Identifying potential dietary treatments for inherited metabolic disorders using Drosophila nutrigenomics.

Inherited metabolic disorders are a group of genetic conditions that can cause severe neurological impairment and child mortality. Uniquely, these disorders respond to dietary treatment; however, this option remains largely unexplored because of low disorder prevalence and the lack of a suitable paradigm for testing diets. Here, we screened 35 Drosophila amino acid disorder models for disease-diet interactions and found 26 with diet-altered development and/or survival. Using a targeted multi-nutrient array, we examine the interaction in a model of isolated sulfite oxidase deficiency, an infant-lethal disorder. We show that dietary cysteine depletion normalizes their metabolic profile and rescues development, neurophysiology, behavior, and lifelong fly survival, thus providing a basis for further study into the pathogenic mechanisms involved in this disorder. Our work highlights the diet-sensitive nature of metabolic disorders and establishes Drosophila as a valuable tool for nutrigenomic studies for informing potential dietary therapies.

Selective activation of Gαob by an adenosine A1 receptor agonist elicits analgesia without cardiorespiratory depression.

The development of therapeutic agonists for G protein-coupled receptors (GPCRs) is hampered by the propensity of GPCRs to couple to multiple intracellular signalling pathways. This promiscuous coupling leads to numerous downstream cellular effects, some of which are therapeutically undesirable. This is especially the case for adenosine A1 receptors (A1Rs) whose clinical potential is undermined by the sedation and cardiorespiratory depression caused by conventional agonists. We have discovered that the A1R-selective agonist, benzyloxy-cyclopentyladenosine (BnOCPA), is a potent and powerful analgesic but does not cause sedation, bradycardia, hypotension or respiratory depression. This unprecedented discrimination between native A1Rs arises from BnOCPA's unique and exquisitely selective activation of Gob among the six Gαi/o subtypes, and in the absence of ß-arrestin recruitment. BnOCPA thus demonstrates a highly-specific Gα-selective activation of the native A1R, sheds new light on GPCR signalling, and reveals new possibilities for the development of novel therapeutics based on the far-reaching concept of selective Gα agonism.

Positive allosteric mechanisms of adenosine A1 receptor-mediated analgesia.

The adenosine A1 receptor (A1R) is a promising therapeutic target for non-opioid analgesic agents to treat neuropathic pain1,2. However, development of analgesic orthosteric A1R agonists has failed because of a lack of sufficient on-target selectivity as well as off-tissue adverse effects3. Here we show that [2-amino-4-(3,5-bis(trifluoromethyl)phenyl)thiophen-3-yl)(4-chlorophenyl)methanone] (MIPS521), a positive allosteric modulator of the A1R, exhibits analgesic efficacy in rats in vivo through modulation of the increased levels of endogenous adenosine that occur in the spinal cord of rats with neuropathic pain. We also report the structure of the A1R co-bound to adenosine, MIPS521 and a Gi2 heterotrimer, revealing an extrahelical lipid-detergent-facing allosteric binding pocket that involves transmembrane helixes 1, 6 and 7. Molecular dynamics simulations and ligand kinetic binding experiments support a mechanism whereby MIPS521 stabilizes the adenosine-receptor-G protein complex. This study provides proof of concept for structure-based allosteric drug design of non-opioid analgesic agents that are specific to disease contexts.

A Positive Allosteric Modulator of the Adenosine A1 Receptor Selectively Inhibits Primary Afferent Synaptic Transmission in a Neuropathic Pain Model.

In the spinal cord and periphery, adenosine inhibits neuronal activity through activation of the adenosine A1 receptor (A1R), resulting in antinociception and highlighting the potential of therapeutically targeting the receptor in the treatment of neuropathic pain. This study investigated the changes in adenosine tone and A1R signaling, together with the actions of a novel A1R positive allosteric modulator (PAM), VCP171 [(2-amino-4-(3-(trifluoromethyl)phenyl)thiophen-3-yl)(phenyl)methanone], on excitatory and inhibitory neurotransmission at spinal cord superficial dorsal horn synapses in a rat partial nerve-injury model of neuropathic pain. In the absence of A1R agonists, superfusion of the A1R antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 1 µM), produced a significantly greater increase in electrically evoked α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor-mediated synaptic current (eEPSC) amplitude in both lamina I and II neurons from nerve-injured animals than in controls, suggesting that endogenous adenosine tone is increased in the dorsal horn. Inhibitory GABAergic and glycinergic synaptic currents were also significantly increased by DPCPX in controls but there was no difference after nerve injury. The A1R agonist, N6-cyclopentyladenosine, produced greater inhibition of eEPSC amplitude in lamina II but not lamina I of the spinal cord dorsal horn in nerve-injured versus control animals, suggesting a functional increase in A1R sensitivity in lamina II neurons after nerve injury. The A1R PAM, VCP171, produced a greater inhibition of eEPSC amplitude of nerve-injury versus control animals in both lamina I and lamina II neurons. Enhanced adenosine tone and A1R sensitivity at excitatory synapses in the dorsal horn after nerve injury suggest that new generation PAMs of the A1R can be effective treatments for neuropathic pain.

A comparison of the anti-inflammatory and immuno-stimulatory activities of orf virus and ovine interleukin-10.

Orf virus causes pustular skin lesions (orf) in sheep, goats and humans. The virus encodes an interleukin-10 (orfvIL-10) that is identical in amino acid composition to ovine IL-10 (ovIL-10) over the C terminal two-thirds of the polypeptide, but not in the N terminal third. The immuno-suppressive and immuno-stimulatory activities of orfvIL-10 and ovIL-10 were compared. Both orfvIL-10 and ovIL-10 inhibited TNF-alpha and IL-8 cytokine production from stimulated ovine macrophages and keratinocytes and IFN-gamma and GM-CSF production from peripheral blood lymphocytes. OrfvIL-10 and ovIL-10 co-stimulated both ovine and murine mast cell proliferation in conjunction with IL-3 (ovine) or IL-4 (murine). Isoleucine at position 87 (Ile(87)) of the mature human IL-10 (huIL-10) has been reported as essential for the immuno-stimulatory activity of huIL-10. In spite of the differences in amino acids within the N-terminal third of orfvIL-10 compared with ovIL-10 and substitution of Ile(87) with Ala(87) in ovIL-10, these variants of ovIL-10 and orfvIL-10 all co-stimulated mast cell proliferation and inhibited macrophage IL-8 production. As ovIL-10 and orfvIL-10 have a similar structure to huIL-10 and conserved receptor-binding residues, it was concluded that Ile(87) is not essential for IL-10 immuno-stimulatory activity. Finally, ovine keratinocytes do not express ovIL-10. This might explain why orf virus has evolved a viral IL-10.

Orf virus immuno-modulation and the host immune response.

Orf virus encodes a range of immuno-modulatory genes that interfere with host anti-virus immune and inflammatory effector mechanisms. The function of these reflects the pathogenesis of orf. The orf virus interferon resistance protein (OVIFNR) and virus IL-10 (vIL-10) inhibit interferon production and activity. In addition the vIL-10 suppresses inflammatory cytokine production by activated macrophages and keratinocytes. The virus GM-CSF inhibitory factor (GIF) is a novel virus protein that binds to and inhibits the biological activity of GM-CSF and IL-2. Together, these immuno-modulators target key effector mechanisms of host anti-virus immunity to allow time for virus replication in epidermal cells.

Orf virus-encoded interleukin-10 stimulates the proliferation of murine mast cells and inhibits cytokine synthesis in murine peritoneal macrophages.

Orf virus (ORFV) is the type species of the parapoxvirus genus and produces cutaneous pustular lesions in sheep, goats and humans. The genome encodes a polypeptide with remarkable homology to interleukin-10 (IL-10), particularly ovine IL-10, and also to IL-10-like proteins encoded by Epstein-Barr virus (EBV) and equine herpesvirus. IL-10 is a pleiotropic cytokine that can exert either immunostimulatory or immunosuppressive effects on many cell types. We have expressed and purified C-terminal FLAG and His(6)-tagged versions of ORFV-IL-10 and shown that ORFV-IL-10 costimulates murine mast cells (MC/9) and inhibits tumour necrosis factor-alpha synthesis in activated mouse peritoneal macrophages. Our results demonstrate that although ORFV-IL-10 is structurally similar to EBV-IL-10 it has evolved a different spectrum of activities. EBV-IL-10 does not stimulate the proliferation of thymocytes or mast cells whereas ORFV-IL-10 has both of these activities. Recent studies show that the critical difference in molecular structure of human IL-10 and EBV-IL-10, which may be the basis of their functional differences, is linked to a single amino acid substitution. Consistent with the activity spectrum reported here for ORFV-IL-10, the viral gene encodes the critical amino acid seen in human IL-10. Although the ORFV-IL-10 gene has clearly undergone significant evolutionary change at the nucleotide level compared with ovine IL-10, it has largely retained the polypeptide structure and functional characteristics of its ovine counterpart, suggesting that mutations of the gene to a potentially more potent immunosuppressive form may compromise the co-existence of host and virus.