Drug Combination Studies of the Dipeptide Nitrile CD24 with Curcumin: A New Strategy to Synergistically Inhibit Rhodesain of Trypanosoma brucei rhodesiense

Abstract

Rhodesain is a cysteine protease that is crucial for the life cycle of Trypanosoma brucei rhodesiense, a parasite causing the lethal form of Human African Trypanosomiasis. CD24 is a recently developed synthetic inhibitor of rhodesain, characterized by a nanomolar affinity towards the trypanosomal protease (Ki = 16 nM), and acting as a competitive inhibitor. In the present work, we carried out a combination study of CD24 with curcumin, the multitarget nutraceutical obtained from Curcuma longa L., which we demonstrated to inhibit rhodesain in a non-competitive manner. By applying the Chou and Talalay method, we obtained an initial additive effect at IC50 (fa = 0.5, Combination Index = 1), while for the most relevant fa values, ranging from 0.6 to 1, i.e., from 60% to 100% of rhodesain inhibition, we obtained a combination index < 1, thus suggesting that an increasingly synergistic action occurred for the combination of the synthetic inhibitor CD24 and curcumin. Furthermore, the combination of the two inhibitors showed an antitrypanosomal activity better than that of CD24 alone (EC50 = 4.85 µM and 10.1 µM for the combination and CD24, respectively), thus suggesting the use of the two inhibitors in combination is desirable.

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

“…If we compare the obtained values of CI with those obtained for the combination CD24 + curcumin [33] (Table 3), it is possible to observe an initial additive effect at the IC 50, followed by a slight-to-moderate synergism at 60-70% and a synergistic action from 80% to 100% rhodesain inhibition. Thus, it is clear that the combination of CD34 + curcumin resulted in a much stronger synergistic action, especially at 100% enzyme inhibition, where a very strong synergism was observed, thus assessing an improved combination strategy for CD34 + curcumin with respect to that of CD24 + the same nutraceutical.…”

“…Curcumin was purchased from Sigma Aldrich. Since the range of active concentrations for curcumin was known by us [33], a preliminary screening with rhodesain was performed only for CD34 at 20 µM; since it inhibited rhodesain by more than 80%, it passed the initial screening and was characterized in detail. An equivalent amount of DMSO was used as negative control.…”

“…Recently, we developed a new class of dipeptide nitrile able to react with the catalytic cysteine of rhodesain by Pinner reaction, giving a reversible thiomidate adduct, thus identifying a novel lead compound, i.e., the dipeptide nitrile CD24, which showed a nanomolar binding affinity towards rhodesain (K i = 16 nM), coupled to a good antiparasitic activity (i.e., EC 50 = 10.1 ± 0.5 µM) [29]. Considering our know-how in drug combinations [30][31][32], we recently carried out a combination study on the lead compound CD24 with curcumin [33] (Figure 1), a potent multitarget nutraceutical extracted from Curcuma longa L., which we previously demonstrated to inhibit rhodesain in a non-competitive manner [31]. Following the rules of Chou and Talalay [34,35], we previously obtained an additive effect at the IC 50 for this combination.…”