American Society for Pharmacology and Experimental Therapeutics Division for Pharmacology Education at EB2022-Meeting report.

The American Society for Pharmacology and Experimental Therapeutics (ASPET) held its annual meeting at the Experimental Biology 2022 conference in Philadelphia, PA on April 2-5, 2022. The authors provide a synopsis and discussion of each of the four sessions presented at the meeting under the ASPET Division for Pharmacology Education (DPE).

Repeated stressor exposure regionally enhances beta-adrenergic receptor-mediated brain IL-1ß production.

It has been proposed that increased brain cytokines during repeated stressor exposure can contribute to neuropathological changes that lead to the onset of depression. Previous studies demonstrate that norepinephrine acting via beta-adrenergic receptors (ß-ARs) mediate brain IL-1 production during acute stressor exposure. The aim of the current studies was to examine how the regulation of brain cytokines by adrenergic signaling might change following repeated stressor exposure. Fischer rats were exposed to four days of chronic mild stress and 24h after the last stressors ß-AR expression, norepinephrine turnover, and ß-AR-mediated induction of brain IL-1 were measured in limbic areas (e.g. hypothalamus, hippocampus, amygdala, and prefrontal cortex) and brainstem. Repeated stressor exposure resulted in decreases in ß-AR expression (B(max)) measured by saturation binding curves in many limbic brain areas, while an increase was observed in the brainstem. This coincided with significant increases in norepinephrine turnover in the prefrontal cortex, hypothalamus, and amygdala, a significant increase in norepinephrine turnover was not observed in the hippocampus or brainstem. Stress increased overall IL-1 production in the amygdala (both basal and stimulated). While stress did not affect basal IL-1 levels in any other brain area, central administration of isoproterenol (a ß-AR agonist) augmented IL-1 production in the hypothalamus of stressed animals. These data indicate that repeated stressor exposure results in brain area specific enhancements in ß-AR-mediated IL-1 production and extends current knowledge of stress-induced enhancement of brain cytokine beyond sensitized response to immunological stimuli.

The adenosine 2A receptor agonist GW328267C improves lung function after acute lung injury in rats.

There is a significant unmet need for treatments of patients with acute lung injury (ALI) and/or acute respiratory distress syndrome (ARDS). The primary mechanism that leads to resolution of alveolar and pulmonary edema is active vectorial Na(+) and Cl(-) transport across the alveolar epithelium. Several studies have suggested a role for adenosine receptors in regulating this fluid transport in the lung. Furthermore, these studies point to the A(2A) subtype of adenosine receptor (A(2A)R) as playing a role to enhance fluid transport, suggesting that activation of the A(2A)R may enhance alveolar fluid clearance (AFC). The current studies test the potential therapeutic value of the A(2A)R agonist GW328267C to accelerate resolution of alveolar edema and ALI/ARDS in rats. GW328267C, at concentrations of 10(-5) M to 10(-3) M, instilled into the airspaces, increased AFC in control animals. GW328267C did not increase AFC beyond that produced by maximal ß-adrenergic stimulation. The effect of GW328267C was inhibited by amiloride but was not affected by cystic fibrosis transmembrane conductance regulator inhibition. The drug was tested in three models of ALI, HCl instillation 1 h, LPS instillation 16 h, and live Escherichia coli instillation 2 h before GW328267C instillation. After either type of injury, GW328267C (10(-4) M) decreased pulmonary edema formation and restored AFC, measured 1 h after GW328267C instillation. These findings show that GW328267C has beneficial effects in experimental models of ALI and may be a useful agent for treating patients with ALI or prophylactically to prevent ALI.

3D-Quantitative structure-activity relationship and docking studies of the tachykinin NK3 receptor.

The tachykinin NK(3) receptor (NK(3)R) is a novel drug target for schizophrenia and drug abuse. Since few non-peptide antagonists of this G protein-coupled receptor are available, we have initiated this study to gain a better understanding of the structure-activity relationships of NK(3) antagonist compounds. We developed a 3D comparative molecular similarity index analysis (CoMSIA) model that gave cross-validated PLS values with q(2) >0.5 which were validated using a test set. We also describe the development of a homology model of the NK(3)R. The model was then used to develop a pharmacophore for docked ligands. This pharmacophore showed two aromatic, two hydrogen donor and one acceptor/aromatic points. These data will be useful for future structure-based drug discovery of ligands for the NK(3)R.

Virtual screening to identify novel antagonists for the G protein-coupled NK3 receptor.

The NK(3) subtype of tachykinin receptor is a G protein-coupled receptor that is a potential therapeutic target for several neurological diseases, including schizophrenia. In this study, we have screened compound databases for novel NK(3) receptor antagonists using a virtual screening protocol of similarity analysis. The lead compound for this study was the potent NK(3) antagonist talnetant. Compounds of the quinoline type found in the virtual screen were additionally evaluated in a comparative molecular field analysis model to predict activity a priori to testing in vitro. Selected members of this latter set were tested for their ability to inhibit ligand binding to the NK(3) receptor as well as to inhibit senktide-induced calcium responses in cells expressing the human NK(3) receptor. Two novel compounds were identified that inhibited NK(3) receptor agonist binding, with potencies in the nM range and antagonized NK(3) receptor-mediated increases in intracellular calcium. These results demonstrate the utility of similarity analysis in identifying novel antagonist ligands for neuropeptide receptors.

Acute nicotine and phencyclidine increase locomotor activity of the guinea pig with attenuated potencies relative to their effects on rat or mouse.

Behavioral assays of the responses to psychomotor stimulants can be used to model certain aspects of CNS pathologies such as psychosis and addiction. However, species-dependent differences in the effects of neuromodulators in these assays can confound the interpretation of the results. The goal of this study was to determine the utility of the guinea pig as a model for assessing the behavioral actions of nicotinic receptor agonists and NMDA receptor antagonists. In the present study, the locomotor activity of adult male guinea pigs was measured, prior to and following an acute injection of nicotine, MK-801 or phencyclidine. Each animal received a single dose of the drug. Nicotine produced a dose-dependent increase in activity with an ED(50) of 1.5mg/kg. Phencyclidine also increased activity, with an ED(50) of 3.4 mg/kg. Nicotine produced increases in locomotion in all individual subjects tested, whereas at the maximally-effective dose of phencyclidine, only a fraction of the animals had locomotor activation. There was no change in activity in response to a single dose of MK-801 (0.5mg/kg). Haloperidol had a significant inhibitory effect on locomotor activity independent of the stimulant administered. Thus, both phencyclidine and nicotine are psychomotor stimulants when given to guinea pigs, although the intensity of the response and the potencies of these drugs are lower than in mice or rats under otherwise similar conditions.

The role of the N-terminal and mid-region residues of substance P in regulating functional selectivity at the tachykinin NK1 receptor.

Previous studies have shown that tachykinin peptide ligands of the tachykinin NK1 receptor exhibit functional selectivity with respect to signal activation and desensitization. The differences are most dramatic between the naturally occurring peptides substance P (RPKPQQFFGLM-NH2) and ranatachykinin C (HNPASFIGLM-NH2). To understand the structural features of the peptides that underlie these differences, four peptide analogs have been designed and tested. The analogs were designed to assess the major structural differences between substance P and ranatachykinin C, including the role of the N-terminal Arg and the substitution of the mid-region Glns with Ala and Ser (Q5 replaced with A and/or Q6 replaced with S). Receptor binding, receptor activation of intracellular calcium fluxes, and receptor desensitization of the rat tachykinin NK1 receptor were quantified for each ligand. All of the peptides bound to the rat tachykinin NK1 receptor with high affinity, produced robust calcium signal activation, and led to agonist-induced receptor desensitization. It was found that deletion of the N-terminal Arg of substance P or replacement of either or both Q5 and Q6 altered the functional selectivity of substance P based on the relationship of receptor binding to receptor activation and activation to desensitization. When considered in light of our previously published nuclear magnetic resonance structure data, the data presented herein suggest that the one, five and six positions of the substance P backbone are key structural residues that govern the relative degree of tachykinin peptide-mediated receptor signaling and desensitization.

Conformational comparisons of a series of tachykinin peptide analogs.

Previous studies have shown differences in the biological activity and the structure of two naturally occurring tachykinin peptides, substance P (SP, RPKPQQFFGLM-NH2) and ranatachykinin C (RTKC, HNPASFIGLM-NH2). To further understand the basis for these differences, four analogs that selectively incorporate the amino acid differences between SP and RTKC have been synthesized for study. The four peptide analogs studied have the following amino acid sequences: SP2-11, also known as des-Arg SP (PKPQQFFGLM-NH2); Q5A-SP (RPKPAQFFGLM-NH2); Q6S-SP (RPKPQSFFGLM-NH2); and Q5AQ6S-SP (RPKPASFFGLM-NH2). Nuclear magnetic resonance spectroscopy and molecular modeling calculations were performed on SP, RTKC, SP2-11, Q5A-SP, Q6S-SP, and Q5AQ6S-SP to compare their conformational differences and similarities in the presence of the membrane mimetic system sodium dodecyl sulfate. The molecular modeling data of the analogs Q5A-SP and Q6S-SP show residues 1-3 have a random conformation and residues 4-8 have a helical structure, while the C-terminus contains a poly C7 conformation that is similar to SP but different from RTKC. The molecular modeling data of the analogs SP2-11 and Q5AQ6S-SP show a continuous helix conformation for residues 4-11 at the C-terminus, which is different from SP but similar to RTKC. These structural differences are related to the functional differences of binding of the peptides at the SP receptor (NK1).

Functional selectivity of NK1 receptor signaling: peptide agonists can preferentially produce receptor activation or desensitization.

A cascade of events follows binding of an agonist ligand to the tachykinin NK(1) receptor. These events include activation of multiple signal transduction pathways as well as cellular modulation of receptor function by the process of desensitization. This study examines the differences in the abilities of naturally occurring peptide agonist ligands of the tachykinin NK(1) receptor to preferentially direct signaling through the receptor to produce signal activation versus receptor desensitization. The differential effects of tachykinin peptides with respect to ligand competition binding, receptor-G protein coupling, intracellular Ca(2+) elevations, and receptor desensitization have been measured. In relation to its potency in competition binding studies, substance P produces desensitization at lower concentrations, whereas higher concentrations are required to elicit a Ca(2+) response. In contrast to this, a related peptide, ranatachykinin C, is more effective at activating a Ca(2+) response relative to its ability to produce desensitization. This difference in functional selectivity is conserved for an amphibian and two mammalian ortholog tachykinin receptors. The present study demonstrates that peptide agonist ligands of NK(1) receptors can preferentially produce signal activation or desensitization.

Functional selectivity, ligand-directed trafficking, conformation-specific agonism: what's in a name?

Research on the design of compounds to selectively affect specific subsets of signals downstream of receptors has burgeoned lately, and several reports discussed at Experimental Biology 2005 indicate progress is being made in the understanding of what makes a drug functionally selective. Different conformations adopted by receptors after associating with specific ligands can determine which intracellular signaling pathways get activated and which do not. The appeal of such specific compounds is enormous when one considers that many disease states might require the subtle manipulation of some (or even one) but not all downstream events stemming from specific receptor activation. Additionally, a better understanding of functional selectivity would likely improve the drug delivery process: if compounds are screened through several functional assays appropriately designed to look for compounds exhibiting a high degree of selectivity, then many potential lead compounds might not be as frequently overlooked.

Analysis of fluorescently labeled substance P analogs: binding, imaging and receptor activation.

BACKGROUND: Substance P (SP) is a peptide neurotransmitter found in central and peripheral nerves. SP is involved in the control of smooth muscle, inflammation and nociception. The amino acid sequence of SP is Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2. Five different forms of fluorescently labeled SP have recently been synthesized, in which Alexa 488, BODIPY Fl, fluorescein, Oregon Green 488 or tetramethylrhodamine has been covalently linked to SP at Lys3. Here, these novel analogs are characterized as to their ligand binding, receptor activation and fluorescence labeling properties. RESULTS: Competition binding studies, using radiolabeled [125I] SP, revealed that all of the labeled forms of SP, except for Alexa 488-SP, effectively competed with radiolabeled SP for binding at the rat SP receptor. With the exception of Alexa 488-SP, all of the SP analogs produced Ca++ elevations and fluorescence labeling of the SP receptor expressed in Chinese hamster ovary cells. In SP-responsive neurons, BODIPY Fl-SP and Oregon Green 488-SP were as effective as unlabeled SP in producing a reduction of the M-type K+ current. Fluorescein-SP produced variable results, while tetramethylrhodamine-SP was less potent and Alexa 488-SP was less effective on intact neurons. CONCLUSIONS: The above results show that fluorescent labeling of SP altered the biological activity and the binding properties of the parent peptide. Oregon Green 488 and BODIPY FL-SP are the most useful fluorophores for labeling SP without affecting its biological activity. Given these results, these probes can now be utilized in further investigations of the mechanisms of SPR function, including receptor localization, internalization and recycling.