Exploring novel targets of sitagliptin for type 2 diabetes mellitus: Network pharmacology, molecular docking, molecular dynamics simulation, and SPR approaches.

Background: Diabetes has become a serious global public health problem. With the increasing prevalence of type 2 diabetes mellitus (T2DM), the incidence of complications of T2DM is also on the rise. Sitagliptin, as a targeted drug of DPP4, has good therapeutic effect for T2DM. It is well known that sitagliptin can specifically inhibit the activity of DPP4 to promote insulin secretion, inhibit islet ß cell apoptosis and reduce blood glucose levels, while other pharmacological mechanisms are still unclear, such as improving insulin resistance, anti-inflammatory, anti-oxidative stress, and anti-fibrosis. The aim of this study was to explore novel targets and potential signaling pathways of sitagliptin for T2DM. Methods: Firstly, network pharmacology was applied to find the novel target most closely related to DPP4. Semi-flexible molecular docking was performed to confirm the binding ability between sitagliptin and the novel target, and molecular dynamics simulation (MD) was carried to verify the stability of the complex formed by sitagliptin and the novel target. Furthermore, surface-plasmon resonance (SPR) was used to explored the affinity and kinetic characteristics of sitagliptin with the novel target. Finally, the molecular mechanism of sitagliptin for T2DM was predicted by the enrichment analysis of GO function and KEGG pathway. Results: In this study, we found the cell surface receptor-angiotensin-converting enzyme 2 (ACE2) most closely related to DPP4. Then, we confirmed that sitagliptin had strong binding ability with ACE2 from a static perspective, and the stability of sitagliptin-ACE2 complex had better stability and longer binding time than BAR708-ACE2 in simulated aqueous solution within 50 ns. Significantly, we have demonstrated a strong affinity between sitagliptin and ACE2 on SPR biosensor, and their kinetic characteristics were "fast binding/fast dissociation". The guiding significance of clinical administration: low dose can reach saturation, but repeated administration was needed. Finally, there was certain relationship between COVID-19 and T2DM, and ACE2/Ang-(1-7)/Mas receptor (MasR) axis may be the important pathway of sitagliptin targeting ACE2 for T2DM. Conclusion: This study used different methods to prove that ACE2 may be another novel target of sitagliptin for T2DM, which extended the application of ACE2 in improving diabetes mellitus.

Multivesicular Liposomes for Glucose-Responsive Insulin Delivery.

An intelligent insulin delivery system is highly desirable for diabetes management. Herein, we developed a novel glucose-responsive multivesicular liposome (MVL) for self-regulated insulin delivery using the double emulsion method. Glucose-responsive MVLs could effectively regulate insulin release in response to fluctuating glucose concentrations in vitro. Notably, in situ released glucose oxidase catalyzed glucose enrichment on the MVL surface, based on the combination of (3-fluoro-4-((octyloxy)carbonyl)phenyl)boronic acid and glucose. The outer MVL membrane was destroyed when triggered by the local acidic and H2O2-enriched microenvironment induced by glucose oxidase catalysis in situ, followed by the further release of entrapped insulin. Moreover, the Alizarin red probe and molecular docking were used to clarify the glucose-responsive mechanism of MVLs. Utilizing chemically induced type 1 diabetic rats, we demonstrated that the glucose-responsive MVLs could effectively regulate blood glucose levels within a normal range. Our findings suggest that glucose-responsive MVLs with good biocompatibility may have promising applications in diabetes treatment.

Development of a sensitive monoclonal antibody-based enzyme-linked immunosorbent assay for the antimalaria active ingredient artemisinin in the Chinese herb Artemisia annua L.

Artemisinin is an endoperoxide sesquiterpene lactone isolated from the Chinese medicinal plant Artemisia annua L. It has been widely used in South-East Asia and Africa as an effective drug against sensitive and multidrug-resistant Plasmodium falciparum malaria. A monoclonal antibody (mAb), designated as 3H2, was generated with artesunate-bovine serum albumin conjugate as the immunogen. mAb 3H2 was used to develop a highly sensitive and specific indirect competitive enzyme-linked immunosorbent assay (icELISA) for artemisinin. The concentration of analyte producing 50% of inhibition (IC(50)) and the working range of the icELISA were 1.3 and 0.2-5.8 ng/mL, respectively. The mAb 3H2 recognized the artemisinin analogs artesunate, dihydroartemisinin, and artemether with cross-reactivity of 650%, 57%, and 3%, respectively, but negligibly recognized deoxyartemisinin and the artemisinin precursors arteannuin B and artemisinic acid. The average recoveries of artemisinin fortified in A. annua samples at concentrations from 156 to 5,000 microg/g determined by icELISA ranged from 91% to 98%. The icELISA was applied for the determination of artemisinin in different wild A. annua samples and the results were confirmed by high-performance liquid chromatography (HPLC) analysis. The correlation coefficient (R(2)) between the two assays was larger than 0.99, demonstrating a good agreement between the icELISA and HPLC results. This ELISA is suitable for quality assurance of A. annua L. materials.

Monoclonal antibody-based enzyme linked immunosorbent assay for the analysis of jasmonates in plants.

Methyl jasmonate (MeJA) and its free-acid form, jasmonic acid (JA) are naturally occurring plant growth regulators widely distributed in higher plants. In order to improve the sensitivity for the analysis of MeJA at low levels in small amounts of plant samples, a monoclonal antibody (MAb) (designated as MAb 3E(5)D(7)C(4)B(6)) against MeJA was derived from a JA-bovine serum albumin (BSA) conjugate as an immunogen. The antibody belongs to the IgG(1) subclass with a kappa type light chain and has a dissociation constant of approximately 6.07 x 10(-9) M. MAb3E(5)D(7)C(4)B(6) is very specific to MeJA. It was used to develop a direct competitive enzyme-linked immunosorbent assay (dcELISA), conventional and simplified indirect competitive ELISAs (icELISA). JA was derivatized into MeJA for the ELISA analysis. The IC(50) value and detection range for MeJA were, respectively, 34 and 4-257 ng/mL by the conventional icELISA, 21 and 3-226 ng/mL by the simplified icELISA and 5.0 and 0.7-97.0 ng/mL by the dcELISA. The dcELISA was more sensitive than either the conventional or simplified icELISA. The assays were used to measure the content of jasmonates as MeJA in tobacco leaves under drought stress or inoculated with tobacco mosaic virus and tomato leaves inoculated with tomato mosaic virus or Lirioinyza sativae Blanchard as compared with the corresponding healthy leaves. The increased jasmonates content indicated its role in response to the drought stress and pathogens.

Development of a monoclonal antibody-based enzyme-linked immunosorbent assay for the herbicide chlorimuron-ethyl.

Hybridomas secreting a monoclonal antibody (mAb) against the herbicide chlorimuron-ethyl (CE) were produced by fusing the mouse myeloma cell line (SP2/0) with splenocytes from a mouse immunized against the conjugate of the sulfonamide moiety of CE and bovine serum albumin (BSA). The mAb, designated 1F5C5A10, had very weak affinity with metsulfuron, ethametsulfuron, pyrazosulfuron, bensulfuron, and chlorsulfuron. Two mAb-based indirect competitive enzyme-linked immunosorbent assays (icELISA) were developed. A conventional icELISA (icELISA-I) showed a concentration of half-maximum inhibition (IC(50)) of 11.6 ng/mL with a dynamic range of 1.6-84 ng/mL. A simplified icELISA (icELISA-II) had an IC(50) of 28.7 ng/mL and a dynamic range of 2.2-372 ng/mL. The two assays were tested on spiked water and soil samples. CE (1-500 ng/mL) fortified in water samples could be analyzed directly without any sample preparation by both immunoassays with an average recovery between 74 and 114%. icELISA-II, but not icELISA-I, was able to accurately analyze the herbicide residues in the crude soil extracts with recoveries between 99 and 129% without obvious matrix effects due to its lesser amount of sample used. In contrast to icELISA-I, icELISA-II is more convenient, whereas it consumes more reagents of coating antigen and goat anti-mouse IgG-peroxidase.

[Resonance light scattering study on interaction of solochrome cyanine R with protein and light scattering determination of trace protein].

The resonance light scattering (RLS) of Solochrome Cyanine R(SCR) is greatly enhanced by proteins. Based on this phenomenon, a novel method for the determination of protein by using SCR as a labeling agent was developed. In the pH 4.0 solution the enhanced intensity of RLS at 400 nm is proportional to the concentration of protein. The linear range for bovine serum albumin (BSA) is 0-5.0 mg x L(-1) and the limit of detection is 44.4 microg x L(-1). The method is simple, rapid, sensitive, stable, and tolerant of many foreign substances. It has been used to determine proteins in human urine samples with satisfactory results.