Publications by authors named "Ross A D Bathgate"

177 Publications

Design, synthesis and pharmacological evaluation of tricyclic derivatives as selective RXFP4 agonists.

Bioorg Chem 2021 Mar 2;110:104782. Epub 2021 Mar 2.

School of Pharmacy, Fudan University, Shanghai 201203, China; School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China; University of Chinese Academy of Sciences, Beijing 100049, China; School of Basic Medical Sciences, Fudan University, Shanghai 200032, China; The National Center for Drug Screening and CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences (CAS), Shanghai 201203, China. Electronic address:

Relaxin family peptide receptors (RXFPs) are the potential therapeutic targets for neurological, cardiovascular, and metabolic indications. Among them, RXFP3 and RXFP4 (formerly known as GPR100 or GPCR142) are homologous class A G protein-coupled receptors with short N-terminal domain. Ligands of RXFP3 or RXFP4 are only limited to endogenous peptides and their analogues, and no natural product or synthetic agonists have been reported to date except for a scaffold of indole-containing derivatives as dual agonists of RXFP3 and RXFP4. In this study, a new scaffold of tricyclic derivatives represented by compound 7a was disclosed as a selective RXFP4 agonist after a high-throughput screening campaign against a diverse library of 52,000 synthetic and natural compounds. Two rounds of structural modification around this scaffold were performed focusing on three parts: 2-chlorophenyl group, 4-hydroxylphenyl group and its skeleton including cyclohexane-1,3-dione and 1,2,4-triazole group. Compound 14b with a new skeleton of 7,9-dihydro-4H-thiopyrano[3,4-d][1,2,4]triazolo[1,5-a]pyrimidin-8(5H)-one was thus obtained. The enantiomers of 7a and 14b were also resolved with their 9-(S)-conformer favoring RXFP4 agonism. Compared with 7a, compound 9-(S)-14b exhibited 2.3-fold higher efficacy and better selectivity for RXFP4 (selective ratio of RXFP4 vs. RXFP3 for 9-(S)-14b and 7a were 26.9 and 13.9, respectively).
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http://dx.doi.org/10.1016/j.bioorg.2021.104782DOI Listing
March 2021

Human Insulin-like Peptide 5 (INSL5). Identification of a Simplified Version of Two-Chain Analog A13.

ACS Med Chem Lett 2020 Dec 19;11(12):2455-2460. Epub 2020 Oct 19.

Florey Institute of Neuroscience and Mental Health, School of Biomedical Science, Department of Biochemistry and Molecular Biology and School of Chemistry, The University of Melbourne, Parkville, VIC 3052, Australia.

The receptor for insulin-like peptide 5 (INSL5), RXFP4, is a potential pharma target for treating human conditions such as constipation, anorexia, and obesity. However, since INSL5 has a complex structure of two chains and three disulfide bonds, its synthesis has proven to be extremely difficult via either chemical or recombinant approaches. Previous studies led to the engineering of a high yielding simplified INSL5 analog, named analog 13 (A13), which retains native INSL5-like activity. The focus of this study is to further simplify the structure of A13 by truncating the N-terminal residues of the B-chain. We have found that the first six residues at the N-terminus of A13 are not important for RXFP4 binding and cAMP potency. The most minimized active structure of INSL5 identified in this study is A13: B7-24 which will be an important research tool to study the physiological role of RXFP4 and a template for further modification to improve its pharmacokinetic properties.
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http://dx.doi.org/10.1021/acsmedchemlett.0c00435DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7734792PMC
December 2020

Effects of C-Terminal B-Chain Modifications in a Relaxin 3 Agonist Analogue.

ACS Med Chem Lett 2020 Nov 22;11(11):2336-2340. Epub 2020 Oct 22.

The Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Parkville, Victoria 3052, Australia.

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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http://dx.doi.org/10.1021/acsmedchemlett.0c00456DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7667869PMC
November 2020

Exploring the Use of Helicogenic Amino Acids for Optimising Single Chain Relaxin-3 Peptide Agonists.

Biomedicines 2020 Oct 14;8(10). Epub 2020 Oct 14.

School of Biomedical Sciences, Faculty of Medicine, The University of Queensland, Brisbane, QLD 4072, Australia.

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--xylene. However, in contrast to previously reported dicarba variants, neither were found to promote structure and RXFP3 activity.
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http://dx.doi.org/10.3390/biomedicines8100415DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7602263PMC
October 2020

A Chemogenetic Tool that Enables Functional Neural Circuit Analysis.

Cell Rep 2020 09;32(11):108139

Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville, VIC 3010, Australia; Department of Physiology, University of Melbourne, Parkville, VIC 3010, Australia. Electronic address:

Chemogenetics enables manipulation of neuronal activity in experimental animals. While providing information about the transduced neuron expressing a ligand-activated molecule, chemogenetics does not provide understanding about the antecedent circuit that drives that neuron's activity. For current approaches, this is not feasible, because the activating molecules are not genetically encoded. The insect allatostatin/allatostatin receptor system, a highly specific, powerful inhibitory chemogenetic approach, has this advantage, because the ligand, being a peptide, is genetically encoded. We developed viral vector-based systems to express biologically active allatostatin in neurons in vivo and allatostatin receptors in subpopulations of postsynaptic neurons. We demonstrate that activity-dependent release of allatostatin induces inhibition of allatostatin receptor-expressing neurons. We validate the approach in the vagal viscerosensory system where inhibitory, rather than the usual excitatory, viscerosensory input leads to sustained decreases in baroreceptor reflex sensitivity and bodyweight.
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http://dx.doi.org/10.1016/j.celrep.2020.108139DOI Listing
September 2020

Conformational Changes in Tyrosine 11 of Neurotensin Are Required to Activate the Neurotensin Receptor 1.

ACS Pharmacol Transl Sci 2020 Aug 29;3(4):690-705. Epub 2020 Apr 29.

The Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Parkville, Victoria 3010, Australia.

Cell-cell communication via endogenous peptides and their receptors is vital for controlling all aspects of human physiology and most peptides signal through G protein-coupled receptors (GPCRs). Disordered peptides bind GPCRs through complex modes for which there are few representative crystal structures. The disordered peptide neurotensin (NT) is a neuromodulator of classical neurotransmitters such as dopamine and glutamate, through activation of neurotensin receptor 1 (NTS). While several experimental structures show how NT binds NTS, details about the structural dynamics of NT during and after binding NTS, or the role of peptide dynamics on receptor activation, remain obscure. Here saturation transfer difference (STD) NMR revealed that the binding mode of NT fragment NT10-13 is heterogeneous. Epitope maps of NT10-13 at NTS suggested that tyrosine 11 (Y11) samples other conformations to those observed in crystal structures of NT-bound NTS. Molecular dynamics (MD) simulations confirmed that when NT is bound to NTS, residue Y11 can exist in two χ rotameric states, gauche plus (g) or gauche minus (g). Since only the g Y11 state is observed in all the structures solved to date, we asked if the g state is important for receptor activation. NT analogues with Y11 replaced with 7-OH-Tic were synthesized to restrain the dynamics of the side chain. P(OH-TIC)IL bound NTS with the same affinity as NT10-13 but did not activate NTS, instead acted as an antagonist. This study highlights that flexibility of Y11 in NT may be required for NT activation of NTS.
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http://dx.doi.org/10.1021/acsptsci.0c00026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7432660PMC
August 2020

Probing the correlation between ligand efficacy and conformational diversity at the α-adrenoreceptor reveals allosteric coupling of its microswitches.

J Biol Chem 2020 05 17;295(21):7404-7417. Epub 2020 Apr 17.

Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville 3052, VIC, Australia; Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville 3052, VIC, Australia. Electronic address:

G protein-coupled receptors (GPCRs) use a series of conserved microswitches to transmit signals across the cell membrane via an allosteric network encompassing the ligand-binding site and the G protein-binding site. Crystal structures of GPCRs provide snapshots of their inactive and active states, but poorly describe the conformational dynamics of the allosteric network that underlies GPCR activation. Here, we analyzed the correlation between ligand binding and receptor conformation of the α-adrenoreceptor, a GPCR that stimulates smooth muscle contraction in response to binding noradrenaline. NMR of [CH]methionine-labeled α-adrenoreceptor variants, each exhibiting differing signaling capacities, revealed how different classes of ligands modulate the conformational equilibria of this receptor. [CH]Methionine residues near the microswitches exhibited distinct states that correlated with ligand efficacies, supporting a conformational selection mechanism. We propose that allosteric coupling among the microswitches controls the conformation of the α-adrenoreceptor and underlies the mechanism of ligand modulation of GPCR signaling in cells.
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http://dx.doi.org/10.1074/jbc.RA120.012842DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7247315PMC
May 2020

The anti-fibrotic actions of relaxin are mediated through AT R-associated protein phosphatases via RXFP1-AT R functional crosstalk in human cardiac myofibroblasts.

FASEB J 2020 06 16;34(6):8217-8233. Epub 2020 Apr 16.

Cardiovascular Disease Program, Monash Biomedicine Discovery Institute, Clayton, VIC, Australia.

Fibrosis is a hallmark of several cardiovascular diseases. The relaxin family peptide receptor 1 (RXFP1) agonist, relaxin, has rapidly occurring anti-fibrotic actions which are mediated through RXFP1 and angiotensin II receptor crosstalk on renal and cardiac myofibroblasts. Here, we investigated whether this would allow relaxin to indirectly activate angiotensin II type 2 receptor (AT R)-specific signal transduction in primary human cardiac myofibroblasts (HCMFs). The anti-fibrotic effects of recombinant human relaxin (RLX; 16.8 nM) or the AT R-agonist, Compound 21 (C21; 1 μM), were evaluated in TGF-β1-stimulated HCMFs, in the absence or presence of an RXFP1 antagonist (1 μM) or AT R antagonist (0.1 μM) to confirm RXFP1-AT R crosstalk. Competition binding for RXFP1 was determined. Western blotting was performed to determine which AT R-specific protein phosphatases were expressed by HCMFs; then, the anti-fibrotic effects of RLX and/or C21 were evaluated in the absence or presence of pharmacological inhibition (NSC95397 (1 μM) for MKP-1; okadaic acid (10 nM) for PP2A) or siRNA-knockdown of these phosphatases after 72 hours. The RLX- or C21-induced increase in ERK1/2 and nNOS phosphorylation, and decrease in α-SMA (myofibroblast differentiation) and collagen-I expression by HCMFs was abrogated by pharmacological blockade of RXFP1 or the AT R, confirming RXFP1-AT R crosstalk in these cells. HCMFs were found to express AT R-dependent MKP-1 and PP2A phosphatases, while pharmacological blockade or siRNA-knockdown of either phosphatase also abolished RLX and/or C21 signal transduction in HCMFs (all P < .05 vs RLX or C21 alone). These findings demonstrated that RLX can indirectly activate AT R-dependent phosphatase activity in HCMFs by signaling through RXFP1-AT R crosstalk, which have important therapeutic implications for its anti-fibrotic actions.
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http://dx.doi.org/10.1096/fj.201902506RDOI Listing
June 2020

High-throughput screening campaign identifies a small molecule agonist of the relaxin family peptide receptor 4.

Acta Pharmacol Sin 2020 Oct 31;41(10):1328-1336. Epub 2020 Mar 31.

The National Center for Drug Screening and CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences (CAS), Shanghai, 201203, China.

Relaxin/insulin-like family peptide receptor 4 (RXFP4) is a class A G protein-coupled receptor (GPCR), and insulin-like peptide 5 (INSL5) is its endogenous ligand. Although the precise physiological role of INSL5/RXFP4 remains elusive, a number of studies have suggested it to be a potential therapeutic target for obesity and other metabolic disorders. Since selective agonists of RXFP4 are scarcely available and peptidic analogs of INSL5 are hard to make, we conducted a high-throughput screening campaign against 52,000 synthetic and natural compounds targeting RXFP4. Of the 109 initial hits discovered, only 3 compounds were confirmed in secondary screening, with JK0621-D008 displaying the best agonism at human RXFP4. Its S-configuration stereoisomer (JK1) was subsequently isolated and validated by a series of bioassays, demonstrating a consistent agonistic effect in cells overexpressing RXFP4. This scaffold may provide a valuable tool to further explore the biological functions of RXFP4.
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http://dx.doi.org/10.1038/s41401-020-0390-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7608467PMC
October 2020

Development of Relaxin-3 Agonists and Antagonists Based on Grafted Disulfide-Stabilized Scaffolds.

Front Chem 2020 18;8:87. Epub 2020 Feb 18.

Faculty of Medicine, School of Biomedical Sciences, The University of Queensland, Brisbane, QLD, Australia.

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 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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http://dx.doi.org/10.3389/fchem.2020.00087DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7039932PMC
February 2020

Colokinetic effect of an insulin-like peptide 5-related agonist of the RXFP4 receptor.

Neurogastroenterol Motil 2020 05 27;32(5):e13796. Epub 2020 Jan 27.

Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville, Vic., Australia.

Background: Insulin-like peptide 5 (INSL5) is a hormone stored in colonic enteroendocrine cells that also contain the unrelated hormones, GLP-1 and PYY. It acts at the relaxin family peptide 4, RXFP4, receptor. RXFP4 is expressed by enteric neurons in the colon, and it has been speculated that INSL5, through its action on enteric neurons, might be involved in the control of colonic contractions. Similar to insulin and relaxin, INSL5 consists of A and B peptide chains linked by three disulfide bonds, two between the chains and one intrinsic to the A chain. Because of its complex structure, it is difficult to synthesize and to prepare peptide analogues to investigate its roles. We have recently developed a potent simplified peptide analogue, INSL5-A13 (INSL5 analogue 13).

Methods: In the present work, we have investigated the actions of INSL5-A13 in mice. We investigated the ability of INSL5-A13 to increase the speed of emptying of a bead from the colon, after expulsion had been slowed by the peripherally restricted opioid agonist, loperamide (1 mg/kg).

Key Results: INSL5-A13 was a full agonist at the mouse RXFP4 expressed in HEK cells, with an EC50 of ~9 nmol/L. INSL5-A13 caused an acceleration of colorectal bead propulsion in mice constipated by loperamide in the dose range 0.2 to 60 µg/kg, with an EC50 of ~6 µg/kg in vivo. It also accelerated bead propulsion in untreated mice. Bead expulsion was not accelerated in RXFP4-/- mice.

Conclusion And Inferences: Our data suggest that RXFP4 agonists could be useful in the treatment of constipation.
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http://dx.doi.org/10.1111/nmo.13796DOI Listing
May 2020

Targeted viral vector transduction of relaxin-3 neurons in the rat using a novel cell-type specific promoter.

IBRO Rep 2020 Jun 13;8:1-10. Epub 2019 Dec 13.

The Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Victoria, Australia.

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 (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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http://dx.doi.org/10.1016/j.ibror.2019.11.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6928288PMC
June 2020

Engineering of chimeric peptides as antagonists for the G protein-coupled receptor, RXFP4.

Sci Rep 2019 11 28;9(1):17828. Epub 2019 Nov 28.

Florey Institute for Neuroscience & Mental Health, University of Melbourne, Melbourne, VIC, Australia.

Insulin-like peptide 5 (INSL5) is a very important pharma target for treating human conditions such as anorexia and diabetes. However, INSL5 with two chains and three disulfide bridges is an extremely difficult peptide to assemble by chemical or recombinant means. In a recent study, we were able to engineer a simplified INSL5 analogue 13 which is a relaxin family peptide receptor 4 (RXFP4)-specific agonist. To date, however, no RXFP4-specific antagonist (peptide or small molecule) has been reported in the literature. The focus of this study was to utilize the non-specific RXFP3/RXFP4 antagonist ΔR3/I5 as a template to rationally design an RXFP4 specific antagonist. Unexpectedly, we demonstrated that ΔR3/I5 exhibited partial agonism at RXFP4 when expressed in CHO cells which is associated with only partial antagonism of INSL5 analogue activation. In an attempt to improve RXFP4 specificity and antagonist activity we designed and chemically synthesized a series of analogues of ΔR3/I5. While all the chimeric analogues still demonstrated partial agonism at RXFP4, one peptide (Analogue 17) exhibited significantly improved RXFP4 specificity. Importantly, analogue 17 has a simplified structure which is more amenable to chemical synthesis. Therefore, analogue 17 is an ideal template for further development into a specific high affinity RXFP4 antagonist which will be an important tool to probe the physiological role of RXFP4/INSL5 axis.
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http://dx.doi.org/10.1038/s41598-019-53707-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6882824PMC
November 2019

Coatings Releasing the Relaxin Peptide Analogue B7-33 Reduce Fibrotic Encapsulation.

ACS Appl Mater Interfaces 2019 Dec 2;11(49):45511-45519. Epub 2019 Dec 2.

CSIRO Manufacturing , Research Way , Clayton , VIC 3168 , Australia.

The development of antifibrotic materials and coatings that can resist the foreign body response (FBR) continues to present a major hurdle in the advancement of current and next-generation implantable medical devices, biosensors, and cell therapies. From an implant perspective, the most important issue associated with the FBR is the prolonged inflammatory response leading to a collagenous capsule that ultimately blocks mass transport and communication between the implant and the surrounding tissue. Up to now, most attempts to reduce the capsule thickness have focused on providing surface coatings that reduce protein fouling and cell attachment. Here, we present an approach that is based on the sustained release of a peptide drug interfering with the FBR. In this study, the biodegradable polymer poly(lactic--glycolic) acid (PLGA) was used as a coating releasing the relaxin peptide analogue B7-33, which has been demonstrated to reduce organ fibrosis in animal models. While in vitro protein quantification was used to demonstrate controlled release of the antifibrotic peptide B7-33 from PLGA coatings, an in vitro reporter cell assay was used to demonstrate that B7-33 retains activity against the relaxin family peptide receptor 1 (RXFP1). Subcutaneous implantation of PLGA-coated polypropylene samples in mice with and without the peptide demonstrated a marked reduction in capsule thickness (49.2%) over a 6 week period. It is expected that this novel approach will open the door to a range of new and improved implantable medical devices.
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http://dx.doi.org/10.1021/acsami.9b17859DOI Listing
December 2019

AT1R-AT2R-RXFP1 Functional Crosstalk in Myofibroblasts: Impact on the Therapeutic Targeting of Renal and Cardiac Fibrosis.

J Am Soc Nephrol 2019 11 11;30(11):2191-2207. Epub 2019 Sep 11.

Department of Biochemistry and Molecular Biology, and

Background: Recombinant human relaxin-2 (serelaxin), which has organ-protective actions mediated its cognate G protein-coupled receptor relaxin family peptide receptor 1 (RXFP1), has emerged as a potential agent to treat fibrosis. Studies have shown that serelaxin requires the angiotensin II (AngII) type 2 receptor (ATR) to ameliorate renal fibrogenesis and . Whether its antifibrotic actions are affected by modulation of the AngII type 1 receptor (ATR), which is expressed on myofibroblasts along with RXFP1 and ATR, is unknown.

Methods: We examined the signal transduction mechanisms of serelaxin when applied to primary rat renal and human cardiac myofibroblasts , and in three models of renal- or cardiomyopathy-induced fibrosis .

Results: The ATR blockers irbesartan and candesartan abrogated antifibrotic signal transduction of serelaxin RXFP1 and . Candesartan also ameliorated serelaxin's antifibrotic actions in the left ventricle of mice with cardiomyopathy, indicating that candesartan's inhibitory effects were not confined to the kidney. We also demonstrated in a transfected cell system that serelaxin did not directly bind to ATRs but that constitutive ATR-RXFP1 interactions could form. To potentially explain these findings, we also demonstrated that renal and cardiac myofibroblasts expressed all three receptors and that antagonists acting at each receptor directly or allosterically blocked the antifibrotic effects of either serelaxin or an ATR agonist (compound 21).

Conclusions: These findings have significant implications for the concomitant use of RXFP1 or ATR agonists with ATR blockers, and suggest that functional interactions between the three receptors on myofibroblasts may represent new targets for controlling fibrosis progression.
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http://dx.doi.org/10.1681/ASN.2019060597DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6830801PMC
November 2019

Using the novel HiBiT tag to label cell surface relaxin receptors for BRET proximity analysis.

Pharmacol Res Perspect 2019 08 30;7(4):e00513. Epub 2019 Jul 30.

Florey Institute of Neuroscience and Mental Health and Florey Department of Neuroscience and Mental Health Parkville Victoria Australia.

Relaxin family peptide 1 (RXFP1) is the receptor for relaxin a peptide hormone with important therapeutic potential. Like many G protein-coupled receptors (GPCRs), RXFP1 has been reported to form homodimers. Given the complex activation mechanism of RXFP1 by relaxin, we wondered whether homodimerization may be explicitly required for receptor activation, and therefore sought to determine if there is any relaxin-dependent change in RXFP1 proximity at the cell surface. Bioluminescence resonance energy transfer (BRET) between recombinantly tagged receptors is often used in GPCR proximity studies. RXFP1 targets poorly to the cell surface when overexpressed in cell lines, with the majority of the receptor proteins sequestered within the cell. Thus, any relaxin-induced changes in RXFP1 proximity at the cell surface may be obscured by BRET signal originating from intracellular compartments. We therefore, utilized the newly developed split luciferase system called HiBiT to specifically label the extracellular terminus of cell surface RXFP1 receptors in combination with mCitrine-tagged receptors, using the GABA heterodimer as a positive control. This demonstrated that the BRET signal detected from RXFP1-RXFP1 proximity at the cell surface does not appear to be due to stable physical interactions. The fact that there is also no relaxin-mediated change in RXFP1-RXFP1 proximity at the cell surface further supports these conclusions. This work provides a basis by which cell surface GPCR proximity and expression levels can be specifically studied using a facile and homogeneous labeling technique such as HiBiT.
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http://dx.doi.org/10.1002/prp2.513DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6667744PMC
August 2019

An apically located hybrid guanylate cyclase-ATPase is critical for the initiation of Ca signaling and motility in .

J Biol Chem 2019 05 16;294(22):8959-8972. Epub 2019 Apr 16.

From the The Walter and Eliza Hall Institute of Medical Research, Parkville, Melbourne, Victoria 3052, Australia,

Protozoan parasites of the phylum Apicomplexa actively move through tissue to initiate and perpetuate infection. The regulation of parasite motility relies on cyclic nucleotide-dependent kinases, but how these kinases are activated remains unknown. Here, using an array of biochemical and cell biology approaches, we show that the apicomplexan parasite expresses a large guanylate cyclase (TgGC) protein, which contains several upstream ATPase transporter-like domains. We show that TgGC has a dynamic localization, being concentrated at the apical tip in extracellular parasites, which then relocates to a more cytosolic distribution during intracellular replication. Conditional TgGC knockdown revealed that this protein is essential for acute-stage tachyzoite growth, as TgGC-deficient parasites were defective in motility, host cell attachment, invasion, and subsequent host cell egress. We show that TgGC is critical for a rapid rise in cytosolic [Ca] and for secretion of microneme organelles upon stimulation with a cGMP agonist, but these deficiencies can be bypassed by direct activation of signaling by a Ca ionophore. Furthermore, we found that TgGC is required for transducing changes in extracellular pH and [K] to activate cytosolic [Ca] flux. Together, the results of our work implicate TgGC as a putative signal transducer that activates Ca signaling and motility in .
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http://dx.doi.org/10.1074/jbc.RA118.005491DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6552420PMC
May 2019

Expression and Purification of a Functional E. coli CH-Methionine-Labeled Thermostable Neurotensin Receptor 1 Variant for Solution NMR Studies.

Methods Mol Biol 2019 ;1947:31-55

Department of Biochemistry and Molecular Biology, The University of Melbourne, Parkville, VIC, Australia.

Escherichia coli (E. coli) is the most widely used expression host for recombinant proteins due to high expression yields and straightforward molecular cloning. Directed evolution of G protein-coupled receptors (GPCRs) has made several of these difficult to express membrane proteins amenable to prokaryotic expression. Here, we describe a protocol for near complete CH-methionine labeling of a thermostable neurotensin receptor 1 (enNTS) variant in E. coli for solution NMR-based dynamics studies. Our expression strategy utilizes methionine biosynthesis pathway inhibition forcing E. coli to incorporate exogenous methionine with 96% efficiency at expression levels of 2.6 mg enNTS per liter of expression culture containing 50 mg of CH-methionine. We also provide a 3-step purification protocol that produces final yields of 0.6 mg of functional Apo-state enNTS.
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http://dx.doi.org/10.1007/978-1-4939-9121-1_3DOI Listing
August 2019

Chronic activation of the relaxin-3 receptor on GABA neurons in rat ventral hippocampus promotes anxiety and social avoidance.

Hippocampus 2019 10 19;29(10):905-920. Epub 2019 Mar 19.

Discovery Science, The Florey Institute of Neuroscience and Mental Health, Parkville, Victoria, Australia.

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 G -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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http://dx.doi.org/10.1002/hipo.23089DOI Listing
October 2019

Diazepam is not a direct allosteric modulator of α-adrenoceptors, but modulates receptor signaling by inhibiting phosphodiesterase-4.

Pharmacol Res Perspect 2019 02 26;7(1):e00455. Epub 2018 Dec 26.

The Florey Institute of Neuroscience and Mental Health University of Melbourne Parkville Vic Australia.

α- and α-adrenoceptors (ARs) are G protein-coupled receptors (GPCRs) that are activated by adrenaline and noradrenaline to modulate smooth muscle contraction in the periphery, and neuronal outputs in the central nervous system (CNS). α- and α-AR are clinically targeted with antagonists for hypertension and benign prostatic hyperplasia and are emerging CNS targets for treating neurodegenerative diseases. The benzodiazepines midazolam, diazepam, and lorazepam are proposed to be positive allosteric modulators (PAMs) of α-ARs. Here, using thermostabilized, purified, α- and α-ARs, we sought to identify the benzodiazepine binding site and modulatory mechanism to inform the design of selective PAMs. However, using a combination of biophysical approaches no evidence was found for direct binding of several benzodiazepines to purified, stabilized α- and α-ARs. Similarly, in cell-based assays expressing unmodified α- and α-ARs, benzodiazepine treatment had no effect on fluorescent ligand binding, agonist-stimulated Ca release, or G protein activation. In contrast, several benzodiazepines positively modulated phenylephrine stimulation of a cAMP response element pathway by α- and α-ARs; however, this was shown to be caused by off-target inhibition of phosphodiesterases, known targets of diazepam. This study highlights how purified, stabilized GPCRs are useful for validating allosteric ligand binding and that care needs to be taken before assigning new targets to benzodiazepines.
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http://dx.doi.org/10.1002/prp2.455DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6306559PMC
February 2019

Multi-Component Mechanism of H2 Relaxin Binding to RXFP1 through NanoBRET Kinetic Analysis.

iScience 2019 Jan 10;11:93-113. Epub 2018 Dec 10.

Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Parkville, VIC 3052, Australia; Department of Biochemistry and Molecular Biology, The University of Melbourne, Parkville, VIC, Australia. Electronic address:

The peptide hormone H2 relaxin has demonstrated promise as a therapeutic, but mimetic development has been hindered by the poorly understood relaxin receptor RXFP1 activation mechanism. H2 relaxin is hypothesized to bind to two distinct ECD sites, which reorientates the N-terminal LDLa module to activate the transmembrane domain. Here we provide evidence for this model in live cells by measuring bioluminescence resonance energy transfer (BRET) between nanoluciferase-tagged RXFP1 constructs and fluorescently labeled H2 relaxin (NanoBRET). Additionally, we validate these results using the related RXFP2 receptor and chimeras with an inserted RXFP1-binding domain utilizing NanoBRET and nuclear magnetic resonance studies on recombinant proteins. We therefore provide evidence for the multi-component molecular mechanism of H2 relaxin binding to RXFP1 on the full-length receptor in cells. Also, we show the utility of NanoBRET real-time binding kinetics to reveal subtle binding complexities, which may be overlooked in traditional equilibrium binding assays.
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http://dx.doi.org/10.1016/j.isci.2018.12.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6309025PMC
January 2019

Real-time examination of cAMP activity at relaxin family peptide receptors using a BRET-based biosensor.

Pharmacol Res Perspect 2018 10 24;6(5):e00432. Epub 2018 Sep 24.

Florey Institute of Neuroscience and Mental Health The University of Melbourne Parkville Victoria Australia.

Relaxin family peptide (RXFPs) 1-4 receptors modulate the activity of cyclic adenosine monophosphate (cAMP) to produce a range of physiological functions. RXFP1 and RXFP2 increase cAMP via Gα, whereas RXFP3 and RXFP4 inhibit cAMP via Gα. RXFP1 also shows a delayed increase in cAMP downstream of Gα. In this study we have assessed whether the bioluminescence resonance energy transfer (BRET)-based biosensor CAMYEL (cAMP sensor using YFP-Epac-Rluc), which allows real-time measurement of cAMP activity in live cells, will aid in understanding ligand- and cell-specific RXFP signaling. CAMYEL detected concentration-dependent changes in cAMP activity at RXFP1-4 in recombinant cell lines, using a variety of ligands with potencies comparable to those seen in conventional cAMP assays. We used RXFP2 and RXFP3 antagonists to demonstrate that CAMYEL detects dynamic changes in cAMP by reversing cAMP activation or inhibition respectively, with real-time addition of antagonist after agonist stimulation. To demonstrate the utility of CAMYEL to detect cAMP activation in native cells expressing low levels of RXFP receptor, we cloned CAMYEL into a lentiviral vector and transduced THP-1 cells, which express low levels of RXFP1. THP-1 CAMYEL cells demonstrated robust cAMP activation in response to relaxin. However, the CAMYEL assay was unable to detect the Gα-mediated phase of RXFP1 cAMP activation in PTX-treated THP-1 cells or HEK293A cells with knockout of Gα. Our data demonstrate that cytoplasmically-expressed CAMYEL efficiently detects real-time cAMP activation by Gα or inhibition by Gα but may not detect cAMP generated in specific intracellular compartments such as that generated by Gα upon RXFP1 activation.
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http://dx.doi.org/10.1002/prp2.432DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6153321PMC
October 2018

Binding conformation and determinants of a single-chain peptide antagonist at the relaxin-3 receptor RXFP3.

J Biol Chem 2018 10 21;293(41):15765-15776. Epub 2018 Aug 21.

From the Faculty of Medicine, School of Biomedical Sciences, University of Queensland, Brisbane, Queensland 4072, Australia,

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 Arg Additional contributors to binding include amino acid residues that are important also for relaxin-3 binding, including Arg, Ile, and Ile Intriguingly, amino acid residues that are not exposed in native relaxin-3, including Phe and Ala, 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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http://dx.doi.org/10.1074/jbc.RA118.002611DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6187623PMC
October 2018

Distinct but overlapping binding sites of agonist and antagonist at the relaxin family peptide 3 (RXFP3) receptor.

J Biol Chem 2018 10 21;293(41):15777-15789. Epub 2018 Aug 21.

From the Florey Institute of Neuroscience and Mental Health,

The relaxin-3 neuropeptide activates the relaxin family peptide 3 (RXFP3) receptor to modulate stress, appetite, and cognition. RXFP3 shows promise as a target for treating neurological disorders, but realization of its clinical potential requires development of smaller RXFP3-specific drugs that can penetrate the blood-brain barrier. Designing such drugs is challenging and requires structural knowledge of agonist- and antagonist-binding modes. Here, we used structure-activity data for relaxin-3 and a peptide RXFP3 antagonist termed R3 B1-22R to guide receptor mutagenesis and develop models of their binding modes. RXFP3 residues were alanine-substituted individually and in combination and tested in cell-based binding and functional assays to refine models of agonist and antagonist binding to active- and inactive-state homology models of RXFP3, respectively. These models suggested that both agonists and antagonists interact with RXFP3 via similar residues in their B-chain central helix. The models further suggested that the B-chain Trp inserts into the binding pocket of RXFP3 and interacts with Trp and Lys, the latter through a salt bridge with the C-terminal carboxyl group of Trp in relaxin-3. R3 B1-22R, which does not contain Trp, used a non-native Arg residue to form cation-π and salt-bridge interactions with Trp and Glu in RXFP3, explaining a key contribution of Arg to affinity. Overall, relaxin-3 and R3 B1-22R appear to share similar binding residues but may differ in binding modes, leading to active and inactive RXFP3 conformational states, respectively. These mechanistic insights may assist structure-based drug design of smaller relaxin-3 mimetics to manage neurological disorders.
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http://dx.doi.org/10.1074/jbc.RA118.002645DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6187618PMC
October 2018

Gram scale preparation of clozapine -oxide (CNO), a synthetic small molecule actuator for muscarinic acetylcholine DREADDs.

MethodsX 2018 23;5:257-267. Epub 2018 Mar 23.

School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Victoria 3010, Australia.

Chemogenetics uses engineered proteins that are controlled by small molecule actuators, allowing functional studies of proteins with temporal and dose control, and include Designer Receptors Exclusively Activated by Designer Drugs (DREADDs). One major class of DREADDs are mutated muscarinic receptors that are unresponsive to acetylcholine, and are activated by administration of clozapine -oxide (CNO). However, CNO is available in only small amounts and large scale studies involving animals and multiple cohorts are prohibitively expensive for many investigators. The precursor, clozapine, is also expensive when purchased from specialist suppliers. Here we report: •A simple extraction method of clozapine from commercial tablets;•A simple preparation of CNO from clozapine, and for the first time its single-crystal X-ray structure; and•That the CNO prepared by this method specifically activates the DREADD receptor hM3Dq . This method provides large quantities of CNO suitable for large-scale DREADD applications that is identical to commercial material.
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http://dx.doi.org/10.1016/j.mex.2018.03.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6053635PMC
March 2018

Optimization and CH methionine labeling of a signaling competent neurotensin receptor 1 variant for NMR studies.

Biochim Biophys Acta Biomembr 2018 Jun 26;1860(6):1372-1383. Epub 2018 Mar 26.

The Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Parkville, Victoria 3010, Australia; Department of Biochemistry and Molecular Biology, The University of Melbourne, Parkville, Victoria 3010, Australia. Electronic address:

Neurotensin is a 13-residue peptide that acts as a neuromodulator of classical neurotransmitters such as dopamine and glutamate in the mammalian central nervous system, mainly by activating the G protein-coupled receptor (GPCR), neurotensin receptor 1 (NTS). Agonist binding to GPCRs shifts the conformational equilibrium of the transmembrane helices towards distinct, thermodynamically favorable conformations that favor effector protein interactions and promotes cell signaling. The introduction of site specific labels for NMR spectroscopy has proven useful for investigating this dynamic process, but the low expression levels and poor stability of GPCRs is a hindrance to solution NMR experiments. Several thermostabilized mutants of NTS have been engineered to circumvent this, with the crystal structures of four of these published. The conformational dynamics of NTS however, has not been thoroughly investigated with NMR. It is generally accepted that stabilized GPCRs exhibit attenuated signaling, thus we thoroughly characterized the signaling characteristics of several thermostabilized NTS variants to identify an optimal variant for protein NMR studies. A variant termed enNTS exhibited the best combination of signaling capability and stability upon solubilization with detergents. enNTS was subsequently labeled with CH-methionine in E. coli and purified to homogeneity in the absence of bound ligands. Using solution NMR spectroscopy we observed several well dispersed CH-methionine resonances, many of which exhibited chemical shift changes upon the addition of the high affinity agonist peptide, NT8-13. Thus, enNTS represents a novel tool for investigating ligand induced conformational changes in NTS to gain insights into the molecular mechanisms underlying neurotensin signaling.
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http://dx.doi.org/10.1016/j.bbamem.2018.03.020DOI Listing
June 2018

Determinants of Ligand Subtype-Selectivity at α-Adrenoceptor Revealed Using Saturation Transfer Difference (STD) NMR.

ACS Chem Biol 2018 04 22;13(4):1090-1102. Epub 2018 Mar 22.

The Florey Institute of Neuroscience and Mental Health , The University of Melbourne , 30 Royal Parade , Parkville , Victoria 3052 , Australia.

α- and α-adrenoceptors (α-AR and α-AR) are closely related G protein-coupled receptors (GPCRs) that modulate the cardiovascular and nervous systems in response to binding epinephrine and norepinephrine. The GPCR gene superfamily is made up of numerous subfamilies that, like α-AR and α-AR, are activated by the same endogenous agonists but may modulate different physiological processes. A major challenge in GPCR research and drug discovery is determining how compounds interact with receptors at the molecular level, especially to assist in the optimization of drug leads. Nuclear magnetic resonance spectroscopy (NMR) can provide great insight into ligand-binding epitopes, modes, and kinetics. Ideally, ligand-based NMR methods require purified, well-behaved protein samples. The instability of GPCRs upon purification in detergents, however, makes the application of NMR to study ligand binding challenging. Here, stabilized α-AR and α-AR variants were engineered using Cellular High-throughput Encapsulation, Solubilization, and Screening (CHESS), allowing the analysis of ligand binding with Saturation Transfer Difference NMR (STD NMR). STD NMR was used to map the binding epitopes of epinephrine and A-61603 to both receptors, revealing the molecular determinants for the selectivity of A-61603 for α-AR over α-AR. The use of stabilized GPCRs for ligand-observed NMR experiments will lead to a deeper understanding of binding processes and assist structure-based drug design.
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http://dx.doi.org/10.1021/acschembio.8b00191DOI Listing
April 2018

INSL5 activates multiple signalling pathways and regulates GLP-1 secretion in NCI-H716 cells.

J Mol Endocrinol 2018 04;60(3):213-224

Drug Discovery BiologyMonash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia.

Insulin-like peptide 5 (INSL5) is a newly discovered gut hormone expressed in colonic enteroendocrine L-cells but little is known about its biological function. Here, we show using RT-qPCR and hybridisation that mRNA is highly expressed in the mouse colonic mucosa, colocalised with proglucagon immunoreactivity. In comparison, mRNA for RXFP4 (the cognate receptor for INSL5) is expressed in various mouse tissues, including the intestinal tract. We show that the human enteroendocrine L-cell model NCI-H716 cell line, and goblet-like colorectal cell lines SW1463 and LS513 endogenously express Stimulation of NCI-H716 cells with INSL5 produced phosphorylation of ERK1/2 (Thr/Tyr), AKT (Thr and Ser) and S6RP (Ser) and inhibited cAMP production but did not stimulate Ca release. Acute INSL5 treatment had no effect on GLP-1 secretion mediated by carbachol or insulin, but modestly inhibited forskolin-stimulated GLP-1 secretion in NCI-H716 cells. However, chronic INSL5 pre-treatment (18 h) increased basal GLP-1 secretion and prevented the inhibitory effect of acute INSL5 administration. LS513 cells were found to be unresponsive to INSL5 despite expressing Another enteroendocrine L-cell model, mouse GLUTag cells did not express detectable levels of and were unresponsive to INSL5. This study provides novel insights into possible autocrine/paracrine roles of INSL5 in the intestinal tract.
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http://dx.doi.org/10.1530/JME-17-0152DOI Listing
April 2018

The relaxin receptor as a therapeutic target - perspectives from evolution and drug targeting.

Pharmacol Ther 2018 07 17;187:114-132. Epub 2018 Feb 17.

Department of Biochemistry & Molecular Biology, The University of Melbourne, Victoria, Australia; Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Victoria, Australia. Electronic address:

The peptide relaxin was first identified as an important circulating hormone during pregnancy over 90 years ago. Research over many years defined the numerous biological roles that relaxin plays throughout pregnancy in many mammalian species. These important biological actions have led to the testing of relaxin as a therapeutic agent for a number of indications. The discovery of the relaxin receptor, RXFP1, in 2002 facilitated the better understanding of the cellular targets of relaxin, its mechanism of action and enabled the development of relaxin mimetics and screening for small molecule agonists. Additionally, the rapid expansion of the genome databases and bioinformatics tools has significantly advanced our understanding of the evolution of the relaxin/RXFP1 signaling system. It is now clear that the relaxin-RXFP1 signaling axis is far more ancient than previously appreciated with important roles for invertebrate relaxin-like peptides in reproductive and non-reproductive functions. This review summarizes these advances as well as developments in drug targeting of RXFP1. Hence the complex mode of activation of RXFP1 is discussed as is the discovery and development of a peptide mimetic and small molecule agonist. Detailed signaling studies are summarized which highlight the cell specific signaling of a peptide mimetic and biased signaling of a small molecule agonist. These studies highlight the complexities of targeting peptide GPCRs such as RXFP1.
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http://dx.doi.org/10.1016/j.pharmthera.2018.02.008DOI Listing
July 2018

A Novel Ultra-Stable, Monomeric Green Fluorescent Protein For Direct Volumetric Imaging of Whole Organs Using CLARITY.

Sci Rep 2018 01 12;8(1):667. Epub 2018 Jan 12.

Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria, 3010, Australia.

Recent advances in thick tissue clearing are enabling high resolution, volumetric fluorescence imaging of complex cellular networks. Fluorescent proteins (FPs) such as GFP, however, can be inactivated by the denaturing chemicals used to remove lipids in some tissue clearing methods. Here, we solved the crystal structure of a recently engineered ultra-stable GFP (usGFP) and propose that the two stabilising mutations, Q69L and N164Y, act to improve hydrophobic packing in the core of the protein and facilitate hydrogen bonding networks at the surface, respectively. usGFP was found to dimerise strongly, which is not desirable for some applications. A point mutation at the dimer interface, F223D, generated monomeric usGFP (muGFP). Neurons in whole mouse brains were virally transduced with either EGFP or muGFP and subjected to Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining/In situ hybridization-compatible Tissue-hYdrogel (CLARITY) clearing. muGFP fluorescence was retained after CLARITY whereas EGFP fluorescence was highly attenuated, thus demonstrating muGFP is a novel FP suitable for applications where high fluorescence stability and minimal self-association are required.
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http://dx.doi.org/10.1038/s41598-017-18045-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5766548PMC
January 2018