Study of the interactions of a novel monoclonal antibody, mAb059c, with the hPD-1 receptor.

Programmed cell death 1 (PD-1) monoclonal antibodies have been approved by regulatory agencies for the treatment of various types of cancer, and the mechanism involves the restoration of T cell functions. We report herein the X-ray crystal structure of a fully human monoclonal antibody mAb059c fragment antigen-binding (Fab) in complex with the PD-1 extracellular domain (ECD) at a resolution of 1.70 Å. Structural analysis indicates 1) an epitope, comprising fragments from the C'D, BC and FG loops of PD-1, contributes to mAb059c interaction, 2) an unique conformation of the C'D loop and a different orientation of R86 enabling the capture of PD-1 by the antibody complementarity determining region (CDR) and the formation of one salt-bridge contact - ASP101(HCDR3):ARG86(PD-1), and 3) the contact of FG with light chain (LC) CDR3 is maintained by a second salt-bridge and two backbone hydrogen bonds. Interface analysis reveals that N-glycosylation sites 49, 74 and 116 on PD-1 do not contact mAb059c; while N58 in the BC loop is recognized by mAb059c heavy chain CDR1 and CDR2. Mutation of N58 attenuated mAb059c binding to PD-1. These findings and the novel anti-PD-1 antibody will facilitate better understanding of the mechanisms of the molecular recognition of PD-1 receptor by anti-PD-1 mAb and, thereby, enable the development of new therapeutics with an expanded spectrum of efficacy for unmet medical needs.

Swainsonine induces apoptosis of rat cardiomyocytes via mitochondria­mediated pathway.

Swainsonine is an Astragalus membranaceus extract. It is indole, alkaloid, and soluble in water. Its effect on rat cardiomyocytes apoptosis, and the mechanisms underlying that effect, were investigated by inducing apoptosis in H9c2 cells. This was detected by MTT assay, Annexin V-FITC/propidium iodide double staining and western blotting. Flow cytometry and fluorescence microscopy were used to confirm swainsonine's effect on mitochondrial membrane potential and levels of reactive oxygen species, while an ATP-dependent bioluminescence assay kit served to find the ATP contents. Assessment was also carried out for peroxisome proliferator activated receptor γ co-activator 1α (PGC-1α) expression levels as well as those of such apoptosis-associated proteins as Cytochrome c, Caspase-3, B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax). Overall, indications were that swainsonine may have the potential to inhibit viability of cells, decrease expression of PGC-1α, induce mitochondrial dysfunction, upregulate Cytochrome c, Bax and Caspase-3, and downregulate Bcl-2. The suggestion would be that apoptosis may be induced through signalling pathways in H9c2 cells mediated by mitochondria.

Novel substituted pyrazolone derivatives as AMP-activated protein kinase activators to inhibit lipid synthesis and reduce lipid accumulation in ob/ob mice.

Non-alcoholic fatty liver disease (NAFLD) is a clinical syndrome characterized by hepatic steatosis. NAFLD is closely linked to obesity, insulin resistance and dyslipidemia. AMP-activated protein kinase (AMPK) functions as an energy sensor and plays a central role in regulating lipid metabolism. In this study, we identified a series of novel pyrazolone AMPK activators using a homogeneous time-resolved fluorescence assay (HTRF) based on the AMPKα2ß1γ1 complex. Compound 29 (C29) is a candidate compound that directly activated the kinase domain of AMPK with an EC50 value of 2.1-0.2 µmol/L and acted as a non-selective activator of AMPK complexes. Treatment of HepG2 cells with C29 (20, 40 µmol/L) dose-dependently inhibited triglyceride accumulation. Chronic administration of C29 (10, 30 mg/kg every day, po, for 5 weeks) significantly improved lipid metabolism in both the liver and the plasma of ob/ob mice. These results demonstrate that the AMPK activators could be part of a novel treatment approach for NAFLD and associated metabolic disorders.

Discovery, synthesis, and structure-activity relationships of 20(S)-protopanaxadiol (PPD) derivatives as a novel class of AMPKα2ß1γ1 activators.

Adenosine 5'-monophosphate-activated protein kinase (AMPK) has been demonstrated as a promising drug target due to its regulatory function in glucose and lipid metabolism. 20(S)-protopanoxadiol (PPD) was firstly identified from high throughput screening as a small molecule activator of AMPK subtype α2ß1γ1. In order to enhance its potency on AMPK, a series of PPD derivatives were synthesized and evaluated. Structure-activity relationship study showed that the amine derivatives at the 24-position (groups I-VI) can improve the potency (EC50: 0.7-2.3 µM) and efficacy (fold: 2.5-3.8). Among them, compounds 12 and 13 exhibited the best potency (EC50: 1.2 and 0.7 µM) and efficacy (fold: 3.7 and 3.8). Further study suggested the mechanism of AMPK activation may functioned at the allosteric position, resulting the inhibition of the lipid synthesis in HepG2 cell model.

Novel small-molecule AMPK activator orally exerts beneficial effects on diabetic db/db mice.

AMP-activated protein kinase (AMPK), which is a pivotal guardian of whole-body energy metabolism, has become an attractive therapeutic target for metabolic syndrome. Previously, using a homogeneous scintillation proximity assay, we identified the small-molecule AMPK activator C24 from an optimization based on the original allosteric activator PT1. In this paper, the AMPK activation mechanism of C24 and its potential beneficial effects on glucose and lipid metabolism on db/db mice were investigated. C24 allosterically stimulated inactive AMPK α subunit truncations and activated AMPK heterotrimers by antagonizing autoinhibition. In primary hepatocytes, C24 increased the phosphorylation of AMPK downstream target acetyl-CoA carboxylase dose-dependently without changing intracellular AMP/ATP ratio, indicating its allosteric activation in cells. Through activating AMPK, C24 decreased glucose output by down-regulating mRNA levels of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) in primary hepatocytes. C24 also decreased the triglyceride and cholesterol contents in HepG2 cells. Due to its improved bioavailability, chronic oral treatment with multiple doses of C24 significantly reduced blood glucose and lipid levels in plasma, and improved the glucose tolerance of diabetic db/db mice. The hepatic transcriptional levels of PEPCK and G6Pase were reduced. These results demonstrate that this orally effective activator of AMPK represents a novel approach to the treatment of metabolic syndrome.

[Intervening effect of naoxintong on anti-platelet treatment with aspirin].

OBJECTIVE: To investigate the effect of small dosage aspirin on platelet biochemical indexes in patients with cardio-cerebrovascular diseases and the intervening action of Naoxintong (NXT). METHODS: The blood levels of P-selectin (P), thrombin B2 (TXB2), and platelet aggregation (PAG) induced by arachidonic acid (AA) and adenosine diphosphate (ADP) were determined in 145 patients with cardio-cerebrovascular diseases (diabetes mellitus, hypertension, coronary heart disease and cerebral infarction), after they were medicated with aspirin 100 mg per day for 7 days. Then they were randomly assigned to the aspirin group and the NXT group Both groups took aspirin 100 mg per day continually, but to patients in the NXT group, NXT 9 tablets per day was given additionally. The blood levels of above-mentioned biochemical indexes were re-examined 1 month after medication. RESULTS: The first determination showed the plasma level of P-selectin and TXB2 concentration were positively correlated with PAG, either induced by AA (r = 0.449, P < 0.01 and r = 0.576, P < 0.01) or by ADP (r = 0.525, P < 0.01; r = 0.501, P < 0.01). Positive correlation also showed between plasma level of P-selectin and TXB2 (r = 0.610, P < 0.01). There was no significant difference of all the three indexes between the two groups (P > 0.05). Re-examination showed that levels of the 3 indexes significantly decreased in both groups (P < 0.01), and all were lower in the NXT group than in the aspirin group respectively (P < 0.05). There was no significant difference in the incidence of adverse reaction between two groups (P > 0.05). CONCLUSION: The anti-platelet effect of one-week administration of aspirin for patients with cardio-cerebrovascular diseases can not be optimal, the combination with NXT could enhance the effect without increase of adverse reaction.