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

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.


Introduction
Human African Trypanosomiasis (HAT), also known as sleeping sickness, is a parasitic disease widespread in sub-Saharan Africa, where it represents a relevant cause of death [1]. HAT is induced by two subspecies of Trypanosoma: T. brucei gambiense, which is able to cause the chronic form of HAT and widespread in western and central Africa, and T. b. rhodesiense, which is common in eastern and southern Africa, and responsible for the rapid-onset high death rate HAT [2].
Current HAT therapy is based on a few dated drugs with a limited spectrum of action, toxicity, and problems related to the parenteral route of administration. At present, the first-line treatment of the gambiense HAT is based on nifurtimox-eflornithine combination therapy (NECT), in which nifurtimox is used off-label, since it was approved for Chagas disease [3]. Recently, a new orally administered molecule, i.e., fexinidazole, was introduced in therapy, with an improvement of patient compliance; however, its approval is limited to the gambiense form of HAT [4,5]. From this perspective, there is an urgent need to identify new targets to develop new drugs that are also active on the lethal rhodesiense form of HAT.
Several strategies have been put in place for the development of novel antitrypanosomal agents [6][7][8][9]. In this scenario, rhodesain, the main cysteine protease of T. b. rhodesiense, 2 of 9 is an attractive target, since it mediates essential processes for parasite survival and disease progression; thus, it has become one of the main targets for the drug discovery process of new antitrypanosomal agents [10,11].
Rhodesain owes its importance to its various functions: (a) it is responsible for the crossing of the blood-brain barrier of the human host [12], thus inducing the neurological stage of HAT; (b) it is involved in the elusion of the host immune system, since it takes part in the turnover of variant surface glycoproteins of the trypanosome coat and degrades the host immunoglobulins [13,14]; (c) last, it shows a significant proteolytic activity in lysosomes, since it is involved in the degradation of intracellularly transported host proteins, as well as in parasite proteins. For these reasons rhodesain is considered an important target for HAT treatment [10,11].
More recently, starting from consistent literature data that demonstrated the ability of the nitrile function to react with the catalytic cysteine of rhodesain and also of several cathepsins [26][27][28], we have developed a new class of dipeptide nitriles, as potent rhodesain inhibitors [29].
Within the most interesting compounds, the dipeptide nitrile CD24 (Figure 1) showed the highest binding affinity towards rhodesain (K i = 16 nM), coupled with a good antiparasitic activity, i.e., EC 50 = 10.1 ± 0.5 µM. We also proved that CD24 directly binds to the active site of rhodesain, acting as competitive inhibitor [29]. need to identify new targets to develop new drugs that are also active on the lethal rhodesiense form of HAT. Several strategies have been put in place for the development of novel antitrypanosomal agents [6][7][8][9]. In this scenario, rhodesain, the main cysteine protease of T. b. rhodesiense, is an attractive target, since it mediates essential processes for parasite survival and disease progression; thus, it has become one of the main targets for the drug discovery process of new antitrypanosomal agents [10,11].
Rhodesain owes its importance to its various functions: (a) it is responsible for the crossing of the blood-brain barrier of the human host [12], thus inducing the neurological stage of HAT; (b) it is involved in the elusion of the host immune system, since it takes part in the turnover of variant surface glycoproteins of the trypanosome coat and degrades the host immunoglobulins [13,14]; (c) last, it shows a significant proteolytic activity in lysosomes, since it is involved in the degradation of intracellularly transported host proteins, as well as in parasite proteins. For these reasons rhodesain is considered an important target for HAT treatment [10,11].
More recently, starting from consistent literature data that demonstrated the ability of the nitrile function to react with the catalytic cysteine of rhodesain and also of several cathepsins [26][27][28], we have developed a new class of dipeptide nitriles, as potent rhodesain inhibitors [29].
Within the most interesting compounds, the dipeptide nitrile CD24 (Figure 1) showed the highest binding affinity towards rhodesain (Ki = 16 nM), coupled with a good antiparasitic activity, i.e., EC50 = 10.1 ± 0.5 μM. We also proved that CD24 directly binds to the active site of rhodesain, acting as competitive inhibitor [29]. Considering our expertise in drug-combinations [30][31][32], we carried out a combination study of the novel identified lead compound CD24 with curcumin ( Figure 1), a multitarget nutraceutical obtained from Curcuma longa L., which we demonstrated to inhibit rhodesain in a non-competitive manner [31].
Our aim was to evaluate, according to the Chou and Talalay method [33,34], if additive or synergistic effects occur in rhodesain inhibition, when we combine the synthetic inhibitor CD24 and the nutraceutical curcumin, considering that there can be many advantages of drug combinations, e.g., reduced risk of toxicity for the human host by reduction of individual dose or the use of lower amounts of poorly soluble compounds [35].

Results and Discussion
CD24 and curcumin were tested against recombinant rhodesain by using Cbz-Phe-Arg-AMC as a fluorogenic substrate [36]. We initially carried out a screening at 100 μM, 1 μM, and 0.1 μM, to evaluate the range of activity of the two inhibitors. CD24 and curcumin were then separately tested in two independent experiments, each performed in duplicate. Seven different concentrations were selected for CD24 and curcumin, starting from the minimum dose required to inhibit the enzyme, to that necessary to fully suppress Considering our expertise in drug-combinations [30][31][32], we carried out a combination study of the novel identified lead compound CD24 with curcumin ( Figure 1), a multitarget nutraceutical obtained from Curcuma longa L., which we demonstrated to inhibit rhodesain in a non-competitive manner [31].
Our aim was to evaluate, according to the Chou and Talalay method [33,34], if additive or synergistic effects occur in rhodesain inhibition, when we combine the synthetic inhibitor CD24 and the nutraceutical curcumin, considering that there can be many advantages of drug combinations, e.g., reduced risk of toxicity for the human host by reduction of individual dose or the use of lower amounts of poorly soluble compounds [35].

Results and Discussion
CD24 and curcumin were tested against recombinant rhodesain by using Cbz-Phe-Arg-AMC as a fluorogenic substrate [36]. We initially carried out a screening at 100 µM, 1 µM, and 0.1 µM, to evaluate the range of activity of the two inhibitors. CD24 and curcumin were then separately tested in two independent experiments, each performed in duplicate. Seven different concentrations were selected for CD24 and curcumin, starting from the minimum dose required to inhibit the enzyme, to that necessary to fully suppress the rhodesain activity. In more detail, we used concentrations in the range 0.05-20 µM and 5-100 µM for CD24 and curcumin, respectively.
IC 50 values were calculated from dose response-curves, as shown in Figure 2: 0.2 ± 0.01 µM for CD24 and 12.3 ± 0.9 µM for curcumin. the rhodesain activity. In more detail, we used concentrations in the range 0.05-20 μM and 5-100 μM for CD24 and curcumin, respectively. IC50 values were calculated from dose response-curves, as shown in Figure 2: 0.2 ± 0.01 μM for CD24 and 12.3 ± 0.9 μM for curcumin. In a subsequent experiment, five data points were established for both compounds (1/4 × IC50, 1/2 × IC50, IC50, 2 × IC50, and 4 × IC50, Table 1), with the aim of evaluating if a synergistic, additive, or antagonist effect occurred in the combination study of the inhibitors. In this assay, the combination of CD24 and curcumin (molar ratio 1:62) provided an IC50 value of 5.6 ± 0.4 μM. We then converted each dose-response curve into a median effect plot, which was obtained by plotting on the y-axis the log (fa/fu) versus the log (D) on the x-axis ( Figure 3). In the median effect plot the maximum response corresponds to 1, instead of the 100 of the dose-response curve. Therefore fa + fu = 1, where fa corresponds to the "affected fraction", i.e., the percentage of enzyme that has been inhibited, while fu is the unaffected fraction, i.e., the residual enzyme activity. The slope of the straight line of each median effect plot is the "m value"; in detail, CD24 showed m1 = 0.9757 and curcumin m2 = 2.6066, while for the combination assay, we found m1,2 = 2.6705, with a molar ratio CD24/curcumin of 1:62. In a subsequent experiment, five data points were established for both compounds (1/4 × IC 50 , 1/2 × IC 50 , IC 50 , 2 × IC 50 , and 4 × IC 50 , Table 1), with the aim of evaluating if a synergistic, additive, or antagonist effect occurred in the combination study of the inhibitors. In this assay, the combination of CD24 and curcumin (molar ratio 1:62) provided an IC 50 value of 5.6 ± 0.4 µM. We then converted each dose-response curve into a median effect plot, which was obtained by plotting on the y-axis the log (f a /f u ) versus the log (D) on the x-axis ( Figure 3). In the median effect plot the maximum response corresponds to 1, instead of the 100 of the dose-response curve. Therefore f a + f u = 1, where f a corresponds to the "affected fraction", i.e., the percentage of enzyme that has been inhibited, while f u is the unaffected fraction, i.e., the residual enzyme activity. The slope of the straight line of each median effect plot is the "m value"; in detail, CD24 showed m 1 = 0.9757 and curcumin m 2 = 2.6066, while for the combination assay, we found m 1,2 = 2.6705, with a molar ratio CD24/curcumin of 1:62. the rhodesain activity. In more detail, we used concentrations in the range 0.05-20 μM and 5-100 μM for CD24 and curcumin, respectively. IC50 values were calculated from dose response-curves, as shown in Figure 2: 0.2 ± 0.01 μM for CD24 and 12.3 ± 0.9 μM for curcumin. In a subsequent experiment, five data points were established for both compounds (1/4 × IC50, 1/2 × IC50, IC50, 2 × IC50, and 4 × IC50, Table 1), with the aim of evaluating if a synergistic, additive, or antagonist effect occurred in the combination study of the inhibitors. In this assay, the combination of CD24 and curcumin (molar ratio 1:62) provided an IC50 value of 5.6 ± 0.4 μM. We then converted each dose-response curve into a median effect plot, which was obtained by plotting on the y-axis the log (fa/fu) versus the log (D) on the x-axis (Figure 3). In the median effect plot the maximum response corresponds to 1, instead of the 100 of the dose-response curve. Therefore fa + fu = 1, where fa corresponds to the "affected fraction", i.e., the percentage of enzyme that has been inhibited, while fu is the unaffected fraction, i.e., the residual enzyme activity. The slope of the straight line of each median effect plot is the "m value"; in detail, CD24 showed m1 = 0.9757 and curcumin m2 = 2.6066, while for the combination assay, we found m1,2 = 2.6705, with a molar ratio CD24/curcumin of 1:62.  Starting from the assessment that CD24 is a competitive inhibitor of rhodesain [29], while curcumin acts as non-competitive rhodesain inhibitor [31], as previously demonstrated by our research group, and with the aim of determining the inhibitory effect given by the combination of CD24 and curcumin, we used the Chou-Talalay method to evaluate the multiple drug effects [33,34].
In more detail, we calculated the combination index (CI), which expresses the nature of the inhibition towards the target enzyme when two drugs are tested in combination.
In particular, it is well known that a CI > 1, CI = 1, and CI < 1 generally correspond to an antagonistic, additive, and synergistic effect, respectively [33,34]. The CI for mutually non-exclusive drugs, which act independently, was calculated as follows: where (IC 50 ) 1 and (IC 50 ) 2 were already obtained using dose-response curves, while the D 1 and D 2 , able to induce a specific percentage of rhodesain inhibition were obtained using a median effect equation.
Grafit software was used to determine the CI, ranging from 50% to 100%, of rhodesain inhibition ( Figure 4). Starting from the IC 50 , which is normally taken to determine the activity of a novel inhibitor, we observed an initial additive effect, since CI resulted = 1, according to Chou's rules [33,34,37]. 5.0.1.3; Erithacus Software Limited, East Grinstead, West Sussex, UK), we established th which were able to induce each percentage of rhodesain inhibition by means of dian effect equation D = IC50 [fa/fu] 1/m [33,34].
Starting from the assessment that CD24 is a competitive inhibitor of rhodes while curcumin acts as non-competitive rhodesain inhibitor [31], as previously strated by our research group, and with the aim of determining the inhibitory effe by the combination of CD24 and curcumin, we used the Chou-Talalay method to e the multiple drug effects [33,34].
In more detail, we calculated the combination index (CI), which expresses th of the inhibition towards the target enzyme when two drugs are tested in combin In particular, it is well known that a CI > 1, CI = 1, and CI < 1 generally corres an antagonistic, additive, and synergistic effect, respectively [33,34]. The CI for m non-exclusive drugs, which act independently, was calculated as follows: where (IC50)1 and (IC50)2 were already obtained using dose-response curves, whil and D2, able to induce a specific percentage of rhodesain inhibition were obtained median effect equation.
Grafit software was used to determine the CI, ranging from 50% to 1 rhodesain inhibition (Figure 4). Starting from the IC50, which is normally taken t mine the activity of a novel inhibitor, we observed an initial additive effect, sinc sulted = 1, according to Chou's rules [33,34,37]. Interestingly, for the most significant fa values, which ranged from 0.6 to 1 (i 60% to 100% of rhodesain inhibition), an increasing synergistic effect was detecte CD24 and curcumin were used in combination (Table 2). Interestingly, for the most significant f a values, which ranged from 0.6 to 1 (i.e., from 60% to 100% of rhodesain inhibition), an increasing synergistic effect was detected when CD24 and curcumin were used in combination (Table 2). Considering our previously recorded activity of CD24 [29] and curcumin [31] alone against T. brucei brucei (Table 3), we decided to test the two inhibitors in combination (molar ratio 1:1) by obtaining EC 50 = 4.85 ± 0.02 µM ( Figure 5). Overall, the obtained data led us to assume that the use of our synthetic inhibitor CD24 in combination with curcumin led to an improvement of its antitrypanosomal activity (EC 50s = 10.1 ± 0.5 µM [29] vs. 4.85 ± 0.02 µM), thus suggesting a fruitful use of the drugs in combination. 2.38 ± 0.88 --Considering our previously recorded activity of CD24 [29] and curcu against T. brucei brucei (Table 3), we decided to test the two inhibitors in com lar ratio 1:1) by obtaining EC50 = 4.85 ± 0.02 μM ( Figure 5). Overall, the obt us to assume that the use of our synthetic inhibitor CD24 in combination led to an improvement of its antitrypanosomal activity (EC50s = 10.1 ± 0.5 μ ± 0.02 μM), thus suggesting a fruitful use of the drugs in combination. Table 3. Activity against T. brucei brucei and HEK293 cells and selectivity index ( curcumin alone and of the combination CD24 + curcumin.

Compounds
T. b. brucei EC50 μM HEK293 EC50 μM CD24 [29] 10.1 ± 0.5 >70 Curcumin [31] 3.12 ± 0.43 >70 CD24 + curcumin 4.85 ± 0.02 >70 Fexinidazole [38] 2.38 ± 0.88 - A classic isobologram analysis was performed to evaluate which of the CD24 and curcumin, used in a molar ratio 1:1, was able to produce a sy when combined against T. b. brucei ( Figure 6). If the combination data po hypothenuse (i.e., the dose of 16.66 μM), an additive effect was indicated. I tion data points fell on the lower left (e.g., all the doses ranging from 0.06 μ a synergism was indicated. While for the sole combination point that fel right (i.e., 33.33 μM), an antagonistic effect was indicated. A classic isobologram analysis was performed to evaluate which of the used doses of CD24 and curcumin, used in a molar ratio 1:1, was able to produce a synergistic effect when combined against T. b. brucei (Figure 6). If the combination data points fell on the hypothenuse (i.e., the dose of 16.66 µM), an additive effect was indicated. If the combination data points fell on the lower left (e.g., all the doses ranging from 0.06 µM to 8.33 µM) a synergism was indicated. While for the sole combination point that fell on the upper right (i.e., 33.33 µM), an antagonistic effect was indicated. Finally, the cytotoxicity of CD24, curcumin alone, and CD24 in combination with curcumin was assessed towards HEK293 cell lines, by using the range of concentrations 70-0.5 μM. In both cases, no cytotoxic effects were observed up to 70 μM.
All in all, the nutraceutical showed the highest selectivity index (SI), while the synthetic inhibitor alone showed the lowest SI. The combination of CD24 + curcumin showed a SI slightly lower than that of curcumin, thus signifying a productive use of the inhibitors in combination, considering their strong synergistic action against rhodesain. Finally, the cytotoxicity of CD24, curcumin alone, and CD24 in combination with curcumin was assessed towards HEK293 cell lines, by using the range of concentrations 70-0.5 µM. In both cases, no cytotoxic effects were observed up to 70 µM.
All in all, the nutraceutical showed the highest selectivity index (SI), while the synthetic inhibitor alone showed the lowest SI. The combination of CD24 + curcumin showed a SI slightly lower than that of curcumin, thus signifying a productive use of the inhibitors in combination, considering their strong synergistic action against rhodesain.

Antitrypanosomal Activity Assay
The parasites used in this study were culture-adapted T. b. brucei 449, descendants of the Lister strain 427 [40]. Cytotoxic activity of the combination of CD24 with curcumin in 1:1 molar ratio against T. b. brucei was determined using the ATPlite assay, as described previously [29,36,41,42]. Stock solution of the CD24 and curcumin in DMSO was prepared by mixing the compounds in a 1:1 molar ratio, for a final concentration of 10 mM. This stock was then used to perform serial dilutions in culture media. The final concentrations applied on the cells were 33.33 µM, 16.66 µM, 8.33 µM, 4.16 µM, 2.08 µM, 1.04 µM, 0.52 µM, 0.26 µM, 0.13 µM, and 0.06 µM.

Cytotoxicity Evaluation
HEK293 cells were cultured in high glucose DMEM medium with L-glutamine, supplemented with 10% FCS, 20 U/mL penicillin, and 20 µg/mL streptomycin, at 37 • C and 5% CO 2 . Cytotoxic activities of CD24 and CD24/curcumin combination (in 1:1 molar ratio) were assayed using resazurin staining, as described previously [43]. Briefly, prepared compound or compound mix stocks in DMSO were subjected to seven consecutive 1:2 dilution steps in DMSO. The resulting eight dilutions of the compound, ranging from 7 mM to 0.055 mM, were further diluted 1:100 with addition to the wells of poly-lysine-coated 48 well plates containing cells that had been seeded at 60,000 cells/well and incubated for 24 h at 37 • C. The cells were incubated with the compounds for 21 h at 37 • C, after which the culture medium in each well was exchanged for medium supplemented with resazurin (15 µg/mL). After 3 h at 37 • C incubation with the resazurin-containing medium, an aliquot of 100 µL was removed from each well and transferred into a black, clear-bottom 96 well plate, and the fluorescence was measured (excitation: 540-14, emission: 590-20) using a CLARIOstar Plus plate reader (BMG Labtech, Ortenberg, Germany). Cells treated with DMSO alone were used as control. The assay was performed in duplicate.

Statistical Analyses
The statistical analysis of the data was performed using the one-way test (ANOVA) with Dunnett's multiple comparison test, considering significant differences of p < 0.05 with respect to the percentage of rhodesain inhibition of curcumin, CD24, and curcumin + CD24. The analyses were performed with GraphPAD Prism 6 (GraphPad software Inc., San Diego, California). Results are expressed as the arithmetic mean ± standard deviation (SD).

Conclusions
In summary, in this study, starting from the single activities of CD24 and curcumin against rhodesain, we investigated the activity of their combination, concluding that at IC 50 an initial additive effect was observed (CI = 1), while for the most significant f a values, i.e., those ranging from 0.6 to 1 (corresponding to the range 60-100% of rhodesain inhibition), an increasingly synergistic action was observed. Moreover, at cellular level we obtained, with the combination synthetic inhibitor + nutraceutical, an antitrypanosomal activity in the low micromolar range and a selectivity index better than that exhibited by CD24 alone; thus, for all the described reasons, their use in combination is desirable.