Antiparasitic effect of a fraction enriched in tight-binding protease inhibitors isolated from the Caribbean coral Plexaura homomalla

Identification of Tight-Binding Plasmepsin II and Falcipain 2 Inhibitors in Aqueous Extracts of Marine Invertebrates by the Combination of Enzymatic and Interaction-Based Assays

“…Typical affinity profiles were obtained for the evaluation of P. homomalla (TCA) and X. muta (heat) clarified extracts on a Plm II-Sepharose matrix (Figure 4A,B), with the eluted fraction showing ~6-fold and 10-fold increase in the specific inhibitory activity, respectively [46]. Interaction of P. homomalla (heat) and S. helianthus (heat) positive extracts with clan CA family C1 enzymes were confirmed in the same way by the use of a Papain-Sepharose matrix (Figure 4C,D) [53]. In contrast, both affinity resins failed to concentrate specific inhibitory activity for P. constellatum (TCA), X. muta (TCA) and P. nigra (TCA) extracts (Figure S1, Supplementary Material), confirming the absence of tight-binding inhibitors of the target enzymes.…”

“…Further kinetic analysis of association and dissociation data allowed the determination of apparent kinetic constants k ass app (1.98 × 10 6 M −1 ·s −1 ), k diss app (2.67 × 10 −3 s −1 ) and k diss app / k ass app (1.35 × 10 −9 M) for the interaction of the inhibitor with immobilized FP2, confirming the occurrence of tight-binding inhibitor in the extract [53]. Similar results were obtained for Plm II, with the interaction displaying slower association ( k a app = 6.19 × 10 4 M −1 ·s −1 ) and dissociation rates ( k d app = 5.96 × 10 −3 s −1 ) than in the previous case.…”

“…Previous to the large-scale evaluation of more than 60 marine invertebrate extracts from Cuban coasts, we performed a validation experiment with five reference extracts, previously characterized in our lab as positives (+) or negatives (−) for inhibition against Plm II, FP2 and other related enzymes [46,47,53]: Plexaura homomalla (Plm II+/FP2+), Phallusia nigra (Plm II−/FP2−), Stichodactyla helianthus (Plm II+/FP2+), Xestospongia muta (Plm II+/FP2−) and Polyclinum constellatum (Plm II−/FP2−).…”

“…To confirm the binding, the formed complexes are then dissociated to regenerate the faded ion signal corresponding to the inhibitor [51,52]. Analysis of the positive P. homomalla (heat) extract confirmed the existence of several molecules interacting specifically with Sepharose-immobilized Papain (Figure 6A), corresponding to m / z + of 5973.8, 6074.2, 7357.7, 14711.0 and 14736.9 [53]. A similar analysis using Pepsin-Sepharose allowed us to identify a molecule ( m / z + 5974.4) specifically interacting with Pepsin-like aspartic proteases (Figure 6B), confirming in both cases the occurrence of binding partners for the targets that may be responsible for the inhibitory activity detected in the reference extract.…”

Screening of Protease Inhibitory Activity in Aqueous Extracts of Marine Invertebrates from Cuban Coast

“…The presence of cysteine protease inhibitors in marine invertebrates have been previously reported [53] [54], although this source of biomolecules have been less explored. Recently, a fraction enriched in tight-binding protease inhibitors was isolated from the Caribbean coral P. homomalla showing Falcipain 2 and Cruzipain inhibitory activity at the nanomolar level [55]. A high number of extracts screened in this work displayed inhibitory activity against the model enzyme papain, and were further confirmed to inhibit also the therapeutic target cathepsin K. In the case of this particular enzyme none inhibitor molecule coming from marine invertebrates has been reported.…”