Carrie Haskell-Luevano

Design of peptides, proteins, and peptidomimetics in chi space

Peptide and protein biological activities depend on their three dimensionals structures in the free state and when interacting with their receptors/acceptors. The backbone conformations such as alpha-helix, beta-sheet, beta-turn, and so forth provide critical templates for the three-dimensional structure, but the overall shape and intrinsic stereoelectronic properties of the peptide or protein important for molecular recognition, signal transduction, enzymatic specificity, immunomodulation, and other biological effects depend on arrangement of the side chain groups in three-dimensional chi space (their chi 1, chi 2, etc. torsional angles). In this paper we explore approaches to the de novo design of polypeptides and peptidomimetics with biased or specific conformational/topographical properties in chi space. We consider computational and experimental methods that can be used to examine the effects of specific structural modifications in constraining side chain groups of amino acid residues and their similarities in chi space to the natural amino acids to evaluate what sort of mimetics are likely to mimic normal amino acids. We then examine some of the asymmetric synthetic methods that are being developed to obtain the amino acid mimetics. Finally, we consider selected examples in the literature where these specialized amino acids have been incorporated in biologically active peptides and the specific insights they have provided regarding the topographical requirements for bioactive peptide potency, selectivity, and other biochemical and pharmacological properties. Constraints in chi space show great promise as useful tools in peptide, protein, and peptidomimetic de novo design of structures and pharmacophores with specific stereostructural, biochemical and biological properties.

Agouti-related protein functions as an inverse agonist at a constitutively active brain melanocortin-4 receptor.

Agouti-related protein (AGRP) is one of two naturally occurring antagonists of G-Protein coupled receptors (GPCRs) identified to date, and has been physiologically implicated in regulating food intake, body weight, and energy homeostasis. AGRP has been identified in vitro, as competitively antagonizing the brain melanocortin-4 (MC4R) and melanocortin-3 (MC3R) receptors, and when over expressed in transgenic mice, results in an obese phenotype. Emerging data propose that AGRP has additional targets in the hypothalamus and/or physiologically functions via a mechanism in addition to competitive antagonism of alpha-MSH at the brain melanocortin receptors. We report data herein supporting an alternative mechanism for AGRP involvement in feeding behavior. A constitutively active MC4R has been generated which possess EC(50) values for melanocortin agonists (alpha-MSH, NDP-MSH, and MTII) and a pA2 value for the synthetic peptide antagonist SHU9119 identical to the wildtype receptor, but increases basal activity to 50% maximal response. AGRP possesses inverse agonist activity at this constitutively active MC4R. These data support the hypothesis for an additional physiological mechanism for AGRP action in feeding behavior and energy homeostasis.

The Proopiomelanocortin System

ABSTRACT: POMC (31,000 MW) is localized to the pituitary, brain, skin, and other peripheral sites. The particular enzyme profile present within a cell dictates the nature of the hormonal ligand (melanocortin) synthesized and secreted: melanotropic peptides (α‐MSH β‐lipotropin, λ‐MSH), corticotropin (ACTH), several endorphins (e.g., met‐enkephalin). These POMC‐derived peptides mediate their actions through typical seven‐spanning membrane receptors (MCRs; MCR1, 2, 3, 4, and 5). A specific melanocortin acting on a specific MCR regulates a particular biological response; for example, α‐MSH on MCR1 increases melanogenesis within melanocytes, ACTH on MCR2 increases cortisol production within adrenal zona fasciculata cells. Within the brain melanocortins regulate satiety (MCR4) and erectile activity (MCR?). MCRs have been localized by melanocortin macromolecular probes, for example, fluorescent to human epidermal melanocytes and also to keratinocytes, suggesting that systemic melanocortins or localized POMC products might regulate these integumental cellular elements in synchrony to enhance skin pigmentation and/or immunological responses. Superpotent, prolonged acting melanotropic peptides have been synthesized and their application in clinical medicine has been demonstrated. MCR antagonists have been used to discover and further delineate other roles of melanocortin ligands. For example, melanocortin‐induced satiety can be antagonized by a melanocortin antagonist. Defects in melanocortin ligand biosynthesis, secretion, and melanocortin receptor function can lead to a diverse number of pathological states.

Molecular Basis for the Interaction of [Nle4,d-Phe7]Melanocyte Stimulating Hormone with the Human Melanocortin-1 Receptor (Melanocyte α-MSH Receptor)

The melanocortin-1 receptor (MC1R) is a seven-transmembrane (TM) G-protein-coupled receptor whose natural ligands are the melanocortin peptides, adrenocorticotropic hormone, and α-, β-, and γ- melanocyte stimulating hormone (MSH). To test a previously constructed three-dimensional model of the molecular interaction between the long-acting, superpotent α-MSH analog [Nle4,d-Phe7]MSH (NDP-MSH) and the human MC1R we examined the effects of site-directed receptor mutagenesis on the binding affinity and potency of NDP-MSH. In addition, we also examined the effects of these same mutations on the binding affinity and potency of the structurally related agonists α-MSH, γ-MSH, and Ac-Nle4-cyclic-[Asp5,His6,d-Phe7,Arg8,Trp9,Lys10]NH2(MT-II). Mutagenesis of acidic receptor residues Glu94 in TM2 and Asp117 or Asp121in TM3 significantly altered the binding affinity and potency of all four agonists suggesting that these receptor residues are important to the ligand-receptor interactions of all. A disproportionate change in agonist potency versus affinity observed with simultaneous mutation of these acidic residues (mutant constructs D117A/D121A or E94A/D117A/D121A) or introduction of a single positive charge (mutant construct D121K) also implicates these residues in receptor activation. In addition, results from the individual mutation of aromatic receptor residues Phe175, Phe196, and Phe257, and simultaneous mutation of multiple TM4, -5, and -6 tyrosine and phenylalanine residues suggests that aromatic-aromatic ligand-receptor interactions also participate in binding these melanocortins to the MC1R. These experiments appear to have identified some of the critical receptor residues involved in the ligand-receptor interactions between these melanocortins and the hMC1R.

α-MSH tripeptide analogs activate the melanocortin 1 receptor and reduce UV-induced DNA damage in human melanocytes

One skin cancer prevention strategy that we are developing is based on synthesizing and testing melanocortin analogs that reduce and repair DNA damage resulting from exposure to solar ultraviolet (UV) radiation, in addition to stimulating pigmentation. Previously, we reported the effects of tetrapeptide analogs of α‐melanocortin (α‐MSH) that were more potent and stable than the physiological α‐MSH, and mimicked its photoprotective effects against UV‐induced DNA damage in human melanocytes. Here, we report on a panel of tripeptide analogs consisting of a modified α‐MSH core His6‐d‐Phe7‐Arg8, which contained different N‐capping groups, C‐terminal modifications, or arginine mimics. The most potent tripeptides in activating cAMP formation and tyrosinase of human melanocytes were three analogs with C‐terminal modifications. The most effective C‐terminal tripeptide mimicked α‐MSH in reducing hydrogen peroxide generation and enhancing nucleotide excision repair following UV irradiation. The effects of these three analogs required functional MC1R, as they were absent in human melanocytes that expressed non‐functional receptor. These results demonstrate activation of the MC1R by tripeptide melanocortin analogs. Designing small analogs for topical delivery should prove practical and efficacious for skin cancer prevention.

Melanoma prevention strategy based on using tetrapeptide α-MSH analogs that protect human melanocytes from UV-induced DNA damage and cytotoxicity

Melanoma is the deadliest form of skin cancer, with no cure for advanced disease. We propose a strategy for melanoma prevention based on using analogs of α‐melanocyte stimulating hormone (α‐MSH) that function as melanocortin 1 receptor (MC1R) agonists. Treatment of human melanocytes with α‐MSH results in stimulation of eumelanin synthesis, reduction of apoptosis that is attributable to reduced hydrogen peroxide generation and enhanced repair of DNA photoproducts. These effects should contribute to genomic stability of human melanocytes, thus preventing their malignant transformation to melanoma. Based on these findings, we synthesized and tested the effects of 3 tetrapeptide α‐MSH analogs, Ac‐His‐D‐Phe‐Arg‐Trp‐NH2, n‐Pentadecanoyl‐ and 4‐Phenylbutyryl‐His‐D‐Phe‐Arg‐Trp‐NH2, on cultured human melanocytes. The latter two analogs were more potent than the former, or α‐MSH, in stimulating the activity of tyrosinase, thus melanogenesis, reducing apoptosis and release of hydrogen peroxide and enhancing repair of DNA photoproducts in melanocytes exposed to UV radiation (UVR). The above analogs are MC1R agonists, as their effects were abrogated by an analog of agouti signaling protein, the physiological MC1R antagonist, and were absent in melanocytes expressing loss‐of‐function MC1R. Analogs, such as 4‐Phenylbutyryl‐His‐D‐Phe‐Arg‐Trp‐NH2 with prolonged and reversible effects, can potentially be developed into topical agents to prevent skin photocarcinogenesis, particularly melanoma.—Abdel‐Malek, Z. A., Kadekaro, A. L., Kavanagh, R. J., Todorovic, A., Koikov, L. N., McNulty, J. C., Jackson, P. J., Millhauser, G. L., Schwemberger, S., Babcock, G., Haskell‐Luevano, C., Knittel, J. J. Melanoma prevention strategy based on using tetrapeptide α‐MSH analogs that protect human melanocytes from UV‐induced DNA damage and cytotoxicity. FASEB J. 20, E888–E896 (2006)

Voluntary exercise prevents the obese and diabetic metabolic syndrome of the melanocortin-4 receptor knockout mouse

Exercise is a mechanism for maintenance of body weight in humans. Morbidly obese human patients have been shown to possess single nucleotide polymorphisms in the melanocortin‐4 receptor (MC4R). MC4R knockout mice have been well characterized as a genetic model that possesses phenotypic metabolic disorders, including obesity, hyperphagia, hyperinsulinemia, and hyperleptinemia, similar to those observed in humans possessing dysfunctional hMC4Rs. Using this model, we examined the effect of voluntary exercise of MC4R knockout mice that were allowed access to a running wheel for a duration of 8 wk. Physiological parameters that were measured included body weight, body composition of fat and lean mass, food consumption, body length, and blood levels of cholesterol and nonfasted glucose, insulin, and leptin. At the termination of the experiment, hypothalamic mRNA expression levels of neuropeptide Y (NPY), agouti‐related protein (AGRP), proopiomelanocortin (POMC), cocaine‐and amphetamine‐regulated transcript (CART), orexin, brain‐derived neurotropic factor (BDNF), phosphatase with tensin homology (Pten), melanocortin‐3 receptor (MC3R), and NPY‐Y1R were determined. In addition, islet cell distribution and function in the pancreas were examined. In the exercising MC4R knockout mice, the pancreatic islet cell morphology and other physiological parameters resembled those observed in the wild‐type littermate controls. Gene expression profiles identified exercise as having a significant effect on hypothalamic POMC, orexin, and MC3R levels. Genotype had a significant effect on AGRP, POMC, CART, and NPY‐Y1R, with an exercise and genotype interaction effect on NPY gene expression. These data support the hypothesis that voluntary exercise can prevent the genetic predisposition of melanocortin‐4 receptor‐associated obesity and diabetes.—Haskell‐Luevano, C., Schaub, J. W., Andreasen, A., Haskell, K. R., Moore, M. C., Koerper, L. M., Rouzaud, F., Baker, H. V., Millard, W. J., Walter, G., Litherland, S. A., Xiang, Z. Voluntary exercise prevents the obese and diabetic metabolic syndrome of the melanocortin‐4 receptor knockout mouse. FASEB J. 23, 642–655 (2009)

Backbone cyclic peptidomimetic melanocortin-4 receptor agonist as a novel orally administrated drug lead for treating obesity.

The tetrapeptide sequence His-Phe-Arg-Trp, derived from melanocyte-stimulating hormone (alphaMSH) and its analogs, causes a decrease in food intake and elevates energy utilization upon binding to the melanocortin-4 receptor (MC4R). To utilize this sequence as an effective agent for treating obesity, we improved its metabolic stability and intestinal permeability by synthesizing a library of backbone cyclic peptidomimetic derivatives. One analog, peptide 1 (BL3020-1), was selected according to its selectivity in activating the MC4R, its favorable transcellular penetration through enterocytes and its enhanced intestinal metabolic stability. This peptide was detected in the brain following oral administration to rats. A single oral dose of 0.5 mg/kg in mice led to reduced food consumption (up to 48% vs the control group) that lasted for 5 h. Repetitive once daily oral dosing (0.5 mg/kg/day) for 12 days reduced weight gain. Backbone cyclization was shown to produce a potential drug lead for treating obesity.

Progress in the development of melanocortin receptor selective ligands

The melanocortin pathway consists of endogenous agonists, antagonists, G-protein coupled receptors (GPCRs), and auxiliary proteins. This pathway has been identified to participate physiologically in numerous biological pathways including energy homeostasis, pigmentation, sexual function, inflammation, cardiovascular function, adrenal function, sebaceous gland lipid production, just to list a few. During this past decade, a clear link between the melanocortin-4 receptor (MC4R) and obesity, in both mice and humans via the regulation of food intake and energy homeostasis, has made this pathway the target of many academic and industrial research endeavors in attempts to develop potent and selective MC4R small molecules as anti-obesity therapeutic agents. Herein, we attempt to summarize the known proteins that constitute the melanocortin system and discuss advances in peptide and non-peptide drug discovery.

A review of melanocortin receptor small molecule ligands

The melanocortin system (MC) is implicated in the regulation of a variety of physiological pathways including pigmentation, steroid function, energy homeostasis, food intake, obesity, cardiovascular, sexual function, and normal gland regulation. The melanocortin system consists of five receptors identified to date (MC1-5R), melanocortin agonists derived from the pro-opiomelanocortin prohormone (POMC) and two naturally existing antagonists. Melanocortin receptor ligand structure-activity studies have been performed since the 1960s, primarily focused on the pigmentation aspect of physiology. During the 1990s, the melanocortin-4 receptor was identified to play a significant physiological role in the regulation of both food intake and obesity. Subsequently, a concerted drug design effort has focused on the design and discovery of melanocortin receptor small molecules. Herein, we present an overview of melanocortin receptor heterocyclic small molecules.