Glycerol kinase of African trypanosomes possesses an intrinsic phosphatase activity

Balogun, E.O.; Inaoka, Daniel Ken; Shiba, T.; Tokuoka, Suzumi M.; Tokumasu, Fuyuki; Sakamoto, Kimitoshi; Kido, Yasutoshi; Michels, Paul A.M.; Watanabe, Yoh-ichi; Harada, Shigeharu; Kita, Kiyoshi

doi:10.1016/j.bbagen.2017.07.028

Cited by 8 publications

(8 citation statements)

References 45 publications

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“…In contrast, the search for a TGK-specific inhibitor has not been as successful, partly because of the lack of a suitable assay compatible with high-throughput screening. However, we recently discovered that TbgGK possesses an intrinsic phosphatase activity that shares the same common catalytic site as the essential kinase activity (33). The phosphatase assay method that we have described in this paper is high-throughput compatible, and we have utilized this assay system to screen a kinase-focused combinatorial library of 5802 compounds from which a hit rate of 1.7% was recorded ( Fig.…”

Section: Discussionmentioning

confidence: 99%

Discovery of trypanocidal coumarins with dual inhibition of both the glycerol kinase and alternative oxidase of Trypanosoma brucei brucei

Balogun¹,

Inaoka

Shiba

et al. 2019

The FASEB Journal

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African trypanosomiasis, sleeping sickness in humans or nagana in animals, is a potentially fatal neglected tropical disease and a threat to 65 million human lives and 100 million small and large livestock animals in sub‐Saharan Africa. Available treatments for this devastating disease are few and have limited efficacy, prompting the search for new drug candidates. Simultaneous inhibition of the trypanosomal glycerol kinase (TGK) and trypanosom alalternative oxidase (TAO) is considered a validated strategy toward the development of new drugs. Our goal is to develop a TGK‐specific inhibitor for coadministration with ascofuranone (AF), the most potent TAO inhibitor. Here, we report on the identification of novel compounds with inhibitory potency against TGK. Importantly, one of these compounds (compound 17) and its derivatives (17a and 17b) killed trypanosomes even in the absence of AF. Inhibition kinetics revealed that derivative 17b is a mixed‐type and competitive inhibitor for TGK and TAO, respectively. Structural data revealed the molecular basis of this dual inhibitory action, which, in our opinion, will aid in the successful development of a promising drug to treat trypanosomiasis. Although the EC50 of compound 17b against trypanosome cells was 1.77 μM, it had no effect on cultured human cells, even at 50 μM.—Balogun, E. O., Inaoka, D. K., Shiba, T., Tsuge, C., May, B., Sato, T., Kido, Y., Nara, T., Aoki, T., Honma, T., Tanaka, A., Inoue, M., Matsuoka, S., Michels, P. A. M., Watanabe, Y.‐I., Moore, A. L., Harada, S., Kita, K. Discovery of trypanocidal coumarins with dual inhibition of both the glycerol kinase and alternative oxidase of Trypanosoma brucei brucei. FASEB J. 33, 13002–13013 (2019). http://www.fasebj.org

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Section: Discussionmentioning

confidence: 99%

Discovery of trypanocidal coumarins with dual inhibition of both the glycerol kinase and alternative oxidase of Trypanosoma brucei brucei

Balogun¹,

Inaoka

Shiba

et al. 2019

The FASEB Journal

Self Cite

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“…T . brucei glycerol kinase is unique in its bidirectional activity, and is known to lack the classical allosteric regulation [50]. According to canonical biochemistry, glycolysis and GNG cannot operate simultaneously, and the exclusive regulatory mechanisms are well known in mammalian systems.…”

Section: Discussionmentioning

confidence: 99%

Gluconeogenesis using glycerol as a substrate in bloodstream-form Trypanosoma brucei

et al. 2018

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Bloodstream form African trypanosomes are thought to rely exclusively upon glycolysis, using glucose as a substrate, for ATP production. Indeed, the pathway has long been considered a potential therapeutic target to tackle the devastating and neglected tropical diseases caused by these parasites. However, plasma membrane glucose and glycerol transporters are both expressed by trypanosomes and these parasites can infiltrate tissues that contain glycerol. Here, we show that bloodstream form trypanosomes can use glycerol for gluconeogenesis and for ATP production, particularly when deprived of glucose following hexose transporter depletion. We demonstrate that Trypanosoma brucei hexose transporters 1 and 2 (THT1 and THT2) are localized to the plasma membrane and that knockdown of THT1 expression leads to a growth defect that is more severe when THT2 is also knocked down. These data are consistent with THT1 and THT2 being the primary routes of glucose supply for the production of ATP by glycolysis. However, supplementation of the growth medium with glycerol substantially rescued the growth defect caused by THT1 and THT2 knockdown. Metabolomic analyses with heavy-isotope labelled glycerol demonstrated that trypanosomes take up glycerol and use it to synthesize intermediates of gluconeogenesis, including fructose 1,6-bisphosphate and hexose 6-phosphates, which feed the pentose phosphate pathway and variant surface glycoprotein biosynthesis. We used Cas9-mediated gene knockout to demonstrate a gluconeogenesis-specific, but fructose-1,6-bisphosphatase (Tb927.9.8720)-independent activity, converting fructose 1,6-bisphosphate into fructose 6-phosphate. In addition, we observed increased flux through the tricarboxylic acid cycle and the succinate shunt. Thus, contrary to prior thinking, gluconeogenesis can operate in bloodstream form T. brucei. This pathway, using glycerol as a physiological substrate, may be required in mammalian host tissues.

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“…Trypanosome glycerol kinase catalyzes not only the forward reaction (ATP-dependent glycerol phosphorylation) but also the reverse reaction. Glycerol kinase is important for trypanosome survival and might be a promising drug target ( Balogun et al, 2017 ). Succinate dehydrogenase is a functional member of both the Krebs cycle and the aerobic respiratory chain.…”

Section: Discussionmentioning

confidence: 99%

Identification and Analysis of the Tegument Protein and Excretory-Secretory Products of the Carcinogenic Liver Fluke Clonorchis sinensis

Shi

Liang

et al. 2020

Front. Microbiol.

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Liver fluke proteins, including excretory-secretory products (ESPs) and tegument proteins, are critical for the pathogenesis, nutrient metabolism, etiology and immune response of liver cancer. To understand the functions of various proteins in Clonorchis sinensis physiology and human clonorchiasis, the ESPs and tegument proteins of C. sinensis were identified. Supernatants containing ESPs from adult C. sinensis after culture for 6 h were harvested and concentrated. The tegument was detached using a freeze/thaw method and successively extracted using various extraction buffers. The outer surface proteins of C. sinensis were labeled with biotin, and the biotinylated proteins were purified. The ESP, tegument and labeled outer surface proteins were identified and analyzed by high-resolution LC-MS/MS. The identified proteins were compared with those of other flukes, and the protein functions associated with pathogenesis, carcinogenesis and potential vaccine antigens and drug targets were predicted and analyzed. A total of 175 proteins were identified after the 6-h culture of C. sinensis ESPs. A total of 352 tegument proteins were identified through sequential solubilization of the isolated teguments, and a subset of these proteins were localized to the surface membrane of the tegument by labeling with biotin. Thirty identified proteins, including annexins, actin and tetraspanins, were identified as potential immunomodulators and promising vaccine antigens. Interestingly, among the 352 tegument proteins, as many as 155 were enzymes, and most were oxidoreductases, hydrolases or transferases. A comparison of the outer surface proteins of C. sinensis with those of other flukes indicated that flukes have some common outer surface proteins, such as actin, tetraspanin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and annexin. Granulin, thioredoxin peroxiredoxin, carbonyl reductase 1 and cystatin were identified in the C. sinensis proteome and predicted to be related to liver

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Glycerol kinase of African trypanosomes possesses an intrinsic phosphatase activity

Cited by 8 publications

References 45 publications

Discovery of trypanocidal coumarins with dual inhibition of both the glycerol kinase and alternative oxidase of Trypanosoma brucei brucei

Discovery of trypanocidal coumarins with dual inhibition of both the glycerol kinase and alternative oxidase of Trypanosoma brucei brucei

Gluconeogenesis using glycerol as a substrate in bloodstream-form Trypanosoma brucei

Identification and Analysis of the Tegument Protein and Excretory-Secretory Products of the Carcinogenic Liver Fluke Clonorchis sinensis

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