Margaret L. Guthrie

Anticancer activity of VDR-coregulator inhibitor PS121912.

AbstractPurposePS121912 has been developed as selective vitamin D receptor (VDR)-coregulator inhibitor starting from a high throughput screening campaign to identify new agents that modulate VDR without causing hypercalcemia. Initial antiproliferative effects of PS121912 were observed that are characterized herein to enable future in vivo investigation with this molecule.Methods Antiproliferation and apoptosis were determined using four different cancer cell lines (DU145, Caco2, HL-60 and SKOV3) in the presence of PS121912, 1,25-(OH)2D3, or a combination of 1,25-(OH)2D3 and PS121912. VDR si-RNA was used to identify the role of VDR during this process. The application of ChIP enabled us to determine the involvement of coregulator recruitment during transcription, which was investigated by RT-PCR with VDR target genes and those affiliated with cell cycle progression. Translational changes of apoptotic proteins were determined with an antibody array. The preclinical characterization of PS121912 includes the determination of metabolic stability and CYP3A4 inhibition.ResultsPS121912 induced apoptosis in all four cancer cells, with HL-60 cells being the most sensitive. At sub-micromolar concentrations, PS121912 amplified the growth inhibition of cancer cells caused by 1,25-(OH)2D3 without being antiproliferative by itself. A knockout study with VDR si-RNA confirmed the mediating role of VDR. VDR target genes induced by 1,25-(OH)2D3 were down-regulated with the co-treatment of PS121912. This process was highly dependent on the recruitment of coregulators that in case of CYP24A1 was SRC2. The combination of PS121912 and 1,25-(OH)2D3 reduced the presence of SRC2 and enriched the occupancy of corepressor NCoR at the promoter site. E2F transcription factors 1 and 4 were down-regulated in the presence of PS121912 and 1,25-(OH)2D3 that in turn reduced the transcription levels of cyclin A and D, thus arresting HL-60 cells in the S or G2/M phase. In addition, proteins with hematopoietic functions such as cyclin-dependent kinase 6, histone deacetylase 9 and transforming growth factor beta 2 and 3 were down-regulated as well. Elevated levels of P21 and GADD45, in concert with cyclin D1, also mediated the antiproliferative response of HL-60 in the presence of 1,25-(OH)2D3 and PS121912. Studies at higher concentration of P121912 identified a VDR-independent pathway of antiproliferation that included the enzymatic and transcriptional activation of caspase 3/7.ConclusionOverall, we conclude that PS121912 behaves like a VDR antagonist at low concentrations but interacts with more targets at higher concentrations leading to apoptosis mediated by caspase 3/7 activation. In addition, PS121912 showed an acceptable metabolic stability to enable in vivo cancer studies.

Identification of a novel, fast-acting GABAergic antidepressant

Current pharmacotherapies for depression exhibit slow onset, side effects and limited efficacy. Therefore, identification of novel fast-onset antidepressants is desirable. GLO1 is a ubiquitous cellular enzyme responsible for the detoxification of the glycolytic byproduct methylglyoxal (MG). We have previously shown that MG is a competitive partial agonist at GABA-A receptors. We examined the effects of genetic and pharmacological inhibition of GLO1 in two antidepressant assay models: the tail suspension test (TST) and the forced swim test (FST). We also examined the effects of GLO1 inhibition in three models of antidepressant onset: the chronic FST (cFST), chronic mild stress (CMS) paradigm and olfactory bulbectomy (OBX). Genetic knockdown of Glo1 or pharmacological inhibition using two structurally distinct GLO1 inhibitors (S-bromobenzylglutathione cyclopentyl diester (pBBG) or methyl-gerfelin (MeGFN)) reduced immobility in the TST and acute FST. Both GLO1 inhibitors also reduced immobility in the cFST after 5 days of treatment. In contrast, the serotonin reuptake inhibitor fluoxetine (FLX) reduced immobility after 14, but not 5 days of treatment. Furthermore, 5 days of treatment with either GLO1 inhibitor blocked the depression-like effects induced by CMS on the FST and coat state, and attenuated OBX-induced locomotor hyperactivity. Finally, 5 days of treatment with a GLO1 inhibitor (pBBG), but not FLX, induced molecular markers of the antidepressant response including brain-derived neurotrophic factor (BDNF) induction and increased phosphorylated cyclic-AMP response-binding protein (pCREB) to CREB ratio in the hippocampus and medial prefrontal cortex (mPFC). Our findings indicate that GLO1 inhibitors may provide a novel and fast-acting pharmacotherapy for depression.

Development of GABAA Receptor Subtype-Selective Imidazobenzodiazepines as Novel Asthma Treatments

Recent studies have demonstrated that subtype-selective GABAA receptor modulators are able to relax precontracted human airway smooth muscle ex vivo and reduce airway hyper-responsiveness in mice upon aerosol administration. Our goal in this study was to investigate systemic administration of subtype-selective GABAA receptor modulators to alleviate bronchoconstriction in a mouse model of asthma. Expression of GABAA receptor subunits was identified in mouse lungs, and the effects of α4-subunit-selective GABAAR modulators, XHE-III-74EE and its metabolite XHE-III-74A, were investigated in a murine model of asthma (ovalbumin sensitized and challenged BALB/c mice). We observed that chronic treatment with XHE-III-74EE significantly reduced airway hyper-responsiveness. In addition, acute treatment with XHE-III-74A but not XHE-III-74EE decreased airway eosinophilia. Immune suppressive activity was also shown in activated human T-cells with a reduction in IL-2 expression and intracellular calcium concentrations [Ca(2+)]i in the presence of GABA or XHE-III-74A, whereas XHE-III-74EE showed only partial reduction of [Ca(2+)]i and no inhibition of IL-2 secretion. However, both compounds significantly relaxed precontracted tracheal rings ex vivo. Overall, we conclude that the systemic delivery of a α4-subunit-selective GABAAR modulator shows good potential for a novel asthma therapy; however, the pharmacokinetic properties of this class of drug candidates have to be improved to enable better beneficial systemic pharmacodynamic effects.

Alleviation of Multiple Asthmatic Pathologic Features with Orally Available and Subtype Selective GABAA Receptor Modulators

We describe pharmacokinetic and pharmacodynamic properties of two novel oral drug candidates for asthma. Phenolic α4β3γ2 GABAAR selective compound 1 and acidic α5β3γ2 selective GABAAR positive allosteric modulator compound 2 relaxed airway smooth muscle ex vivo and attenuated airway hyperresponsiveness (AHR) in a murine model of asthma. Importantly, compound 2 relaxed acetylcholine contracted human tracheal airway smooth muscle strips. Oral treatment of compounds 1 and 2 decreased eosinophils in bronchoalveolar lavage fluid in ovalbumin sensitized and challenged mice, thus exhibiting anti-inflammatory properties. Additionally, compound 1 reduced the number of lung CD4+ T lymphocytes and directly modulated their transmembrane currents by acting on GABAARs. Excellent pharmacokinetic properties were observed, including long plasma half-life (up to 15 h), oral availability, and extremely low brain distribution. In conclusion, we report the selective targeting of GABAARs expressed outside the brain and demonstrate reduction of AHR and airway inflammation with two novel orally available GABAAR ligands.

A Novel Orally Available Asthma Drug Candidate That Reduces Smooth Muscle Constriction and Inflammation by Targeting GABAA Receptors in the Lung

We describe lead compound MIDD0301 for the oral treatment of asthma based on previously developed positive allosteric α5β3γ2 selective GABAA receptor (GABAAR) ligands. MIDD0301 relaxed airway smooth muscle at single micromolar concentrations as demonstrated with ex vivo guinea pig tracheal rings. MIDD0301 also attenuated airway hyperresponsiveness (AHR) in an ovalbumin murine model of asthma by oral administration. Reduced numbers of eosinophils and macrophages were observed in mouse bronchoalveolar lavage fluid without changing mucous metaplasia. Importantly, lung cytokine expression of IL-17A, IL-4, and TNF-α were reduced for MIDD0301-treated mice without changing antiinflammatory cytokine IL-10 levels. Automated patch clamp confirmed amplification of GABA induced current mediated by α1-3,5β3γ2 GABAARs in the presence of MIDD0301. Pharmacodynamically, transmembrane currents of ex vivo CD4+ T cells from asthmatic mice were potentiated by MIDD0301 in the presence of GABA. The number of CD4+ T cells observed in the lung of MIDD0301-treated mice were reduced by an oral treatment of 20 mg/kg b.i.d. for 5 days. A half-life of almost 14 h was demonstrated by pharmacokinetic studies (PK) with no adverse CNS effects when treated mice were subjected to sensorimotor studies using the rotarod. PK studies also confirmed very low brain distribution. In conclusion, MIDD0301 represents a safe and improved oral asthma drug candidate that relaxes airway smooth muscle and attenuates inflammation in the lung leading to a reduction of AHR at a dosage lower than earlier reported GABAAR ligands.