Docking for fragment inhibitors of AmpC beta-lactamase.

Fragment screens for new ligands have had wide success, notwithstanding their constraint to libraries of 1,000-10,000 molecules. Larger libraries would be addressable were molecular docking reliable for fragment screens, but this has not been widely accepted. To investigate docking's ability to prioritize fragments, a library of >137,000 such molecules were docked against the structure of beta-lactamase. Forty-eight fragments highly ranked by docking were acquired and tested; 23 had K(i) values ranging from 0.7 to 9.2 mM. X-ray crystal structures of the enzyme-bound complexes were determined for 8 of the fragments. For 4, the correspondence between the predicted and experimental structures was high (RMSD between 1.2 and 1.4 A), whereas for another 2, the fidelity was lower but retained most key interactions (RMSD 2.4-2.6 A). Two of the 8 fragments adopted very different poses in the active site owing to enzyme conformational changes. The 48% hit rate of the fragment docking compares very favorably with "lead-like" docking and high-throughput screening against the same enzyme. To understand this, we investigated the occurrence of the fragment scaffolds among larger, lead-like molecules. Approximately 1% of commercially available fragments contain these inhibitors whereas only 10(-7)% of lead-like molecules do. This suggests that many more chemotypes and combinations of chemotypes are present among fragments than are available among lead-like molecules, contributing to the higher hit rates. The ability of docking to prioritize these fragments suggests that the technique can be used to exploit the better chemotype coverage that exists at the fragment level.

Active nuclear receptors exhibit highly correlated AF-2 domain motions.

Nuclear receptor ligand binding domains (LBDs) convert ligand binding events into changes in gene expression by recruiting transcriptional coregulators to a conserved activation function-2 (AF-2) surface. While most nuclear receptor LBDs form homo- or heterodimers, the human nuclear receptor pregnane X receptor (PXR) forms a unique and essential homodimer and is proposed to assemble into a functional heterotetramer with the retinoid X receptor (RXR). How the homodimer interface, which is located 30 A from the AF-2, would affect function at this critical surface has remained unclear. By using 20- to 30-ns molecular dynamics simulations on PXR in various oligomerization states, we observed a remarkably high degree of correlated motion in the PXR-RXR heterotetramer, most notably in the four helices that create the AF-2 domain. The function of such correlation may be to create "active-capable" receptor complexes that are ready to bind to transcriptional coactivators. Indeed, we found in additional simulations that active-capable receptor complexes involving other orphan or steroid nuclear receptors also exhibit highly correlated AF-2 domain motions. We further propose a mechanism for the transmission of long-range motions through the nuclear receptor LBD to the AF-2 surface. Taken together, our findings indicate that long-range motions within the LBD scaffold are critical to nuclear receptor function by promoting a mobile AF-2 state ready to bind coactivators.

Structural basis of human pregnane X receptor activation by the hops constituent colupulone.

Hops extracts are used to alleviate menopausal symptoms and as an alternative to hormone replacement therapy, but they can produce potentially harmful drug-drug interactions. The nuclear xenobiotic receptor pregnane X receptor (PXR) is promiscuously activated by a range of structurally distinct chemicals. It has a key role in the transcriptional regulation of genes that encode xenobiotic metabolism enzymes. In this study, hops extracts are shown to induce the expression of numerous drug metabolism and excretion proteins. The beta-bitter acid colupulone is demonstrated to be a bioactive component and direct activator of human PXR. The 2.8-A resolution crystal structure of the ligand binding domain of human PXR in complex with colupulone was elucidated, and colupulone was observed to bind in a single orientation stabilized by both van der Waals and hydrogen bonding contacts. The crystal structure also indicates that related alpha- and beta-bitter acids have the capacity to serve as PXR agonists as well. Taken together, these results reveal the structural basis for drug-drug interactions mediated by colupulone and related constituents of hops extracts.

Activated pregnenolone X-receptor is a target for ketoconazole and its analogs.

PURPOSE: Variations in biotransformation and elimination of microtubule-binding drugs are a major cause of unpredictable side effects during cancer therapy. Because the orphan receptor, pregnenolone X-receptor (PXR), coordinately regulates the expression of paclitaxel metabolizing and transport enzymes, controlling this process could improve therapeutic outcome. EXPERIMENTAL DESIGN: In vitro RNA-, protein-, and transcription-based assays in multiple cell lines derived from hepatocytes and PXR wild-type and null mouse studies were employed to show the effects of ketoconazole and its analogues on ligand-activated PXR-mediated gene transcription and translation. RESULTS: The transcriptional activation of genes regulating biotransformation and transport by the liganded human nuclear xenobiotic receptor, PXR, was inhibited by the commonly used antifungal ketoconazole and related azole analogs. Mutations at the AF-2 surface of the human PXR ligand-binding domain indicate that ketoconazole may interact with specific residues outside the ligand-binding pocket. Furthermore, in contrast to that observed in PXR (+/+) mice, genetic loss of PXR results in increased (preserved) blood levels of paclitaxel. CONCLUSIONS: These studies show that some azole compounds repress the coordinated activation of genes involved in drug metabolism by blocking PXR activation. Because loss of PXR maintains blood levels of paclitaxel upon chronic dosing, ketoconazole analogues may also serve to preserve paclitaxel blood levels on chronic dosing of drugs. Our observations may facilitate new strategies to improve the clinical efficacy of drugs and to reduce therapeutic side effects.

The nuclear xenobiotic receptor pregnane X receptor: recent insights and new challenges.

The nuclear receptor pregnane X receptor (PXR) plays a key but structurally enigmatic role in human biology. This ligand-regulated transcription factor responds to a diverse array of chemically distinct ligands, including many endogenous compounds and clinical drugs, and regulates the expression of a critical set of protective gene products involved in xenobiotic and endobiotic metabolism. The structural basis of this receptor's remarkable and unique promiscuity is just now coming into focus. We examine the importance of mobile regions novel to the nuclear receptor ligand-binding domain fold in the ability of PXR to respond to a variety of small and large agonists. We also review the functional roles played by PXR in numerous biological pathways and outline emerging areas for the future examination of this key nuclear xenobiotic receptor.

Differentiation of AmpC beta-lactamase binders vs. decoys using classification kNN QSAR modeling and application of the QSAR classifier to virtual screening.

The use of inaccurate scoring functions in docking algorithms may result in the selection of compounds with high predicted binding affinity that nevertheless are known experimentally not to bind to the target receptor. Such falsely predicted binders have been termed 'binding decoys'. We posed a question as to whether true binders and decoys could be distinguished based only on their structural chemical descriptors using approaches commonly used in ligand based drug design. We have applied the k-Nearest Neighbor (kNN) classification QSAR approach to a dataset of compounds characterized as binders or binding decoys of AmpC beta-lactamase. Models were subjected to rigorous internal and external validation as part of our standard workflow and a special QSAR modeling scheme was employed that took into account the imbalanced ratio of inhibitors to non-binders (1:4) in this dataset. 342 predictive models were obtained with correct classification rate (CCR) for both training and test sets as high as 0.90 or higher. The prediction accuracy was as high as 100% (CCR = 1.00) for the external validation set composed of 10 compounds (5 true binders and 5 decoys) selected randomly from the original dataset. For an additional external set of 50 known non-binders, we have achieved the CCR of 0.87 using very conservative model applicability domain threshold. The validated binary kNN QSAR models were further employed for mining the NCGC AmpC screening dataset (69653 compounds). The consensus prediction of 64 compounds identified as screening hits in the AmpC PubChem assay disagreed with their annotation in PubChem but was in agreement with the results of secondary assays. At the same time, 15 compounds were identified as potential binders contrary to their annotation in PubChem. Five of them were tested experimentally and showed inhibitory activities in millimolar range with the highest binding constant K(i) of 135 microM. Our studies suggest that validated QSAR models could complement structure based docking and scoring approaches in identifying promising hits by virtual screening of molecular libraries.