Computational Methods. In this research, four published agonists, partial agonists and antagonists of various NRs were selected as lead compounds. Tamoxifen [12] is an antagonist of estrogen receptors that has been used for more than 30 years to treat breast cancer in women. Raloxifene [13], an oral selective estrogen receptor modulator (SERM), has estrogenic actions on bone and anti-estrogenic actions on the uterus and breast. SID-7969543 [14] is an NIH probe that shows antagonist properties against SF-1 but no effect on LRH-1. The last selected compound MFA-1, a steroid analog and agonist of the farnesoid X receptor (FXR), was obtained from PDB: 3BEJ [15].
Computational Methods. The crystal structure of TLR4-MD2-dimer complexed with Xx X. coli LPS (PDB code: 3FXI) was recently released.[74] Based on this crystal structure, Meningococcal LOS was docked into human TLR4/MD-2 dimer by Autodock 4.0. [75] Because the ligand Meningococcal LOS has large numbers of rotatable bonds, it is hard to dock this ligand with all rotatable bond setting to be flexible. Here, we tried to make four chains which are buried in the human MD-2 cavity to be rigid, while the other two chains (R2 and R2’) which locate near the TLR4/MD-2 interface are flexible. The AutoGrid calculation was run with 70 points (separated by 0.503 A) in each spatial dimension. AutoDock was run using the Lamarckian genetic algorithm, producing 50 structures, which were evaluated in the analysis step. Once all the docking steps are finished, the MM-GBSA scores for each docking poses are estimated by performing the Prime MMGBSA.
Computational Methods. Efforts to improve the potency of 1 employed the X-ray structure of the GLP/BIX-01294 complex as a template for structure-based design. Both the protein structure and preliminary docking models suggested three sectors of the ligand as best suited for structural modifications: a) the diazepine ring, b) the piperidine ring benzyl appendage, and c) the C7-methoxy groups (Figure 6.1).
Computational Methods. Quantification of PP values A single-cell sorting assay was used to measure PP, the probability that an individual genome segment from an infectious virion is successfully delivered and replicated within the infected cell. The technical details are described in the “Experimental Methods” section. Here we describe the mathematical analysis used to calculate PP from the experimental data. The presence or absence of different viral genome segments was measured by qRT-PCR, with each well of a 96-well plate representing the viral RNA that was present in the cell that was initially sorted into the plate. Given the MOI of Pan/99-WT virus used in the experiments, an appreciable number of xxxxx are expected to receive two or more viral genomes, and so a mathematical adjustment is needed to estimate the probability of each genome segment being delivered by a signle virion. Using the relationship between MOI and the fraction of cells infected from Poisson statistics, i.e., f = 1 – e–MOI, the probability of the ith segment being present in a singly infected cell, or PP,i can be calculated from the 96-well plate using the following equation: −ln (1 − ) ln (1 − D) V,D = D = D = MOI[\ −ln (1 − [\) ln (1 − ) where A is the number of Helper+ xxxxx, B is the number of WT+ xxxxx (containing any WT segment), and Ci is the number of xxxxx positive for the WT segment in question. Xxxxx that were negative for Helper virus segments were excluded from analysis. For each experimental replicate, the geometric mean PP value was calculated to represent an average PP.
