Specific binding sites for muramyl peptides on murine macrophages

Two radiolabeled (125I) muramyl peptide derivatives of high specific activity were prepared: a tripeptide with an iodinated C-terminal tyrosine methyl ester (Ligand I), and a muramyl tripeptide with a C-terminal lysine derivatized with Bolton-Hunter reagent (Ligand II). These were used to characterize binding of muramyl peptides to monolayers of murine macrophages. Saturable high-affinity binding to resident, caseinate-elicited, and Listeria-activated peritoneal cells was observed with both radioligands. Binding affinities varied with the state of activation of the macrophages, and KD values ranged from 48 +/- 33 pM (for resident macrophages, Ligand I) to 1020 +/- 90 pM (for activated macrophages, Ligand II). Specific binding sites were also found on a macrophage-derived cell line. The ability of several unlabeled muramyl peptides to compete with Ligands I and II for their binding sites was tested. Competition was stereospecific and correlated with known biological activities of these compounds (i.e., immunoadjuvanticity, pyrogenicity, and somnogenicity). The sites identified here for Ligands I and II may mediate some of the effects that muramyl peptides have previously been demonstrated to have on macrophages.     

Immunological activities of muramyl peptides

Muramyl peptides are endowed with numerous modulatory effects on the immune and nervous systems. Studies with synthetic muramyl dipeptide (MDP), the smallest unit of bacterial cell walls that can replace Mycobacteria in Freund's complete adjuvant, revealed that this glycopeptide can regulate several functions of cells involved in the immune response. The adjuvanticity of MDP and the MDP-induced activation of macrophages against tumors were found to be potentiated in vitro and in vivo with monoclonal anti-MDP antibodies. When used on immunoadsorbent columns, the anti-MDP antibodies removed the somnogenic and pyrogenic activities contained in supernatants of stimulated rabbit peritoneal macrophages. Based on these data a hypothesis is put forward to explain the immuno- and neuro-modulatory effects of muramyl peptides.     

Functional continuum of regulatory peptides (RPs): vector model of RP-effects representation

During the past decades, bioactive (regulatory) peptides have been identified as the major players in the regulation of many important biological processes. Dozens of peptides have found their application as pharmaceutical agents, which further stimulated research in this field making it one of the most rapidly developing areas on the edge of biological science and medicine. However, the fast accumulation of enormous amounts of experimental data has revealed a great difficulty in their analysis and demanded the development of a systematic approach for generalization of the obtained information. We propose a new computer-based algorithm for studying biological activities of regulatory peptides and their groups based on their representation as vectors in n -dimensional functional space. Our method allows the rapid analysis of databases containing thousands of polyfunctional regulatory peptides with overlapping spectra of physiological activity. The described method permits to perform several types of correlations which, when applied to the large databases, could reveal new important information about the system of regulatory peptides. It can select the groups of peptides with similar physiological role (peptide constellations) and search for the optimal peptide combinations with predetermined spectrum of effects and minimal side effects for their further pharmacological application. It can also reveal the role of regulatory peptides in induction of chain physiological reactions.     

Muramyl Peptides as Possible Endogenous Immunopharmacological Mediators

Synthetic N-acetylmuramyl-L -alanyl-d-isoglutamine, also called MDP for muramyl dipeptide, is a copy of a fragment of bacterial peptidoglycan. Soon after the recognition of MDP as being the minimal subunit responsible for the activity of Freund's complete adjuvant, a great number of derivatives were synthesized. Because of their very low molecular weight it was hoped that they could retain selectively certain of the numerous effects produced by complex bacterial agents. Evidence was gathered showing MDP's direct effect on lymphocytes and on macrophages. The ensuing studies reviewed that MDP and several of its derivatives have marked immunopharmacological and neuropharmacological activities. Thus, besides being adjuvants, they are capable of producing hyperthermia by acting directly on thermoregulation centers or by inducing in vivo and in vitro endogenous pyrogens (EP). More recently, Krueger et al have shown that slow-wave sleep (SWS) factor was a muramyl peptide of a molecular weight close to 1,000 daltons. They have also shown that MDP and several of its synthetic analogs had a somnogenic activity. It has previously been hypothesized that several of the immunological activities of the muramyl peptides could be due to biological mimicry with endogenous products. Recent observations argue in favor of the presence of an MDP bacterial structure in mammalian mediators which increase slow-wave sleep and/or produce fever. The implications of these findings will be discussed.     

Modulation of natural killer activity by muramyl peptides: relationship with adjuvant and anti-infectious properties

Natural killer (NK) activity of spleen cells was studied in DBA/2 mice, 24 and 72 h after intravenous injection of various muramyl peptides: muramyl dipeptide (MDP) and derivatives which are both adjuvant-active and able to increase resistance against Klebsiella pneumoniae; derivatives which are adjuvant-active but devoid of anti-infectious properties; derivatives which are anti-infectious but devoid of adjuvant activity, and derivatives which are devoid of both activities such as the stereoisomer MDP[D-Ala]1. An early increase in NK activity was observed 24 h after injection of all nonadjuvant derivatives, whatever their effect on infection. A stimulation of natural cytotoxicity was always induced 72 h after injection of MDP and derivatives able to protect mice against Klebsiella pneumoniae infection. So, even if the reverse was not true, there seems to exist some correlation between the anti-infectious effect of muramyl peptides and the late increase in NK activity. The modulation of NK activity by muramyl peptides appeared to be independent of interferon production. Moreover, inhibition of the stimulatory effect by a cell cycle-specific drug, hydroxyurea, observed 72 h after MDP suggests a requirement for proliferation.     

Endogenous opiates: 1980

Vast amounts of research have been done that have attempted to delineate the pharmacological and physiological effects of the endogenous opiate peptides. A great deal of knowledge has also been accumulated in a limited time span concerning the types and locations of the opiate receptors and peptides, as well as their functions. In 1980, reports were made concerning the effects of these peptides on analgesia, on tolerance and dependence, on activity, on learning and memory, on schizophrenia and other types of emotional disturbances, and on physiological responses such as eating and drinking, cardiovascular responses, and sexual function. Additional understanding was also gained concerning their interactions with neurotransmitters, other neuropeptides, and hormones. These and other studies published only in 1980 are reviewed in this paper, which is the third of an annual series.     

Muramyl dipeptide analogues as potentiators of the antitumor action of endotoxin

Summary The potentiation of endotoxin-induced necrosis and regression of solid syngeneic Meth A tumors in mice previously observed following administration of N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) was investigated further by use of various muramyl peptide analogues and two unrelated synthetic adjuvants, viz. the pluronic polyol L121 and dimethyldioctadecylammonium bromide (DDA) instead of MDP. All agents were administered in aqueous solution by the IV route. None of the muramyl peptide analogues nor L121 or DDA had any strong antitumor action of their own. Two 6-O-acylated muramyl peptides (L2-MDP and B30-MDP) and muramyl dipeptide stearoyllysine [MDP-Lys (L18)] clearly potentiated endotoxin-induced necrosis and regression. In contrast, MDP with L- instead of D-isoglutamine was completely inactive. Optimal activity of B30-MDP and MDP-Lys (L18) was only achieved by adding of suitable amounts of a nonionic surfactant. L121 and DDA could not replace muramyl peptides as potentiating agent. The combination of endotoxin, MDP, and L121 caused complete tumor regression in all mice, but was highly toxic.On the basis of the data in the literature on the biological response-modifying activities of the agents used it is concluded that the potentiating activity of muramyl peptides cannot yet be related to their immunoadjuvant action or their capacity to activate macrophages or to enhance nonspecific bacterial resistance.The work described in this paper was supported by grant UUKC 82-15 from the Koningin Wilhelmina Fonds, The Netherlands Cancer Organization     

Muramyl peptides in mammalian tissues and their effects at the cellular level

Muramyl peptides (MPs), presumably breakdown products of bacterial cell walls, have been found in the brain, liver, and kidney of the rat. They exert multiple physiological effects on higher animals as immunoadjuvants, activators of macrophages, pyrogens, antitumor agents, inducers of contractility of smooth muscle, and promoters of slow-wave sleep, as well as nonspecific protectors of animals against infection. Structure-function relationships of these substances have been extensively studied, especially with respect to somnogenicity. In the role an intact muramyl ring is required, and the 1,6-anhydro form is active. The presence of free carboxyls or amides on the glutamyl and diaminopimelyl entities have important effects. The stereochemistry is crucial: the alanine adjacent to the N-acetylmuramyl entity must be L, and the glutamate must be D. Studies were carried out with murine macrophages to establish mechanisms of action of these glycopeptides. There are two populations of binding sites for MPs on those cells. When compounds of different structure are compared, binding ability correlates with pyrogenic and somnogenic activity. Serotonin competes with these agents for binding sites. Binding of that substance induces at least one macrophage response characteristic of the binding of MP.     

Synthesis and biological activities of neurokinin alpha and beta

Two novel neuropeptides, neurokinin alpha and beta isolated from porcine spinal cord, were announced. We have synthesized neurokinin alpha as Asp-Met-His-Asp-Phe-Phe-Val-Gly-Leu-Met-NH2, which were 98-99% pure by HPLC. Assays on the isolated guinea pig ilium showed neurokinin alpha to have 81% and neurokinin beta to have 65% of the activity of Substance P. Knowledge of these three related peptides having a common activity opens new considerations of their intrinsic physiological roles in neurotransmission versus pharmacological activities, and reappraisal of the diverse activities of Substance P including that in the inflammatory response of the eye.     

Peptide length requirement for experimental allergic encephalomyelitis in guinea pigs

Three peptides overlapping the tryptophan region of bovine CNS myelin basic protein were synthesized by the solid phase procedure of Merrifield. These were the nonapeptide H-Phe-Ser-Trp-Gly-Ala-Glu-Gly-Gln-Lys-OH, the octapeptide H-Ser-Trp-Gly-Ala-Glu-Gly-Gln-Lys-OH, and the heptapeptide H-Trp-Gly-Ala-Glu-Gly-Gln-Lys-OH. They were tested for encephalitogenic activity in guinea pigs with either Freund's complete adjuvant containing M. tuberculosis or muramyl dipeptide in incomplete Freund's adjuvant at doses of 10 microgram per animal. The results show that deletion of one or two residues from the amino-terminal end of the nonapeptide destroyed the ability of the shorter peptides to induce clinical but not histological signs of EAE.     

Potential therapeutic value of muramyl peptides for modulating human immunologic responses

The clinical potential and the limitations of synthetic muramyl peptides have been suggested through extensive work describing their immunomodulating properties and toxicology. The intent of this paper is to offer the clinician a summary of these studies and to introduce the reader to the biological effects associated with administration of muramyl peptides.     

Mechanisms of physiological and pharmacological sex hormone action on the mammalian liver

Androgen and oestrogen receptors have been demonstrated in mammalian liver, but since it is generally accepted that they are probably non-functional at endogenous steroid concentrations, it is not apparent how they mediate physiological influences on this organ. Nor is it certain to what extent pharmacological actions of sex hormones reflect overstimulation of physiological routes or whether alternative mechanisms become available once threshold values have been reached. In this presentation an attempt has been made to answer some of these questions using data obtained from a study of the regulation of the activities of microsomal 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSDH) and 5 alpha-reductase in rat liver. Androgens exert their primary physiological and pharmacological influences at the level of the hypothalamus. Oestrogens can elicit three different types of effect-physiological, antiandrogenic and pharmacological--of which the first two involve primary effects on the pituitary. Hepatic oestrogen receptors only become activated when oestrogen concentrations reach pharmacological levels. Progestins probably have no physiological influence on the livers of non-pregnant rats. Their pharmacological actions may either be traced back to secondary androgenic (e.g. medroxyprogesterone acetate, levonorgestrel) or oestrogenic (e.g. norethynodrel, lynestrenol) properties, involving the routes described above, or to independent effects on the central nervous system (e.g. cyproterone acetate modulation of 5 alpha-reductase activity).     

Antihistamine, anticholinergic and cardiovascular effects of 2-substituted-4-phenylquinoline derivatives

Eleven new derivatives of 4-phenylquinoline, having various substituents at the 2-position of the quinoline ring, were previously synthesized. The antidepressant activities of these derivatives were demonstrated by their antagonism to reserpine-induced hypothermia in mice. The ED50 values were found to be in the range of 12-42 mg/kg (imipramine is 21.0 mg/kg). In the present work, comparative studies of the effects of these new drugs on the cholinergic and histaminergic (H1) systems, as well as their effects on the cardiovascular system, are presented. Both imipramine and trazodone were utilized as standards representing typical and atypical antidepressant drugs, respectively. All these new compounds have very low antihistaminic (H1) activities, as compared to imipramine. In addition, a clear cut separation of the antidepressant activity from the antihistaminic (H1) activity was observed. These compounds have weak anticholinergic (atropine-like properties) activity as compared to imipramine, using the isolated guinea-pig ileum. Animal studies of the cardiac toxicity of these compounds showed reduced lethality for some of them as compared to imipramine. Arrhythmias commonly associated with imipramine were absent for most of these compounds. The effects of these compounds on the heart conduction were determined by electrocardiographic studies. Although some of these compounds do not interfere with heart conduction, as compared to imipramine most were inferior to trazodone. Correlation between the pharmacological activities and structural modifications of these derivatives has also been observed.     

The biological activities of fish peptides and methods of their isolation

Fish, like other aquatic organisms, are a potential source of structurally diverse bioactive compounds. Studies of the pharmacological effects of fish peptides have revealed their antihypertensive, immunomodulatory, antioxidant, antitumor, and antimicrobial activities. Analysis of the literature data confirms that fish can be used not only for nutritional purposes, but also as a source of unique peptides with a broad spectrum of biological activities. Further investigations will allow the inclusion of fish peptides as acting agents in modern medicinal drugs.     

The Wellcome Foundation lecture, 1982. Opioid peptides and their receptors

The remarkable feature of the opioid system is the complexity of its ligands and their interactions with the mu-, delta- and kappa-binding sites. The three endogenous opioid precursors give rise to more than ten opioid fragments. The fragments of pro-opiocortin and pro-enkephalin have affinities mainly to the mu- and delta-binding sites and those of pro-dynorphin have a preference for the kappa-binding site. It is important to realize that some of the larger fragments may have pharmacological actions that are of a non-opioid character. As the endogenous opioid peptides bind to more than one of the types of binding sites, it was necessary to obtain synthetic compounds that bind almost exclusively at one site. There are now agonists for which this aim has been achieved but we still require antagonists that are exclusively selective for only one opioid site. The results obtained with opioid peptides or non-peptides having such qualities would be the physiological basis for a correlation of the binding at mu-, delta- and kappa-receptors with their pharmacological effects. Furthermore, since almost all endogenous opioid ligands are degraded by peptidases, it is necessary to synthesize non-toxic inhibitors of those peptidases that play a role in opioid transmission. Related to this problem is the need to develop methods for the study of the release of various endogenous opioid peptides under physiological conditions.     

Function and therapeutic potential of host defence peptides.

Cationic host defence (antimicrobial) peptides are an important component of the innate immune systems of a wide variety of plants, animals, and bacteria. Although most of these compounds have direct antimicrobial activities under specific conditions, a greater appreciation for the diversity of functions of these molecules is beginning to develop in the field. In addition to their directly antimicrobial activities, they also have a broad spectrum of activity on the host immune system, with both pro-inflammatory and anti-inflammatory effects being invoked. Increasingly sophisticated approaches to understand the role of host defence peptides in modulating innate immunity are already serving to guide the development of novel therapeutics.     

Bioactive peptides from marine organisms: a short overview

Marine organisms are an immense source of new biologically active compounds. These compounds are unique because the aqueous environment requires a high demand of specific and potent bioactive molecules. Diverse peptides with a wide range of biological activities have been discovered, including antimicrobial, antitumoral, and antiviral activities and toxins amongst others. These proteins have been isolated from different phyla such as Porifera, Cnidaria, Nemertina, Crustacea, Mollusca, Echinodermata and Craniata. Purification techniques used to isolate these peptides include classical chromatographic methods such as gel filtration, ionic exchange and reverse-phase HPLC. Multiple in vivo and in vitro bioassays are coupled to the purification process to search for the biological activity of interest. The growing interest to study marine natural products results from the discovery of novel pharmacological tools including potent anticancer drugs now in clinical trials. This review presents examples of interesting peptides obtained from different marine organisms that have medical relevance. It also presents some of the common methods used to isolate and characterize them.     

Affinities of gidazepam and its analogs for mitochondrial benzodiazepine receptors

Mitochondrial benzodiazepine receptors (MBRs) participate in many physiological processes, such as calcium flow regulation, proliferative and respiratory cell functions, mitochondrial steroidogenesis and adaptational reactions to stress. We have found that the selective anxiolytic gidazepam has a higher affinity for CNS MBRs as compared to central benzodiazepine receptors. The ability of gidazepam to bind to MBRs probably underlies a wide spectrum of its pharmacological effects. We have studied affinities of gidazepam analogs for CNS MBRs in search for the ligands possessing higher affinity and selectivity. The experiments were carried out with male Wistar rats weighing between 200-220 g. Affinities of the investigated compounds were assessed on their ability to displace radioligand Ro5-4864 from its specific binding sites on MBRs of rat brain. Within the series of tested compounds three substances comparable on affinity with Ro5-4864 were found. Experimental results have shown that the presence of chlorine atom in o-position of 5-phenyl substituent leads to a 10 to 15-fold increase in affinity for CNS MBRs. We have also found that the essential contribution in affinity of the investigated series is brought by lipophilicity of substituent in IN-position. Our data may be useful in design and synthesis of novel potent selectively acting ligands of CNS MBRs.     

Tracheal cytotoxin structural requirements for respiratory epithelial damage in pertussis

The respiratory epithelial pathology of pertussis (whooping cough) can be reproduced by tracheal cyto-toxin (TCT), a disaccharide-tetrapeptide released by Bordetella pertussis. TCT is a muramyl peptide, a class of peptidoglycan-derived compounds which have many biological activities including adjuvanticity, somnogenicity, pyrogenicity, and cytotoxicity. The structural requirements for muramyl peptides to produce some of these biological effects have been partially characterized. Using in vitro assays with respiratory epithelial cells and tissue, we have previously determined that the disaccharide moiety of TCT is not involved in toxicity and that the side-chain functional groups of diaminopimelic acid (A₂pm) are crucial for toxicity. In this study, we determine the importance of every amino acid, functional group and chiral centre in the peptide portion of TCT. Although lactyl tetrapeptides are the most toxic of the TCT fragments, producing dose-response curves identical to TCT, the smallest analogues of TCT which are active in our assay are of the form X-γ-(d )-Glu-meso-A₂pm, where X may be an amino acid or a blocking group. Within this active substructure, main-chain chirality and all functional groups are essential for toxicity. This definition of the core region of TCT indicates that the TCT interaction site is unlike almost all other muramyl peptide interaction sites for which structure-activity data are available.     

Effect of a synthetic adjuvant on the induction of primary immune responses in T cell-depleted spleen cultures.

N-acetylmuramyl-L-alanyl-D-isoglutamine (muramyl dipeptide) stimulates in vitro primary immune responses to SRBC in T cell-depleted (nude) spleen cultures. The stimulation of immune responses by muramyl peptide was antigen dependent. A microculture system was used to compare the T cell-replacing activities of several structural analogues of muramyl dipeptide and to compare the activity of muramyl dipeptide to helper T cells. In a limiting dilution analysis with excess helper T cells or muramyl dipeptide, the frequency of B cell precursors that respond to SRBC was similar, ranging from 1.5 to 5 X 10(-5). Decreasing the cell density in microcultures did not affect the efficiency of B cell precursor responses in the presence of muramyl dipeptide. Muramyl dipeptide was examined for mitogenic activity in spleen cell cultures. In serum-free medium, muramyl dipeptide stimulates slight (3-fold) increases in DNA synthetic activity. In medium supplemented with 5 to 20% fetal calf serum, muramyl dipeptide showed no significant mitogenic activity. There are a number of possible explanations for the T cell-replacing activity of muramyl dipeptide. The most likely is that muramyl dipeptide interacts directly with B cells to mimic the helper T cell signal in the inductive stimulus.