Identifying robust and reliable volatile organic compounds in human sebum for biomarker discovery.

Sebum from sebaceous glands is a rich source of volatile organic compounds (VOCs) that can readily be sampled non-invasively from the surface of skin. The VOC profiles of sebum can then be used to obtain information regarding different medical conditions including diabetes and Parkinson's Disease. However, the effects of sampling approaches and environmental factors on sebum VOC profiles are not established and the confident attribution of VOCs to disease states needs to be free of extraneous influences such as sampling materials and preparatory conditions. Here, we investigated a more standardised skin swab sampling approach for profiling sebum VOCs from healthy human subjects using thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS). Using a standard GC-MS method for the chemical analysis of sebum swabs, a surprisingly high number of VOCs originate from 'blank' medical swab material alone (up to 74 VOCs) and from the ambient environment (up to 29 VOCs) based on control experiments. We found that heat-treatment of medical swabs prior to GC-MS reduced the number of VOCs detected from 'blank' swabs and improved the reproducibility of VOC profiling, however significant VOC absorption can still occur from environmental exposure to ambient air. VOCs identified in 'blank' swabs consisted predominantly of hydrocarbons, esters, and silicon-based compounds and depended strongly on the material used (cotton and polyester-rayon). Environmental VOCs found to absorb to swabs from the ambient air during sampling included 1-butylheptyl-benzene and hexadecanoic acid methyl ester as well as exogenous VOCs such as isopropyl palmitate and isopropyl myristate. In contrast, sebum VOCs consisted primarily of esters, alcohols, ketones, and aldehydes. 23 and 18 VOCs were identified in sebum collected using polyester-rayon and cotton-based medical swabs, respectively, with 14 VOCs common to both swabs. The effect of subject bathing prior to sebum sampling had minimal impact on the VOC profiles. However, individual differences owing to external factors such as skin type, diet, and exercise will likely influence sebum production. This study highlights the importance of using rigorous controls in sebum sampling, and recommendations are provided for future research involving sebum VOC analysis. For example, the use of sebum sample replicates across multiple days, and the use of control swabs during sample collection is required to confirm the origin and reliability of sebum VOCs. It is anticipated that these recommendations in conjunction with a library of well-established VOCs from medical swabs will further strengthen biomarker identification resulting from sebum VOC analysis.

Design and optimisation of dendrimer-conjugated Bcl-2/xL inhibitor, AZD0466, with improved therapeutic index for cancer therapy.

Dual Bcl-2/Bcl-xL inhibitors are expected to deliver therapeutic benefit in many haematological and solid malignancies, however, their use is limited by tolerability issues. AZD4320, a potent dual Bcl-2/Bcl-xL inhibitor, has shown good efficacy however had dose limiting cardiovascular toxicity in preclinical species, coupled with challenging physicochemical properties, which prevented its clinical development. Here, we describe the design and development of AZD0466, a drug-dendrimer conjugate, where AZD4320 is chemically conjugated to a PEGylated poly-lysine dendrimer. Mathematical modelling was employed to determine the optimal release rate of the drug from the dendrimer for maximal therapeutic index in terms of preclinical anti-tumour efficacy and cardiovascular tolerability. The optimised candidate is shown to be efficacious and better tolerated in preclinical models compared with AZD4320 alone. The AZD4320-dendrimer conjugate (AZD0466) identified, through mathematical modelling, has resulted in an improved therapeutic index and thus enabled progression of this promising dual Bcl-2/Bcl-xL inhibitor into clinical development.

Evaluating liver uptake and distribution of different poly(2-methyl-2-oxazoline) modified lysine dendrimers following intravenous administration.

We report on the synthesis of four poly(2-methyl-2-oxazoline) modified lysine dendrimers with different residual groups or modifications on the dendrimer core, including: amino groups (positive charge), carboxyl groups (negative charge), and two drug molecules, one of which has a high log P. We looked at the in vivo distribution amongst three main liver cell types: hepatocytes, liver sinusoidal endothelial cells (LSECs) and Kupffer cells (KCs) and found differences in cell distribution and uptake concentrations dependent on these residual groups. In particular, the amino-functional polymer showed greater uptake by the hepatocytes whilst the carboxyl-functionalised polymer exhibited greater uptake by KCs and LSECs. These findings provide insight into which professional scavenger cells of the liver remove these types of nanoparticles from the bloodstream and we describe some of the design criteria to consider when creating novel drug delivery systems.

Parkinson's disease-linked human PARK9/ATP13A2 maintains zinc homeostasis and promotes α-Synuclein externalization via exosomes.

α-Synuclein plays a central causative role in Parkinson's disease (PD). Increased expression of the P-type ATPase ion pump PARK9/ATP13A2 suppresses α-Synuclein toxicity in primary neurons. Our data indicate that ATP13A2 encodes a zinc pump; neurospheres from a compound heterozygous ATP13A2(-/-) patient and ATP13A2 knockdown cells are sensitive to zinc, whereas ATP13A2 over-expression in primary neurons confers zinc resistance. Reduced ATP13A2 expression significantly decreased vesicular zinc levels, indicating ATP13A2 facilitates transport of zinc into membrane-bound compartments or vesicles. Endogenous ATP13A2 localized to multi-vesicular bodies (MVBs), a late endosomal compartment located at the convergence point of the endosomal and autophagic pathways. Dysfunction in MVBs can cause a range of detrimental effects including lysosomal dysfunction and impaired delivery of endocytosed proteins/autophagy cargo to the lysosome, both of which have been observed in cells with reduced ATP13A2 function. MVBs also serve as the source of intra-luminal nanovesicles released extracellularly as exosomes that can contain a range of cargoes including α-Synuclein. Elevated ATP13A2 expression reduced intracellular α-Synuclein levels and increased α-Synuclein externalization in exosomes >3-fold whereas ATP13A2 knockdown decreased α-Synuclein externalization. An increased export of exosome-associated α-Synuclein may explain why surviving neurons of the substantia nigra pars compacta in sporadic PD patients were observed to over-express ATP13A2. We propose ATP13A2's modulation of zinc levels in MVBs can regulate the biogenesis of exosomes capable of containing α-Synuclein. Our data indicate that ATP13A2 is the first PD-associated gene involved in exosome biogenesis and indicates a potential neuroprotective role of exosomes in PD.

Discovery of AZD3514, a small-molecule androgen receptor downregulator for treatment of advanced prostate cancer.

Removal of the basic piperazine nitrogen atom, introduction of a solubilising end group and partial reduction of the triazolopyridazine moiety in the previously-described lead androgen receptor downregulator 6-[4-(4-cyanobenzyl)piperazin-1-yl]-3-(trifluoromethyl)[1,2,4]triazolo[4,3-b]pyridazine (1) addressed hERG and physical property issues, and led to clinical candidate 6-(4-{4-[2-(4-acetylpiperazin-1-yl)ethoxy]phenyl}piperidin-1-yl)-3-(trifluoromethyl)-7,8-dihydro[1,2,4]triazolo[4,3-b]pyridazine (12), designated AZD3514, that is being evaluated in a Phase I clinical trial in patients with castrate-resistant prostate cancer.

Compounds from an unbiased chemical screen reverse both ER-to-Golgi trafficking defects and mitochondrial dysfunction in Parkinson's disease models.

alpha-Synuclein (alpha-syn) is a small lipid-binding protein involved in vesicle trafficking whose function is poorly characterized. It is of great interest to human biology and medicine because alpha-syn dysfunction is associated with several neurodegenerative disorders, including Parkinson's disease (PD). We previously created a yeast model of alpha-syn pathobiology, which established vesicle trafficking as a process that is particularly sensitive to alpha-syn expression. We also uncovered a core group of proteins with diverse activities related to alpha-syn toxicity that is conserved from yeast to mammalian neurons. Here, we report that a yeast strain expressing a somewhat higher level of alpha-syn also exhibits strong defects in mitochondrial function. Unlike our previous strain, genetic suppression of endoplasmic reticulum (ER)-to-Golgi trafficking alone does not suppress alpha-syn toxicity in this strain. In an effort to identify individual compounds that could simultaneously rescue these apparently disparate pathological effects of alpha-syn, we screened a library of 115,000 compounds. We identified a class of small molecules that reduced alpha-syn toxicity at micromolar concentrations in this higher toxicity strain. These compounds reduced the formation of alpha-syn foci, re-established ER-to-Golgi trafficking and ameliorated alpha-syn-mediated damage to mitochondria. They also corrected the toxicity of alpha-syn in nematode neurons and in primary rat neuronal midbrain cultures. Remarkably, the compounds also protected neurons against rotenone-induced toxicity, which has been used to model the mitochondrial defects associated with PD in humans. That single compounds are capable of rescuing the diverse toxicities of alpha-syn in yeast and neurons suggests that they are acting on deeply rooted biological processes that connect these toxicities and have been conserved for a billion years of eukaryotic evolution. Thus, it seems possible to develop novel therapeutic strategies to simultaneously target the multiple pathological features of PD.

Alpha-synuclein is part of a diverse and highly conserved interaction network that includes PARK9 and manganese toxicity.

Parkinson's disease (PD), dementia with Lewy bodies and multiple system atrophy, collectively referred to as synucleinopathies, are associated with a diverse group of genetic and environmental susceptibilities. The best studied of these is PD. alpha-Synuclein (alpha-syn) has a key role in the pathogenesis of both familial and sporadic PD, but evidence linking it to other predisposition factors is limited. Here we report a strong genetic interaction between alpha-syn and the yeast ortholog of the PD-linked gene ATP13A2 (also known as PARK9). Dopaminergic neuron loss caused by alpha-syn overexpression in animal and neuronal PD models is rescued by coexpression of PARK9. Further, knockdown of the ATP13A2 ortholog in Caenorhabditis elegans enhances alpha-syn misfolding. These data provide a direct functional connection between alpha-syn and another PD susceptibility locus. Manganese exposure is an environmental risk factor linked to PD and PD-like syndromes. We discovered that yeast PARK9 helps to protect cells from manganese toxicity, revealing a connection between PD genetics (alpha-syn and PARK9) and an environmental risk factor (PARK9 and manganese). Finally, we show that additional genes from our yeast screen, with diverse functions, are potent modifiers of alpha-syn-induced neuron loss in animals, establishing a diverse, highly conserved interaction network for alpha-syn.

Alpha-synuclein blocks ER-Golgi traffic and Rab1 rescues neuron loss in Parkinson's models.

Alpha-synuclein (alphaSyn) misfolding is associated with several devastating neurodegenerative disorders, including Parkinson's disease (PD). In yeast cells and in neurons alphaSyn accumulation is cytotoxic, but little is known about its normal function or pathobiology. The earliest defect following alphaSyn expression in yeast was a block in endoplasmic reticulum (ER)-to-Golgi vesicular trafficking. In a genomewide screen, the largest class of toxicity modifiers were proteins functioning at this same step, including the Rab guanosine triphosphatase Ypt1p, which associated with cytoplasmic alphaSyn inclusions. Elevated expression of Rab1, the mammalian YPT1 homolog, protected against alphaSyn-induced dopaminergic neuron loss in animal models of PD. Thus, synucleinopathies may result from disruptions in basic cellular functions that interface with the unique biology of particular neurons to make them especially vulnerable.

BASP1 is a transcriptional cosuppressor for the Wilms' tumor suppressor protein WT1.

The Wilms' tumor suppressor protein WT1 is a transcriptional regulator that plays a key role in the development of the kidneys. The transcriptional activation domain of WT1 is subject to regulation by a suppression region within the N terminus of WT1. Using a functional assay, we provide direct evidence that this requires a transcriptional cosuppressor, which we identify as brain acid soluble protein 1 (BASP1). WT1 and BASP1 associate within the nuclei of cells that naturally express both proteins. BASP1 can confer WT1 cosuppressor activity in transfection assays, and elimination of endogenous BASP1 expression augments transcriptional activation by WT1. BASP1 is present in the developing nephron structures of the embryonic kidney and, coincident with that of WT1, its expression is restricted to the highly specialized podocyte cells of the adult kidney. Taken together, our results show that BASP1 is a WT1-associated factor that can regulate WT1 transcriptional activity.

A role of protein kinase C mu in signalling from the human adenosine A1 receptor to the nucleus.

1 Stimulation of adenosine A(1) receptors produced a stimulation of c-fos promoter-regulated gene transcription in Chinese hamster ovary (CHO)-A1 cells expressing the human A(1) receptor. Gene transcription was monitored using a luciferase-based reporter gene (pGL3). 2 This response to the A(1) receptor agonist N(6)-cyclopentyladenosine (CPA) was sensitive to inhibition by pertussis toxin, the MEK-1 inhibitor PD 98059 and by the phosphatidylinositol-3-kinase inhibitors wortmannin and LY 294002. The response was also completely abolished by the protein kinase C (PKC) inhibitor Ro-31-8220. 3 Several isoforms of PKC can be detected in CHO-A1 cells (alpha, delta, epsilon, micro, iota, zeta), but only PKC alpha, PKC delta and PKC were downregulated by prolonged treatment with phorbol ester. The c-fos-regulated luciferase response to A(1) agonists was not, however, inhibited by 24 h pretreatment with the phorbol esters phorbol 12,13-dibutyrate (PDBu). This observation, together with the fact that a significant attenuation (40%) of the c-fos-luciferase response to PDBu and A(1) agonist was produced by low concentrations of the PKC inhibitor Gö 6976 suggests a role for PKC micro. 4 Stimulation of CHO-A1 cells with CPA stimulated the activation of endogenous PKC micro as measured by autophosphorylation. This was rapid, occurred within 1-2 min, but returned to basal levels after 30 min. Furthermore, transient expression of a constitutively active form of PKC micro resulted in a significant increase in c-fos-regulated gene expression. 5 Taken together, these data suggest that PKC micro plays an important role in the ability of the adenosine A(1) receptor to signal to the nucleus.