Natural metabolites from Tabebuia aurea reveal isoform-specific and cooperative modulation of cathepsins B and L

The development of isoform-selective inhibitors for homologous cysteine proteases remains challenging due to structural similarity and overlapping specificities. Metabolites isolated from Tabebuia aurea were investigated as modulators of Cathepsins B and L. Bioassay-guided fractionation yielded three compounds: 2,3-DH-5-HMB (1), veratric acid (2), and iridoid specioside (3). Enzymatic assays identified veratric acid as the most active compound (IC₅₀ = 4.23 ± 0.17 μM for Cathepsin B and 1.28 ± 0.03 μM for Cathepsin L). Steady-state kinetic analyses revealed marked mechanistic divergence between isoforms. Against Cathepsin B, compounds 1 and 2 acted as competitive inhibitors with strong positive cooperativity, quantitatively captured by the cooperativity factor β (0.00041 and 0.0129, respectively), corresponding to 2400-fold and 78-fold increases in affinity upon binding of a second inhibitor molecule. For compound 2, where extreme cooperativity precluded independent estimation of microscopic parameters, the quadratic cooperativity parameter γ (0.273 μM^-2) enabled robust quantification of the cooperative effect. In contrast, all three compounds inhibited Cathepsin L via a simple linear non-competitive mechanism (α ≈ 1), reflecting its more accessible active-site architecture. Cellular assays confirmed selective antiproliferative activity of veratric acid in K562 leukemia cells (EC₅₀ = 12.47 μM) while sparing non-tumor HaCaT cells. Molecular docking and molecular dynamics simulations of 1:1 complexes provided structural context for the observed isoform-specific binding modes. These results demonstrate that structurally simple phenolic compounds can induce fundamentally different inhibition mechanisms in closely related enzymes, establishing a structure-mechanism framework for isoform-selective modulation of cysteine proteases.