Current and emerging approaches to noncompetitive AR inhibition.

The androgen receptor (AR) has been shown to be a key determinant in the pathogenesis of castration-resistant prostate cancer (CRPC). The current standard of care therapies targets the ligand-binding domain of the receptor and can afford improvements to life expectancy often only in the order of months before resistance occurs. Emerging preclinical and clinical compounds that inhibit receptor activity via differentiated mechanisms of action which are orthogonal to current antiandrogens show promise for overcoming treatment resistance. In this review, we present an authoritative summary of molecules that noncompetitively target the AR. Emerging small molecule strategies for targeting alternative domains of the AR represent a promising area of research that shows significant potential for future therapies. The overall quality of lead candidates in the area of noncompetitive AR inhibition is discussed, and it identifies the key chemotypes and associated properties which are likely to be, or are currently, positioned to be first in human applications.

Recurrent deletions of ULK4 in schizophrenia: a gene crucial for neuritogenesis and neuronal motility.

Although many pathogenic copy number variations (CNVs) are associated with neuropsychiatric diseases, few of them have been functionally characterised. Here we report multiple schizophrenia cases with CNV abnormalities specific to unc-51-like kinase 4 (ULK4), a serine/threonine kinase gene. Deletions spanning exons 21-34 of ULK4 were present in 4 out of 3391 schizophrenia patients from the International Schizophrenia Consortium, but absent in 3181 controls. Deletions removing exons 33 and 34 of the large splice variant of ULK4 also were enriched in Icelandic schizophrenia and bipolar patients compared with 98,022 controls (P = 0.0007 for schizophrenia plus bipolar disorder). Combining the two cohorts gives a P-value less than 0.0001 for schizophrenia, or for schizophrenia plus bipolar disorder. The expression of ULK4 is neuron-specific and developmentally regulated. ULK4 modulates multiple signalling pathways that include ERK, p38, PKC and JNK, which are involved in stress responses and implicated in schizophrenia. Knockdown of ULK4 disrupts the composition of microtubules and compromises neuritogenesis and cell motility. Targeted Ulk4 deletion causes corpus callosum agenesis in mice. Our findings indicate that ULK4 is a rare susceptibility gene for schizophrenia.

Synthesis and Evaluation of Small Molecule Inhibitors of the Androgen Receptor N-Terminal Domain.

The androgen receptor (AR) is central to prostate cancer pathogenesis and has been extensively validated as a drug target. However, small-molecule anti-androgen therapies remain limited due to resistance and will eventually fail to suppress tumor growth, resulting in progression to castration-resistant prostate cancer (CRPC). The intrinsically disordered N-terminal domain (NTD) is crucial for AR transactivation and has been investigated as a suitable target in the presence of ligand binding domain mutations. A screening campaign identified biaryl isoxazole compound 7 as a weak inhibitor of the AR NTD. A library of biaryl analogues were synthesized, and their biological activities were assessed in a VCaP cell-based luciferase reporter gene assay. A structure-activity relationship (SAR) study revealed that indazole analogue 16 exhibited increased potency and favorable physicochemical properties with a benchmarked pharmacokinetic profile, providing a suitable starting point for further optimization of 16 as a CRPC therapeutic in the presence of AR mutations.

Development of a High-Throughput Screening Assay for Small-Molecule Inhibitors of Androgen Receptor Splice Variants.

The role of the androgen receptor (AR) in the progression of prostate cancer (PCa) is well established and competitive inhibition of AR ligand binding domain (LBD) has been the mainstay of antiandrogen therapies for advanced and metastatic disease. However, the efficacy of such drugs is often limited by the emergence of resistance, mediated through point mutations and receptor splice variants lacking the AR-LBD. As a result, the prognosis for patients with malignant, castrate-resistant disease remains poor. The amino terminal domain (NTD) of the AR has been shown to be critical for AR function. Its modular activation function (AF-1) is important for both gene regulation and participation in protein-protein interactions. However, due to the intrinsically disordered structure of the domain, its potential as a candidate for therapeutic intervention has been generally overlooked. In this article, we describe the design and development of a functional cell-based assay aimed at identifying small-molecule inhibitors of the AR-NTD. We demonstrate the suitability of the assay for high-throughput screening platforms and validate two initial hits emerging from a small, targeted, library screen in PCa cells.

Amiloride-sensitive channels are a major contributor to mechanotransduction in mammalian muscle spindles.

We investigated whether channels of the epithelial sodium/amiloride-sensitive degenerin (ENaC/DEG) family are a major contributor to mechanosensory transduction in primary mechanosensory afferents, using adult rat muscle spindles as a model system. Stretch-evoked afferent discharge was reduced in a dose-dependent manner by amiloride and three analogues - benzamil, 5-(N-ethyl-N-isopropyl) amiloride (EIPA) and hexamethyleneamiloride (HMA), reaching > or = 85% inhibition at 1 mm. Moreover, firing was slightly but significantly increased by ENaC delta subunit agonists (icilin and capsazepine). HMA's profile of effects was distinct from that of the other drugs. Amiloride, benzamil and EIPA significantly decreased firing (P < 0.01 each) at 1 microm, while 10 microm HMA was required for highly significant inhibition (P < 0.0001). Conversely, amiloride, benzamil and EIPA rarely blocked firing entirely at 1 mm, whereas 1 mm HMA blocked 12 of 16 preparations. This pharmacology suggests low-affinity ENaCs are the important spindle mechanotransducer. In agreement with this, immunoreactivity to ENaC alpha, beta and gamma subunits was detected both by Western blot and immunocytochemistry. Immunofluorescence intensity ratios for ENaC alpha, beta or gamma relative to the vesicle marker synaptophysin in the same spindle all significantly exceeded controls (P < 0.001). Ratios for the related brain sodium channel ASIC2 (BNaC1alpha) were also highly significantly greater (P < 0.005). Analysis of confocal images showed strong colocalisation within the terminal of ENaC/ASIC2 subunits and synaptophysin. This study implicates ENaC and ASIC2 in mammalian mechanotransduction. Moreover, within the terminals they colocalise with synaptophysin, a marker for the synaptic-like vesicles which regulate afferent excitability in these mechanosensitive endings.

The multi-functional role of sphingosylphosphorylcholine.

The sphingomyelin metabolite, sphingosylphosphorylcholine (SPC) has been the subject of much recent interest and controversy. Studies have indicated that SPC naturally occurs in plasma and a constituent of lipoproteins. Synthesis is also increased in some pathological conditions. Research has demonstrated that SPC is a potentially important lipid mediator of cell type specific functions in major tissues, such as heart, blood vessels, skin, brain and immune system. These effects are regulated via a number of different intracellular signalling cascades, also dependent upon cell type. Initial reports identifying high affinity SPC receptors at first appeared to reinforce the physiological relevance of this sphingolipid. However, these studies have now been retracted. Some SPC effects have been shown be occur via plasma membrane receptors for the related sphingolipid, sphingosine 1-phosphate (S1P). Despite a lack of well-defined receptor signal transduction mechanisms and sparse pharmacological data, several key characteristics of SPC are now emerging. SPC can act as a mitogen in several different cell types and in certain circumstances, may also be a pro-inflammatory mediator. In this review, these actions of SPC are discussed with a view to understanding the potential physiological relevance of this sphingolipid.

Tissue control of androgen action: The ups and downs of androgen receptor expression.

The hormone testosterone plays crucial roles during male development and puberty and throughout life, as an anabolic regulator of muscle and bone structure and function. The actions of testosterone are mediated, primarily, through the androgen receptor, a member of the nuclear receptor superfamily. The androgen receptor gene is located on the X-chromosome and receptor levels are tightly controlled both at the level of transcription of the gene and post-translationally at the protein level. Sp1 has emerged as the major driver of expression of the androgen receptor gene, while auto-regulation by androgens is associated with both positive and negative regulation in a possible cell-selective manner. Research into the networks of positive and negative regulators of the androgen receptor gene are vital in order to understand the temporal and spatial control of receptor levels and the consequences for healthy aging and disease. A clear understanding of the multiple transcription factors participating in regulation of the androgen receptor gene will likely aid in the development and application of hormone therapies to boast or curb receptor activity.

Regulation of Androgen Receptor Activity by Transient Interactions of Its Transactivation Domain with General Transcription Regulators.

The androgen receptor is a transcription factor that plays a key role in the development of prostate cancer, and its interactions with general transcription regulators are therefore of potential therapeutic interest. The mechanistic basis of these interactions is poorly understood due to the intrinsically disordered nature of the transactivation domain of the androgen receptor and the generally transient nature of the protein-protein interactions that trigger transcription. Here, we identify a motif of the transactivation domain that contributes to transcriptional activity by recruiting the C-terminal domain of subunit 1 of the general transcription regulator TFIIF. These findings provide molecular insights into the regulation of androgen receptor function and suggest strategies for treating castration-resistant prostate cancer.

Control of cortex development by ULK4, a rare risk gene for mental disorders including schizophrenia.

Schizophrenia is a debilitating familial neuropsychiatric disorder which affects 1% of people worldwide. Although the heritability for schizophrenia approaches 80% only a small proportion of the overall genetic risk has been accounted for, and to date only a limited number of genetic loci have been definitively implicated. We have identified recently through genetic and in vitro functional studies, a novel serine/threonine kinase gene, unc-51-like kinase 4 (ULK4), as a rare risk factor for major mental disorders including schizophrenia. Now using the approach of in utero gene transfer we have discovered that Ulk4 plays a key modulatory role in corticogenesis. Knockdown of Ulk4 leads to significantly decreased cell proliferation in germinal zones and profound deficits in radial migration and neurite ramification. These abnormalities can be reversed successfully by Ulk4 gene supplementation. Ulk4 also regulated acetylation of α-tubulin, an important post-translational modification of microtubules. We conclude that Ulk4 plays an essential role in normal brain development and when defective, the risk of neurodevelopmental disorders such as schizophrenia is increased.

An Sp1 Modulated Regulatory Region Unique to Higher Primates Regulates Human Androgen Receptor Promoter Activity in Prostate Cancer Cells.

Androgen receptor (AR) mediated signalling is necessary for normal development of the prostate gland and also drives prostate cancer (PCa) cell growth and survival, with many studies showing a correlation between increased receptor levels and therapy resistance with progression to fatal castrate recurrent PCa (CRPC). Although it has been held for some time that the transcription factor Sp1 is the main stimulator of AR gene transcription, comprehensive knowledge of the regulation of the AR gene remains incomplete. Here we describe and characterise in detail two novel active regulatory elements in the 5'UTR of the human AR gene. Both of these elements contain overlapping binding sites for the positive transcription factor Sp1 and the repressor protein pur-α. Aberrant cell signalling is characteristic of PCa and the transcriptional activity of the AR promoter in PCa cells is dependent upon the relative amounts of the two transcription factors. Together with our corroboration of the dominant role of Sp1, the findings support the rationale of targeting this transcription factor to inhibit tumour progression. This should be of particular therapeutic relevance in CRPC where the levels of the repressor pur-α are reduced.

Negative regulation of the androgen receptor gene through a primate-specific androgen response element present in the 5' UTR.

The androgen receptor (AR) is a widely expressed ligand-activated transcription factor which mediates androgen signalling by binding to androgen response elements (AREs) in normal tissue and prostate cancer (PCa). Within tumours, the amount of AR plays a crucial role in determining cell growth, resistance to therapy and progression to fatal castrate recurrent PCa in which prostate cells appear to become independent of androgenic steroids. Despite the pivotal role of the AR in male development and fertility and all stages of PCa development, the mechanisms governing AR expression remain poorly understood. In this work, we describe an active nonconsensus androgen response element (ARE) in the 5' UTR of the human AR gene. The ARE represses transcription upon binding of activated AR, and this downregulation is relieved by disruption of the regulatory element through mutation. Also, multiple species comparison of the genomic region reveals that this ARE is specific to primates, leading to the conclusion that care must be exercised when elucidating the operation of the human AR in PCa based upon rodent promoter studies.

Sphingosylphosphorylcholine is a proinflammatory mediator in cerebral arteries.

Inflammation has an important function in the development of cerebral vasospasm after subarachnoid hemorrhage (SAH); however, the mediators of this inflammatory response have not been clearly identified. In this study, we have investigated the potential function of two sphingolipids, which occur naturally in plasma and serum, sphingosylphosphorylcholine (SPC) and sphingosine 1-phosphate (S1P), to act as proinflammatory mediators in cerebral artery vascular smooth muscle (VSM) cells. In rat cerebral arteries, SPC but not S1P activated p38 mitogen-activated protein kinase (MAPK). Using transcription factor arrays, two proinflammatory transcription factors activated by SPC in cerebral arteries were identified--nuclear factor-κB and CCAAT-enhancer-binding protein. Both these transcription factors were activated by SPC in a p38MAPK-dependent manner. To determine whether this contributed to vascular inflammation, an inflammatory protein array was performed, which showed that SPC increased release of the chemokine monocyte chemoattractant protein-1 (MCP-1) in cultured rat VSM cells. This increase in MCP-1 expression was confirmed in cerebral arteries. The S1P did not increase MCP-1 release. Taken together, our results suggest that SPC, but not S1P, can act as a proinflammatory mediator in cerebral arteries. This may contribute to inflammation observed after SAH and may be part of the initiating event in vasospasm.

A phospholipase Cγ1-activated pathway regulates transcription in human vascular smooth muscle cells.

AIMS: Growth factor-induced repression of smooth muscle (SM) cell marker genes is an integral part of vascular SM (VSM) cell proliferation. This is partly regulated via translocation of extracellular signal-regulated kinase 1/2 (ERK1/2) to the nucleus which activates the transcription factor Elk-1. The mediators involved in ERK1/2 nuclear translocation in VSM cells are unknown. The aim of this study is to examine the mechanisms which regulate growth factor-induced nuclear translocation of ERK1/2 and gene expression in VSM cells. METHODS AND RESULTS: In cultured human VSM cells, phospholipase C (PLC)γ1 expression was required for platelet-derived growth factor (PDGF)-induced ERK1/2 nuclear translocation, Elk-1 phosphorylation, and subsequent repression of SM α-actin gene expression. The mechanisms of a role for PLCγ1 in ERK1/2 nuclear localization were further examined by investigating interacting proteins. The ERK1/2-binding phosphoprotein, protein enriched in astrocytes-15 (PEA-15), was phosphorylated by PDGF and this phosphorylation required activation of PLCγ1. In cells pre-treated with PEA-15 siRNA, ERK1/2 distribution significantly increased in the nucleus and resulted in decreased SM α-actin expression and increased VSM cell proliferation. Overexpression of PEA-15 increased ERK1/2 localization in the cytoplasm. The regulatory role of PEA-15 phosphorylation was assessed. In VSM cells overexpressing a non-phosphorylatable form of PEA-15, PDGF-induced ERK1/2 nuclear localization was inhibited. CONCLUSION: These results suggest that PEA-15 phosphorylation by PLCγ1 is required for PDGF-induced ERK1/2 nuclear translocation. This represents an important level of phenotypic control by directly affecting Elk-1-dependent transcription and ultimately SM cell marker protein expression in VSM cells.

Spatial compartmentalization of tumor necrosis factor (TNF) receptor 1-dependent signaling pathways in human airway smooth muscle cells. Lipid rafts are essential for TNF-alpha-mediated activation of RhoA but dispensable for the activation of the NF-kappaB and MAPK pathways.

Tumor necrosis factor (TNF)-alpha-induced activation of RhoA, mediated by TNF receptor 1 (TNFR1), is a prerequisite step in a pathway that leads to increased 20-kDa light chain of myosin (MLC20) phosphorylation and airway smooth muscle contraction. In this study, we have investigated the proximal events in TNF-alpha-induced RhoA activation. TNFR1 is localized to both lipid raft and nonraft regions of the plasma membrane in primary human airway smooth muscle cells. TNF-alpha engagement of TNFR1 recruited the adaptor proteins TRADD, TRAF-2, and RIP into lipid rafts and activated RhoA, NF-kappaB, and MAPK pathways. Depletion of cholesterol from rafts with methyl-beta-cyclodextrin caused a redistribution of TNFR1 to nonraft plasma membrane and prevented ligand-induced RhoA activation. By contrast, TNF-alpha-induced activation of NF-kappaB and MAPKs was unaffected by methyl-beta-cyclodextrin indicating that, in airway smooth muscle cells, activation of these pathways occurred independently of lipid rafts. Targeted knockdown of caveolin-1 completely abrogated TNF-alpha-induced RhoA activation, identifying this raft-resident protein as a positive regulator of the activation process. The signaling adaptors TRADD and RIP were also found to be necessary for ligand-induced RhoA activation. Taken together, our results suggest that in airway smooth muscle cells, spatial compartmentalization of TNFR1 provides a mechanism for generating distinct signaling outcomes in response to ligand engagement and define a mechanistic role for lipid rafts and caveolin-1 in TNF-alpha-induced activation of RhoA.

Tumor necrosis factor-alpha-induced activation of RhoA in airway smooth muscle cells: role in the Ca2+ sensitization of myosin light chain20 phosphorylation.

Tumor necrosis factor-alpha (TNF), an inflammatory cytokine, has a potentially important role in the pathogenesis of bronchial asthma and may contribute to airway hyper-responsiveness. Recent evidence has revealed that TNF can increase the Ca(2+) sensitivity of agonist-stimulated myosin light chain(20) (MLC(20)) phosphorylation and contractility in guinea pig airway smooth muscle (ASM). In the present study, the potential intracellular pathways responsible for this TNF-induced Ca(2+) sensitization were investigated. In permeabilized cultured guinea pig ASM cells, recombinant human TNF stimulated an increase in Ca(2+)-activated MLC(20) phosphorylation under Ca(2+) "clamp" conditions. This increased MLC(20) phosphorylation was inhibited by preincubation with the Rho-kinase inhibitor Y27632. TNF also increased the proportion of GTP-bound RhoA, as measured using rhotekin Rho-binding domain, in a time course compatible with a role in the TNF-induced Ca(2+) sensitization. In cultured human ASM cells, recombinant human TNF also activated RhoA with a similar time course. In addition, TNF stimulated phosphorylation of the regulatory subunit of the myosin phosphatase, which was inhibited by Y27632. Although human ASM cells expressed both receptor subtypes, TNF-R1 and TNF-R2, the activation of RhoA was predominantly via stimulation of the TNF-R1, although RhoA did not immunoprecipitate with the TNF-R1. In conclusion, the TNF-induced increase in the Ca(2+) sensitivity of MLC(20) phosphorylation is through stimulation of the TNF-R1 receptor and via a RhoA/Rho-kinase pathway leading to inhibition of the myosin light chain phosphatase. This intracellular mechanism may contribute to TNF-induced airway hyper-responsiveness.