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. 2024 Sep 5;29(17):4224.
doi: 10.3390/molecules29174224.

Structure-Aided Computational Design of Triazole-Based Targeted Covalent Inhibitors of Cruzipain

Affiliations

Structure-Aided Computational Design of Triazole-Based Targeted Covalent Inhibitors of Cruzipain

Juan Pablo Cerutti et al. Molecules. .

Abstract

Cruzipain (CZP), the major cysteine protease present in T. cruzi, the ethiological agent of Chagas disease, has attracted particular attention as a therapeutic target for the development of targeted covalent inhibitors (TCI). The vast chemical space associated with the enormous molecular diversity feasible to explore by means of modern synthetic approaches allows the design of CZP inhibitors capable of exhibiting not only an efficient enzyme inhibition but also an adequate translation to anti-T. cruzi activity. In this work, a computer-aided design strategy was developed to combinatorially construct and screen large libraries of 1,4-disubstituted 1,2,3-triazole analogues, further identifying a selected set of candidates for advancement towards synthetic and biological activity evaluation stages. In this way, a virtual molecular library comprising more than 75 thousand diverse and synthetically feasible analogues was studied by means of molecular docking and molecular dynamic simulations in the search of potential TCI of CZP, guiding the synthetic efforts towards a subset of 48 candidates. These were synthesized by applying a Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) centered synthetic scheme, resulting in moderate to good yields and leading to the identification of 12 hits selectively inhibiting CZP activity with IC50 in the low micromolar range. Furthermore, four triazole derivatives showed good anti-T. cruzi inhibition when studied at 50 μM; and Ald-6 excelled for its high antitrypanocidal activity and low cytotoxicity, exhibiting complete in vitro biological activity translation from CZP to T. cruzi. Overall, not only Ald-6 merits further advancement to preclinical in vivo studies, but these findings also shed light on a valuable chemical space where molecular diversity might be explored in the search for efficient triazole-based antichagasic agents.

Keywords: Chagas disease; Cruzipain; computer-aided drug design; targeted covalent inhibitors; triazole derivatives.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
(a) 3D structure of CZP (PDB 3IUT [22]). (b) 3D structure of CZP active site, highlighting the S1’ (red), S1 (green), S2 (blue), S3 (purple) and catalytic (yellow) subsites. The catalytic triad and oxyanion hole are represented in licorice.
Figure 2
Figure 2
Peptidomimetic (Ts-370) and peptide-based (K777) promising CZP inhibitors reported in literature, with poor translation to T. cruzi inhibitory activity [32,45].
Figure 3
Figure 3
Synthetic core scheme for the synthesis of 1,4-disubstituted 1,2,3-triazole-based potential inhibitors of CZP.
Figure 4
Figure 4
Summary of the docking-based vHTS results focused on R1 with CZP.
Figure 5
Figure 5
(a) Summary of the docking-based vHTS results for the 1,2,3-triazoles obtained by combinatorial synthesis. Clusters of optimal (green box, upper right) and less favored (fuchsia box, bottom right) synthesized compounds are highlighted. (b) Chemical structure of the most promising triazole-derivatives identified by means of vHTS and synthesized. (c) Docking pose of Ald-6 (licorice), highlighting its interaction with Asp161 (CPK).
Figure 6
Figure 6
Synthetic core scheme for the synthesis of Es- and Ald-derived 1,4-disubstituted 1,2,3-triazoles. * The yields of Ald-15, Ald-16 and Ald-24 are not included in the range since they could not be isolated.
Figure 7
Figure 7
Mechanism of CZP inhibition by Es-15, evaluated in triplicate. (a) Michaelis–Menten plot. The curves correspond to the fitting of a mixed-model of inhibition to data. (b) Plot of the Kmapp for varying Es-15 concentrations (c) Lineweaver–Burk reciprocal plot.
Figure 8
Figure 8
Jump dilution assay results for (a) Ald-6 and (b) Ald-10. The inhibitors were incubated at 10 × IC50 with 100 × [CZP] for 30 min, followed by 100× dilution and 2 h monitoring. The ligand behavior during the first 15 min of the assay is depicted in the magnifications on the top left. The mean and SEM for three replicates are shown.
Figure 9
Figure 9
(a) Interaction pattern identified for Ald-6:CZP. The alternative poses of the R1 substituent are represented (gray). (b) MM-MD snapshots of Ald-6 (licorice), highlighting the alternative poses of its R1 when interacting with Ser64 and Asp161 (CPK). (c) Distance plots of R1-n-butylamino of Ald-6 with CZP-Asp161 and Ser64.
Figure 10
Figure 10
Chemical space of the triazole-based derivatives feasible to be synthesized with the biologically evaluated ones colored according to their IC50 against CZP. The subplot on the right shows a magnification of the most populated cluster.
Figure 11
Figure 11
(a) Mean ± SD of T. cruzi viability quantified in triplicate ([Inh.] = 50 μM) 96 h after cell seeding (72 h p.i.) by β-gal activity assay and expressed as % of remaining infection. ** [Ald-4] = 20 μM. (b) Percentage of uninfected Vero cell viability after 96h incubation with triazole-based CZP inhibitors (50 μM), quantified by Alamar blue assay.

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