Design, synthesis, and biological evaluation of 4-arylmethyl-1-phenylpyrazole and 4-aryloxy-1-phenylpyrazole derivatives as novel androgen receptor antagonists

A series of 4-arylmethyl-1-phenylpyrazole and 4-aryloxy-1-phenylpyrazole compounds B were designed, synthesized, and evaluated for their potential as new-generation androgen receptor (AR) antagonists therapeutically effective against castration-resistant prostate cancer (CRPC). Introduction of a bulky amide substituent (R(2)) to the terminal aryl ring of the 4-arylmethyl group favored the reduction of agonistic activity and improved the pharmacokinetic (PK) properties. Similarly, introduction of a bulky substituent in the 4-aryloxy derivatives also resulted in improved PK properties. Compounds 28 h and 44b exhibited potent antitumor effects against a CRPC model of LNCaP-hr cell line in a mouse xenograft model. On the contrary, bicalutamide showed only partial suppression of tumor growth. These results suggest that the novel pyrazole derivatives are new-generation AR antagonists, different from the 'first-generation' antagonists such as bicalutamide in a CRPC treatment model.     

How do nitrogen and phosphorus deficiencies affect strigolactone production and exudation?

Plants exude strigolactones (SLs) to attract symbiotic arbuscular mycorrhizal fungi in the rhizosphere. Previous studies have demonstrated that phosphorus (P) deficiency, but not nitrogen (N) deficiency, significantly promotes SL exudation in red clover, while in sorghum not only P deficiency but also N deficiency enhances SL exudation. There are differences between plant species in SL exudation under P- and N-deficient conditions, which may possibly be related to differences between legumes and non-legumes. To investigate this possibility in detail, the effects of N and P deficiencies on SL exudation were examined in Fabaceae (alfalfa and Chinese milk vetch), Asteraceae (marigold and lettuce), Solanaceae (tomato), and Poaceae (wheat) plants. In alfalfa as expected, and unexpectedly in tomato, only P deficiency promoted SL exudation. In contrast, in Chinese milk vetch, a leguminous plant, and in the other non-leguminous plants examined, N deficiency as well as P deficiency enhanced SL exudation. Distinct reductions in shoot P levels were observed in plants grown under N deficiency, except for tomato, in which shoot P level was increased by N starvation, suggesting that the P status of the shoot regulates SL exudation. There seems to be a correlation between shoot P levels and SL exudation across the species/families investigated.     

The role of X/Y linker region and N-terminal EF-hand domain in nuclear translocation and Ca2+ oscillation-inducing activities of phospholipase Czeta, a mammalian egg-activating factor

Sperm-specific phospholipase C-zeta (PLCzeta) causes intracellular Ca(2+) oscillations and thereby egg activation and is accumulated into the formed pronucleus (PN) when expressed in mouse eggs by injection of cRNA encoding PLCzeta, which consists of four EF-hand domains (EF1-EF4) in the N terminus, X and Y catalytic domains, and C-terminal C2 domain. Those activities were analyzed by expressing PLCzeta mutants tagged with fluorescent protein Venus by injection of cRNA into unfertilized eggs or 1-cell embryos after fertilization. Nuclear localization signal (NLS) existed at 374-381 in the X/Y linker region. Nuclear translocation was lost by replacement of Arg(376), Lys(377), Arg(378), Lys(379), or Lys(381) with glutamate, whereas Ca(2+) oscillations were conserved. Nuclear targeting was also absent for point mutation of Lys(299) and/or Lys(301) in the C terminus of X domain, or Trp(13), Phe(14), or Val(18) in the N terminus of EF1. Ca(2+) oscillation-inducing activity was lost by the former mutation and was remarkably inhibited by the latter. A short sequence 374-383 fused with Venus showed active translocation into the nucleus of COS-7 cells, but 296-309 or 1-19 did not. Despite the presence of these special regions, both activities were deprived by deletion of not only EF1 but also EF2-4 or C2 domain. Thus, PLCzeta is driven into the nucleus primarily by the aid of NLS and putative regulatory sites, but coordinated three-dimensional structure, possibly formed by a folding in the X/Y linker and close EF/C2 contact as in PLCdelta1, seems to be required not only for enzymatic activity but also for nuclear translocation ability.     

Identification of human UDP-glucuronosyltransferase isoform(s) responsible for the C-glucuronidation of phenylbutazone

Glucuronidation is a major metabolic pathway in the biotransformation of many xenobiotics and endogeneous compounds. There have been many studies on the formation of O-, N- or S-glucuronides and identification of the UDP-glucuronosyltransferase (UGT) isoforms responsible for the formation of these glucuronides. However, there is no information available on which UGT isoform(s) catalyzes C-glucuronidation. In the present study, 16 human UGTs (UGTs 1A1, 1A3, 1A4, 1A5, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B10, 2B11, 2B15, 2B17 and 2B28) were cloned and expressed in baculovirus-infected insect cells and investigated to determine their C-glucuronidating activity toward phenylbutazone (PB). Among the UGT isoforms investigated, only UGT1A9 catalyzed PB C-glucuronidation. Human liver and kidney microsomes, which are well known to express UGT1A9, had C-glucuronidating activity toward PB. However, the jejunum, which did not express UGT1A9, had no C-glucuronidating activity. These results demonstrate for the first time that PB C-glucuronidation is catalyzed by only UGT1A9.     

SWTY--a general peptide probe for homogeneous solution binding assay of 14-3-3 proteins

Dimeric 14-3-3 proteins exert diverse functions in eukaryotes by binding to specific phosphorylated sites on diverse target proteins. Critical to the physiological function of 14-3-3 proteins is the wide range of binding affinity to different ligands. The existing information of binding affinity is mainly derived from nonhomogeneous-based methods such as surface plasmon resonance and quantitative affinity precipitation. We have developed a fluorescence anisotropy peptide probe using a genetically isolated 14-3-3-binding SWTY motif. The synthetic 5-(and-6)-carboxyfluorescein(FAM)-RGRSWpTY-COOH peptide, when bound to 14-3-3 proteins, exhibits a seven-fold increase in fluorescence anisotropy. Different from the existing assays for 14-3-3 binding, this homogeneous assay tests the interaction directly in solution. Hence it permits more accurate determination of the dissociation constants of 14-3-3 binding molecules. Protocols for a simple mix-and-read format have been developed to evaluate 14-3-3 protein interactions using either purified recombinant 14-3-3 fusion proteins or native 14-3-3s in crude cell lysate. Optimal assay conditions for high-throughput screening for modulators of 14-3-3 binding have been determined.     

Codon reassignment in the Escherichia coli genetic code

Most organisms, from Escherichia coli to humans, use the ‘universal’ genetic code, which have been unchanged or ‘frozen’ for billions of years. It has been argued that codon reassignment causes mistranslation of genetic information, and must be lethal. In this study, we successfully reassigned the UAG triplet from a stop to a sense codon in the E. coli genome, by eliminating the UAG-recognizing release factor, an essential cellular component, from the bacterium. Only a few genetic modifications of E. coli were needed to circumvent the lethality of codon reassignment; erasing all UAG triplets from the genome was unnecessary. Thus, UAG was assigned unambiguously to a natural or non-natural amino acid, according to the specificity of the UAG-decoding tRNA. The result reveals the unexpected flexibility of the genetic code.     

Genetic encoding of non‐natural amino acids in Drosophila melanogaster Schneider 2 cells

Insect cells are useful for the high‐yield production of recombinant proteins including chemokines and membrane proteins. In this study, we developed an insect cell‐based system for incorporating non‐natural amino acids into proteins at specific sites. Three types of promoter systems were constructed, and their efficiencies were compared for the expression of the prokaryotic amber suppressor tRNA^Tyr in Drosophila melanogaster Schneider 2 cells. When paired with a variant of Escherichia coli tyrosyl‐tRNA synthetase specific for 3‐iodo‐L ‐tyrosine, the suppressor tRNA transcribed from the U6 promoter most efficiently incorporated the amino acid into proteins in the cells. The transient and stable introductions of these prokaryotic molecules into the insect cells were then compared in terms of the yield of proteins containing non‐natural amino acids, and the “transient” method generated a sevenfold higher yield. By this method, 4‐azido‐L ‐phenylalanine was incorporated into human interleukin‐8 at a specific site. The yield of the azido‐containing IL‐8 was 1 μg/1 mL cell culture, and the recombinant protein was successfully labeled with a fluorescent probe by the Staudinger–Bertozzi reaction.