Alex H. Y.
Chan‡
a,
Imam
Fathoni‡
b,
Terence C. S.
Ho
ac,
Kevin J.
Saliba
b and
Finian J.
Leeper
*a
aYusuf Hamied Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK. E-mail: fjl1@cam.ac.uk
bResearch School of Biology, The Australian National University, Canberra, ACT 2601, Australia
cNorwich Medical School, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, UK
First published on 7th June 2022
A series of derivatives of a triazole analogue of thiamine has been synthesised. When tested as inhibitors of porcine pyruvate dehydrogenase, the benzoyl ester derivatives proved to be potent thiamine pyrophosphate (TPP) competitive inhibitors, with the affinity of the most potent analogue (Ki = 54 nM) almost matching the affinity of TPP itself. When tested as antiplasmodials, most of the derivatives showed modest activity (IC50 value >60 μM), except for a 4′-N-benzyl derivative, which has an IC50 value in the low micromolar range. This activity was not affected by increasing the extracellular concentration of thiamine in the culture medium for any of the compounds (except a modest increase in the IC50 for the unfunctionalized benzoyl ester), nor by overexpressing thiamine pyrophosphokinase in the parasite, making it unlikely to be due to an effect on thiamine transport or metabolism.
In Plasmodium, thiamine 1 (Fig. 1) is converted into its active form, thiamine pyrophosphate (TPP) 2, by the enzyme thiamine pyrophosphokinase (TPK). TPP is a cofactor of several enzymes, catalyzing diverse reactions in essential biochemical pathways.5 Previous studies showed that oxythiamine 4, a thiamine analogue widely used as a probe in chemical biology and pharmacological studies, can be converted by TPK into the antimetabolite oxythiamine pyrophosphate (OxPP) 5 within the parasite. OxPP then goes on to interact with, and likely inhibit, at least two TPP-dependent enzymes, validating thiamine utilisation as a viable antimalarial drug target.6 With the knowledge that TPP-dependent enzymes play a key role in metabolism, several small-molecule inhibitors that demonstrate antimicrobial activities have recently been reported. For example, He and coworkers developed several pyruvate dehydrogenase complex (PDHc) inhibitors for targeting cyanobacteria,7–9 and the groups of Freel Meyers10 and Hirsch11 independently developed inhibitors of 1-deoxy-D-xylulose 5-phosphate synthase (DXPS) from E. coli and Deinococcus radiodurans.
In this study, we attempted to develop drug-like TPP-dependent enzyme inhibitors as antimalarial agents because, to our knowledge, this has not been explored in any detail. Our design of inhibitors for TPP-dependent enzymes was inspired by a very potent inhibitor, triazole pyrophosphate 7 reported in 2008 by Erixon et al.12 In enzyme assays using Zymomonas mobilis pyruvate decarboxylase (zmPDC) (KD of TPP: ∼350 nM), 7 was found to bind about 10000 times tighter than TPP (Ki ∼30 pM). The proposed reason12 for such a tight binding behavior was that the neutral triazole ring, in place of the positively charged thiazolium ring of TPP, mimics the activated ylide form 3 of TPP. So, the triazole pyrophosphate captures the strong interactions between the enzyme and the TPP ylide, which help to stabilize this high energy intermediate. Despite the high binding affinity towards TPP-dependent enzymes, 7 itself can hardly be of medicinal use. The polyanionic nature of the pyrophosphate motif under physiological conditions prevents the molecule from crossing lipophilic biological membranes, so it lacks activity in cell-based assays.13 Triazole 6 (without the pyrophosphate tail) binds zmPDC about 1000 times weaker than TPP (Ki: about 300 μM).12 This shows that for this enzyme, most of the binding energy of TPP to the active site comes from the ionic interactions between the pyrophosphate motif and the coordinated Mg2+. Therefore, the pyrophosphate motif contributes greatly to binding, but makes the molecule too polar to cross membranes for cellular activity.
Since 2008, research has focused on modifications of the pyrophosphate motif, aiming to discover neutral substituents that enable sufficient binding. The rationale is that even if a substituent cannot capture all the binding energy of the pyrophosphate motif, its binding energy added to those of the neutral triazole and aminopyrimidine motifs can together yield an inhibitor with good binding affinity. Furthermore, the overall neutral species should be membrane-permeable and so able to reach its target. In recent years, He et al. have employed this approach to develop potent inhibitors derived from the triazole scaffold (e.g.9 and 10).7,8 These inhibitors slowed the growth rate of cyanobacteria through inhibition of PDHc. In this study, a similar approach is adopted to tackle malaria.
All compounds were tested for inhibition of TPP-dependent porcine PDHc E1. As shown in Table 1, 6, 12a–g and 13a and b all showed inhibitory activity, whereas 15 showed none. 12b showed the highest inhibition. IC50 values were determined for 12b along with 12a and 12g to assess the effect of the chloro-substituent on the benzoyl group (Fig. S5†). Indeed, the p-chloro ester 12b was nearly two-fold more potent an inhibitor than the unsubstituted 12a and three-fold more potent than p-nitro ester 12g. To investigate the mode of inhibition by compounds 12 and 13, the TPP concentration was varied and the results demonstrated that all these compounds are competitive inhibitors with respect to TPP (see Table S1†). The reported KD for TPP in pig heart pyruvate dehydrogenase is 50 ± 10 nM,14 which allows the Ki of the tested compounds to be calculated from their IC50 values (see Table 1) giving nanomolar Ki values.
Compound | Inhibitiona,b (%) | IC50a,c (μM) | K I , (nM) |
---|---|---|---|
a Data are the means of measurements in three technical replicates ± SEM. b Percentage inhibition determined for compounds at 250 μM with [TPP] = 50 μM. c IC50 values for selected analogues determined at [TPP] = 10 μM. d K i is based on the previously reported KD for TPP of 50 nM.14 | |||
6 | 60 ± 7 | — | — |
12a | 72 ± 3 | 20 ± 3 | 100 ± 15 |
12b | 81 ± 2 | 11 ± 2 | 54 ± 12 |
12c | 72 ± 3 | — | — |
12d | 61 ± 3 | — | — |
12e | 73 ± 2 | — | — |
12f | 55 ± 5 | — | — |
12g | 56 ± 5 | 36 ± 4 | 182 ± 20 |
13a | 75 ± 3 | — | — |
13b | 71 ± 4 | — | — |
15 | 0 | — | — |
Analogues 12a–g, 13a and b and 15 were then evaluated against P. falciparum 3D7 (chloroquine-sensitive) strain at varying thiamine concentrations (thiamine-free, 2.97 μM thiamine, the concentration of thiamine present in the RPMI-1640 medium in which malaria parasites are maintained, and 297 μM thiamine). Research by Wrenger et al. showed that removing thiamine from the medium that is used to culture P. falciparum only negatively affects the parasite growth after 13 days.15 As shown in Table 2 and Fig. S6,† most compounds displayed some antiplasmodial activity with IC50 values in the range 62–343 μM, but compound 15 showed much higher potency with a low micromolar IC50 value. However, its antiplasmodial activity (as well as that of the other compounds, except perhaps for compound 12a) is unlikely to be related to thiamine utilisation because the activity is independent of the concentration of thiamine in the medium. The IC50 of compound 12a is slightly increased when tested in the replete medium (IC50 >200 μM, p = 0.031) but, because the change in activity is relatively small (compared to that of oxythiamine6), it was not deemed worthy of further investigation. Oxythiamine is likely to be transported into cells and certainly needs to be pyrophosphorylated for effective inhibition of TPP-dependent enzymes.6 Given that oxythiamine is a thiamine analogue, both of these processes are likely to be antagonised by excess thiamine in the medium. Given that compounds 12 and 13 are TPP (rather than thiamine) mimics and are likely to be able to diffuse across the cell membrane (since they are neutral and sufficiently non-polar), excess extracellular thiamine may not be able to antagonise their antiplasmodial activity, even if they do target PDHc or a related TPP-dependent enzyme. Establishing whether this is the case requires additional experimental work.
Compound | IC50 valuesa (μM ± SEM) | Cytotoxicityb (μM) | Selectivity index | ||
---|---|---|---|---|---|
Thiamine free | 2.97 μM thiamine | 297 μM thiamine | |||
a Data are the means of three independent experiments, each carried out in triplicate. b Cytotoxicity against human foreskin fibroblast cells. The compounds were tested at up to the highest possible concentration depending on their solubility. | |||||
12a | 128 ± 13 | 136 ± 18 | >200 | >200 | >1.6 |
12b | 107 ± 16 | 125 ± 22 | 122 ± 6 | — | — |
12c | 63 ± 15 | 62 ± 17 | 79 ± 5 | >200 | >3.2 |
12d | 339 ± 31 | 347 ± 20 | 343 ± 33 | — | — |
12e | 75 ± 6 | 85 ± 4 | 82 ± 8 | — | — |
12f | >25 | >25 | >25 | — | — |
12g | 135 ± 15 | 139 ± 9 | 143 ± 10 | — | — |
13a | 168 ± 26 | >200 | >200 | — | — |
13b | >200 | >200 | >200 | — | — |
15 | 2.2 ± 0.2 | 2.3 ± 0.1 | 2.7 ± 0.01 | 98 ± 7 | 45 |
Compounds 12a, 12c, and 15 were chosen for further investigation; 15 and 12c because they showed the best antiplasmodial activity and 12a, with the unsubstituted phenyl ring, was included for comparison with 12c to determine the effect of the chloro substituent of 12c. When tested against human foreskin fibroblast (HFF) cells (Table 2 and Fig. S7†), 12a and 12c did not show any cytotoxicity at the highest concentration tested, while 15 was cytotoxic only when tested at its highest concentration (200 μM). Taking all the data in Table 2 together, 15 demonstrated the most potent antiplasmodial activity (parasite IC50: ∼2 μM) and has high selectivity towards Plasmodium versus human cells (∼45-fold).
P. falciparum parasites expressing a GFP-tagged PfTPK (PfTPK+-GFP), in addition to the endogenous PfTPK, were generated (Fig. S8†) to investigate further whether the antiplasmodial activity of the compounds is related to thiamine utilisation. Parasites overexpressing TPK (PfTPK-strep) were previously shown to be more sensitive to oxythiamine compared to the wild-type parasites.612a, 12c and 15 were tested in the presence or absence of thiamine (2.97 μM). Overexpression of PfTPK increased the sensitivity of the parasites to oxythiamine when compared to the mock (parasites with empty plasmid) and wild-type parasites, but had no effect on the sensitivity towards compounds 12a, 12c or 15 (Fig. S9†). These results provide additional evidence that the antiplasmodial activity of these compounds is not due to inhibition of TPK, nor to deacylation (for 12a and c) or dealkylation (for 15) followed by pyrophosphorylation by TPK to produce a TPP analogue that inhibits TPP-dependent enzymes in Plasmodium.
Due to the structural similarity between the thiamine analogues and trimethoprim 8 (Fig. 1), a known inhibitor of the folate pathway in bacteria, we also explored the possibility that the compounds inhibited parasite proliferation by interfering with folate metabolism in P. falciparum. To do this we tested the in vitro antiplasmodial effect of 15 and sulfadoxine (which suppresses the folate pathway in P. falciparum), in the presence of either 2.2 μM (the concentration present in RPMI-1640 medium) or 220 μM of folic acid.16 The result (Fig. S10†) revealed that there was no difference in the parasite's sensitivity towards 15 upon changing the folic acid concentration in the medium, whereas the sensitivity towards sulfadoxine, was diminished. This is consistent with the antiplasmodial activity of 15 being unrelated to folate utilisation.
Second, we discover 15 as a potential antimalarial agent, which suppresses parasite proliferation at single-digit micromolar levels and is highly selective towards the parasite. Cell-based experiments suggest that antagonism of thiamine or folate utilisation are not its mode of antiplasmodial action, and its molecular target remains undetermined. A search of ChEMBL resulted in 10 compounds with at least 40% similarity but, among these, only compound 16 (see Scheme 1) had been tested against P. falciparum, with an IC50 of 18.5 μM.20 As with 15, the mode of action of 16 is unknown.
The molecular properties of 15 are within Lipinski's and Veber's rules21 (in as far as these rules apply to antiparasitic drugs22) and thus it is predicted to be drug-like and orally available. In the future, should the molecular target of 15 be elucidated, further structure-based drug design may be available to optimize the ligand–target interactions. Based on its antiplasmodial potency and selectivity, coupled with its drug-like molecular properties, 15 is a useful lead compound with the potential to be developed as an antimalarial agent.
Footnotes |
† Electronic supplementary information (ESI) available: Synthetic and biological methods, NMR spectra (Fig. S1–S4), IC50 curves. See DOI: https://doi.org/10.1039/d2md00085g |
‡ These authors contributed equally. |
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