Rayna M. Bauzo

Implication of the melanocortin-3 receptor in the regulation of food intake

The melanocortin system is well recognized to be involved in the regulation of food intake, body weight, and energy homeostasis. To probe the role of the MC(3) in the regulation of food intake, JRH322-18 a mixed MC(3) partial agonist/antagonist and MC(4) agonist tetrapeptide was examined in wild type (WT) and melanocortin 4 receptor (MC(4)) knockout mice and shown to reduce food intake in both models. In the wild type mice, 2.0 nmol of JRH322-18 statistically reduced food intake 4h post icv treatment into satiated nocturnally feeding wild type mice. The same dose in the MC(4)KO mice significantly reduced cumulative food intake 24h post treatment. Conditioned taste aversion as well as activity studies supports that the decreased food intake was not due to visceral illness. Since these studies resulted in loss-of-function results, the SHU9119 and agouti-related protein (AGRP) melanocortin receptor antagonists were administered to wild type as well as the MC(3) and MC(4) knockout mice in anticipation of gain-of-function results. The SHU9119 ligand produced an increase in food intake in the wild type mice as anticipated, however no effect was observed in the MC(3) and MC(4) knockout mice as compared to the saline control. The AGRP ligand however, produced a significant increase in food intake in the wild type as well as the MC(3) and MC(4) knockout mice and it had a prolonged affect for several days. These data support the hypothesis that the MC(3) plays a subtle role in the regulation of food intake, however the mechanism by which this is occurring remains to be determined.

Preadolescent tobacco smoke exposure leads to acute nicotine dependence but does not affect the rewarding effects of nicotine or nicotine withdrawal in adulthood in rats

Epidemiological studies indicate that parental smoking increases the risk for smoking in children. However, the underlying mechanisms by which parental smoking increases the risk for smoking are not known. The aim of these studies was to investigate if preadolescent tobacco smoke exposure, postnatal days 21-35, affects the rewarding effects of nicotine and nicotine withdrawal in adult rats. The rewarding effects of nicotine were investigated with the conditioned place preference procedure. Nicotine withdrawal was investigated with the conditioned place aversion procedure and intracranial self-stimulation (ICSS). Elevations in brain reward thresholds in the ICSS paradigm reflect a dysphoric state. Plasma nicotine and cotinine levels in the preadolescent rats immediately after smoke exposure were 188 ng/ml and 716 ng/ml, respectively. Preadolescent tobacco smoke exposure led to the development of nicotine dependence as indicated by an increased number of mecamylamine-precipitated somatic withdrawal signs in the preadolescent tobacco smoke exposed rats compared to the control rats. Nicotine induced a similar place preference in adult rats that had been exposed to tobacco smoke or air during preadolescence. Furthermore, mecamylamine induced place aversion in nicotine dependent rats but there was no effect of preadolescent tobacco smoke exposure. Finally, preadolescent tobacco smoke exposure did not affect the elevations in brain reward thresholds associated with precipitated or spontaneous nicotine withdrawal. These studies indicate that passive exposure to tobacco smoke during preadolescence leads to the development of nicotine dependence but preadolescent tobacco smoke exposure does not seem to affect the rewarding effects of nicotine or nicotine withdrawal in adulthood.

Chimeric NDP-MSH and MTII melanocortin peptides with agouti-related protein (AGRP) Arg-Phe-Phe amino acids possess agonist melanocortin receptor activity

Agouti-related protein (AGRP) is one of only two known endogenous antagonists of G-protein coupled receptors (GPCRs). Specifically, AGRP antagonizes the brain melanocortin-3 and -4 receptors involved in energy homeostasis, regulation of feeding behavior, and obesity. Alpha-melanocyte stimulating hormone (alpha-MSH) is one of the known endogenous agonists for these receptors. It has been hypothesized that the Arg-Phe-Phe (111-113) human AGRP amino acids may be mimicking the melanocortin agonist Phe-Arg-Trp (7-9) residue interactions with the melanocortin receptors that are important for both receptor molecular recognition and stimulation. To test this hypothesis, we generated thirteen chimeric peptide ligands based upon the melanocortin agonist peptides NDP-MSH (Ac-Ser-Tyr-Ser-Nle4-Glu-His-DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH2) and MTII (Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-NH2). In these chimeric ligands, the agonist DPhe-Arg-Trp amino acids were replaced by the AGRP Arg-Phe-Phe residues, and resulted in agonist activity at the mouse melanocortin receptors (mMC1R and mMC3-5Rs), supporting the hypothesis that the AGRP antagonist ligand Arg-Phe-Phe residues mimic the agonist Phe-Arg-Trp amino acids. Interestingly, the Ac-Ser-Tyr-Ser-Nle4-Glu-His-Arg-DPhe-Phe-Gly-Lys-Pro-Val-NH2 peptide possessed 7 nM mMC1R agonist potency, and is 850-fold selective for the mMC1R versus the mMC3R, 2300-fold selective for the mMC1R versus the mMC4R, and 60-fold selective for the MC1R versus the mMC5R, resulting in the discovery of a new peptide template for the design of melanocortin receptor selective ligands.

Elongation studies of the human agouti-related protein (AGRP) core decapeptide (Yc[CRFFNAFC]Y) results in antagonism at the mouse melanocortin-3 receptor

The agouti-related protein (AGRP) is an endogenous antagonist of the brain melanocortin receptors (MC3R and MC4R) and is believed to be involved in feeding behavior and energy homeostasis. Previous results identified that the human AGRP decapeptide Yc[CRFFNAFC]Y binds to the MC3R and MC4R and acts as an antagonist at the MC4R but not at the MC3R. We have synthesized the amidated version of this decapeptide as well as performed elongation studies at both the N-and C-terminus of the monocyclic hAGRP(109-118) peptide. This study was designed to identify monocyclic peptide fragments of the hAGRP(86-132) to determine the minimal active monocyclic sequence necessary for antagonism at the MC3R. For binding studies, radiolabeled 125I-NDP-MSH versus 125I-hAGRP(86-132) were utilized to determine if there were differences in the ability of the AGRP fragments prepared herein to competitively displace the 125I-NDP-MSH versus AGRP(86-132) radiolabel. The binding IC(50) values of radiolabeled hAGRP(86-132) versus NDP-MSH were identical within experimental error, supporting the hypothesis that AGRP and NDP-MSH interact with overlapping binding epitopes at the MC3R and MC4R. The most notable results include identification of the TAYc[CRFFNAFC]YAR-NH(2) (pA(2)=6.1, K(i)=790nM, mMC3R) and the Yc[CRFFNAFC]YARKL-NH(2) (pA(2)=6.2, K(i)=630nM, mMC3R) peptides as the minimal monocyclic AGRP-based fragments possessing antagonist pharmacology at the MC3R. Interestingly, extension of the AGRP(109-118) decapeptide at both the N- and C-terminus by two amino acids conferred detectable mMC3R antagonism, while retaining high nanomolar MC4R antagonist and micromolar MC1R agonist pharmacological properties. These data support the hypothesis that elongation of the hAGRP(109-118) decapeptide results in antagonism at the MC3R while retaining MC1R agonist activity and MC4R antagonist activity.

Urea small molecule agonists on mouse melanocortin receptors

A series of urea compounds based on the tripeptide Phe-Trp-Lys were synthesized and pharmacologically characterized at the mouse melanocortin receptors. The results include identification of novel melanocortin receptor agonists with potencies ranging from nanomolar to micromolar.

Animal models of nicotine withdrawal: intracranial self-stimulation and somatic signs of withdrawal.

Tobacco addiction is one of the leading causes of preventable death worldwide. Despite the negative health outcomes of tobacco use and a desire to quit, there is a low success rate of maintaining abstinence. Nicotine, the main psychoactive component of tobacco smoke, is mildly rewarding and maintains smoking behavior. Nicotine withdrawal induces somatic symptoms that may contribute to smoking behavior. However, it has been hypothesized that the negative affective signs are of greater motivational significance in contributing to relapse and continued tobacco use than the somatic symptoms of nicotine withdrawal (Markou and Koob (Eds.) Intracranial self-stimulation thresholds as a measure of reward, Vol. 2, Oxford University Press, New York, 1993; Koob et al. Semin Neurosci 5: 351-358, 1993). Intracranial self-stimulation (ICSS) has been established as a method to assess the bivalent properties of nicotine exposure and withdrawal from acute and chronic nicotine administration. Thus, ICSS provides a means to measure the negative affective aspects of nicotine withdrawal in animal models and may contribute to the understanding of the neurobiological bases of nicotine dependence and the development of effective treatment strategies to facilitate nicotine abstinence.

Repeated pre-exposure to tobacco smoke potentiates subsequent locomotor responses to nicotine and tobacco smoke but not amphetamine in adult rats

These studies investigated if pre-exposure to tobacco smoke affects the locomotor response to tobacco smoke, nicotine, and amphetamine in adult rats. The rats were habituated to an open field for 3-4 days and then exposed to tobacco smoke for 2h/day for 13-14 days. The effect of exposure to tobacco smoke on locomotor activity was investigated after 1, 7, and 14 days of smoke exposure and after one 2-hour exposure session that followed a 3-week off period. The effects of tobacco smoke on the locomotor responses to nicotine (0.04 and 0.4 mg/kg, base) and amphetamine (0.1 and 0.5mg/kg) were investigated on day 14, one day after the last smoke exposure session. The locomotor response to tobacco smoke was increased after 7 and 14 days of smoke exposure and after one exposure session after the 3-week off-period. The acute administration of the high dose of nicotine (0.4 mg/kg) led to a brief period of hypoactivity that was followed by a period of hyperactivity. Pre-exposure to tobacco smoke attenuated the nicotine-induced hypoactivity and potentiated the nicotine-induced hyperactivity. The low dose of nicotine (0.04 mg/kg) did not affect locomotor activity in the control rats but increased the total distance traveled in the tobacco smoke exposed rats. Exposure to tobacco smoke did not affect the locomotor response to amphetamine. These findings indicate that exposure to tobacco smoke leads to tolerance to the depressant effects of nicotine and potentiates the stimulant effects of nicotine and tobacco smoke.

Methods in tobacco abuse: proteomic changes following second-hand smoke exposure.

Smoking is one of the leading preventable causes of disease, disability, and death in the USA and leads to more than 400,000 preventable deaths per year. Nicotine is the major alkaloid present in tobacco smoke, and many of the negative effects of smoking are attributed to nicotine. Nicotine is not only the addictive component of tobacco smoke, but also highly associated with carcinogenesis and induces oxidative stress. Furthermore, the administration of nicotine via subcutaneous mini-osmotic pumps or by injection is an established method in preclinical studies for this area of research. Thus, preclinical research on the negative effects of tobacco smoke and tobacco addiction has focused primarily on the effects of nicotine. However, there are over 4,500 components found in tobacco smoke, many of which are highly toxic. Other components may also contribute to the addictive properties of tobacco smoke. Furthermore, the negative effects of tobacco smoke are not isolated to the smoker but can have negative effects to those exposed to the secondhand smoke (SHS) stream. SHS exposure is the third leading cause of preventable death. Approximately 38,000 deaths per year are attributed to SHS exposure in the USA. SHS exposure increases the risk of heart disease by approximately 30% and is associated with increased risk of stroke, cancer, type II diabetes, as well as pulmonary disease. Thus, methods of administering tobacco smoke in a controlled environment will further our understanding of tobacco addiction and the role tobacco smoke in other disease states. Moreover, combining smoke exposure with proteomics can lead to the discovery of biomarkers that can be potentially useful tools in screening, early diagnosis, prevention, and treatment of diseases caused by SHS.

A critical role for the melanocortin 4 receptor in stress‐induced relapse to nicotine seeking in rats

Tobacco addiction is characterized by a lack of control over smoking and relapse after periods of abstinence. Smoking cessation leads to a dysphoric state that contributes to relapse to smoking. After the acute withdrawal phase, exposure to stressors increases the risk for relapse. Blockade of melanocortin 4 (MC4) receptors has anxiolytic and antidepressant‐like effects in animal models. The aim of these studies was to investigate the role of MC4 receptors in the dysphoria associated with nicotine withdrawal and stress‐induced reinstatement of nicotine seeking. To study stress‐induced reinstatement, rats self‐administered nicotine for 16 days and then nicotine seeking was extinguished by substituting saline for nicotine. Nicotine seeking was reinstated by intermittent footshock stress. The intracranial self‐stimulation (ICSS) procedure was used to assess the negative mood state associated with nicotine withdrawal. Elevations in the ICSS thresholds are indicative of a dysphoric state. The selective MC4 receptor antagonists HS014 and HS024 prevented stress‐induced reinstatement of extinguished nicotine seeking. Drug doses that prevented stress‐induced relapse did not affect responding for food pellets, which indicates that the drugs did not induce sedation or motor impairments. In the ICSS experiments, the nicotinic acetylcholine receptor antagonist mecamylamine elevated the ICSS thresholds of the nicotine‐dependent rats. Pre‐treatment with HS014 or HS024 did not prevent the elevations in ICSS thresholds. These studies indicate that MC4 receptors play a critical role in stress‐induced reinstatement of nicotine seeking, but these receptors may not play a role in the dysphoria associated with acute nicotine withdrawal.

A Novel Solid Phase Approach to Thioether Cyclized Peptides: Discovery of Mouse Melanocortin-1 Receptor Agonists

The β-turn 1 is both one of the most abundant motifs of peptide and protein secondary structure, and one of the key recognition elements in biological systems. In recent years the design of conformationally constrained peptidomimetics and small molecule β-turn mimics has been persued with significant effort. Such peptidomimetic scaffolds can provide potential drug leads, as well as a better understanding of molecular recognition. We have prepared a range of novel heterocyclic peptidomimetics with the general structure 2 (Scheme 1, Figure 1, Table 1).