Potential anti-liver cancer targets and mechanisms of kaempferitrin based on network pharmacology, molecular docking and experimental verification.

AIM: Kaempferitrin is an active component in Chenopodium ambrosioides, showing medicinal functions against liver cancer. This study aimed to identify the potential targets and pathways of kaempferitrin against liver cancer using network pharmacology and molecular docking, and verify the essential hub targets and pathway in mice model of SMMC-7721 cells xenografted tumors and SMMC-7721 cells. METHODS: Kaempferitrin therapeutical targets were obtained by searching SwissTargetPrediction, PharmMapper, STITCH, DrugBank, and TTD databases. Liver cancer specific genes were obtained by searching GeneCards, DrugBank, TTD, OMIM, and DisGeNET databases. PPI network of "kaempferitrin-targets-liver cancer" was constructed to screen the hub targets. GO, KEGG pathway and MCODE clustering analyses were performed to identify possible enrichment of genes with specific biological subjects. Molecular docking and molecular dynamics simulation were employed to determine the docking pose, potential and stability of kaempferitrin with hub targets. The potential anti-liver cancer mechanisms of kaempferitrin, as predicted by network pharmacology analyses, were verified by in vitro and in vivo experiments. RESULTS: 228 kaempferitrin targets and 2186 liver cancer specific targets were identified, of which 50 targets were overlapped. 8 hub targets were identified through network topology analysis, and only SIRT1 and TP53 had a potent binding activity with kaempferitrin as indicated by molecular docking and molecular dynamics simulation. MCODE clustering analysis revealed the most significant functional module of PPI network including SIRT1 and TP53 was mainly related to cell apoptosis. GO and KEGG enrichment analyses suggested that kaempferitrin exerted therapeutic effects on liver cancer possibly by promoting apoptosis via p21/Bcl-2/Caspase 3 signaling pathway, which were confirmed by in vivo and in vitro experiments, such as HE staining of tumor tissues, CCK-8, qRT-PCR and Western blot. CONCLUSION: This study provided not only insight into how kaempferitrin could act against liver cancer by identifying hub targets and their associated signaling pathways, but also experimental evidence for the clinical use of kaempferitrin in liver cancer treatment.

Biochemical and molecular characterization of a novel porphobilinogen synthase from Corynebacterium glutamicum.

Corynebacterium glutamicum porphobilinogen synthase (PBGS) is a metal enzyme with a hybrid active site metal binding sequence. In this study, the porphobilinogen synthase gene of C. glutamicum was cloned and heterogeneously expressed in Escherichia coli. C. glutamicum PBGS was purified, and its enzymatic characteristics were analyzed. The results showed that C. glutamicum PBGS is a Zn2+-dependent enzyme, and Mg2+ has allosteric regulation. The allosteric Mg2+ plays a vital role in forming the quaternary structure of C. glutamicum PBGS. Based on the ab initio predictive structure modeling of the enzyme and the molecular docking model of 5-aminolevulinic acid (5-ALA), 11 sites were selected for site-directed mutagenesis. When the hybrid active site metal binding site of C. glutamicum PBGS is converted into a cysteine-rich motif (Zn2+-dependent) or an aspartic acid-rich motif (Mg2+/K+-dependent), the enzyme activity is basically lost. Four residues, D128, C130, D132, and C140, in the metal binding site, were the binding sites of Zn2+ and the active center of the enzyme. The band migration, from the native PAGE, of five variants with mutations in the center of enzyme activity was the same as that of the variant enzymes as purified, individually adding two metal ion chelating agents. Their Zn2+ active center structures were abnormal, and the quaternary structure equilibrium was altered. The destroyed active center affects the construction of its quaternary structure. The quaternary structural balance between octamer and hexamer through dimers was regulated by the allosteric regulation of C. glutamicum PBGS. The enzyme activity was also affected by the change of the active site lid structure and (α ß)8-barrel structure caused by mutation. Structural changes in the variants were analyzed to understand C. glutamicum PBGS better.

Biochemical characterization of acyl-coenzyme A synthetases involved in mycobacterial steroid side-chain catabolism and molecular design: synthesis of an anti-mycobacterial agent.

The metabolism of host cholesterol by Mycobacterium tuberculosis is an important factor for both its virulence and pathogenesis. However, the rationale for this cholesterol metabolism has not been fully understood yet. In the present study, we characterized several previously undescribed acyl-CoA synthetases that are involved in the steroid side-chain degradation in Mycobacterium smegmatis, and an analogue of intermediate from steroid degradation, 5'-O-(lithocholoyl sulfamoyl) adenosine (LCA-AMS), was successfully designed and synthesized to be used as a specific anti-mycobacterial agent. The acyl-CoA synthetases exhibited strong preferences for the length of side chain. FadD19 homologs, including FadD19 (MSMEG_5914), FadD19-2 (MSMEG_2241), and FadD19-4 (MSMEG_3687), are unanimously favorable cholesterol with a C8 alkanoate side chain. FadD17 (MSMEG_5908) and FadD1 (MSMEG_4952) showed high preferences for steroids, containing a C5 alkanoate side chain. FadD8 (MSMEG_1098) exhibited specific activity toward cholestenoate with a C8 alkanoate side chain. An acylsulfamoyl analogue of lithocholate, 5'-O-(lithocholoyl sulfamoyl) adenosine (LCA-AMS), was designed and synthesized. As expected, the intermediate analogue not only specifically inhibited those steroid-activated acyl-CoA synthetases, but also selectively inhibited the growth of mycobacterial species, including M. tuberculosis, M. smegmatis, and Mycobacterium neoaurum. Overall, our research advanced our understanding of mycobacterial steroid degradation and provided new insights to develop novel mechanism-based anti-mycobacterial agents.

Gordonia neofelifaecis sp. nov., a cholesterol side-chain-cleaving actinomycete isolated from the faeces of Neofelis nebulosa.

A cholesterol side-chain-cleaving bacterial strain, AD-6(T), was isolated from fresh faeces of a clouded leopard (Neofelis nebulosa) and was studied using a polyphasic taxonomic approach. 16S rRNA gene sequence analysis showed that the novel strain formed a distinct subline within the genus Gordonia, its closest neighbours being the type strains of Gordonia cholesterolivorans, Gordonia sihwensis and Gordonia hydrophobica, with sequence similarity values of 98.2, 97.8 and 97.6 %, respectively. The gyrB gene sequence of strain AD-6(T) exhibited similarities of 77-91 % with those of the type strains of recognized species of the genus Gordonia, being most similar to the type strains of G. sihwensis, G. hydrophobica and Gordonia hirsuta (91, 87 and 84 % similarity, respectively). The results of whole-cell fatty acid analyses and DNA-DNA relatedness data readily distinguished the new isolate from its nearest neighbours. Strain AD-6(T) is therefore considered to represent a novel species of the genus Gordonia, for which the name Gordonia neofelifaecis sp. nov. is proposed. The type strain is AD-6(T) (=NRRL B-59395(T)=CCTCC AB-209144(T)).