Rosmarinic Acid Present in Lepechinia floribunda and Lepechinia meyenii as a Potent Inhibitor of the Adenylyl Cyclase gNC1 from Giardia lamblia

Abstract

Giardiasis is a parasitosis caused by Giardia lamblia with significant epidemiological and clinical importance due to its high prevalence and pathogenicity. The lack of optimal therapies for treating this parasite makes the development of new effective chemical entities an urgent need. In the search for new inhibitors of the adenylyl cyclase gNC1 obtained from G. lamblia, 14 extracts from Argentinian native plants were screened. Lepechinia floribunda and L. meyenii extracts exhibited the highest gNC1 inhibitory activity, with IC₅₀ values of 9 and 31 µg/mL, respectively. In silico studies showed rosmarinic acid, a hydroxycinnamic acid present in both mentioned species, to be a promising anti-gNC1 compound. This result was confirmed experimentally, with rosmarinic acid showing an IC₅₀ value of 10.1 µM. Theoretical and experimental findings elucidate the molecular-level mechanism of rosmarinic acid, pinpointing the key interactions stabilizing the compound-enzyme complex and the binding site. These results strongly support that rosmarinic acid is a promising scaffold for developing novel compounds with inhibitory activity against gNC1, which could serve as potential therapeutic agents to treat giardiasis.

Keywords: Giardia lamblia; Lepechinia floribunda; Lepechinia meyenii; adenylyl cyclase inhibitors; molecular modeling; rosmarinic acid.

Figure 1

Adenylyl cyclase activity of the catalytic domain of gNC1-301 incubated with the target extracts at 250 μg/mL. 2-Catechol estrogen (2-CE) at 50 µM was used as a positive control. The data are representative of three different experiments. Significant differences between treatments and the negative control were determined by using the two-tailed paired Student’s t-test (** p < 0.01, * p < 0.05).

Figure 2

Chemical structures of of p-coumaric acid (1), caffeic acid (2), rosmarinic acid (3), carnosol (4), rosmanol (5), carnosic acid (6), 2-catechol estrogen (7) and AMJ-147 (8).

Figure 3

Histogram of the scores from the best docking poses of compounds 1–6. The reference compound 7 was included as control. Bars colored in orange represent compounds with lower scores that were not further studied and bars colored in blue represent compounds with higher scores.

Figure 4

Stability evaluation of the docking complexes. (a) Root mean square fluctuations (RMSF) of the compound’s atoms. (b) Distance between the docked compound and its position along the MD simulation.

Figure 5

Binding energy decomposition plot showing the contribution of individual residues of the active site to the overall binding energy of the gNC1/compound complex.

Figure 6

Determination of adenylyl cyclase activity of the catalytic domain of gNC1 incubated in the presence of the indicated concentrations of compound 3 expressed on a log scale. Significant differences between treatments and the negative control were determined by using the two-tailed paired Student’s t-test (* p < 0.001). Data are representative of three independent experiments.

Figure 7

“RMSD_mdlovofit”. Flexibility analysis of compound 3/gNC1 complex in molecular dynamics simulations. (a) Fractional alignment plot. Blue line represents the RMSD value as a function of the fraction of the atoms (θ) considered in the alignment. (b) Time-dependent structural deviation; the RMSD of the 75% least mobile atoms is represented by the blue line, while the RMSD of all residues is denoted by the orange line. (c) Molecular superposition of aligned trajectory frames colored according to the algorithm automatic classification; least mobile regions are shown in blue and most mobile regions in red.

Figure 8

Atomic fluctuations of compound 3 bound to both gNC1 active sites.

Figure 9

Molecular structure of compound 3/gNC1 complex with both active sites occupied. (a) Compound 3 is shown as sticks colored according to the root mean square fluctuation (RMSF) values. (b) Polar interactions (hydrogen and ionic bonds). Calcium atoms are represented as green spheres. Interacting residues are depicted as gray sticks, while the molecules of compound 3 located into both ATP sites are shown as yellow sticks.

Figure 10

Residue decomposition analysis highlighting the most significant contributions to inhibitor binding at each active site.

Figure 11

Inhibitory effect of rosmarinic acid (3) on gNC1 adenylyl cyclase activity. The assay was performed by increasing the concentrations of ATP in the presence of the dissolution solvent as a control (black filled circles), 5 µM of compound 3 (cyan filled squares), and 10 µM of compound 3 (red filled triangles). The values of K_0.5, Vmax and Hill coefficient are indicated in Table 4. The data are representative of three different experiments with similar results.