to MK-0916 was generally well tolerated. Conclusions These findings indicate that 11β-hydroxysteroid dehydrogenase type 1 is effectively inhibited in human subjects by doses of MK-0916 that are well tolerated.

Sitagliptin is an orally active, highly selective dipeptidyl peptidase IV (DPP-4) inhibitor for treatment of type 2 diabetes mellitus. This randomized, open-label, 2-part, 2-period crossover study assessed pharmacokinetics/pharmacodynamics of warfarin in the presence/absence of multiple-dose sitagliptin. Twelve participants received treatments A and B separated by >7-day washout: treatment A involved coadministration of sitagliptin 200 mg/d for 11 days (days 1-11) and warfarin 30 mg on day 5, and treatment B involved warfarin 30 mg alone on day 1. R(+) warfarin, S(-) warfarin, and international normalized ratio (INR) were assayed predose and up to 168 hours postdose. The geometric mean ratios (GMRs; warfarin + sitagliptin/warfarin alone) (90% confidence intervals [CIs]) were 0.99 (0.95, 1.03) and 0.95 (0.90, 1.02) for the AUC(0-infinity) of R(+) and S(-) warfarin, respectively. GMRs (warfarin + sitagliptin/warfarin alone) (90% CIs) were 0.89 (0.86, 0.93) and 0.89 (0.86, 0.92) for the C(max) of R(+) and S(-) warfarin, respectively. INR AUC(0-168 h) and INR(max) GMRs were 1.01 (0.96, 1.06) and 1.08 (1.00, 1.17), respectively. Coadministration of sitagliptin and warfarin was generally well tolerated. Pharmacokinetics (AUC for R(+) and S(-) warfarin) and pharmacodynamics (INR of R(+) or S(-) warfarin) were not meaningfully altered following coadministration of multiple-dose sitagliptin and single-dose warfarin, indicating that no dosage adjustment for warfarin is necessary when coadministered with sitagliptin.

Background Some drugs that are actively taken up into the liver exhibit greater than dose proportional increases in plasma exposure, although human liver-to-plasma concentration ratios have rarely been evaluated. Understanding these relationships has implications for drug concentrations at the target site for certain classes of compounds, such as direct-acting antivirals, targeted towards HCV. Methods Treatment-experienced, chronic HCV non-cirrhotic patients ( n=3) received vaniprevir (600 mg or 300 mg twice daily) on days 1–3 and (600 mg or 300 mg single dose) on day 4. Core needle biopsy was performed at 6 or 12 h post-dose on day 4. Blood samples were collected pre-dose on days 1 and 4, and for 24 h post-dose on day 4. The primary study objective was the hepatic concentration of vaniprevir at 6 and 12 h post-dose. Results Vaniprevir plasma pharmacokinetic parameters increased in a greater than dose-proportional manner between the 300 mg and 600 mg doses, with approximately fivefold increases in AUC0–12 and Cmax associated with a twofold increase in dose (AUC0–12, 10.6 μM/h to 59.5 μM/h; Cmax, 2.60 μM to 13.5 μM). In the 300 mg and 600 mg dose groups, mean liver concentrations of vaniprevir were 84.6 μM and 169 μM at 6 h post-dose, and 29.4 μM and 53.7 μM at 12 h post-dose. Liver concentrations were higher than plasma with liver-to-plasma concentration ratios of approximately 20–280. Conclusions These data confirm higher vaniprevir concentrations in human liver compared with plasma and demonstrate that measurement of human liver drug concentration using needle biopsy is feasible.

Rationale Prostaglandin D 2 (PGD 2 ) is released from mast cells during the allergic response. Since PGD 2 has been shown to induce nasal congestion in humans, we investigated the distribution of hematopoietic Prostaglandin D synthase (PGDS) and the two PGD 2 receptors, DP and CRTH2 in human nasal mucosa from healthy subjects and subjects suffering from polyposis, a severe form of chronic rhinosinusitis. Methods DP mRNA expression was detected by in situ hybridization while PGDS, CRTH2 and various leukocyte markers expression was revealed by immunohistochemistry. Results In the normal mucosa, PGDS was only detected in few resident mast cells while CRTH2 was undetectable. In contrast, DP receptor mRNA was detected in epithelial goblet cells, serous glands and in the vasculature. In the nasal mucosa of subjects suffering from polyposis: 1) PGDS was detected in mast cells and other large infiltrating inflammatory cells, 2) both DP mRNA and CRTH2 were detected in eosinophils and 3) CRTH2 was detected on a subset of infiltrating T-cells. Although DP mRNA could not be detected in the T-cells invading the nasal mucosa, it was found to be expressed in the T-cells present in the lymph node and the thymus from normal individuals. Conclusions This study indicates that cells capable of producing PGD 2 are present in the nasal mucosa and that both PGD 2 receptors, DP and CRTH2, might play a role in inflammatory disease of the upper airways.

FOSAMAX® PLUS D is a once-weekly alendronate (ALN) 70-mg / vitamin D3 (vit-D3) 5600 IU combination tablet developed by Merck & Co., Inc. A single-dose, open-label, randomized, 2-period, crossover study was conducted to test bioequivalence between FOSAMAX® PLUS D combination tablets and co-administration of corresponding doses of ALN 70-mg (FOSAMAX®) and vit-D3 5600 IU (2x 2800 IU) as individual tablets in healthy male and female Asian subjects to support registration in Taiwan. Results: the 90%CI of the GMR for ALN AUC0-∞ fell within the pre-specified bioequivalence bounds of 0.8 to 1.25, but slightly exceeded 1.25 for ALN AUC0-last and Cmax. The 90% CI of the GMR for vit-D3, unadjusted for endogenous vit-D3, was also contained within the pre-specified bioequivalence bounds. Thus, the combination tablet was bioequivalent based on ALN AUC0-∞ and vit-D3 unadjusted for endogenous levels. However, it was not bioequivalent to co-administration with respect to ALN AUC0-last and Cmax . In conclusion, the small d...

This study demonstrates the localization of the prostaglandin (PG)D2 receptor (DP) within the mucous-secreting globlet cells of the human colon by in situ hybridization, which suggests a role for DP in mucous secretion. Selective high affinity ligands were used, therefore, to evaluate DP regulation of mucous secretion in LS174T human colonic adenocarcinoma cells. The expression of hDP in LS174T cells was confirmed at the mRNA level by reverse transcriptase-polymerase chain reaction, and at the protein level by radioligand binding assays and signal transduction (cyclic AMP accumulation) assays. PGD2 and the highly selective DP-specific agonist L-644,698 ((4-(3-(3-(3-hydroxyoctyl)-4-oxo-2-thiazolidinyl) propyl) benzoic acid) (racemate)), but not PGE2 competed for [3H]-PGD2-specific binding to LS174T cell membranes (Ki values of 0.4 nM and 7 nM, respectively). The DP-specific agonists PGD2, PGJ2, BW245C (5-(6-carboxyhexyl)-1-(3-cyclohexyl-3-hydroxypropylhydantoin)), and L-644,698 showed similar potencies in stimulating cyclic AMP accumulation (EC50 values: 45–90 nM) and demonstrated the expected rank order of potency. PGE2 also elicited cyclic AMP production in this cell line (EC50 value: 162 nM). The activation of cyclic AMP production by PGD2 and L-644,698, but not PGE2, was inhibited by the selective DP antagonist BW A868C. Thus, PGD2 and L-644,698 act through hDP in LS174T cells. PGD2, L-644,698 and PGE2 (an established mucin secretagogue) potently stimulated mucin secretion in LS174T cells in a concentration-dependent manner (EC50

1. A human embryonic kidney cell line [HEK 293(EBNA)] stably expressing the human recombinant prostaglandin D2 (PGD2) receptor (hDP) has been characterized with respect to radioligand binding and signal transduction properties by use of prostanoids and prostanoid analogues. Radioligand binding studies included saturation analyses, the effects of nucleotide analogues, the initial rate of ligand-receptor association and equilibrium competition assays. In addition, adenosine 3':5'-cyclic monophosphate (cyclic AMP) generation in response to ligand challenge was also measured, as this is the predominant hDP signalling pathway. 2. L-644,698 ((4-(3-(3-(3-hydroxyoctyl)-4-oxo-2-thiazolidinyl) propyl) benzoic acid) (racemate)) was identified as a novel ligand having high affinity for hDP with an inhibitor constant (Ki) of 0.9 nM. This Ki value was comparable to the Ki values obtained in this study for ligands that have previously shown high affinity for DP: PGD2 (0.6 nM), ZK 110841 (0.3 nM), BW245C (0.4 nM), and BW A868C (2.3 nM). 3. L-644,698 was found to be a full agonist with an EC50 value of 0.5 nM in generating cyclic AMP following activation of hDP. L-644,698 is, therefore, comparable to those agonists with known efficacy at the DP receptor (EC50): PGD2 (0.5 nM), ZK 110841 (0.2 nM) and BW245C (0.3 nM). 4. L-644,698 displayed a high degree of selectivity for hDP when compared to the family of cloned human prostanoid receptors: EP1 (> 25,400 fold), EP2 (approximately 300 fold), EP3-III (approximately 4100 fold), EP4 (approximately 10000 fold), FP (> 25,400 fold), IP (> 25,400 fold) and TP (> 25,400 fold). L-644,698 is, therefore, one of the most selective DP agonists as yet described. 5. PGJ2 and delta12-PGJ2, two endogenous metabolites of PGD2, were also tested in this system and shown to be effective agonists with Ki and EC50 values in the nanomolar range for both compounds. In particular, PGJ2 was equipotent to known DP specific agonists with a Ki value of 0.9 nM and an EC50 value of 1.2 nM.

Prostanoids exert significant effects on circulatory beds. They play a role in the response of the vasculature to adjustments in perfusion pressure and oxygen and carbon dioxide tension, and they mediate the actions of numerous factors. The role of prostanoids in governing circulation of the perinate is suggested to surpass that in the adult. Prostanoids are abundantly generated in the perinate. They have been implicated in autoregulation of blood flow as studied in brain and eyes. Prostaglandins are also dominant regulators of ductus arteriosus tone. The effects of these autacoids are mediated through specific G protein-coupled receptors. In addition to the pharmacological characterization of the prostanoid receptors, important advances in understanding the biology of these receptors have been made in the last decade. Their cloning and the development of animals with disrupted genes of these receptors have been very informative. The involvement of prostanoid receptors in the developing subject, especially on brain and ocular vasculature and on ductus arteriosus, has also begun to be investigated; the expression of these receptors changes with development. Some but not all of the ontogenic changes in these receptors are attributed to homologous regulation. Interestingly, in the process of elucidating their effects, functional perinuclear prostaglandin E2receptors have been uncovered. This article reviews prostanoid receptors and addresses implications on the developing subject with attention to vascular physiology.

We report the cloning, functional expression and cell-specific localization of the rat homologue of the prostaglandin D2 receptor (DP). In situ hybridization, utilizing multiple digoxigenin-labelled riboprobes and their complementary sense controls, was performed to determine the detailed distribution of DP receptor mRNA in the central nervous system and the gastrointestinal tract. Within the brain, the leptomeninges and choroid plexus expressed DP receptor mRNA. Transcripts detected in the spinal cord were localized to the sensory and motor neurons of the dorsal and ventral horns, respectively, suggesting a role for the DP receptor in the modulation of central nervous system processes, including pain transmission. Within the gastrointestinal tract (stomach, duodenum, ileum and colon) signals were highly localized to the mucous-secreting goblet cells and the columnar epithelium. These findings suggest a novel biological role for prostaglandin D2-mediated activity at the DP receptor, namely mucous secretion. In addition, radioligand binding assays (saturation analyses and equilibrium competition assays) and functional assays (measuring cAMP accumulation) were performed to characterize the recombinant rat DP receptor expressed in human embryonic kidney (HEK) 293(EBNA) cells. A single site of binding (K(D) = 14 nM, Bmax = 115 fmol/mg protein) was measured for prostaglandin D2-specific binding to the rat DP receptor. Prostaglandin D2 proved to be a potent agonist at the rat DP receptor (EC50 = 5 nM). The rank order of efficacy for DP receptor specific agonists [prostaglandin D2 = prostaglandin J2 = BW 245C (5-(6-carboxyhexyl)-1-(3-cyclohexyl-3-hydroxypropylhydantoin)) > L-644,698 ((4-(3-(3-(3-hydroxyoctyl)-4-oxo-2-thiazolidinyl) propyl) benzoic acid) (racemate)] reflected the affinity with which the ligands bound to the receptor.

1. A human embryonic kidney cell line [HEK 293(EBNA)] stably expressing the human recombinant prostaglandin D2 (PGD2) receptor (hDP) has been characterized with respect to radioligand binding and signal transduction properties by use of prostanoids and prostanoid analogues. Radioligand binding studies included saturation analyses, the effects of nucleotide analogues, the initial rate of ligand-receptor association and equilibrium competition assays. In addition, adenosine 3':5'-cyclic monophosphate (cyclic AMP) generation in response to ligand challenge was also measured, as this is the predominant hDP signalling pathway. 2. L-644,698 ((4-(3-(3-(3-hydroxyoctyl)-4-oxo-2-thiazolidinyl) propyl) benzoic acid) (racemate)) was identified as a novel ligand having high affinity for hDP with an inhibitor constant (Ki) of 0.9 nM. This Ki value was comparable to the Ki values obtained in this study for ligands that have previously shown high affinity for DP: PGD2 (0.6 nM), ZK 110841 (0.3 nM), BW245C (0.4 nM), and BW A868C (2.3 nM). 3. L-644,698 was found to be a full agonist with an EC50 value of 0.5 nM in generating cyclic AMP following activation of hDP. L-644,698 is, therefore, comparable to those agonists with known efficacy at the DP receptor (EC50): PGD2 (0.5 nM), ZK 110841 (0.2 nM) and BW245C (0.3 nM). 4. L-644,698 displayed a high degree of selectivity for hDP when compared to the family of cloned human prostanoid receptors: EP1 (25,400 fold), EP2 (approximately 300 fold), EP3-III (approximately 4100 fold), EP4 (approximately 10000 fold), FP (25,400 fold), IP (25,400 fold) and TP (25,400 fold). L-644,698 is, therefore, one of the most selective DP agonists as yet described. 5. PGJ2 and delta12-PGJ2, two endogenous metabolites of PGD2, were also tested in this system and shown to be effective agonists with Ki and EC50 values in the nanomolar range for both compounds. In particular, PGJ2 was equipotent to known DP specific agonists with a Ki value of 0.9 nM and an EC50 value of 1.2 nM.