Abstract
The screening of extracts from marine organisms is a widely used strategy to discover new drug leads. A common problem in the screening process is the generation of false positive hits through unspecific effects from the complex chemical composition of the crude extracts. In this study, we explored a combination of a fluorescence resonance energy transfer (FRET) based activity assay and a surface plasmon resonance (SPR) based binding assay to avoid this problem. An aqueous extract was prepared from rest raw material of the Norwegian spring spawning herring, and further fractionated by methanol solubility and solid phase extraction. FRET based activity assays were used to determine the influence of each extract on the activity of different proteases. Several extracts showed more than 50% inhibition. The inhibition mechanisms were elucidated by SPR based competition experiments with known inhibitors. For the secreted aspartic proteases 1, 2, 3 and HIV-1 protease, the results indicated that some extracts contain inhibitors interacting specifically with the active site of the enzymes. The study shows that a combination of an activity assay and an SPR based binding assay is a powerful tool to identify potent inhibitors in marine extracts. Furthermore, the study shows that marine vertebrates offer an interesting source for new bioactive compounds, although they have rarely been explored for this purpose.
Highlights
Small organic molecules produced by marine organisms are a vast source for novel bioactive compounds and drugs leads [1]
We explored extracts from the Norwegian spring spawning herring for inhibitors of the proteases SAP1, 2 and 3 from Candida albicans, HIV-1 protease, pepsin, BACE1 and human cytomegalovirus (HCMV)
We showed that the combination of an activity assay and an surface plasmon resonance (SPR) based binding assay is a powerful tool for screening marine extracts for protease inhibitors, since it allows the identification of false positive hits
Summary
Small organic molecules produced by marine organisms are a vast source for novel bioactive compounds and drugs leads [1]. A widely used type of screening assay to identify bioactive compounds inhibiting proteases, an important class of drug targets, are fluorescence resonance energy transfer (FRET) based activity assays due to the simple design of substrates, the high sensitivity of the read out and the real time monitoring of cleavage [8]. A more recently developed type of screening assay to study protease inhibitors involves the analysis of binding to the target, using surface plasmon resonance spectroscopy (SPR) [9,10,11] Such assays enable the elucidation of the interaction mechanism and the discrimination between specific and unspecific interactions. The FRET based activity assay allowed the identification of extracts inhibiting the proteases, whereas the SPR based binding assay elucidated the mechanism causing the inhibition In this way it was possible to identify extracts containing promising protease inhibitors
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