KIRA8 attenuates non-alcoholic steatohepatitis through inhibition of the IRE1α/XBP1 signalling pathway.

Endoplasmic reticulum (ER) stress is enhanced in non-alcoholic steatohepatitis (NASH). Among three signalling pathways, the IRE1α/XBP1 signalling pathway is strongly implicated in the pathogenesis of NASH but its significance is still largely uncharacterised. In this report, we constructed a hepatocyte-specific XBP1-Luciferase knock-in mouse model that allows in vivo monitoring of the IRE1α/XBP1 activity in hepatocytes. Using this mouse model, we found that IRE1α/XBP1 was activated within hepatocytes during the pathogenesis of NASH. Significantly, a specific IRE1α kinase-inhibiting RNase attenuator, KIRA8, attenuated NASH in mice. In conclusion, our hepatocyte-specific XBP1 splicing reporter mouse represents a valid model for research and drug development of NASH, which showed that the IRE1α-induced XBP splicing is potentiated in hepatocytes during pathogenesis of NASH. Furthermore, we carried out the proof-of-concept study to demonstrate that the allosteric IRE1α RNase inhibitor serves as a promising therapeutic agent for the treatment of NASH.

The natural compound rutaecarpine promotes white adipocyte browning through activation of the AMPK-PRDM16 axis.

The prevalence of obesity is increasing globally and is associated with many metabolic disorders, such as type 2 diabetes and cardiovascular diseases. In recent years, a number of studies suggest that promotion of white adipose browning represents a promising strategy to combat obesity and its related metabolic disorders. The aim of this study was to identify compounds that induce adipocyte browning and elucidate their mechanism of action. Among the 500 natural compounds screened, a small molecule named Rutaecarpine, was identified as a positive regulator of adipocyte browning both in vitro and in vivo. KEGG pathway analysis from RNA-seq data suggested that the AMPK signaling pathway was regulated by Rutaecarpine, which was validated by Western blot analysis. Furthermore, inhibition of AMPK signaling mitigated the browning effect of Rutaecaripine. The effect of Rutaecaripine on adipocyte browning was also abolished upon deletion of Prdm16, a downstream target of AMPK pathway. In collusion, Rutaecarpine is a potent chemical agent to induce adipocyte browning and may serve as a potential drug candidate to treat obesity.

Regulation of human 4-hydroxy-2-oxoglutarate aldolase by pyruvate and α-ketoglutarate: implications for primary hyperoxaluria type-3.

4-hydroxy-2-oxoglutarate aldolase (HOGA1) is a mitochondrial enzyme that plays a gatekeeper role in hydroxyproline metabolism. Its loss of function in humans causes primary hyperoxaluria type 3 (PH3), a rare condition characterised by excessive production of oxalate. In this study, we investigated the significance of the associated oxaloacetate decarboxylase activity which is also catalysed by HOGA1. Kinetic studies using the recombinant human enzyme (hHOGA1) and active site mutants showed both these dual activities utilise the same catalytic machinery with micromolar substrate affinities suggesting that both are operative in vivo. Biophysical and structural studies showed that pyruvate was a competitive inhibitor with an inhibition constant in the micromolar range. By comparison α-ketoglutarate was a weak inhibitor with an inhibition constant in the millimolar range and could only be isolated as an adduct with the active site Lys196 in the presence of sodium borohydride. These studies suggest that pyruvate inhibits HOGA1 activity during gluconeogenesis. We also propose that loss of HOGA1 function could increase oxalate production in PH3 by decreasing pyruvate availability and metabolic flux through the Krebs cycle.

Synthesis of isotopically labelled αCGRP8-37 and its lipidated analogue.

α-Calcitonin gene related peptide (αCGRP) inhibitors are important medicinal targets due to their ability to produce antimigraine effects, thus, the discovery of long-acting αCGRP inhibitors is of significant interest. Herein we report the synthesis of an isotopically labelled version of the well-known CGRP receptor antagonist, αCGRP8-37 , as well as lipidated αCGRP8-37 with comparable antagonistic activity. These isotopically labelled peptides can be employed in assays to determine the metabolic stability of the lipidated αCGRP8-37 and compare this with the stability of known αCGRP8-37 .

Dihydrodipicolinate Synthase: Structure, Dynamics, Function, and Evolution.

Enzymes are usually comprised of multiple subunits and more often than not they are made up of identical subunits. In this review we examine lysine biosynthesis and focus on the enzyme dihydrodipicolinate synthase in terms of its structure, function and the evolution of its varied number of subunits (quaternary structure). Dihydrodipicolinate synthase is the first committed step in the biosynthesis of lysine, which occurs naturally in plants, bacteria, archaea and fungi, but is not synthesized in mammals. In bacteria, there have been four separate pathways identified from tetrahydrodipicolinate to meso-diaminopimelate, which is the immediate precursor to lysine. Dihydrodipicolinate synthases from many bacterial and plant species have been structurally characterised and the results show considerable variability with respect to their quaternary structure, hinting at their evolution. The oligomeric state of the enzyme plays a key role, both in catalysis and in the allosteric regulation of the enzyme by lysine. While most bacteria and plants have tetrameric enzymes, where the structure of the dimeric building blocks is conserved, the arrangement of the dimers differs. We also review a key development in the field, namely the discovery of a human dihydrodipicolinate synthase-like enzyme, now known as 4-hydroxy-2-oxoglutarate aldolase . This discovery complicates the rationale underpinning drug development against bacterial dihydrodipicolinate synthases, since genetic errors in 4-hydroxy-2-oxoglutarate aldolase cause the disease Primary Hyperoxaluria Type 3 and therefore compounds that are geared towards the inhibition of bacterial dihydrodipicolinate synthase may be toxic to mammalian cells.

Solid-Phase Thiol-Ene Lipidation of Peptides for the Synthesis of a Potent CGRP Receptor Antagonist.

We report a new method herein coined SP-CLipPA (solid-phase cysteine lipidation of a peptide or amino acid) for the synthesis of mono-S-lipidated peptides. This technique utilizes thiol-ene chemistry for conjugation of a vinyl ester to a free thiol of a semiprotected, resin-bound peptide. Advantages of SP-CLipPA include: ease of handling, conversions of up to 91 %, by-product removal by simple filtration, and a single purification step. Additionally, the desired lipidated products show high chromatographic separation from impurities, thus facilitating RP-HPLC purification. To showcase the utility of SP-CLipPA, we synthesized a potent calcitonin gene-related peptide (CGRP) receptor antagonist peptide in excellent yield and purity. This peptide, selected from a series of lipidated analogues of CGRP8-37 and CGRP7-37 , has potential for the treatment of migraine.

Rutin suppresses human-amylin/hIAPP misfolding and oligomer formation in-vitro, and ameliorates diabetes and its impacts in human-amylin/hIAPP transgenic mice.

Pancreatic islet ß-cells secrete the hormones insulin and amylin, and defective ß-cell function plays a central role in the pathogenesis of type-2 diabetes (T2D). Human amylin (hA, also termed hIAPP) misfolds and forms amyloid aggregates whereas orthologous mouse amylin does neither. Furthermore, hA elicits apoptosis in cultured ß-cells and ß-cell death in ex-vivo islets. In addition, hA-transgenic mice that selectively express hA in their ß-cells, manifest ß-cell apoptosis and progressive islet damage that leads to diabetes closely resembling that in patients with T2D. Aggregation of hA is thus linked to the causation of diabetes. We employed time-dependent thioflavin-T spectroscopy and ion-mobility mass spectrometry to screen potential suppressors of hA misfolding for anti-diabetic activity. We identified the dietary flavonol rutin as an inhibitor of hA-misfolding and measured its anti-diabetic efficacy in hA-transgenic mice. In vitro, rutin bound hA, suppressed misfolding, disaggregated oligomers and reverted hA-conformation towards the physiological. In hA-transgenic mice, measurements of glucose, fluid-intake, and body-weight showed that rutin-treatment slowed diabetes-progression by lowering of rates of elevation in blood glucose (P = 0.030), retarding deterioration from symptomatic diabetes to death (P = 0.014) and stabilizing body-weight (P < 0.0001). In conclusion, rutin treatment suppressed hA-aggregation in vitro and doubled the lifespan of diabetic mice (P = 0.011) by a median of 69 days compared with vehicle-treated control-diabetic hA-transgenic mice.

Structure and function of human xylulokinase, an enzyme with important roles in carbohydrate metabolism.

D-Xylulokinase (XK; EC 2.7.1.17) catalyzes the ATP-dependent phosphorylation of d-xylulose (Xu) to produce xylulose 5-phosphate (Xu5P). In mammals, XK is the last enzyme in the glucuronate-xylulose pathway, active in the liver and kidneys, and is linked through its product Xu5P to the pentose-phosphate pathway. XK may play an important role in metabolic disease, given that Xu5P is a key regulator of glucose metabolism and lipogenesis. We have expressed the product of a putative human XK gene and identified it as the authentic human d-xylulokinase (hXK). NMR studies with a variety of sugars showed that hXK acts only on d-xylulose, and a coupled photometric assay established its key kinetic parameters as K(m)(Xu) = 24 ± 3 µm and k(cat) = 35 ± 5 s(-1). Crystal structures were determined for hXK, on its own and in complexes with Xu, ADP, and a fluorinated inhibitor. These reveal that hXK has a two-domain fold characteristic of the sugar kinase/hsp70/actin superfamily, with glycerol kinase as its closest relative. Xu binds to domain-I and ADP to domain-II, but in this open form of hXK they are 10 Å apart, implying that a large scale conformational change is required for catalysis. Xu binds in its linear keto-form, sandwiched between a Trp side chain and polar side chains that provide exquisite hydrogen bonding recognition. The hXK structure provides a basis for the design of specific inhibitors with which to probe its roles in sugar metabolism and metabolic disease.

NAD+ dependent UPRmt activation underlies intestinal aging caused by mitochondrial DNA mutations.

Aging in mammals is accompanied by an imbalance of intestinal homeostasis and accumulation of mitochondrial DNA (mtDNA) mutations. However, little is known about how accumulated mtDNA mutations modulate intestinal homeostasis. We observe the accumulation of mtDNA mutations in the small intestine of aged male mice, suggesting an association with physiological intestinal aging. Using polymerase gamma (POLG) mutator mice and wild-type mice, we generate male mice with progressive mtDNA mutation burdens. Investigation utilizing organoid technology and in vivo intestinal stem cell labeling reveals decreased colony formation efficiency of intestinal crypts and LGR5-expressing intestinal stem cells in response to a threshold mtDNA mutation burden. Mechanistically, increased mtDNA mutation burden exacerbates the aging phenotype of the small intestine through ATF5 dependent mitochondrial unfolded protein response (UPRmt) activation. This aging phenotype is reversed by supplementation with the NAD+ precursor, NMN. Thus, we uncover a NAD+ dependent UPRmt triggered by mtDNA mutations that regulates the intestinal aging.

Proteomic Analysis of Honey: Peptide Profiling as a Novel Approach for New Zealand Manuka (Leptospermum scoparium) Honey Authentication.

New Zealand manuka (Leptospermum scoparium) honey is a premium food product. Unfortunately, its high demand has led to "not true to label" marketed manuka honey. Robust methods are therefore required to determine authenticity. We previously identified three unique nectar-derived proteins in manuka honey, detected as twelve tryptic peptide markers, and hypothesized these could be used to determine authenticity. We invoked a targeted proteomic approach based on parallel reaction-monitoring (PRM) to selectively monitor relative abundance of these peptides in sixteen manuka and twenty six non-manuka honey samples of various floral origin. We included six tryptic peptide markers derived from three bee-derived major royal jelly proteins as potential internal standards. The twelve manuka-specific tryptic peptide markers were present in all manuka honeys with minor regional variation. By comparison, they had negligible presence in non-manuka honeys. Bee-derived peptides were detected in all honeys with similar relative abundance but with sufficient variation precluding their utility as internal standards. Manuka honeys displayed an inverse relationship between total protein content and the ratio between nectar- to bee-derived peptide abundance. This trend reveals an association between protein content on possible nectar processing time by bees. Overall, these findings demonstrate the first successful application of peptide profiling as an alternative and potentially more robust approach for manuka honey authentication.

Cold-Induced Reprogramming of Subcutaneous White Adipose Tissue Assessed by Single-Cell and Single-Nucleus RNA Sequencing.

Adipose browning has demonstrated therapeutic potentials in several diseases. Here, by conducting transcriptomic profiling at the single-cell and single-nucleus resolution, we reconstituted the cellular atlas in mouse inguinal subcutaneous white adipose tissue (iWAT) at thermoneutrality or chronic cold condition. All major nonimmune cells within the iWAT, including adipose stem and progenitor cells (ASPCs), mature adipocytes, endothelial cells, Schwann cells, and smooth muscle cells, were recovered, allowing us to uncover an overall and detailed blueprint for transcriptomes and intercellular cross-talks and the dynamics during white adipose tissue brown remodeling. Our findings also unravel the existence of subpopulations in mature adipocytes, ASPCs, and endothelial cells, as well as new insights on their interconversion and reprogramming in response to cold. The adipocyte subpopulation competent of major histocompatibility complex class II (MHCII) antigen presentation is potentiated. Furthermore, a subcluster of ASPC with CD74 expression was identified as the precursor of this MHCII+ adipocyte. Beige adipocytes are transdifferented from preexisting lipid generating adipocytes, which exhibit developmental trajectory from de novo differentiation of amphiregulin cells (Aregs). Two distinct immune-like endothelial subpopulations are present in iWAT and are responsive to cold. Our data reveal fundamental changes during cold-evoked adipose browning.

Androgen Receptor is a Negative Regulator of PRDM16 in Beige Adipocyte.

PRDM16 (PR domain containing protein 16) serves as a dominant activator of brown and beige adipocyte. However, mechanisms underlying the regulation of PRDM16 expression are incompletely understood. A Prdm16 luciferase knockin reporter mouse model is generated, enabling high throughput monitoring of Prdm16 transcription. Single clonal analysis reveals high heterogeneity of Prdm16 expression in the inguinal white adipose tissue (iWAT) cells. Amongst all transcription factors, androgen receptor (Ar) shows the strongest negative correlation with Prdm16. A sex dimorphism for PRDM16 mRNA expression is present in human WAT, with female individuals exhibiting increased expression than males. Androgen-AR signaling mobilization suppresses Prdm16 expression, accompanied by attenuated beiging in beige adipocytes, but not in brown adipose tissue. The suppressive effect of androgens on beiging is abolished upon overexpression of Prdm16. Cleavage under targets and tagmentation mapping reveals direct binding of AR within the intronic region of Prdm16 locus, whereas no direct binding is detected on Ucp1 and other browning-related genes. Adipocyte-selective deletion of Ar potentiates beige cell biogenesis whereas adipocyte-specific overexpression of AR attenuates white adipose beiging. This study highlights an essential role of AR in negative regulation of PRDM16 in WAT and provides an explanation for the observed sex difference in adipose beiging.

The Leptospermum scoparium (Manuka)-Specific Nectar and Honey Compound 3,6,7-Trimethyllumazine (LepteridineTM) That Inhibits Matrix Metalloproteinase 9 (MMP-9) Activity.

3,6,7-trimethyllumazine (Lepteridine™) is a newly discovered natural pteridine derivative unique to Manuka (Leptospermum scoparium) nectar and honey, with no previously reported biological activity. Pteridine derivative-based medicines, such as methotrexate, are used to treat auto-immune and inflammatory diseases, and Manuka honey reportedly possesses anti-inflammatory properties and is used topically as a wound dressing. MMP-9 is a potential candidate protein target as it is upregulated in recalcitrant wounds and intestinal inflammation. Using gelatin zymography, 40 µg/mL LepteridineTM inhibited the gelatinase activities of both pro- (22%, p < 0.0001) and activated (59%, p < 0.01) MMP-9 forms. By comparison, LepteridineTM exerted modest (~10%) inhibition against a chromogenic peptide substrate and no effect against a fluorogenic peptide substrate. These findings suggest that LepteridineTM may not interact within the catalytic domain of MMP-9 and exerts a negligible effect on the active site hydrolysis of small soluble peptide substrates. Instead, the findings implicate fibronectin II domain interactions by LepteridineTM which impair gelatinase activity, possibly through perturbed tethering of MMP-9 to the gelatin matrix. Molecular modelling analyses were equivocal over interactions at the S1' pocket versus the fibronectin II domain, while molecular dynamic calculations indicated rapid exchange kinetics. No significant degradation of synthetic or natural LepteridineTM in Manuka honey occurred during simulated gastrointestinal digestion. MMP-9 regulates skin and gastrointestinal inflammatory responses and extracellular matrix remodelling. These results potentially implicate LepteridineTM bioactivity in Manuka honey's reported beneficial effects on wound healing via topical application and anti-inflammatory actions in gastrointestinal disorder models via oral consumption.

Purification, crystallization and preliminary crystallographic analysis of human dihydrodipicolinate synthase-like protein (DHDPSL).

Human dihydrodipicolinate synthase-like protein (DHDPSL) is a gene product of unknown function. It is homologous to bacterial pyruvate-dependent aldolases such as dihydrodipicolinate synthase (DHDPS), which functions in lysine biosynthesis. However, it cannot have this function and instead is implicated in a genetic disorder that leads to excessive production of oxalate and kidney-stone formation. In order to better understand its function, DHDPSL was expressed as an MBP-fusion protein and crystallized using an in situ proteolysis protocol. Two crystal forms were obtained, both of which diffracted X-rays to approximately 2.0 Å resolution. One of these, belonging to space group P6(2)22 or P6(4)22 with unit-cell parameters a = b = 142.9, c = 109.8 Å, α = ß = 90, γ = 120°, was highly reproducible and suitable for structure determination by X-ray crystallography.

Use of a repetitive seeding protocol to obtain diffraction-quality crystals of a putative human D-xylulokinase.

In mammals, the enzyme D-xylulokinase (XK; EC 2.7.1.17) catalyses the last step of the glucuronate-xylulose pathway, in which the ketopentose sugar D-xylulose is phosphorylated to yield D-xylulose 5-phosphate (Xu5P). Xu5P is also a metabolite of the pentose phosphate pathway and acts as a signalling molecule that regulates lipogenesis and glycolysis in the liver. To date, no eukaryotic XK has been structurally characterized. A putative human XK was expressed in Escherichia coli aided by molecular chaperones, purified and crystallized. A seeding procedure involving repeated rounds of seeding was developed and proved to be essential for obtaining diffraction-quality crystals. Preliminary X-ray diffraction analysis was performed using synchrotron radiation. This resulted in the collection of a complete diffraction data set to 2.7 Šresolution from a crystal belonging to the trigonal space group P3(1) or P3(2) with unit-cell parameters a = b = 101.87, c = 158.85 Å.

Bimatoprost promotes satiety and attenuates body weight gain in rats fed standard or obesity-promoting diets.

Bimatoprost is a synthetic prostamide F2α analog that down-regulates adipogenesis in vitro. This effect has been attributed to participation in a negative feedback loop that regulates anandamide-induced adipogenesis. A follow-on investigation has now been conducted into the broader metabolic effects of bimatoprost using rats under both normal state and obesity-inducing conditions. Chronic bimatoprost administration attenuated weight gain in a dose dependent-manner in rats fed either standard [max effect -7%] or obesity-promoting diets [max effect -23%] over a 9-10 week period. Consistent with these findings, bimatoprost promoted satiety as measured by decreased food intake [max effect, -7%], gastric emptying [max effect, -33-50%] and decreased circulating concentrations of the gut hormones, ghrelin and GLP-1 [max effect, -33-50%]. Additionally, subcutaneous, and visceral fat mass were distinctly affected by treatment [-30% diet independent]. Taken together, these results suggest that bimatoprost regulates energy homeostasis through promoting satiety and a decrease in food intake. These newly reported activities of bimatoprost reveal an additional method of metabolic disease intervention for potential therapeutic exploitation.

Discovery of JND003 as a New Selective Estrogen-Related Receptor α Agonist Alleviating Nonalcoholic Fatty Liver Disease and Insulin Resistance.

Nonalcoholic fatty liver disease (NAFLD) is one of the most prevalent forms of chronic liver diseases and is causally linked to hepatic insulin resistance and reduced fatty acid oxidation. Therapeutic treatments targeting both hepatic insulin resistance and lipid oxidative metabolism are considered as feasible strategies to alleviate this disease. Emerging evidence suggests Estrogen-Related Receptor alpha (ERRα), the first orphan nuclear receptor identified, as a master regulator in energy homeostasis by controlling glucose and lipid metabolism. Small molecules improving the functions of ERRα may provide a new option for management of NAFLD. In the present study, by using liver-specific Errα knockout mouse (Errα-LKO), we showed that liver-specific deletion of ERRα exacerbated diet-evoked fatty liver, hepatic and systemic insulin resistance in mice. A potent and selective ERRα agonist JND003 (7) was also discovered. In vitro and in vivo investigation demonstrated that the compound enhanced the transactivation of ERRα downstream target genes, which was accompanied by improved insulin sensitivity and fatty liver symptoms. Furthermore, the therapeutic effects were completely abolished in Errα-LKO mice, indicative of its on-target efficacy. Our study thus suggests that hepatic ERRα is a viable target for NAFLD and that ERRα agonist may serve as an intriguing pharmacological option for management of metabolic diseases.

Structure-Based Discovery and Optimization of Furo[3,2-c]pyridin-4(5H)-one Derivatives as Potent and Second Bromodomain (BD2)-Selective Bromo and Extra Terminal Domain (BET) Inhibitors.

Pan-bromodomain and extra terminal (Pan-BET) inhibitors show profound efficacy but exhibit pharmacology-driven toxicities in clinical trials. The development of domain-selective BET inhibitors to separate efficacy and toxicity is urgently needed. Herein, we report a series of furo[3,2-c]pyridin-4(5H)-one derivatives as novel BD2-selective BET inhibitors. The representative compound 8l (XY153) potently bound to BRD4 BD2 with an half-maximum inhibitory concentration (IC50) value of 0.79 nM and displayed 354-fold selectivity over BRD4 BD1. Besides, 8l exhibited 6-fold BRD4 BD2 domain selectivity over other BET BD2 domains. Compound 8l displayed potent antiproliferative activity against multiple tumor cell lines, especially MV4-11 (IC50 = 0.55 nM), while showing weak cytotoxicity against the normal lung fibroblast cell line. It highlights the safety profile of this series of BD2 inhibitors. 8l also demonstrated good metabolic stability in vitro. These data indicate that 8l may serve as a new and valuable lead compound for the development of potential therapeutics against acute myeloid leukemia (AML).

Comparative transcriptomic analysis of rabbit interscapular brown adipose tissue whitening under physiological conditions.

Interscapular brown adipose tissue (iBAT) of both rabbits and humans exhibits a similar whitening phenomenon under physiological conditions. However, a detailed characterization of iBAT whitening in them is still lacking. Here, we chose rabbits as a model to gain a better understanding of the molecular signature changes during the whitening process of iBAT by transcriptomic analysis of rabbit iBAT at day 1, day 14, 1 month and 4 months after birth. We applied non-invasive MRI imaging to monitor the whitening process and correlated these changes with analysis of morphological, histological and molecular features. Principal component analysis (PCA) of differentially expressed genes delineated three major phases for the whitening process as Brown, Transition and Whitened BAT phases. RNA-sequencing data revealed that whitening of iBAT was an orchestrated process where multiple types of cells and tissues participated in a variety of physiological processes including neovascularization, formation of new nervous networks and immune regulation. Several key metabolic and signalling pathways contributed to whitening of iBAT, and immune cells and immune regulation appeared to play an overarching role.

Lipidated Calcitonin Gene-Related Peptide (CGRP) Peptide Antagonists Retain CGRP Receptor Activity and Attenuate CGRP Action In Vivo.

Signaling through calcitonin gene-related peptide (CGRP) receptors is associated with pain, migraine, and energy expenditure. Small molecule and monoclonal antibody CGRP receptor antagonists that block endogenous CGRP action are in clinical use as anti-migraine therapies. By comparison, the potential utility of peptide antagonists has received less attention due to suboptimal pharmacokinetic properties. Lipidation is an established strategy to increase peptide half-life in vivo. This study aimed to explore the feasibility of developing lipidated CGRP peptide antagonists that retain receptor antagonist activity in vitro and attenuate endogenous CGRP action in vivo. CGRP peptide analogues based on the archetypal CGRP receptor antagonist, CGRP8-37, were palmitoylated at the N-terminus, position 24, and near the C-terminus at position 35. The antagonist activities of the lipidated peptide analogues were tested in vitro using transfected Cos-7 cells expressing either the human or mouse CGRP receptor, amylin subtype 1 (AMY1) receptor, adrenomedullin (AM) receptors, or calcitonin receptor. Antagonist activities were also evaluated in SK-N-MC cells that endogenously express the human CGRP receptor. Lipidated peptides were then tested for their ability to antagonize endogenous CGRP action in vivo using a capsaicin-induced dermal vasodilation (CIDV) model in C57/BL6J mice. All lipidated peptides except for the C-terminally modified analogue retained potent antagonist activity compared to CGRP8-37 towards the CGRP receptor. The lipidated peptides also retained, and sometimes gained, antagonist activities at AMY1, AM1 and AM2 receptors. Several lipidated peptides produced robust inhibition of CIDV in mice. This study demonstrates that selected lipidated peptide antagonists based on αCGRP8-37 retain potent antagonist activity at the CGRP receptor and are capable of inhibition of endogenous CGRP action in vivo. These findings suggest that lipidation can be applied to peptide antagonists, such as αCGRP8-37 and are a potential strategy for antagonizing CGRP action.