Drug Synergism: Studies of Combination of RK-52 and Curcumin against Rhodesain of Trypanosoma brucei rhodesiense

Information ABSTRACT: Rhodesain is an enzyme essential for the life of Trypanosoma brucei rhodesiense, a parasite causing a rapid-onset form of Human African Trypanosomiasis. RK-52 is a synthetic inhibitor of rhodesain, characterized by an impressive k second value ( k second = 67000 × 10 3 M − 1 min − 1 ) and by a picomolar a ﬃ nity toward the trypanosomal protease ( K i = 38 pM). Di ﬀ erently, curcumin, the golden multitarget nutraceutical obtained from Curcuma longa L., was proven to inhibit rhodesain non-competitively with an IC 50 of 7.75 μ M. In the present study, we carried out studies of a combination of RK-52 and curcumin toward rhodesain, by applying the Chou and Talalay approach, which led us to obtain a combination index <1 for the most relevant f a values, which means a potent synergistic e ﬀ ect for the reduction of rhodesain activity from 40% to

H uman African Trypanosomiasis (HAT) is a parasitic disease occurring in sub-Saharan Africa, inducing mortality despite a significant decrease of novel cases recently having been observed. 1 HAT is induced by two subspecies of Trypanosoma: T. brucei gambiense, common in western and central Africa, able to cause a chronic HAT, and T. brucei rhodesiense, widespread in eastern and southern Africa, responsible for a rapid-onset high death rate HAT. 2 In the hemolymphatic stage of HAT, the parasite invades the bloodstream and then the lymph nodes and spleen inducing illness, muscle aches, and fatigue. If the parasite crosses the blood−brain barrier (BBB) a neurological stage occurs characterized by sleep disorders and death. 3 At present HAT therapy is based on only a few available drugs: pentamidine and suramin for the first-stage HAT, while the second-stage HAT is treated with eflornithine, melarsoprol, and nifurtimox. However, melarsoprol shows a high neurotoxicity, while eflornithine, whose use is safer, is active only against T. b. gambiense. 4 Thus, the first-line therapy for the treatment of the second-stage HAT is based on the coadministration of nifurtimox and eflornithine; more recently, in 2018, fexinidazole was introduced in therapy because of its activity against T. brucei gambiense. 5−7 Thus, new molecular targets have to be identified to develop innovative effective drugs for HAT therapy.
In this research area rhodesain, the key cysteine protease for the survival of T. brucei rhodesiense, is certainly a promising target to treat rhodesiense trypanosomiasis, the most lethal form of HAT. 8,9 Rhodesain is essential to permit the parasite to cross the BBB reaching the central nervous system, thus inducing the second-stage HAT; it takes part in the turnover of variant surface glycoproteins of the trypanosome coat 10   Letter pubs.acs.org/acsmedchemlett evasion of the host immune system by degrading host immunoglobulins. 11 Moreover, rhodesain is predominant also in the lysosomes where it exerts its protease activity; thus, it is reasonable to consider the trypanosomal protease a promising target for HAT treatment. 12 In this context, in the past decade our research team has been working on the discovery of new rhodesain inhibitors as a novel strategy for HAT therapy, 13−20 leading to the identification of a potent irreversible rhodesain inhibitor, i.e. RK-52 (k second = 67000 × 10 3 M −1 min −1 ) ( Figure 1), endowed with a picomolar affinity toward trypanosomal protease (K i = 38 pM). 21 In the same field, we demonstrated also that curcumin (Figure 1), the golden multitarget nutraceutical obtained from Curcuma longa L., possesses an antitrypanosomal activity due to rhodesain inhibition (IC 50 value of 7.75 μM). 22   In this study, we now discuss studies of a combination of RK-52 and curcumin, by applying the Chou and Talalay approach, 23,24 to evaluate if additive or synergistic effects occur in rhodesain inhibition.
RK-52 was synthesized as previously reported by our group. 21 Curcumin was purchased from Sigma-Aldrich.
Rhodesain was obtained as previously described by Caffrey et al. 25 Curcumin and RK-52 were then separately tested in three independent experiments, each performed in duplicate.
Biological evaluation against rhodesain was carried out using Cbz-Phe-Arg-AMC as fluorogenic substrate. IC 50 values have been obtained from dose response-curves shown in Figure 2: 7.75 ± 1.53 μM for curcumin 22 and 0.009 ± 0.0008 μM for RK-52. Subsequently, five data points for both compounds (1/ 4 × IC 50 , 1/2 × IC 50 , IC 50 , 2 × IC 50 , and 4 × IC 50 , Table 1) have been selected, with the aim to verify the possible synergistic, additive, or antagonist effect in the combination assay of the inhibitors. In this case, the combination of RK-52 and curcumin provided an IC 50 value of 3.05 ± 0.21 μM.
The median effect equation described by Chou states that f a /f u = (D/D m ) m , where f a and f u represent the affected and the unaffected fractions, respectively, of the target enzyme, by the dose D; D m corresponds to IC 50 , able to produce the median effect, while m represents the Hill-type coefficient, which means the sigmoidal trend (or S-shaped) of the dose-reponse curve. 23,24 The median effect plot was generated plotting the three dose−response curves as log (f a /f u ), on the y-axis, with respect to log (D), on the x-axis (Figure 3). This allowed us to obtain the IC 50 values of the three different evaluations, together with the related m coefficients. In detail, curcumin showed IC 50 = 7.75 μM and m 1 = 1.4681, RK-52 IC 50 = 0.009 μM and m 2 = 1.7841 while for the combination assay we found IC 50 = 3.05 μM and m 1,2 = 2.0305, with a molar ratio RK-52/curcumin of 1:861.
In order to determine the inhibitory effect given by curcumin and RK-52, the multiple drug effect evaluation reported in the literature by Chou and Talalay was used. 23,24 In this contest, the combination index (CI) provides the nature of the inhibition toward the target enzyme, when two drugs are evaluated simultaneously. In particular, three effects have been demonstrated for CI > 1, CI = 1, and CI < 1, which correspond to an antagonistic, additive, and synergistic impact, respectively.
The CI for mutually exclusive drugs was calculated as follows:    where (D 50 ) 1 and (D 50 ) 2 represent the IC 50 values for curcumin and RK-52 when they are evaluated alone, while (D) 1 and (D) 2 are the concentrations of both compounds able to produce 50% of rhodesain inhibition when they are used in combination. Grafit software was used to determine the CI (Figure 4). The obtained values show a clear synergistic effect when the most significant f a is ranging from 0.40 to 0.90, while for f a = 0.10 and f a ranging from 0.20 to 0.30, an antagonist and additive effect was recorded, respectively (Table 2).
Finally, since a synergism at all the relevant fa values was observed between the two rhodesain inhibitors we further investigated if the inhibition is competitive with respect to the substrate by determining the IC 50 in dependence of five substrate concentration (5, 10, 15, 20, and 25 μM).
In the case of RK-52 (Figure 5a) we found linearly increasing IC 50 values with increasing substrate concentration proving that RK-52 binds to the active site. On the contrary, in the case of curcumin (Figure 5b) with increasing substrate concentration no variation of IC 50 was observed thus suggesting that curcumin binds to an allosteric site.
The comparable activity of curcumin with respect to RK-52 against T. b. brucei, with respect to the very different activity against rhodesain, could be explained considering that the antitrypanosomal activity of curcumin might be due, in addition to the inhibition of rhodesain, to the interaction with other proteases that can be found in the trypanosomal intracellular environment.
We have also to consider that the activity against T. b. brucei is also related to the capability of a drug to cross the cellular membranes, which is certainly more easy for low molecular weight molecules such as curcumin, with respect to RK-52.
All in all, in this study starting from the activities of RK-52 and curcumin against rhodesain, we investigated the activity of their combination, obtaining for the most significant f a values (from 0.40 to 0.90) a synergistic effect for rhodesain inhibition. The combination curcumin + RK-52 showed an IC 50 value againt T. b. brucei of 4.64 μM; thus, it is reasonable in future studies to further investigate the overall reduction of toxicity of the combination with respect to the single inhibitors, which is normally the main reason for the use of drug combinations. orcid.org/0000-0002-8942-797X Complete contact information is available at: https://pubs.acs.org/10.1021/acsmedchemlett.9b00635

Author Contributions
All authors approved the content of the manuscript.

Funding
This study was economically supported by the "Fondo per il finanziamento delle attivitàbase di ricerca" (FFABR 2017) financed by MIUR.