No abstract

The ligand-binding characteristics of rat and human CYP2D isoforms, i.e., rat CYP2D1-4 and human CYP2D6, were investigated by measuring IC(50) values of 11 known CYP2D6 ligands using 7-methoxy-4-(aminomethyl)coumarin (MAMC) as substrate. Like CYP2D6, all rat CYP2D isozymes catalyzed the O-demethylation of MAMC with K(m) and V(max) values ranging between 78 and 145 microM and 0.048 and 1.122 min(-1), respectively. To rationalize observed differences in the experimentally determined IC(50) values, homology models of the CYP2D isoforms were constructed. A homology model of CYP2D6 was generated on the basis of crystallized rabbit CYP2C5 and was validated on its ability to reproduce binding orientations corresponding to metabolic profiles of the substrates and to remain stable during unrestrained molecular dynamics simulations at 300 K. Twenty-two active site residues, sharing up to 59% sequence identity, were identified in the CYP2D binding pockets and included CYP2D6 residues Phe120, Glu216, and Asp301. Electrostatic potential calculations displayed large differences in the negative charge of the CYP2D active sites, which was consistent with observed differences in absolute IC(50) values. MD studies on the binding mode of sparteine, quinidine, and quinine in CYP2D2 and CYP2D6 furthermore concurred well with experimentally determined IC(50) values and metabolic profiles. The current study thus provides new insights into differences in the active site topology of the investigated CYP2D isoforms.

A novel scoring algorithm based on molecular interaction fingerprints (IFPs) was comparatively evaluated in its scaffold hopping efficiency against four virtual screening standards (GlideXP, Gold, ROCS, and a Bayesian classifier). Decoy databases for the two targets under examination, adenosine deaminase and retinoid X receptor alpha, were obtained from the Directory of Useful Decoys and were further enriched with approximately 5% of active ligands. Structure and ligand-based methods were used to generate the ligand poses, and a Tanimoto metric was chosen for the calculation of the similarity interaction fingerprint between the reference ligand and the screening database. Database enrichments were found to strongly depend on the pose generator algorithm. In spite of these dependencies, enrichments using molecular IFPs were comparable to those obtained with GlideXP, Gold, ROCS, and the Bayesian classifier. More interestingly, the molecular IFP scoring algorithm outperformed these methods at scaffold hopping enrichment, regardless of the pose generator algorithm.

The granulocyte colony-stimulating factor receptor (G-CSF-R) forms a tetrameric complex with G-CSF containing two ligand and two receptor molecules. The Nterminal Ig-like domain of the G-CSF-R is required for receptor dimerization, but it is not known whether it binds G-CSF or interacts elsewhere in the complex. Alanine scanning mutagenesis was used to show that residues in the Ig-like domain of the G-CSF-R (Phe 75 , Gln 87 , and Gln 91 ) interact with G-CSF. This binding site for G-CSF overlapped with the binding site of a neutralizing anti-G-CSF-R antibody. A model of the Ig-like domain showed that the binding site is very similar to the viral interleukin-6 binding site (site III) on the Ig-like domain of gp130, a related receptor. To further characterize the G-CSF-R complex, exposed and inaccessible regions of monomeric and dimeric ligand-receptor complexes were mapped with monoclonal antibodies. The results showed that the E helix of G-CSF was inaccessible in the dimeric but exposed in the monomeric complex, suggesting that this region binds to the Ig-like domain of the G-CSF-R. In addition, the N terminus of G-CSF was exposed to antibody binding in both complexes. These data establish that the dimerization interface of the complete receptor complex is different from that in the x-ray structure of a partial complex. A model of the tetrameric G-CSF⅐G-CSF-R complex was prepared, based on the viral interleukin-6⅐gp130 complex, which explains these and previously published data. The granulocyte colony-stimulating factor receptor (G-CSF-R)1 is a transmembrane protein that is expressed predominantly on cells of the neutrophil lineage and is important in transmitting signals for their proliferation, differentiation, and function (1, 2). The extracellular region comprises six structural domains: an N-terminal Ig-like domain followed by five fibronectin type III (FNIII) domains (3) (Fig. 1A). The first two of the FNIII domains (called D2 and D3) form the cytokine receptor homology (CRH) module, which contains four cysteine residues in D2 and a WSXWS motif in D3. These features of the CRH module are conserved in members of the class 1 cytokine receptor family (4). This domain structure is also found in the closest homologue of the G-CSF-R, gp130, which is the shared signal transducing receptor chain of the interleukin (IL)-6 family of cytokines (5). The G-CSF-R and gp130 share 46% sequence similarity in the extracellular region (6). The ligands for the class 1 cytokine receptors have a conserved 4-␣-helical bundle structure but little sequence similarity (7).In the gp130 family of ligands and receptors, three binding interfaces have been identified (reviewed in Ref. 8). For example, IL-6 interacts with the IL-6 receptor at site I and gp130 at site II. A second gp130 receptor interacts at site III, resulting in a hexameric complex of two components each of IL-6, IL-6 receptor, and gp130 (9). On the IL-6 family of ligands, site I is located at the end of the D helix, site II on the A and C helices, and site III at the N...