ABSTRACT
The caseinolytic protease (Clp) system plays an essential role in the protein homeostasis of the malaria parasite, particularly at the stage of apicoplast development. The inhibition of this protein is known to have a lethal effect on the parasite and is therefore considered an interesting avenue for antimalaria drugs discovery. The catalytic activity of the Clp system is modulated by its proteolytic subunit (ClpP), which belongs to the serine protease family member and is therefore extensively studied for further inhibitors development. Among many inhibitors, the group of ß-lactone is known to be a specific inhibitor for ClpP. Nevertheless, other groups of lactones have never been studied. This study aims to characterize the catalytic properties of ClpP of Plasmodium knowlesi (Pk-ClpP) and the inhibition properties of a δ-lactone hyptolide against this protein. Accordingly, a codon-optimized synthetic gene encoding Pk-ClpP was expressed in Escherichia coli BL21(DE3) and purified under a single step of Ni2+-affinity chromatography, yielding a 2.20 mg from 1 L culture. Meanwhile, size-exclusion chromatography indicated that Pk-ClpP migrated primarily as homoheptameric with a size of 205 kDa. The specific activity of pure Pk-ClpP was 0.73 U µg-1, with a catalytic efficiency kcat/KM of 0.05 µM-1 s-1, with optimum temperature and pH of 50 °C and 7.0-7.5, respectively. Interestingly, hyptolide, a member of δ-lactone, was shown to inhibit Pk-ClpP with an IC50 value of 17.36 ± 1.44 nM. Structural homology modelling, secondary structure prediction, and far-UV CD spectra revealed that helical structures dominate this protein. In addition, the structural homology modeling showed that this protein forms a barrel-shaped homoheptamer. Docking simulation revealed that the inhibition was found to be a competitive inhibition in which hyptolide was able to dock into the catalytic site and block the substrate. The competitiveness of hyptolide is due to the higher binding affinity of this molecule than the substrate.
Subject(s)
Plasmodium knowlesi , Catalytic Domain , Escherichia coli , Lactones/pharmacology , Serine ProteasesABSTRACT
Proteolytic subunit of the caseinolytic protease system of Plasmodium knowlesi (Pk-ClpP; EC 3.4.21.92) is considered a viable target for antimalarial drug development to eradicate P. knowlesi malaria infection in Malaysia and Southeast Asian region. Inhibition of this system leads to a disruption in the protein homeostasis molecular machinery and therefore be lethal for the parasite. While plants are considered excellent sources of bioactive compounds exhibiting inhibition activity towards Pk-ClpP, many local medicinal plants remain unexplored. This article expands the data collected from the inhibition properties of the methanolic extract of Asystasia gangetica (Chinese Violet), Alstonia scholaris (Pulai Tree), Piper retrofractum (Javanese Long Pepper) and Smallanthus sonchifolius (Yacon) towards Pk-ClpP. These plants are widely found in Malaysia and Indonesia and have been traditionally used in various medical treatments. The present dataset showed that the extracts contained phenolic and flavonoid compounds in various concentrations, whereby S. sonchifolius was found to have the lowest content of phenolic and flavonoid contents, while A. gangetica and A. scholaris were statistically comparable, yet higher than P. retrofactum and S. sonchifolus. Further inhibition data assay towards Pk-ClpP revealed that A. gangetica, A. scholaris and P. retrofactum demonstrated remarkable inhibition activity with IC50 values of 39.06 ± 1.98, 48.92 ± 1.52, and 87.63 ± 3.55, respectively. However, the inhibition activity of these extracts was significantly lower than a serine protease inhibitor of phenylmethylsulfonyl fluoridenone (PMSF). Meanwhile, S. sonchifolus did not exhibit significant inhibition activity towards Pk-ClpP. In addition, Pk-ClpP was not inhibited by a cysteine protease inhibitor of E64.
