Green fluorescent protein and related proteins carry chromophores formed within the protein from their own amino acids. Corresponding synthetic compounds are non-fluorescent in solution due to photoinduced isomerization of the benzylideneimidiazolidinone core. Restriction of this internal rotation by binding to host molecules leads to pronounced, up to three orders of magnitude, increase of fluorescence intensity. This property allows using GFP chromophore analogs as fluorogenic dyes to detect metal ions, proteins, nucleic acids, and other hosts. For example, RNA aptamer named Spinach, which binds to and activates fluorescence of some GFP chromophores, was proved to be a unique label for live-cell imaging of specific RNAs, endogenous metabolites and target proteins. Chemically locked GFP chromophores are brightly fluorescent and represent potentially useful dyes due to their small size and high water solubility.
We described a set of novel histone deacetylase inhibitors (HDACi) equipped with either an antagonist or an agonist of the estrogen receptor (ER) to confer selective activity against breast cancers. These bifunctional compounds potently inhibit HDAC at nanomolar concentrations, and either agonize or antagonize ERα and ERβ. The ER antagonist activities of tamoxifen-HDACi conjugates (Tam-HDACi) are nearly identical to those of tamoxifen. Conversely, ethynyl-estradiol HDACi conjugates (EED-HDACi) have attenuated ER agonist activities relative to the parent ethynyl-estradiol. In silico docking analysis provides structural basis for the trends of ER agonism/antagonism and ER subtype selectivity. Excitingly, lead Tam-HDACi conjugates show anticancer activity that is selectively more potent against MCF-7 (ERα positive breast) compared to MDA-MB-231 (triple negative breast cancer), DU145 (prostate cancer) or Vero (non-cancerous cell line). This dual-targeting approach illustrates the utility of designing small molecules with an emphasis on cell-type selectivity, not merely improved potency, working towards a higher therapeutic index at the earliest stages of drug development.
The nuclear receptor retinoid X receptor (RXR) is a ligand-activated transcription factor. To create receptors for a new ligand, a structure-based approach was used to generate a library of Ϸ380,000 mutant RXR genes. To discover functional variants within the library, we used chemical complementation, a method of protein engineering that uses the power of genetic selection. Wild-type RXR has an EC50 of 500 nM for 9-cis retinoic acid (9cRA) and an EC50 of >10 M for the synthetic retinoid-like compound LG335 in yeast. T he human retinoid X receptor ␣ (RXR) is a ligand-activated transcription factor of the nuclear receptor superfamily. RXR plays an important role in morphogenesis and differentiation and serves as a dimerization partner for other nuclear receptors (1). Like most nuclear receptors, RXR has two structural domains, the DNA-binding domain (DBD) and the ligand-binding domain (LBD), which are connected by a flexible hinge region. The DBD contains two zinc modules, which bind a sequence of six bases (2). The LBD binds and activates transcription in response to multiple ligands including phytanic acid, docosahexaenoic acid and 9-cis retinoic acid (9cRA) (Fig. 5, which is published as supporting information on the PNAS web site) (3-6). RXR is a modular protein; the DBD and LBD function independently. Therefore, the LBD can be fused to other DBDs and retain function. A conformational change is induced in the LBD upon ligand-binding, which initiates recruitment of coactivators and the basal transcription machinery resulting in transcription of the target gene (7).Nuclear receptors have evolved to bind and activate transcription in response to a variety of small-molecule ligands. The known ligands for nuclear receptors are chemically diverse and include steroid and thyroid hormones, vitamin D, prostaglandins, fatty acids, leukotrienes, retinoids, antibiotics, and other xenobiotics. Evolutionarily closely related receptors (e.g., thyroid hormone receptor and retinoic acid receptor) bind different ligands, whereas some members of distant subfamilies (e.g., RXR and retinoic acid receptor) bind the same ligand (8). This diversity of ligand-receptor interactions demonstrates the versatility of the fold for ligandbinding and suggests that it should be possible to engineer LBDs with a large range of novel specificities.The crystal structure of RXR bound to 9cRA elucidates important hydrophobic and polar interactions in the LBD binding pocket (9). There are 20 hydrophobic and polar amino acids within 4.4 Å of the bound 9cRA (Fig. 6, which is published as supporting information on the PNAS web site). RXR is a good candidate for creating variants that bind different ligands through site-directed mutagenesis, because side-chain atoms, not main-chain atoms, contribute the majority of the ligand contacts (10-13). A library of RXR LBDs with all 20 amino acids at each of the 20 positions in the ligand-binding pocket screened against multiple compounds could potentially produce many new ligand-receptor pairs. However, the numb...
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