Structural basis of nucleoside and nucleoside drug selectivity by concentrative nucleoside transporters

Johnson, Z.L.; Lee, Jun-Ho; Lee, Kiyoun; Lee, Minhee; Kwon, Deok-Ho; Hong, Jiyong; Lee, Seok‐Yong

doi:10.7554/elife.03604

Cited by 55 publications

(56 citation statements)

References 59 publications

(93 reference statements)

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“…Furthermore, more recent structural analysis of two other CPA family members, PaNhaP and MjNhaP1, suggest that only slight conformational changes are required for transport 32,33 . The structure of a bacterial concentrative nucleoside transporter, VcCNT, also reveals the hallmarks of an elevator-type transporter, although its structure has so far only been captured in an inward-facing state 34,35 . Interestingly, repeat-swap modeling of VcCNT predicts an elevator-type movement for this protein though this prediction has not yet been experimentally tested 26 .…”

Section: Discussionmentioning

confidence: 99%

The bacterial dicarboxylate transporter VcINDY uses a two-domain elevator-type mechanism

Mulligan

Fenollar–Ferrer

Fitzgerald

et al. 2016

Nat Struct Mol Biol

122

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Secondary transporters use alternating access mechanisms to couple uphill substrate movement to downhill ion flux. Most known transporters utilize a “rocking bundle” motion, where the protein moves around an immobile substrate binding site. However, the glutamate transporter homolog, GltPh, translocates its substrate binding site vertically across the membrane, an “elevator” mechanism. Here, we used the “repeat swap” approach to computationally predict the outward-facing state of the Na+/succinate transporter VcINDY, from Vibrio cholerae. Our model predicts a substantial “elevator”-like movement of vcINDY’s substrate binding site, with a vertical translation of ~15 Å and a rotation of ~43°; multiple disulfide crosslinks which completely inhibit transport provide experimental confirmation and demonstrate that such movement is essential. In contrast, crosslinks across the VcINDY dimer interface preserve transport, revealing an absence of large scale coupling between protomers.

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Section: Discussionmentioning

confidence: 99%

The bacterial dicarboxylate transporter VcINDY uses a two-domain elevator-type mechanism

Mulligan

Fenollar–Ferrer

Fitzgerald

et al. 2016

Nat Struct Mol Biol

122

View full text Add to dashboard Cite

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“…Complex nucleoside derivatives are also useful as antibiotics [18]. Therapies based on oligonucleotides and aptamers are also under development [19,20]. Various other nucleoside drugs, such as kinase inhibitors, can mimic a substrate and compete for a common binding site on their targets.…”

Section: Conventional Targets Of Nucleosides and Small Nucleotidesmentioning

confidence: 99%

Polypharmacology of conformationally locked methanocarba nucleosides

Tosh

Toti

Ciancetta

2017

Drug Discovery Today

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A single molecular scaffold can be adapted to interact with diverse targets, either separately or simultaneously. Nucleosides and nucleotides in which ribose is substituted with bicyclo[3.1.0]hexane are an example of a versatile drug-like scaffold for increasing selectivity at their classical targets: kinases, polymerases, adenosine and P2 receptors. Also, by applying structure-based functional group manipulations, rigidified adenosine derivatives can be repurposed to satisfy pharmacophoric requirements of various GPCRs, ion channels, enzymes and transporters, initially detected as off-target activities. Recent examples include 5HT2B serotonin receptor antagonists and novel dopamine transporter allosteric modulators. This directable target diversity establishes rigid nucleosides as privileged scaffolds.

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“…The crystal structure of the Vibrio cholerae concentrative nucleoside transporter (PDBid: 3TIJ) [6] in the PBD was identified as a potential template. Other, vcCNT pdbs structures were published later by the same group [12]. All three hCNTs showed identities above 35% compared to the 3TIJ vcCNT structure, which was considered good for building homology models (the rule of thumb being that a sequence homology of 30% or above is sufficient).…”

Section: Methodsmentioning

confidence: 90%

“…A total of 172 compounds were selected according to docking score and drug-likeness for biological testing, with phlorizin being used as the standard hCNT inhibitor control, which was tested at 250 μM, its IC 50 against hCNT1 [18]. Compounds were tested at 10 μM concentration, and were screened against all individual three hCNTs stably expressed in PKNTD cell lines [12–14]. After biological screening, we obtained 14 novel selective (Figure 8) non-nucleoside hCNT1 inhibitors (Table 3), an 8% success rate.…”

Section: Resultsmentioning

confidence: 99%

Homology Modeling of Human Concentrative Nucleoside Transporters (hCNTs) and Validation by Virtual Screening and Experimental Testing to Identify Novel hCNT1 Inhibitors

2017

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Objective The nucleoside transporter family is an emerging target for cancer, viral and cardiovascular diseases. Due to the difficulty in the expression, isolation and crystallization of membrane proteins, there is a lack of structural information on any of the mammalian and for that matter the human proteins. Thus the objective of this study was to build homology models for the three cloned concentrative nucleoside transporters hCNT1, hCNT2 and hCNT3 and validate them for screening towards the discovery of much needed inhibitors and probes. Methods The recently reported crystal structure of the Vibrio cholerae concentrative nucleoside transporter (vcCNT), has satisfactory similarity to the human CNT orthologues and was thus used as a template to build homology models of all three hCNTs. The Schrödinger modeling suite was used for the exercise. External validation of the homology models was carried out by docking a set of recently reported known hCNT1 nucleoside class inhibitors at the putative binding site using induced fit docking (IDF) methodology with the Glide docking program. Then, the hCNT1 homology model was subsequently used to conduct a virtual screening of a 360,000 compound library, and 172 compounds were obtained and biologically evaluated for hCNT 1, 2 and 3 inhibitory potency and selectivity. Results Good quality homology models were obtained for all three hCNTs as indicated by interrogation for various structural parameters and also external validated by docking of known inhibitors. The IDF docking results showed good correlations between IDF scores and inhibitory activities; particularly for hCNT1. From the top 0.1% of compounds ranked by virtual screening with the hCNT1 homology model, 172 compounds selected and tested for against hCNT1, hCNT2 and hCNT3, yielded 14 new inhibitors (hits) of (i.e., 8% success rate). The most active compound exhibited an IC50 of 9.05 μM, which shows a greater than 25-fold higher potency than phlorizin the standard CNT inhibitor (IC50 of 250 μM). Conclusion We successfully undertook homology modeling and validation for all human concentrative nucleoside transporters (hCNT 1, 2 and 3). The proof-of-concept that these models are promising for virtual screening to identify potent and selective inhibitors was also obtained using the hCNT1 model. Thus we identified a novel potent hCNT1 inhibitor that is more potent and more selective than the standard inhibitor phlorizin. The other hCNT1 hits also mostly exhibited selectivity. These homology models should be useful for virtual screening to identify novel hCNT inhibitors, as well as for optimization of hCNT inhibitors.

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Structural basis of nucleoside and nucleoside drug selectivity by concentrative nucleoside transporters

Cited by 55 publications

References 59 publications

The bacterial dicarboxylate transporter VcINDY uses a two-domain elevator-type mechanism

The bacterial dicarboxylate transporter VcINDY uses a two-domain elevator-type mechanism

Polypharmacology of conformationally locked methanocarba nucleosides

Homology Modeling of Human Concentrative Nucleoside Transporters (hCNTs) and Validation by Virtual Screening and Experimental Testing to Identify Novel hCNT1 Inhibitors

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