针对5-脂氧合酶(5-LOX)和mPGES1促炎靶点的天然产物文库筛选以用于CRC阻断

Colorectal Cancer (CRC) remains the leading cause of cancer-related mortality worldwide. Inflammation is a key hallmark of many cancers, including CRC. Pro-inflammatory lipid mediators play a key role, while COX-2 inhibiting NSAIDs are promising, their chronic use is linked with unwanted side-effects. In this context, mechanistic studies suggest that targeting microsomal prostaglandin synthase-1 (mPGES-1) and 5-lipoxygenase (5-LOX) with natural products (NP) presents a valuable opportunity to intercept CRC and mitigate those side effects. Here we aimed to perform high-throughput screening (HTS) of NCI NPs library (~500,000 semi-purified fractions) to identify potential inhibitors of 5-LOX and mPGES1; and further validate the purified compounds using secondary assays. To establish the enzyme activity inhibitory assays, first we developed a stable Human Embryonic Kidney (HEK) 293 cell lines with mPGES-1 and 5-LOX overexpression as well as 5-LOX overexpressing insect cells. Proteins expression was confirmed using western blotting. Lysates from human 5-LOX expressed in Sf9 insect cells and HEK-293 cells, were used to generate an assay format in 384-well microplates suitable for HTS. In this assay format, lysates are preincubated with inhibitors for 20 minutes, stimulated with arachidonic acid (AA) for 5 minutes. The production of free radicals, as a result of the conversion of AA to 5HPETE and LTA4 by 5-LOX activity, is detected upon the addition of 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA). The non-fluorescent H2DCFDA when oxidized by the free radicals, generates a highly fluorescent compound, which can be measured to quantify enzyme activity. The reaction was stopped after 15 minutes upon the addition of acetonitrile. Finally, enzyme activity was calculated by measuring “total relative fluorescence units (RFU) at 485-nm excitation and 530-nm emission spectra. The assay has been optimized using a final volume of 15ul and has a Z' Factor score of 0.65 and a S/B of 3.5 in 384-well microplates. The 5-LOX activity was completely inhibited by 20uM NDGA, a known inhibitor of 5-LOX. Additionally, a pre-plated NCI library of semi-purified NP fractions (5mg/ml stock in DMSO) has been assessed to validate the assay and identify potential fractions that demonstrate 5-LOX inhibitory activity. The fractions with the most promising activity based on this evaluation will be presented at the meeting. We have also optimized the mPGES1 activity assay using HEK293 cell line overexpressing COX2 and mPGES-1. In this assay format, treatment with AA results in elevated levels of PGE2, as detected in a PGE2 HTRF assay. In summary, these optimized assays will be employed for large scale robotic HTS of NCI NP library to discover and develop safer inhibitors of proinflammatory targets mPGES1 and 5-LOX for intercepting inflammation associated cancers. (Funded by NCI-UG3CA290310-01).