Carboxyl-terminal sequences in APOA5 are important for suppressing ANGPTL3/8 activity.

Apolipoprotein AV (APOA5) lowers plasma triglyceride (TG) levels by binding to the angiopoietin-like protein 3/8 complex (ANGPTL3/8) and suppressing its capacity to inhibit lipoprotein lipase (LPL) catalytic activity and its ability to detach LPL from binding sites within capillaries. However, the sequences in APOA5 that are required for suppressing ANGPTL3/8 activity have never been defined. A clue to the identity of those sequences was the presence of severe hypertriglyceridemia in two patients harboring an APOA5 mutation that truncates APOA5 by 35 residues ("APOA5Δ35"). We found that wild-type (WT) human APOA5, but not APOA5Δ35, suppressed ANGPTL3/8's ability to inhibit LPL catalytic activity. To pursue that finding, we prepared a mutant mouse APOA5 protein lacking 40 C-terminal amino acids ("APOA5Δ40"). Mouse WT-APOA5, but not APOA5Δ40, suppressed ANGPTL3/8's capacity to inhibit LPL catalytic activity and sharply reduced plasma TG levels in mice. WT-APOA5, but not APOA5Δ40, increased intracapillary LPL levels and reduced plasma TG levels in Apoa5-/- mice (where TG levels are high and intravascular LPL levels are low). Also, WT-APOA5, but not APOA5Δ40, blocked the ability of ANGPTL3/8 to detach LPL from cultured cells. Finally, an antibody against a synthetic peptide corresponding to the last 26 amino acids of mouse APOA5 reduced intracapillary LPL levels and increased plasma TG levels in WT mice. We conclude that C-terminal sequences in APOA5 are crucial for suppressing ANGPTL3/8 activity in vitro and for regulating intracapillary LPL levels and plasma TG levels in vivo.

Decoding the role of angiopoietin-like protein 4/8 complex-mediated plasmin generation in the regulation of LPL activity.

After feeding, adipose tissue lipoprotein lipase (LPL) activity should be maximized, therefore the potent LPL-inhibitory activity of angiopoietin-like protein 4 (ANGPTL4) must be blocked by ANGPTL8 through formation of ANGPTL4/8 complexes. ANGPTL4/8 tightly binds and protects LPL but also partially inhibits its activity. Recently, we demonstrated ANGPTL4/8 also binds tissue plasminogen activator (tPA) and plasminogen to generate plasmin that cleaves ANGPTL4/8 to restore LPL activity. Although fully active LPL in the fat postprandially is desirable, ANGPTL4/8 removal could subject LPL to profound inhibition by ANGPTL3/8 (the most potent circulating LPL inhibitor), inhibition by other LPL inhibitors like ANGPTL4, ANGPTL3, and ApoC3 or interfere with ApoC2-mediated LPL activation. To understand better these potential paradoxes, we examined LPL inhibition by ANGPTL3/8, ANGPTL4, ANGPTL3, and ApoC3 and LPL stimulation by ApoC2 in the presence of ANGPTL4/8 + tPA + plasminogen. Remarkably, ANGPTL3/8-mediated LPL inhibition was almost completely blocked, with the mechanism being cleavage of fibrinogen-like domain-containing ANGPTL3 present in the ANGPTL3/8 complex. The LPL-inhibitory effects of ANGPTL4, ANGPTL3, and ApoC3 were also largely reduced in the presence of ANGPTL4/8 + tPA + plasminogen. In contrast, the ability of ApoC2 to stimulate LPL activity was unaffected by ANGPTL4/8-mediated plasmin generation. Together, these results explain how plasmin generated by increased postprandial ANGPTL4/8 levels in adipose tissue enables maximal LPL activity by preventing ANGPTL3/8, ANGPTL4, ANGPTL3, and ApoC3 from inhibiting LPL, while permitting ApoC2-mediated LPL activation to occur.

Inverse association between apolipoprotein C-II and cardiovascular mortality: role of lipoprotein lipase activity modulation.

AIMS: Apolipoprotein C-II (ApoC-II) is thought to activate lipoprotein lipase (LPL) and is therefore a possible target for treating hypertriglyceridemia. Its relationship with cardiovascular risk has not been investigated in large-scale epidemiologic studies, particularly allowing for apolipoprotein C-III (ApoC-III), an LPL antagonist. Furthermore, the exact mechanism of ApoC-II-mediated LPL activation is unclear. METHODS AND RESULTS: ApoC-II was measured in 3141 LURIC participants of which 590 died from cardiovascular diseases during a median (inter-quartile range) follow-up of 9.9 (8.7-10.7) years. Apolipoprotein C-II-mediated activation of the glycosylphosphatidylinositol high-density lipoprotein binding protein 1 (GPIHBP1)-LPL complex was studied using enzymatic activity assays with fluorometric lipase and very low-density lipoprotein (VLDL) substrates. The mean ApoC-II concentration was 4.5 (2.4) mg/dL. The relationship of ApoC-II quintiles with cardiovascular mortality exhibited a trend toward an inverse J-shape, with the highest risk in the first (lowest) quintile and lowest risk in the middle quintile. Compared with the first quintile, all other quintiles were associated with decreased cardiovascular mortality after multivariate adjustments including ApoC-III as a covariate (all P < 0.05). In experiments using fluorometric substrate-based lipase assays, there was a bell-shaped relationship for the effect of ApoC-II on GPIHBP1-LPL activity when exogenous ApoC-II was added. In ApoC-II-containing VLDL substrate-based lipase assays, GPIHBP1-LPL enzymatic activity was almost completely blocked by a neutralizing anti-ApoC-II antibody. CONCLUSION: The present epidemiologic data suggest that increasing low circulating ApoC-II levels may reduce cardiovascular risk. This conclusion is supported by the observation that optimal ApoC-II concentrations are required for maximal GPIHBP1-LPL enzymatic activity.

Angiopoietin-like protein 4/8 complex-mediated plasmin generation leads to cleavage of the complex and restoration of LPL activity.

Triglyceride (TG) metabolism is highly regulated by angiopoietin-like protein (ANGPTL) family members [Y. Q. Chen et al., J. Lipid Res. 61, 1203-1220 (2020)]. During feeding, ANGPTL8 forms complexes with the fibrinogen-like domain-containing protein ANGPTL4 in adipose tissue to decrease ANGPTL3/8- and ANGPTL4-mediated lipoprotein lipase (LPL)-inhibitory activity and promote TG hydrolysis and fatty acid (FA) uptake. The ANGPTL4/8 complex, however, tightly binds LPL and partially inhibits it in vitro. To try to reconcile the in vivo and in vitro data on ANGPTL4/8, we aimed to find novel binding partners of ANGPTL4/8. To that end, we performed pulldown experiments and found that ANGPTL4/8 bound both tissue plasminogen activator (tPA) and plasminogen, the precursor of the fibrinolytic enzyme plasmin. Remarkably, ANGPTL4/8 enhanced tPA activation of plasminogen to generate plasmin in a manner like that observed with fibrin, while minimal plasmin generation was observed with ANGPTL4 alone. The addition of tPA and plasminogen to LPL-bound ANGPTL4/8 caused rapid, complete ANGPTL4/8 cleavage and increased LPL activity. Restoration of LPL activity in the presence of ANGPTL4/8 was also achieved with plasmin but was blocked when catalytically inactive plasminogen (S760A) was added to tPA or when plasminogen activator inhibitor-1 was added to tPA + plasminogen, indicating that conversion of plasminogen to plasmin was essential. Together, these results suggest that LPL-bound ANGPTL4/8 mimics fibrin to recruit tPA and plasminogen to generate plasmin, which then cleaves ANGPTL4/8, enabling LPL activity to be increased. Our observations thus reveal a unique link between the ANGPTL4/8 complex and plasmin generation.

A sensitive and drug tolerant assay for detecting anti-AAV9 antibodies using affinity capture elution.

Adeno-associated virus (AAV) based gene therapies are gaining significant momentum as a novel therapeutic modality. However, a yet unsolved concern for using AAV as a vector is the high potential to elicit humoral and cellular responses, which are often exacerbated by pre-existing immunity due to exposure to wild type AAV. Therefore, characterization of pre-existing and treatment emergent anti-AAV antibodies is of great importance to the development of AAV based gene therapies. In this project, a sensitive and drug tolerant total antibody (TAb) assay was developed using recombinant AAV9-GFP (green fluorescent protein) as a surrogate AAV9. The assay format was affinity capture and elution (ACE) with ruthenium labeled AAV9-GFP as detection. Upon evaluation, three commercial anti-AAV9 monoclonal antibodies (clones HI17, HI35, and HL2374) were chosen and mixed at equal concentrations as positive control material. The assay sensitivity was estimated to be 11.2 ng/mL. Drug tolerance was estimated to be 5.4 × 10E10 DRP/mL AAV9-GFP at 100 ng/mL anti-AAV9 antibodies and to be at least 1 × 10E11 DRP/mL at 500 ng/mL and 250 ng/mL anti-AAV9 antibodies. The assay showed desirable specificity and precision. Using this TAb assay, significant pre-existing antibodies were detected from normal human sera.

Structure-Based Design of Active-Site-Directed, Highly Potent, Selective, and Orally Bioavailable Low-Molecular-Weight Protein Tyrosine Phosphatase Inhibitors.

Protein tyrosine phosphatases constitute an important class of drug targets whose potential has been limited by the paucity of drug-like small-molecule inhibitors. We recently described a class of active-site-directed, moderately selective, and potent inhibitors of the low-molecular-weight protein tyrosine phosphatase (LMW-PTP). Here, we report our extensive structure-based design and optimization effort that afforded inhibitors with vastly improved potency and specificity. The leading compound inhibits LMW-PTP potently and selectively (Ki = 1.2 nM, >8000-fold selectivity). Many compounds exhibit favorable drug-like properties, such as low molecular weight, weak cytochrome P450 inhibition, high metabolic stability, moderate to high cell permeability (Papp > 0.2 nm/s), and moderate to good oral bioavailability (% F from 23 to 50% in mice), and therefore can be used as in vivo chemical probes to further dissect the complex biological as well as pathophysiological roles of LMW-PTP and for the development of therapeutics targeting LMW-PTP.

An anti-ANGPTL3/8 antibody decreases circulating triglycerides by binding to a LPL-inhibitory leucine zipper-like motif.

Triglycerides (TG) are required for fatty acid transport and storage and are essential for human health. Angiopoietin-like-protein 8 (ANGPTL8) has previously been shown to form a complex with ANGPTL3 that increases circulating TG by potently inhibiting LPL. We also recently showed that the TG-lowering apolipoprotein A5 (ApoA5) decreases TG levels by suppressing ANGPTL3/8-mediated LPL inhibition. To understand how LPL binds ANGPTL3/8 and ApoA5 blocks this interaction, we used hydrogen-deuterium exchange mass-spectrometry and molecular modeling to map binding sites of LPL and ApoA5 on ANGPTL3/8. Remarkably, we found that LPL and ApoA5 both bound a unique ANGPTL3/8 epitope consisting of N-terminal regions of ANGPTL3 and ANGPTL8 that are unmasked upon formation of the ANGPTL3/8 complex. We further used ANGPTL3/8 as an immunogen to develop an antibody targeting this same epitope. After refocusing on antibodies that bound ANGPTL3/8, as opposed to ANGPTL3 or ANGPTL8 alone, we utilized bio-layer interferometry to select an antibody exhibiting high-affinity binding to the desired epitope. We revealed an ANGPTL3/8 leucine zipper-like motif within the anti-ANGPTL3/8 epitope, the LPL-inhibitory region, and the ApoA5-interacting region, suggesting the mechanism by which ApoA5 lowers TG is via competition with LPL for the same ANGPTL3/8-binding site. Supporting this hypothesis, we demonstrate that the anti-ANGPTL3/8 antibody potently blocked ANGPTL3/8-mediated LPL inhibition in vitro and dramatically lowered TG levels in vivo. Together, these data show that an anti-ANGPTL3/8 antibody targeting the same leucine zipper-containing epitope recognized by LPL and ApoA5 markedly decreases TG by suppressing ANGPTL3/8-mediated LPL inhibition.

Angiopoietin-like protein 4 (ANGPTL4) is an inhibitor of endothelial lipase (EL) while the ANGPTL4/8 complex has reduced EL-inhibitory activity.

We previously demonstrated that angiopoietin-like protein 8 (ANGPTL8) forms ANGPTL3/8 and ANGPTL4/8 complexes that increase with feeding to direct fatty acids (FA) toward adipose tissue through differential modulation of lipoprotein lipase (LPL) activity. Each complex correlated inversely with high density lipoprotein cholesterol (HDL) in control subjects. We thus investigated ANGPTL3/8 and ANGPTL4/8 levels in type 2 diabetes patients, who can present with decreased HDL. While ANGPTL3/8 levels in type 2 diabetes patients were similar to those previously observed in normal controls, ANGPTL4/8 levels were roughly twice as high as those in control subjects. Concentrations of ANGPTL3/8 and ANGPTL4/8 in type 2 diabetes patients were inversely correlated with HDL, with the correlation being significant for ANGPTL4/8. We therefore measured the ability of the various ANGPTL proteins and complexes to inhibit endothelial lipase (EL), the enzyme which hydrolyzes phospholipids (PL) in HDL. While confirming ANGPTL3 as an EL inhibitor, we found that ANGPTL4 was a more potent EL inhibitor than ANGPTL3. Interestingly, we observed that while ANGPTL3/8 had increased EL-inhibitory activity compared to ANGPTL3 alone, ANGPTL4/8 exhibited decreased potency in inhibiting EL compared to ANGPTL4 alone. Together, these results show for the first time that ANGPTL4 is a more potent EL inhibitor than ANGPTL3 and suggest a possible reason for why ANGPTL4/8 levels are correlated inversely with HDL.

The Angiopoietin-Like Protein 3 and 8 Complex Interacts with Lipoprotein Lipase and Induces LPL Cleavage.

Lipoprotein lipase (LPL) is the key enzyme that hydrolyzes triglycerides from triglyceride-rich lipoproteins. Angiopoietin-like proteins (ANGPTL) 3, 4, and 8 are well-characterized protein inhibitors of LPL. ANGPTL8 forms a complex with ANGPTL3, and the complex is a potent endogenous inhibitor of LPL. However, the nature of the structural interaction between ANGPTL3/8 and LPL is unknown. To probe the conformational changes in LPL induced by ANGPTL3/8, we found that HDX-MS detected significantly altered deuteration in the lid region, ApoC2 binding site, and furin cleavage region of LPL in the presence of ANGPTL3/8. Supporting this HDX structural evidence, we found that ANGPTL3/8 inhibits LPL enzymatic activities and increases LPL cleavage. ANGPTL3/8-induced effects on LPL activity and LPL cleavage are much stronger than those of ANGPTL3 or ANGPTL8 alone. ANGPTL3/8-mediated LPL cleavage is blocked by both an ANGPTL3 antibody and a furin inhibitor. Knock-down of furin expression by siRNA significantly reduced ANGPT3/8-induced cleavage of LPL. Our data suggest ANGPTL3/8 promotes furin-mediated LPL cleavage.

ApoA5 lowers triglyceride levels via suppression of ANGPTL3/8-mediated LPL inhibition.

Triglyceride (TG) molecules represent the major storage form of fatty acids, and TG metabolism is essential to human health. However, the mechanistic details surrounding TG metabolism are complex and incompletely elucidated. Although it is known that angiopoietin-like protein 8 (ANGPTL8) increases TGs through an ANGPTL3/8 complex that inhibits LPL, the mechanism governing ApoA5, which lowers TGs, has remained elusive. Current hypotheses for how ApoA5 acts include direct stimulation of LPL, facilitation of TG-containing particle uptake, and regulation of hepatic TG secretion. Using immunoprecipitation-MS and Western blotting, biolayer interferometry, functional LPL enzymatic assays, and kinetic analyses of LPL activity, we show that ApoA5 associates with ANGPTL3/8 in human serum and most likely decreases TG by suppressing ANGPTL3/8-mediated LPL inhibition. We also demonstrate that ApoA5 has no direct effect on LPL, nor does it suppress the LPL-inhibitory activities of ANGPTL3, ANGPTL4, or ANGPTL4/8. Importantly, ApoA5 suppression of ANGPTL3/8-mediated LPL inhibition occurred at a molar ratio consistent with the circulating concentrations of ApoA5 and ANGPTL3/8. Because liver X receptor (LXR) agonists decrease ApoA5 expression and cause hypertriglyceridemia, we investigated the effect of the prototypical LXR agonist T0901317 on human primary hepatocytes. We observed that T0901317 modestly stimulated hepatocyte ApoA5 release, but markedly stimulated ANGPTL3/8 secretion. Interestingly, the addition of insulin to T0901317 attenuated ApoA5 secretion, but further increased ANGPTL3/8 secretion. Together, these results reveal a novel intersection of ApoA5 and ANGPTL3/8 in the regulation of TG metabolism and provide a possible explanation for LXR agonist-induced hypertriglyceridemia.

Application of a novel drug-tolerant target assay for measuring target engagement when only one epitope remains after therapeutic antibodies bind their targets.

The measurement of proteins with a limited number of available non-overlapping epitopes recognizable by antibodies represents a common challenge for the development of drug-tolerant clinical biomarker assays. For target proteins with two dominant epitopes, only one epitope remains when the other is occupied by the therapeutic antibody. Alternative strategies for overcoming this obstacle have been described in the literature; however, these methods have potential limitations. We have developed a novel method for measuring target engagement when only one epitope remains after therapeutic antibodies bind their analytes. The method combines Affinity Capture Elution (ACE) followed by simultaneous capture and detection of the protein of interest. This novel method has been named ACE-Sandwich. The application of this method is not dependent on the immunoglobulin G subclass of the therapeutic antibody, nor does this method require sample pretreatment. Furthermore, the ACE-Sandwich method is highly sensitive, reproducible, and tolerant to high concentrations of therapeutic antibody.

Angiopoietin-like protein 4(E40K) and ANGPTL4/8 complex have reduced, temperature-dependent LPL-inhibitory activity compared to ANGPTL4.

We previously demonstrated that angiopoietin-like 8 (ANGPTL8) forms a localized complex with ANGPTL4 to reduce its lipoprotein lipase (LPL)-inhibitory activity and enable increased postprandial uptake of fatty acids (FA) into adipose tissue. Because prolonged cold exposure may increase adipose tissue FA uptake and decrease circulating triglycerides (TG) by reducing ANGPTL4 expression and inducing ANGPTL8 expression (and thus ANGPTL4/8 expression), we investigated the effect of temperature on ANGPTL4 and ANGPTL4/8 LPL-inhibitory activities in vitro. As the ANGPTL4(E40K) mutation results in decreased TG, we also characterized ANGPTL4(E40K) and ANGPTL4(E40K)/8 complex LPL-inhibitory activities. Interestingly, while ANGPTL3, ANGPTL3/8, and ANGPTL4 showed similar LPL inhibition at 37 °C and 22 °C, the already reduced LPL-inhibitory activity of ANGPTL4/8 at 37 °C was even more decreased at 22 °C. At 37 °C, ANGPTL4(E40K) manifested decreased LPL-inhibitory activity compared to ANGPTL4/8, while ANGPTL4(E40K)/8 had even further reduced potency. Remarkably, ANGPTL4/8, ANGPTL4(E40K), and ANGPTL4(E40K)/8 were each actually capable of stimulating LPL activity at 22 °C. Together, these results indicate that ANGPTL4/8 stimulation of LPL activity at low temperatures may represent an additional mechanism for further increasing adipose tissue FA uptake during cold exposure, beyond that already occurring due to decreased ANGPTL4 expression and increased ANGPTL8 expression. In addition, because ANGPTL4(E40K) has decreased LPL-inhibitory activity compared to ANGPTL4/8, our findings also suggest why ANGPTL4(E40K) carriers have decreased circulating TG levels.

Angiopoietin-like protein 8 differentially regulates ANGPTL3 and ANGPTL4 during postprandial partitioning of fatty acids.

Angiopoietin-like protein (ANGPTL)8 has been implicated in metabolic syndrome and reported to regulate adipose FA uptake through unknown mechanisms. Here, we studied how complex formation of ANGPTL8 with ANGPTL3 or ANGPTL4 varies with feeding to regulate LPL. In human serum, ANGPTL3/8 and ANGPTL4/8 complexes both increased postprandially, correlated negatively with HDL, and correlated positively with all other metabolic syndrome markers. ANGPTL3/8 also correlated positively with LDL-C and blocked LPL-facilitated hepatocyte VLDL-C uptake. LPL-inhibitory activity of ANGPTL3/8 was >100-fold more potent than that of ANGPTL3, and LPL-inhibitory activity of ANGPTL4/8 was >100-fold less potent than that of ANGPTL4. Quantitative analyses of inhibitory activities and competition experiments among the complexes suggested a model in which localized ANGPTL4/8 blocks the LPL-inhibitory activity of both circulating ANGPTL3/8 and localized ANGPTL4, allowing lipid sequestration into fat rather than muscle during the fed state. Supporting this model, insulin increased ANGPTL3/8 secretion from hepatocytes and ANGPTL4/8 secretion from adipocytes. These results suggest that low ANGPTL8 levels during fasting enable ANGPTL4-mediated LPL inhibition in fat tissue to minimize adipose FA uptake. During feeding, increased ANGPTL8 increases ANGPTL3 inhibition of LPL in muscle via circulating ANGPTL3/8, while decreasing ANGPTL4 inhibition of LPL in adipose tissue through localized ANGPTL4/8, thereby increasing FA uptake into adipose tissue. Excessive caloric intake may shift this system toward the latter conditions, possibly predisposing to metabolic syndrome.

Integration of phage and yeast display platforms: A reliable and cost effective approach for binning of peptides as displayed on-phage.

Hundreds of target specific peptides are routinely discovered by peptide display platforms. However, due to the high cost of peptide synthesis only a limited number of peptides are chemically made for further analysis. Here we describe an accurate and cost effective method to bin peptides on-phage based on binding region(s), without any requirement for peptide or protein synthesis. This approach, which integrates phage and yeast display platforms, requires display of target and its alanine variants on yeast. Flow cytometry was used to detect binding of peptides on-phage to the target on yeast. Once hits were identified, they were synthesized to confirm their binding region(s) by HDX (Hydrogen deuterium exchange) and crystallography. Moreover, we have successfully shown that this approach can be implemented as part of a panning process to deplete non-functional peptides. This technique can be applied to any target that can be successfully displayed on yeast; it narrows down the number of peptides requiring synthesis; and its utilization during selection results in enrichment of peptide population against defined binding regions on the target.

Folium nelumbinis (Lotus leaf) volatile-rich fraction and its mechanisms of action against melanogenesis in B16 cells.

This study was aimed at determining the influence of Folium nelumbinis (Lotus leaf) extracts on melanogenesis in vitro models of melanoma cell line. The anticancer activity of four fractions, including petroleum ether (PEE), n-hexane (HE), ethanol (EE), and ethyl acetate (EAE) from F. nelumbinis on B16 cell lines (C57BL/6J melanoma cell), were evaluated after 24 and 48 h treatment. Results showed that PEE as well as volatile-rich fractions of linolenic acid and linolenic acid ethyl ester significantly (p < 0.05) reduced tyrosinase activity and melanin content in B16 melanoma cells model. Meanwhile, PEE and its primarily contained compound triggered apoptosis of B16 cells in a dose-dependent way. These results demonstrated that PEE possessed effective activities against melanin and tyrosinase generations through the induction of apoptosis. Moreover, a relation between the volatile-rich fractions of F. nelumbinis and the anticancer effects was demonstrated as well.

Sonchus oleraceus Linn extract enhanced glucose homeostasis through the AMPK/Akt/ GSK-3ß signaling pathway in diabetic liver and HepG2 cell culture.

The extracts of S. oleraceus Linn (SOL) and its main phenolic compounds have shown anti-diabetic effects, but their underlying mechanisms for glucose homeostasis remain unclear. The aim of this study is to evaluate the anti-diabetic mechanism of SOL by using the streptozocin (STZ) induced diabetic rat model. When diabetic rats were fed with SOL at a dose of 400 mg/kg/day for 6 weeks, the concentrations of triglycerides (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C) were reduced by 43%, 22%, and 16%, respectively. Meanwhile, it was also found that daily feeding of SOL to diabetic rats led to a decrease in plasma glucose level by approximately 23%. Positive effects were observed on glucose homeostasis due to the down-regulation of AMPK/Akt/GSK-3ß pathway, as indicated by the suppressions of adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK), protein kinase (Akt) phosphorylation, glycogen synthase kinase 3 beta (GSK-3ß), and the hepatic insulin resistance. In HepG2 cells, AMPK, Akt and GSK-3ß showed a consistent transcript regulation. SOL at dose of 400 mg/kg/day feeding for 6 weeks showed a positive effect comparable to metformin.

Aurora A-Selective Inhibitor LY3295668 Leads to Dominant Mitotic Arrest, Apoptosis in Cancer Cells, and Shows Potent Preclinical Antitumor Efficacy.

Although Aurora A, B, and C kinases share high sequence similarity, especially within the kinase domain, they function distinctly in cell-cycle progression. Aurora A depletion primarily leads to mitotic spindle formation defects and consequently prometaphase arrest, whereas Aurora B/C inactivation primarily induces polyploidy from cytokinesis failure. Aurora B/C inactivation phenotypes are also epistatic to those of Aurora A, such that the concomitant inactivation of Aurora A and B, or all Aurora isoforms by nonisoform-selective Aurora inhibitors, demonstrates the Aurora B/C-dominant cytokinesis failure and polyploidy phenotypes. Several Aurora inhibitors are in clinical trials for T/B-cell lymphoma, multiple myeloma, leukemia, lung, and breast cancers. Here, we describe an Aurora A-selective inhibitor, LY3295668, which potently inhibits Aurora autophosphorylation and its kinase activity in vitro and in vivo, persistently arrests cancer cells in mitosis, and induces more profound apoptosis than Aurora B or Aurora A/B dual inhibitors without Aurora B inhibition-associated cytokinesis failure and aneuploidy. LY3295668 inhibits the growth of a broad panel of cancer cell lines, including small-cell lung and breast cancer cells. It demonstrates significant efficacy in small-cell lung cancer xenograft and patient-derived tumor preclinical models as a single agent and in combination with standard-of-care agents. LY3295668, as a highly Aurora A-selective inhibitor, may represent a preferred approach to the current pan-Aurora inhibitors as a cancer therapeutic agent.

Assessment and Clinical Relevance of Serum IL-19 Levels in Psoriasis and Atopic Dermatitis Using a Sensitive and Specific Novel Immunoassay.

Because development of reliable biomarkers in psoriasis and atopic dermatitis has lagged behind therapeutic progress, we created a blood-based test to fill the void in objective methods available for dermatological assessments. Our novel interleukin-19 (IL-19) immunoassay was initially tested to determine concentrations of IL-19 serum levels, then correlated with the psoriasis activity and severity index (PASI) in psoriasis, and the eczema area and severity index (EASI) in atopic dermatitis. Not only was IL-19 increased in psoriasis and correlated to PASI, but ixekizumab administration led to rapid, sustained IL-19 decreases to normal levels, with decreases at 2-weeks correlating with PASI improvement at 16-weeks. IL-19 increased upon ixekizumab withdraw, prior to relapse, and decreased following re-treatment. In baricitinib- and etanercept-treated psoriasis patients, IL-19 decreases also correlated with improvement. Many patients with limited skin disease, including genital psoriasis and psoriatic arthritis patients, also had increased IL-19, which was reduced to normal levels upon ixekizumab treatment, correlating with PASI improvement. We also measured IL-19 in baricitinib-treated atopic dermatitis patients. In atopic dermatitis, IL-19 was significantly elevated, correlated with EASI scores, and decreased with skin improvement. Therefore, measurement of serum IL-19 provides clinicians with an objective disease-activity assessment tool for psoriasis and atopic dermatitis patients.

Modulation of TARP γ8-Containing AMPA Receptors as a Novel Therapeutic Approach for Chronic Pain.

Nonselective glutamate α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonists are efficacious in chronic pain but have significant tolerability issues, likely arising from the ubiquitous expression of AMPA receptors in the central nervous system (CNS). Recently, LY3130481 has been shown to selectively block AMPA receptors coassembled with the auxiliary protein, transmembrane AMPA receptor regulatory protein (TARP) γ8, which is highly expressed in the hippocampus but also in pain pathways, including anterior cingulate (ACC) and somatosensory cortices and the spinal cord, suggesting that selective blockade of γ8/AMPA receptors may suppress nociceptive signaling with fewer CNS side effects. The potency of LY3130481 on recombinant γ8-containing AMPA receptors was modulated by coexpression with other TARPs; γ2 subunits affected activity more than γ3 subunits. Consistent with these findings, LY3130481 had decreasing potency on receptors from rat hippocampal, cortical, spinal cord, and cerebellar neurons that was replicated in tissue from human brain. LY3130481 partially suppressed, whereas the nonselective AMPA antagonist GYKI53784 completely blocked, AMPA receptor-dependent excitatory postsynaptic potentials in ACC and spinal neurons in vitro. Similarly, LY3130481 attenuated short-term synaptic plasticity in spinal sensory neurons in vivo in response to stimulation of peripheral afferents. LY3130481 also significantly reduced nocifensive behaviors after intraplantar formalin that was correlated with occupancy of CNS γ8-containing AMPA receptors. In addition, LY3130481 dose-dependently attenuated established gait impairment after joint damage and tactile allodynia after spinal nerve ligation, all in the absence of motor side effects. Collectively, these data demonstrate that LY3130481 can suppress excitatory synaptic transmission and plasticity in pain pathways containing γ8/AMPA receptors and significantly reduce nocifensive behaviors, suggesting a novel, effective, and safer therapy for chronic pain conditions.

Characterization of a novel AICARFT inhibitor which potently elevates ZMP and has anti-tumor activity in murine models.

AICARFT is a folate dependent catalytic site within the ATIC gene, part of the purine biosynthetic pathway, a pathway frequently upregulated in cancers. LSN3213128 is a potent (16 nM) anti-folate inhibitor of AICARFT and selective relative to TS, SHMT1, MTHFD1, MTHFD2 and MTHFD2L. Increases in ZMP, accompanied by activation of AMPK and cell growth inhibition, were observed with treatment of LY3213128. These effects on ZMP and proliferation were dependent on folate levels. In human breast MDA-MB-231met2 and lung NCI-H460 cell lines, growth inhibition was rescued by hypoxanthine, but not in the A9 murine cell line which is deficient in purine salvage. In athymic nude mice, LSN3213128 robustly elevates ZMP in MDA-MB-231met2, NCI-H460 and A9 tumors in a time and dose dependent manner. Significant tumor growth inhibition in human breast MDA-MB231met2 and lung NCI-H460 xenografts and in the syngeneic A9 tumor model were observed with oral administration of LSN3213128. Strikingly, AMPK appeared activated within the tumors and did not change even at high levels of intratumoral ZMP after weeks of dosing. These results support the evaluation of LSN3213128 as an antineoplastic agent.