Chemical Probes for Protein α-N-Terminal Methylation

While protein α-N-terminal methylation has been known for nearly four decades since it was first uncovered on bacteria ribosomal proteins L33, the function of this modification is still not entirely understood. Recent discoveries have demonstrated α-N-terminal methylation is essential to stabilize the interactions between regulator of chromosome condensation 1 (RCC1) and chromatin during mitosis, to localize and enhance the interaction of centromere proteins (CENPs) with chromatin, and to facilitate the recruitment of DNA damage-binding protein 2 (DDB2) to DNA damage foci. Identification of N-terminal methyltransferase 1 (NTMT1) unveiled the eukaryotic methylation writer for protein α-N-termini. In addition, NTMT2 that shares over 50% sequence similarity, has been identified as another mammalian protein α-N-terminal methylation writer. Knockdown of NTMT1 results in mitotic defects and sensitizes chemotherapeutic agents in breast cancer cell lines, while NTMT1 knockout mice showed premature aging. Additionally, NTMT1 has been shown to be overexpressed in a colorectal and melanoma tumor tissues, and in lung and liver cancer cell lines. Given the vast array of clinical relevance, chemical probes and inhibitors for NTMT1 are vital to elucidate information about the function and downstream process of protein α-N-terminal methylation. Therefore, 47 peptidomimetic compounds have been synthesized that target NTMT1. These peptide-based compounds range from three to six amino acids in length and the top 5 compounds have 3- to 300- fold selectivity for NTMT1 compared to other methyltransferases. An inhibition mechanism study has also been performed to verify the inhibitors are targeting the NTMT1 peptide binding site. Seven compounds have an IC50 of less than 5 µM and our top inhibitor, BM-47, has an IC50 of 0.32 µM ± 0.06 for NTMT1. To further elucidate information about the NTMTs and their downstream effects, we utilized photoaffinity probes to target these enzymes. Our 6 photoaffinity probes exhibited in a dose- and time-dependent manner. Probe labeling has been shown to be driven by recognition and selectively and competitively label the NTMT writers in a complex cellular mixture. Our results also provided the first indication of substrate preferences among NTMT1/2. Methylated photoaffinity probes were also synthesized to identify novel proteins that recognize a methylated N-terminus and shed light on the function of α-N-terminal methylation

The anti-sepsis activity of the components of Huanglian Jiedu Decoction with high lipid A-binding affinity

Huanglian Jiedu Decoction (HJD), one of the classic recipes for relieving toxicity and fever, is a common method for treating sepsis in China. However, the effective components of HJD have not yet been identified. This experiment was carried out to elucidate the effective components of HJD against sepsis. Thus, seven fractions from HJD were tested using a biosensor to test their affinity for lipid A. The components obtained that had high lipid A-binding fractions were further separated, and their affinities to lipid A were assessed with the aid of a biosensor. The levels of LPS in the blood were measured, and pathology experiments were conducted. The LPS levels and mRNA expression analysis of TNF-α and IL-6 of the cell supernatant and animal tissue were evaluated to investigate the molecular mechanisms. Palmatine showed the highest affinity to lipid A and was evaluated by in vitro and in vivo experiments. The results of the in vitro and in vivo experiments indicated that the levels of LPS, TNF-α and IL-6 of the palmatine group were significantly lower than those of the sepsis model group (p \u3c 0.01). The group treated with palmatine showed strong neutralizing LPS activity in vivo. The palmatine group exhibited stronger protective activity on vital organs compared to the LPS-induced animal model. This verifies that HJD is a viable treatment option for sepsis given that there are multiple components in HJD that neutralize LPS, decrease the release of IL-6 and TNF-α induced by LPS, and protect vital organs

Identification of the Metabolic Enzyme Involved Morusin Metabolism and Characterization of Its Metabolites by Ultraperformance Liquid Chromatogaphy Quadrupole Time-of-Flight Mass Spectrometry (UPLC/Q-TOF-MS/MS)

Morusin, the important active component of a traditional Chinese medicine, Morus alba L., has been shown to exhibit many vital pharmacological activities. In this study, six recombinant CYP450 supersomes and liver microsomes were used to perform metabolic studies. Chemical inhibition studies and screening assays with recombinant human cytochrome P450s were also used to characterize the CYP450 isoforms involved in morusin metabolism. The morusin metabolites identified varied greatly among different species. Eight metabolites of morusin were detected in the liver microsomes from pigs (PLMs), rats (RLMs), and monkeys (MLMs) by LC-MS/MS and six metabolites were detected in the liver microsomes from humans (HLMs), rabbits (RAMs), and dogs (DLMs). Four metabolites (M1, M2, M5, and M7) were found in all species and hydroxylation was the major metabolic transformation. CYP1A2, CYP2C9, CYP2D6, CYP2E1, CYP3A4, and CYP2C19 contributed differently to the metabolism of morusin. Compared to other CYP450 isoforms, CYP3A4 played the most significant role in the metabolism of morusin in human liver microsomes. These results are significant to better understand the metabolic behaviors of morusin among various species

Identification of the Metabolic Enzyme Involved Morusin Metabolism and Characterization of Its Metabolites by Ultraperformance Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometry (UPLC/Q-TOF-MS/MS)

Morusin, the important active component of a traditional Chinese medicine, Morus alba L., has been shown to exhibit many vital pharmacological activities. In this study, six recombinant CYP450 supersomes and liver microsomes were used to perform metabolic studies. Chemical inhibition studies and screening assays with recombinant human cytochrome P450s were also used to characterize the CYP450 isoforms involved in morusin metabolism. The morusin metabolites identified varied greatly among different species. Eight metabolites of morusin were detected in the liver microsomes from pigs (PLMs), rats (RLMs), and monkeys (MLMs) by LC-MS/MS and six metabolites were detected in the liver microsomes from humans (HLMs), rabbits (RAMs), and dogs (DLMs). Four metabolites (M1, M2, M5, and M7) were found in all species and hydroxylation was the major metabolic transformation. CYP1A2, CYP2C9, CYP2D6, CYP2E1, CYP3A4, and CYP2C19 contributed differently to the metabolism of morusin. Compared to other CYP450 isoforms, CYP3A4 played the most significant role in the metabolism of morusin in human liver microsomes. These results are significant to better understand the metabolic behaviors of morusin among various species