1,2,4-thiadiazol-5(4H)-ones: a new class of selective inhibitors of Trypanosoma cruzi triosephosphate isomerase. Study of the mechanism of inhibition

Figures & data

Figure 1.  Chemical structure of 1,2,4-thiadiazol derivatives and the previously studied¹² 1,3,4-oxathiazol. Derivatives 1 and 2 were also analyzed against HsTIM showing at least 10-fold selectivity for TcTIM.

Table 1.  Classic inhibition assay of Tc, Hs, Lm and Tb TIMs to evaluate the selectivity of the inhibitor compounds.

Compound	Inhibition
	IC50 (µM)		% of inhibition at 100 µM
	TcTIM	HsTIM	LmTIM	TbTIM
1	3.5 ± 1^a (6.0)^b	~100^a (59.0)^c	2.0	16.0
2	10 ± 1^a (200.0)^b	>100^a (0.0)^c	0.0	0.0

^aFrom the reference Alvarez et al.¹².

^bThe values in parentheses correspond to the IC₉₀ (µM).

^cThe values in parenthesis correspond to the percentage of inhibition of enzyme activity at 100 µM.

Figure 2.  Effect of compounds 1 on the activity of TcTIM. (a) Lineweaver–Burk plot of TcTIM alone and TcTIM+compound 1 (3.5 µM). (b) TcTIM activity dependence on compound 1 concentration.

Figure 3.  Effect of compound 1 on TcTIM stability. (a) Effect of compound 1 on TcTIM stability. The enzyme was incubated at the indicated concentrations for 2 h at 37°C with (▪) and without (•;) 3.5 µM compound 1 and the activity of each of the samples was measured. (b) Effect of compound 1 on the formation of active TcTIM from GdnHCl unfolded monomers. The experiment was performed as described in the Methods section. Activity was measured 15 (•) and 60 (▪) min after the denaturing mixture was diluted one hundred fold.

Figure 4.  Calculated electronic distribution for compounds 1–4. (a) Potential electrostatic molecular maps. The solid arrow shows the reaction centre (sulfur 1, S1) and the dotted arrow points the protonated centre (nitrogen 2, N2). (b) HOMO maps (red-blue) superimposed to electron density bond maps (silver). The solid arrows show the contribution of S1 and N2 to the HOMO only in derivative 1. The S1-N2 bond lengths for each compound are also shown.

Figure 5.  Structural similarities of the analyzed compounds (1–4).

Table 2.  Classic inhibition assay, using modified TcTIMs, to evaluate the selectivity of the inhibitor 1.

Compound	Inhibition
	IC50 (µM)	% of inhibition at 100 µM
	TcTIMC15A	TcTIM 2,3,5-8 (chimera)
1	1.5 ± 1	19
2	ns^a	4

^ans, not studied.

Figure 6.  Stereoview of TcTIM showing the regions of TbTIM (in red) that were grafted in TcTIM to generate the chimera studied herein.

Figure 7.  T. cruzi growth inhibition assays. (a) Dose-response curves showing growth inhibition of T. cruzi by compound 1 incorporated into phospholipid (PL). (b) Dose-response curves showing growth inhibition of T. cruzi by compound 1 incorporated into PL and the reference drugs, Nifurtimox (Nfx) and Benznidazole (Bnz).

Alvarez G, Aguirre-López B, Varela J, Cabrera M, Merlino A, López GV et al. Massive screening yields novel and selective Trypanosoma cruzi triosephosphate isomerase dimer-interface-irreversible inhibitors with anti-trypanosomal activity. Eur J Med Chem 2010;45:5767–5772.

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