Opioids assays

Potenza MN, Gold SJ, Roby-Shcmkowitz A, et al Effects of regulators of G protein-signaling proteins on the functional response of the mu-opioid receptor in a mel-anophore-based assay. J Pharmacol Exp Ther 291 482 91, 1999 Quaglio G, Talamini G, Lechi A, et al Study of 2708 heroin-related deaths in northeastern Italy 1985—98 to establish the main causes of death. Addiction 96 1127— 1137, 2001... 

Because the psychotomimetic benzomorphans, classed as sigma opioids, inhibit binding of 3H-PCP and show PCP-like actions in several behavioral assays, it has been suggested that PCP and sigma opioids act through the same binding sites. However, recent work by a number of investigators (Su 1982 Tam 1983 Martin et al. 

Agonistic and Antagonistic Effects of PCP-Derivatives and Sigma Opioids in PCP Behavioral and Receptor Assays... 

The purpose of these studies was to determine whether stereotyped behavior and ataxia induced by PCP-like drugs and sigma opioids is mediated by PCP receptors. Also, we wanted to investigate whether sigma opioid and PCP receptors are the same receptors using behavioral and radioreceptor assays. 

In 1995 the dipeptide H-Dmt-Tic-OH was reported to be a 6-opioid antagonist with unprecedented 6-receptor affinity (K = 0.022 nM) and 6 receptor selectivity (Kf/Kf = 150,000) [47]. However, in a direct comparison under identical assay conditions, this compound showed about 30 times lower 6-antagonist potency and 6 times lower 6-receptor selectivity... 

Not many nonpeptide agonists are yet available, but such compounds have been described — for example, in the angiotensin, CCK, and opioid receptor systems. In fact, for a few receptors, such as the somatostatin, ghrelin, and complement C5A receptors, basically all compounds found by screening using binding assays are agonists. In contrast, for the majority of receptors for which... 

It should be noted that much of the early SAR data on opioids was generated using whole-animal analgesic assays rather than with isolated tissue preparations rich in receptor subtypes. 

Progress in the molecular characterization of opioid receptors has been slower than for other cell-surface receptors and, to date, none has been sequenced or cloned and the second messengers mediating opioid actions are still unknown. The literature in this area has been reviewed in 1990 by Lo and Smith [11], who cite three main problems with the opioid receptor it is difficult to solubilize, there are no simple biochemical assays to test the functional integrity of an isolated receptor extract and there are at least three receptor subtypes (designated as mu, kappa and delta). 

Compound No. Opioid receptor binding (IC onM) Smooth muscle assay (ICso nM) ... 

Library Screening and Competition Assays for /<-Opioid Receptors... 

Fig. 7.11 Structures of compounds used in competitive MS binding assays for //-opioid receptors.

Table 7.3 Nonbound morphine in MS binding assays at /i-opioid receptors [65],...

Fig. 7.12 Schematic flowchart of the competitive MS binding assay for //-opioid receptors inciuding iiberation of bound marker and test compounds, respectiveiy. After incubation of the target (/t-opioid receptor) in the presence of the marker (morphine) and a compound iibrary, the binding sampies are centrifuged to separate bound from nonbound marker. Subsequentiy, the nonbound marker in the resuiting supernatant is quantified by LC-ESI-MS/MS without further sampie preparation (route 1).

To this end, the pellets remaining from the competitive MS binding assay were, after several washing steps, resuspended in binding buffer and incubated with a great excess of competitor (50 pM (+)-methadone) to liberate the unknown bound ligand (as well as the bound marker). Then the supernatants obtained by centrifugation were analyzed by LC-ESl-MS/MS. In addition to morphine as the marker, naloxone was identified as the hit that had been searched for. Thereby, the relative concentrations of marker (2.93 nM) and hit (2.30 nM) pointed to the fact that the hit had a similar affinity to the //-opioid receptor as the marker [65]. 

This strategy was realized in a competitive MS binding assay examining the affinity of naloxone for //-opioid receptors (with morphine as marker under the conditions described above for library screening). The respective experiments led... 

Fig. 7.13 Representative binding curve obtained by nonlinear regression from a competitive MS binding assay for //-opioid receptors, in which naloxone competes with morphine as marker. The points describe nonbound morphine quantified by LC-ESl-MS/MS at concentrations of nonbound naloxone determined simultaneously by LC-ESl-MS/MS.

These peptides were characterized in vitro and found to be powerful opioid agonists in the mouse vas deferens (MVD) and guinea pig ileum (GPI) assay. In vivo (rat tail-flick) they are only active when administered directly to the brain - a general limitation of simple linear peptides consisting of natural L-amino acids - but with less potency and shorter duration of action than morphine (Casy and Parfitt, 1986). 

In contrast, a low dose of the Ca2+ channel agonist BAY K 8644, a dihydropyridine derivative, antagonizes the antinociceptive effect of p-opioids. This is in agreement with results from Smith and Stevens (1995), who reported that Ca2+, when administered i.c.v., antagonizes morphine-induced antinociception in the mouse tail flick assay. The dose - response curve of morphine is shifted to the right by i.c.v. administration of calcium ions. 

---