Biophysical characterization of a binding site for TLQP-21, a naturally occurring peptide which induces resistance to obesity.

Recently, we demonstrated that TLQP-21 triggers lipolysis and induces resistance to obesity by reducing fat accumulation [1]. TLQP-21 is a 21 amino acid peptide cleavage product of the neuroprotein VGF and was first identified in rat brain. Although TLQP-21 biological activity and its molecular signaling is under active investigation, a receptor for TLQP-21 has not yet been characterized. We now demonstrate that TLQP-21 stimulates intracellular calcium mobilization in CHO cells. Furthermore, using Atomic Force Microscopy (AFM), we also provide evidence of TLQP-21 binding-site characteristics in CHO cells. AFM was used in force mapping mode equipped with a cantilever suitably functionalized with TLQP-21. Attraction of this functionalized probe to the cell surface was specific and consistent with the biological activity of TLQP-21; by contrast, there was no attraction of a probe functionalized with biologically inactive analogues. We detected interaction of the peptide with the binding-site by scanning the cell surface with the cantilever tip. The attractive force between TLQP-21 and its binding site was measured, statistically analyzed and quantified at approximately 40 pN on average, indicating a single class of binding sites. Furthermore we observed that the distribution of these binding sites on the surface was relatively uniform.

Ghrelin control of GH secretion and feeding behaviour: the role of the GHS-R1a receptor studied in vivo and in vitro using novel non-peptide ligands.

Energy homeostasis is controlled by a complex regulatory system of molecules that affect food intake and that are critical for maintaining a stable body weight during life. Ghrelin is a peptide of 28 amino acid synthesized predominantly by the stomach and the gut, which activate the type 1a growth hormone (GH) secretagogue receptor (GHS-R1a), a G-protein coupled receptor. The acylated form of ghrelin potently stimulates GH secretion both in vitro and in vivo in several animal species, including humans. Beside the endocrine effect, ghrelin shows also extraendocrine activities, including stimulation of feeding behaviour. Several classes of small synthetic peptide and non-peptide ligands of the GHS-R1a have been described and are able to release GH and stimulate food intake. However, in time, it appeared that the stimulating effects on GH secretion could be divorced from those on food intake, suggesting that more than a single receptor might be involved. Several experimental data have even questioned the physiological role of ghrelin in the control of GH secretion and energy metabolism. By using novel agonists, partial agonists, and antagonists for the GHS-R1a receptor, we have studied whether the stimulation of this receptor could account for the purported physiological role of ghrelin. Our results demonstrate that the ability to bind in vitro the GHS-R1a is not predictive of the in vivo biological activity of the compounds and that the endocrine and extraendocrine effects could be mediated also by receptors different from the GHS-R1a.

Imaging heterogeneity of peptide delivery and binding in solid tumors using SPECT imaging and MRI.

BACKGROUND: As model system, a solid-tumor patient-derived xenograft (PDX) model characterized by high peptide receptor expression and histological tissue homogeneity was used to study radiopeptide targeting. In this solid-tumor model, high tumor uptake of targeting peptides was expected. However, in vivo SPECT images showed substantial heterogeneous radioactivity accumulation despite homogenous receptor distribution in the tumor xenografts as assessed by in vitro autoradiography. We hypothesized that delivery of peptide to the tumor cells is dictated by adequate local tumor perfusion. To study this relationship, sequential SPECT/CT and MRI were performed to assess the role of vascular functionality in radiopeptide accumulation. METHODS: High-resolution SPECT and dynamic contrast-enhanced (DCE)-MRI were acquired in six mice bearing PC295 PDX tumors expressing the gastrin-releasing peptide (GRP) receptor. Two hours prior to SPECT imaging, animals received 25 MBq (111)In(DOTA-(ßAla)2-JMV594) (25 pmol). Images were acquired using multipinhole SPECT/CT. Directly after SPECT imaging, MR images were acquired on a 7.0-T dedicated animal scanner. DCE-MR images were quantified using semi-quantitative and quantitative models. The DCE-MR and SPECT images were spatially aligned to compute the correlations between radioactivity and DCE-MRI-derived parameters over the tumor. RESULTS: Whereas histology, in vitro autoradiography, and multiple-weighted MRI scans all showed homogenous tissue characteristics, both SPECT and DCE-MRI showed heterogeneous distribution patterns throughout the tumor. The average Spearman's correlation coefficient between SPECT and DCE-MRI ranged from 0.57 to 0.63 for the "exchange-related" DCE-MRI perfusion parameters. CONCLUSIONS: A positive correlation was shown between exchange-related DCE-MRI perfusion parameters and the amount of radioactivity accumulated as measured by SPECT, demonstrating that vascular function was an important aspect of radiopeptide distribution in solid tumors. The combined use of SPECT and MRI added crucial information on the perfusion efficiency versus radiopeptide uptake in solid tumors and showed that functional tumor characteristics varied locally even when the tissue appeared homogenous on current standard assessment techniques.

Arginine 197 of the cholecystokinin-A receptor binding site interacts with the sulfate of the peptide agonist cholecystokinin.

The knowledge of the binding sites of G protein-coupled cholecystokinin receptors represents important insights that may serve to understand their activation processes and to design or optimize ligands. Our aim was to identify the amino acid of the cholecystokinin-A receptor (CCK-AR) binding site in an interaction with the sulfate of CCK, which is crucial for CCK binding and activity. A three-dimensional model of the [CCK-AR-CCK] complex was built. In this model, Arg197 was the best candidate residue for a ionic interaction with the sulfate of CCK. Arg197 was exchanged for a methionine by site-directed mutagenesis. Wild-type and mutated CCK-AR were transiently expressed in COS-7 cells for pharmacological and functional analysis. The mutated receptor on Arg197 did not bind the agonist radioligand 125I-BH-[Thr, Nle]-CCK-9; however, it bound the nonpeptide antagonist [3H]-SR27,897 as the wild-type receptor. The mutant was approximately 1,470- and 3,200-fold less potent than the wild-type CCK-AR to activate G proteins and to induce inositol phosphate production, respectively. This is consistent with the 500-fold lower potency and 800-fold lower affinity of nonsulfated CCK relative to sulfated CCK on the wild-type receptor. These data, together with those showing that the mutated receptor failed to discriminate nonsulfated and sulfated CCK while it retained other pharmacological features of the CCK-AR, strongly support an interaction between Arg197 of the CCK-AR binding site and the sulfate of CCK. In addition, the mutated CCK-AR resembled the low affinity state of the wild-type CCK-AR, suggesting that Arg197-sulfate interaction regulates conformational changes of the CCK-AR that are required for its physiological activation.

Immunologic identification of both plasma and human renal inactive renin as prorenin.

Antibodies were raised against a synthetic tetradecapeptide which is a component of the non-renin portion (prosegment) of human renin precursor. Inactive renin from human kidney and plasma strongly adsorbed to a gel coupled to immunoglobulins purified from such an antiserum. These results suggest that renal and circulating inactive human renins contain in their structure the prosegment of prorenin.

Aldehydic peptides inhibiting renin.

Aldehydic peptides in which the C terminal residue is leucinal or phenylalaninal were synthesized. These compounds exhibited potent inhibition of renin activity and appeared to be precursors of transition state analogues for renin-catalysed amide bond hydrolysis.

Analysis of inactive renin by renin profragment monoclonal antibodies.

Two peptides were synthesized, corresponding to the sequences (-19 to -7) and (-26 to -17) of the prorenin prosegment. Monoclonal antibodies were raised to these sequences and used to characterize human plasma inactive renin. Only anti (-19 to -7) reacted with inactive renin, as measured by direct assay or affinity chromatography. The data were used to evaluate two possible inactive renin stuctures: plasma inactive renin is a truncated prorenin lacking the prosegment N-terminal portion; its spatial conformation masks the N-terminal extremity, preventing interaction of this region with specific antibodies.

Synthesis and biological evaluation of bombesin constrained analogues.

Analogues of bombesin which incorporate dipeptide or turn mimetics have been synthesized. One of them (compound 11) containing a seven-membered lactam ring revealed a good affinity for GRP/BN receptors on rat pancreatic acini (K(i) value of 1.7 +/- 0.4 nM) and on Swiss 3T3 cells (K(i) value of 1.0 +/- 0.2 nM). On the basis of this observation, antagonists containing the same dipeptide mimic were obtained by modification of the C-terminal part of the bombesin analogues. The most potent constrained compounds (15 and 17) were able to antagonize 1 nM bombesin-stimulated amylase secretion from rat pancreatic acini with high potency (K(i) = 21 +/- 3 and 3.3 +/- 1.0 nM, respectively) and 10(-7) M bombesin-stimulated ¿(3)Hthymidine incorporation into Swiss 3T3 cells (K(i) = 7.8 +/- 2. 0 and 0.5 +/- 0.1 nM, respectively).

Variations in dystrophin complex in red and white caudal muscles from Torpedo marmorata.

We present an up-to-date study on the nature, at the protein level, of various members of the dystrophin complex at the muscle cell membrane by comparing red and white caudal muscles from Torpedo marmorata. Our investigations involved immunodetection approaches and Western blotting analysis. We determined the presence or absence of different molecules belonging to the dystrophin family complex by analyzing their localization and molecular weight. Specific antibodies directed against dystrophin, i.e., DRP2 alpha-dystrobrevin, beta-dystroglycan, alpha-syntrophin, alpha-, beta-, gamma-, and delta-sarcoglycan, and sarcospan, were used. The immunofluorescence study (confocal microscopy) showed differences in positive immunoreactions at the sarcolemmal membrane in these slow-type and fast-type skeletal muscle fibers. Protein extracts from T. marmorata red and white muscles were analyzed by Western blotting and confirmed the presence of dystrophin and associated proteins at the expected molecular weights. Differences were confirmed by comparative immunoprecipitation analysis of enriched membrane preparations with anti-beta-dystroglycan polyclonal antibody. These experiments revealed clear complex or non-complex formation between members of the dystrophin system, depending on the muscle type analyzed. Differences in the potential function of these various dystrophin complexes in fast or slow muscle fibers are discussed in relation to previous data obtained in corresponding mammalian tissues. (J Histochem Cytochem 49:857-865, 2001)

Comparative study of in vitro and in vivo activities of bombesin pseudopeptide analogs modified on the C-terminal dipeptide fragment.

Analogs of bombesin in which the peptide bond between the two last amino acid residues were replaced by a pseudopeptide bond mimicking the transition state analog were evaluated. These compounds were able to recognize the bombesin receptor on isolated rat pancreatic acini with high potency (Ki from 0.60 +/- 0.27 nM to 4.3 +/- 2.3 nM). Although they were devoid of agonist activity, they were able to antagonize bombesin-induced amylase secretion in this model, with potencies in accordance with their affinities (IC50 from 1.6 +/- 0.3 nM to 10.0 +/- 1.7 nM). When tested in vivo in the anesthetized rat, these bombesin receptor antagonists exhibited high potency in inhibiting bombesin-induced pancreatic secretion (H-DPhe-Gln-Trp-Ala-Val-Gly-His-NH-CH[CH2-CH(CH3)2]-CHOH-(CH 2)3-CH3, JMV845, was among the most potent compounds with ED50 of 7.82 +/- 2.89 nM in inhibiting bombesin-induced protein secretion). The results of this study showed that replacing the peptide bond between the two last amino acid residues in bombesin by mimicking the transition state analog resulted in in vitro and in vivo potent bombesin receptor antagonists.

JMV641: a potent bombesin receptor antagonist that inhibits Swiss 3T3 cell proliferation.

The peptides of the bombesin family are involved in stimulation of mitogenesis in various cell lines, including cancerous cell lines. Bombesin receptor antagonists are of great interest to inhibit this proliferation. We have synthesized a potent bombesin receptor antagonist, e.g., compound JMV641 [H-DPhe-Gln-Trp-Ala-Val-Gly-His-NH-*CH[CH2-CH(CH3)2]-**CHOH- (CH2)3-CH3 [*(S); **92% of (S) isomer], in which a pseudopeptide bond mimicking the transition state analogue replaced the peptide bond between the two C-terminal residues. This compound was highly potent to dose-dependently inhibit binding of 125I-GRP to Swiss 3T3 cells (IC50 = 0.85 +/- 0.15 nM) and bombesin-stimulated Swiss 3T3 proliferation (pA2 = 8.78). However, compound JMV641 can inhibit bombesin-induced AP-1 regulated genes that are nuclear messengers mediating the actions of signal transduction pathways stimulated by growth factors.

A synthetic glycine-extended bombesin analogue interacts with the GRP/bombesin receptor.

alpha-amidation of a peptide (which takes place from a glycine-extended precursor) is required to produce biologically active amidated hormones, such as gastrin-releasing peptide (GRP)/Pyr-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH(2) (bombesin). It was shown that glycine-extended gastrin mediates mitogenic effects on various cell lines by interacting with a specific receptor, different from the classical CCK(1) or CCK(2) receptors. On the basis of this observation, we have extended the concept of obtaining active glycine-extended forms of others amidated peptides to produce new active analogues. In this study, we have tested the biological behaviour of a synthetic analogue of the glycine-extended bombesin (para-hydroxy-phenyl-propionyl-Gln-Trp-Ala-Val-Gly-His-Leu-Met-Gly-OH or JMV-1458) on various in vitro models. We showed that compound JMV-1458 was able to inhibit specific (3-[125I]iodotyrosyl(15)) GRP ([125I]GRP) binding in rat pancreatic acini and in Swiss 3T3 cells with K(i) values of approximately 10(-8) M. In isolated rat pancreatic acini, we found that JMV-1458 induced inositol phosphates production and amylase secretion in a dose-dependent manner. In Swiss 3T3 cells, the glycine-extended bombesin analogue dose-dependently produced [3H]thymidine incorporation. By using potent GRP/bombesin receptor antagonists, we showed that this synthetic glycine-extended bombesin analogue induces its biological activities via the classical GRP/bombesin receptor.

A C-terminal cyclic 8-13 neurotensin fragment analog appears less exposed to neprilysin when it crosses the blood-brain barrier than the cerebrospinal fluid-brain barrier in mice.

A C-terminal cyclic 8-13 neurotensin fragment analog. JMV 1193, a direct agonist of central neurotensin receptors, is able to cross both the cerebrospinal fluid-brain barrier and the blood-brain barrier. When administered intracerebroventricularly (i.c.v.), its hypothermic effect was potentiated by the enkephalinase inhibition induced either by thiorphan (simultaneous intracerebroventricular administration of 10 micrograms) or by the thiorphan prodrug. acetorphan (intravenous (i.v.) administration of 10 mg/kg). Such a potentiation was not observed when both JMV 1193 and acetorphan were administered intravenously. Therefore it appears that the sensitivity of JMV 1193 to enkephalinase depends on its route of administration. It is exposed to this peptidase after i.c.v. injection (when crossing the cerebrospinal fluid-brain barrier), while it is not after i.v. administration (when crossing the blood-brain barrier).

Dystrophin and dystrophin-associated protein in muscles and nerves from monkey.

Since all organs (i.e. skeletal, cardiac, smooth muscles and sciatic nerve) are never only taken from a single patient, all these tissues were obtained from one cynomolgus monkey, a model closely resembling humans. This work describes an up-to-date reinvestigation of the dystrophin-glycoprotein complex and related molecules in various monkey tissues such those cited above. We used monoclonal and polyclonal antibodies produced in our laboratory, which are directed against dystrophin, utrophin, short-dystrophin products, alpha-dystrobrevin, beta-dystroglycan, alpha-syntrophin, alpha-, beta-, gamma-, delta-, epsilon-sarcoglycan, and sarcospan. For each molecule, we determined their molecular weight and tissue localization. Regardless of the tissue analyzed, at least one dystrophin or utrophin as full-length molecule and one short-dystrophin product or dystrobrevin as proteins belonging to the dystrophin superfamily were found. Beta-dystroglycan, beta and delta sarcoglycans were always detected, while other sarcoglycans varied from all to only three components. Epsilon sarcoglycan appears to be specific to smooth muscle, which is devoid of alpha sarcoglycan. Sarcospan is only absent from sciatic nerve structures. Among the different muscles investigated in this study, short dystrophin products are only present in cardiac muscle. All of these findings are summarized in one table, which highlight in one single animal the variability of the dystrophin-glycoprotein complex components in relation with the organ studied. This statement is important because any attempt to estimate protein restoration needs in each study the knowledge of the expected components that should be considered normal.

Renin inhibition by synthetic peptides related to mouse and human renin prosegments.

Recently the nucleotide sequences of the genes coding for mouse and human renins have been elucidated and the primary structures of the corresponding renin precursors deduced. The existence of a prosegment suggests that renin is biosynthesized as an inactive zymogen prorenin. Three peptides related to the region 11-19 of the mouse submaxillary prosegment have been synthesized and tested as inhibitors of pure mouse renin acting on a synthetic porcine substrate. Inhibition of renin activity was effective, with IC50s in the 10(-6) M range. Butyloxycarbonyl-leucyl-lysyl-lysyl-methionyl-proline methyl ester (15-19) was the most potent inhibitor with a K value of 2.3 X 10(-6) M at 37 degrees C in 0.5 M citrate/phosphate buffer (pH 6.0). Peptides 16-20 and 9-20 of the human renin prosegment and peptides 11-19 and 15-19 of the mouse prosegment were tested on human plasma renin activity and found to be inhibitory, with IC50s of 3 to 5 X 10(-4) M. These results demonstrate that the renin prosegment is a renin inhibitor and support the hypothesis that prorenin is an inactive zymogen.

Immunological approach to blockade of the renin-substrate reaction.

To block the renin-substrate reaction by immunological tools is a long-standing dream. Since 1951 active immunization and the passive transfer of antirenin antisera have been successfully carried out in different species and in different experimental models of hypertension and normotension. These studies indicated that renin is a powerful immunogenic protein, capable of breaking down self-tolerance in different species. In this initial period the most significant results were obtained with hog renin. Passive transfer of antisera raised against hog renin or active immunization with hog renin was able to decrease blood pressure in renovascular or essential hypertension in dogs. Renin was semipurified and injected without adjuvant, however, since there was no method for determining plasma renin activity. Recently, complete purification of murine and human renin has allowed an extension of this approach, using the passive transfer of antirenin polyclonal antisera or monoclonal antibodies. Active immunization against pure human renin was successful in normotensive marmosets. This immunization with a nearly homologous renin in a primate model induced a significant decrease in blood pressure, associated with a complete disappearance of plasma renin activity. Unfortunately this powerful immunization was associated with an autoimmune disease that is specific for the kidney, related to self-recognition of the production site of renin by antibodies and lymphocytes. Similar results were reported with the use of mouse submandibular gland renin as an immunogen in spontaneously hypertensive rats (SHR). This manipulation decreased blood pressure in SHR to a level near that of normotensive Wistar-Kyoto control rats. However, again the animals showed a severe autoimmune disease of the kidney.(ABSTRACT TRUNCATED AT 250 WORDS)

Triazole GHS-R1a antagonists JMV4208 and JMV3002 attenuate food intake, body weight, and adipose tissue mass in mice.

The only peripherally released orexigenic hormone, ghrelin, plays a key role in food intake and body weight regulation. Antagonizing the ghrelin receptor, GHS-R1a, represents a promising approach for anti-obesity therapy. In our study, two novel GHS-R1a antagonists JMV4208 and JMV3002, which are trisubstituted 1,2,4-triazoles, decreased food intake in fasted lean mice in a dose-dependent manner, with ED50 values of 5.25 and 2.05 mg/kg, respectively. Both compounds were stable in mouse blood, with half-lives of 90 min (JMV4208) and 60 min (JMV3002), and disappeared from the blood 8h after administration. Fourteen days of treatment with the ghrelin antagonists (20 mg/kg twice a day) decreased food intake, body weight and adipose tissue mass in mice with diet-induced obesity (DIO). These results are likely attributable to an impact on food intake reduction and an attenuated expression of the lipogenesis-promoting enzymes (acetyl-CoA carboxylase 1 in subcutaneous fat and fatty acid synthase in subcutaneous and intraperitoneal fat). The decrease in fat mass negatively impacted circulating leptin levels. These data suggest that JMV4208 and JMV3002 could be useful therapeutic agents for the treatment of obesity.

Ghrelin agonist JMV 1843 increases food intake, body weight and expression of orexigenic neuropeptides in mice.

Ghrelin and agonists of its receptor GHS-R1a are potential substances for the treatment of cachexia. In the present study, we investigated the acute and long term effects of the GHS R1a agonist JMV 1843 (H Aib-DTrp-D-gTrp-CHO) on food intake, body weight and metabolic parameters in lean C57BL/6 male mice. Additionally, we examined stability of JMV 1843 in mouse blood serum. A single subcutaneous injection of JMV 1843 (0.01-10 mg/kg) increased food intake in fed mice in a dose-dependent manner, up to 5-times relative to the saline-treated group (ED(50)=1.94 mg/kg at 250 min). JMV 1843 was stable in mouse serum in vitro for 24 h, but was mostly eliminated from mouse blood after 2 h in vivo. Ten days of treatment with JMV 1843 (subcutaneous administration, 10 or 20 mg/kg/day) significantly increased food intake, body weight and mRNA expression of the orexigenic neuropeptide Y and agouti-related peptide in the medial basal hypothalamus and decreased the expression of uncoupling protein 1 in brown adipose tissue. Our data suggest that JMV 1843 could have possible future uses in the treatment of cachexia.

Ghrelin agonists impact on Fos protein expression in brain areas related to food intake regulation in male C57BL/6 mice.

Many peripheral substances, including ghrelin, induce neuronal activation in the brain. In the present study, we compared the effect of subcutaneously administered ghrelin and its three stable agonists: Dpr(3)ghr ([Dpr(N-octanoyl)(3)] ghrelin) (Dpr - diaminopropionic acid), YA GHRP-6 (H-Tyr-Ala-His-DTrp-Ala-Trp-DPhe-Lys-NH(2)), and JMV1843 (H-Aib-DTrp-D-gTrp-CHO) on the Fos expression in food intake-responsive brain areas such as the hypothalamic paraventricular (PVN) and arcuate (ARC) nuclei, the nucleus of the solitary tract (NTS), and area postrema (AP) in male C57BL/6 mice. Immunohistochemical analysis showed that acute subcutaneous dose of each substance (5mg/kg b.w.), which induced a significant food intake increase, elevated Fos protein expression in all brain areas studied. Likewise ghrelin, each agonist tested induced distinct Fos expression overall the PVN. In the ARC, ghrelin and its agonists specifically activated similarly distributed neurons. Fos occurrence extended from the anterior (aARC) to middle (mARC) ARC region. In the latter part of the ARC, the Fos profiles were localized bilaterally, especially in the ventromedial portions of the nucleus. In the NTS, all substances tested also significantly increased the number of Fos profiles in neurons, which also revealed specific location, i.e., in the NTS dorsomedial subnucleus (dmNTS) and the area subpostrema (AsP). In addition, cells located nearby the NTS, in the AP, also revealed a significant increase in number of Fos-activated cells. These results demonstrate for the first time that ghrelin agonists, regardless of their different chemical nature, have a significant and similar activating impact on specific groups of neurons that can be a part of the circuits involved in the food intake regulation. Therefore there is a real potency for ghrelin agonists to treat cachexia and food intake disorders. Thus, likewise JMV1843, the other ghrelin agonists represent substances that might be involved in trials for clinical purposes.

On the central mechanism underlying ghrelin's chronic pro-obesity effects in rats: new insights from studies exploiting a potent ghrelin receptor antagonist.

In the present study, we explore the central nervous system mechanism underlying the chronic central effects of ghrelin with respect to increasing body weight and body fat. Specifically, using a recently developed ghrelin receptor antagonist, GHS-R1A (JMV2959), we investigate the role of GHS-R1A in mediating the effects of ghrelin on energy balance and on hypothalamic gene expression. As expected, in adult male rats, chronic central treatment with ghrelin for 14 days, when compared to vehicle-treated control rats, resulted in an increased body weight, lean mass and fat mass (assessed by dual X-ray absorptiometry), dissected white fat pad weight, cumulative food intake, food efficiency, respiratory exchange ratio and a decrease of energy expenditure. Co-administration of the ghrelin receptor antagonist JMV2959 suppressed/blocked the majority of these effects, with the notable exception of ghrelin-induced food intake and food efficiency. The hypothesis emerging from these data, namely that GHS-R1A mediates the chronic effects of ghrelin on fat accumulation, at least partly independent of food intake, is discussed in light of the accompanying data regarding the hypothalamic genes coding for peptides and receptors involved in energy balance regulation, which were found to have altered expression in these studies.