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

Abstract

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.

Keywords: Anticancer agents; Antiviral agents; Concentrative nucleoside transporter; Docking; High throughput virtual screening; Hit enrichment; Homology modeling.

Figure 1

Structures of known nucleoside analogue hCNT inhibitors used in further validation of the homology models.

Figure 2

The sequence alignments between hCNTs and vcCNT (sequence identities are: hCNT1 36%; hCNT2 37%; hCNT3 39%). Red color indicates identical residues, orange color indicates similar residues and black color indicates important amino acids at vcCNT binding site.

Figure 3

Overlay of homology models of hCNT1 (Green), hCNT2 (Turquoise) and hCNT3 (Red). Bound Na ion is shown as a pink sphere. The uridine pose in the nucleoside binding site of hCNT1 is shown in green sticks, while the major site (amino acids within 3Å) residues it interacted with are shown in green wires and labeled.

Figure 4

Ramachandran plots of homology models. (A) hCNT1, (B) hCNT2 and (C) hCNT3. Favored (98%) regions are demarkated by blue lines, and allowed regions by purple lines. Good amino acids are denoted by black circles and outlier amino acids are denoted by magenta circles.

Figure 5

Plots of pIC50 values against induced fit (IDF) docking scores of nucleoside analogue hCNT inhibitors docked in the homology models during the validation process.

Figure 6

Docking complex model hCNT1-MeThPmR (red) at the binding site (amino acids within 3 Å of bound uridine in hCNT1are displayed. Uridine is also overlayed on the pyrrolo-cytidine and adenosine of the PDBid 4PD8 (green) and PDBid 4PD9 (yellow) structures, respectively.

Figure 7

The virtual screening protocol used in the study.

Figure 8

hCNT1, 2 and 3 inhibitory activities and selectivity of the 14 hits identified from the combined virtual screening and biological testing. Compounds HM1- HM152, were tested at a concentration of 10 μM, while phlorizin was tested at 250 μM its IC₅₀ for hCNT1. The data are the mean ± sem for three experiments.

Figure 9

Dose-response curve for the most potent hit identified, compound HM50.

Figure 10

Docking complex model of hCNT1-HM50 (purple) and overlayed pose on adenosine of 4PD9 (yellow). Amino acids within 3Å of docked HM50 are displayed.