On the intrinsic reactivity of highly potent trypanocidal cruzain inhibitors

Abstract

The cysteine protease cruzipain is considered to be a validated target for therapeutic intervention in the treatment of Chagas disease. Hence, peptidomimetic cruzipain inhibitors having a reactive group (known as warhead) are subject to continuous studies to discover novel antichagasic compounds. Here, we evaluated how different warheads for a set of structurally similar related compounds could inhibit the activity of cruzipain and, ultimately, their trypanocidal effect. We first investigated in silico the intrinsic reactivity of these compounds by applying the Fukui index to correlate it with the enzymatic affinity. Then, we evaluated their potency against T. cruzi (Y and Tulahuen strains), which revealed the reversible cruzain inhibitor Neq0656 as a better trypanocidal agent (EC^Y.strain ₅₀ = 0.1 μM; SI = 58.4) than the current drug benznidazole (EC^Y.strain ₅₀ = 5.1 μM; SI > 19.6). We also measured the half-life time by HPLC analysis of three lead compounds in the presence of glutathione and cysteine to experimentally assess their intrinsic reactivity. Results clearly illustrated the reactivity trend for the warheads (azanitrile > aldehyde > nitrile), where the aldehyde displayed an intermediate intrinsic reactivity. Therefore, the aldehyde bearing peptidomimetic compounds should be subject for in-depth evaluation in the drug discovery process.

Fig. 1. 2D schematic representation of the compounds presented in this study. Blue color depicts the difference in warhead structures.

Fig. 2. Schematic representation of the warheads investigated using electrophilicity indices. Carbon (in blue) reacts with the thiolate nucleophile of cysteine. The Fukui index of electrophilicity was calculated for the carbon represented in blue.

Scheme 1. Synthetic scheme for the synthesis of compounds Neq0646 and Neq0673. Conditions: (a) TFAA, NH₄NO₃, rt, 2 h; (b) 6 M HCl, SnCl₂, 1.5 h, rt; (c) Trp-OH or Phe-OH, 1 M NaOH, 0 °C, 0.5 h; (d) EDC, HOBt, THF, rt, 18 h.

Fig. 3. The putative linear equation and the coefficient of determination obtained through the linear correlation between ω_c⁺ and pK_i^Cz.