Phospholipase C (PLC) enzymes hydrolyze phosphatidylinositol lipids to produce second messengers, including inositol‐1,4,5‐triphosphate (IP3) and diacylgycerol (DAG), which increase intracellular calcium and activate protein kinase C (PKC), respectively. PLCɛ contributes to cardiac hypertrophy and contractility, as well as to oncogenic and inflammatory signaling pathways following activation of G protein‐coupled receptors and receptor tyrosine kinases. PLCɛ shares a conserved core with the PLC superfamily, but the roles of individual domains in regulation of activity and membrane binding have not been established. We used functional assays to show that the PLCɛ PH domain significantly increases basal lipase activity, but is dispensable for stability. We provide the first structural insights into domain organization of PLCɛ using small‐angle X‐ray scattering (SAXS) and electron microscopy (EM) to reveal that the PH domain is conformationally heterogeneous in solution. Comparisons of the PLCɛ solution structure to that of the closely‐related PLCβ enzyme demonstrate that the PLCβ PH domain is also mobile in solution, in contrast to previously reported crystal structures. We propose that the dynamic nature of the PLC PH domain and resulting conformational heterogeneity contributes to subfamily‐specific differences in activity and regulation by G proteins. We are now using cryo‐EM to expand on these findings and obtain higher resolution structures. Support or Funding Information This work is supported by the Purdue Center for Cancer Research, AHA grant 16SDG29930017, NIH NLHBI 1R01HL141076‐01 to A.M.L., and Purdue College of Science Staff and Administrative & Professional Staff Advisory Council Professional Development awards to E.E.G. SAXS experiments used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE‐AC02‐06CH11357. This project was supported by grant 9 P41 GM103622 from the National Institute of General Medical Sciences of the National Institutes of Health. Use of the Pilatus 3 1M detector was provided by grant 1S10OD018090‐01 from NIGMS. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Background and Purpose:Mitragyna speciosa extract and kratom alkaloids decrease alcohol consumption in mice at least in part through actions at the δ-opioid receptor (δOR). However, the most potent opioidergic kratom alkaloid, 7-hydroxymitragynine, exhibits rewarding properties and hyperlocomotion presumably due to preferred affinity for the mu opioid receptor (µOR). We hypothesized that opioidergic kratom alkaloids like paynantheine and speciogynine with reduced µOR potency could provide a starting point for developing opioids with an improved therapeutic window to treat alcohol use disorder.Experimental Approach: We characterized paynantheine, speciociliatine, and four novel kratom-derived analogs for their ability to bind and activate δOR, µOR, and κOR. Select opioids were assessed in behavioral assays in male C57BL/6N WT and δOR knockout mice.Key Results: Paynantheine (10 mg∙kg−1, i.p.) produced aversion in a limited conditioned place preference (CPP) paradigm but did not produce CPP with additional conditioning sessions. Paynantheine did not produce robust antinociception but did block morphine-induced antinociception and hyperlocomotion. Yet, at 10 and 30 mg∙kg−1 doses (i.p.), paynantheine did not counteract morphine CPP. 7-hydroxypaynantheine and 7-hydroxyspeciogynine displayed potency at δOR but limited µOR potency relative to 7-hydroxymitragynine in vitro, and dose-dependently decreased voluntary alcohol consumption in WT but not δOR in KO mice. 7-hydroxyspeciogynine has a maximally tolerated dose of at least 10 mg∙kg−1 (s.c.) at which it did not produce significant CPP neither alter general locomotion nor induce noticeable seizures.Conclusion and Implications: Derivatizing kratom alkaloids with the goal of enhancing δOR potency and reducing off-target effects could provide a pathway to develop novel lead compounds to treat alcohol use disorder with an improved therapeutic window.

As tool compounds to study cardiac ischemia, the endogenous δ-opioid receptors (δOR) agonist Leu 5 -enkephalin and the more metabolically stable synthetic peptide (d-Ala 2 , d-Leu 5 )-enkephalin are frequently employed. However, both peptides have similar pharmacological profiles that restrict detailed investigation of the cellular mechanism of the δOR's protective role during ischemic events. Thus, a need remains for δOR peptides with improved selectivity and unique signaling properties for investigating the specific roles for δOR signaling in cardiac ischemia. To this end, we explored substitution at the Phe 4 position of Leu 5 -enkephalin for its ability to modulate receptor function and selectivity. Peptides were assessed for their affinity to bind to δORs and µ-opioid receptors (µORs) and potency to inhibit cAMP signaling and to recruit β-arrestin 2. Additionally, peptide stability was measured in rat plasma. Substitution of the meta-position of Phe 4 of Leu 5 -enkephalin provided high-affinity ligands with varying levels of selectivity and bias at both the δOR and µOR and improved peptide stability, while substitution with picoline derivatives produced lower-affinity ligands with G protein biases at both receptors. Overall, these favorable substitutions at the meta-position of Phe 4 may be combined with other modifications to Leu 5 -enkephalin to deliver improved agonists with finely tuned potency, selectivity, bias and drug-like properties.

µ Opioid receptors agonists provide potent and effective acute analgesia; however, their therapeutic window narrows considerably upon repeated administration, such as required for treating chronic pain. In contrast, bifunctional µ/δ...

Purpose Opioids have been the main factor for drug overdose deaths in the United States. Current naloxone delivery systems are effective in mitigating the opioid effects only for hours. Naloxone-loaded poly(lactide-co-glycolide) (PLGA) microparticles were prepared as quick-and long-acting naloxone delivery systems to extend the naloxone effect as an opioid antidote. Methods The naloxone-PLGA microparticles were made using an emulsification solvent extraction approach with different formulation and processing parameters. Two PLGA polymers with the lactide:glycolide (L:G) ratios of 50:50 and 75:25 were used, and the drug loading was varied from 21% to 51%. Two different microparticles of different sizes with the average diameters of 23 μm and 50 μm were produced using two homogenization-sieving conditions. All the microparticles were critically characterized, and three of them were evaluated with β-arrestin recruitment assays. Results The naloxone encapsulation efficiency (EE) was in the range of 70-85%. The EE was enhanced when the theoretical naloxone loading was increased from 30% to 60%, the L:G ratio was changed from 50:50 to 75:25, and the average size of the particles was reduced from 50 μm to 23 μm. The in vitro naloxone release duration ranged from 4 to 35 days. Reducing the average size of the microparticles from 50 μm to 23 μm helped eliminate the lag phase and obtain the steadystate drug release profile. The cellular pharmacodynamics of three selected formulations were evaluated by applying DAMGO, a synthetic opioid peptide agonist to a μ-opioid receptor, to recruit β-arrestin 2. Conclusions Naloxone released from the three selected formulations could inhibit DAMGO-induced β-arrestin 2 recruitment. This indicates that the proposed naloxone delivery system is adequate for opioid reversal during the naloxone release duration. KEY WORDS β-arrestin 2 inhibition . DAMGO . drug loading . encapsulation efficiency . naloxone . opioid overdose . PLGA microparticles . zero-order release