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Andrew L Gundlach - One of the best experts on this subject based on the ideXlab platform.
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Involvement of the Nucleus Incertus and Relaxin-3/RXFP3 Signaling System in Explicit and Implicit Memory
Frontiers in Neuroanatomy, 2021Co-Authors: Isis Gil-miravet, Andrew L Gundlach, Héctor Albert-gascó, Francisco Ros-bernal, Aroa Mañas-ojeda, Esther Castillo-gómez, Francisco E. Olucha-bordonauAbstract:Telencephalic cognitive and emotional circuits/functions are strongly modulated by subcortical inputs. The main focus of past research on the nature of this modulation has been on the widespread monoamine projections to the telencephalon. However, the nucleus incertus (NI) of the pontine tegmentum provides a strong GABAergic and peptidergic innervation of the hippocampus, basal forebrain, amygdala, prefrontal cortex, and related regions; and represents a parallel source of ascending modulation of cognitive and emotional domains. NI GABAergic neurons express multiple peptides, including neuromedin-B, cholecystokinin, and Relaxin-3, and receptors for stress and arousal transmitters, including corticotrophin-releasing factor and orexins/hypocretins. A functional relationship exists between NI neurons and their associated peptides, Relaxin-3 and neuromedin-B, and hippocampal theta rhythm, which in turn, has a key role in the acquisition and extinction of declarative and emotional memories. Furthermore, RXFP3, the cognate receptor for Relaxin-3, is a Gi/o protein-coupled receptor, and its activation inhibits the cellular accumulation of cAMP and induces phosphorylation of ERK, processes associated with memory formation in the hippocampus and amygdala. Therefore, this review summarizes the role of NI transmitter systems in relaying stress- and arousal-related signals to the higher neural circuits and processes associated with memory formation and retrieval.
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Targeted viral vector transduction of Relaxin-3 neurons in the rat nucleus incertus using a novel cell-type specific promoter
IBRO reports, 2019Co-Authors: Alexander D. Wykes, Andrew L GundlachAbstract:Modern neuroscience utilizes transgenic techniques extensively to study the activity and function of brain neural networks. A key feature of this approach is its compatibility with molecular methods for selective transgene expression in neuronal circuits of interest. Until now, such targeted transgenic approaches have not been applied to the extensive circuitry involving the neuropeptide, Relaxin-3. Pharmacological and gene knock-out studies have revealed Relaxin-3 signalling modulates interrelated behaviours and cognitive processes, including stress and anxiety, food and alcohol consumption, and spatial and social memory, highlighting the potential of this system as a therapeutic target. In the present study, we aimed to identify a promoter sequence capable of regulating cell-type specific transgene expression from an adeno-associated viral (AAV) vector in Relaxin-3 neurons of the rat nucleus incertus (NI). In parallel to Relaxin-3 promoter sequences, we also tested an AAV vector containing promoter elements for the tropomyosin receptor kinase A (TrkA) gene, as TrkA is co-expressed with Relaxin-3 in rat NI neurons. Stereotaxic injection of an mCherry-expressing AAV vector revealed widespread non-specific TrkA promoter (880 bp) activity in and adjacent to the NI at 8 weeks post-treatment. In contrast, mCherry expression was successfully restricted to Relaxin-3 NI neurons with 98% specificity using a 1736 bp Relaxin-3 promoter. In addition to detailed anatomical mapping of NI Relaxin-3 networks, illustrated here in association with GABAergic medial septum neurons, this method for targeted transgene delivery offers a versatile tool for ongoing preclinical studies of Relaxin-3 circuitry.
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Chronic activation of the Relaxin-3 receptor on GABA neurons in rat ventral hippocampus promotes anxiety and social avoidance.
Hippocampus, 2019Co-Authors: Valeria Rytova, David Hawkes, Despina E. Ganella, Andrew L GundlachAbstract:Anxiety disorders are highly prevalent in modern society and better treatments are required. Key brain areas and signaling systems underlying anxiety include prefrontal cortex, hippocampus, and amygdala, and monoaminergic and peptidergic systems, respectively. Hindbrain GABAergic projection neurons that express the peptide, Relaxin-3, broadly innervate the forebrain, particularly the septum and hippocampus, and Relaxin-3 acts via a Gi/o -protein-coupled receptor known as the Relaxin-family peptide 3 receptor (RXFP3). Thus, Relaxin-3/RXFP3 signaling is implicated in modulation of arousal, motivation, mood, memory, and anxiety. Ventral hippocampus (vHip) is central to affective and cognitive processing and displays a high density of Relaxin-3-positive nerve fibers and RXFP3 binding sites, but the identity of target neurons and associated effects on behavior are unknown. Therefore, in adult, male rats, we assessed the neurochemical nature of hippocampal RXFP3 mRNA-expressing neurons and anxiety-like and social behavior following chronic RXFP3 activation in vHip by viral vector expression of an RXFP3-selective agonist peptide, R3/I5. RXFP3 mRNA detected by fluorescent in situ hybridization was topographically distributed across the hippocampus in somatostatin- and parvalbumin-mRNA expressing GABA neurons. Chronic RXFP3 activation in vHip increased anxiety-like behavior in the light-dark box and elevated-plus maze, but not the large open-field test, and reduced social interaction with a conspecific stranger. Our data reveal disruptive effects of persistent RXFP3 signaling on hippocampal GABA networks important in anxiety; and identify a potential therapeutic target for anxiety disorders that warrants further investigation in relevant preclinical models.
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Involvement of Serotonergic and Relaxin-3 Neuropeptide Systems in the Expression of Anxiety-like Behavior.
Neuroscience, 2018Co-Authors: Adam J. Lawther, Andrew L Gundlach, Andrew Flavell, Stephen Kent, Christopher A. Lowry, Matthew W. HaleAbstract:Abstract Anxiety-related defensive behavior is controlled by a distributed network of brain regions and interconnected neural circuits. The dorsal raphe nucleus (DR), which contains the majority of forebrain-projecting serotonergic neurons, is a key brain region involved in fear states and anxiety-related behavior via modulation of this broad neural network. Evidence suggests that Relaxin-3 neurons in the nucleus incertus (NI) may also interact with this network, however, the potential role of the NI in the control of anxiety-related defensive behavior requires further investigation. In this study, we examined the response of an anxiety-related neuronal network, including serotonergic neurons in the DR and Relaxin-3-containing neurons in the NI, to administration of an anxiogenic drug and exposure to an aversive environment. We administered an anxiogenic dose of the adenosine receptor antagonist, caffeine (50 mg/kg, i.p.), or vehicle, to adult male Wistar rats and 30 min later exposed them to either an elevated plus-maze (EPM) or a home cage environment. Administration of caffeine and exposure to the EPM activated a broad network of brain regions involved in control of anxiety-like behaviors, including serotonergic neurons in the DR, as measured using c-Fos immunohistochemistry. However, only exposure to the EPM activated Relaxin-3-containing neurons in the NI, and activation of these neurons was not correlated with changes in anxiety-like behavior. These data suggest activation of the NI Relaxin-3 system is associated with expression of behavior in tests of anxiety, but may not be directly involved in the approach-avoidance conflict inherent in anxiety-related defensive behavior in rodents.
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Modulation of forebrain function by nucleus incertus and Relaxin-3/RXFP3 signaling.
CNS neuroscience & therapeutics, 2018Co-Authors: Francisco E. Olucha-bordonau, Ana María Sánchez-pérez, Héctor Albert-gascó, Francisco Ros-bernal, Valeria Rytova, Emma K E Ong-pålsson, Andrew L GundlachAbstract:The nucleus incertus (NI) in the pontine tegmentum sends ascending projections to the midbrain, hypothalamus, amygdala, basal forebrain, hippocampus, and prefrontal cortex, and has a postulated role in modulating several forebrain functions. A substantial population of GABAergic NI neurons expresses the neuropeptide, Relaxin-3, which acts via the Gi/o -protein-coupled receptor, RXFP3, present throughout the forebrain target regions. Broad and specific manipulations of these systems by activation or inhibition of the NI or modulating RXFP3 signaling have revealed key insights into the likely influence of the NI/Relaxin-3/RXFP3 system on modalities including arousal, feeding, stress responses, anxiety and addiction, and attention and memory. This range of actions corresponds to a likely impact of NI/(Relaxin-3) projections on multiple integrated circuits, but makes it difficult to draw conclusions about a generalized function for this network. This review will focus on the key physiological process of oscillatory theta rhythm and the neural circuits that promote it during behavioral activation, highlighting the ability of NI and Relaxin-3/RXFP3 signaling systems to modulate these circuits. A better understanding of these mechanisms may provide a way to therapeutically adjust malfunction of forebrain activity present in several pathological conditions.
John D. Wade - One of the best experts on this subject based on the ideXlab platform.
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Effects of C-Terminal B-Chain Modifications in a Relaxin 3 Agonist Analogue.
ACS medicinal chemistry letters, 2020Co-Authors: Praveen Praveen, John D. Wade, K Johan Rosengren, Julien Tailhades, Mengjie Liu, Mohammed Akhter HossainAbstract:The receptor for the neuropeptide Relaxin 3, Relaxin family peptide 3 (RXFP3) receptor, is an attractive pharmacological target for the control of eating, addictive, and psychiatric behaviors. Several structure-activity relationship studies on both human Relaxin 3 (containing 3 disulfide bonds) and its analogue A2 (two disulfide bonds) suggest that the C-terminal carboxylic acid of the tryptophan residue in the B-chain is important for RXFP3 activity. In this study, we have added amide, alcohol, carbamate, and ester functionalities to the C-terminus of A2 and compared their structures and functions. As expected, the C-terminal amide form of A2 showed lower binding affinity for RXFP3 while ester and alcohol substitutions also demonstrated lower affinity. However, while these analogues showed slightly lower binding affinity, there was no significant difference in activation of RXFP3 compared to A2 bearing a C-terminal carboxylic acid, suggesting the binding pocket is able to accommodate additional atoms.
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Central injection of Relaxin-3 receptor (RXFP3) antagonist peptides reduces motivated food seeking and consumption in C57BL/6J mice
Behavioural brain research, 2014Co-Authors: Craig M Smith, John D. Wade, Berenice E Chua, Andrew W Walker, Fazel Shabanpoor, Mohammad Akhter Hossain, Cary Zhang, Mouna Haidar, David Hawkes, K Johan RosengrenAbstract:Behavioural arousal in mammals is regulated by various interacting central monoamine- and peptide-neurotransmitter/receptor systems, which function to maintain awake, alert and active states required for performance of goal-directed activities essential for survival, including food seeking. Existing anatomical and functional evidence suggests the highly-conserved neuropeptide, Relaxin-3, which signals via its cognate Gi/o-protein coupled receptor, RXFP3, contributes to behavioural arousal and feeding behaviour in rodents. In studies to investigate this possibility further, adult male C57BL/6J mice were treated with the selective RXFP3 antagonist peptides, R3(B1-22)R/I5(A) and R3(B1-22)R, and motivated food seeking and consumption was assessed as a reflective output of behavioural arousal. Compared to vehicle treatment, intracerebroventricular (icv) injection of RXFP3 antagonists reduced: (i) food anticipatory activity before meal time during food restriction; (ii) consumption of highly palatable food; (iii) consumption of regular chow during the initial dark phase, and; (iv) consumption of regular chow after mild (∼4-h) food deprivation. Effects were not due to sedation and appeared to be specifically mediated via antagonism of Relaxin-3/RXFP3 signalling, as RXFP3 antagonist treatment did not alter locomotor activity in wild-type mice or reduce palatable food intake in Relaxin-3 deficient (knock-out) mice. Notably, in contrast to similar studies in the rat, icv injection of RXFP3 agonists and infusion into the paraventricular hypothalamic nucleus did not increase food consumption in mice, suggesting species differences in Relaxin-3/RXFP3-related signalling networks. Together, our data provide evidence that endogenous Relaxin-3/RXFP3 signalling promotes motivated food seeking and consumption, and in light of the established biological and translational importance of other arousal systems, Relaxin-3/RXFP3 networks warrant further experimental investigation.
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Relaxin-3/RXFP3 system regulates alcohol-seeking
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Philip Ryan, John D. Wade, Andrew L Gundlach, Mohammed Akhter Hossain, K Johan Rosengren, Hanna E. Kastman, Elena Krstew, Leonid Churilov, Andrew J LawrenceAbstract:Relapse and hazardous drinking represent the most difficult clinical problems in treating patients with alcohol use disorders. Using a rat model of alcohol use and alcohol-seeking, we demonstrated that central administration of peptide antagonists for Relaxin family peptide 3 receptor (RXFP3), the cognate receptor for the highly conserved neuropeptide, Relaxin-3, decreased self-administration of alcohol in a dose-related manner and attenuated cue- and stress-induced reinstatement following extinction. By comparison, RXFP3 antagonist treatment did not significantly attenuate self-administration or reinstatement of sucrose-seeking, suggesting a selective effect for alcohol. RXFP3 is densely expressed in the stress-responsive bed nucleus of the stria terminalis, and bilateral injections of RXFP3 antagonist into the bed nucleus of the stria terminalis significantly decreased self-administration and stress-induced reinstatement of alcohol, suggesting that this brain region may, at least in part, mediate the effects of RXFP3 antagonism. RXFP3 antagonist treatment had no effect on general ingestive behavior, activity, or procedural memory for lever pressing in the paradigms assessed. These data suggest that Relaxin-3/RXFP3 signaling regulates alcohol intake and relapse-like behavior, adding to current knowledge of the brain chemistry of reward-seeking.
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Chemical synthesis and orexigenic activity of rat/mouse Relaxin-3
Amino Acids, 2013Co-Authors: Mohammed Akhter Hossain, Craig M Smith, Andrew L Gundlach, Elena Büchler, Ross A D Bathgate, Philip J. Ryan, John D. WadeAbstract:The insulin-like peptide, Relaxin-3 was first identified just a decade ago via a genomic database search and is now recognized to be a key neuropeptide with several roles including the regulation of arousal, stress responses and neuroendocrine homeostasis. It also has significant potential as a drug to treat stress and obesity. Its actions are mediated via its cognate G protein-coupled receptor, RXFP3, which is found in abundant numbers in the brain. However, much remains to be determined with respect to the mechanism of neurological action of this peptide. Consequently, the chemical synthesis of the rat and mouse (which share identical primary structures) two-chain, three disulfide peptide was undertaken and the resulting peptide subjected to detailed in vitro and in vivo assay. Use of efficient solid-phase synthesis methods provided the two regioselectively S-protected A- and B-chains which were readily combined via sequential disulfide bond formation. The synthetic rat/mouse Relaxin-3 was obtained in high purity and good overall yield. It demonstrated potent orexigenic activity in rats in that central intracerebroventricular infusion led to significantly increased food intake and water drinking.
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Relaxin 3 innervation of the intergeniculate leaflet of the rat thalamus neuronal tract tracing and in vitro electrophysiological studies
European Journal of Neuroscience, 2013Co-Authors: Anna Blasiak, John D. Wade, Andrew L Gundlach, Tomasz Blasiak, M H Lewandowski, Mohammed Akhter HossainAbstract:Behavioural state is controlled by a range of neural systems that are sensitive to internal and external stimuli. The Relaxin-3 and Relaxin family peptide receptor 3 (RXFP3) system has emerged as a putative ascending arousal network with putative involvement in regulation of stress responses, neuroendocrine control, feeding and metabolism, circadian activity and cognition. Relaxin-3/γ-aminobutyric acid neuron populations have been identified in the nucleus incertus, pontine raphe nucleus, periaqueductal grey (PAG) and an area dorsal to the substantia nigra. Relaxin-3-positive fibres/terminals densely innervate arousal-related structures in the brainstem, hypothalamus and limbic forebrain, but the functional significance of the heterogeneous Relaxin-3 neuron distribution and its inputs to specific brain areas are unclear. Therefore, in this study, we used neuronal tract-tracing and immunofluorescence staining to explore the source of the dense Relaxin-3 innervation of the intergeniculate leaflet (IGL) of the thalamus, a component of the neural circadian timing system. Confocal microscopy analysis revealed that Relaxin-3-positive neurons retrogradely labelled from the IGL were predominantly present in the PAG and these neurons expressed corticotropin-releasing factor receptor-like immunoreactivity. Subsequently, whole-cell patch-clamp recordings revealed heterogeneous effects of RXFP3 activation in the IGL by the RXFP3 agonist, Relaxin-3 B-chain/insulin-like peptide-5 A-chain (R3/I5). Identified, neuropeptide Y-positive IGL neurons, known to influence suprachiasmatic nucleus activity, were excited by R3/I5, whereas neurons of unidentified neurotransmitter content were either depolarized or displayed a decrease in action potential firing and/or membrane potential hyperpolarization. Our data identify a PAG to IGL Relaxin-3/RXFP3 pathway that might convey stress-related information to key elements of the circadian system and influence behavioural state rhythmicity.
Zhan-yun Guo - One of the best experts on this subject based on the ideXlab platform.
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A negatively charged transmembrane aspartate residue controls activation of the Relaxin-3 receptor RXFP3
Archives of biochemistry and biophysics, 2016Co-Authors: Yu Liu, Xiaoxia Shao, Ya-li Liu, Lei Zhang, Zhan-yun GuoAbstract:Relaxin-3 is an insulin/Relaxin superfamily neuropeptide involved in the regulation of food intake and stress response via activation of its cognate receptor RXFP3, an A-class G protein-coupled receptor (GPCR). In recent studies, a highly conserved ExxxD motif essential for binding of Relaxin-3 has been identified at extracellular end of the second transmembrane domain (TMD2) of RXFP3. For most of the A-class GPCRs, a highly conserved negatively charged Asp residue (Asp(2.50) using Ballesteros-Weinstein numbering and Asp128 in human RXFP3) is present at the middle of TMD2. To elucidate function of the conserved transmembrane Asp128, in the present work we replaced it with other residues and the resultant RXFP3 mutants all retained quite high ligand-binding potency, but their activation and agonist-induced internalization were abolished or drastically decreased. Thus, the negatively charged transmembrane Asp128 controlled transduction of agonist-binding information from the extracellular region to the intracellular region through maintaining RXFP3 in a metastable state for efficient conformational change induced by binding of an agonist.
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Identification of hydrophobic interactions between Relaxin-3 and its receptor RXFP3: implication for a conformational change in the B-chain C-terminus during receptor binding.
Amino acids, 2016Co-Authors: Xiaoxia Shao, Ya-li Liu, Jia-hui Wang, Dian Wei, Zhan-yun GuoAbstract:Relaxin-3 is an insulin/Relaxin superfamily neuropeptide implicated in the regulation of food intake and stress response via activation of the G protein-coupled receptor RXFP3. Their electrostatic interactions have been recently identified, and involves three positively charged B-chain residues (B12Arg, B16Arg, and B26Arg) of Relaxin-3 and two negatively charged residues (Glu141 and Asp145) in a highly conserved ExxxD motif at the extracellular end of the second transmembrane domain of RXFP3. To investigate their hydrophobic interactions, in the present work we deleted the highly conserved B-chain C-terminal B27Trp residue of Relaxin-3, and mutated four highly conserved aromatic residues (Phe137, Trp138, Phe146, and Trp148) around the ExxxD motif of RXFP3. The resultant [∆B27W]Relaxin-3 exhibited approximately tenfold lower binding potency and ~1000-fold lower activation potency towards wild-type RXFP3, confirming its importance for Relaxin-3 function. Although the RXFP3 mutants could be normally trafficked to cell membrane, they had quite different activities. [F137A]RXFP3 could normally distinguish wild-type Relaxin-3 and [∆B27W]Relaxin-3 in binding and activation assays, whereas [W138A]RXFP3 lost most of this capability, suggesting that the Trp138 residue of RXFP3 forms hydrophobic interactions with the B27Trp residue of Relaxin-3. The hydrophobic Trp138 residue and the formerly identified negatively charged Glu141 and Asp145 residues in the highly conserved WxxExxxD motif may thus form a functional surface that is important for interaction with Relaxin-3. We hypothesize that the Relaxin-3 B-chain C-terminus changes from the original folding-back conformation to an extended conformation during binding with RXFP3, to allow its B27Trp and B26Arg residues to interact with the Trp138 and Glu141 residues of RXFP3, respectively.
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The electrostatic interactions of Relaxin-3 with receptor RXFP4 and the influence of its B-chain C-terminal conformation.
The FEBS journal, 2014Co-Authors: Xin-yi Wang, Xiaoxia Shao, Weijie Zhang, Yu-qi Guo, Ya-li Liu, Zhan-yun GuoAbstract:Relaxin-3 (also known as insulin-like peptide 7) is an insulin/Relaxin-superfamily peptide hormone that can bind and activate three Relaxin-family peptide receptors: RXFP3, RXFP4, and RXFP1. Recently, we identified key electrostatic interactions between Relaxin-3 and its cognate receptor RXFP3 by using a charge-exchange mutagenesis approach. In the present study, the electrostatic interactions between Relaxin-3 and RXFP4 were investigated with the same approach. Mutagenesis of the negatively charged extracellular residues of human RXFP4 identified a conserved EXXXD(100–104) motif that is essential for RXFP4 activation by Relaxin-3. Mutagenesis of the conserved positively charged Arg residues of Relaxin-3 demonstrated that B12Arg, B16Arg and B26Arg were all involved in the binding and activation of RXFP4, especially B26Arg. The activity complementation between the mutant ligands and the mutant receptors suggested two probable electrostatic interaction pairs: Glu100 of RXFP4 versus B26Arg of Relaxin-3, and Asp104 of RXFP4 versus both B12Arg and B16Arg of Relaxin-3. For interaction with the essential EXXXD motifs of both RXFP3 and RXFP4, a folding-back conformation of the Relaxin-3 B-chain C-terminus seems to be critical, because it brings B26Arg sufficiently close to B12Arg and B16Arg. To test this hypothesis, we replaced the conserved B23Gly-B24Gly dipeptide of Relaxin-3 with an Ala-Ser dipeptide that occupied the corresponding position of insulin-like peptide 5 and resulted in an extended helical conformation. The mutant Relaxin-3 showed a significant decrease in receptor-activation potency towards both RXFP3 and RXFP4, suggesting that a folding-back conformation of the B-chain C-terminus was important for Relaxin-3 to efficiently interact with the EXXXD motifs of both receptors. Structured digital abstract RXFP4 and Relaxin-3 physically interact by competition binding (1, 2, 3, 4, 5, 6, 7, 8, 9)
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The highly conserved negatively charged Glu141 and Asp145 of the G-protein-coupled receptor RXFP3 interact with the highly conserved positively charged arginine residues of Relaxin-3.
Amino acids, 2014Co-Authors: Weijie Zhang, Xiaoxia Shao, Xin-yi Wang, Yu-qi Guo, Ya-li Liu, Xiao Luo, Xue-juan Gao, Zhan-yun GuoAbstract:Relaxin-3 is a newly identified insulin/Relaxin superfamily peptide that plays a putative role in the regulation of food intake and stress response by activating its cognate G-protein-coupled receptor RXFP3. Relaxin-3 has three highly conserved arginine residues, B12Arg, B16Arg and B26Arg. We speculated that these positively charged arginines may interact with certain negatively charged residues of RXFP3. To test this hypothesis, we first replaced the negatively charged residues in the extracellular domain of RXFP3 with arginine, respectively. Receptor activation assays showed that arginine replacement of Glu141 or Asp145, especially Glu141, significantly decreased the sensitivity of RXFP3 to wild-type Relaxin-3. In contrast, arginine replacement of other negatively charged extracellular residues had little effect. Thus, we deduced that Glu141 and Asp145, locating at the extracellular end of the second transmembrane domain, played a critical role in the interaction of RXFP3 with Relaxin-3. To identify the ligand residues interacting with the negatively charged EXXXD motif of RXFP3, we replaced the three conserved arginines of Relaxin-3 with negatively charged glutamate or aspartate, respectively. The mutant Relaxin-3s retained the native structure, but their binding and activation potencies towards wild-type RXFP3 were decreased significantly. The compensatory effects of the mutant Relaxin-3s towards mutant RXFP3s suggested two probable interaction pairs during ligand–receptor interaction: Glu141 of RXFP3 interacted with B26Arg of Relaxin-3, meanwhile Asp145 of RXFP3 interacted with both B12Arg and B16Arg of Relaxin-3. Based on these results, we proposed a Relaxin-3/RXFP3 interaction model that shed new light on the interaction mechanism of the Relaxin family peptides with their receptors.
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Design, recombinant expression and convenient A-chain N-terminal europium-labelling of a fully active human Relaxin-3 analogue.
The FEBS journal, 2012Co-Authors: Weijie Zhang, Xiaoxia Shao, Xin-yi Wang, Xiao Luo, Ge Song, Zhan-yun GuoAbstract:Relaxin-3 (also known as INSL7) is a recently identified neuropeptide belonging to the insulin/Relaxin superfamily. It plays a putative role in the regulation of food intake, in the stress response and in reproduction by activating the G-protein-coupled receptor, RXFP3. In a previous study, we prepared 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)/Eu3+-labelled human Relaxin-3 as a tracer for the study of ligand–receptor interactions, which necessitated a complicated site-specific labelling strategy because human Relaxin-3 contains four primary amine moieties, all of which react with the primary amine-specific modification reagent. To simplify the labelling procedure, in the present study we created an easily labelled, recombinant analogue of human Relaxin-3 with only one primary amine moiety at the A-chain N-terminus. The analogue retained full activity and could be easily labelled by various functional probes at the A-chain N-terminus. The DOTA/Eu3+-labelled analogue retained high binding affinity for its cognate receptor, RXFP3, and thus represents a useful, nonradioactive and stable tracer for studying the interaction of RXFP3 with various natural or synthetic ligands. This new analogue is also a suitable template for the design of other Relaxin-3 analogues that can be easily labelled with the DOTA/Eu3+ moiety and used to study binding activity and interactions with various RXFP3 analogues in the future.
K Johan Rosengren - One of the best experts on this subject based on the ideXlab platform.
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Effects of C-Terminal B-Chain Modifications in a Relaxin 3 Agonist Analogue.
ACS medicinal chemistry letters, 2020Co-Authors: Praveen Praveen, John D. Wade, K Johan Rosengren, Julien Tailhades, Mengjie Liu, Mohammed Akhter HossainAbstract:The receptor for the neuropeptide Relaxin 3, Relaxin family peptide 3 (RXFP3) receptor, is an attractive pharmacological target for the control of eating, addictive, and psychiatric behaviors. Several structure-activity relationship studies on both human Relaxin 3 (containing 3 disulfide bonds) and its analogue A2 (two disulfide bonds) suggest that the C-terminal carboxylic acid of the tryptophan residue in the B-chain is important for RXFP3 activity. In this study, we have added amide, alcohol, carbamate, and ester functionalities to the C-terminus of A2 and compared their structures and functions. As expected, the C-terminal amide form of A2 showed lower binding affinity for RXFP3 while ester and alcohol substitutions also demonstrated lower affinity. However, while these analogues showed slightly lower binding affinity, there was no significant difference in activation of RXFP3 compared to A2 bearing a C-terminal carboxylic acid, suggesting the binding pocket is able to accommodate additional atoms.
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Exploring the Use of Helicogenic Amino Acids for Optimising Single Chain Relaxin-3 Peptide Agonists.
Biomedicines, 2020Co-Authors: Han Siean Lee, Mohammed Akhter Hossain, Richard J. Clark, Shu Hui Wang, James T. Daniel, K Johan RosengrenAbstract:Relaxin-3 is a highly conserved two-chain neuropeptide that acts through its endogenous receptor the Relaxin Family Peptide-3 (RXFP3) receptor. The ligand/receptor system is known to modulate several physiological processes, with changes in food intake and anxiety-levels the most well studied in rodent models. Agonist and antagonist analogues based on the native two-chain peptide are costly to synthesise and not ideal drug leads. Since RXFP3 interacting residues are found in the Relaxin B-chain only, this has been the focus of analogue development. The B-chain is unstructured without the A-chain support, but in single-chain variants structure can be induced by dicarba-based helical stapling strategies. Here we investigated whether alternative helical inducing strategies also can enhance structure and activity at RXFP3. Combinations of the helix inducing α-aminoisobutyric acid (Aib) were incorporated into the sequence of the Relaxin-3 B-chain. Aib residues at positions 13, 17 and 18 partially reintroduce helicity and activity of the Relaxin-3 B-chain, but other positions are generally not suited for modifications. We identify Thr21 as a putative new receptor contact residue important for RXFP3 binding. Cysteine residues were also incorporated into the sequence and cross-linked with dichloroacetone or α, α'-dibromo-m-xylene. However, in contrast to previously reported dicarba variants, neither were found to promote structure and RXFP3 activity.
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Development of Relaxin-3 Agonists and Antagonists Based on Grafted Disulfide-Stabilized Scaffolds.
Frontiers in chemistry, 2020Co-Authors: Han Siean Lee, Linda M. Haugaard-kedström, Richard J. Clark, Michael Postan, Angela Song, K Johan RosengrenAbstract:Relaxin-3 is a neuropeptide with important roles in metabolism, arousal, learning and memory. Its cognate receptor is the Relaxin family peptide-3 (RXFP3) receptor. Relaxin-3 agonist and antagonist analogs have been shown to be able to modulate food intake in rodent models. The Relaxin-3 B-chain is sufficient for receptor interactions, however, in the absence of a structural support, linear Relaxin-3 B-chain analogs are rapidly degraded and thus unsuitable as drug leads. In this study, two different disulfide-stabilized scaffolds were used for grafting of important Relaxin-3 B-chain residues to improve structure and stability. The use of both Veronica hederifolia Trypsin inhibitor (VhTI) and apamin grafting resulted in agonist and antagonist analogs with improved helicity. VhTI grafted peptides showed poor binding and low potency at RXFP3, on the other hand, apamin variants retained significant activity. These variants also showed improved half-life in serum from ~5 min to >6 h, and thus are promising RXFP3 specific pharmacological tools and drug leads for neuropharmacological diseases.
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Binding conformation and determinants of a single-chain peptide antagonist at the Relaxin-3 receptor RXFP3.
The Journal of biological chemistry, 2018Co-Authors: Linda M. Haugaard-kedström, Mohammed Akhter Hossain, Han Siean Lee, Angela Song, Maryon V. Jones, Vishaal Rathod, K Johan RosengrenAbstract:The neuropeptide Relaxin-3 and its receptor Relaxin family peptide receptor-3 (RXFP3) play key roles in modulating behavior such as memory and learning, food intake, and reward seeking. A linear Relaxin-3 antagonist (R3 B1-22R) based on a modified and truncated Relaxin-3 B-chain was recently developed. R3 B1-22R is unstructured in solution; thus, the binding conformation and determinants of receptor binding are unclear. Here, we have designed, chemically synthesized, and pharmacologically characterized more than 60 analogues of R3 B1-22R to develop an extensive understanding of its structure-activity relationships. We show that the key driver for affinity is the nonnative C-terminal Arg23 Additional contributors to binding include amino acid residues that are important also for Relaxin-3 binding, including Arg12, Ile15, and Ile19 Intriguingly, amino acid residues that are not exposed in native Relaxin-3, including Phe14 and Ala17, also interact with RXFP3. We show that R3 B1-22R has a propensity to form a helical structure, and modifications that support a helical conformation are functionally well-tolerated, whereas helix breakers such as proline residues disrupt binding. These data suggest that the peptide adopts a helical conformation, like Relaxin-3, upon binding to RXFP3, but that its smaller size allows it to penetrate deeper into the orthosteric binding site, creating more extensive contacts with the receptor.
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Development of a Single-Chain Peptide Agonist of the Relaxin-3 Receptor Using Hydrocarbon Stapling
Journal of medicinal chemistry, 2016Co-Authors: Keiko Hojo, Andrew L Gundlach, Mohammed Akhter Hossain, Hanna E. Kastman, Fazel Shabanpoor, Julien Tailhades, Lilian L. L. Wong, Emma E. K. Ong-pålsson, K Johan RosengrenAbstract:Structure–activity studies of the insulin superfamily member, Relaxin-3, have shown that its G protein-coupled receptor (RXFP3) binding site is contained within its central B-chain α-helix and this helical structure is essential for receptor activation. We sought to develop a single B-chain mimetic that retained agonist activity. This was achieved by use of solid phase peptide synthesis together with on-resin ruthenium-catalyzed ring closure metathesis of a pair of judiciously placed i,i+4 α-methyl, α-alkenyl amino acids. The resulting hydrocarbon stapled peptide was shown by solution NMR spectroscopy to mimic the native helical conformation of Relaxin-3 and to possess potent RXFP3 receptor binding and activation. Alternative stapling procedures were unsuccessful, highlighting the critical need to carefully consider both the peptide sequence and stapling methodology for optimal outcomes. Our result is the first successful minimization of an insulin-like peptide to a single-chain α-helical peptide agonist ...
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Effects of C-Terminal B-Chain Modifications in a Relaxin 3 Agonist Analogue.
ACS medicinal chemistry letters, 2020Co-Authors: Praveen Praveen, John D. Wade, K Johan Rosengren, Julien Tailhades, Mengjie Liu, Mohammed Akhter HossainAbstract:The receptor for the neuropeptide Relaxin 3, Relaxin family peptide 3 (RXFP3) receptor, is an attractive pharmacological target for the control of eating, addictive, and psychiatric behaviors. Several structure-activity relationship studies on both human Relaxin 3 (containing 3 disulfide bonds) and its analogue A2 (two disulfide bonds) suggest that the C-terminal carboxylic acid of the tryptophan residue in the B-chain is important for RXFP3 activity. In this study, we have added amide, alcohol, carbamate, and ester functionalities to the C-terminus of A2 and compared their structures and functions. As expected, the C-terminal amide form of A2 showed lower binding affinity for RXFP3 while ester and alcohol substitutions also demonstrated lower affinity. However, while these analogues showed slightly lower binding affinity, there was no significant difference in activation of RXFP3 compared to A2 bearing a C-terminal carboxylic acid, suggesting the binding pocket is able to accommodate additional atoms.
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Exploring the Use of Helicogenic Amino Acids for Optimising Single Chain Relaxin-3 Peptide Agonists.
Biomedicines, 2020Co-Authors: Han Siean Lee, Mohammed Akhter Hossain, Richard J. Clark, Shu Hui Wang, James T. Daniel, K Johan RosengrenAbstract:Relaxin-3 is a highly conserved two-chain neuropeptide that acts through its endogenous receptor the Relaxin Family Peptide-3 (RXFP3) receptor. The ligand/receptor system is known to modulate several physiological processes, with changes in food intake and anxiety-levels the most well studied in rodent models. Agonist and antagonist analogues based on the native two-chain peptide are costly to synthesise and not ideal drug leads. Since RXFP3 interacting residues are found in the Relaxin B-chain only, this has been the focus of analogue development. The B-chain is unstructured without the A-chain support, but in single-chain variants structure can be induced by dicarba-based helical stapling strategies. Here we investigated whether alternative helical inducing strategies also can enhance structure and activity at RXFP3. Combinations of the helix inducing α-aminoisobutyric acid (Aib) were incorporated into the sequence of the Relaxin-3 B-chain. Aib residues at positions 13, 17 and 18 partially reintroduce helicity and activity of the Relaxin-3 B-chain, but other positions are generally not suited for modifications. We identify Thr21 as a putative new receptor contact residue important for RXFP3 binding. Cysteine residues were also incorporated into the sequence and cross-linked with dichloroacetone or α, α'-dibromo-m-xylene. However, in contrast to previously reported dicarba variants, neither were found to promote structure and RXFP3 activity.
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binding conformation and determinants of a single chain peptide antagonist at the Relaxin 3 receptor rxfp3
Journal of Biological Chemistry, 2018Co-Authors: Mohammed Akhter Hossain, Angela Song, Maryon V. Jones, Vishaal Rathod, Linda M Haugaardkedstrom, Ross A D Bathgate, Johan K RosengrenAbstract:The neuropeptide Relaxin-3 and its receptor Relaxin family peptide receptor-3 (RXFP3) play key roles in modulating behavior such as memory and learning, food intake and reward-seeking. A linear Relaxin-3 antagonist (R3 B1-22R) based on a modified and truncated Relaxin-3 B-chain was recently developed. R3 B1-22R is unstructured in solution, thus the binding conformation and determinants of receptor binding are unclear. Here we have designed, chemically synthesized and pharmacologically characterized more than 60 analogues of R3 B1-22R to develop an extensive understanding of its structure-activity relationships. We show that the key driver for affinity is the non-native C-terminal Arg23. Additional contributors to binding include amino acid residues that are important also for Relaxin-3 binding, including Arg12, Ile15 and Ile19. Intriguingly, amino acid residues that are not exposed in native Relaxin-3, including Phe14 and Ala17, also interact with RXFP3. We show that R3 B1-22R has a propensity to form a helical structure and modifications that support a helical conformation are functionally well tolerated while helix breakers such as proline residues disrupt binding. These data suggest the peptide adopts a helical conformation, like Relaxin-3, upon binding to RXFP3 but that its smaller size allows it to penetrate deeper into the orthosteric binding site creating more extensive contacts with the receptor.
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Binding conformation and determinants of a single-chain peptide antagonist at the Relaxin-3 receptor RXFP3.
The Journal of biological chemistry, 2018Co-Authors: Linda M. Haugaard-kedström, Mohammed Akhter Hossain, Han Siean Lee, Angela Song, Maryon V. Jones, Vishaal Rathod, K Johan RosengrenAbstract:The neuropeptide Relaxin-3 and its receptor Relaxin family peptide receptor-3 (RXFP3) play key roles in modulating behavior such as memory and learning, food intake, and reward seeking. A linear Relaxin-3 antagonist (R3 B1-22R) based on a modified and truncated Relaxin-3 B-chain was recently developed. R3 B1-22R is unstructured in solution; thus, the binding conformation and determinants of receptor binding are unclear. Here, we have designed, chemically synthesized, and pharmacologically characterized more than 60 analogues of R3 B1-22R to develop an extensive understanding of its structure-activity relationships. We show that the key driver for affinity is the nonnative C-terminal Arg23 Additional contributors to binding include amino acid residues that are important also for Relaxin-3 binding, including Arg12, Ile15, and Ile19 Intriguingly, amino acid residues that are not exposed in native Relaxin-3, including Phe14 and Ala17, also interact with RXFP3. We show that R3 B1-22R has a propensity to form a helical structure, and modifications that support a helical conformation are functionally well-tolerated, whereas helix breakers such as proline residues disrupt binding. These data suggest that the peptide adopts a helical conformation, like Relaxin-3, upon binding to RXFP3, but that its smaller size allows it to penetrate deeper into the orthosteric binding site, creating more extensive contacts with the receptor.
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Development of a Single-Chain Peptide Agonist of the Relaxin-3 Receptor Using Hydrocarbon Stapling
Journal of medicinal chemistry, 2016Co-Authors: Keiko Hojo, Andrew L Gundlach, Mohammed Akhter Hossain, Hanna E. Kastman, Fazel Shabanpoor, Julien Tailhades, Lilian L. L. Wong, Emma E. K. Ong-pålsson, K Johan RosengrenAbstract:Structure–activity studies of the insulin superfamily member, Relaxin-3, have shown that its G protein-coupled receptor (RXFP3) binding site is contained within its central B-chain α-helix and this helical structure is essential for receptor activation. We sought to develop a single B-chain mimetic that retained agonist activity. This was achieved by use of solid phase peptide synthesis together with on-resin ruthenium-catalyzed ring closure metathesis of a pair of judiciously placed i,i+4 α-methyl, α-alkenyl amino acids. The resulting hydrocarbon stapled peptide was shown by solution NMR spectroscopy to mimic the native helical conformation of Relaxin-3 and to possess potent RXFP3 receptor binding and activation. Alternative stapling procedures were unsuccessful, highlighting the critical need to carefully consider both the peptide sequence and stapling methodology for optimal outcomes. Our result is the first successful minimization of an insulin-like peptide to a single-chain α-helical peptide agonist ...