Light activated recombination.

Many genes elicit their actions through their expression in precise spatial patterns in tissues. Photoregulated expression systems offer a means to remotely pattern gene expression in tissues. Using currently available photopatterning methods, gene expression is only transient. Herein is described a general method to permanently alter a cell's genome under the control of light. The photocaged estrogen receptor (ER) antagonists, nitroveratryl-hydroxytamoxifen (Nv-HTam) and nitroveratryl-hydroxytamoxifen aziridine (Nv-HTaz), mediate exposure-dependent recombination in cells expressing the Cre-ER, a fusion of the site-specific recombinase Cre and ER. Both Nv-HTam and Nv-HTaz only activate recombination by Cre-ER after exposure to light. When released only intracellularly, the covalent-modifying Taz can mediate significant amounts of recombination in an exposure-dependent manner. Nv-HTaz and Cre-ER represent perhaps the first compound that can be used to photopattern gene expression through recombination.

Design and synthesis of complementing ligands for mutant thyroid hormone receptor TRbeta(R320H): a tailor-made approach toward the treatment of resistance to thyroid hormone.

The thyroid hormone receptors (TR) are ligand-dependant transcription factors that regulate key genes involved in metabolic regulation, thermogenesis and development. Resistance to thyroid hormone (RTH) is a genetic disease associated with mutations to TRbeta that lack or show reduced responsiveness to thyroid hormone (triiodothyronine). Previously we reported that the neutral alcohol-based thyromimetic HY-1 can selectively restore activity to a functionally impaired form of TR associated with RTH without over-stimulating TRalpha, which has been associated with undesirable side effects. Two new series of tetrazole and thiazolidinedione based ligands were evaluated for their ability to recover potency and efficacy to three of the most common RTH-associated mutants, TRbeta(R320C), TRbeta(R320H), and TRbeta(R316H), in cell based assays. A new thiazolidinedione based ligand AH-9 was identified, which has near wild-type potency (EC(50)=0.54 nM) to TRbeta(R320C) and TRbeta(R320H). Significantly, AH-9 is equipotent toward TRalpha(wt), TRbeta(wt), TRbeta(R320C), and TRbeta(R320H), suggesting that AH-9 may have the potential to restore the normal homeostatic balance of thyroid hormone actions in patients or models harboring these mutations.

Mutant-selective thyromimetics for the chemical rescue of thyroid hormone receptor mutants associated with resistance to thyroid hormone.

The thyroid hormone receptors (TRs) are ligand-dependent transcription factors that control the expression of multiple genes involved in development and homeostasis in response to thyroid hormone (triiodothyronine, T3). Mutations to TRbeta that reduce or abolish ligand-dependent transactivation function are associated with resistance to thyroid hormone (RTH), an autosomal dominant human genetic disease. A series of neutral alcohol-based compounds, based on the halogen-free thyromimetic GC-1, have been designed, synthesized, and evaluated in cell-based assays for their ability to selectively rescue three of the most common RTH-associated mutations (i.e., Arg320 --> Cys, Arg320 --> His, and Arg316 --> His) that affect the basic carboxylate-binding arginine cluster of TRbeta. Several analogues show improved potency and activity in the mutant receptors relative to the parent compound GC-1. Most significantly, two of these mutant-complementing thyromimics show high potency and activity with a strong preference for the mutant receptors over wild-type TRalpha(wt), that is associated with the cardiotoxic actions of T3. The compounds were evaluated in reporter gene assays using the four common thyroid hormone response elements, DR4, PAL, F2 (LAP), and TSH, and show activities and selectivites consistent with their unique potential as agents to selectively rescue thyroid function to these RTH-associated mutants.

Light-activated transcription and repression by using photocaged SERMs.

Recently developed methods to regulate the spatial and temporal patterning of genes in a light-directed manner hold promise as powerful tools for exploring the function of genes that act through their unique spatiotemporal patterning. To further explore the application of photocaged ligands of nuclear receptors to control gene expression patterning, the actions of photocaged analogues of selective estrogen-receptor modulators (SERMs) have been evaluated. Photocaged derivatives of hydroxytamoxifen (NB-Htam) and guanidine tamoxifen (NB-Gtam) have been synthesized that selectively antagonize ER alpha- and ER beta-mediated transcription at classic estrogen response elements (EREs) in response to light. When present only intracellularly, Htam and Gtam provide a similar transient repression response. When SERMs are allowed to diffuse out of the cell, transcription is recovered at a similar rate for Htam and Gtam (6.4 and 5.6 h(-1)), but is notably faster than is observed with the covalently binding SERM tamoxifen aziridine (Taz) (3.8 h(-1)). This suggests that the duration of agonist action is controlled by ligand off-rates/diffusion and not by receptor turnover. Gtam activates ER beta-mediated transcription at AP1 sites in a similar way to what has previously been reported for Htam. NB-Gtam and NB-Tam provide a light-activated transcription response at AP1-driven reporters, thus illustrating the unique ability of photocaged SERMs to simultaneously mediate light-activated transcription and repression.

Functionally orthogonal ligand-receptor pairs for the selective regulation of gene expression generated by manipulation of charged residues at the ligand-receptor interface of ER alpha and ER beta.

The reengineering of protein-small molecule interfaces represents a powerful tool of chemical biology. For many applications it is necessary to engineer receptors so that they do not interact with their endogenous ligands but are highly responsive to designed ligand analogues, which in turn do not interact with endogenous proteins. The chemical design strategy used to reengineer protein-small molecule interfaces is particularly challenging for interfaces involving relatively plastic receptor binding sites and therefore presents a unique challenge in molecular design. In this study we explore the scope and limitations of a new strategy for manipulating polar/charged residues across the ligand receptor interface of estradiol (E2) and the estrogen receptor (ER). Carboxylate-functionalized E2 analogues can activate ER alpha(Glu353-->Ala) and ER beta(Glu305-->Ala) with very large selectivites, demonstrating that this design strategy is extendable to other members of the steroid hormone receptor family. Neutral E2 analogues were found to complement ER alpha(E353A) with similar potencies but with generally lower selectivities. This suggests that the high selectivity observed with ligand-receptor pairs generated by exchanging charged residues across ligand-receptor interfaces is only due in part to their complementary shapes and that appropriate introduction of charged functionality on the ligand can provide substantial enhancement of selectivity by decreasing the engineered ligands affinity for the endogenous receptor. Attempts to modify the cationic residues by complementing Arg394-->Ala or Arg394-->Glu were not successful.