Recently, it was suggested that curcumin is an irreversible inhibitor of rhodesain, a cathepsin L-like cysteine protease found in the lysosome of the protozoan parasite Trypanosoma brucei. However, dilution of rhodesain incubated with curcumin with curcumin free buffer resulted in an immediate recovery of enzymatic activity. This finding clearly demonstrates that curcumin is in fact a reversible inhibitor of rhodesain

Steverding, Dietmar

Dietmar Steverding

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Comments on “Drug combination studies of curcumin and genistein against rhodesain of
                    Trypanosoma brucei rhodesiense

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1  Comments on “Drug combination studies of 1 curcumin and genistein against rhodesain of 2 Trypanosoma brucei rhodesiense” 3  4 Dietmar Steverding  5  6 Bob Champion Research and Education Building, Norwich Medical 7 School, University of East Anglia, Norwich, United Kingdom 8  9 CONTACT: Dietmar Steverding  d.steverding@uea.ac.uk 10   11 2  ABSTRACT 12 Recently, it was suggested that curcumin is an irreversible inhibitor of rhodesain, a 13 cathepsin L-like cysteine protease found in the lysosome of the protozoan parasite 14 Trypanosoma brucei. However, dilution of rhodesain incubated with curcumin with 15 curcumin free buffer resulted in an immediate recovery of enzymatic activity. This 16 finding clearly demonstrates that curcumin is in fact a reversible inhibitor of rhodesain. 17  18 KEYWORDS 19 Rhodesain; curcumin; reversible inhibitor 20   21 3  Dear Editor, 22  23 I would like to comment on the article “Drug combination studies of curcumin and 24 genistein against rhodesain of Trypanosoma brucei rhodesiense” by Ettari et al. 25 (2018). In their article, the authors claim that curcumin and genistein are irreversible 26 inhibitors of rhodesain (the major lysosomal cysteine protease of T. brucei) and that 27 both compounds in combination act synergistically in inactivating the enzyme. 28 However, it is not possible that two irreversible inhibitors can interact synergistically in 29 inhibiting the activity of an enzyme. By definition, irreversible inhibitors chemically 30 modify their target enzyme by reacting with the active-site amino acid residue. 31 Accordingly, two compounds irreversibly inhibiting an enzyme cannot influence each 32 other on their binding of the catalytic amino acid residue. This is only possible if one 33 of the compounds binds the enzyme at a different site. Thus, the binding of one 34 compound could increase the affinity for the other compound and vice versa resulting 35 in a synergetic effect of the combination of the two compounds. 36 The evidence provided by the authors to support that curcumin and genistein are 37 irreversible inhibitors of rhodesain are unconvincing. The irreversible inhibition of the 38 compounds were based (i) on a time-dependent loss of enzyme activity and (ii) on the 39 lack of recovery of enzymatic activity after dilution. However, the time-dependent 40 inhibition was only marginal and the enzyme was never completely inhibited, even at 41 higher inhibitor concentrations. Likewise, the dilution factor in the dilution experiment 42 was minimal (only 1:2), so that the final inhibitor concentration was still very high. In 43 the case of genistein, the high IC50 value (500 µM) observed for the inhibition of 44 rhodesain activity is quite unusual for an irreversible inhibitor. Generally, irreversible 45 inhibitors of T. brucei cysteine proteases have IC50 values below 1 µM and those with 46 IC50 values > 100 µM are usually considered inactive (Steverding et al. 20112). 47 The lack of convincing evidence whether curcumin and genistein are irreversible 48 inhibitors of rhodesain prompted me to perform own experiments. Based on available 49 reagents, I carried out dilution experiments with curcumin (Figure 1). Incubation of 50 4  rhodesain with 10 µM curcumin resulted in an enzymatic rate of 2.74 RFU/min. When 51 the reaction mixture was diluted 1:4 with measuring buffer containing 10 µM curcumin, 52 the enzymatic rate dropped to 0.74 RFU/min, which was 27% of the activity before 53 dilution and thus in agreement with an one in four dilution of the amount of enzyme 54 (the expected activity would have been 25%). In contrast, when the reaction mixture 55 was diluted 1:4 with measuring buffer lacking curcumin, the enzymatic rate increased 56 to 12.82 RFU/min, which was 4.68 and 17.32 times higher compared with the starting 57 mixture and after dilution in the presence of 10 µM curcumin, respectively. Were 58 curcumin an irreversible inhibitor of rhodesain, no difference in the enzymatic rate 59 between the dilution in the presence and absence of 10 µM curcumin would be 60 expected. As the enzymatic rate after dilution in curcumin free measuring buffer 61 increased substantially (despite a 4-fold reduction in enzyme concentration), thus it 62 can be concluded that curcumin is a reversible inhibitor of rhodesain and not an 63 irreversible one. 64 The lesson, here, is: in order to determine the mode of action of inhibitors, one 65 needs to carry out the right experiments and to interpret the obtained data correctly. 66 The best way to do this would be to perform rigorous kinetic enzyme analysis. 67  68 Disclosure statement 69 The author reports no conflict of interest. 70  71 Acknowledgements 72 I thank Professor Conor R. Caffrey (Center for Discovery and Innovation in Parasitic 73 Diseases, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of 74 California San Diego) for providing rhodesain, and Dr Kevin Tyler (Norwich Medical 75 School, University of East Anglia) for critical reading of the manuscript. 76  77 ORCID 78 Dietmar Steverding  https://orcid.org/0000-0002-0050-7771 79 5   80 References 81 Ettari R, Previti S, Maiorana S, Allegra A, Schirmeister T, Grasso S, Zappalà M. 2018. 82 Drug combination studies of curcumin and genistein against rhodesain of 83 Trypanosoma brucei rhodesiense. Nat Prod Res. In press. 84 Steverding D, Sexton DW, Wang X, Gehrke SS, Wagner GK, Caffrey CR. 2012. 85 Trypanosoma brucei: chemical evidence that cathepsin L is essential for survival 86 and a relevant drug target. Int J Parasitol. 42:481-488. 87  88 Figure Legends 89 Figure 1. Reversibility of curcumin inhibition on rhodesain. Rhodesain (67 ng) was 90 incubated in measuring buffer (100 mM citrate, pH 5.0, 2 mM DTT and 10 µM 91 benzyloxycarbonyl-phenylalanyl-arginyl-7-amido-4-methyl coumarin (Z-FR-AMC) as 92 fluorogenic substrate) in the presence of 10 µM curcumin in a final volume of 2 ml at 93 room temperature. The release of 7-amino-4-methylcoumarin (AMC) was recorded 94 with a BIORAD VersaFluor fluorometer using excitation and emission wavelengths of 95 360 nm and 460 nm every min (triangles). After 20 min incubation, the reaction mixture 96 was diluted 1:4 with measuring buffer either containing 10 µM curcumin (squares) or 97 the equivalent amount of DMSO (circles), and release of AMC was continued to be 98 recorded. Note that the drop in RFU after dilution with measuring buffer containing 10 99 µM curcumin is due to the dilution of released AMC. Note that the increase in RFU 100 after dilution with measuring buffer lacking curcumin is due to the fact that curcumin 101 shifts RFU into negative values. Therefore, by diluting curcumin containing buffer with 102 curcumin free buffer, RFU increases. RFU, relative fluorescence unit. 103   104 6  Figure 1 105  106  107  108 01002003004000 10 20 30 40RFUTime (min)m = 2.74 RFU/minm = 0.74 RFU/minm = 12.82 RFU/min

Comments on “Drug combination studies of curcumin and genistein against rhodesain of Trypanosoma brucei rhodesiense”

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Comments on “Drug combination studies of curcumin and genistein against rhodesain of Trypanosoma brucei rhodesiense”

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