We employed a structural bioinformatics approach to develop novel peptides with predicted affinity to the binding site for negative allosteric modulators (NAMs) of metabotropic glutamate receptor 5 (mGluR5). Primary screening in zebrafish (Danio rerio) revealed a stimulatory effect of two peptides, LCGM-10 and LCGM-15. Target validation studies using calcium ion flux imaging and a luciferase reporter assay confirmed mGluR5 as the target. LCGM-10 showed greater potency than LCGM-15; it was comparable to that of the mGluR5 NAM 2-methyl-6-(phenylethynyl) pyridine (MPEP). Rodent behavioral screening in the open field and elevated plus maze revealed increased locomotor activity in both tests after acute LCGM-10 treatment, supported by further analysis of home cage spontaneous locomotor activity (SLA). The stimulating effect of a single LCGM-10 administration on SLA was evident up to 60 min after administration and was not accompanied by hypokinetic rebound observed for caffeine. According to our results, LCGM-10 has therapeutic potential to treat hypo- and dyskinesias of various etiologies. Further investigation of LCGM-10 effects in the delay discounting model of impulsive choice in rats revealed reduced trait impulsivity after single and chronic administrations, suggesting potential implication for attention deficit hyperactivity disorder, obsessive compulsive disorder, and addictions.
The development of novel treatments normalizing metabotropic glutamate receptor 5 (mGluR5) function in the central nervous system (CNS) is of great importance. mGluR5 is expressed at high levels in several brain regions and is involved in a multitude of brain-related illnesses including fragile X syndrome (FXS), depression, Parkinson’s disease, Alzheimer’s disease, attention deficit hyperactivity disorder (ADHD), and addictions. Because direct-acting agonists produce substantial adverse effects and eventually lead to profound receptor desensitization, the development of allosteric modulators has been at the forefront of G protein–coupled receptor (GPCR) drug development. Negative allosteric modulators (NAMs) and positive allosteric modulators (PAMs) will only modulate receptor activity in the presence of the endogenous agonist, which is not possible with orthosteric ligands and enables more specific control of the tissue response. The non-competitive mechanism of action of NAMs makes them relatively unaffected by high concentrations of glutamate that may be present in disease states (e.g., stroke, epilepsy, neuropathic pain, etc.).
The development of new drugs with an allosteric mode of action has been greatly enhanced by advances in X-ray crystallography and cryo-electron microscopy, which have provided databases of high-resolution GPCR structures in complex with ligands and intracellular effectors for the docking studies. Here we employed computational modeling to search for peptides that interact with the NAM site of mGluR5. We focused on peptides as potential therapeutics because of their safety and tolerability profiles, which are superior to those of other small molecules. We identified four novel, previously undescribed peptides with predicted affinity to the mGluR5 NAM site based on in silico docking studies and revealed that two of them, LCGM-10 and LCGM-15, have a potential stimulating effect in zebrafish. mGluR5 modulation by LCGM-10 in a wide range of concentrations was supported by calcium ion flux imaging and a luciferase assay, using known mGluR5 agonists and antagonists. Several mGluR5 NAMs have been investigated for the treatment of drug addiction, FXS, Parkinson’s disease L-dopa-induced dyskinesia, and depression, showing promising results in animal models. Several mGluR5 non-competitive antagonists have been tested for potential efficacy in clinical trials, including mavoglurant (FXS, cocaine use disorder, L-dopa induced dyskinesias, obsessive compulsive disorder [OCD], and Huntington’s disease), basimglurant (major depressive disorder), GET 73 (alcohol use disorder), and ADX10059 (gastroesophageal reflux disease, dental anxiety, and migraine). The efficacy of mGluR5 antagonists has been reported in trials with patients with gastroesophageal reflux; however, data from patients with Parkinson’s disease or FXS have not been as robust as hoped. Fenobam was approved for use as an anxiolytic prior to its recognition as a mGluR5 NAM.
In this study, in vivo behavioral characterization of the LCGM-10 and LCGM-15 peptides revealed hyperlocomotion in intact animals in the standard behavioral paradigms of open field (OF) and elevated plus maze (EPM), as well as in the home cage conditions. Additionally, in the delay discounting model of impulsivity, we found that single and chronic LCGM-10 treatment potently reduced impulsivity in rats. Our results suggest the need for additional studies of LCGM-10 as a potential treatment for hypo-and dyskinesias as well as for ADHD, OCD, and pathological conditions associated with impulsive behavior (drug addiction and gambling).
Wildtype D. rerio (128 fish, shortfin phenotype, 6–8 months of age, male to female ratio 50: 50) were kept in a ZebTEC recirculating system (Tecniplast S.p.a, Buguggiate, Italy), and housed under a 14/10-h photoperiod (lights on at 08: 00 and off at 22: 00). The system parameters were maintained automatically with water set at 28°C, pH 6.8–7.5, 550–700 mOsm/L, and constant aeration. Feeding was carried out twice a day with special food for fish (Special Diet Services, Scientific Fish Food, SDS 300–400). In total, 30 male Sprague Dawley rats were used in the behavioral screening study, and 53 male Wistar rats in the locomotion study (N = 23) and the delay discounting test (N = 30). The animals were housed under constant environmental conditions (12-h photoperiod at 22 ± 2°C) with ad libitum access to food and water. All animal experiments were conducted in accordance with the European Directive 2010/63/EU of the European Parliament (Council of 22 September 2010 on the protection of animals used for scientific purposes) and were approved by the local Bioethics Commissions. All manipulations with animals were carried out at the end of the 14-day adaptation period.
LCGM peptides (supplied by Lactocore Inc., synthesized by Peptide 2.0 Inc. [Herndon, VA, United States], 98% purity) were administered at a dose of 1 mg/kg (intraperitoneal, prepared in saline on the day of the experiment). The peptides had the following amino acid composition: LCGM-2 (AGAS = AlaGlyAlaSer), LCGM-5 (DSGH = AspSerGlyHis), LCGM-10 (KEDV = LysGluAspVal), and LCGM-15 peptide (RAHE = ArgAlaHisGlu). The control groups were treated in parallel with vehicle alone (saline). For drug injection, the fish were anesthetized briefly by placing them in 10°C water. The vehicle controls were tested in parallel (four control groups in total, one for each peptide-treated group).
Behavioral testing was initiated 10 min after drug injection, starting with the novel tank test (NTT) and followed immediately thereafter by the light–dark box (LDB) test. The NTT was adapted from Maximino et al., 2013 with the behavior video-recorded and the data processed using EthoVision XT14 (Noldus, Netherlands). The distance traveled; speed; the number of visits to the bottom, middle, and top thirds of the aquarium; and the times spent in each of these zones during the initial 5 min in the tank were recorded. A decreased time spent at the surface of the NTT apparatus reflects a reduction in exploratory behavior or increased hiding motivation. The behavior in the LDB test, adapted from Maximino et al., 2011, was video recorded and processed using EthoVision XT14 (Noldus). The fish were added to the center zone of a three-zone aquarium and allowed to adapt for 1–2 min before removal of the septa separating the center from the flanking zones. The time spent and the number of visits to the light and dark flanking zones and the latency to enter the lit zone were recorded during a 5-min session. Stress of fish is associated with increased time spent in the dark compartment (scototaxis). All behavioral testing was done in the light phase using 500 lux illumination. The experiment was performed by the Institute of Mitoengineering of MSU on a contract basis.
For statistical evaluation, experimental raw data were converted into Z-scores as followed: (1) for each control group mean (μ) and standard deviation (σ) were calculated; (2) Z-score for each fish from the peptide-treated group was found with formula: z=x−μ σ, where x is the observed value in the peptide-treated group, μ and σ are values of the corresponding control group.
We administered LCGM peptides intranasally. Peptides were dissolved in saline at a concentration appropriate for the dosage. For administration, the rat was held in a horizontal position, with its head slightly tilted back. Then, using an automatic laboratory pipette, no more than 10 μL of the peptide solution was carefully introduced into each nostril of the rat. After visually confirming that the entire volume of liquid entered the animal’s nose, the rats were returned to their home cage. The total volume administered did not exceed 20 μL per rat and was calculated based on the animal’s weight. This route of administration has several advantages for short peptides, such as rapid systemic drug absorption and the potential to bypass the blood–brain barrier more effectively and access the central nervous system. For patients, this method is relatively noninvasive and limits the side effects associated with peripheral administration of substances.
