Insulin-regulated Aminopeptidase Research Articles

The Discovery of New Inhibitors of Insulin-Regulated Aminopeptidase by a High-Throughput Screening of 400,000 Drug-like Compounds.

With the ambition to identify novel chemical starting points that can be further optimized into small drug-like inhibitors of insulin-regulated aminopeptidase (IRAP) and serve as potential future cognitive enhancers in the clinic, we conducted an ultra-high-throughput screening campaign of a chemically diverse compound library of approximately 400,000 drug-like small molecules. Three biochemical and one biophysical assays were developed to enable large-scale screening and hit triaging. The screening funnel, designed to be compatible with high-density microplates, was established with two enzyme inhibition assays employing either fluorescent or absorbance readouts. As IRAP is a zinc-dependent enzyme, the remaining active compounds were further evaluated in the primary assay, albeit with the addition of zinc ions. Rescreening with zinc confirmed the inhibitory activity for most compounds, emphasizing a zinc-independent mechanism of action. Additionally, target engagement was confirmed using a complementary biophysical thermal shift assay where compounds causing positive/negative thermal shifts were considered genuine binders. Triaging based on biochemical activity, target engagement, and drug-likeness resulted in the selection of 50 qualified hits, of which the IC50 of 32 compounds was below 3.5 µM. Despite hydroxamic acid dominance, diverse chemotypes with biochemical activity and target engagement were discovered, including non-hydroxamic acid compounds. The most potent compound (QHL1) was resynthesized with a confirmed inhibitory IC50 of 320 nM. Amongst these compounds, 20 new compound structure classes were identified, providing many new starting points for the development of unique IRAP inhibitors. Detailed characterization and optimization of lead compounds, considering both hydroxamic acids and other diverse structures, are in progress for further exploration.

Intrathecal Injections of Angiotensin IV and Oxytocin Conjugates Induce Antihyperalgesia and Antiallodynia in Both Sexes of Rats

Our previous studies have established that intrathecal oxytocin (OT) and angiotensin IV (Ang IV) injections induce antihyperalgesia and antiallodynia in rodents. Ang IV, a renin-angiotensin system hexapeptide, acts as an endogenous inhibitor that inhibits the oxytocin-degrading enzyme insulin-regulated aminopeptidase (IRAP). The pain inhibitory effects by Ang IV were found to be through its inhibition on IRAP to potentiate the effect of OT. However, these effects were found to be with a significant sex difference, which could be partially due to the higher expression of IRAP at the spinal cords of female. Therefore, we synthesized Ang IV and OT conjugates connected with a peptide bond and tested for their effects on hyperalgesia and allodynia. Carrageenan-induced hyperalgesia and partial sciatic nerve ligation (PSNL) were performed using rat models. Conjugates Ang IV-OT (Ang IV at the N-terminal) and OT-Ang IV (OT at the N-terminal) were synthesized and intrathecally injected into male and female rats. Our results showed that Ang IV-OT exhibited prominent antihyperalgesia in male rats, particularly during hyperalgesia recovery, whereas OT-Ang IV was more effective during development stage. Ang IV-OT showed clear antihyperalgesia in female rats, but OT-Ang IV had no significant effect. Notably, both conjugates alleviated neuropathic allodynia in male rats; however, OT-Ang IV had no effect in female rats, whereas Ang IV-OT induced significant antiallodynia. In conclusion, Ang IV-OT has greater therapeutic potential for treating hyperalgesia and allodynia than OT-Ang IV. Its effects were not affected by sex, unlike those of OT and OT-Ang IV, extending its possible clinical applications.

Inhibition of Insulin-Regulated Aminopeptidase by Imidazo [1,5-α]pyridines-Synthesis and Evaluation.

Inhibition of insulin-regulated aminopeptidase (IRAP) has been shown to improve cognitive functions in several animal models. Recently, we performed a screening campaign of approximately 10,000 compounds, identifying novel small-molecule-based compounds acting as inhibitors of the enzymatic activity of IRAP. Here we report on the chemical synthesis, structure-activity relationships (SAR) and initial characterization of physicochemical properties of a series of 48 imidazo [1,5-α]pyridine-based inhibitors, including delineation of their mode of action as non-competitive inhibitors with a small L-leucine-based IRAP substrate. The best compound displays an IC50 value of 1.0 µM. We elucidate the importance of two chiral sites in these molecules and find they have little impact on the compound's metabolic stability or physicochemical properties. The carbonyl group of a central urea moiety was initially believed to mimic substrate binding to a catalytically important Zn2+ ion in the active site, although the plausibility of this binding hypothesis is challenged by observation of excellent selectivity versus the closely related aminopeptidase N (APN). Taken together with the non-competitive inhibition pattern, we also consider an alternative model of allosteric binding.

Mechanisms of Allosteric Inhibition of Insulin-Regulated Aminopeptidase

Inhibition of Insulin-Regulated Aminopeptidase is being actively explored for the treatment of several human diseases and several classes of inhibitors have been developed although no clinical applications have been reported yet. Here, we combine enzymological analysis with x-ray crystallography to investigate the mechanism employed by two of the most studied inhibitors of IRAP, an aryl sulfonamide and a 2-amino-4H-benzopyran named HFI-419. Although both compounds have been hypothesized to target the enzyme's active site by competitive mechanisms, we discovered that they instead target previously unidentified proximal allosteric sites and utilize non-competitive inhibition mechanisms. X-ray crystallographic analysis demonstrated that the aryl sulfonamide stabilizes the closed, more active, conformation of the enzyme whereas HFI-419 locks the enzyme in a semi-open, and likely less active, conformation. HFI-419 potency is substrate-dependent and fails to effectively block the degradation of the physiological substrate cyclic peptide oxytocin. Our findings demonstrate alternative mechanisms for inhibiting IRAP through allosteric sites and conformational restricting and suggest that the pharmacology of HFI-419 may be more complicated than initially considered. Such conformation-specific interactions between IRAP and small molecules can be exploited for the design of more effective second-generation allosteric inhibitors.

Development of a sandwich ELISA to detect circulating, soluble IRAP as a potential disease biomarker

There is growing interest in the use of the enzyme, insulin regulated aminopeptidase (IRAP), as a biomarker for conditions such as cardio-metabolic diseases and ischemic stroke, with upregulation in its tissue expression in these conditions. However, quantification of circulating IRAP has been hampered by difficulties in detecting release of the truncated, soluble form of this enzyme into the blood stream. The current study aimed to develop a sandwich ELISA using novel antibodies directed towards the soluble portion of IRAP (sIRAP), to improve accuracy in detection and quantification of low levels of sIRAP in plasma. A series of novel anti-IRAP antibodies were developed and found to be highly specific for sIRAP in Western blots. A sandwich ELISA was then optimised using two distinct antibody combinations to detect sIRAP in the low nanogram range (16–500 ng/ml) with a sensitivity of 9 ng/ml and intra-assay variability < 10%. Importantly, the clinical validity of the ELISA was verified by the detection of significant increases in the levels of sIRAP throughout gestation in plasma samples from pregnant women. The specific and sensitive sandwich ELISA described in this study has the potential to advance the development of IRAP as a biomarker for certain diseases.

Inhibition of insulin-regulated aminopeptidase confers neuroprotection in a conscious model of ischemic stroke

Stroke is a leading cause of mortality and morbidity with a paucity of effective pharmacological treatments. We have previously identified insulin-regulated aminopeptidase (IRAP) as a potential target for the development of a new class of drugs for the treatment of stroke, as global deletion of this gene in mice significantly protected against ischemic damage. In the current study, we demonstrate that small molecular weight IRAP inhibitors reduce infarct volume and improve neurological outcome in a hypertensive animal model of ischemic stroke. The effects of two structurally distinct IRAP inhibitors (HFI419 or SJM164) were investigated in a model of stroke where the middle cerebral artery was transiently occluded with endothelin-1 in the conscious spontaneously hypertensive rat. IRAP inhibitor was administered into the lateral ventricle at 2 or 6 h after stroke, with subsequent doses delivered at 24, 48 and 70 h post-stroke. Functional outcomes were assessed prior to drug treatment, and on day 1 and 3 post-stroke. Histological analyses and neuroinflammatory cytokine profiling were conducted at 72 and 24 h post-stroke respectively. IRAP inhibitor treatment following stroke significantly reduced infarct volume and improved neurological and motor deficits. These protective effects were maintained even when the therapeutic window was extended to 6 h. Examination of the cellular architecture at 72 h post-stroke demonstrated that IRAP expression was upregulated in CD11b positive cells and activated astrocytes. Furthermore, IRAP inhibitor treatment significantly increased gene expression for interleukin 6 and C–C motif chemokine ligand 2 in the ischemic core. This study provides proof-of-principle that selective inhibition of IRAP activity with two structurally distinct IRAP inhibitors reduces infarct volume and improves functional outcome even when the first dose is administered 6 h post-stroke. This is the first direct evidence that IRAP inhibitors are a class of drug with potential use in the treatment of ischemic stroke.

Insulin-regulated aminopeptidase (IRAP) is required for appropriate dilution of urine after an acute water load

Study Objective: Understand the response to acute water load in mice lacking the insulin-regulated aminopeptidase (IRAP). Hypothesis: IRAP degrades vasopressin in vivo, therefore, mice lacking IRAP would have impaired ability to dilute urine and to excrete water. Methods: Age matched 8–10-week-old IRAP WT and KO male mice were used for all experiments. Immunofluorescence and immunoblotting were performed on kidneys at baseline and after 1-hr acute water load (2ml sterile water/IP). Urine/serum electrolytes and urine osmolality were measured before and after water load. Urine was collected from IRAP WT and KO mice before and after 1-hr administration of OPC 31260 (10mg/kg/IP) and furosemide (2.5mg/ml/IP) for urine osmolality and electrolyte measurements. Results: IRAP is expressed in the glomerulus, thick ascending loop of Henle, distal tubule, connecting and collecting ducts. At baseline, IRAP WT and KO mice have similar systolic blood pressure (122 ± 6.6 vs 128 ± 1.9 mmHg), glomerular filtration rate (251 ± 20 vs 254 ± 16 uL/min), and serum Na + levels (142 ± 1.9 vs 143 ± 2.6 mmol/L), but KO mice have reduced proteinuria (3.3 ± 0.57 vs 4.5 ± 1.6 mg/mg). At baseline, IRAP KO have elevated serum copeptin levels (111 ± 43 vs 53 ± 31 pg/mL) and urine osmolality compared to respective IRAP WT (2506 ± 310 vs 1007 ± 212 mOsm/L). Urine osmolality decreased in IRAP KO after administration of vasopressin type 2 receptor antagonist, OPC 31260 (473 ± 341 vs 2479 ± 215 mOsm/L) and loop diuretic, furosemide (517 ± 96 vs 2750 ± 414 mOsm/L). After an acute water load, IRAP KO have lower serum Na + levels (127 ± 3.8 vs 134 ±3.1 mmol/L) and increased urine osmolality compared to IRAP WT (2741 ± 400 vs 577 ± 274 mOsm/L). Additionally, after an acute water load, IRAP KO mice also have higher protein abundance of the Na + /K + /2Cl - (NKCC2) co-transporter (0.36 ± 0.04 vs 0.20 ± 0.03 AU) and phosphorylation of water channel, AQP2 (0.032 ± 0.005 vs 0.025 ± 0.003 AU). Conclusions: IRAP is required to dilute urine and to increase water excretion in response to an acute water load due to persistent activation of NKCC2 and AQP2. Funding Sources: Harold Amos Medical Faculty Development Award - Robert Wood Johnson Foundation; Vanderbilt Center for Kidney Disease This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Comparative study of Dapagliflozin versus Glimepiride effect on insulin regulated aminopeptidase (IRAP) and interleukin-34 (IL-34) in patient with type 2 diabetes mellitus

Type 2 diabetes mellitus (T2DM) is one of the most common diseases, that managed by several medications such as Glimepiride and Dapagliflozin. This study aims to compare the effects of Dapagliflozin versus Glimepiride on glycemic control, insulin resistance, and biomarkers as (extracellular domain of insulin regulated aminopeptidase) IRAPe, (interleukin-34) IL-34, and (N-terminal pro b-type natriuretic peptide) NT-proBNP. This study included 60 type 2 diabetic patients, who are randomized to receive either Glimepiride 4 mg/day (group 1) or Dapagliflozin 10 mg/day (group 2). Blood samples were collected at baseline and after 3 months of treatment for biochemical analysis. Additionally, HOMA-IR is calculated. After 3 months of receiving the intervention, there is no significant difference between the effects of Glimepiride and Dapagliflozin on FBG, PPBG, HbA1C%, fasting insulin, and HOMA-IR. The difference between both groups is significant for IL-34 (p = 0.002) and non-significant for IRAPe (p = 0.12) and NT-Pro BNP (p = 0.68). Both Glimepiride and Dapagliflozin significantly improve glycemic control, and HOMA-IR with no significant difference between them. Both drugs significantly improved the level of NT-proBNP. Dapagliflozin has a borderline significant effect on IRAPe but not IL-34, and Glimepiride has significant effect on IL-34 but not IRAPe.Clinical Trial Registration: This trial was registered on clinicaltrial.gov (NCT04240171).

Insulin-regulated aminopeptidase is required for water excretion in response to acute hypotonic stress.

The objective of this study was to understand the response of mice lacking insulin-regulated aminopeptidase (IRAP) to an acute water load. For mammals to respond appropriately to acute water loading, vasopressin activity needs to decrease. IRAP degrades vasopressin in vivo. Therefore, we hypothesized that mice lacking IRAP have an impaired ability to degrade vasopressin and, thus, have persistent urinary concentration. Age-matched 8- to 12-wk-old IRAP wild-type (WT) and knockout (KO) male mice were used for all experiments. Blood electrolytes and urine osmolality were measured before and 1 h after water load (∼2 mL sterile water via intraperitoneal injection). Urine was collected from IRAP WT and KO mice for urine osmolality measurements at baseline and after 1 h administration of the vasopressin type 2 receptor antagonist OPC-31260 (10 mg/kg ip). Immunofluorescence and immunoblot analysis were performed on kidneys at baseline and after 1 h acute water load. IRAP was expressed in the glomerulus, thick ascending loop of Henle, distal tubule, connecting duct, and collecting duct. IRAP KO mice had elevated urine osmolality compared with WT mice due to higher membrane expression of aquaporin 2 (AQP2), which was restored to that of controls after administration of OPC-31260. IRAP KO mice developed hyponatremia after an acute water load because they were unable to increase free water excretion due to increased surface expression of AQP2. In conclusion, IRAP is required to increase water excretion in response to an acute water load due to persistent vasopressin stimulation of AQP2.NEW & NOTEWORTHY Insulin-regulated aminopeptidase (IRAP) degrades vasopressin, but its role in urinary concentration and dilution is unknown. Here, we show that IRAP-deficient mice have a high urinary osmolality at baseline and are unable to excrete free water in response to water loading. These results reveal a novel regulatory role for IRAP in urine concentration and dilution.

Mast cell–mediated inflammation relies on insulin-regulated aminopeptidase controlling cytokine export from the Golgi

On activation, mast cells rapidly release preformed inflammatory mediators from large cytoplasmic granules via regulated exocytosis. This acute degranulation is followed by a late activation phase involving synthesis and secretion of cytokines, growth factors, and other inflammatory molecules via the constitutive pathway that remains ill defined. We investigated the role for an insulin-responsive vesicle-like endosomal compartment, marked by insulin-regulated aminopeptidase (IRAP), in the secretion of TNF-α and IL-6 in mast cells and macrophages. Murine knockout (KO) mouse models (IRAP-KO and kit-Wsh/sh) were used to study inflammatory disease models and to measure and mechanistically investigate cytokine secretion and degranulation in bone marrow-derived mast cells invitro. IRAP-KO mice are protected from TNF-α-dependent kidney injury and inflammatory arthritis. In the absence of IRAP, TNF-α and IL-6 but not IL-10 fail to be efficiently secreted. Moreover, chemical targeting of IRAP endosomes reduced proinflammatory cytokine secretion. Mechanistically, impaired TNF-α export from the Golgi and reduced colocalization of vesicle-associated membrane protein (VAMP) 3-positive TNF-α transport vesicles with syntaxin 4 (aka Stx4) was observed in IRAP-KO mast cells, while VAMP8-dependent exocytosis of secretory granules was facilitated. IRAP plays a novel role in mast cell-mediated inflammation through the regulation of exocytic trafficking of cytokines.

Alzheimer's disease: Is there a relationship between brain renin-angiotensin system, estradiol and glucose transporter-4 (GLUT-4)?

&lt;abstract&gt; &lt;p&gt;One of the diseases more related to the continuous aging of the population is Alzheimer's disease, which is a type of dementia currently without either effective diagnosis biomarkers or treatments. Its higher prevalence in women makes it necessary to study pathways/systems that could participate and/or be involved in its development, as well as those that could be affected by hormonal factors, which, in this case, are estradiol levels. In this sense, one of the systems under study that is gaining special relevance in the scientific community is the brain renin-angiotensin system and its regulatory proteolytic enzymes. This system is strongly modulated by estrogens, and it is also connected with the cerebral glucose metabolism through the angiotensin IV receptor, also recognized as the insulin-regulated aminopeptidase (IRAP). Due to the fact that the cerebral glucose metabolism is highly compromised in patients with Alzheimer's disease, it is necessary to know the elements of the systems and their functions in this process, namely, the cerebral renin-angiotensin system, estradiol and IRAP, an enzyme and receptor co-localized in brain tissue with the insulin-dependent glucose transporter 4 (GLUT4). Knowledge of the connection between them could shed light on the molecular mechanisms of this disease and also provide new diagnostic and therapeutic targets.&lt;/p&gt; &lt;/abstract&gt;

Inhibition of insulin-regulated aminopeptidase increases dendritic spine density in rat primary hippocampal cells

Inhibition of insulin-regulated aminopeptidase increases dendritic spine density in rat primary hippocampal cells

Akt-mediated mitochondrial metabolism regulates proplatelet formation and platelet shedding post vasopressin exposure

BackgroundPlatelet shedding from mature megakaryocytes (MKs) in thrombopoiesis is the critical step for elevating circulating platelets fast and efficiently, however, the underlying mechanism is still not well-illustrated, and the therapeutic targets and candidates are even less. ObjectivesIn order to investigate the mechanisms for platelet shedding after vasopressin treatment and find new therapeutic targets for thrombocytopenia. MethodsPlatelet production was evaluated both in vivo and in vitro after arginine vasopressin (AVP) administration. The underlying biological mechanism of AVP-triggered thrombopoiesis were then investigated by a series of molecular and bioinformatics techniques. Resultsit is observed that proplatelet formation and platelet shedding in the final stages of thrombopoiesis promoted by AVP, an endogenous hormone, can quickly increases peripheral platelets. This rapid elevation is thus able to speed up platelet recovery after radiation as expected. The mechanism analysis reveal that proplatelet formation and platelet release from mature MKs facilitated by AVP is mainly mediated by Akt-regulated mitochondrial metabolism. In particular, phosphorylated Akt regulates mitochondrial metabolism through driving the association of hexokinase-2 with mitochondrial voltage dependent anion channel-1 in AVP-mediated thrombopoiesis. Further studies suggest that this interaction is stabilized by IκBα, the expression of which is controlled by insulin-regulated membrane aminopeptidase. Conclusionthese data demonstrate that phosphorylated Akt-mediated mitochondrial metabolism regulates platelet shedding from MKs in response to AVP, which will provide new therapeutic targets and further drug discovery clues for thrombocytopenia treatment.

Fetal pancreas size and maternal serum biomarkers glycated albumin and insulin-regulated aminopeptidase provide no potential for early prediction of gestational diabetes mellitus.

We aimed to determine the predictive values of fetal pancreas size and maternal serum biomarkers glycated albumin (GA) and insulin-regulated aminopeptidase (IRAP) for gestational diabetes mellitus (GDM). In this prospective observational study including 109 pregnant women, the fetal pancreas size and maternal serum biomarkers GA and IRAP were measured at the gestational age of 20-22weeks and later at the gestational age of 24-28weeks, in 19 participants of them, GDM was confirmed with the 75-g oral glucose tolerance test (OGTT) and the fetal pancreas size was measured in all the participants again. The median fetal pancreas sizes were significantly higher in women with or without GDM when measured at the 24-28weeks of pregnancy compared to those at the 20-22weeks of pregnancy (p < 0.05). At both of the 20-22 and 24-28weeks of pregnancy, the median values of fetal pancreas sizes in the women with or without GDM were found comparable (p > 0.05). There were no significant differences between pregnant women with or without GDM regarding maternal serum biomarkers GA and IRAP (p > 0.05). Multivariate logistic regression analysis revealed no meaningful association of study parameters with the development of GDM. The fetal pancreas size and maternal serum biomarkers GA and IRAP provide no potential for early prediction of GDM at the 20-22weeks of gestation. Further studies, including serial measurement of these parameters during the second and third trimesters of GDM pregnancies, may clarify their role in the antenatal care of women with GDM. NCT05392231.

Insulin-regulated aminopeptidase contributes to setting the intensity of FcR-mediated inflammation.

The function of intracellular trafficking in immune-complex triggered inflammation remains poorly understood. Here, we investigated the role of Insulin-Regulated Amino Peptidase (IRAP)-positive endosomal compartments in Fc receptor (FcR)-induced inflammation. Less severe FcγR-triggered arthritis, active systemic anaphylaxis and FcεRI-triggered passive systemic anaphylaxis were observed in IRAP-deficient versus wild-type mice. In mast cells FcεRI stimulation induced rapid plasma membrane recruitment of IRAP-positive endosomes. IRAP-deficient cells exhibited reduced secretory responses, calcium signaling and activating SykY519/520 phosphorylation albeit receptor tyrosine phosphorylation on β and γ subunits was not different. By contrast, in the absence of IRAP, SHP1-inactivating phosphorylation on Ser591 that controls Syk activity was decreased. Ex-vivo cell profiling after FcγR-triggered anaphylaxis confirmed decreased phosphorylation of both SykY519/520 and SHP-1S591 in IRAP-deficient neutrophils and monocytes. Thus, IRAP-positive endosomal compartments, in promoting inhibition of SHP-1 during FcR signaling, control the extent of phosphorylation events at the plasma membrane and contribute to setting the intensity of immune-complex triggered inflammatory diseases.

Hydrogen Peroxide Induced Toxicity Is Reversed by the Macrocyclic IRAP-Inhibitor HA08 in Primary Hippocampal Cell Cultures.

Angiotensin IV (Ang IV), a metabolite of Angiotensin II, is a bioactive hexapeptide that inhibits the insulin-regulated aminopeptidase (IRAP). This transmembrane zinc metallopeptidase with many biological functions has in recent years emerged as a new pharmacological target. IRAP is expressed in a variety of tissues and can be found in high density in the hippocampus and neocortex, brain regions associated with cognition. Ang IV is known to improve memory tasks in experimental animals. One of the most potent IRAP inhibitors known today is the macrocyclic compound HA08 that is significantly more stable than the endogenous Ang IV. HA08 combines structural elements from Ang IV and the physiological substrates oxytocin and vasopressin, and binds to the catalytic site of IRAP. In the present study we evaluate whether HA08 can restore cell viability in rat primary cells submitted to hydrogen peroxide damage. After damaging the cells with hydrogen peroxide and subsequently treating them with HA08, the conceivable restoring effects of the IRAP inhibitor were assessed. The cellular viability was determined by measuring mitochondrial activity and lactate dehydrogenase (LDH) release. The mitochondrial activity was significantly higher in primary hippocampal cells, whereas the amount of LDH was unaffected. We conclude that the cell viability can be restored in this cell type by blocking IRAP with the potent macrocyclic inhibitor HA08, although the mechanism by which HA08 exerts its effects remains unclear.

The emerging multifunctional roles ofERAP1, ERAP2 and IRAP between antigen processing and renin-angiotensin system modulation.

The Endoplasmic Reticulum Aminopeptidase 1 and 2 (ERAP1 and ERAP2) and Insulin Regulated Aminopeptidase (IRAP) are three M1 zinc metalloproteases whose role in antigen processing is the refining of peptidome either in the Endoplasmic reticulum (ERAP1 and ERAP2), or in the endosomes (IRAP). However, other novel and distinct functions are emerging. Here, we focus specifically on ERAP2. This gene has a peculiar evolutionary history, being absent in rodents and undergoing in humans to a balanced selection of two haplotypes, one of which not expressing the full length ERAP2. These observations suggest that its role in antigen presentation is not essential. An additional, less investigated role is in the regulation of the Renin Angiotensin System (RAS). ERAP1 and ERAP2 cleave Angiotensin II (Ang II) into Ang III and IV, which counteract the action of Ang II whereas IRAP is itself the receptor for Ang IV. We have recently reported that macrophages, independently from the haplotype, express and release a N-terminus ERAP2 “short” form which directly binds IRAP and the two molecules are co-expressed in the endosomes and on the cell membrane. This new evidence suggests that the maintenance of the ERAP2 gene in humans could be due to its activity in the regulation of the RAS system, possibly as an Ang IV agonist. Its role in the immune-mediated diseases as well as in disorders more specifically related to an imbalance of the RAS system, including hypertension, pre-eclampsia but also viral infections such as COVID-19, is discussed here.

Discovery of Selective Nanomolar Inhibitors for Insulin-Regulated Aminopeptidase Based on α-Hydroxy-β-amino Acid Derivatives of Bestatin.

The oxytocinase subfamily of M1 zinc aminopeptidases comprises emerging drug targets, including the ER-resident aminopeptidases 1 and 2 (ERAP1 and ERAP2) and insulin-regulated aminopeptidase (IRAP); however, reports on clinically relevant inhibitors are limited. Here we report a new synthetic approach of high diastereo- and regioselectivity for functionalization of the α-hydroxy-β-amino acid scaffold of bestatin. Stereochemistry and mechanism of inhibition were investigated by a high-resolution X-ray crystal structure of ERAP1 in complex with a micromolar inhibitor. By exploring the P1 side-chain functionalities, we achieve significant potency and selectivity, and we report a cell-active, low-nanomolar inhibitor of IRAP with >120-fold selectivity over homologous enzymes. X-ray crystallographic analysis of IRAP in complex with this inhibitor suggest that interactions with the GAMEN loop is an unappreciated key determinant for potency and selectivity. Overall, our results suggest that α-hydroxy-β-amino acid derivatives may constitute useful chemical tools and drug leads for this group of aminopeptidases.

Estrogen can abolish oxytocin-induced spinal anti-hyperalgesia

Our previous study verified a sex difference of anti-hyperalgesia in rats and anti-allodynia in mice induced by intrathecal oxytocin (OT). In the model of intraplantar carrageenan-induced inflammatory hyperalgesia, intrathecal OT injection induced a substantial anti-hyperalgesia in male rats even at a low dose (0.125 nmol). In contrast, female rats only responded to an extremely high dose (1.25 nmol). This sex difference concurs with a lower expression of OT receptors and higher expression of insulin-regulated aminopeptidase (IRAP; OT degrading enzyme) in the spinal cords of female rats. In this study, we further determined the role of female hormones in this sex difference by using ovariectomized rats. Our results show that a low dose of intrathecal OT caused a significant anti-hyperalgesia effect in ovariectomized female rats, similar to that seen in male rats. Ovariectomy did not cause any change of paw edema except at the late stage of convalescence when compared with the sham-operated group. Ovariectomy-induced faster recovery from edema but did not affect the severity of hyperalgesia. Moreover, there was a similar amount of IRAP expression in ovariectomized and sham rats. When estradiol (E2) was given together with OT, OT-induced anti-hyperalgesia was abolished at the developmental stage of hyperalgesia in ovariectomized rats. These results show an inhibitory role of female hormones generated from ovaries (mainly estrogen) in the sex difference of anti-hyperalgesia induced by OT. This study suggests the feasibility of a novel OT-based remedy to treat hyperalgesia in men and in menopausal women no receiving hormonal supplements.

Omega-3 polyunsaturated fatty acids promote SNAREs mediated GLUT4 vesicle docking and fusion

Glucose homeostasis imbalance and insulin resistance (IR) are major contributors to the incidence of type 2 diabetes. Omega-3 polyunsaturated fatty acids (PUFAs) are key ingredients for maintaining cellular functions and improving insulin sensitivity. However, how omega-3 PUFAs modulate the dynamic process of glucose transport at the cellular level remains unclear. Here we unraveled eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) may regulate the glucose transporter 4 (GLUT4) vesicle trafficking in both normal and IR adipocytes. Both omega-3 PUFAs significantly increase glucose consumption within a range of 10–32% in the basal state. Furthermore, both EPA (200 μM) and DHA (100 μM) may significantly promote the serine/threonine protein kinase (Akt) phosphorylation by 70% and 40% in the physiological state of adipocytes, respectively. Both omega-3 PUFAs significantly advanced the Akt phosphorylation in a dose-dependent way and showed a ∼2-fold increase at the dose of 200 μM in the IR pathological state. However, they could not up-regulate the expression of GLUT4 and insulin-regulated aminopeptidase protein. We further revealed that both omega-3 PUFAs dynamically promote insulin-stimulated GLUT4 vesicle translocation and soluble N-ethylmaleimide-sensitive factor attachment protein receptor mediated vesicle docking and fusion to the plasma membrane via specifically modulating the expression of vesicle-associated membrane protein 2. Understanding the mechanisms by which omega-3 PUFAs modulate cellular metabolism and IR in peripheral tissues may provide novel insights into the potential impact of omega-3 PUFAs on the metabolic function and the management of IR.