Reversing binding sensitivity to A147T translocator protein

The translocator protein (TSPO) is a target for the development of neuroinflammation imaging agents. Clinical translation of TSPO PET ligands, such as [11C]DPA-713, has been hampered by the presence of a common polymorphism (A147T TSPO), at which all second-generation TSPO ligands lose affinity. Little is known about what drives binding at A147T compared to WT TSPO. This study aimed to identify moieties in DPA-713, and related derivatives, that influence binding at A147T compared to WT TSPO. We found changes to the nitrogen position and number in the heterocyclic core influences affinity to WT and A147T to a similar degree. Hydrogen bonding groups in molecules with an indole core improve binding at A147T compared to WT, a strategy that generated compounds that possess up to ten-times greater affinity for A147T. These results should inform the future design of compounds that bind both A147T and WT TSPO for use in neuroinflammation imaging.The work was supported by the National Health and Medical Research Council of Australia (APP1132524, APP1136241 and APP1154692)

Pharmacological evaluation of novel bioisosteres of an adamantanyl benzamide P2X7 receptor antagonist

Adamantanyl benzamide 1 was identified as a potent P2X7R antagonist but failed to progress further due to poor metabolic stability. We describe the synthesis and SAR of a series of bioisosteres of benzamide 1 to explore improvements in the pharmacological properties of this lead. Initial efforts investigated a series of heteroaromatic bioisosteres, which demonstrated improved physicochemical properties but reduced P2X7R antagonism. Installation of bioisosteric fluorine on the adamantane bridgeheads was well tolerated and led to a series of bioisosteres with improved physicochemical properties and metabolic stability. Trifluorinated benzamide 34 demonstrated optimal physicochemical parameters, superior metabolic stability (ten times longer than lead benzamide 1), and an improved physicokinetic profile and proved effective in the presence of several known P2X7R polymorphisms

Radiosynthesis of (R,S)-[18 F]GE387: A Potential PET Radiotracer for Imaging Translocator Protein 18 kDa (TSPO) with Low Binding Sensitivity to the Human Gene Polymorphism rs6971.

Translocator protein (TSPO) is a biomarker of neuroinflammation, which is a hallmark of many neurodegenerative diseases and has been exploited as a positron emission tomography (PET) target. Carbon-11-labelled PK11195 remains the most applied agent for imaging TSPO, despite its short-lived isotope and low brain permeability. Second-generation radiotracers show variance in affinity amongst subjects (low-, mixed-, and high-affinity binders) caused by the genetic polymorphism (rs6971) of the TSPO gene. To overcome these limitations, a new structural scaffold was explored based on the TSPO pharmacophore, and the analogue with a low-affinity binder/high-affinity binder (LAB/HAB) ratio similar (1.2 vs. 1.3) to that of (R)-[11 C]PK11195 was investigated. The synthesis of the reference compound was accomplished in six steps and 9 % overall yield, and the precursor was prepared in eight steps and 8 % overall yield. The chiral separation of the reference and precursor compounds was performed using supercritical fluid chromatography with >95 % ee. The absolute configuration was determined by circular dichroism. Optimisation of reaction conditions for manual radiolabelling revealed acetonitrile as a preferred solvent at 100 °C. Automation of this radiolabelling method provided R and S enantiomers in respective 21.3±16.7 and 25.6±7.1 % decay-corrected yields and molar activities of 55.8±35.6 and 63.5±39.5 GBq μmol-1 (n=3). Injection of the racemic analogue into a healthy rat confirmed passage through the blood-brain barrier.This work was supported by Medical Research Council (UK) grant awards RG46503 (LM, MH, XZ) and RG70550 (EF), National Institute of Health Research (UK), Cambridge Biomedical Research Unit in Dementia (EF, NKR) and the Herchel Smith Fellowship programme (LQ)

Identification of novel ATP and interleukin-10 sources in the spinal cord

Chronic pain has a large individual and societal impact. Although effective treatments for acute pain exist, a specific and effective treatment for chronic pain is still to be discovered. Recent use of genetic techniques has revealed that spinal ATP plays a role in the generation of allodynia and hyperalgesia, two symptoms of chronic pain. Furthermore, it has been shown that the symptoms of chronic pain can be alleviated by increasing spinal levels of the anti-inflammatory cytokine interleukin-10 (IL-10). These observations suggest that decreasing the spinal release of ATP and increasing the spinal release of IL-10 may be novel approaches to chronic pain treatment. Yet knowledge of the endogenous sources of spinal ATP and IL—10 is limited. Therefore, the aim of this thesis was to identify novel sources of ATP and IL—10 in the spinal cord. The first series of experiments (Chapter 3) investigated whether spinal cord astrocytes could be a source“ of ATP in response to the nociceptive neurotransmitters glutamate and substance P. Glutamate stimulated ATP release from cultured spinal cord astrocytes and this release was greatly potentiated by substance P, even though substance P alone did not elicit ATP release. Substance P also potentiated glutamateinduced inward currents without alone causing such currents. Glutamate, when applied on its own, acted exclusively through AMPA receptors to stimulate Ca2+ influx-dependent ATP release. However when substance P was co—applied with glutamate, ATP release could be elicited by activation of NMDA and metabotropic glutamate receptors. ‘ ATP release resulting from AMPA and kainate receptor stimulation was not affected by substance P. Activation of neurokinin receptor subtypes, protein kinase C and phospholipase A2, C and D were required for substance P to bring about its effects. The second series of experiments (Chapter 4) explored whether spinal cord astrocytes could be a source of IL-10 in response to glutamate and stimulation of the Toll-like receptor 4 (TLR4), which is known to occur in chronic pain. TLR4 stimulation increased IL—lO release from cultured spinal cord astrocytes. TLR4-stimulated IL—lO release was enhanced in the presence of glutamate. Glutamate potentiated TLR4— stimulated IL—lO release by upregulating IL-10 mRNA and worked synergistically through metabotropic glutamate receptor groups I, II and possibly III. The third series of experiments (Chapter 5) examined whether spinal cord microglia could be a source of IL-10 in response to glutamate and stimulation of the TLR4. TLR4 stimulation enhanced both IL—10 transcription and translation, resulting in an increase in IL-lO release from cultured spinal cord microglia. Glutamate significantly increased TLR4-stimulated IL-lO release from microglia by binding NMDA, AMPA, metabotropic glutamate receptors and possibly kainate receptors and enhancing TLR4-induced IL-lO mRNA expression. The results of this thesis suggest that astrocytes may be a major source of ATP in the spinal cord on activation of nerve fibres that co-release substance P and glutamate. The results of this thesis also suggest that when glutamate and TLR4 agonists are released into the spinal cord after noxious stimulation, astrocytes and microglia may be a major source of IL-10. These results implicate spinal cord glia in both pronociceptive and anti-nociceptive responses to pain—related substances

Flexible analogues of WAY-267,464 : synthesis and pharmacology at the human oxytocin and vasopressin 1ₐ receptors

A previously identified, non-peptidic oxytocin (OT) receptor agonist WAY-267,464 (1) and nine novel derivatives (3, 4a-7a, 4b-7b) were synthesised and evaluated in vitro with the aim of systematically exploring hydrogen bonding interactions and ligand flexibility. All analogues were subjected to competition radioligand binding assays at human oxytocin (OT) and arginine vasopressin 1ₐ (V1ₐ) receptors. Physiological activity was determined using whole cell IP1 accumulation assays. Under these conditions, WAY-267,464 had higher affinity for the V1ₐ receptor compared to the OT receptor (8.5x more selective) with poor functional selectivity (2x selective for OT receptor agonism over V1ₐ receptor antagonism). Methylation of the resorcinol moiety (3) reversed the OT receptor pharmacological profile, removing agonist activity and inducing antagonist activity, without altering V1a receptor pharmacology. All flexible tethered derivatives removed OT receptor affinity and activity resulting in the generation of highly selective V1ₐ receptor ligands.11 page(s

Levels of glial cell line-derived neurotrophic factor are decreased, but fibroblast growth factor 2 and cerebral dopamine neurotrophic factor are increased in the hippocampus in parkinson's disease

Growth factors can facilitate hippocampus-based learning and memory and are potential targets for treatment of cognitive dysfunction via their neuroprotective and neurorestorative effects. Dementia is common in Parkinson's disease (PD), but treatment options are limited. We aimed to determine if levels of growth factors are altered in the hippocampus of patients with PD, and if such alterations are associated with PD pathology. Enzyme-linked immunosorbent assays were used to quantify seven growth factors in fresh frozen hippocampus from 10 PD and nine age-matched control brains. Western blotting and immunohistochemistry were used to explore cellular and inflammatory changes that may be associated with growth factor alterations. In the PD hippocampus, protein levels of glial cell line-derived neurotrophic factor were significantly decreased, despite no evidence of neuronal loss. In contrast, protein levels of fibroblast growth factor 2 and cerebral dopamine neurotrophic factor were significantly increased in PD compared to controls. Levels of the growth factors epidermal growth factor, heparin-binding epidermal growth factor, brain-derived neurotrophic factor and mesencephalic astrocyte-derived neurotrophic factor did not differ between groups. Our data demonstrate changes in specific growth factors in the hippocampus of the PD brain, which potentially represent targets for modification to help attenuate cognitive decline in PD. These data also suggest that multiple growth factors and direction of change needs to be considered when approaching growth factors as a potential treatment for cognitive decline.Peer reviewe

Synthesis of 7,7 '-Linked-Bis-Indoles Via 7-Tryptamines

The synthesis of 7-tryptamines was accomplished via the reduction of 7-nitrovinylindoles which were developed by the condensation of indole-7-carbaldehydes with nitromethane and ammonium acetate. 7-Tryptamines were subsequently used for the construction of 2,3-disubstituted and 3 -substituted 7,7'-linked-bis-indoles.University of New South Wales; Turkish GovernmentTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK); New South Wales Government, National Collaborative Research Infrastructure StrategyAustralian GovernmentDepartment of Industry, Innovation and ScienceWe thank the University of New South Wales and the Turkish Government for their financial support. Mass spectrometric analysis for this work was carried out at the Bioanalytical Mass Spectrometry Facility, UNSW and was supported in part by infrastructure funding from the New South Wales Government as part of its coinvestment in the National Collaborative Research Infrastructure Strateg

Determination and reduction of translocator protein (TSPO) ligand rs6971 discrimination

The 18 kDa translocator protein (TSPO) is a target for development of diagnostic imaging agents for glioblastoma and neuroinflammation. Clinical translation of TSPO imaging agents has been hindered by the presence of a polymorphism, rs6971, which causes a non-conservative substitution of alanine for threonine at amino acid residue 147 (TSPO A147T). Disclosed brain-permeant second-generation TSPO ligands bind TSPO A147T with reduced affinity compared to the wild type protein (TSPO WT). Efforts to develop a TSPO ligand that binds TSPO WT and TSPO A147T with similarly high affinity have been hampered by a lack of knowledge about how ligand structure differentially influences interaction with the two forms of TSPO. To gain insight, we have established human embryonic kidney cell lines stably over-expressing human TSPO WT and TSPO A147T, and tested how modifications of a novel N-alkylated carbazole scaffold influence affinity to both TSPO isoforms. Most of the new analogues developed in this study showed high affinity to TSPO WT and a 5–6-fold lower affinity to TSPO A147T. Addition of electron-withdrawing substituents yielded analogues with highest affinity for TSPO A147T without decreasing affinity for TSPO WT. This knowledge can be used to inform further development of non-discriminating TSPO ligands for use as diagnostic markers for glioblastoma and neuroinflammation irrespective of rs6971