Protease inhibition activity of semi-synthetic derivatives of Piperine isolated from Piper tuberculatum (Piperaceae) from Brazilian Flora

Protease inhibition assays have been used as a model for screening fractions and secondary metabolites from plant species with biological activity. As part of our research on Brazilian flora species, we aimed at exploring the effects of piperine isolated from Piper tuberculatum and of 20 of it's semi-synthetic derivatives. FRET spectroscopy (350nm excitation, 450nm emission) were used for inhibition analyses of aspartate protease pepsin (17 nM, 1 mM acetate buffer, pH 4.4) and serinic protease subtilisin (37 nM, 1 mM phosphate buffer, pH 7.5). The substrate used was Arg-Glu-(EDANS)-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-Lys-(DALBCYL)-Arg (EDANS-DABCYL) (2 uM).

Fig.1

The most effective semi-synthetic amide derivatives were AM1, AM3 and AM7, showing potent and selective inhibitory activity on aspartate protease pepsin, as shown in table 1. The experiments with the serinic protease subtilisin showed that only the compound AM 7 inhibit the enzyme activity. In conclusion, the results showed that the amides tested were selective for the inhibition of the aspartate protease. The bioactive semi-synthetic compounds will be submitted to further assays using other asparte proteases as HIV protease, rennin and beta-secretase.

Table 1: Results of piperine and its semi-synthetic derivatives in the protease inhibition assay

Pepsin
Concentration (ug/mL)	10	1
	% Inhibition
Piperine	65.03	54.12
AM 1	65.47	33.30
AM 7	52.89	37.98
AM 3	62.84	11.38

Subtilisin
Piperine	nd	31.85
AM 1	–3.45	–2.04
AM 7	50.71	32.28
AM 3	–16.49	–7.37

Acknowledgements: FAPESP, CAPES and CNPq for financial support.