Dipeptide Nitrile CD34 with Curcumin: A New Improved Combination Strategy to Synergistically Inhibit Rhodesain of Trypanosoma brucei rhodesiense

Abstract

Rhodesain is the main cysteine protease of Trypanosoma brucei rhodesiense, the parasite causing the acute lethal form of Human African Trypanosomiasis. Starting from the dipeptide nitrile CD24, the further introduction of a fluorine atom in the meta position of the phenyl ring spanning in the P3 site and the switch of the P2 leucine with a phenylalanine led to CD34, a synthetic inhibitor that shows a nanomolar binding affinity towards rhodesain (K_i = 27 nM) and an improved target selectivity with respect to the parent dipeptide nitrile CD24. In the present work, following the Chou and Talalay method, we carried out a combination study of CD34 with curcumin, a nutraceutical obtained from Curcuma longa L. Starting from an affected fraction (f_a) of rhodesain inhibition of 0.5 (i.e., the IC₅₀), we observed an initial moderate synergistic action, which became a synergism for f_a values ranging from 0.6 to 0.7 (i.e., 60-70% inhibition of the trypanosomal protease). Interestingly, at 80-90% inhibition of rhodesain proteolytic activity, we observed a strong synergism, resulting in 100% enzyme inhibition. Overall, in addition to the improved target selectivity of CD34 with respect to CD24, the combination of CD34 + curcumin resulted in an increased synergistic action with respect to CD24 + curcumin, thus suggesting that it is desirable to use CD34 and curcumin in combination.

Keywords: combination studies; curcumin; cysteine protease; dipeptide nitrile; rhodesain inhibitors.

Figure 1

Structures of CD24, CD34, and curcumin.

Scheme 1

Reagents and conditions: (a) EDCI, HOBt, DIPEA, dry DCM, 0 °C to rt., on.

Figure 2

Dose-response curves for rhodesain inhibition by CD34 (a), curcumin (b), and CD34 + curcumin in combination (c). Each experiment was performed twice, each in duplicate, with *** p < 0.001 vs. no inhibitor.

Figure 3

Median Effect Plot for CD34 (a), curcumin (b), and CD34+ curcumin in combination (molar ratio 1:70) (c). D is the dose, and f_a and f_u the dose-affected and dose-unaffected fractions of rhodesain activity, respectively.

Figure 4

Computer-generated graphical presentation of the combination index (CI) vs. the fraction affected (f_a), i.e., the effect of reduction in rhodesain activity exerted by a mixture of CD34–curcumin (molar ratio 1:70). The purple circles indicate the trend of the combination index.