A docking-based structural analysis of geldanamycin-derived inhibitor binding to human or Leishmania Hsp90

Abstract

Leishmaniasis is a neglected disease that affects millions of individuals around the world. Regardless of clinical form, treatment is based primarily on the use of pentavalent antimonials. However, such treatments are prolonged and present intense side effects, which lead to patient abandonment in many cases. The search for chemotherapeutic alternatives has become a priority. Heat Shock Protein 90 (Hsp90) inhibitors have recently come under investigation due to antiparasitic activity in Plasmodium sp., Trypanosoma sp. and Leishmania sp. Some of these inhibitors, such as geldanamycin and its analogs, 17-AAG and 17-DMAG, bind directly to Hsp90, thereby inhibiting its activity. Previous studies have demonstrated that different parasite species are more susceptible to some of these inhibitors than host cells. We hypothesized that this increased susceptibility may be due to differences in binding of Hsp90 inhibitors to Leishmania protein compared to host protein. Based on the results of the in silico approach used in the present study, we propose that geldanamycin, 17-AAG and 17-DMAG present an increased tendency to bind to the N-terminal domain of Leishmania amazonensis Hsp83 in comparison to human Hsp90. This could be partially explained by differences in intermolecular interactions between each of these inhibitors and Hsp83 or Hsp90. The present findings demonstrate potential for the use of these inhibitors in the context of anti-Leishmania therapy.

Figure 1

L. amazonensis presents greater susceptibility to Hsp90 inhibitors than the differentiated THP-1 cell-line. L. amazonensis axenic promastigotes and differentiated THP-1 cells were treated with (a) GA, (b) 17-AAG or (c) 17-DMAG in a 12-step serial dilution assay. Graphs depict IC₅₀ (L. amazonensis) and CC₅₀ (THP-1 cell-line) values for each respective inhibitor. Mann Whitney test ***p = 0.001, **p = 0.0043.

Figure 2

Intracellular viability of L. amazonensis-infected THP-1 cells treated with 17-DMAG. THP-1 cells (5 × 10⁵ per well) were plated in RPMI medium with PMA (100 nM) to promote differentiation into macrophages, then infected with L. amazonensis promastigotes (10:1) for 6 h at 35 °C. After washing to remove non-internalized parasites, cells were treated with 17-DMAG at concentrations of 6.25 nM, 12.5 nM, 25 nM, 50 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM and 400 nM (five replicates per concentration) for 72 h at 35 °C. Cells were washed, Schneider’s complete medium was added and, after four days at 24 °C, parasites were counted in a Neubauer chamber. Mann Whitney test **p < 0.01.

Figure 3

L. amazonensis Hsp83 binding site with inhibitors and interaction types. (a) L. amazonensis Hsp83 binding site with GA (orange), 17-AAG (blue) and 17-DMAG (magenta). (b) GA (orange), (c) 17-AAG (blue) and (d) 17-DMAG (magenta) and the Hsp83 residues that make hydrophobic contacts (green) and hydrogen bonds (red).

Figure 4

Homo sapiens Hsp90 binding site with inhibitors and interaction types. (a) Homo sapiens Hsp90 binding site with GA (orange), 17-AAG (blue) and 17-DMAG (magenta). (b) GA (orange), (c) 17-AAG (blue) and (d) 17-DMAG (magenta) and the Hsp90 residues that formed hydrophobic contacts (green), salt bridges (cyan) and hydrogen bonds (red).