Discovery of Novel Proline-Based Neuropeptide FF Receptor Antagonists

Abstract

The neuropeptide FF (NPFF) system has been implicated in a number of physiological processes including modulating the pharmacological activity of opioid analgesics and several other classes of drugs of abuse. In this study, we report the discovery of a novel proline scaffold with antagonistic activity at the NPFF receptors through a high throughput screening campaign using a functional calcium mobilization assay. Focused structure-activity relationship studies on the initial hit 1 have resulted in several analogs with calcium mobilization potencies in the submicromolar range and modest selectivity for the NPFF1 receptor. Affinities and potencies of these compounds were confirmed in radioligand binding and functional cAMP assays. Two compounds, 16 and 33, had good solubility and blood-brain barrier permeability that fall within the range of CNS permeant candidates without the liability of being a P-glycoprotein substrate. Finally, both compounds reversed fentanyl-induced hyperalgesia in rats when administered intraperitoneally. Together, these results point to the potential of these proline analogs as promising NPFF receptor antagonists.

Validation of a High-Throughput Calcium Mobilization Assay for the Human Trace Amine-Associated Receptor 1

“…33 Since then, our group has developed numerous calcium mobilization assays for multiple targets and found that pretreating cells for 15 min prior to compound addition and simultaneous data acquisition dramatically reduces the initial cellular response to DMSO. [35][36][37][38] Thus, when this pretreatment protocol was applied to the hTAAR1 assay during the initial development stage, a concentration of 1% DMSO did not elevate the baseline (baseline RFU = 243 for 1% DMSO, compared with 250 RFU for 0% DMSO).…”

“…Apparent K e values were calculated using the equation K e = [L]/((A′/A) -1), where [L] is the concentration of test compound, A′ is the EC 50 value of PEA in the presence of antagonist, and A is the EC 50 value of PEA in the absence of antagonist. 35,37,[39][40][41][42][43] K e values were considered valid when the ER was at least 4. In the curve shift assay, antagonist 1 has a K e value of 110 nM, while antagonist 2 has a K e value of 159 nM.…”

Neuropeptide FF and Its Receptors: Therapeutic Applications and Ligand Development

“…Finally, NPFF has been demonstrated to play a role in the development of opioid tolerance, and opioid-induced hyperalgesia. Pharmacological blockade of NPFFR1/2 prevented the development of opioid-induced hyperalgesia and analgesic tolerance. − Blocking NPFFRs with antiserum or antisense , or NPFFR antagonist RF9 ( 14 , Table , Figure ) reversed morphine tolerance in rodents. Two hypotheses have been proposed to explain the development of opioid tolerance and hyperalgesia .…”

“…Nguyen and colleagues reported NPFFR antagonists, 39 and 40 (compounds 16 and 33 in the original paper), with a distinct proline scaffold emerging from a high throughput screening campaign using CHO cells stably expressing NPFFRs . Both compounds showed antagonist activities at both NPFFRs in calcium mobilization and cAMP assays.…”

Patent Highlights August–September 2018

“…The baseline mean paw withdrawal threshold, which had decreased from 24.2 ± 1.8 g to 5.7 ± 0.8 after fentanyl treatment, was essentially restored by both candidate compounds at doses of 10-32 mg/kg. For the peer review companion paper see Nguyen et al [23] Published: 23 The miR-210 has been identified as one of the major micro-RNA species induced by hypoxia. It is a regulator of several cellular functions (e.g., angiogenesis, neurogenesis after stroke and induction of erythrocyte development), including many that are not directly related to hypoxia [24].…”