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Biomedical subjects

John T Koh

Publications and source records attributed to John T Koh.

7 recordsLinked to original sources
PubMed2004

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. These findings are in contrast to a closely related system, which uses a caged retinoic acid receptor agonist, which provides only a short transcription response. Comparison of the two systems, show that the duration of transcription response is not controlled by the rate of diffusion of free ligand out of the cell, but perhaps by the duration of ligand-induced transcription/stability of the active transcription complex.

Gene Expression Profiling↗
PubMed2004

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.

Gene Expression Regulation↗
PubMed2002

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. The missense mutation Arg274 --> Leu causes a >1000-fold reduction in 1,25(OH)(2)D(3) responsiveness and is, therefore, no longer regulated by physiological concentrations of the hormone. In this study, computer-aided molecular design was used to generate a focused library of nonsteroidal analogues of the VDR agonist LG190155 that were uniquely designed to complement the Arg274 --> Leu associated with hVDRR. Half of the designed analogues exhibit substantial activity in the hVDRR-associated mutant, whereas none of the structurally similar control compounds exhibited significant activity. The seven most active designed analogues were more than 16 to 526 times more potent than 1,25(OH)(2)D(3) in the mutant receptor (EC(50) = 3.3-121 nM). Significantly, the analogues are selective for the nuclear VDR and did not stimulate cellular calcium influx, which is associated with activation of the membrane-associated vitamin D receptor.

Biphenyl Compounds↗
PubMed2002

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). The three analogues were 17 to 286 times more potent than 1,25(OH)2D3 with the mutant in cell-based assays and did not substantially activate cellular calcium influx.

Calcium↗
PubMed2002

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.

Cells, Cultured↗
PubMed2002

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.

Binding Sites↗