Identification and Validation of Active Ingredient in Cerebrotein Hydrolysate-I Based on Pharmacokinetic and Pharmacodynamic Studies.

Cerebrotein hydrolysate-1 (CH-1), a mixture of small peptides, polypeptides, and various amino acids derived from porcine brain, has been widely used in the treatment of cerebral injury. However, the bioactive composition and pharmacokinetics of CH-1 are still unexplored because of their complicated composition and relatively tiny amounts in vivo. Herein, NanoLC Orbitrap Fusion Lumos Tribrid Mass Spectrometer was firstly used to qualitatively analyze the components of CH-1. A total of 1347 peptides were identified, of which 43 peptides were characterized by high mass spectrometry (MS) intensity and identification accuracy. We then innovatively synthesized four main peptides for activity verification, and the results suggested that Pep72 (NYEPPTVVPGGDL) had the strongest neuroprotective effect on both in vivo and in vitro models. Next, a quantitative method for Pep72 was established based on liquid chromatography tandem mass spectrometry (LC-MS/MS) with the aid of Skyline software and then used in its pharmacokinetic studies. The results revealed that Pep72 had a high elimination rate and low exposure in rats. In addition, a hCMEC/D3-based in vitro model was built and firstly used to investigate the transport of Pep72. We found that Pep72 had extremely low blood-brain barrier permeability and was not a substrate of efflux transporters. The biotransformation of Pep72 in rat fresh plasma and tissues was investigated to explore the contradiction between pharmacokinetics and efficacy. A total of 11 main metabolites were structurally identified, with PGGDL and EPPTVPGGDL being the main metabolites of Pep72. Notably, metalloproteinase and cysteine protease were confirmed to be the main enzymes mediating Pep72 metabolism in rat tissues. SIGNIFICANCE STATEMENT: The NanoLC Orbitrap Fusion Lumos Tribrid Mass Spectrometer was firstly applied to discover the components of CH-1, and one main peptide Pep72 (NYEPPTVVPGGDL) was innovatively synthesized and firstly found to have the strongest neuroprotective effect among 1347 peptides identified from CH-1. Our study is the first time to identify and verify the active ingredient of CH-1 from the perspective of pharmacokinetics and pharmacodynamics, and provides a systematic technical platforms and strategies for the active substance research of other protein hydrolysates.

Strategies for mapping protein hydrolysate profiles and pharmacokinetics based on non-targeted proteomics combining skyline-aided quantitative techniques.

Numerous works have been focused on the bioactivities of protein hydrolysates (PHs) and their application in food or drug formulations, but their composition and pharmacokinetics have never been addressed due to their complex constitutes, short half-life, extremely low concentrations and lack of authentic standards. The present study aims to develop systematic analytical strategy and technical platform with optimized sample preparation, separation and detection protocols for PHs. Lineal peptides (LPs), extraction of the spleen of healthy pigs or calves, were used as cases. First, solvents with polarity gradients were used to globally extract peptides of LP from biological matrix. Non-targeted proteomics based on a high-resolution MS system was used to establish a reliable qualitative analysis workflow for PHs. Based on the developed approach, 247 unique peptides were identified using NanoLC-Orbitrap-MS/MS, and then further verified on the MicroLC-Q-TOF/MS system. In the quantitative analysis workflow, Skyline software was used to predict and optimize the LC-MS/MS detection parameters of LPs followed by investigating the linearity and precision of the developed analytical assay. Note worthily, we innovatively prepared calibration curves by sequential dilution of LP solution to overcome the bottleneck of lacking authentic standards and complex PH composition. All the peptides exhibited good linearity and precision in biological matrix. The established qualitative and quantitative assays were successfully applied to study the distribution characteristics of LPs in mice, and would be conductive to systematically map the profile and pharmacokinetics of peptides in various PHs in vivo and in vitro.

Human dendritic cell targeting peptide can be targeted to porcine dendritic cells to improve antigen capture efficiency to stimulate stronger immune response.

Dendritic cells (DCs) play a key role in the natural recognition of pathogens and subsequent activation of adaptive immune responses due to their potent antigen-presenting ability. Dendritic cell-targeting peptide (DCpep) is strongly targeted to DCs, which often express antigens, to enhance the efficacy of vaccines. Our previous study showed that recombinant Lactobacillus expressing human DCpep could significantly induce stronger immune responses than recombinant Lactobacillus without DCpep, but the mechanism remains unclear. In this study, the mechanism by which DCpep enhances the immune response against recombinant Lactobacillus was explored. Fluorescence-labeled human DCpep was synthesized to evaluate the binding ability of human DCpep to porcine monocyte-derived dendritic cells (Mo-DCs) and DCs of the small intestine. The effects of Mo-DC function induced by recombinant Lactobacillus expressing human DCpep fused with the porcine epidemic diarrhea virus (PEDV) core neutralizing epitope (COE) antigen were also investigated. The results showed that human DCpep bind to porcine DCs, but not to porcine small intestinal epithelial cells. Human DCpep can also improve the capture efficiency of recombinant Lactobacillus by Mo-DCs, promote the maturation of dendritic cells, secrete more cytokines, and enhance the ability of porcine DCs to activate T-cell proliferation. Taken together, these results promote advanced understanding of the mechanism by which DCpep enhances immune responses. We found that some DCpeps are conserved between humans and pigs, which provides a theoretical basis for the development of a DC-targeted vaccine.

Design, synthesis and biological evaluation of novel 2-phenyl pyrimidine derivatives as potent Bruton's tyrosine kinase (BTK) inhibitors.

BTK is an effective target for the treatment of B-cell malignant tumors and autoimmune diseases. In this work, a series of 2-phenyl pyrimidine derivatives were prepared and their preliminary in vitro activities on B-cell leukemia cells as well as the BTK enzyme were determined. The results showed that compound 11g displayed the best inhibitory activity on BTK with an inhibition rate of 82.76% at 100 nM and excellent anti-proliferation activity on three B-cell leukemia lines (IC50 = 3.66 µM, 6.98 µM, and 5.39 µM against HL60, Raji and Ramos, respectively). Besides, the flow cytometry analysis results indicated that 11g inhibited the proliferation of the Raji cells in a dose- and time-dependent manner, and blocked the Ramos cells at the G0/G1 phase, which is in accordance with the positive control ibrutinib. The mechanism investigation demonstrated that 11g could inhibit the phosphorylation of BTK and its downstream substrate phospholipase γ2 (PLCγ2). All these results showed that 11g was a promising lead compound that merited further optimization as a novel class of BTK inhibitor for the treatment of B-cell lymphoblastic leukemia.

Optimization of novel benzofuro[3,2-b]pyridin-2(1H)-one derivatives as dual inhibitors of BTK and PI3Kδ.

BTK and PI3Kδ play crucial roles in the progression of leukemia, and studies confirmed that the dual inhibition against BTK and PI3Kδ could provide superior anticancer agents to single targeted therapies. Herein, a new series of novel benzofuro[3,2-b]pyridin-2(1H)-one derivatives were optimized based on a BTK/PI3Kδ inhibitor 2 designed by our group. Biological studies clarified that compound 6f exhibited the most potent inhibitory activity (BTK: IC50 = 74 nM; PI3Kδ: IC50 = 170 nM) and better selectivity than 2. Moreover, 6f significantly inhibited the proliferation of Raji and Ramos cells with IC50 values of 2.1 µM and 2.65 µM respectively by blocking BTK and PI3K signaling pathways. In brief, 6f possessed of the potency for further optimization as an anti-leukemic drug by inhibiting BTK and PI3Kδ kinase.