John T. Koh

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 ERα‐ and ERβ‐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 ERβ‐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.

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. A variety of chemical design strategies have been used to reengineer ligand-receptor interfaces. The advantages and limitations of various strategies, which involve the manipulation of hydrophobic, polar, and charged residues, are compared. New design strategies and potential applications of ligand-receptor engineering are also discussed.

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. This revealed unique motility dynamics of drug-treated and adhesion molecule-transfected cells and, thus, this is a considerable improvement over current methods of measurement and analysis. Several fluorescent vital labeling methods commonly used for end-point analyses (GFP expression, DiO lipophilic dye, and Qtracker nanocrystals) were found to be useful for time-lapse studies under specific conditions that are described. To illustrate one application, fluorescently labeled tumor cells were seeded onto cell monolayers expressing ectopic adhesion molecules, and this resulted in consistently reduced tumor cell migration velocities. These highly quantitative time-lapse analysis methods will promote the creation of new cell motility assays and increase the resolution and accuracy of existing assays.

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. Methods to date have been limited by the small fraction of irradiated cells that have expression levels significantly above uninduced background and have not been shown to affect a defined biological response. The tetracycline-dependent transactivator/transrepressor system, RetroTET-ART, combined with a photocaged form of doxycycline (NvOC-Dox) can be used to form photolithographic patterns of induced expression wherein up to 85% of the patterned cells show expression levels above uninduced regions. The efficiency and inducibility of the RetroTET-ART system allows one to quantitatively measure the limits of resolution and the relative induction levels mediated by a small molecule photocaged effector for the first time. Well-defined patterns of reporter genes were reproducibly formed within 6-36 h with feature sizes as small as 300 microm. After photo-patterning, NvOC-Dox can be rapidly removed, rendering cells photoinsensitive and allowing one to monitor GFP product formation in real time. Patterned co-expression of the cell surface ligand ephrin A5 on cell monolayers creates well-defined patterns that are sufficient to direct and segregate co-cultured cells via either attractive or repulsive signaling cues. The ability to direct the arrangement of cells on living cell monolayers through the action of light may serve as a model system for engineering artificial tissues.

Highly efficient synthesis of 1-thioglycosides in solution and solid phase using iminophosphorane bases

Disaccharides of 1-thioglycosides, an important class of glycomimics, can be synthesized by direct S-alkylation in exceptionally high yields when iminophosphorane bases are employed. The reaction conditions employed appear to be general and stereospecific. Axial and equatorial 4-triflates and primary tosylates of alkyl pyranosides provided excellent yields of thio-disaccharides without substantial elimination products. The iminophosphorane bases also proved to be useful in solid support-bound couplings of thioglycosides though with lower efficiency.

Selective Chemical Rescue of a Thyroid‐Hormone‐Receptor Mutant, TRβ(H435Y), Identified in Pituitary Carcinoma and Resistance to Thyroid Hormone

The thyroid hormone receptors (TRs) are ligand-dependent transcriptional regulators that control critical genes in development and homeostasis in response to triiodothyronine (T3). As an important regulator of differentiation, TRb has been shown to be mutated in a high percentage of certain cancer types, including kidney, pituitary, liver, and thyroid cancer. These spontaneous TRb mutations cause the reduction or loss of TR function in a similar way to germline TRb mutants associated with inheritable genetic disease resistance to thyroid hormone (RTH). Paradoxically, RTH patients do not appear to be predisposed to these forms of cancer, although, in a few cases, identical TRb mutants have been identified in cancer and RTH. As part of our studies exploring applications of chemical rescue by small-molecule complementation, we previously examined how mutations to the TRb “His-Phe switch” motif, which mediates ligand-dependent transactivation response, can dramatically impair receptor function. Herein, we describe a new strategy to rescue a naturally occurring TRb mutant, His435!Tyr, by reorienting hydrogen-bonding interactions at the ligand–receptor interface. As TRb(H435Y) has been found in both RTH and pituitary carcinoma, our results serve perhaps as the first example of chemical rescue that targets a mutant protein involved in multiple disease states. Upon ligand binding, TR undergoes a conformational change that involves the repositioning of helix 12 to form a coactivator-binding interface (Figure 1a). For most nuclear receptors, the hormone does not make direct contact with helix 12, but rather interacts with residues on helix 11. These residues make contacts with helix 12 through a His-Trp or a His-Phe switch, which transduces ligand binding into a transcription response. Mutations to the His-Phe switch of TRb have been associated with dramatic (320–> 5000fold) reductions in ligand potency. 9] The high-resolution crystal structures of T3-bound TRb and TRa suggest that His435 forms a hydrogen bond with the 4’-OH group of T3 and participates simultaneously in aryl– aryl interactions with the Phe459 residue of helix 12 (Figure 2, left). We demonstrated previously that 4’-alkoxy derivatives of the thyroid-hormone-receptor agonist GC-1 have greater potency and efficacy with TRb(H435A) than the natural hormone T3; however, these analogues are ineffective in rescuing the activity of the His435 mutants TRb(H435Y) and TRb(H435L), which are known to be associated with RTH and cancer. Whereas TRb(His435L) is inactive at all T3 concentrations tested ( 5 mm), T3 is a full agonist (100% efficacy) for TRb(H435Y), although it is 390 times less potent with this mutant than with wild-type (wt) TRb. These results suggest that TRb(H435Y) retains its intrinsic ability to mediate ligand-dependent transcription response but requires extreme supraphysiological concentrations of T3 that would not be tolerated in vivo owing to the overstimulation of wildtype TRs. As in other studies in which the thyroid hormone receptor was targeted, the delicate balance of TR activity within the hypothalamic-pituitary-thyroid axis emphasizes the need for a ligand with subtype selectivity. Molecular modeling of TRb(H435Y) suggested that the Tyr435 side chain is still able to engage Phe459 through aryl– aryl interactions (Figure 2, right). Although the phenol hydroxy group of tyrosine is capable of forming a hydrogen bond, it is not appropriately positioned to interact with receptor-bound T3. We reasoned that appropriately designed hormone analogues may be able to rescue potency to TRb(H435Y) selectively by restoring hydrogen-bonding/ aryl–aryl interactions of the His-Phe switch through the creation of a novel Tyr-Phe switch. This strategy presented a unique challenge, as the side chain of tyrosine is considerably longer than that of histidine; therefore, it was necessary to introduce a hydrogen-bonding group while making the overall ligand structure smaller. As an initial approach, we reasoned that the outer phenyl ring of T3 could be replaced by a pyridyl ring (Figure 1b, right). For ease of synthesis and product stability, we chose to make analogues of the halogenfree thyromimetic GC-1 rather than analogues of T3 itself. We could then vary the alkyl substituent at the 3’-position with the aim of optimizing the hydrogen-bond geometry and hydrophobic contacts of the 3’ substituent (Scheme 1). As a control, we also synthesized a “phenyl” analogue of GC-1, QH9, in which the phenol hydroxy group has been replaced by a hydrogen atom. Pyridyl analogues were derived from the corresponding 2-substituted 4-cyanopyridines by the nucleophilic addition to 4-cyanopyridine of alkyl radicals generated by silver-promoted radical decarboxylation of the corresponding carboxylic acids. This method provided efficient access to the 2-alkyl pyridine series of ligands (see Scheme S1 in the Supporting Information). [*] A. Q. Hassan, J. T. Koh Department of Chemistry and Biochemistry, University of Delaware Newark, DE 19716 (USA) E-mail: johnkoh@udel.edu

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. Whereas the T3 or GC-1 based conjugates did not bind TRbeta with high affinity, the NH-2 inspired fluorescein-conjugate JZ01 showed low nanomolar affinity for TRbeta and could be used as a nonradiometric probe for ligand binding. A related analogue JZ07 was a potent TR antagonist that is 13-fold selective for TRbeta over TRalpha. JZ01 localizes in the nuclei of TRbeta expressing cells and may serve as a prototype for other TR-targeting conjugates.

Difluoromethyl analogs of the natural hormone 1α,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.

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. Here, we describe the synthesis and in vitro assessment of a photocaged VDR agonist specific to a mutant NHR that is associated with vitamin D‐resistant rickets. The results provide insight into the utility of the agonist as a potential tool for photoinduced gene patterning.

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. The analog, DRL527, represses basal AP-1 gene expression and antagonizes Raloxifene stimulated AP-1 expression. Therefore DRL527 has a unique, previously unreported, ERE/AP-1 activity profile.