Characterizing the Pregnane X Receptor’s Interactions and Biophysical Properties

Pregnane X Receptor is a ligand-activated transcription factor critical in protecting tissues from xenobiotics and endobiotics. PXR is shown to interact with GRIP- 1 and PGC-1α on DR-4 and XREM-CYP3A4 promoters. Experiments with full-length PXR have been limited by the inability to produce it. This study reports production of full-length PXR from Spodoptera frugiperda and use in peptide phage display experiments. PXR LBD was also mapped using peptide phage display. Sequencing results demonstrate a conserved motif consistent with class II nuclear receptor boxes but adding an additional residue, a polar residue in the -3 position. There is a novel intermolecular β-sheet mediating homodimerization in all PXR LBD structures. Mammalian two-hybrid studies demonstrated that a mutant of PXR that disrupts the homodimerization interface and eliminates basal transcriptional activity is unable to recruit SRC-1. Thermal denaturation studies of other PXR LBD mutants that affect basal transcriptional activity show changes in overall protein stability

Human PXR Forms a Tryptophan Zipper-Mediated Homodimer †

The human nuclear receptor pregnane X receptor (PXR) responds to a wide variety of potentially harmful chemicals and coordinates the expression of genes central to xenobiotic and endobiotic metabolism. Structural studies reveal that the PXR ligand binding domain (LBD) uses a novel sequence insert to form a homodimer unique to the nuclear receptor superfamily. Terminal β-strands from each monomeric LBD interact in an ideal antiparallel fashion to bury potentially exposed surface β-strands, generating a ten-stranded intermolecular β-sheet. Conserved tryptophan and tyrosine residues lock across the dimer interface and provide the first tryptophan-zipper (Trp-Zip) interaction observed in a native protein. We show using analytical ultracentrifugation that the PXR LBD forms a homodimer in solution. We further find that removal of the interlocking aromatic residues eliminates dimer formation but does not affect PXR's ability to interact with DNA, RXRα, or ligands. Disruption of the homodimer significantly reduces receptor activity in transient transfection experiments, however, and effectively eliminates the receptor's recruitment of the transcriptional coactivator SRC-1 both in vitro and in vivo. Taken together, these results suggest that the unique Trp-Zip-mediated PXR homodimer plays a role in the function of this nuclear xenobiotic receptor

A novel class of achiral \u3cem\u3eseco\u3c/em\u3e-analogs of CC-1065 and the duocarmycins: design, synthesis, DNA binding, and anticancer properties

The synthesis, DNA binding properties, and in vitro and in vivo anticancer activity of fifteen achiral seco-cyclopropylindoline (or achiral seco-CI) analogs (5a-o) of CC-1065 and the duocarmycins are described. The achiral seco-CI analogs contain a 4-hydroxyphenethyl halide moiety that is attached to a wide range of indole, benzimidazole, pyrrole, and pyridyl-containing noncovalent binding components. The 4-hydroxyphenethyl halide moiety represents the simplest mimic of the seco-cyclopropylpyrroloindoline (seco-CPI) pharmacophore found in the natural products, and it lacks a chiral center. The sequence and minor groove specificity of the achiral compounds was ascertained using a Taq DNA polymerase stop assay and a thermal induced DNA cleavage experiment using either a fragment of pBR322 or pUC18 plasmid DNA. For example, seco-CI-InBf (5a) and seco-CI-TMI (5c) demonstrated specificity for AT-rich sequences, particularly by reacting with the underlined adenine-N3 position of 5\u27-AAAAA(865)-3\u27. This is also the sequence that CC-1065 and adozelesin prefer to alkylate. The achiral seco-CI compounds were subjected to cytotoxicity studies against several human (K562, LS174T, PC3, and MCF-7) and murine cancer cell lines (L1210 and P815). Following continuous drug exposure, the achiral compounds were found to be cytotoxic, with IC(50) values in the muM range. Interestingly, the carbamate protected compound 5p was significantly less cytotoxic than agent 5c, supporting the hypothesis that loss of HCl and formation of a spiro2,5]cyclopropylcyclohexadienone intermediate is necessary for biological activity. The achiral seco-CI compounds 5a and 5c were submitted to the National Cancer Institute for further cytotoxicity screening against a panel of 60 different human cancer cell lines. Both compounds showed significant activity, particularly against several solid tumor cell lines. Flow cytometry studies of P815 cells that were incubated with compound 5c at its IC(50) concentration for 24h showed induction of apoptosis in a large percentage of cells. Compounds 5a and 5c were selected by the NCI for an in vivo anticancer hollow-fiber test, and received composite scores of 18 and 22, respectively. These two compounds were subsequently evaluated for in vivo anticancer activity against the growth of a human advanced stage SC UACC-257 melanoma in skid mice. At a dose of 134 mg/kg administered IP, compound 5c gave a T/C value of 40% (for day 51), and the median number of days of doubling tumor growth was 27.7, versus 15.8 for untreated animals. For compound 5a, at 200mg/kg, the T/C was 58% and the median number of days of doubling tumor growth was 20.0 versus 8.7 for untreated animals. At these doses no toxicity or weight loss was observed for either compound. Furthermore, compound 5c was not toxic to murine bone marrow cell growth in culture, at a dose that was toxic for the previously reported seco-CBI (cyclopropylbenzoindoline)-TMI (4)

COVID-19 vaccine effectiveness against symptomatic infection with SARS-CoV-2 BA.1/BA.2 lineages among adults and adolescents in a multicentre primary care study, Europe, December 2021 to June 2022

International audienceBackgroundScarce European data in early 2021 suggested lower vaccine effectiveness (VE) against SARS-CoV-2 Omicron lineages than previous variants.AimWe aimed to estimate primary series (PS) and first booster VE against symptomatic BA.1/BA.2 infection and investigate potential biases.MethodsThis European test-negative multicentre study tested primary care patients with acute respiratory symptoms for SARS-CoV-2 in the BA.1/BA.2-dominant period. We estimated PS and booster VE among adults and adolescents (PS only) for all products combined and for Comirnaty alone, by time since vaccination, age and chronic condition. We investigated potential bias due to correlation between COVID-19 and influenza vaccination and explored effect modification and confounding by prior SARS-CoV-2 infection.ResultsAmong adults, PS VE was 37% (95% CI: 24–47%) overall and 60% (95% CI: 44–72%), 43% (95% CI: 26–55%) and 29% (95% CI: 13–43%) < 90, 90–179 and ≥ 180 days post vaccination, respectively. Booster VE was 42% (95% CI: 32–51%) overall and 56% (95% CI: 47–64%), 22% (95% CI: 2–38%) and 3% (95% CI: −78% to 48%), respectively. Primary series VE was similar among adolescents. Restricting analyses to Comirnaty had little impact. Vaccine effectiveness was higher among older adults. There was no signal of bias due to correlation between COVID-19 and influenza vaccination. Confounding by previous infection was low, but sample size precluded definite assessment of effect modification.ConclusionPrimary series and booster VE against symptomatic infection with BA.1/BA.2 ranged from 37% to 42%, with similar waning post vaccination. Comprehensive data on previous SARS-CoV-2 infection would help disentangle vaccine- and infection-induced immunity