LPA receptor-promoted intracellular Ca2+ mobilization was measured for LPA receptors in stably expressed cells as previously described.11, 20 Briefly, cells were plated in 96-well plates and cultured overnight. and purinergic like receptors).1-4 These receptors play pathological and physiological roles regulating cell proliferation, migration, and survival.5 LPA receptors have been implicated in diseases that include breast cancer and cardiovascular disease.5-8 Bioactive molecules targeting LPA receptors have explored due to the potential therapeutic value of such compounds.9-18 The majority of these compounds have been evaluated only at receptors in the EDG subclass, LPA1-3, due to their earlier association with LPA signaling. Among LPA receptors in the EDG subclass, both LPA2 and LPA3 have restricted expression patterns, whereas LPA1 is broadly expressed.6 LPA2 is found in the nervous tissue, kidney, testis, lung, and prostate. LPA3 is found in the testis, prostate, JNJ 303 heart, brain, lung, and kidney. Selective bioactive molecules for these receptors offer therapeutic potential for several diseases.5, 6, 17, 19 During previous virtual screening efforts, several novel antagonists were identified.11 Identification of these antagonists JNJ 303 relied upon a rhodopsin-based homology model of the LPA3 receptor previously developed to study interactions of the binding site with known LPA antagonists.20 Based on these interactions, we defined a three-point pharmacophore consisting of an anionic and two hydrophobic sites. Matches to the pharmacophore were further analyzed using flexible docking before JNJ 303 selection of candidates for experimental screening. Antagonists identified from virtual screening using the pharmacophore and subsequent similarity searching were diverse in structure and had nanomolar potency (Table 1 and Figure 1 panel B).11 These differed from previously reported LPA antagonists which had lipid-like structures (Figure 1 panel A).9, 21 Open in a separate window Figure 1 Structures of reported lipid (panel A) JNJ 303 and non-lipid (panel B) LPA antagonists. Similarity search target, compound 5 (panel C). Table 1 Previously reported LPA antagonists identified using structure-based approaches. NE no effect thead th align=”left” valign=”top” rowspan=”1″ colspan=”1″ Antagonists /th th colspan=”5″ align=”center” valign=”top” rowspan=”1″ IC50 (nM) /th th align=”left” valign=”top” rowspan=”1″ colspan=”1″ /th th align=”center” valign=”top” rowspan=”1″ colspan=”1″ LPA1 /th th align=”center” valign=”top” rowspan=”1″ colspan=”1″ LPA2 /th th align=”center” valign=”top” rowspan=”1″ colspan=”1″ LPA3 /th th align=”center” valign=”top” rowspan=”1″ colspan=”1″ LPA4 /th th align=”center” valign=”top” rowspan=”1″ colspan=”1″ LPA5 /th /thead Compound 1NENE24 br / Imax = 69.8%NENECompound 222022NENENECompound 3NE355 br / Imax = 53.3%30 br / Imax = 81.7%NENECompound 42735491230NENECompound 5NENE4504 br / Imax = 50%NENE Open in a separate window The structure activity relationship (SAR) has been previously reviewed and is briefly summarized here. LPA antagonists were initially developed via changes to the polar head group and altering the Mouse monoclonal to BID length and number of hydrophobic chains present in the glycerol backbone.21 This is the case for DGPP, a nonselective LPA antagonist. DGPP possesses a pyrophosphate headgroup and two hydrocarbon chains rather than a phosphate headgroup and one hydrocarbon chain, as in LPA. Many of the earlier LPA antagonists were described prior to the identification of the newest LPA GPCR family members. Thus the true selectivity of these compounds remains an open question. Compound 1 is the only reported LPA3-selective compound to be tested on the LPA1-5 receptors11, instead of only LPA1-3. Compound 5 (Figure 1 panel C) emerged as a selective partial antagonist from virtual screening optimizing an LPA2 and LPA3 antagonist. In this present work we use our virtual screening workflow to rapidly identify additional LPA3 antagonists. Two compounds with 1.5- and 6-fold improvements in potency and improved efficacy from 50% to 100% inhibition of the LPA response over compound 5 were identified; however selectivity was reduced. 2. Results & Discussion 2.1. Database searching identifies similar analogs Compound 5 was found to be a weak selective LPA3 antagonist (IC50=4504 nM). while screening using the pthalamide scaffold. We began to search for more potent and efficacious LPA3 antagonists by 2D similarity searching using compound 5 for our search query. Similarity searching at an 80% threshold produced 183 matches. Compounds were selected for docking that maintained three moieties from compound 5; the phenyl ring, phthalimide, and the anionic group. This reduced the JNJ 303 183 matches to 42 compounds for virtual screening. The structures were then downloaded and prepared for docking. 2.2. Flexible docking The ligands were flexibly docked into the inactive LPA3 model using Autodock3.0.20, 22 Ligands were evaluated and chosen for experimental screening based on overlap of docked poses with compound 5. The docked complexes were analyzed for interactions analogous to those made compound 5 and any improved interactions (table 2). Figure 2 shows the receptor interactions from our structure-based pharmacophore comparing compound 5 and two of the 42 docked matches. The docked complex of compound 5 in panel A illustrates the minimum interactions that were set as requirement for each matches to have to justify experimental screening. These.