Optimization of 4,6-Disubstituted Pyrido[3,2-d]pyrimidines as Dual MNK/PIM Inhibitors to Inhibit Leukemia Cell Growth.

Mitogen-activated protein kinase-interacting kinases (MNKs) and provirus integration in maloney murine leukemia virus kinases (PIMs) are downstream enzymes of cell proliferation signaling pathways associated with the resistance of tyrosine kinase inhibitors. MNKs and PIMs have complementary effects to regulate cap-dependent translation of oncoproteins. Dual inhibitors of MNKs and PIMs have not been developed. We developed a novel 4,6-disubstituted pyrido[3,2-d]pyrimidine compound 21o with selective inhibition of MNKs and PIMs. The IC50's of 21o to inhibit MNK1 and MNK2 are 1 and 7 nM and those to inhibit PIM1, PIM2, and PIM3 are 43, 232, and 774 nM, respectively. 21o inhibits the growth of myeloid leukemia K562 and MOLM-13 cells with GI50's of 2.1 and 1.2 µM, respectively. 21o decreases the levels of p-eIF4E and p-4EBP1, the downstream products of MNKs and PIMs, as well as cap-dependent proteins c-myc, cyclin D1, and Mcl-1. 21o inhibits the growth of MOLM-13 cell xenografts without causing evident toxicity. 21o represents an innovative dual MNK/PIM inhibitor with a good pharmacokinetic profile.

Application of sulfoximines in medicinal chemistry from 2013 to 2020.

In recent years, interest in sulfoximine chemistry has been greatly increased. For example, at least three sulfoximine containing drugs BAY 1143572, BAY 1251152 and AZD6738 have entered the clinic. Despite the increasing interest in sulfoximines and their chemistry, the routine application of this structure in drug discovery is still hampered due to limited experience in physicochemical and in vitro parameters of sulfoximines. Therefore, we reviewed all relevant articles from 2013 to the present in terms of potency and pharmacokinetic properties in order to support the addition of the sulfoximine component to the toolbox of medicinal chemists.

The pharmacological efficacy of the anti-IL17 scFv and sTNFR1 bispecific fusion protein in inflammation mouse stimulated by LPS.

Acute lung injury (ALI) is still a leading cause of morbidity and mortality in critically ill patients. Recently, our study found that a bispecific fusion protein treatment can ameliorate the lung injury induced by LPS. However, the molecular mechanisms which bispecific fusion protein ameliorates acute lung injury remain unclear. In this study, we found that the bispecific fusion protein treatment inhibited the nuclear transcription of NF-κB in confocal laser scanning fluorescence microscopy, the bispecific fusion protein exert protective effects in the cell model of ALI induced by lipopolysaccharide (LPS) via inhibiting the nuclear factor κB (NF-κB) signaling pathway and mediate inflammation. Moreover, the treatment of the bispecific fusion protein show its efficacy in animal models stimulated by LPS, the results of real-time PCR and ELISA demonstrate that bispecific fusion protein treatment effectively inhibited the over-expression of inflammatory cytokines(tumor necrosis factor α, interleukin 1ß and interleukin 17). In addition, LPS-challenged mice exhibited significant lung injury characterized by the deterioration of histopathology, which was meliorated by bispecific fusion protein treatment. Collectively, these results demonstrate that bispecific fusion protein treatment ameliorates LPS-induced ALI through reducing inflammatory cytokines and lung inflammation, which may be associated with the decreased the nuclear transcription of NF-κB. The bispecific fusion protein may be useful as a novel therapy to treat ALI.

A recombinant avian antibody against VP2 of infectious bursal disease virus protects chicken from viral infection.

A stable cell-line was established that expressed the recombinant avian antibody (rAb) against the infectious bursal disease virus (IBDV). rAb exhibited neutralization activity to IBDV-B87 strain in DF1 cells. The minimum rAb concentration required for inhibition of the cytopathic effect (CPE) was 1.563µg/mL. To test the efficacy of rAb, a 168-h cohabitation challenge experiment was performed to transmit the disease from the chickens challenged with vvIBDV (HLJ0504 strain) to three test groups of chickens, i.e. (1) chickens treated with rAb, (2) chickens treated with yolk antibody, and (3) non-treatment chickens. The survival rates of chickens treated with rAb, yolk antibody and without treatment were 73%, 67% and 20%, respectively. Another batch of chickens was challenged with IBDV (BC6/85 strain) and then injected with rAb (1.0mg/kg) 6, 24 and 36h post-challenge. Non-treatment chickens had 100% morbidity, whereas those administered with rAb exhibited only 20% morbidity. Morbidity was evaluated using clinical indicators and bursal histopathological section. This study provides a new approach to treating IBDV and the rAb represents a promising candidate for this IBDV therapy.

Screening scFv antibodies against infectious bursal disease virus by co-expression of antigen and antibody in the bacteria display system.

We have previously reported an antigen and antibody co-expression (AAC) technology to demonstrate the interaction between a known antigen and antibody. To validate the co-expression system for screening antibody libraries, a single chain fragment variable(scFv)antibody library was constructed from chickens immunized with the VP2 protein of infectious bursal disease virus (IBDV). The genes of both VP2 and scFv antibodies were inserted into the pBFD-Ab-Ag vector. The co-expression library was subjected to three rounds of screening by flow cytometry (FCM) using a polyclonal antibody against VP2 through a bacteria display system. We achieved enrichment of scFv specific for IBDV. 110 individual clones were initially selected and sequenced. Twenty clones were selected based on fluorescence intensity by FCM. The scFv antibodies were expressed by pET-27b in E.coli and purified. The specificity and affinity of the selected scFv antibodies were confirmed by western blotting assay and ELISA analysis. What's more, the neutralizing capacity was measured with IBDV-B87(100 TCID50) in vitro. Four scFvs (clone 8(1), Y8, L10 and L7) showed significant neutralizing capacity. Two of the four scFvs (clone 8(1) and Y8) demonstrated a higher neutralizing activity to IBDV-B87 and the titers were 16,384 and 8,192, respectively. The two scFvs had higher neutralizing capacity than those obtained in previous studies. We demonstrated that the AAC technology could be applied to screen antibody libraries without baiting antigen to make antibody screening process easier and obtain scFvs with higher neutralizing capacity.

The bispecific antibody aimed at the vicious circle of IL-1ß and IL-17A, is beneficial for the collagen-induced rheumatoid arthritis of mice through NF-κB signaling pathway.

Rheumatoid arthritis (RA) is a chronic, systemic and autoimmune disease with overexpression inflammation cytokines. The biological therapy targeting these inflammatory cytokines has been applied for the clinic. Drugs aimed at a single target are ineffective in some patients with RA. However, double-target and multi-target drugs have huge advantages in therapy. Interleukin-1ß (IL-1ß) and Interleukin-17A (IL-17A) are the keys to inflammatory factors in RA. The bispecific antibody(BsAb)against both human IL-1ß and human IL-17A was formed and expressed in E. coli. The binding specificity and efficiency of the BsAb was tested by enzyme-linked immunosorbent assay, Western blotting and several cells experiments including THP-1, 3T3-L1 and L929 in vitro. Different doses of BsAb (5, 2, 0.8mg/kg) were compared in collagen-induced arthritis (CIA) mice, with Adalimumab and Dexamethasone as the positive control. The effects on mice were determined by the degree of arthritis severity, cytokines levels, the level of IgG against CII and rheumatoid factor level in serum, the transcription levels of relative cytokines in the spleen, and histological damage. Furthermore, the activation of NF-κB was analyzed by Western blotting. In conclusion, BsAb can bind with IL-1ß and IL-17A to alleviate the severity of arthritis, to decrease the levels of inflammation cytokines in serum, to down-regulate the expression of IL-1ß, IL-2, IL-6, IL-17A, TNF-α, IFN-γ, and MMP-3, to up-regulate the expression of IL-10, to relieve histological damage and to inhibit bone destruction. BsAb inhibited nuclear translocation of the p65 subunit and cytoplasm IκB-α degradation by blocking IL-1ß and IL-17A, the upstream of NF-κB pathway. High doses of BsAb had a more beneficial effect on CIA mice than Adalimumab and Dexamethasone. Thus, these results indicate that BsAb can be regarded as an ideal candidate for RA therapy.

A novel function for fibroblast growth factor 21: stimulation of NADPH oxidase-dependent ROS generation.

Fibroblast growth factor 21 (FGF-21) is a major paracrine and endocrine regulator of metabolic homeostasis. Here we demonstrate that FGF-21 is also a potent mediator of innate immunity. Double-staining flow cytometry identified neutrophils and monocytes as the main sources of FGF-21 among circulating leukocytes. Functional assays showed that FGF-21 stimulates phagocytosis and production of reactive oxygen species in neutrophil-like HL-60 cells and monocytic THP-1 cells. The mechanism of action of FGF-21 was observed to involve FGF receptor activation, signal transduction through the PI3K/Akt pathway, and stimulation of NADPH oxidase activity. This study indicates that FGF-21 could be an attractive target for the management of inflammatory disorders.