Novel, natural allosteric inhibitors and enhancers of Candida rugosa lipase activity.

Candida rugosa lipase (CRL) is an enzyme commonly used in medicinal and biotechnological applications. Allosteric modulators of CRL could aid in modifying lipase-related diseases as well as improving biotechnological processes. Thus, a combinatorial approach of computational in-silico and high-throughput in-vitro screening was used to identify allosteric modulators of CRL. The screening of natural product libraries resulted in 132 compounds of which 53 were tested in-vitro. Subsequently, four inhibitors and three enhancers were identified of which rutin and cynaroside represented the strongest inhibitors of CRL activity (IC50: 227 ± 26 µM and 446 ± 15 µM, respectively) and NP-008496 the strongest enhancer (EC50: 425 ± 18 µM). All three compounds were predicted to bind the same allosteric site suggesting a common mechanism. Therefore, the present study demonstrated a reliable work-flow, identified an allosteric site of CRL and determined inhibitors and enhancers with numerous potential medical and biotechnological applications.

A fast, miniaturised in-vitro assay developed for quantification of lipase enzyme activity.

The discovery of allosteric modulators is a multi-disciplinary approach, which is time- and cost-intensive. High-throughput screening combined with novel computational tools can reduce these factors. Thus, we developed an enzyme activity assay, which can be included in the drug discovery work-flow subsequent to the in-silico library screening. While the in-silico screening yields in the identification of potential allosteric modulators, the developed in-vitro assay allows for the characterisation of them. Candida rugosa lipase (CRL), a glyceride hydrolysing enzyme, has been selected for the pilot development. The assay conditions were adjusted to CRL's properties including pH, temperature and substrate specificity for two different substrates. The optimised assay conditions were validated and were used to characterise Tropolone, which was identified as an allosteric modulator. In conclusion, the assay is a reliable, reproducible, and robust tool, which can be streamlined with in-silico screening and incorporated in an automated high-throughput screening workflow.