Antagonizing the Androgen Receptor with a Biomimetic Acyltransferase.

The Androgen Receptor (AR) remains the leading target of advanced prostate cancer therapies. Thiosalicylamide analogs have previously been shown to act in cells as acyltransfer catalysts that are capable of transferring cellular acetate, presumably from acetyl-CoA, to HIV NCp7. Here we explore if the cellular acetyl-transfer activity of thiosalicylamides can be redirected to other cellular targets guided by ligands for AR.

Photocaged DNA provides new levels of transcription control.

Spot lit: photocaged nucleic acids have been used to regulate gene expression through the action of light. Whereas most methods target mRNAs, DNA decoys have recently been used to target DNA transcription by targeting specific DNA-transcription-factor interactions. This has allowed researchers to "turn-off" transcription through the action of light on caged nucleic acids for the first time.

A mutant selective anti-estrogen is a pure antagonist on EREs and AP-1 response elements.

Estrogen receptors (ERs) regulate gene transcription through classic estrogen response elements (EREs) as well as AP-1 responsive genes. The common SERMs Raloxifene, Tamoxifen, and ICI164384 function as ER antagonists on EREs but as ERbeta agonists/partial agonists on AP-1 responsive genes. While developing a mutant selective analog of Raloxifene, that is an antagonist of ERalpha(E353A), we discovered an antagonist of wild-type ERalpha and ERbeta that is also an antagonist of ERbeta/AP-1 response.

Light-activated gene expression directs segregation of co-cultured cells in vitro.

Light-directed gene patterning methods have been described as a means to regulate gene expression in a spatially and temporally controlled manner. Several methods have been reported that use photocaged forms of small molecule effectors to control ligand-dependent transcription factors. Whereas these methods offer many advantages including high specificity and transient light-sensitivity, the free diffusion of the uncaged effector can limit both the magnitude and resolution of localized gene induction.

Automated time-lapse microscopy and high-resolution tracking of cell migration.

We describe a novel fully automated high-throughput time-lapse microscopy system and evaluate its performance for precisely tracking the motility of several glioma and osteoblastic cell lines. Use of this system revealed cell motility behavior not discernable with conventional techniques by collecting data (1) from closely spaced time points (minutes), (2) over long periods (hours to days), (3) from multiple areas of interest, (4) in parallel under several different experimental conditions. Quantitation of true individual and average cell velocity and path length was obtained with high spatial and temporal resolution in "scratch" or "wound healing" assays.

Development of a thyroid hormone receptor targeting conjugate.

Molecular conjugates of hormone receptor-ligands with molecular probes or functional domains are finding diverse applications in chemical biology. Whereas many examples of hormone conjugates that target steroid hormone receptors have been reported, practical ligand conjugates that target the nuclear thyroid hormone receptor (TRbeta) are lacking. TR-targeting conjugate scaffolds based on the ligands GC-1 and NH-2 and the natural ligand triiodothyronine (T3) were synthesized and evaluated in vitro and in cellular assays.

The new biomimetic chemistry: artificial transcription factors.

While many research programs have focused on the challenge of developing small molecules that can inhibit protein-protein interactions, some researchers have taken the problem one step further by attempting to develop small molecules that mimic the essential features of an entire protein. An area of particular interest has been in the field of artificial transcription factors (ATFs), where the essential function of some transcription factors is to recruit and promote the assembly of a larger transcription complex, leading to the expression of a gene of interest. The goal of synthesizing small-molecule ATFs holds promise as a means to independently control the expression of genes such as those that are misregulated in cancer and disease.

Photocaged agonist for an analogue-specific form of the vitamin D receptor.

Nuclear hormone receptors (NHRs) represent a diverse class of ligand-dependent transcriptional regulators. NHRs that have been rendered functionally inactive due to mutations that abrogate proper ligand binding can often be rescued by appropriately designed hormone analogues. The analogue-specific receptor-ligand pairs provide an ideal platform from which to develop new chemogenomic tools for the spatial and temporal control of gene expression.

Circumventing anti-androgen resistance by molecular design.

Nuclear/steroid hormone receptors (NHRs) function as ligand-dependent transcriptional regulators of diverse sets of genes involved in development and homeostasis. Mutations to the androgen receptor (AR), a member of NHRs, have been linked to the failure of hormonal therapy in the treatment of prostate cancer (PCa). It has been proposed that AR mutations such as Thr877 → Ala, Trp741 → Leu and Trp741 → Cys that cause anti-androgens such as flutamide and bicalutamide to function as agonists are likely associated with anti-androgen withdrawal syndrome in PCa therapy.

Difluoromethyl analogs of the natural hormone 1alpha,25-dihydroxyvitamin D3: Design, synthesis, and preliminary biological evaluation.

Three new Vitamin D analogs 3-5 incorporating a -CHF(2) group as an -OH surrogate have been prepared. Two of these new analogs (3 and 5) are strongly antiproliferative toward murine keratinocytes and are approximately 50 times less calciuric in vivo than the natural hormone calcitriol. The transcriptional activity of the 25-CHF(2) analog 3 is higher than that of the 1-CHF(2) analog 4.

Chemical biology of steroid and nuclear hormone receptors.

The nuclear hormone receptors are ligand-gated transcription factors that modulate gene expression by directly acting upon genomic DNA, and have been of profound interest across all biological disciplines. Recent advancements in this area have included the expansion of transgene activation through ligand-receptor engineering, drug development from structural design and the exploitation of innate ligand-specific associations towards developing novel conditional protein-based recombinant and diagnostic tools. These advancements come on the heels of exciting new modes of hormone action that challenge and expand upon the classic paradigms of hormone receptor function.

A functionally orthogonal ligand-receptor pair created by targeting the allosteric mechanism of the thyroid hormone receptor.

Nuclear receptors are ligand-dependent transcription factors that are of interest as potential tools to artificially regulate gene expression. Ligand binding induces a conformational change involving helix-12 which forms part of the dimerization interface used to bind transcriptional coactivators. When triiodothyronine (T3) binds the thyroid hormone receptor (TR) it indirectly contacts helix-12 through intermediary residues His(435) and Phe(451) termed a His-Phe switch.

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.

Ligand-receptor engineering and its application towards the complementation of genetic disease and target identification.

In some instances, small molecules can restore function to proteins that are impaired by genetic mutations. There are now many examples where non-specific molecules or specific ligands can act as chemical chaperones to fold proteins or stabilize folded proteins harboring genetic mutations. In contrast a few recent examples have shown that functionally impaired proteins that are stably folded can be "functionally rescued" by appropriate small molecules.

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.

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.

Photo-caged agonists of the nuclear receptors RARgamma and TRbeta provide unique time-dependent gene expression profiles for light-activated gene patterning.

Light-activated gene expression systems hold promise as new tools for studying spatial and temporal gene patterning in multicellular systems. Photo-caged forms of nuclear receptor agonists have recently been shown to mediate photo-dependent transcription in mammalian cells, however, because intracellularly released agonists can rapidly diffuse out of cells, the photo-initiated transcription response is only transient and limited to only a few hours in reported examples. Herein we describe a photo-caged thyroid hormone receptor agonist that provides a robust 36 h transcription response to a single irradiation event.

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.

Rational design of vitamin D3 analogues which selectively restore activity to a vitamin D receptor mutant associated with rickets.

[formula: see text] Vitamin D3-resistant rickets (VDRR) is associated with mutations to the Vitamin D receptor (VDR) which effect ligand-dependent transactivation. Some VDRR associated mutants directly disrupt ligand binding. Using the reported VDR-1,25-dihydroxy vitamin D3 (1,25(OH)2D3) cocrystal structure, three 1,25(OH)2D3 analogues were designed to uniquely complement the rickets associated mutant VDR(Arg274-->Leu).

Structure-based design of selective agonists for a rickets-associated mutant of the vitamin d receptor.

The nuclear and steroid hormone receptors function as ligand-dependent transcriptional regulators of diverse sets of genes associated with development and homeostasis. Mutations to the vitamin D receptor (VDR), a member of the nuclear and steroid hormone receptor family, have been linked to human vitamin D-resistant rickets (hVDRR) and result in high serum 1,25(OH)(2)D(3) concentrations and severe bone underdevelopment. Several hVDRR-associated mutants have been localized to the ligand binding domain of VDR and cause a reduction in or loss of ligand binding and ligand-dependent transactivation function.

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.

Engineering selectivity and discrimination into ligand-receptor interfaces.

The reengineering of protein-ligand (or enzyme-substrate) interfaces using a combination of chemical and genetic methods has become an increasingly common technique to create new tools to manipulate and study biological systems. Many applications of ligand receptor engineering require that the engineered ligand and receptor function independently of endogenous ligands and receptors. Engineered ligands must selectively interact with modified receptors, and modified receptors must effectively discriminate against endogenous ligands.