PTP1B Inhibitors Research Articles

Germacrane sesquiterpenes from leaves of Eupatorium chinense inhibit protein tyrosine phosphatase

Three new germacrane-type sesquiterpene lactones (1–3) were isolated alongside seven known related congeners (4–10) from the leaves of Eupatorium chinense L. (Compositae). The planar structures of 1–3 were elucidated by their spectroscopic data, including 1D and 2D NMR spectra. The relative and absolute configurations of 1–3 were determined using NOESY experiments and electronic circular dichroism analyses. Compounds 1, 4, 5, and 7 inhibited protein tyrosine phosphatase (PTP) 1B activity with IC50 values of 25, 11, 28, and 24 μM, respectively. Among these, compound 4 exhibited an inhibitory effect on T-cell PTP (TCPTP) with an IC50 value of 25 μM. To our knowledge, this is the first study demonstrating the PTP inhibitory activity of the germacrane sesquiterpenes. The results show that compound 4 acts as an inhibitor of both PTP1B and TCPTP.

Curcumin and Its New Derivatives: Correlation between Cytotoxicity against Breast Cancer Cell Lines, Degradation of PTP1B Phosphatase and ROS Generation.

Breast cancer is the most common cancer of women—it affects more than 2 million women worldwide. PTP1B phosphatase can be one of the possible targets for new drugs in breast cancer therapy. In this paper, we present new curcumin derivatives featuring a 4-piperidone ring as PTP1B inhibitors and ROS inducers. We performed cytotoxicity analysis for twelve curcumin derivatives against breast cancer MCF-7 and MDA-MB-231 cell lines and the human keratinocyte HaCaT cell line. Furthermore, because curcumin is a known antioxidant, we assessed antioxidant effects in its derivatives. For the most potent cytotoxic compounds, we determined intracellular ROS and PTP1B phosphatase levels. Moreover, for curcumin and its derivatives, we performed real-time microscopy to observe the photosensitizing effect. Finally, computational analysis was performed for the curcumin derivatives with an inhibitory effect against PTP1B phosphatase to assess the potential binding mode of new inhibitors within the allosteric site of the enzyme. We observed that two tested compounds are better anticancer agents than curcumin. Moreover, we suggest that blocking the -OH group in phenolic compounds causes an increase in the cytotoxicity effect, even at a low concentration. Furthermore, due to this modification, a higher level of ROS is induced, which correlates with a lower level of PTP1B.

Tridepsides from the endophytic fungus colletotrichum gloeosporioides associated with a toxic medicinal plant tylophora ovata

Abstract Introduction Bioactive secondary metabolites from the microbes living in frigid, toxic or other extreme environments are emerging as a new medicinal resource. Here, we report the discovery of new antidiabetic and anti-inflammatory compounds with novel structures from endophytic fungi hosted toxic medicinal plant. Methods The endophytic fungus isolated from toxic plants was fermented and extracted. The obtained extracts were purified with preparative HPLC to yield pure compounds. The purified compounds were examined by PTP1b inhibition and NO inhibition assays to evaluate their bioactivities. Results One new tridepsides (Compound 1), one new benzeneacetic acid derivative (Compound 3) and five known compounds (Compounds 2 and 4-7) were isolated from the ethyl acetate extract of Colletotrichum gloeosporioides, an endophytic fungus obtained from a toxic medicinal plant Tylophora ovata. Their structures were determined by spectroscopic data (1D and 2D NMR, HRESIMS) analyses. Compound 2 showed significant inhibitory activity against PTP1b with an IC50 value of 0.84 μM. Compounds 2 and 3 exhibited moderate inhibitory activities against the NO (nitric oxide) release in LPS-induced RAW 264.7 cells at 10 μM with percent inhibition of 39% and 33%, respectively. Conclusion The Compound 2 has potent PTP1b inhibitory effect indicating its antidiabetic potential and thus might be considered a lead compound for antidiabetic drug development.

Dual High-Resolution α-Glucosidase and PTP1B Inhibition Profiling Combined with HPLC-PDA-HRMS-SPE-NMR Analysis for the Identification of Potentially Antidiabetic Chromene Meroterpenoids from Rhododendron capitatum.

Thirteen previously undescribed chromene meroterpenoids, capitachromenic acids A-M (3-6, 7a, 7b, 8a, 8b, 9a, 9b, 10a, 10b, and 11b), were identified from an ethyl acetate extract of Rhododendron capitatum, using dual high-resolution α-glucosidase and PTP1B inhibition profiling in combination with HPLC-PDA-HRMS-SPE-NMR. In addition, one known chromene meroterpenoid, daurichromenic acid (15), and its biosynthetic precursor, grifolic acid (12), two C-methylated flavanones, (2S)-5,7,4'-trihydroxy-8-methylflavanone (1) and farrerol (2), and two triterpenoids, oleanolic acid (14a) and ursolic acid (14b), were identified. New structures were elucidated by extensive 1D and 2D NMR analysis, and absolute configurations of new chromene meroterpenoids were assigned by analysis of their ECD spectra on the basis of the empirical chromane helicity rule and from Rh2(OCOCF3)4-induced ECD spectra by applying the bulkiness rule. Compounds 5, 9a, 9b, 12, and 15 showed α-glucosidase inhibitory activity with IC50 values ranging from 8.0 to 93.5 μM, while compounds 3, 5, 8b, 9a, 9b, 10b, 11b, 12, and 15 showed PTP1B inhibitory activity with IC50 values ranging from 2.5 to 68.1 μM.

Protein tyrosine phosphatase 1B inhibition improves endoplasmic reticulum stress‑impaired endothelial cell angiogenic response: A critical role for cell survival.

Endoplasmic reticulum (ER) stress contributes to endothelial dysfunction, which is the initial step in atherogenesis. Blockade of protein tyrosine phosphatase (PTP)1B, a negative regulator of insulin receptors that is critically located on the surface of ER membrane, has been found to improve endothelial dysfunction. However, the role of ER stress and its related apoptotic sub‑pathways in PTP1B‑mediated endothelial dysfunction, particularly its angiogenic capacity, have not yet been fully elucidated. Thus, the present study aimed to investigate the impact of PTP1B suppression on ER stress‑mediated impaired angiogenesis and examined the contribution of apoptotic signals in this process. Endothelial cells were exposed to pharmacological ER stressors, including thapsigargin (TG) or 1,4‑dithiothreitol (DTT), in the presence or absence of a PTP1B inhibitor or small interfering (si)RNA duplexes. Then, ER stress, angiogenic capacity, cell cycle, apoptosis and the activation of key apoptotic signals were assessed. It was identified that the inhibition of PTP1B prevented ER stress caused by DTT and TG. Moreover, ER stress induction impaired the activation of endothelial nitric oxide synthase (eNOS) and the angiogenic capacity of endothelial cells, while PTP1B inhibition exerted a protective effect. The results demonstrated that blockade or knockdown of PTP1B prevented ER stress‑induced apoptosis and cell cycle arrest. This effect was associated with reduced expression levels of caspase‑12 and poly (ADP‑Ribose) polymerase1. PTP1B blockade also suppressed autophagy activated by TG. The current data support the critical role of PTP1B in ER stress‑mediated endothelial dysfunction, characterized by reduced angiogenic capacity, with an underlying mechanism involving reduced eNOS activation and cell survival. These findings provide evidence of the therapeutic potential of targeting PTP1B in cardiovascular ischemic conditions.

Rearranged Diels-Alder Adducts and Prenylated Flavonoids as Potential PTP1B Inhibitors from Morus nigra.

Two novel rearranged Diels-Alder adducts, morunigrines A (1) and B (2), and four new prenylated flavonoids, morunigrols A-D (3-6), were isolated from the twigs of Morus nigra, together with four known prenylated phenolic compounds, including two flavonoids (7 and 8) and two 2-arylbenzofurans (9 and 10). Morunigrines A (1) and B (2) are a novel class of Diels-Alder adducts with unprecedented carbon skeletons featuring a rearranged chalcone-stilbene/2-arylbenzofuran core decorated with a unique methylbiphenyl moiety. The structures of the new compounds were assigned by analysis of spectroscopic data. The absolute configuration of compound 6 was determined by the measurement of specific rotation. A plausible biogenetic pathway for 1 and 2 is also proposed. Compounds 1 and 2 exhibited more potent protein tyrosine phosphatase 1B inhibitory activity with IC50 values of 1.8 ± 0.2 and 1.3 ± 0.3 μM, respectively, than that of the positive control oleanolic acid (IC50, 2.5 ± 0.1 μM).

Pyrrolo[1,2-a]quinoxal-5-inium salts and 4,5-dihydropyrrolo[1,2-a]quinoxalines: Synthesis, activity and computational docking for protein tyrosine phosphatase 1B

Protein tyrosine phosphatase (PTP1B) is an interesting therapeutical target for diabetes, obesity, heart disease and cancer. As such, inhibition of PTP1B using orally administered drugs is still being pursued by academia and pharmaceutical companies. The failure of catalytic-site inhibitors led to the focus in this field being switched to allosteric inhibitors. To date, the non-competitive inhibitors that have reached clinical trials target the site formed by the α3/α6/α7 tunnel or the site found in a disordered C-terminal non-catalytic segment. Herein, pyrrolo[1,2-a]quinoxal-5-inium salts and 4,5-dihydropyrrolo[1,2-a]quinoxalines are synthesized from pyrrolo[1,2-a]quinoxalines by alkylation and reduction, respectively. These compounds showed no toxicity in HepG2 cells and exhibited inhibitory activity against PTP1B, with inhibition percentages of between 37% and 53% at 1 μM and activities (IC50) of between 0.25 and 1.90 μM. The inhibitory activity against T-cell protein tyrosine phosphatase (TC-TPT) was also assayed, with 4,5-dihydropyrrolo[1,2-a]quinoxalines being found to be slightly more active and selective. Compounds from the two series behave as insulin mimetics since they exhibit enhancement of glucose uptake in C2C12 cells. Computational docking studies provide information about the putative binding mode for both series and the preference for the α3/α6/α7 allosteric tunnel.

On the Inhibitability of Natural Products Isolated from Tetradium ruticarpum towards Tyrosine Phosphatase 1B (PTP1B) and α-Glucosidase (3W37): An In Vitro and In Silico Study.

Folk experiences suggest natural products in Tetradium ruticarpum can be effective inhibitors towards diabetes-related enzymes. The compounds were experimentally isolated, structurally elucidated, and tested in vitro for their inhibition effects on tyrosine phosphatase 1B (PTP1B) and α-glucosidase (3W37). Density functional theory and molecular docking techniques were utilized as computational methods to predict the stability of the ligands and simulate interaction between the studied inhibitory agents and the targeted proteins. Structural elucidation identifies two natural products: 2-heptyl-1-methylquinolin-4-one (1) and 3-[4-(4-methylhydroxy-2-butenyloxy)-phenyl]-2-propenol (2). In vitro study shows that the compounds (1 and 2) possess high potentiality for the inhibition of PTP1B (IC50 values of 24.3 ± 0.8, and 47.7 ± 1.1 μM) and α-glucosidase (IC50 values of 92.1 ± 0.8, and 167.4 ± 0.4 μM). DS values and the number of interactions obtained from docking simulation highly correlate with the experimental results yielded. Furthermore, in-depth analyses of the structure–activity relationship suggest significant contributions of amino acids Arg254 and Arg676 to the conformational distortion of PTP1B and 3W37 structures overall, thus leading to the deterioration of their enzymatic activity observed in assay-based experiments. This study encourages further investigations either to develop appropriate alternatives for diabetes treatment or to verify the role of amino acids Arg254 and Arg676.

Polyprenylated acylphloroglucinol meroterpenoids with PTP1B inhibition from Hypericum forrestii

Three new polyprenylated acylphloroglucinol meroterpenoids, hyperiforins A–C (1–3), were isolated from Hypericum forrestii (Chittenden) N. Robson, together with twelve known analogues (4–15). Their structures were established by extensive physical and spectroscopic data analysis. Compounds 1, 2, 5, 7, and 13–15 showed potent inhibitory effects on protein tyrosine phosphatase 1B with IC50 values from 6.63 ± 2.40 to 14.21 ± 3.51 μM.

Tyrosine phosphatase PTP1B impairs presynaptic NMDA receptor-mediated plasticity in a mouse model of Alzheimer's disease

Mutations in the beta-amyloid protein (APP) cause familial Alzheimer's disease. In hAPP-J20 mice expressing mutant APP, pharmacological inhibition or genetic ablation of the tyrosine phosphatase PTP1B prevents CA3 hippocampus neuron loss and cognitive decline. However, how targeting PTP1B affects the cellular mechanisms underlying these cognitive deficits remains unknown. Changes in synaptic strength at the hippocampus can affect information processing for learning and memory. While prior studies have focused on post-synaptic mechanisms to account for synaptic deficits in Alzheimer's disease models, presynaptic mechanisms may also be affected. Here, using whole cell patch-clamp recording, coefficient of variation (CV) analysis suggested a profound presynaptic deficit in long-term potentiation (LTP) of CA3:CA1 synapses in hAPP-J20 mice. While the membrane-impermeable ionotropic NMDA receptor (NMDAR) blocker norketamine in the post-synaptic recording electrode had no effect on LTP, additional bath application of the ionotropic NMDAR blockers MK801 could replicate the deficit in LTP in wild type mice. In contrast to LTP, the paired-pulse ratio and short-term facilitation (STF) were aberrantly increased in hAPP-J20 mice. These synaptic deficits in hAPP-J20 mice were associated with reduced phosphorylation of NMDAR GluN2B and the synaptic vesicle recycling protein NSF (N-ethylmaleimide sensitive factor). Phosphorylation of both proteins, together with synaptic plasticity and cognitive function, were restored by PTP1B ablation or inhibition by the PTP1B-selective inhibitor Trodusquemine. Taken together, our results indicate that PTP1B impairs presynaptic NMDAR-mediated synaptic plasticity required for spatial learning in a mouse model of Alzheimer's disease. Since Trodusquemine has undergone phase 1/2 clinical trials to treat obesity, it could be repurposed to treat Alzheimer's disease.

Discovery of Novel PTP1B Inhibitors Derived from the BH3 Domain of Proapoptotic Bcl-2 Proteins with Antidiabetic Potency.

BH3 peptide analogues are generally believed to exhibit great potency as cancer therapeutics via targeting antiapoptotic Bcl-2 proteins. Here, we describe the synthesis and identification of a new class of palmitoylated peptide BH3 analogues derived from the core region (h1-h4) of BH3 domains of proapoptotic Bcl-2 proteins and as alternative PTP1B inhibitors with antidiabetic potency in vitro and in vivo. PTP1B inhibitors are attractive for treatment of type 2 diabetes. We design the analogues using a simple lipidation approach and discovered novel lead analogues with promising antidiabetic potency in vitro and in vivo. The results presented here expanded the alternative target and function for the BH3 peptide analogues from one member Bim to other members of the proapoptotic Bcl-2 proteins and emphasize their therapeutic potential in T2DM. Furthermore, our findings may provide new proof of the regulatory function of Bcl-2 family proteins in mitochondrial nutrient and energy metabolism.

Ketamine's schizophrenia-like effects are prevented by targeting PTP1B

Subanesthetic doses of ketamine induce schizophrenia-like behaviors in mice including hyperlocomotion and deficits in working memory and sensorimotor gating. Here, we examined the effect of in vivo ketamine administration on neuronal properties and endocannabinoid (eCB)-dependent modulation of synaptic transmission onto layer 2/3 pyramidal neurons in brain slices of the prefrontal cortex, a region tied to the schizophrenia-like behavioral phenotypes of ketamine. Since deficits in working memory and sensorimotor gating are tied to activation of the tyrosine phosphatase PTP1B in glutamatergic neurons, we asked whether PTP1B contributes to these effects of ketamine. Ketamine increased membrane resistance and excitability of pyramidal neurons. Systemic pharmacological inhibition of PTP1B by Trodusquemine restored these neuronal properties and prevented each of the three main ketamine-induced behavior deficits. Ketamine also reduced mobilization of eCB by pyramidal neurons, while unexpectedly reducing their inhibitory inputs, and these effects of ketamine were blocked or occluded by PTP1B ablation in glutamatergic neurons. While ablation of PTP1B in glutamatergic neurons prevented ketamine-induced deficits in memory and sensorimotor gating, it failed to prevent hyperlocomotion (a psychosis-like phenotype). Taken together, these results suggest that PTP1B in glutamatergic neurons mediates ketamine-induced deficits in eCB mobilization, memory and sensorimotor gating whereas PTP1B in other cell types contributes to hyperlocomotion. Our study suggests that the PTP1B inhibitor Trodusquemine may represent a new class of fast-acting antipsychotic drugs to treat schizophrenia-like symptoms.

Enhancement of Endothelialization by Topographical Features Is Mediated by PTP1B-Dependent Endothelial Adherens Junctions Remodeling.

Endothelial Cells (ECs) form cohesive cellular lining of the vasculature and play essential roles in both developmental processes and pathological conditions. Collective migration and proliferation of endothelial cells (ECs) are key processes underlying endothelialization of vessels as well as vascular graft, but the complex interplay of mechanical and biochemical signals regulating these processes are still not fully elucidated. While surface topography and biochemical modifications have been used to enhance endothelialization in vitro, thus far such single-modality modifications have met with limited success. As combination therapy that utilizes multiple modalities has shown improvement in addressing various intractable and complex biomedical conditions, here, we explore a combined strategy that utilizes topographical features in conjunction with pharmacological perturbations. We characterized EC behaviors in response to micrometer-scale grating topography in concert with pharmacological perturbations of endothelial adherens junctions (EAJ) regulators. We found that the protein tyrosine phosphatase, PTP1B, serves as a potent regulator of EAJ stability, with PTP1B inhibition synergizing with grating topographies to modulate EAJ rearrangement, thereby augmenting global EC monolayer sheet orientation, proliferation, connectivity, and collective cell migration. Our data delineates the crosstalk between cell-ECM topography sensing and cell-cell junction integrity maintenance and suggests that the combined use of grating topography and PTP1B inhibitor could be a promising strategy for promoting collective EC migration and proliferation.

Identification of PTP1B regulators from Cymbopogon citratus and its enrichment analysis for diabetes mellitus.

PTP1B is identified as the insulin signaling pathway downregulator; involved in pancreatic β-cell apoptosis. Further, it associates in regulating multiple pathways in diabetes mellitus; kindled us to identify the binding affinity of bioactives from Cymbopogon citratus by targeting PTP1B and identify the probably associated with it; further identifying the probable pathways involved in diabetes mellitus. In this regard, ChEBI database was used to retrieve bio-actives from C. citrates and 3D structures for the same were obtained from the PubChem database. The energy of bioactives was minimized and converted into ligand and the docking was carried using autodock 4.0 against PTP1B. Further, multiple characters of bio-actives like drug-likeness score, ADMET profile, probable adverse effects, and boiled egg model for bioavailability were also studied. Swertiajaponin was predicted for the highest drug-likeness score i.e. 0.26. However, swertiajaponin was predicted with the highest probable side effect of nephrotoxicity with pharmacological activity of 0.478. Similarly, swertiajaponin was predicted for the highest binding affinity with PTP1B with the binding energy of -8.3kcal/mol. Likewise, KEGG identified 80 pathways associated with PTP1B modulation in which 7 pathways were involved in diabetes mellitus in which FoxO signaling pathway was predicted to have the least false discovery rate by modulating 7 genes. Swertiajaponin could act as the potent inhibitor of PTP1B; scored highest druglikeness score but possessed minimum GIT absorptivity; further, PTP1B was identified to be linked with multiple pathways that are concerned with diabetes mellitus.

Water Extract of Mungbean (Vigna radiata L.) Inhibits Protein Tyrosine Phosphatase-1B in Insulin-Resistant HepG2 Cells.

The present study aimed to investigate the effects of mungbean water extract (MWE) on insulin downstream signaling in insulin-resistant HepG2 cells. Whole seed mungbean was extracted using boiling water, mimicking a traditional cooking method. Vitexin and isovitexin were identified in MWE. The results showed that MWE inhibited protein tyrosine phosphatase (PTP)-1B (IC50 = 10 μg/mL), a negative regulator of insulin signaling. MWE enhanced cellular glucose uptake and altered expression of genes involved in glucose metabolism, including forkhead box O1 (FOXO1), phosphoenolpyruvate carboxykinase (PEPCK), and glycogen synthase kinase (GSK)-3β in the insulin-resistant HepG2 cells. In addition, MWE inhibited both α-amylase (IC50 = 36.65 mg/mL) and α-glucosidase (IC50 = 3.07 mg/mL). MWE also inhibited the formation of advanced glycation end products (AGEs) (IC50 = 2.28 mg/mL). This is the first study to show that mungbean water extract increased cellular glucose uptake and improved insulin sensitivity of insulin-resistant HepG2 cells through PTP-1B inhibition and modulating the expression of genes related to glucose metabolism. This suggests that mungbean water extract has the potential to be a functional ingredient for diabetes.

Role of protein tyrosine phosphatase 1B inhibitor in central insulin resistance and associated cognitive deficits

BackgroundProtein tyrosine phosphatase 1B (PTP1B) inhibitors are potential candidates for the treatment of peripheral insulin resistance and diabetes mellitus. Similar to peripheral action within the brain also, PTP1B activation impairs insulin signaling pathways. Activation of PTP1B in brain also accentuates neuroinflammation, oxidative stress and decreases neurotrophic factors in various brain dysfunctions including cognitive decline. ObjectivesThe main objective of our study was to elucidate the role of alendronate, a potent PTP1B inhibitor (blood brain barrier crossing bisphosphonate) in central insulin resistance and associated memory deficits. MethodologyTo induce central insulin resistance, streptozotocin (3 mg/kg) intracerebroventricular (ICV) was administered in two alternate days (1st and 3rd). After 21 days, memory was assessed via using the passive avoidance and Morris water maze paradigm. At the end of behavioral studies, animals were sacrificed to assess a variety of biochemical and molecular parameters in the hippocampus and cerebral cortex region of the brain. Treatment drug alendronate (3 mg/kg/day, p.o) and standard drug donepezil (3 mg/kg/i.p.) were administered from the 3rd day of STZ administration till the end of the study. Inhibition of PTP1B activates phosphoinsotide-3 kinase (PI3 K) (down-stream regulator of insulin signaling pathway).Thus, to illuminate the mechanism of action of alendronate, PI3 K inhibitor, wortmannin was administered in presence of alendronate in one group. ResultsAdministration of alendronate to ICV streprozotocin treated rats resulted in modulation of the insulin signaling pathway and associated behavioral, biochemical and molecular changes in central insulin resistance. However, the protective effect of alendronate was entirely vanished when it was administered in the presence of wortmannin. ConclusionAlendronate can be an important treatment strategy in central insulin signaling pathway dysfunction and associated cognitive deficits. Protective effect of alendronate is via modulation of PI3-K/Akt signaling pathway.

PTP1B Inhibitory and Anti-inflammatory Properties of Constituents from Eclipta prostrata L.

The white-flowered leaves of Eclipta prostrata L. together with leaves of Scoparia dulcis and Cynodon dactylon are mixedly boiled in water and given to diabetic patients resulting in the significant improvement in the management of diabetes. However, the active constituents from this plant for antidiabetic and anti-obesity properties are remaining unclear. Thus, this study was to discover anti-diabetes and anti-obesity activities through protein tyrosine phosphatases (PTP)1B inhibitory effects. We found that the fatty acids (23, 24) showed potent PTP1B inhibition with IC50 values of 2.14 and 3.21 µM, respectively. Triterpenoid-glycosides (12-15) also exhibited strong to moderate PTP1B inhibitory effects, with IC50 values ranging from 10.88 to 53.35 µM. Additionally, active compounds were investigated for their PTP1B inhibitory mechanism and docking analysis. On the other hand, the anti-inflammatory activity from our study revealed that compounds (1-4, 7, 8, 10) displayed the significant inhibition nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Especially, compound 9 showed the potent inhibitory effects in LPS-induced NO production on RAW264.7 cell. Therefore, further Western blot analysis was performed to identify the inhibitory expression including heme oxygenase-1 (HO-1) and inhibitor of kappaB (IκB) phosphorylation.

Chemical profiling and antidiabetic potency of Paeonia delavayi: Comparison between different parts and constituents

Paeonia delavayi (Paeoniaceae), an endemic plant mainly distributed in southwest China, is always used as the substitute of P. suffruticosa due to their morphological and pharmacological similarity. In the previous study, P. suffruticosa was revealed with antidiabetic potency, whereas the chemical difference and antidiabetic property between different parts of P. delavayi has not yet been studied. This paper was designed to clarify the chemical constituents and antidiabetic potency of P. delavayi by LCMS analysis and enzyme inhibition on α-glucosidase, PTP1B, TCPTP, and DPP4. By interpretation of their UV absorptions and MS fragmentations, and/or comparison with reference samples, 57 constituents comprising 15 flavonoids, 10 monoterpene glycosides, eight triterpenoids, seven galloyl glucoses, six N-containing compounds, five gallic acids, two acetophenones, and four other types of compounds were identified from the different parts of P. delavayi. Moreover, two new monoterpene aglycones (42 and 47) and one new noroleanane triterpenoid (51) were speculated by their MS/MS fragmentation rules. Principal component analysis (PCA) suggested the chemical resemblance between root core and root bark which could be well differentiated with the leaves and stems by their characteristic constituents (monoterpene glycosides, flavonoids, and acetophenones). All the four parts (200 μg/mL) showed obvious inhibition on α-glucosidase and PTP1B (81.2%–98.5%), but moderate to weak inhibition on TCPTP and DPP4 (19.5%–34.9%). Nine compounds representing five main types of constituents in Paeonia plants were assayed for their antidiabetic effects, indicating flavonoids and triterpenoids were the main active substances regarding to the four enzymes. Luteolin displayed obvious activity on α-glucosidase, PTP1B, and TCPTP with IC50 values of 94.6, 136.3, and 157.3 μM, and akebonic acid could inhibit α-glucosidase and PTP1B with IC50 values of 73.5 and 57.8 μM. Luteolin and akebonic acid were recognized as competitive inhibitors of α-glucosidase, but anticompetitive and mix-type inhibitors of PTP1B, respectively. Docking study demonstrated akebonic acid as PTP1B (over TCPTP) selective inhibitor by bonding to the catalytic sites (B/C) of PTP1B. This LCMS combined with enzymatic comparison opens new sights for recognizing the chemical profiles and antidiabetic potency of P. delavayi.

MSI-1436 improves EMS adipose derived progenitor stem cells in the course of adipogenic differentiation through modulation of ER stress, apoptosis, and oxidative stress

BackgroundProtein tyrosine phosphatase 1B (PTP1B) is one of the major negative regulators of leptin and insulin signaling, and has been strongly implicated in insulin resistance development in the course of obesity and metabolic syndrome conditions; however, its exact role in controlling adipose tissue biogenesis is still poorly understood.ObjectivesThis investigation aimed to elucidate whether selective inhibition of PTP1B using MSI-1436 compound may improve and restore the defective adipogenicity of ASCs isolated from EMS-affected horses.MethodsEquine ASC EMS cells were cultured under adipogenic conditions in the presence of PTP1B inhibitor and were subsequently tested for expression of the main adipogenic-related genes using RT-qPCR, changes in free fatty acid profiles by means of GC-MS technique, and for mitochondrial dynamics improvement through the analysis of mitochondrial transmembrane potential and oxidative stress.ResultsSelective inhibition of PTP1B in equine ASC EMS cells improved substantially adipogenic differentiation by promoting cellular proliferation and normalizing expression of C/EBPalpha, PPARγ, and Adipoq markers that are critical for proper adipogenesis. Levels of secreted adiponectin and PPARγ were also shown to be increased in MSI-1436-conditioned cells, while total leptin levels markedly dropped under the same conditions. Moreover, MSI-1436 treatment enabled the regulation of metabolic-related transcripts that are crosslink to adipogenesis, namely Akt1, Akt2, and SHBG. The obtained results demonstrated also an obvious reduction in intracellular accumulated ROS and NO, as well as mitigated ER stress through the downregulation of Chop, Perk, Atf6, Ire1, and Xbp1 transcripts upon PTP1B inhibition. Furthermore, general fluctuations in FFA composition of all differentiated groups have been highlighted, where palmitic acid, palmitoleic acid, stearic acid, and linolelaidic acid that are known to be associated with the development of metabolic disorders were found to be normalized upon PTP1B inhibition during adipogenic differentiation.ConclusionThe presented data provides the evidence that the use of PTP1B inhibitor may be successful in controlling and enhancing adipogenic differentiation of impaired equine ASCs affected by metabolic syndrome, and thus offers new insights for the management of obesity through the regulation of adipose tissue dynamics.Graphical abstract

Abstract PO018: Restoring the tumor-suppressed immune response during chemotherapy by targeting Mer:PTP1b interactions

Abstract The development of adaptive immune checkpoint inhibitors, which prevent tumor-mediated inactivation of lymphocytes, has drastically improved outcomes for advanced cancer patients. However, only 20-30% of patients benefit from these treatments and those that do often present moderate toxicity and/or later relapse. After discovering a novel innate immune checkpoint, we have identified a therapeutic strategy to restore the tumor-suppressed immune response during chemotherapy. Here, we show that multiple tumor types (including melanoma, lung, breast and pancreatic cancers) co-cultured with murine primary macrophages suppress the expression of the essential Toll-like Receptor (TLR) signal-transducing adapter protein MyD88 by 57-81% +/- 2-5% (mean +/- SEM). In the absence of MyD88, macrophages are unable to respond to TLR stimulating Damage Associated Molecular Patterns (DAMPs) released by dying tumor cells after chemotherapy, leading to as much as 97% +/- 6% reduction in pro-inflammatory gene expression in our co-culture models. Tumors exert this suppression by activating the macrophage Mer receptor, inducing recruitment of a phosphatase, known as PTP1b, and Stat1 to Mer. Increasing association between PTP1b and Stat1 leads to reduced Stat1 phosphorylation and translocation to the nucleus as well as a downstream reduction in MyD88 expression. Treatment with a pharmacological inhibitor of PTP1b (BVT948) blocks the association of Mer, PTP1b and Stat1, restoring MyD88 expression and responsiveness to chemotherapy-released DAMPs. In chemotherapy-resistant murine syngeneic melanoma models, including B16F10 and GEMM6 (Braf V600E, Pten-/-), we show that combination therapy with BVT948 and Cisplatin or Vemurafenib, respectively, leads to a 47% reduction in tumor growth rates. Treating chemo- and immuno-therapy resistant Lewis Lung Cancer (LLC) tumor-bearing mice with combination BVT948/Cisplatin therapy leads to a similar reduction in tumor growth that is magnified with the addition of adaptive immune checkpoint blockade. In summary, we have described a novel mechanism of cancer-mediated immune suppression that dramatically reduces the immune response during chemotherapy. PTP1b inhibition could represent a novel therapeutic strategy to improve the effectiveness of conventional chemotherapy in patients with advanced malignancies. Citation Format: Nestor Prieto-Dominguez, Eric S. Ubil. Restoring the tumor-suppressed immune response during chemotherapy by targeting Mer:PTP1b interactions [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO018.