Identification of 1,3,8-Triazaspiro[4.5]Decane-2,4-Dione Derivatives as a Novel δ Opioid Receptor-Selective Agonist Chemotype.

δ opioid receptors (DORs) hold potential as a target for neurologic and psychiatric disorders, yet no DOR agonist has proven efficacious in critical phase II clinical trials. The exact reasons for the failure to produce quality drug candidates for the DOR are unclear. However, it is known that certain DOR agonists can induce seizures and exhibit tachyphylaxis. Several studies have suggested that those adverse effects are more prevalent in delta agonists that share the (+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC80)/4-[(αR*)-α-((2S*,5R*)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-hydroxybenzyl]-N,N-diethylbenzamide chemotype. There is a need to find novel lead candidates for drug development that have improved pharmacological properties to differentiate them from the current failed delta agonists. Our objective in this study was to identify novel DOR agonists. We used a ß-arrestin assay to screen a small G-protein coupled receptors (GPCR)-focused chemical library. We identified a novel chemotype of DOR agonists that appears to bind to the orthosteric site based of docking and molecular dynamic simulation. The most potent agonist hit compound is selective for the DOR over a panel of 167 other GPCRs, is slightly biased toward G-protein signaling and has anti-allodynic efficacy in a complete Freund's adjuvant model of inflammatory pain in C57BL/6 male and female mice. The newly discovered chemotype contrasts with molecules like SNC80 that are highly efficacious ß-arrestin recruiters and may suggest this novel class of DOR agonists could be expanded on to develop a clinical candidate drug. SIGNIFICANCE STATEMENT: δ opioid receptors are a clinical target for various neurological disorders, including migraine and chronic pain. Many of the clinically tested delta opioid agonists share a single chemotype, which carries risks during drug development. Through a small-scale high-throughput screening assay, this study identified a novel δ opioid receptor agonist chemotype, which may serve as alternative for the current analgesic clinical candidates.

Histone deacetylase inhibitor decreases hyperalgesia in a mouse model of alcohol withdrawal-induced hyperalgesia.

BACKGROUND: Alcohol withdrawal-induced hyperalgesia (AWH) is characterized as an increased pain sensitivity observed after cessation of chronic alcohol use. Alcohol withdrawal-induced hyperalgesia can contribute to the negative affective state associated with abstinence and can increase susceptibility to relapse. We aimed to characterize pain sensitivity in mice during withdrawal from two different models of alcohol exposure: chronic drinking in the dark (DID) and the Lieber-DeCarli liquid diet. We also investigated whether treatment with a histone deacetylase (HDAC) inhibitor, suberoylanilide hydroxamic acid (SAHA), could ameliorate AWH in mice treated with the Lieber-DeCarli diet. METHODS: Male and female C57BL/6J mice were used for these studies. In the DID model, mice received bottles of 20% ethanol or water during the dark cycle for 4 h per day on four consecutive days per week for 6 weeks. Peripheral mechanical sensitivity was measured weekly the morning of Day 5 using von Frey filaments. In the Lieber-DeCarli model, mice received ethanol (5% v/v) or control liquid diet for 10 days, along with a single binge ethanol gavage (5 g/kg) or control gavage, respectively, on Day 10. Peripheral mechanical sensitivity was measured during the liquid diet administration and at 24 and 72 h into ethanol withdrawal. An independent group of mice that received the Lieber-DeCarli diet were administered SAHA (50 mg/kg, i.p.) during withdrawal. RESULTS: Male mice exhibited mechanical hypersensitivity after consuming ethanol for 5 weeks in the DID procedure. In the Lieber-DeCarli model, ethanol withdrawal led to hyperalgesia in both sexes. Suberoylanilide hydroxamic acid treatment during withdrawal from the ethanol liquid diet alleviated AWH. CONCLUSIONS: These results demonstrate AWH in mice after chronic binge drinking in males and after Lieber-DeCarli liquid diet administration in both sexes. Like previous findings in rats, HDAC inhibition reduced AWH in mice, suggesting that epigenetic mechanisms are involved in AWH.

Protein-Peptide Binding Energetics under Crowded Conditions.

Nearly all biological processes, including strictly regulated protein-protein interactions fundamental in cell signaling, occur inside living cells where the concentration of macromolecules can exceed 300 g/L. One such interaction is between a 7 kDa SH3 domain and a 25 kDa intrinsically disordered region of Son of Sevenless (SOS). Despite its key role in the mitogen-activated protein kinase signaling pathway of all eukaryotes, most biophysical characterizations of this complex are performed in dilute buffered solutions where cosolute concentrations rarely exceed 10 g/L. Here, we investigate the effects of proteins, sugars, and urea, at high g/L concentrations, on the kinetics and equilibrium thermodynamics of binding between SH3 and two SOS-derived peptides using 19F NMR lineshape analysis. We also analyze the temperature dependence, which enables quantification of the enthalpic and entropic contributions. The energetics of SH3-peptide binding in proteins differs from those in the small molecules we used as control cosolutes, demonstrating the importance of using proteins as physiologically relevant cosolutes. Although most of the protein cosolutes destabilize the SH3-peptide complexes, the effects are nongeneralizable and there are subtle differences, which are likely from weak nonspecific interactions between the test proteins and the protein crowders. We also quantify the effects of cosolutes on SH3 translational and rotational diffusion to rationalize the effects on association rate constants. The absence of a correlation between the SH3 diffusion data and the kinetic data in certain cosolutes suggests that the properties of the peptide in crowded conditions must be considered when interpreting energetic effects. These studies have implications for understanding protein-protein interactions in cells and show the importance of using physiologically relevant cosolutes for investigating macromolecular crowding effects.

Rapid Quantification of Protein-Ligand Binding via 19F NMR Lineshape Analysis.

Fluorine incorporation is ideally suited to many NMR techniques, and incorporation of fluorine into proteins and fragment libraries for drug discovery has become increasingly common. Here, we use one-dimensional 19F NMR lineshape analysis to quantify the kinetics and equilibrium thermodynamics for the binding of a fluorine-labeled Src homology 3 (SH3) protein domain to four proline-rich peptides. SH3 domains are one of the largest and most well-characterized families of protein recognition domains and have a multitude of functions in eukaryotic cell signaling. First, we showe that fluorine incorporation into SH3 causes only minor structural changes to both the free and bound states using amide proton temperature coefficients. We then compare the results from lineshape analysis of one-dimensional 19F spectra to those from two-dimensional 1H-15N heteronuclear single quantum coherence spectra. Their agreement demonstrates that one-dimensional 19F lineshape analysis is a robust, low-cost, and fast alternative to traditional heteronuclear single quantum coherence-based experiments. The data show that binding is diffusion limited and indicate that the transition state is highly similar to the free state. We also measured binding as a function of temperature. At equilibrium, binding is enthalpically driven and arises from a highly positive activation enthalpy for association with small entropic contributions. Our results agree with those from studies using different techniques, providing additional evidence for the utility of 19F NMR lineshape analysis, and we anticipate that this analysis will be an effective tool for rapidly characterizing the energetics of protein interactions.