Evaluation and mechanism study of Pien Tze Huang against EV-A71 infection.

Hand, foot, and mouth disease (HFMD) caused by enterovirus A71 (EV-A71) infection, currently lacks specific preventive and therapeutic interventions. Here, we demonstrated that Pien Tze Huang (PZH) could dose-dependently inhibit EV-A71 replication at the cellular level, resulting in significant reductions in EV-A71 virus protein 1 (VP1) expression and viral yields in Vero and human rhabdomyosarcoma cells. More importantly, we confirmed that PZH could protect mice from EV-A71 infection for the first time, with Ribavirin serving as a positive control. PZH treatment reduced EV-A71 VP1 protein expression, viral yields in infected muscles, and improved muscle pathology. Additionally, we conducted a preliminary mechanism study using quantitative proteomics. The results suggested that the suppression of the PI3K/AKT/mTOR and NF-κB signaling pathways may contribute to the anti-EV-A71 activity of PZH. These findings provide strong evidence supporting the potential therapeutic application of PZH for EV-A71 infection management.

Comprehensive 2D-Quantitative Structure-Activity Relationship Study on Monobactam Analogues Against Gram-Negative Bacteria.

Human health has been severely affected by infections resulting from multidrug-resistant (MDR) gram-negative bacteria (GNB). Monobactam antibiotics are known to be effective against such infections. This study aimed to construct a predictive two-dimensional quantitative structure-activity relationship (2D-QSAR) model for the rational design of new monobactams based on the 65 known monobactams against Escherichia coli (Eco) and Klebsiella pneumonia (Kpn) strains using the kernel partial least squares regression (KPLS) algorithm. The total performance of Eco and Kpn KPLS modes was shown as RMSE: 0.681/0.596, R²: 0.946/0.882, Q²: 0.922/0.877, and RMSU: 0.625/0.593. Thirty-four monobactams reported in our lab were chosen as external data to predict their activities against Eco and Kpn using the newly established models, by which the R² between the experimental and predicted values was 0.878 and 0.871, respectively. The models developed and verified in this study provide a powerful design strategy for novel monobactams that are effective against MDR gram-negative bacterial infections.

Identification of a new isomer from a reversible isomerization of ceftriaxone in aqueous solution.

A reversible isomerization of ceftriaxone in aqueous solution was observed, and the structure of the isomer was determined by mass spectrometry and various 1D and 2D NMR techniques. The mechanism of isomerization was also discussed. Finally, molecular docking simulations were performed and the antimicrobial activities of the isomers were measured. This showed that the biological activity of ceftriaxone was stronger than that of its isomer. The results reported in this article may be important to quality control requirements and to the stability of ceftriaxone products.

Isolation, identification and characterization of related substances in furbenicillin.

Furbenicillin is a broad-spectrum semisynthetic penicillin with strong antibacterial activity against Gram-negative bacteria. In this study, three impurities in furbenicillin, including an unknown epimer, were determined. On the basis of a complete analysis of the spectrum (MS, (1)H,(13)C, 2D NMR and CD) and the results of chemical methods, the unknown epimer impurity was identified as 10-epi-furbenicillin (impurity 1). Isolation and structure elucidation of impurity 1 was also reported here for the first time.

Characterization of impurities in cefpodoxime proxetil using LC-MS (n).

Reversed-phase liquid chromatography coupled with electrospray ionization tandem mass spectrometry (ESI-MS/MS) was used to characterize impurities in cefpodoxime proxetil, an ester-modified prodrug. Based on the mechanisms by which cephalosporins are degraded, stress tests were designed and performed. The bulk material and capsule were eluted through a C18 column with formic acid-methanol-water as the mobile phase. In total, 15 impurities were characterized in commercial samples, including 7 known impurities and 8 new impurities. The structures of these unknown compounds were deduced via comparison with the fragmentation patterns of cefpodoxime proxetil. Data from this systematic study will help improve the safety and quality of cefpodoxime proxetil.

Toxic effects of cephalosporins with specific functional groups as indicated by zebrafish embryo toxicity testing.

Cephalosporins, derivatives of 7-aminocephalosporanic acid (7-ACA), are potent antibacterial agents. The toxicity prediction of these compounds is of considerable importance in new drug development. Zebrafish embryo toxicity testing was thought to be suitable for evaluation of the toxic properties of cephalosporins. Here, five kinds of cephalosporins and their isomers were used for investigation of the toxic functional groups of cephalosporins and for further evaluation of the efficacy of zebrafish embryo toxicity testing. Computational chemistry methods were also used to study the conformations of the stereoisomers of cephalosporins in aqueous solution to explore the relationship between the stereoisomers and the experimental results of toxicity tests on zebrafish embryos. Our results suggest that both the C-7 and C-3 substituents of cephalosporins are toxic functional groups. The toxic functional groups increase the toxic reaction of 7-ACA and can induce variable abnormal phenotypes in zebrafish embryo toxicity testing. The embryonic toxicities of cephalosporins were involved in organogenesis, mainly in the development of the cranial nerve, cardiovascular system, notochord and abdomen, and pigment formation; those tissues and organs are derived from ectoderm, mesoderm, and endoderm. The theoretical calculations showed a strong negative correlation between topological polar surface area (TPSA) values and the toxic effect, which indicated that molecular polarity may be crucial to the toxic effects of the isomers of cephalosporins. The concept of toxic functional groups may help us understand the safety differences of cephalosporins.

[Identification of impurity peaks in the HPLC chromatogram by LC-MS and two-dimensional chromatographic correlation spectroscopy].

A novel qualitative analytical method by using two-dimensional chromatographic correlation spectroscopy techniques for recognizing impurity peaks of HPLC methods of quality control and LC-MS chromatographic system was established. The structures of major degradation products of ceftizoxime and cefdinir were identified by LC-MS and MassWorks application; the standard chromatographic and spectral data of the degradation impurities were obtained by high-performance liquid chromatography with diode array detection. The impurity peaks of two-dimensional chromatography were matched by comparison of spectra and calculating correlation coefficients. Peaks in chromatography can be identified accurately and rapidly in different chromatographic systems such as column and mobile phase changed. The method provides a new way and thought to identify the peaks in quality control of impurities without reference impurity substances.

On the isomerisation of cefozopran in solution.

In this study, we first described the two configurations of cephalosporins by cefozopran, then determined their molecular structure and finally evaluated the stability and biological activity of the two configurations. Our results showed that cefozopran existed as two different configurations in the aqueous solution with acetonitrile. Using mass spectrometry with liquid chromatography, nuclear magnetic resonance with liquid chromatography and optical rotation detection technology, we determined the spatial structures of both configurations and the detailed mechanism for change. By molecular docking and determining their antimicrobial activities, we showed that the biological activity of cis-isomer was stronger than that of trans-isomer.

Influence of substituent groups at the 3-position on the mass spectral fragmentation pathways of cephalosporins.

The structural fragment ions of nine cephalosporins were studied by electrospray ionization quadrapole trap mass spectrometry (Q-Trap MS(n)) in positive mode. The influence of substituent groups in the 3-position on fragmentation pathway B, an alpha-cleavage between the C7-C8 single bond, coupled with a [2,4]-trans-Diels-Alder cleavage simultaneously within the six-membered heterocyclic ring, was also investigated. It was found that when the substituent groups were methyl, chloride, vinyl, or propenyl, fragmentations belonging to pathway B were detected; however, when the substituents were heteroatoms such as O, N, or S, pathway B fragmentation was not detected. This suggested that the [M-R(3)](+) ion, which was produced by the bond cleavage within the substituent group at the 3-position, had a key influence on fragmentation pathway B. This could be attributed to the strong electronegativity of the heteroatoms (O, N, S) that favors the production of the [M-R(3)](+) ion. Moreover, having the positive charge of the [M-R(3)](+) ion localized on the nitrogen atom in the 1-position changed the electron density distribution of the heterocyclic structure, which prohibits a [2,4]-reverse-Diels-Alder fragmentation and as a result fragmentation pathway B could not occur. The influence of the substituent group in the 3-position was determined by the intensity ratio (e/d) of ions produced by fragmentation pathway A, a [2,2]-trans-Diels-Alder cleavage within the quaternary lactam ring, including the breaking of the amide bond and the C6-C7 single bond (ion d), and fragmentation pathway B (ion e). The results indicate that the electronegativity of the substituent group was a key influencing factor of pathway B fragmentation intensity, because the intensity ratio (e/d) is higher for a chlorine atom, a vinyl, or a propenyl group than that of a methyl group. This study provided some theoretical basis for the identification of cephalosporin antibiotics and structural analysis of related substances in drugs.