Hanki Lee

Computer‐assisted 1H NMR analysis of the anti‐tuberculosis drug lead ecumicin

Keywords: Oligopeptides; 1H iterative Full Spin Analysis (HiFSA); Ecumicin; Computational Iteration; Structure elucidation

Improvement of the productivity of ecumicin, a novel anti-tuberculosis agent, from new Nonomuraea sp. MJM5123

Ecumicin is a novel anti-tuberculosis agent produced by Nonomuraea sp. MJM5123 as a new strain of actinomycetes. First, in order to increase the cell mass of Nonomuraea sp. MJM5123, we optimized the culture conditions with regard to carbon and nitrogen sources. The cell mass of Nonomuraea sp. MJM5123 increased by approximately twofold when glucose and soybean flour were used as carbon and nitrogen sources, respectively. For maximum production of ecumicin, we optimized the culture conditions by adding amino acids as building blocks for ecumicin, by adding vegetable oils and by controlling the temperature and pH. Ecumicin production was two times higher with the addition of valine as the building blocks for ecumicin compared with the production in the absence of valine. Interestingly, with the addition of 1% corn oil, the production of ecumicin increased by 4.6-fold compared with the production in the absence of corn oil. Finally, by controlling the pH and temperature, we established an optimized culture condition in which Nonomuraea sp. MJM5123 produced 576 mg ecumicin per litre of medium, which is about 50 times higher than in the control medium at 30 °C and pH 7.0.

Residual Complexity Does Impact Organic Chemistry and Drug Discovery: The Case of Rufomyazine and Rufomycin

Residual complexity (RC) involves the impact of subtle but critical structural and biological features on drug lead validation, including unexplained effects related to unidentified impurities. RC commonly plagues drug discovery efforts due to the inherent imperfections of chromatographic separation methods. The new diketopiperazine, rufomyazine (6), and the previously known antibiotic, rufomycin (7), represent a prototypical case of RC that (almost) resulted in the misassignment of biological activity. The case exemplifies that impurities well below the natural abundance of 13C (1.1%) can be highly relevant and calls for advanced analytical characterization of drug leads with extended molar dynamic ranges of >1:1,000 using qNMR and LC-MS. Isolated from an actinomycete strain, 6 was originally found to be active against Mycobacterium tuberculosis with a minimum inhibitory concentration (MIC) of 2 μg/mL and high selectivity. As a part of lead validation, the dipeptide was synthesized and surprisingly found to be inactive. The initially observed activity was eventually attributed to a very minor contamination (0.24% [m/m]) with a highly active cyclic peptide (MIC ∼ 0.02 μM), subsequently identified as an analogue of 7. This study illustrates the serious implications RC can exert on organic chemistry and drug discovery, and what efforts are vital to improve lead validation and efficiency, especially in NP-related drug discovery programs.

Decursin and decursinol angelate improve wound healing by upregulating transcription of genes encoding extracellular matrix remodeling proteins, inflammatory cytokines, and growth factors in human keratinocytes

The coumarins decursin and decursinol angelate, which are found in Angelica gigas Nakai, have a variety of biological functions. Here, we show that treatment with these compounds improves wound healing by HaCaT human keratinocytes. Wound healing was increased by treatment with up to a threshold concentration of decursin, decursinol angelate, a mixture of both, and a nano-emulsion of these compounds, but inhibited by treatment with higher concentrations. Immunoblotting and fluorescence imaging of cells expressing an epidermal growth factor receptor (EGFR) biosensor demonstrated that these compounds did not stimulate wound healing by inducing EGFR phosphorylation. Rather, transcriptional analysis revealed that decursin and decursinol angelate improved wound healing by upregulating the expression of genes encoding extracellular matrix remodeling proteins, inflammatory cytokines, and growth factors.

Structural Sequencing of Oligopeptides Aided by 1H Iterative Full-Spin Analysis

This report describes an approach using 1H NMR iterative full-spin analysis (HiFSA) to extract definitive structural information on unknown peptides from 1D 1H NMR data. By comparing the experimental data and HiFSA fingerprint of a known analogue, it is possible to isolate the characteristic 1H subspectrum of the different amino acids and, thus, elucidate the structure of the peptide. To illustrate this methodology, a comprehensive analysis of five new anti-Mycobacterium tuberculosis peptides (2-6), all analogues of ecumicin (1), was carried out. The method was validated by demonstrating congruence of the HiFSA-based structures with all available data, including MS and 2D NMR. The highly reproducible HiFSA fingerprints of the new ∼1600 amu peptides were generated in this process. Besides oligo-peptides, the HiFSA sequencing approach could be extended to all oligomeric compounds consisting of chains of monomers lacking H-H spin-spin coupling across the moieties. HiFSA sequencing is capable of differentiating complex oligomers that exhibit minor structural differences such as shifted hydoxyl or methyl groups. Because it employs the basic and most sensitive 1D 1H NMR experiment, HiFSA sequencing enables the exploration of peptide analogues up to at least 2000 amu, even with basic contemporary spectrometers and when only sub-milligram amounts of isolates are available.

High-Content Screening of Raw Actinomycete Extracts for the Identification of Antituberculosis Activities

The feasibility and relevance of screening a library of raw actinomycete extracts (ECUM library) for the identification of antituberculosis activities was assessed on 11,088 extracts using a multiple-screening approach. Each extract was first tested at two concentrations against noninfected macrophages as a control, then against Mycobacterium tuberculosis growing in broth medium as well as infecting murine macrophages. The screening results indicated a library of good quality with an apparent low proportion of cytotoxic extracts. A correlation was found between both bacterial assays, but the intracellular assay showed limitations due to low rates of cell survival. Several extracts of interest were highlighted by this multiple screening. A focus on the strain producing the two most effective revealed similarities with known producers of active molecules, suggesting the possibility of selecting relevant extracts using this strategy.