Alterations in antigenic structure of gonadotropins following deglycosylation

Radioimmunological techniques were utilized to probe possible changes in conformation of gonadotropins (human chorionic gonadotropin-hCG; and ovine luteinizing hormone—oLH) following chemical deglycosylation (DG-hCG and DG-LH). All antisera produced in rabbits, rats or mice contained antibodies that were specific to the deglycosylated hormones with the native hormones showing weak and non-parallel cross-reaction (<5%), but with rabbit antibodies to native hormones the deglycosylated hormones were fully reactive. Using hCG, asialo-hCG (A-hCG) and DG-hCG, we have shown that removal of sugars internal to sialic acid is required to produce these specific antibodies. These are in complete agreement with the observations that extensive deglycosylation of these hormones is necessary to induce changes in biological activity at the cellular level. Based on these data, we suggest that chemical deglycosylation results in changes in antigenic structure of these hormones by generation of new determinants or exposure of previously buried sites and these changes are of no consequence to receptor recognition.     

Synthetic glucagon antagonists and partial agonists.

This paper reports the synthesis and the biological activities of six new glucagon analogues. In these compounds N-terminal modifications of the glucagon sequence were made, in most cases combined with changes in the C-terminal region which had been shown previously to enhance receptor affinity. The design of these analogues was based on [Lys17,18,Glu21]glucagon,1 a superagonist, which binds five times better than glucagon to the glucagon receptor, and on the potent glucagon antagonist [D-Phe4,Tyr5,Arg12]glucagon, which does not stimulate adenylate cyclase system even at very high concentrations. The N-terminal modifications involved substitution of His1 by the unnatural conformationally constrained residue, 4,5,6,7-tetrahydro-1H-imidazo[c]pyridine-6-carboxylic acid (Tip) and by desaminohistidine (dHis). In addition we prepared two analogues (6 and 7), in which we deleted the Phe6 residue, which was suggested to be part of a hydrophobic patch and involved in receptor binding. The following compounds were synthesized: [Tip1, Lys17,18,Glu21]glucagon (2); [Tip1,D-Phe4,Tyr5,Arg12,Lys17,18,Glu21]glucagon (3); [dHis1,D-Phe4,Tyr5,Arg12,Lys17,18,Glu21]glucagon (4); [dHis1,Asp3,D-Phe4,Tyr5,Arg12,Lys17,18,Glu21+ ++]glucagon (5); des-Phe6-[Tip1,D-Phe4,Tyr5,Arg12,Glu21]glucagon (6); des-Phe6-[Asp3,D-Phe4,Tyr5,Arg12,Glu21]glucagon (7). The binding potencies of these new analogues relative to glucagon (= 100) are 3.2 (2), 2.9 (3), 10.0 (4), 1.0 (5), 8.5 (6), and 1.7 (7). Analogue 2 is a partial agonist (maximum stimulation of adenylate cyclase (AC) approximately 15% and a potency 8.9% that of glucagon, while the remaining compounds 3-7 are antagonists unable to activate the AC system even at concentrations as high as 10(-5) M. In addition, in competition experiments, analogues 3-7 caused a right-shift of the glucagon stimulated adenylate cyclase dose-response curve.(ABSTRACT TRUNCATED AT 250 WORDS)     

Clinical aspects of dopamine agonists and antagonists.

Dopamine and its specific receptors are widely distributed in man. Body regions where dopaminergic activity is of special pharmacologic interest include the basal ganglions, hypothalamus, chemoreceptor trigger zone, other less well defined areas in the central nervous system, and the renal and cardiovascular systems. The search for dopaminergic agents to modify these systems in disease states has depended heavily on in vitro and in vivo bioassays. These assays involving receptor binding, enzyme activation, smooth muscle and neuronal excitation, and modification of animal behavior have provided physicians with important therapeutic tools. Indeed, the introduction of levodopa for the treatment of Parkinson's disease and of the phenothiazines and related drugs for schizophrenia and psychosis has been a hallmark of neuropharmacologic research. However, the maximal benefits that these drugs may afford have not yet been realized due to an inadequate understanding of disease processes and a relative lack of specificity of drug action.     

Dependence of pharmacological activity of new NMDA agonists and antagonists on their chemical structure

A group of imidazole derivatives was tested for their agonistic and antagonistic activity with respect to NMDA receptors in pyramidal neurons of the rat hippocampus. The data from the experiments using intracerebroventricular injections of the tested agents were compared with those carried out on isolated cells using a patch-clamp technique in the whole-cell configuration. It has been shown that the presence of lipophilic groups in the molecules of the above derivatives determines their ability to be NMDA antagonists.     

Solution structure of the proapoptotic molecule BID: a structural basis for apoptotic agonists and antagonists

Members of the BCL2 family of proteins are key regulators of programmed cell death, acting either as apoptotic agonists or antagonists. Here we describe the solution structure of BID, presenting the structure of a proapoptotic BCL2 family member. An analysis of sequence/structure of BCL2 family members allows us to define a structural superfamily, which has implications for general mechanisms for regulating proapoptotic activity. It appears two criteria must be met for proapoptotic function within the BCL2 family: targeting of molecules to intracellular membranes, and exposure of the BH3 death domain. BID's activity is regulated by a Caspase 8-mediated cleavage event, exposing the BH3 domain and significantly changing the surface charge and hydrophobicity, resulting in a change of cellular localization.     

Functional methods for quantifying agonists and antagonists.

The development of occupancy theory has allowed the formulation of a series of mathematical models that describe the interaction of agonists and antagonists with their receptors, in terms of affinity and efficacy. These models provide a framework for the analysis and interpretation of E/[A] curve data and have proved to be useful tools in quantitative pharmacology. Unfortunately, despite the proven utility of this approach and the widespread availability of powerful computer-based curve-fitting programs [BMDP (41), Microsoft Excel. etc.], which greatly facilitate analysis, the application of mathematical modeling remains the exception rather than the rule in pharmacological studies.     

Melatoninergic receptor agonists and antagonists: therapeutic perspectives

The chronobiotic neurohormone melatonin, synthetized in the pineal gland during darkness periods governs the circadian and seasonal biological rhythms. Physiologically, melatonin regulates the sleep/activity alternance, together with the circadian cycle of body temperature and cortisol secretion, and influences various immune, endocrine and metabolic functions. Dysfunction of the endogenous melatonin secretion is associated with mood and behavioral disorders including body weight. Patients with severe depression exhibit desynchronized and reduced melatonin secretion, in parallel with marked sleep disturbances whereas exogenous melatonin administration and antidepressive drugs restore melatonin secretion. A dysregulated melatonin secretion is also observed in obese subjects. Implication of melatonin in these disorders stimulated the search for melatonin analogues with enhanced antidepressive and body weight control effects. The melatoninergic agonist S 20098, or agomelatin, disclosed a potent antidepressive and anxiolytic activity in preclinical studies, which was confirmed in clinical trials in patients with major depression. The antagonist S 20928 was shown to limit seasonal weight gain in an hibernating rodent model. Thus, development of melatoninergic agonists and antagonists appear as an innovative approach in the treatment of depression and obesity, two major public health problems.     

Design of subtype selective melatonin receptor agonists and antagonists.

Studies of the physiological actions of melatonin have been hindered by the lack of specific, potent and subtype selective agonists and antagonists. In the present study, we describe the utility of a melanophore cell line from Xenopus laevis for exploring structure-activity relationships among novel melatonin analogues and report a novel MT2-selective agonist (IIK7) and MT2-selective receptor antagonist (K185). IIK7 is a potent melatonin receptor agonist in the melanophore model, and in NIH3T3 cells expressing human mt1 and MT2 receptor subtypes. In radioligand binding experiments IIK7 is 90-fold selective for the MT2 subtype. K185 is devoid of agonist activity, but acts as a competitive melatonin antagonist in melanophores. A low concentration (10(-9) M) antagonizes melatonin inhibition of forskolin stimulation of cyclic AMP in NIH3T3 cells expressing human MT2 receptors, but has no effect in cells expressing mt1 receptors. In binding assays, K185 is 140-fold selective for the MT2 subtype.     

Noradrenergic agonists and antagonists: effects on avoidance behaviour in rats

The effects of various agonists and antagonists of both alpha and beta adrenoceptors on the acquisition of avoidance behaviour were investigated in the rat. Clonidine, a selective agonist of alpha 2 adrenoceptors depressed avoidance acquisition whilst yohimbine, an antagonist of these receptors produced an opposite effect. Prazosin which showed postsynaptic alpha 1 adrenoceptor blocking activity reduced avoidance behaviour. A similar effect was produced by propranolol, a non-selective antagonist of beta adrenoceptors. On the other hand, salbutamol preferentially stimulating beta 2 adrenoceptors facilitated avoidance behaviour. In general, the results show a fairly good correlation between avoidance acquisition and efficacy of noradrenergic neurotransmission.     

Influence of carbohydrates on the stability and structure of a hyperglycosylated human interferon alpha mutein

Protein physical and chemical instability is one of the major challenges in the development of biopharmaceuticals during every step of the process, ranging from production to final delivery. This is particularly applicable to human recombinant interferon alpha-2b (rhIFN-α2b), a pleiotropic cytokine currently used worldwide for the treatment of various cancer and chronic viral diseases, which presents a poor stability in solution. In previous studies, we have demonstrated that the introduction of four N-glycosylation sites in order to construct a heavily glycosylated IFN variant (4N-IFN) resulted in a markedly prolonged plasma half-life which was reflected in an enhanced therapeutic activity in mice in comparison with the commercial non-glycosylated rhIFN-α2b (NG-IFN). Herein, we evaluated the influence of glycosylation on the in vitro stability of 4N-IFN towards different environmental conditions. Interestingly, the hyperglycosylated cytokine showed enhanced stability against thermal stress, acid pH and repetitive freeze-thawing cycles in comparison with NG-IFN. Besides, microcalorimetric analysis indicated a much higher melting temperature of 4N-IFN, also demonstrating a higher solubility of this variant as denoted by the absence of precipitation at the end of the experiment, in contrast with the NG-IFN behaviour. Furthermore, far-UV circular dichroism (CD) spectrum of 4N-IFN was virtually superimposed with that of NG-IFN, indicating that the IFN structure was not altered by the addition of carbohydrate moieties. The same conclusion could be inferred from limited proteolysis studies. Our results suggest that glycoengineering could be a useful strategy for protecting rhIFN-α2b from inactivation by various external factors and for overcoming aggregation problems during the production process and storage.     

Irreversible opiate agonists and antagonists: V. Hydrazone and acylhydrazone derivatives of naltrexone

We have synthesized a series of hydrazones and acylhydrazones of naltrexone. These substitutions had modest effects on competition of mu binding but many greatly enhanced the relative potency of the compounds for delta receptors. Increased delta affinity was most prominent with the acylhydrazones. Many of the derivatives elicited a wash-resistant inhibition of binding which was restricted to mu, not delta, binding sites. This wash-resistant inhibition of binding did not correlate with affinity, as determined by IC50 values, implying that the inhibition could not be explained simply by slow rate of dissociation due to increased affinity.     

Design of non-peptide agonists and antagonists at neuropeptide receptors starting from the chemical structure of neuropeptides

Non-peptide small-molecule antagonists for cholecystokinin (CCK)-A and -B receptors, tachykinin NK-1, NK-2 and NK-3 receptors and bombesin BB-1 receptors have been designed and synthesized starting from the chemical structure of the endogenous mammalian neuropeptides cholecystokinin, substance-P and bombesin, respectively. A non-peptide CCK-A agonist, with weak potency but high efficacy, was also identified from the same strategy.     

Effect of histamine receptor agonists and antagonists on the uterine vasculature

Histamine H1 and H2 receptors are known to exist in uterine smooth muscle; however, neither receptor has been clearly identified in the uterine vasculature. In the present study, 12 nonpregnant ewes were chronically instrumented with catheters in the carotid artery, jugular vein, uterine arteries, and electromagnetic flow probes on the uterine arteries for continuous measurement of uterine blood flow. Dose response curves were determined for bolus injections of Histamine (1-10 micrograms), the H1 receptor agonist 2PEA (10-100 micrograms), and the H2 receptor agonist Dimaprit (30-300 micrograms) before H1 receptor blockade with pyrilamine, following H1 receptor blockade, and following H2 receptor blockade with metiamide. Uterine vasodilator responses to histamine and 2PEA were essentially abolished by pyrilamine, while responses to dimaprit were not altered. Following addition of metiamide, responses to histamine were reduced further and responses to dimaprit were abolished. Baseline uterine blood flow was not altered by either H1 or H2 receptor blockade or their combination. Intraarterial bolus injections of the mast cell histamine-releasing compound 48/80 (100-1000 micrograms) had no effect on uterine blood flow. These experiments demonstrate that the uterine vasculature of the ovine contains almost exclusively H1 receptors, does not contain compound 48/80 sensitive mast cells and is not dependent upon endogenous histamine to maintain blood flow.     

Design of non-peptide agonists and antagonists at neuropeptide receptors starting from the chemical structure of neuropeptides

Summary Non-peptide small-molecule antagonists for cholecystokinin (CCK)-A and-B receptors, tachykinin NK-1, NK-2 and NK-3 receptors and bombesin BB-1 receptors have been designed and synthesized starting from the chemical structure of the endogenous mammalian neuropeptides cholecystokinin, substance-P and bombesin, respectively. A non-peptide CCK-A agonist, with weak potency but high efficacy, was also identified from the same strategy.     

Influence of agonists and antagonists on the segmental motion of residues near the agonist binding pocket of the acetylcholine-binding protein

Using the Lymnaea acetylcholine-binding protein as a surrogate of the extracellular domain of the nicotinic receptor, we combined site-directed labeling with fluorescence spectroscopy to assess possible linkages between ligand binding and conformational dynamics. Specifically, 2-[(5-fluoresceinyl)aminocarbonyl]ethyl methanethiosulfonate was conjugated to a free cysteine on loop C and to five substituted cysteines at strategic locations in the subunit sequence, and the backbone flexibility around each site of conjugation was measured with time-resolved fluorescence anisotropy. The sites examined were in loop C (Cys-188 using a C187S mutant), in the beta9 strand (T177C), in the beta10 strand (D194C), in the beta8-beta9 loop (N158C and Y164C), and in the beta7 strand (K139C). Conjugated fluorophores at these locations show distinctive anisotropy decay patterns indicating different degrees of segmental fluctuations near the agonist binding pocket. Ligand occupation and decay of anisotropy were assessed for one agonist (epibatidine) and two antagonists (alpha-bungarotoxin and d-tubocurarine). The Y164C and Cys-188 conjugates were also investigated with additional agonists (nicotine and carbamylcholine), partial agonists (lobeline and 4-hydroxy,2-methoxy-benzylidene anabaseine), and an antagonist (methyllycaconitine). With the exception of the T177C conjugate, both agonists and antagonists perturbed the backbone flexibility of each site; however, agonist-selective changes were only observed at Y164C in loop F where the agonists and partial agonists increased the range and/or rate of the fast anisotropy decay processes. The results reveal that agonists and antagonists produced distinctive changes in the flexibility of a portion of loop F.     

PAF-receptor. III. Conformational and electronic properties of PAF-like agonists and antagonists.

In order to compare electronic and conformational properties of PAF-agonists and PAF-antagonists, 14 analogues structurally related to PAF were studied. A common conformation of the glycerol backbone was present in all agonists and all constrained or flexible antagonists. The distinction between agonists and antagonists appears to be casted on position-2 where the folded conformation of the substituent for agonists should be the most probable. In position-3 the gauche conformation can be adopted by all the analysed compounds. The electrostatic potential well at -30 kcal/mol stretches to the carbonyl group in position-2 in the folded conformation of the agonists. On the contrary, in constrained antagonists, a second negative zone appears around the carbamate group. Given the modelling results, the triethylammonium PAF analogue considered in literature as a weak agonist, was resynthesized and proved to be more potent than previously reported. These experimental results confirm our hypothesis in terms of a common conformation of agonist and antagonist PAF-like molecules.     

Fluorescent and biotinylated probes for B2 bradykinin receptors: agonists and antagonists.

Peptide ligands carrying additional reporter groups are valuable research tools to facilitate biochemical and pharmacological studies of G protein-coupled receptors. B2 bradykinin receptors, widely distributed in mammalian tissues, regulate many physiological systems and are therapeutic targets. Acylation of the amino-terminus of bradykinin (BK) and a B2a-selective antagonist produced ligands derivatized with biotinamidocaproate or 7-Amino-4-methylcoumarin-3-acetate. These fluorescent and biotinylated peptides bound with high affinity to bovine and rodent B2 receptors. Analysis of second messenger production confirmed that fluorescent and biotinylated analogs of BK were B2 receptor agonists whereas derivatives of DArg0[Hyp3,DPhe7,Leu8]BK were BK receptor antagonists. The complimentary properties of these selective receptor probes will be useful in studying B2 receptor localization, expression and desensitization.     

Influence of acetylcholine agonists and antagonists on the swelling of etiolated wheat (Triticum aestivum L.) mesophyll protoplasts

Etiolated wheat (Triticum aestivum L.) mesophyll protoplasts swell within 30 min in darkness after a red light (R) pulse or addition of acetylcholine (ACh), if 0.5 mM CaCl₂ is present in the medium. In addition, ACh is also able to induce swelling in the presence of both 0.1 mM KCl or NaCl. Besides ACh, only carbamylcholine out of the choline derivatives tested was active in induction of swelling in the presence of K⁺ or Na⁺. The K⁺/Na⁺-dependent ACh-induced protoplast swelling was nullified by a ‘calmodulin inhibitor’, but not by Ca²⁺-channel blockers, Li⁺ or VO ₄ ^3- . The antagonists atropine (of muscarine-sensitive ACh receptors, mAChRs) andd-tubocurarine (of nicotine-sensitive ACh receptors, nAChRs) nullified the Ca²⁺ — and the K⁺/Na⁺-dependent protoplast swelling responses, respectively, while having no effect on the Ca²⁺-dependent R-induced swelling response. Moreover, muscarine and nicotine mimicked ACh in the Ca²⁺- and K⁺/Na⁺-dependent swelling responses respectively. Just as is the case in animal cells, the proposed mAChRs appear to be associated with a phosphatidylinositol-dependent pathway, whereas the proposed nAChRs are phosphatidylinositol independent. Similarity between the action of ACh via the proposed mChRs and R via phytochrome in protoplast swelling indicates they share in common signal-transduction pathway. We dedicate this paper to Hans Mohr on the occasion of his 60th birthday We thank the Department of Molecular Biology of the Agricultural University, Wageningen for the use of the photomicroscope and Dr. G. Fassina, Department of Pharmacology, University of Padua, Italy for the gift of nifedipine. These studies were supported by The Foundation for Fundamental Biological Research (BION) which is subsidized by The Netherlands Organization for the Advancement of Research (NWO). A.T. was also supported by: a Research Fellowship from the Agricultural University, Wageningen; a Visitors Fellowship from NWO, the Netherlands; RP II 12.15 from Ministry of Education, Poland.