A thin layer chromatography assay to detect laccase inhibitors

CABEZUDO, IGNACIO; FURLAN, RICARDO L.E.

Rosario

Congreso; 7ma Reunión Internacional de Ciencias Farmacéuticas (RICiFa 2023); 2023

FBIOYF-UNR

Isolation and identification of the molecules responsible forbioactivity within natural extracts is an essential step in natural productbioprospecting. Thin layer chromatography combined with in situ enzymeactivity determination has shown to be an assay format particularly well-suitedfor the effect-directed analysis of complex samples (1). This approachfacilitates the acquisition of activity-profile chromatograms, which assist inpinpointing the active constituents within complex mixtures by observing theinhibition halos they produce (see Figure). Antifungal resistance poses agrowing challenge in the field of medicine and human health. Fungi such as Cryptococcusspp are responsible for most cases of systemic and potentially life-threateningcryptococcosis in immunocompromised individuals. One of the most crucialvirulence traits of these species is the expression of laccase, which inducesthe production of a melanin-like pigment. Recent findings have demonstratedthat inhibiting laccase activity also inhibits the multiplication of this species.Despite the increasing need to identify laccaseinhibitors, an effective method for their detection in mixtures has not beenestablished. We present an assay to detect laccase inhibitors directly on theTLC surface.  After the compounds areseeded and developed using CAMAG automatized TLC equipment, the gel-immobilizedenzyme is deposited on the plate where it catalyses the conversion of colourlessABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), into blue ABTS•+.Laccase inhibitors prevent the formation of ABTS•+ and can,therefore, be detected on the plate as colourless spots against a bluebackground. The assay is compatible with normal and reverse phase plates(depending on the gel used to immobilize the enzyme), and a control assayallowed us to discriminate true inhibitors from radical scavengers. The linearity of the normal phase enzymatic assay wasevaluated by linear regression analysis of different amounts of sodium azide seededmass. The areas of inhibition were quantified using CAMAG scanner at 700 nm. Thedata showed an R2 of 0.933, a detection limit of 0.067 µg and aquantification limit of 0.204 µg. Therefore, the potency of the inhibitors canbe semi-quantified. We conclude that the proposed assay is valid to detectlaccase inhibitors selectively, in different TLC plate phases. These have thepotential to be tested as anti-virulence in resistant fungal strains. (1) Cabezudo, I. et al. Effect-Directed Analysis in Food by Thin-LayerChromatography Assays. Food Chemistry 2022, 132937.(2) Azam, F. et al. In Silico and in Vitro Studies on the Inhibition of LaccaseActivity by Ellagic Acid: Implications in Drug Designing for the Treatment ofCryptococcal Infections. International Journal of Biological Macromolecules2022, 209, 642–654.