A second generation of 1,2,4-oxadiazole derivatives with enhanced solubility for inhibition of 3-hydroxykynurenine transaminase (HKT) from Aedes aegypti

Abstract

The most widely used method for the control of the Aedes aegypti mosquito population is the chemical control method. It represents a time- and cost-effective way to curb several diseases (e.g. dengue, Zika, chikungunya, yellow fever) through vector control. For this reason, the discovery of new compounds with a distinct mode of action from the available ones is essential in order to minimize the rise of insecticide resistance. Detoxification enzymes are an attractive target for the discovery of new insecticides. The kynurenine pathway is an important metabolic pathway, and it leads to the chemically stable xanthurenic acid, biosynthesized from 3-hydroxykynurenine, a precursor of reactive oxygen and nitrogen species, by the enzyme 3-hydroxykynurenine transaminase (HKT). Previously, we have reported the effectiveness of 1,2,4-oxadiazole derivatives acting as larvicides for A. aegypti and AeHKT inhibitors from in vitro and in silico studies. Here, we report the synthesis of new sodium 4-[3-(aryl)-1,2,4-oxadiazol-5-yl] propanoates and the cognate HKT-inhibitory activity. These new derivatives act as competitive inhibitors with IC₅₀ values in the range of 42 to 339 μM. We further performed molecular docking simulations and QSAR analysis for the previously synthesized sodium 4-[3-(aryl)-1,2,4-oxadiazol-5-yl] butanoates reported earlier by our group and the data produced herein. Most of the 1,2,4-oxadiazole derivatives, including the canonical compounds for both series, showed a similar binding mode with HKT. The binding occurs similarly to the co-crystallized inhibitor via anchoring to Arg³⁵⁶ and positioning of the aromatic ring and its substituents outwards at the entry of the active site. QSAR analysis was performed in search of more than 770 molecular descriptors to establish a relationship between the lowest energy conformations and the IC₅₀ values. The five best descriptors were selected to create and validate the model, which exhibited parameters that attested to its robustness and predictability. In summary, we observed that compounds with a para substitution and heavier groups (i.e. CF₃ and NO₂ substituents) had an enhanced HKT-inhibition profile. These compounds comprise a series described as AeHKT inhibitors via enzymatic inhibition experiments, opening the way to further the development of new substances with higher potency against HKT from Aedes aegypti.

Fig. 1. Structures of HKT inhibitors 4-(2-aminophenyl)-4-oxobutyric acid (4-OB) and sodium 4-[3-(4-iodo-phenyl)-[1,2,4] oxadiazol-5-yl]-butanoate.

Fig. 2. Modifications made on oxadiazole derivatives in this work.

Scheme 1. Synthesis of oxadiazole salts tested as AeHKT inhibitors. (i) NH₂OH.HCl and NaHCO₃ (4 eq.), ethanol/H₂O, r.t.; (ii) succinic anhydride (1.5 eq.), neat, 140 °C; (iii) NaOH (1 eq.) and methanol, r.t; (iv) glutaric anhydride (1.5 eq.), neat, 140 °C; (v) NaOH (1 eq.) and methanol, r.t.

Fig. 3. Inhibition mechanism of the canonical compound 4a. (A) Kinetic model of Michaelis–Menten inhibition in the presence and absence of inhibitor 4a. (B) Lineweaver–Burk plot for the enzymatic reaction in the presence and absence of inhibitor 4a.

Fig. 4. The 4-OB ligand binding modes in AgHKT and AeHKT active sites after the redocking procedure. (A) AgHKT in light yellow, showing the PLP cofactor and Arg³⁵⁶ residue (important for substrate and ligand binding and recognition). Co-crystallized 4-OB in orange and the lowest energy conformation from molecular docking calculations in purple. (B) AeHKT in grey, showing the PLP cofactor and Arg³⁵⁶ residue. Co-crystallized 4-OB in green and the lowest energy conformations (dark blue and cyan) from calculations started with the ligand placed outside the active site. Distances in angstroms.

Fig. 5. Calculated binding modes for oxadiazole derivatives in AeHKT active sites via molecular docking simulations. (A) The lowest energy conformation for compounds 4a (red) and 4-OB (dark blue), showing similar interactions with residues Arg³⁵⁶, Ser¹⁵⁴ and Asn⁴⁴. (B) The lowest energy conformation of compound 4n (pink), resulting in a conformation with a 180-degree turn in comparison to the canonical 4a. (C) The lowest energy conformation of compound 6i (blue), which is representative for most compounds of this series except for 6g. (D) All ortho-substituted compounds 4m, 4q and 4s (yellow) exhibited the lowest energy conformations similar to 4n, with the aromatic ring pointing to the inner side of the active site. (E) Compounds 6g and 4i displayed the lowest energy conformation outside of the active site, but still interacting with Arg³⁵⁶.

Fig. 6. Plot of inhibitory activity of the oxadiazole derivative sodium salts (IC₅₀) versus estimated K_I values. Compounds 4a–4s are shown as filled symbols, whereas compounds 6a–6k are shown as empty symbols. The shape of the symbols is equivalent to the position of the substitution (para, meta, ortho or other). The same substituents have matching colors for both series.

Fig. 7. Leave-N-out cross-validation results.

Fig. 8. Y-scrambling results (blue dots: results for scrambled models; red triangle: results from the original model).