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Molecular and Kinetic Characterization of Babesia microti Gray Strain Lactate Dehydrogenase as a Potential Drug Target.

ABSTRACT: Babesia microti is an emerging zoonotic protozoan organism that causes "malaria-like" symptoms that can be fatal in immunocompromised people. Owing to lack of specific therapeutic regiment against the disease, we cloned and characterized B. microti lactate dehydrogenase (BmLDH) as a potential molecular drug receptor. The in vitro kinetic properties of BmLDH enzyme was evaluated using nicotinamide adenine dinucleotide (NAD(+)) as a co-factor and lactate as a substrate. Inhibitory assay was also done using gossypol as BmLDH inhibitor to determine the inhibitory concentration 50 (IC50). The result showed that the 0.99 kbp BmLDH gene codes for a barely soluble 36 kDa protein (332 amino acids) localized in both the cytoplasm and nucleus of the parasite. In vitro enzyme kinetic studies further revealed that BmLDH is an active enzyme with a high catalytic efficiency at optimal pH of 10.2. The K m values of NAD(+) and lactate were 8.7 ± 0.57 mM and 99.9 ± 22.33 mM, respectively. The IC50 value for gossypol was 0.345 ?M, while at 2.5 ?M, gossypol caused 100% inhibition of BmLDH catalytic activity. These findings, therefore, provide initial evidence that BmLDH could be a potential drug target, although further in vivo studies are needed to validate the practical application of lactate dehydrogenase inhibitors against B. microti infection.

SUBMITTER: Vudriko P

PROVIDER: S-EPMC4125376 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Molecular and Kinetic Characterization of Babesia microti Gray Strain Lactate Dehydrogenase as a Potential Drug Target.

Vudriko Patrick P Masatani Tatsunori T Cao Shinuo S Terkawi Mohamad Alla MA Kamyingkird Ketsarin K Mousa Ahmed A AA Adjou Moumouni Paul F PF Nishikawa Yoshifumi Y Xuan Xuenan X

Drug target insights 20140728

Babesia microti is an emerging zoonotic protozoan organism that causes "malaria-like" symptoms that can be fatal in immunocompromised people. Owing to lack of specific therapeutic regiment against the disease, we cloned and characterized B. microti lactate dehydrogenase (BmLDH) as a potential molecular drug receptor. The in vitro kinetic properties of BmLDH enzyme was evaluated using nicotinamide adenine dinucleotide (NAD(+)) as a co-factor and lactate as a substrate. Inhibitory assay was also d ...[more]

PMID: 25125971

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Project description:Babesia bigemina is a tick-borne apicomplexan hemoprotozoan responsible for bovine babesiosis. The current drugs used for bovine babesiosis treatment have several drawbacks, including toxicity, the lack of effectiveness to clear the parasite, and potential to develop resistance. Identifying compounds that target essential and unique parasite metabolic pathways is a rational approach toward finding alternative drug treatments. Based on the genome sequence and transcriptomics analysis, it can be inferred that anaerobic glycolysis is the dominant adenosine triphosphate (ATP) supply for Babesia, and lactate dehydrogenase (LDH) is one of the essential enzymes in this pathway. Furthermore, the Babesia LDH sequence is distinct from its bovine homologue and thus a potential chemotherapeutic target that would result in decreasing the ATP supply to the parasite but not to the host. Gossypol is a known efficient specific inhibitor of LDH in the sensu stricto B. bovis and the sensu lato B. microti, among other related parasites, but no such data are currently available in the sensu stricto B. bigemina parasites. Hereby, we show that the LDH amino acid sequence is highly conserved among sensu stricto but not in sensu lato Babesia spp. A predictive structural analysis of B. bigemina LDH showed the conservation of the key amino acids involved in the binding to gossypol compared to B. bovis. Gossypol has a significant (P < 0.0001) inhibitory effect on the in vitro growth of B. bigemina, with IC50 of 43.97 mM after 72 h of treatment. The maximum IC (IC98) was observed at 60 mM gossypol. However, a significant effect on the viability of cattle PBMC was observed when the cells were cultured with 60 mM (IC98) gossypol compared with DMSO-exposed control cells. Interestingly, B. bigemina cultured at 3% oxygen expresses significantly higher levels of LDH and is more resistant to gossypol than the parasites maintained at ambient conditions containing ~20% oxygen. Altogether, the results suggest the potential of gossypol as an effective drug against B. bigemina infection, but the risk of host toxicity at therapeutic doses should be further evaluated in in vivo studies.

Dataset Information

Molecular and Kinetic Characterization of Babesia microti Gray Strain Lactate Dehydrogenase as a Potential Drug Target.

Publications

Molecular and Kinetic Characterization of Babesia microti Gray Strain Lactate Dehydrogenase as a Potential Drug Target.

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