Journal of Pharmaceutical Negative Results

: 2015  |  Volume : 6  |  Issue : 1  |  Page : 7--10

The prolyl oligopeptidase inhibitor KYP-2047 is not readily bioavailable to bloodstream form trypanosomes and human myelocytic leukemia cells

Chinenye Ajoko, Dietmar Steverding 
 BioMedical Research Centre, Department of Medicine, Norwich Medical School, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, United Kingdom

Correspondence Address:
Dr. Dietmar Steverding
BioMedical Research Centre, Norwich Medical School, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ
United Kingdom


Introduction: Only three drugs are currently available for treatment of the neurological stage of human African sleeping sickness caused by the protozoan parasite Trypanosoma brucei. As these drugs have serious side effects and are difficult to administer, new and safe antitrypanosomal medications are urgently required. Research in recent years has shown that prolyl oligopeptidase (POP) inhibitors are promising trypanocidal agents. As the novel POP inhibitor KYP-2047 can cross the blood-brain barrier, we aimed to test whether this compound would prove to be a promising anti-trypanosomal drug candidate. Materials and Methods: The efficacy of KYP-2047 to inhibit trypanosome and human POP was evaluated with cell lysates using the fluorogenic peptide substrate Suc-Gly-Pro-Leu-Gly-Pro-AMC. The trypanocidal and cytotoxic activity of KYP-2047 was studied in vitro using bloodstream forms of T. brucei and human myelocytic leukemia HL-60 cells. The bioavailability of KYP-2047 to T. brucei and HL-60 cells was determined by incubation of cells with the inhibitor for 24 h, followed by measuring the residual POP activity. Results: KYP-2047 inactivated POP in cell lysates of T. brucei and HL-60 cells with IC 50 values in the mid nanomolar range indicating that the compound is a very potent inhibitor of the trypanosome and human enzyme. However, KYP-2047 did not affect the growth of T. brucei and HL-60 cells. Upon incubation of the cells with 100 μM KYP-2047, POP activity was inhibited between 20% and 80% which is too low to have any effect on cell growth. Conclusion: The absence of trypanocidal and cytotoxic activity of KYP-2047 is due to low bioavailability of the inhibitor to bloodstream forms of T. brucei and human HL-60 cells.

How to cite this article:
Ajoko C, Steverding D. The prolyl oligopeptidase inhibitor KYP-2047 is not readily bioavailable to bloodstream form trypanosomes and human myelocytic leukemia cells.J Pharm Negative Results 2015;6:7-10

How to cite this URL:
Ajoko C, Steverding D. The prolyl oligopeptidase inhibitor KYP-2047 is not readily bioavailable to bloodstream form trypanosomes and human myelocytic leukemia cells. J Pharm Negative Results [serial online] 2015 [cited 2020 Jun 5 ];6:7-10
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Prolyl oligopeptidase (POP), aka prolyl endopeptidase (EC is a serine protease that cleaves small peptides at the carboxyl side of internal proline residues. [1] The enzyme is a potential drug target in the treatment of neurodegenerative disorders (Alzheimer's and Parkinson's disease) and trypanosome infections (Chagas disease and African sleeping sickness). [2],[3] Studies with small-molecule POP inhibitors have demonstrated an increase in the level of cognition-enhancing neuropeptides in the brain, prevention of scopolamine-induced amnesia in rats and cognitive improvements in a monkey model of Parkinson's disease. [4],[5],[6] POP inhibitors have also been shown to inhibit the in vitro growth of Trypanosoma brucei and Trypanosoma cruzi, the causative agents of African sleeping sickness and Chagas disease, respectively. [7],[8] In addition, POP inhibitors block the invasion of T. cruzi into host cells. [9]

The compound KYP-2047 [Figure 1] is currently one of the most potent and selective POP inhibitor available. It has an IC 50 value of 0.2 nM and a Ki value of 0.02 nM determined with pig brain POP. [10] The inhibitor has been shown to be able to cross an in vitro blood-brain barrier model consisting of primary bovine brain microvessel endothelial cells and to penetrate in vivo into the brain in pharmacologically active concentrations. [11] In addition, the compound was able to inhibit effectively the targeted enzyme in the brain when POP activity was measured ex vivo in homogenized perfused brains dissected from rodents treated with a single dose of KYP-2047 (50 μmol/kg i.p.). [11],[12] Moreover, KYP-2047 has been shown to inhibit intracellular POP activity in primary rat cortical neurons in culture indicating that the compound is cell-permeable and reaches its targets within cells. [13] These properties make KYP-2047 a very interesting drug candidate for treatment of late-stage African sleeping sickness, given that the neurological stage of the disease is characterized by the presence of the parasite in the central nervous system.{Figure 1}

The aim of this study was to investigate whether KYP-2047 can inhibit the growth of bloodstream forms of T. brucei and human promyelocytic leukemia HL-60 cells and whether any observed growth arrest is associated with the inactivation of POP.



KYP-2027 (4-phenylbutanoyl-L-prolyl-2(S) -cyanopyrrolidine) was purchased from Merck Millipore (Nottingham, UK). Suc-Gly-Pro-Leu-Gly-Pro-AMC (succinyl-glycyl-L-prolyl-L-leucyl-glycyl-L-proline-4 -methylcoumaryl-7-amide) was from PeptaNova (Sandhausen, Germany). CHAPS (3-[(3-cholamidopropyl) -dimethylammonio]-1-propanesulfonate) was from Fisher Scientific (Loughborough, UK).

Cell culture

Bloodstream forms of T. brucei clone 427-221a [14] and human myeloid leukemia HL-60 cells [15] were grown in Baltz medium [16] and RPMI 1640 medium, [17] respectively. Both media were supplemented with 16.7% heat-inactivated fetal calf serum. T. brucei and HL-60 cells were cultured at 37°C in a humidified atmosphere containing 5% carbon dioxide.

Inhibition of prolyl oligopeptidase in cell lysates and within cells

The effect of KYP-2047 on POP activity in cell lysates and within cells was determined as follows. Bloodstream forms of T. brucei and human HL-60 cells were harvested by centrifugation, washed twice with PBS/1% glucose and lysed in 10 mM Tris, 0.1 mM ethylenediaminetetraacetic acid, pH - 7.0, 2% CHAPS (trypanosomes: 10 7 cells/100 μl; HL-60s: 2 × 10 6 cells/100 μl). Clarified cell extracts were preincubated with various concentration of KYP-2047 (10 − 4 -10 -10 M) in the presence of 10% dimethyl sulfoxide (DMSO) for 30 min at room temperature. Controls were pretreated with 10% DMSO alone for the same period of time. Alternatively, cells were first incubated with 100 μM KYP-2047 in the presence of 1% DMSO at 37°C at an initial cell density of 2 × 10 5 /ml (trypanosomes) and 2.5 × 10 5 /ml (HL-60 cells), respectively. Control cultures were treated with 1% DMSO alone. After 24 h incubation, cells were washed 3 times with PBS/1% glucose and lysed as described above. Then 90 μl of cell lysate was added to 1910 μl of 50 mM HEPES, pH 7.5, 5 mM dithiothreitol containing 10 μM of Suc-Gly-Pro-Leu-Gly-Pro-AMC. After 60 min, the fluorescence of released AMC was measured at excitation and emission wavelength of 360 and 460 nM using a BIORAD VersaFluor fluorometer.

Cell proliferation assay

The effect of KYP-2047 on the growth of bloodstream form trypanosomes and human HL-60 cells was determined by a growth inhibition assay. In brief, cells were seeded in 24-well plates in a final volume of 1 mL with appropriate culture medium containing 100 μM of KYP-2047 dissolved in 100% DMSO. Controls contained DMSO alone. In all experiments, the final DMSO concentration was 1%. The seeding densities were 10 4 /ml trypanosomes and 10 5 /ml HL-60 cells. After 48 h of incubation, live cells were counted using an improved Neubauer hemocytometer. The viability of HL-60 cells was determined by trypan blue exclusion.


First, we determined the half maximal inhibitory concentration (IC 50 ) value of KYP-2047 for T. brucei and human POP. As source for the enzyme we used cell lysates prepared from bloodstream form trypanosomes and myeloid leukemia HL-60 cells. Similar assays have been routinely used in the past to measure POP activity. [10],[11],[12],[13] KYP-2047 showed a dose-dependent effect on the activity of trypanosome and human POP with IC 50 values of 13.3 nM ± 0.5 nM and 32.5 nM ± 3.4 nM [Figure 2]. This result indicates that KYP-2047 is an effective inhibitor for both trypanosome and human POP, although lower IC 50 values in the sub-nanomolar range have been previously reported for other mammalian POPs. [10],[13] The difference may be explained by different experimental conditions (e.g., different preincubation times with inhibitor, different substrates, different buffers, presence of detergent). Compared to the human enzyme, the trypanosomal enzyme is slightly more sensitive to the inhibitor. Similar selectivity values between trypanosome and human POP have been previously reported for other POP inhibitors. [18]{Figure 2}

Next, the effect of KYP-2047 on the growth of bloodstream forms of T. brucei and human myeloid leukemia HL-60 cells was investigated. The inhibitor had no effect on the growth of trypanosomes and HL-60 cells when tested at 100 μM [Figure 3], the concentration at which POP activity in cell lysates was completely blocked [Figure 2]. This result was unexpected as other POP inhibitors have been shown to be trypanocidal and cytotoxic. [7],[8] The observation that KYP-2047 does not impair the proliferation of T. brucei and HL-60 cells can be only explained by an inability of the inhibitor to enter these cells.{Figure 3}

To check whether the lack of trypanocidal and cytotoxic activity of KYP-2047 is due to reduced bioavailability, bloodstream forms of T. brucei and HL-60 cells were incubated with the inhibitor for 24 h following measuring POP activity in cell lysates. At 100 μM, which caused complete inactivation of POP in cell lysates [Figure 2], KYP-2047 inhibited the POP activity in trypanosomes by only 20% and in HL-60 cells by 79% [Figure 4]. This low level of POP inhibition indicates that the inhibitor is not readily bioavailable for these cell types. The reason for this remains unclear, particularly as KYP-2047 with a log P = 1.6 should be sufficiently lipophilic to enter readily cells. [10]{Figure 4}


In order to show an effect on cell growth, the activity of an essential enzyme must be inhibited almost completely. For example, the cysteine protease inhibitor CA-074 at 100 μM blocks the activity of the essential lysosomal protease cathepsin L in bloodstream forms of T. brucei by 98% but the compound displays no trypanocidal effect. [19] Only an inhibition of cathepsin L in trypanosomes by over 99% results in a growth arrest of the parasites. [19] Similarly, the cysteine protease inhibitor Z-FA-DMK at 100 μM inhibits the activity of cathepsin B and L by 96% in HL-60 cells, but slowed down the growth of the cells by only 50%. [20],[21] In agreement with this is our finding that partial inhibition of POP activity in trypanosomes and HL-60 cells did not affect the growth of the cells too. This is supported by previous observation that incomplete ablation of T. brucei POP by RNA interference leaving 20% activity of the enzyme did not produce any phenotype in bloodstream forms of T. brucei. [8]

This study has shown that the POP inhibitor KYP-2047 does not enter bloodstream forms of T. brucei and human myeloid leukemia cells in adequate concentrations that are high enough to inhibit sufficiently the target enzyme in order to have an effect on the growth of these cells. This is in contrast to previous observations that the compound inhibits efficiently POP in the brain and cultured neural cells. [11],[12],[13] This difference may be explained by the fact that trypanosomes and cancer cells are fast dividing cells while neurons are nonreplicating cells. Thus, absorbed substances are diluted through continuous cell division in proliferating cells whereas they may accumulate in neurons over time. In addition, there is also evidence that neurons take up substances at a higher rate than cancer cells or trypanosomes. For example, it has been shown that the uptake of the anticancer drug cisplatin is increased over 2-fold in dorsal root ganglia neuron compared with pheochromocytoma tumor cells. [22] The higher uptake is probably due to the much higher cell surface of neurons compared with other cells. In conclusion, this study has shown that, with respect to inhibitors, results obtained with one cell system cannot always be applied directly to another cell system.


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