A) Two binding poses for AM404: Pose I (blue) and pose II (magenta)

A) Two binding poses for AM404: Pose I (blue) and pose II (magenta). (Schroeder, Atshaves, McIntosh, Gallegos, Storey & Parr, et al., 2007). Furthermore, there is evidence that SCP-2 is expressed in the brain and is particularly enriched in synaptosomal preparations (Avdulov, Chochina, Igbavboa, Warden, Schroeder & Wood, 1999; Myers-Payne, Fontaine, Loeffler, Pu, Rao & Kier, et al., 1996). We found that micromolar concentrations of AEA compete with cholesterol for SCP-2-mediated transfer between vesicles and cell membranes; and docking studies predict that both AEA and 2-AG bind to SCP-2, but that AEA has higher predicted affinity (Liedhegner, Vogt, Sem, Cunningham & Hillard, 2014). These findings support the hypothesis that SCP-2 plays a role in the regulation of the concentrations of the eCBs available to activate the CB1R. To further test this hypothesis, selective, high-affinity inhibitors of eCB binding to SCP-2 are required. In pursuit of that goal, we have utilized an SCP-2 binding assay to determine the affinities of a variety of head group-substituted fatty acids and a second series of compounds that had been shown previously to inhibit binding of lipids to the (mosquito) SCP-2 homologue. Finally, we applied computer-aided drug design (CADD) techniques toward the rational discovery of structurally unique, small-molecule inhibitor leads. 2.?Materials and Methods-NBDS Displacement Assay 2.1. Materials Human recombinant SCP-2 was prepared and purified as previously described (Matsuura, George, Ramachandran, Alvarez, Strauss 3rd & Billheimer, 1993). The fluorescent probe, 12-to identify hits from diverse small-molecule libraries. 3.2. SAR of Head Group-Substituted Fatty Acids The first approach to lead discovery involves investigation of the SAR governing known, endogenous SCP-2 substrates. Sterols and amphiphilic fatty acid derivatives represent the most understood classes of SCP-2 substrates described in the literature (selected representative examples: Schroeder, Myers-Payne, Billheimer & Wood, 1995; Dansen, Westerman, Wouters, Wanders, van Hoek, Gadella & Wirtz, 1999; Atshaves, Jefferson, McIntosh, Gallegos & McCann et al., 2007). The structural features supporting binding to SCP-2 of the lipid portion of fatty acids and sphingolipids have been well-characterized (Gadella & Wirtz, 1994; Stolowich, Frolov, Atshaves, Murphy, Jolly & Billheimer, et al., 1997; Stolowich, Frolov, Petrescu, Scott, Billheimer & Schroeder, 1999), though it was not until Rabbit Polyclonal to CtBP1 recently that carboxylate-substituted fatty acid amides and esters were reported to be transported by SCP-2 (Liedhegner et al., 2014). A large number of head group-modified arachidonate analogues are commercially available, representing an extensive library of AEA and 2-AG analogues from which to generate SAR (Fig. 1A). In addition to AEA and 2-AG, a sample of structurally diverse, head group-modified analogues 3C11 were purchased and evaluated for the ability to compete with NBDS for binding to SCP-2 (Fig. 1A). As a test of the tolerance of the SCP-2 binding site to alternate head group-modified lipids, oleamide (12) and docosahexaenoyl ethanolamide (DHEA, 13) were also evaluated (Fig. 1A). All tested compounds displaced SCP-2 bound NBDS to varying extent. Open in a separate window Fig. 1. Displacement curves of SCPI displacing SCP-2 bound NBDS.After SCP-2 (500 nM) was equilibrated with NBDS (500 nM), it was titrated with increasing amount of SCPI. Panel A, arachidonates; Panel B, SCPI-1 and ?5 analogs; Panel C, HTS cpds. NBDS fluorescence was recorded (Ex = 490 Lemborexant nm, Em max = 528 nm) and corrected as described in Methods. Data were presented as mean SE (n=4). Table 1 provides the structures of compounds 1C11. Analysis of multiple displacement curves (n=4) for each Lemborexant compound (Table 1) shows the relative potencies (EC50, Ki) and efficacies as given in % displacement of NBDS. The eCBs, AEA (1) and 2-AG (2) were the most potent competitors of NBD binding (Ki 1.0 M), though only produced ~55C60% maximal displacement. One possible explanation for the partial displacement of NBDS by 1 and 2 may be poor solubility in buffer under these conditions. Table 1. Displacement of SCP-2-Bound NBDS by Arachidonate Compounds: Maximum % Displacement and SCP-2 with IC50 values between 0.042 and 0.347 M. Of these hits, two stand out as being potentially advantageous for lead development. SCPI-1 (14) is a 2-aminophenyl-4-phenylthiazole, a core scaffold that is present in Lemborexant sphingosine kinase inhibitors (Gustin, Li, Brown, Min, Schmitt & Wanska, et al., 2013) and probes of opioid receptor allosteric binding sites (Burford, Wehrman, Bassoni, OConnell, Banks, Zhang & Alt, 2014). SCPI-5 (18) was the most potent inhibitor of mouse SCP-2.