Nuclear Receptor Pharmacophore Modelling

Human PXR (hPXR) - The structure of the hPXR-LBD has been resolved (16, 20). The hPXR-ligand binding domain is closely related in structure to the vitamin D receptor (VDR), sharing 45% sequence identity; less similarity is shared with RXR,

PPARy, and the estrogen and progesterone receptors. It has been reported that the hPXR-LBD has evolved several structural features that permits it to function as a broad chemical "sensor". These features include a large elliptical ligand binding cavity (1150 A3) that offers few surface features to impact the steric selection of its ligands. This represents a potential problem since there may well not be a defined SAR for hPXR within any one chemical template. This highlights the importance of having structural template diversity for all basic research programs. It should be noted, however, that while paclitaxel (1) is a reasonably potent activator of hPXR, with commensurate induction CYP3A4, CYP2C8 and MDR1 in LS180 cells, its close structural analog, docetaxel (2), is not (42); highlighting some potential at least for hPXR SAR to emerge in the future.

Other features of hPXR include a flexible binding cavity which can accommodate ligands in more than one orientation (three were observed for SR12813), and can bind ligands which are both large (taxol, 854 Da; rifampicin, 823 Da) and small (phenobarbital, 232 Da). Mutation of Asp205 resulted in a marked decrease in basal transcriptional activity of hPXR, highlighting this to be an important determinant of activity. Of the three energetically favorable conformations in which SR12813 binds, only one of twenty eight amino acid residues involved in ligand contact, Phe288, interacted with SR12813 in all three conformations highlighting, in addition to Asp205, the importance of this residue. The most potent ligands defined to date include a constituent in the St. John's Wort herbal antidepressant, hyperforin (Kd = 27 nM, 514 Da), and the hypocholesterolemic compound, SR12813 (Kd = 41 nM, 505 Da) (20, 43).

Using literature data for EC50 values for 12 hPXR ligands a pharmacophore model for hPXR has been developed (44). This pharmacophore was also used to predict the binding affinity for 28 molecules not in the model but known to be hPXR ligands of differing potencies. The pharmacophore distinguished the most potent activators of hPXR (that display >5-fold activation/deactivation), like ecteinascidin, troglitazone, nifedipine, and dexamethasone-t-butylacetate, from poor activators, such as scopoletin and kaempferol. Such a model may offer insights into the SAR of hPXR ligands as they strive to synthesize compounds, or alternative chemical templates, which are devoid of this property.

Human CAR (hCAR) - Several groups have reported on structural comparisons between hCAR and hPXR (18, 43, 45). A 3D model of the ligand binding domain of the hCAR was constructed based on the available X-ray structures of hPXR and VDR (18). The model shows that the size of the ligand binding cavities of hCAR and hPXR are similar, but larger than that of VDR. However, in contrast to hPXR which can bind extremely large ligands such as rifampicin through the flexibility of a surface loop, hCAR would only be expected to bind the smaller hPXR ligands. In support of this observation, it has been reported that helices 6 and 7 provide the walls of the ligand binding pocket in hCAR, but its Helix1-Helix3 insert is too short to reach into the volume occupied by the analogous region in hPXR (43).

Arvl Hydrocarbon Receptor (AhR) - The AhR ligand binding pocket can bind planar ligands with maximal dimensions of 14 x 12 x 5 A which have certain thermodynamic and electronic properties characterized by molecules such as benzo(a)pyrene (3) and p-naphthoflavone (4) (46-48). By using an AhR-reporter gene assay based on green fluorescent protein (49), a more diverse range of ligands has now been identified, characterized by thiabendazole (5) and 5-methyl-2-phenylindole; other substrates are summarized in detail elsewhere (15).

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