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.

PIK3CA regulates development of diabetes retinopathy through the PI3K/Akt/mTOR pathway.

OBJECTIVE: To explore their association with the development of diabetes retinopathy (DR), single nucleotide polymorphism (SNP) mutations were screened out by high-throughput sequencing and validated in patients diagnosed with DR. To understand the role of PIK3CA in the pathogenesis of DR and explore the relationship between PIK3CA,phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR),and DR, the effect of PIK3CA.rs17849079 mutation was investigated in a DR cell model. METHODS: Twelve patients diagnosed with DR at the Qinghai Provincial People's Hospital from September 2020 to June 2021 were randomly selected as the case group, while 12 healthy subjects of similar age and gender who underwent physical examination in Qinghai Provincial People's Hospital physical examination center during the same period were randomly selected as the control group. Blood samples (2 mL) were collected from both groups using EDTA anticoagulant blood collection vessels and frozen at -20°C for future analysis. SNP mutations were detected by high-throughput sequencing, and the shortlisted candidates were subjected by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. The detected SNP candidates were verified by expanding the sample size (first validation: 56 patients in the case group and 58 controls; second validation: 157 patients in the case group and 96 controls). A lentivirus vector carrying mutated or wild-type PIK3CA.rs17849079 was constructed. ARPE-19 cells were cultured in a medium supplemented with 10% fetal bovine serum (FBS) to establish a DR cell model. PIRES2-PIK3CA-MT and PIRES2-PIK3CA-WT vectors were transfected into DR model cells, which were categorized into control, mannitol, model, empty vector, PIK3CA wild-type, and PIK3CA mutant-type groups. Cell activity was detected by the cell counting kit (CCK)-8 assay, and cellular apoptosis was evaluated by flow cytometry. Glucose concentration and levels of cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-1ß were detected using enzyme-linked immunosorbent assay kits. The expression of PIK3CA, AKT1, mTOR, and VEGF genes was detected by real-time quantitative polymerase chain reaction (RT-qPCR), while the expression of PI3K, p-PI3K, AKT1, p-AKT1, mTOR, p-mTOR, and VEGF proteins was detected by western blotting. RESULTS: The mutated SNPs were mainly enriched in the PI3K/AKT pathway, calcium ion pathway, and glutamatergic synaptic and cholinergic synaptic signaling pathways. Seven SNPs, including PRKCE.rs1533476, DNAH11.rs10485983, ERAP1.rs149481, KLHL1.rs1318761, APOBEC3C.rs1969643, FYN.rs11963612, and KCTD1.rs7240205, were not related to the development of DR. PIK3CA.rs17849079 was prone to C/T mutation. The risk of DR increased with the presence of the C allele and decreased in the presence of the T allele. High glucose induced the expression of PIK3CA and VEGF mRNAs as well as the expression of PI3K, p-PI3K, p-AKT1, p-mTOR, and VEGF proteins in ARPE-19 cells, which led to secretion of inflammatory factors TNF-αand IL-1, cell apoptosis, and inhibition of cell proliferation. The PIK3CA.rs17849079 C allele accelerated the progression of DR. These biological effects were inhibited when the C allele of PIK3CA.rs17849079 was mutated to T allele. CONCLUSION: The mutated SNP sites in patients with DR were mainly enriched in PI3K/AKT, calcium ion, and glutamatergic synaptic and cholinergic synaptic signaling pathways. The rs17849079 allele of PIK3CA is prone to C/T mutation where the C allele increases the risk of DR. High glucose activates the expression of PIK3CA and promotes the phosphorylation of PI3K, which leads to the phosphorylation of AKT and mTOR. These effects consequently increase VEGF expression and accelerate the development of DR. The C to T allele mutation in PIK3CA.rs17849079 can play a protective role and reduce the risk of DR.

Correlational Study of Aminopeptidase Activities between Left or Right Frontal Cortex versus the Hypothalamus, Pituitary, Adrenal Axis of Spontaneously Hypertensive Rats Treated with Hypotensive or Hypertensive Agents.

It has been suggested that the neuro-visceral integration works asymmetrically and that this asymmetry is dynamic and modifiable by physio-pathological influences. Aminopeptidases of the renin-angiotensin system (angiotensinases) have been shown to be modifiable under such conditions. This article analyzes the interactions of these angiotensinases between the left or right frontal cortex (FC) and the same enzymes in the hypothalamus (HT), pituitary (PT), adrenal (AD) axis (HPA) in control spontaneously hypertensive rats (SHR), in SHR treated with a hypotensive agent in the form of captopril (an angiotensin-converting enzyme inhibitor), and in SHR treated with a hypertensive agent in the form of the L-Arginine hypertensive analogue L-NG-Nitroarginine Methyl Ester (L-NAME). In the control SHR, there were significant negative correlations between the right FC with HPA and positive correlations between the left FC and HPA. In the captopril group, the predominance of negative correlations between the right FC and HPA and positive correlations between the HPA and left FC was maintained. In the L-NAME group, a radical change in all types of interactions was observed; particularly, there was an inversion in the predominance of negative correlations between the HPA and left FC. These results indicated a better balance of neuro-visceral interactions after captopril treatment and an increase in these interactions in the hypertensive animals, especially in those treated with L-NAME.

Murine cytomegalovirus downregulates ERAAP and induces an unconventional T cell response to self.

The endoplasmic reticulum aminopeptidase associated with antigen processing (ERAAP) plays a crucial role in shaping the peptide-major histocompatibility complex (MHC) class I repertoire and maintaining immune surveillance. While murine cytomegalovirus (MCMV) has multiple strategies for manipulating the antigen processing pathway to evade immune responses, the host has also developed ways to counter viral immune evasion. In this study, we find that MCMV modulates ERAAP and induces an interferon γ (IFN-γ)-producing CD8+ T cell effector response that targets uninfected ERAAP-deficient cells. We observe that ERAAP downregulation during infection leads to the presentation of the self-peptide FL9 on non-classical Qa-1b, thereby eliciting Qa-1b-restricted QFL T cells to proliferate in the liver and spleen of infected mice. QFL T cells upregulate effector markers upon MCMV infection and are sufficient to reduce viral load after transfer to immunodeficient mice. Our study highlights the consequences of ERAAP dysfunction during viral infection and provides potential targets for anti-viral therapies.

Effects of feeding a thermomechanical, enzyme-facilitated, coprocessed yeast and soybean meal on growth performance, organ weights, leg health, and gut development of broiler chickens.

The development of a healthy gut during prestarter and starter phases is crucial to drive chicken's productivity. This study aimed to evaluate the effects of a thermomechanical, enzyme-facilitated, coprocessed yeast and soybean meal (pYSM) on growth performance, organ weights, leg health, and gut development in broiler chickens. A total of 576 as-hatched broiler chicks were randomly allotted to 3 dietary treatments (8 replicates/treatment, 24 chickens/replicate): a control group (C) without the pYSM, a treatment group 1 (T1), in which the pSYM was included at 20, 10, 5, 0, and 0% levels in the prestarter, starter, grower, finisher I, and finisher II feeding phases, respectively, and a treatment group 2 (T2), in which the pSYM was included at 5, 5, 5, 0, and 0% levels in each feeding phase. On d 3 and 10, 16 broilers/treatment were euthanized. The T1 broilers tended to show higher live weight (d 3 and 7) and average daily gain (prestarter and starter phases) than the other groups (P ≤ 0.10). Differently, pYSM-based diets did not influence the growth performance of the other feeding phases and the whole experimental period (P > 0.05). Relative weights of pancreas and liver were also unaffected by pYSM utilization (P > 0.05). Litter quality tended to have higher average scores in C group (P = 0.079), but no differences were observed for leg health (P > 0.05). Histomorphometry of gut, liver, and bursa of Fabricius was not affected by diet (P > 0.05). Gut immunity was driven to an anti-inflammatory pattern, with the reduction of IL-2, INF-γ, and TNF-α in the duodenum of treated birds (d 3, P < 0.05). Also, MUC-2 was greater in the duodenum of C and T2 group when compared to T1 (d 3, P = 0.016). Finally, T1-fed chickens displayed greater aminopeptidase activity in the duodenum (d 3 and 10, P < 0.05) and jejunum (d 3, P < 0.05). Feeding high levels of pYSM (10-20%) to broilers in the first 10 d tended to improve growth performance in the prestarter and starter phases. It also positively downregulated proinflammatory cytokines during the first 3 d, as well as stimulated the aminopeptidase activity in the prestarter and starter periods.

Paradoxical Effects of Endoplasmic Reticulum Aminopeptidase 1 Deficiency on HLA-B27 and Its Role as an Epistatic Modifier in Experimental Spondyloarthritis.

OBJECTIVE: We undertook this study to examine the functional basis for epistasis between endoplasmic reticulum aminopeptidase 1 (ERAP1) and HLA-B27 in experimental spondyloarthritis (SpA). METHODS: ERAP1-knockout rats were created using genome editing and bred with HLA-B27/human ß2 -microglobulin-transgenic (HLA-B27-Tg) rats and HLA-B7-Tg rats. The effects of ERAP1 deficiency on HLA allotypes were determined using immunoprecipitation and immunoblotting, flow cytometry, allogeneic T cell proliferation assays, and gene expression analyses. Animals were examined for clinical features of disease, and tissue was assessed by histology. RESULTS: ERAP1 deficiency increased the ratio of folded to unfolded (ß2 m-free) HLA-B27 heavy chains, while having the opposite effect on HLA-B7. Furthermore, in rats with ERAP1 deficiency, HLA-B27 misfolding was reduced, while free HLA-B27 heavy chain dimers on the cell surface and monomers were increased. The effects of ERAP1 deficiency persisted during up-regulation of HLA-B27 and led to a reduction in endoplasmic reticulum stress. ERAP1 deficiency reduced the prevalence of arthritis in HLA-B27-Tg rats by two-thirds without reducing gastrointestinal inflammation. Dendritic cell abnormalities attributed to the presence of HLA-B27, including reduced allogeneic T cell stimulation and loss of CD103-positive/major histocompatibility complex class II-positive cells, were not rescued by ERAP1 deficiency, while excess Il23a up-regulation was mitigated. CONCLUSION: ERAP1 deficiency reduced HLA-B27 misfolding and improved folding while having opposing effects on HLA-B7. The finding that HLA-B27-Tg rats had partial protection against SpA in this study is consistent with genetic evidence that loss-of-function and/or reduced expression of ERAP1 reduces the risk of ankylosing spondylitis. Functional studies support the concept that the effects of ERAP1 on HLA-B27 and SpA may be a consequence of how peptides affect the biology of this allotype rather than their role as antigenic determinants.

The dual-targeted prolyl aminopeptidase PAP1 is involved in proline accumulation in response to stress and during pollen development.

Plant endosymbiotic organelles such as mitochondria and chloroplasts harbour a wide array of biochemical reactions. As a part of protein homeostasis to maintain organellar activity and stability, unwanted proteins and peptides need to be completely degraded in a stepwise mechanism termed the processing pathway, where at the last stage single amino acids are released by aminopeptidases. Here, we determined the molecular and physiological functions of a prolyl aminopeptidase homologue PAP1 (At2g14260) that is able to release N-terminal proline. Transcript analyses demonstrate that an alternative transcription start site gives rise to two alternative transcripts, generating two in-frame proteins PAP1.1 and PAP1.2. Subcellular localization studies revealed that the longer isoform PAP1.1, which contains a 51 residue N-terminal extension, is exclusively targeted to chloroplasts, while the truncated isoform PAP1.2 is located in the cytosol. Distinct expression patterns in different tissues and developmental stages were observed. Investigations into the physiological role of PAP1 using loss-of-function mutants revealed that PAP1 activity may be involved in proline homeostasis and accumulation, required for pollen development and tolerance to osmotic stress. Enzymatic activity, subcellular location, and expression patterns of PAP1 suggest a role in the chloroplastic peptide processing pathway and proline homeostasis.

The Type of Fat in the Diet Influences Regulatory Aminopeptidases of the Renin-Angiotensin System and Stress in the Hypothalamic-Pituitary-Adrenal Axis in Adult Wistar Rats.

(1) Background: Prolonged feeding with a high-fat diet (HFD) acts as a stressor by activating the functions of the hypothalamic-pituitary-adrenal gland (HPA) stress axis, accompanied of hypertension by inducing the renin-angiotensin-aldosterone system. Angiotensinases enzymes are regulatory aminopeptidases of angiotensin metabolism, which together with the dipeptidyl peptidase IV (DPP-IV), pyroglutamyl- and tyrosyl-aminopeptidase (pGluAP, TyrAP), participate in cognitive, stress, metabolic and cardiovascular functions. These functions appear to be modulated by the type of fat used in the diet. (2) Methods: To analyze a possible coordinated response of aminopeptidases, their activities were simultaneously determined in the hypothalamus, adenohypophysis and adrenal gland of adult male rats fed diets enriched with monounsaturated (standard diet (S diet) supplemented with 20% virgin olive oil; VOO diet) or saturated fatty acids (diet S supplemented with 20% butter and 0.1% cholesterol; Bch diet). Aminopeptidase activities were measured by fluorimetry using 2-Naphthylamine as substrates. (3) Results: the hypothalamus did not show differences in any of the experimental diets. In the pituitary, the Bch diet stimulated the renin-angiotensin system (RAS) by increasing certain angiotensinase activities (alanyl-, arginyl- and cystinyl-aminopeptidase) with respect to the S and VOO diets. DPP-IV activity was increased with the Bch diet, and TyrAP activity decrease with the VOO diet, having both a crucial role on stress and eating behavior. In the adrenal gland, both HFDs showed an increase in angiotensinase aspartyl-aminopeptidase. The interrelation of angiotensinases activities in the tissues were depending on the type of diet. In addition, correlations were shown between angiotensinases and aminopeptidases that regulate stress and eating behavior. (4) Conclusions: Taken together, these results support that the source of fat in the diet affects several peptidases activities in the HPA axis, which could be related to alterations in RAS, stress and feeding behavior.

Harnessing affinity-based protein profiling to reveal a novel target of nintedanib.

Nintedanib (BIBF1120), a triple angiokinase inhibitor, was first approved for idiopathic pulmonary fibrosis (IPF) therapy and is also efficacious for lung carcinoma, and interstitial lung diseases, far beyond its inhibition of VEGFR/PDGFR/FGFR. We identified tripeptidyl-peptidase 1 (TPP1) as one of the direct targets of nintedanib employing the affinity-based protein profiling (AfBPP) technique. This may be a new mechanism for nintedanib's role different from tyrosine kinase inhibition.

Development of quinoline-based hybrid as inhibitor of methionine aminopeptidase 1 from Leishmania donovani.

Methionine aminopeptidase 1 (MetAP1) is a target for drug discovery against many adversaries and a potential antileishmanial target for its role in N-terminal methionine processing. As an effort towards new inhibitor discovery against methionine aminopeptidase 1 from Leishmania donovani (LdMetAP1), we have synthesized a series of quinoline-based hybrids, that is (Z)-5-((Z)-benzylidine)-2-(quinolin-3-ylimino)thiazolidin-4-ones (QYT-4a-i) whose in vitro screening led to the discovery of a novel inhibitor molecule (QYT-4h) against LdMetAP1. The compound QYT-4h showed nearly 20-fold less potency for human MetAP1 and had drug-like features. Time-course kinetic assays suggested QYT-4h acting through a competitive mode by binding to the metal-activated catalytic site. Notably, QYT-4h was most potent against the physiologically relevant Mn(II) and Fe(II) supplemented forms of LdMetAP1 and less potent against Co(II) supplemented form. Surface plasmon resonance and fluorescence spectroscopy demonstrated high affinity of QYT-4h for LdMetAP1. Through molecular modelling and docking studies, we found QYT-4h binding at the LdMetAP1 catalytic pocket occupying both the catalytic and substrate binding sites mostly with hydrogen bonding and hydrophobic interactions which provide structural basis for its promising potency. These results demonstrate the feasibility of employing small-molecule inhibitors for selective targeting of LdMetAP1 which may find use to effectively eliminate leishmaniasis.

Variations in MHC class I antigen presentation and immunopeptidome selection pathways.

Major histocompatibility class I (MHC-I) proteins mediate immunosurveillance against pathogens and cancers by presenting antigenic or mutated peptides to antigen receptors of CD8+ T cells and by engaging receptors of natural killer (NK) cells. In humans, MHC-I molecules are highly polymorphic. MHC-I variations permit the display of thousands of distinct peptides at the cell surface. Recent mass spectrometric studies have revealed unique and shared characteristics of the peptidomes of individual MHC-I variants. The cell surface expression of MHC-I-peptide complexes requires the functions of many intracellular assembly factors, including the transporter associated with antigen presentation (TAP), tapasin, calreticulin, ERp57, TAP-binding protein related (TAPBPR), endoplasmic reticulum aminopeptidases (ERAPs), and the proteasomes. Recent studies provide important insights into the structural features of these factors that govern MHC-I assembly as well as the mechanisms underlying peptide exchange. Conformational sensing of MHC-I molecules mediates the quality control of intracellular MHC-I assembly and contributes to immune recognition by CD8 at the cell surface. Recent studies also show that several MHC-I variants can follow unconventional assembly routes to the cell surface, conferring selective immune advantages that can be exploited for immunotherapy.

N-Benzyl Residues as the P1' Substituents in Phosphorus-Containing Extended Transition State Analog Inhibitors of Metalloaminopeptidases.

Peptidyl enzyme inhibitors containing an internal aminomethylphosphinic bond system (P(O)(OH)-CH2-NH) can be termed extended transition state analogs by similarity to the corresponding phosphonamidates (P(O)(OH)-NH). Phosphonamidate pseudopeptides are broadly recognized as competitive mechanism-based inhibitors of metalloenzymes, mainly hydrolases. Their practical use is, however, limited by hydrolytic instability, which is particularly restricting for dipeptide analogs. Extension of phosphonamidates by addition of the methylene group produces a P-C-N system fully resistant in water conditions. In the current work, we present a versatile synthetic approach to such modified dipeptides, based on the three-component phospha-Mannich condensation of phosphinic acids, formaldehyde, and N-benzylglycines. The last-mentioned component allowed for simple and versatile introduction of functionalized P1' residues located on the tertiary amino group. The products demonstrated moderate inhibitory activity towards porcine and plant metalloaminopeptidases, while selected derivatives appeared very potent with human alanyl aminopeptidase (Ki = 102 nM for 6a). Analysis of ligand-protein complexes obtained by molecular modelling revealed canonical modes of interactions for mono-metallic alanyl aminopeptidases, and distorted modes for di-metallic leucine aminopeptidases (with C-terminal carboxylate, not phosphinate, involved in metal coordination). In general, the method can be dedicated to examine P1'-S1' complementarity in searching for non-evident structures of specific residues as the key fragments of perspective ligands.

A mitochondrial prolyl aminopeptidase PAP2 releases N-terminal proline and regulates proline homeostasis during stress response.

Most mitochondrial proteins are synthesised in the cytosol and targeted into the organelle via N-terminal targeting peptides that are cleaved upon import. The free targeting peptide is subsequently processed in a stepwise manner, with single amino acids released as final products. Here, we have characterised a proline-cleaving aminopeptidase in Arabidopsis thaliana, prolyl aminopeptidase-2 (PAP2, At3g61540). Activity assays show that PAP2 has a preferred activity to hydrolyse N-terminal proline. Protein localisation studies revealed that PAP2 is exclusively targeted to mitochondria. Characterisation of pap2 mutants show defective pollen, enhanced dark-induced senescence and increased susceptibility to abiotic stresses, which are likely attributed to a reduced level of accumulated free proline. Taken together, these results demonstrate the role of PAP2 in proline cleavage from mitochondrial peptides and proline homeostasis, which is required for the development of male gametophyte, tolerance to abiotic stresses, and leaf senescence.

P1' Residue-Oriented Virtual Screening for Potent and Selective Phosphinic (Dehydro) Dipeptide Inhibitors of Metallo-Aminopeptidases.

Designing side chain substituents complementary to enzyme binding pockets is of great importance in the construction of potent and selective phosphinic dipeptide inhibitors of metallo-aminopeptidases. Proper structure selection makes inhibitor construction more economic, as the development process typically consists of multiple iterative preparation/bioassay steps. On the basis of these principles, using noncomplex computation and modeling methodologies, we comprehensively screened 900 commercial precursors of the P1' residues of phosphinic dipeptide and dehydrodipeptide analogs to identify the most promising ligands of 52 metallo-dependent aminopeptidases with known crystal structures. The results revealed several nonproteinogenic residues with an improved energy of binding compared with the best known inhibitors. The data are discussed taking into account the selectivity and stereochemical implications of the enzymes. Using this approach, we were able to identify nontrivial structural elements substituting the recognized phosphinic peptidomimetic scaffold of metallo-aminopeptidase inhibitors.

Identification of an Unconventional Subpeptidome Bound to the Behçet's Disease-associated HLA-B*51:01 that is Regulated by Endoplasmic Reticulum Aminopeptidase 1 (ERAP1).

Human leukocyte antigen (HLA) B*51:01 and endoplasmic reticulum aminopeptidase 1 (ERAP1) are strongly genetically associated with Behçet's disease (BD). Previous studies have defined two subgroups of HLA-B*51 peptidome containing proline (Pro) or alanine (Ala) at position 2 (P2). Little is known about the unconventional non-Pro/Ala2 HLA-B*51-bound peptides. We aimed to study the features of this novel subpeptidome, and investigate its regulation by ERAP1. CRISPR-Cas9 was used to generate an HLA-ABC-triple knockout HeLa cell line (HeLa.ABC-KO), which was subsequently transduced to express HLA-B*51:01 (HeLa.ABC-KO.B51). ERAP1 was silenced using lentiviral shRNA. Peptides bound to HLA-B*51:01 were eluted and analyzed by mass spectrometry. The characteristics of non-Pro/Ala2, Pro2, and Ala2 peptides and their alteration by ERAP1 silencing were investigated. Effects of ERAP1 silencing on cell surface expression of HLA-B*51:01 were studied using flow cytometry. More than 20% of peptides eluted from HLA-B*51:01 lacked Pro or Ala at P2. This unconventional group of HLA-B*51:01-bound peptides was relatively enriched for 8-mers (with relatively fewer 9-mers) compared with the Pro2 and Ala2 subpeptidomes and had similar N-terminal and C-terminal residue usages to Ala2 peptides (with the exception of the less abundant leucine at position Ω). Knockdown of ERAP1 increased the percentage of non-Pro/Ala2 from 20% to ∼40%, increased the percentage of longer (10-mer and 11-mer) peptides eluted from HLA-B*51:01 complexes, and abrogated the predominance of leucine at P1. Interestingly knockdown of ERAP1 altered the length and N-terminal residue usage of non-Ala2&Pro2 and Ala2 but not the Pro2 peptides. Finally, ERAP1 silencing regulated the expression levels of cell surface HLA-B*51 in a cell-type-dependent manner. In conclusion, we have used a novel methodology to identify an unconventional but surprisingly abundant non-Pro/Ala2 HLA-B*51:01 subpeptidome. It is increased by knockdown of ERAP1, a gene affecting the risk of developing BD. This has implications for theories of disease pathogenesis.

MetAP2 inhibitor treatment of high-fat and -fructose-fed dogs: impact on the response to oral glucose ingestion and a hyperinsulinemic hyperglycemic clamp.

We examined the methionine aminopeptidase 2 inhibitor fumagillin in dogs consuming a high-fat and -fructose diet (HFFD). In pilot studies (3 dogs that had consumed HFFD for 3 yr), 8 wk of daily treatment with fumagillin reduced food intake 29%, weight 6%, and the glycemic excursion during an oral glucose tolerance test (OGTT) 44%. A second group of dogs consumed the HFFD for 17 wk: pretreatment (weeks 0-4), treatment with fumagillin (FUM; n = 6), or no drug (Control, n = 8) (weeks 4-12), washout period (weeks 12-16), and fumagillin or no drug for 1 wk (week 17). OGTTs were performed at 0, 4, 11, and 16 wk. A hyperinsulinemic hyperglycemic clamp was performed in week 12; 4 chow-fed dogs underwent identical clamps. Kilocalories per day intake during the treatment period was 2,067 ± 50 (Control) versus 1,824 ± 202 (FUM). Body weights (kg) increased 1.9 ± 0.3 vs. 2.7 ± 0.8 (0-4 wk) and 1.2 ± 0.2 vs. -0.02 ± 0.9 (4-12 wk) in Control versus fumagillin. The OGTT glycemic response was 30% greater in Control versus fumagillin at 11 wk. Net hepatic glucose uptake (NHGU; mg·kg-1·min-1) in the Chow, Control, and fumagillin dogs was ~1.5 ± 0.6, -0.1 ± 0.1, and 0.3 ± 0.4 (with no portal glucose infusion) and 3.1 ± 0.6, 0.5 ± 0.3, and 1.5 ± 0.5 (portal glucose infusion at 4 mg·kg-1·min-1), respectively. Fumagillin improved glucose tolerance and NHGU in HFFD dogs, suggesting methionine aminopeptidase 2 (MetAP2) inhibitors have the potential for improving glycemic control in prediabetes and diabetes.

Seasonal variations in extracellular enzymatic activity in marine snow-associated microbial communities and their impact on the surrounding water.

Seasonal changes of microbial abundance and associated extracellular enzymatic activity in marine snow and in seawater were studied in the northern Adriatic during a three-year period. Marine snow was present during the entire period of investigation, although in higher concentrations during summer than during winter. Microorganisms densely colonized marine snow and aggregate-associated enzymatic activity was substantially higher (up to 105 times) than in seawater. Alkaline phosphatase activity (APA) and aminopeptidase activity in marine snow showed seasonal variations with higher values in late spring-summer than in autumn-winter, probably in response to changes in the quantity and quality of organic matter. The highest cell-specific bacterial activity was found for phosphatase, followed by peptidase, and the lowest was for glucosidases. Differential hydrolysis of marine snow-derived organic matter points to the well-known phosphorus limitation of the northern Adriatic and indicates preferential utilization of phosphorus- and nitrogen-rich organic compounds by microbes, while hydrolysis of polysaccharides seemed to be less important. In oligotrophic conditions during summer, organic matter released from marine snow might represent a significant source of substrate for free-living bacteria in seawater. For the first time microorganisms producing APA in marine snow were identified, revealing that dense populations of bacteria expressed APA, while cyanobacteria did not. Cyanobacteria proliferating in marine snow could benefit from phosphorus release by bacteria and nanoflagellates.

Efficacy and safety of methionine aminopeptidase 2 inhibition in type 2 diabetes: a randomised, placebo-controlled clinical trial.

AIMS/HYPOTHESIS: This multicentre randomised double-blind placebo-controlled clinical trial assessed the efficacy and safety of a methionine aminopeptidase 2 (MetAP2) inhibitor, beloranib, in individuals with obesity (BMI ≥30 kg/m2) and type 2 diabetes (HbA1c 53-97 mmol/mol [7-11%] and fasting glucose <15.6 mmol/l). METHODS: Participants were randomised (via a centralised interactive web response system) to placebo, 1.2 or 1.8 mg beloranib s.c. twice weekly for 26 weeks. Participants, investigators and the sponsor were blinded to group assignment. The primary endpoint was the change in weight from baseline to week 26. The trial was terminated early when beloranib development was stopped because of an imbalance of venous thromboembolism events in beloranib-treated individuals vs placebo that became evident during late-stage development of the drug. RESULTS: In total, 153 participants were randomised, 51 to placebo, 52 to 1.2 mg beloranib and 50 to 1.8 mg beloranib. In participants who completed week 26, the least squares mean ± SE weight change (baseline 111 kg) was -3.1 ± 1.2% with placebo (n = 22) vs -13.5 ± 1.1% and -12.7 ± 1.3% with 1.2 and 1.8 mg beloranib, respectively (n = 25; n = 19; p < 0.0001). The change in HbA1c (baseline 67 mmol/mol [8.3%]) was -6.6 ± 2.2 mmol/mol (-0.6 ± 0.2%) with placebo vs -21.9 ± 2.2 mmol/mol (-2.0 ± 0.2%) or -21.9 ± 3.3 mmol/mol (-2.0 ± 0.3%) with 1.2 or 1.8 mg beloranib (p < 0.0001), respectively. The most common beloranib adverse events were sleep related. One beloranib-treated participant experienced a non-fatal pulmonary embolism. CONCLUSIONS/INTERPRETATION: MetAP2 inhibitors represent a novel mechanism for producing meaningful weight loss and improvement in HbA1c. TRIAL REGISTRATION: ClinicalTrials.gov NCT02324491 FUNDING: The study was funded by Zafgen, Inc.

The Effect of Different Levels of Cu, Zn and Mn Nanoparticles in Hen Turkey Diet on the Activity of Aminopeptidases.

The aim of the study was to estimate the influence of the different levels of Cu, Zn, and Mn nanoparticles on the activity of aminopeptidases in turkey. An experiment was carried out on 144 turkey hen Hybrid Converter. The birds were divided into groups given standard- and nanoparticle-supplementation of different level of copper (Cu 20, 10, 2 mg/kg), zinc (Zn 100, 50, 10 ppm), and manganese (Mn 100, 50, 10 ppm), covering respectively 100%, 50%, and 10% of the physiological demands for those minerals in the diet. The activity of aminopeptidases (alanyl: AlaAP, leucyl: LeuAP and arginyl: ArgAP) after supplementation of minerals was determined in the breast and thigh turkey muscle. The strongest effect of interaction among minerals supplementation form and dose on the activity levels of the aminopeptidases in thigh muscle was observed for nano-Cu already at the lowest dose of 2 mg/kg. In this dose (covering 10% of the birds' demand) nano form of supplementation significantly increased the activity of Ala-, Leu-, and ArgAP (877, 201, and 719, respectively), compared to standard form of supplementation (461, 90.5, and 576, respectively). In turn, in breast muscle, after supplementation covering 10% of the demand with the nano-Cu, nano-Zn, and nano-Mn compared to the standard form, we did not observe any significant difference in the activity levels of any of the investigated aminopeptidases, except for AlaAP under Zn supplementation. Supplementation with the 20 mg/kg of Nano-Cu (100% of demand) and with 10 mg/kg of Nano-Cu (50% of demand) inhibited the activity of all of the three aminopeptidases in thigh muscle. Supplementation of the minerals in nano form into the diet, especially of Cu and Zn in the dose covering 10% of the demand is relevant to maintain homeostasis in turkey muscles, as indicated by the activity of the aminopeptidases.

Divergent profile between hypothalamic and plasmatic aminopeptidase activities in WKY and SHR. Influence of beta-adrenergic blockade.

AIMS: Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) differ in their renin-angiotensin system function and sympathetic tone. The metabolism of angiotensins and vasopressin depends on the action of certain aminopeptidases whose activity may be influenced by the autonomic nervous system. Their regulation may differ between WKY and SHR in hypothalamus and plasma according to the sympathetic tone. We analyzed aminopeptidases responsible for the hydrolysis of certain angiotensins, vasopressin, cholecystokinin or enkephalins in hypothalamus and plasma of WKY and SHR in untreated controls rats and under beta-adrenoceptor blockade. Systolic blood pressure, food intake, water intake and diuresis were measured as parameters modulated by the autonomic nervous system and the above mentioned peptides. MAIN METHODS: Glutamyl-, aspartyl-, cystinyl- and alanyl-aminopeptidase activities were analyzed fluorimetrically in plasma and hypothalamus of control and propranolol-treated (100mg/kg/day administered in drinking water for 1month) WKY and SHR, using arylamide derivatives as substrates. KEY FINDINGS: An opposite response of aminopeptidases to propranolol treatment between plasma and hypothalamus was observed in either WKY and SHR. Furthermore, the behavior of aminopeptidases was inversed between WKY and SHR either in hypothalamus and plasma: while the activity increased in hypothalamus and decreased in plasma of WKY, it decreased in hypothalamus and increased in plasma of SHR. SIGNIFICANCE: These results revealed an inverse response of aminopeptidases between hypothalamus and plasma and also an opposite behavior of these enzymes between WKY and SHR in hypothalamus and plasma. These observations support the involvement of the sympathetic system in the modulation of aminopeptidase activities.