Giardia intestinalis thymidine kinase is a high-affinity enzyme crucial for DNA synthesis and an exploitable target for drug discovery

Krakovka, Sascha; Ranjbarian, Farahnaz; Lujan, Lucas; Saura, Alicia; Larsen, Nicolai Balle; Jiménez-González, Alejandro; Reggenti, Anna; Luján, Hugo D.; Svärd, Staffan G.; Hofer, Anders

doi:10.1016/j.jbc.2022.102028

Cited by 6 publications

(18 citation statements)

References 56 publications

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“…The effect of cladribine on cyst formation is unpublished data from a previous study of the effect of azidothymidine on the encystation process [19]. The two negative controls (Control and DMSO), and the positive control (metronidazole) shown here for comparison are therefore identical with the previous publication, whereas the data on cladribine is new (S5-S6 Fig) . Cells were resuspended to a density of ~2.5 × 10 5 cells and allowed to attach for 3 h in 3 ml Nunc cell culture tubes #156758 (Thermo Fisher Scientific, Waltham, MA, USA) before exchanging the medium into encystation medium.…”

Section: G Intestinalis Cyst Formationsupporting

confidence: 76%

“…HPLC analysis. Nucleotides from enzyme assays (as well as the protein preparations in S9 Fig) were analyzed using a 150 mm × 4.6 mm SunShell C18-WP HPLC column from ChromaNik Technologies Inc (Osaka, Japan) using a modified version of a previous published protocol developed for a multisubstrate assay with all five substrates (dCyd, dUrd, dThd, dGuo and dAdo) [19]. Instead of using a ternary mixture of three aqueous solutions, we changed it to a binary mixture of A and B, with the third component, tetrabuytylammonium bromide (TBA-Br) mixed into the two other solutions instead of using it separately.…”

Section: Methodsmentioning

confidence: 99%

“…The incentive behind the current study was to investigate if also other dNKs in G. intestinalis have developed higher substrate affinities than the host enzymes as an adaptation to survive in the mammalian body. Studies of G. intestinalis, T. brucei and mammalian TK1 members have shown that substrate affinity can be gained by increasing the number of units the enzyme is composed [7,[19][20][21]. However, it has yet not been demonstrated for the non-TK1-like dNKs, which are in most cases dimers [17].…”

Section: _________________________________________________________mentioning

confidence: 99%

“…The TK1 family has a strict substrate specificity adapted to phosphorylate thymidine-like substrates (thymidine and deoxyuridine) and serve the purpose to supply the cell with dTTP [18]. The thymidine kinase from G. intestinalis was recently characterized and this TK1-like enzyme was found to have a ∼10-fold higher affinity to thymidine than the human TK1 [19]. This could be an adaptation to be able to efficiently compete with the host cells and compensate for the lack of de novo dTTP synthesis in G. intestinalis .…”

mentioning

confidence: 99%

“…This could be an adaptation to be able to efficiently compete with the host cells and compensate for the lack of de novo dTTP synthesis in G. intestinalis . The high affinity makes the parasite sensitive to the substrate analogue azidothymidine, which is an efficient antigiardial agent in in vitro cell proliferation assays as well as in G. intestinalis -infected gerbils [19]. The incentive behind the current study was to investigate if also other dNKs in G. intestinalis have developed higher substrate affinities than the host enzymes as an adaptation to survive in the mammalian body.…”

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confidence: 99%

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Giardia intestinalisdeoxyadenosine kinase has a unique tetrameric structure that enables high substrate affinity and makes the parasite sensitive to deoxyadenosine analogues

Ranjbarian,

Rafie,

Shankar

et al. 2023

Preprint

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Giardia intestinalisis a protozoan parasite causing giardiasis, a severe, sometimes even life-threatening, diarrheal disease.Giardiais one of only a few known organisms that lackde novosynthesis of DNA building blocks, and the parasite is therefore completely dependent on salvaging deoxyribonucleosides from the host. The deoxyribonucleoside kinases (dNKs) needed for this salvage are generally divided into two structurally distinct families, thymidine kinase 1 (TK1)-like dNKs and non-TK1-like dNKs. We have characterized theG. intestinalisdeoxyadenosine kinase and found that it, in contrast to previously studied non-TK1-like dNKs, has a tetrameric structure. Deoxyadenosine was the best natural substrate of the enzyme (KM=1.12 μM; Vmax=10.3 μmol·min-1·mg-1), whereas the affinities for deoxyguanosine, deoxyinosine and deoxycytidine were 400-2000 times lower. Deoxyadenosine analogues halogenated at the 2- and/or 2’ s-positions were also potent substrates, with comparable EC50values as the main drug used today, metronidazole, but with the advantage of being usable on metronidazole-resistant parasites. Cryo-EM and 2.1 Å X-ray structures of the enzyme in complex with the product dAMP (and dADP) showed that the tetramer is kept together by extended N- and C-termini that reach across from one canonical dimer to the next in a novel dimer-dimer interaction. Removal of the two termini resulted in lost ability to form tetramers and a 100-fold decreased deoxyribonucleoside substrate affinity. This is the first example of a non-TK1-like dNK that has a higher substrate affinity as the result of a higher oligomeric state. The development of high substrate affinity could be an evolutionary key factor behind the ability of the parasite to survive solely on deoxyribonucleoside salvage.Authors summaryThe human pathogenGiardia intestinalisis one of only a few organisms that lack ribonucleotide reductase and is therefore completely dependent on salvaging deoxyribonucleosides from the host for the supply of DNA building blocks. We have characterized one of theG. intestinalissalvage enzymes, which was named deoxyadenosine kinase based on its substrate specificity. The enzyme also phosphorylated many deoxyadenosine analogues that were equally efficient in preventing parasite growth as the most used drug today, metronidazole, and also usable against metronidazole-resistant parasites. Structural analysis of the enzyme with cryo-EM and X-ray crystallography showed that the enzyme was unique in its family of deoxyribonucleoside kinases by forming a tetramer and mutational analysis showed that tetramerization is a prerequisite for the high substrate affinity of the enzyme. The ability to gain substrate affinity by increasing the number of enzyme subunits could potentially represent an evolutionary pathway that has assisted the parasite to become able to survive entirely on salvage synthesis of DNA building blocks.

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Section: G Intestinalis Cyst Formationsupporting

confidence: 76%

Section: Methodsmentioning

confidence: 99%

Section: _________________________________________________________mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Giardia intestinalisdeoxyadenosine kinase has a unique tetrameric structure that enables high substrate affinity and makes the parasite sensitive to deoxyadenosine analogues

Ranjbarian,

Rafie,

Shankar

et al. 2023

Preprint

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Targeting the nucleotide metabolism of Trypanosoma brucei and other trypanosomatids

Hofer

2023

FEMS Microbiology Reviews

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African sleeping sickness, Chagas disease, and leishmaniasis are life-threatening diseases that together affect millions of people around the world and are caused by different members of the protozoan family Trypanosomatidae. The most-studied member of the family is Trypanosoma brucei, which is spread by tsetse flies and causes African sleeping sickness. Nucleotide metabolism in T. brucei and other trypanosomatids is significantly different from that of mammals and was recognized as a target for chemotherapy already in the 1970s–1980 s. A more thorough investigation of the nucleotide metabolism in recent years has paved the way for identifying nucleoside analogues that can cure T. brucei brain infections in animal models. Specific features of T. brucei nucleotide metabolism include the lack of de novo purine biosynthesis, the presence of very efficient purine transporters, the lack of salvage pathways for CTP synthesis, unique enzyme localizations, and a recently discovered novel pathway for dTTP synthesis. This review describes the nucleotide metabolism of T. brucei, highlights differences and similarities to other trypanosomatids, and discusses how to exploit the parasite-specific features for drug development.

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A Broad-Spectrum Horizontal Transfer Inhibitor Prevents Transmission of Plasmids Carrying Multiple Antibiotic Resistance Genes

Jia,

Zheng,

Yang

et al. 2024

Transboundary and Emerging Diseases

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The dissemination of antimicrobial resistance (AMR) severely degrades the performance of antibiotics and constantly paralyzes the global health system. In particular, plasmid-mediated transfer of antibiotic resistance genes (ARGs) across bacteria is recognized as the primary driver. Therefore, antiplasmid transfer approaches are urgently warranted to resolve this intractable problem. Herein, we demonstrated the potential of azidothymidine (AZT), an FDA-approved anti-HIV drug, as a broad-spectrum horizontal transfer inhibitor to effectively prevent the transmission of multiple ARGs, including mcr-1, blaNDM−5, and tet(X4), both in vitro and in vivo. It was also noteworthy that the inhibitory effect of AZT was proved to be valid within and across bacterial genera under different mating conditions. Mechanistic studies revealed that AZT dissipated bacterial proton motive force, which was indispensable for ATP synthesis and flagellar motility. In addition, AZT downregulated bacterial secretion systems involving general and type IV secretion systems (T4SS). Furthermore, the thymidine kinase, which is associated with DNA synthesis, turned out to be the potential target of AZT. Collectively, our work demonstrates the broad inhibitory effect of AZT in preventing ARGs transmission, opening new horizons for controlling AMR.

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Giardia intestinalis thymidine kinase is a high-affinity enzyme crucial for DNA synthesis and an exploitable target for drug discovery

Cited by 6 publications

References 56 publications

Giardia intestinalisdeoxyadenosine kinase has a unique tetrameric structure that enables high substrate affinity and makes the parasite sensitive to deoxyadenosine analogues

Giardia intestinalisdeoxyadenosine kinase has a unique tetrameric structure that enables high substrate affinity and makes the parasite sensitive to deoxyadenosine analogues

Targeting the nucleotide metabolism of Trypanosoma brucei and other trypanosomatids

A Broad-Spectrum Horizontal Transfer Inhibitor Prevents Transmission of Plasmids Carrying Multiple Antibiotic Resistance Genes

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