Development of novel Vitamin D Receptor-Coactivator Inhibitors.

Nuclear receptor coregulators are master regulators of transcription and selectively interact with the vitamin D receptor (VDR) to modulate cell differentiation, cell proliferation and calcium homeostasis. Herein, we report the syntheses and evaluation of highly potent and selective VDR-coactivator inhibitors based on a recently identified 3-indolylmethanamine scaffold. The most active compound, PS121912, selectively inhibited VDR-mediated transcription among eight other nuclear receptors tested. PS121912 is also selectively disrupting the binding between VDR and the third nuclear receptor interaction domain of the coactivator SRC2. Genetic studies revealed that PS121912 behaves like a VDR antagonist by repressing 1,25-(OH)2D3 activated gene transcription. In addition, PS121912 induced apoptosis in HL-60.

A fluorescence-based high throughput assay for the determination of small molecule-human serum albumin protein binding.

Herein, we describe the development of a fluorescence-based high throughput assay to determine the small molecule binding towards human serum albumin (HSA). This innovative competition assay is based on the use of a novel fluorescent small molecule Red Mega 500 with unique spectroscopic and binding properties. The commercially available probe displays a large fluorescence intensity difference between the protein-bound and protein-unbound state. The competition of small molecules for HSA binding in the presence of probe resulted in low fluorescence intensities. The assay was evaluated with the library of pharmacological active compounds (LOPAC) small molecule library of 1,280 compounds identifying known high protein binders. The small molecule competition of HSA-Red Mega 500 binding was saturable at higher compound concentrations and exhibited IC50 values between 3 and 24 µM. The compound affinity toward HSA was confirmed by isothermal titration calorimetry indicating that the new protein binding assay is a valid high throughput assay to determine plasma protein binding.

Peroxisome proliferation-activated receptor δ agonist GW0742 interacts weakly with multiple nuclear receptors, including the vitamin D receptor.

A high-throughput screening campaign was conducted to identify small molecules with the ability to inhibit the interaction between the vitamin D receptor (VDR) and steroid receptor coactivator 2. These inhibitors represent novel molecular probes for modulating gene regulation mediated by VDR. Peroxisome proliferator-activated receptor (PPAR) δ agonist GW0742 was among the identified VDR-coactivator inhibitors and has been characterized herein as a pan nuclear receptor antagonist at concentrations of > 12.1 µM. The highest antagonist activity for GW0742 was found for VDR and the androgen receptor. Surprisingly, GW0742 behaved as a PPAR agonist and antagonist, activating transcription at lower concentrations and inhibiting this effect at higher concentrations. A unique spectroscopic property of GW0742 was identified as well. In the presence of rhodamine-derived molecules, GW0742 increased the fluorescence intensity and level of fluorescence polarization at an excitation wavelength of 595 nm and an emission wavelength of 615 nm in a dose-dependent manner. The GW0742-inhibited NR-coactivator binding resulted in a reduced level of expression of five different NR target genes in LNCaP cells in the presence of agonist. Especially VDR target genes CYP24A1, IGFBP-3, and TRPV6 were negatively regulated by GW0742. GW0742 is the first VDR ligand inhibitor lacking the secosteroid structure of VDR ligand antagonists. Nevertheless, the VDR-meditated downstream process of cell differentiation was antagonized by GW0742 in HL-60 cells that were pretreated with the endogenous VDR agonist 1,25-dihydroxyvitamin D3.

High-throughput identification of promiscuous inhibitors from screening libraries with the use of a thiol-containing fluorescent probe.

Testing small molecules for their ability to modify cysteine residues of proteins in the early stages of drug discovery is expected to accelerate our ability to develop more selective drugs with lesser side effects. In addition, this approach also enables the rapid evaluation of the mode of binding of new drug candidates with respect to thiol reactivity and metabolism by glutathione. Herein, we describe the development of a fluorescence-based high-throughput assay that allows the identification of thiol-reactive compounds. A thiol-containing fluorescent probe, MSTI, was synthesized and used to evaluate small molecules from the Library of Pharmacologically Active Compounds (LOPAC) collection of bioactive molecules. LOPAC compounds that are known to react with sulfur nucleophiles were identified with this assay, for example, irreversible protease inhibitors, nitric oxide-releasing compounds, and proton-pump inhibitors. The results confirm that both electrophilic and redox reactive compounds can be quickly identified in a high-throughput manner, enabling the assessment of screening libraries with respect to thiol-reactive compounds.

Discovery of the first irreversible small molecule inhibitors of the interaction between the vitamin D receptor and coactivators.

The vitamin D receptor (VDR) is a nuclear hormone receptor that regulates cell proliferation, cell differentiation, and calcium homeostasis. The receptor is activated by vitamin D analogues that induce the disruption of VDR-corepressor binding and promote VDR-coactivator interactions. The interactions between VDR and coregulators are essential for VDR-mediated transcription. Small molecule inhibition of VDR-coregulator binding represents an alternative method to the traditional ligand-based approach in order to modulate the expression of VDR target genes. A high throughput fluorescence polarization screen that quantifies the inhibition of binding between VDR and a fluorescently labeled steroid receptor coactivator 2 peptide was applied to discover the new small molecule VDR-coactivator inhibitors, 3-indolylmethanamines. Structure-activity relationship studies with 3-indolylmethanamine analogues were used to determine their mode of VDR-binding and to produce the first VDR-selective and irreversible VDR-coactivator inhibitors with the ability to regulate the transcription of the human VDR target gene TRPV6.