PhD Candidate, University of Sydney
Synthesis of TSPO Ligands with Binding Sensitivity to Single Nucleotide Polymorphism rs6971
The increasing incidence of pathological conditions associated with neurodegeneration including Alzheimer’s and Parkinson’s disorders have prompted the need for novel therapeutic targets. Since its discovery in 1977 and elucidation of its role in neurosteroid biosynthesis, the 18 kDa translocator protein (formally known as the peripheral benzodiazepine receptor) presents a viable target for pharmaceutical intervention. Furthermore, its upregulation within cancer has attracted considerable attention as a potential biomarker for neoplastic growth or as a novel receptor target for chemotherapy.
Although there are TSPO ligands currently available, their clinical utility 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). 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. Whilst there are several distinctive chemical classes of TSPO ligands, there is no definitive chemotype. Each chemical class has different activity and selectivity profiles, making the characterisation of the TSPO binding sites a promising area of research.
My research reports the synthesis and initial in vitro biological evaluation of over 40 new TSPO analogues in an aim to explore the pharmacophoric model for optimal binding to WT and SNP TSPO. A series of novel TSPO ligands were developed based on the indole, quinazoline and carbazole heterocyclic core. The ligands incorporate either a bicyclic or tricyclic heterocyclic core, an acetamide or amide moiety, and an additional phenyl group.
To assess the potential of these analogues between the two TSPO isoforms, human embryonic kidney cell lines stably over-expressing human TSPO WT and TSPO A147T were established, and the analogues synthesised were tested to determine their binding affinity to both isoforms. Presently, the carbazole acetamides are a class of high affinity TSPO ligands, displaying nanomolar binding affinities to both TSPO isoforms.
Abstract: Translocator protein (TSPO) is upregulated in activated microglia and thus can serve as a marker of neuroinflammation. Recently, a novel radioligand, (11)C-N,N-diethyl-2-[2-(4-methoxyphenyl)-5,7-dimethyl-pyrazolo[1,5-a]pyrimidin-3-yl]-acetamide ((11)C-DPA-713), has been described that binds to TSPO with high affinity. Here, we report the first examination of (11)C-DPA-713 in human subjects using PET.Five healthy controls were studied with PET for 90 min after a bolus injection of high-specific-activity (11)C-DPA-713. For comparison, 2 additional healthy controls were studied with (11)C-R-PK11195. Arterial blood sampling and metabolite analysis were performed to allow the accurate quantification of tracer kinetics. Tracer uptake was evaluated for several brain regions. Tissue time-activity curves were fitted using 1- and 2-tissue-compartment models, with goodness-of-fit tests showing a preference for the 2-tissue model.In the healthy brain, the average plasma-to-tissue clearance and the total volume of distribution were an order of magnitude larger than measured for (11)C-R-PK11195. Accordingly, dose-normalized time-activity curves showed that (11)C-DPA-713 gives a larger brain signal.Studies in patient populations will help determine whether (11)C-DPA-713 provides better sensitivity for evaluating increased TSPO expression. This initial study in humans shows that (11)C-DPA-713 is a promising ligand for evaluating TSPO binding with PET.
Pub.: 21 Jul '09, Pinned: 29 Aug '17
Abstract: Positron emission tomography (PET) imaging with radiotracers that target translocator protein 18 kDa (TSPO) has become a popular approach to assess putative neuroinflammatory processes and associated microglia activation in psychotic illnesses. It remains unclear, however, whether TSPO imaging can accurately capture low-grade inflammatory processes such as those present in schizophrenia and related disorders. Therefore, we evaluated the validity of TSPO as a disease-relevant marker of inflammation using a translational approach, which combined neurodevelopmental and neurodegenerative mouse models with PET imaging in patients with recent-onset schizophrenia and matched controls. Using an infection-mediated neurodevelopmental mouse model, we show that schizophrenia-relevant behavioral abnormalities and increased inflammatory cytokine expression are associated with reduced prefrontal TSPO levels. On the other hand, TSPO was markedly upregulated in a mouse model of acute neurodegeneration and reactive gliosis, which was induced by intrahippocampal injection of kainic acid. In both models, the changes in TSPO levels were not restricted to microglia but emerged in various cell types, including microglia, astrocytes and vascular endothelial cells. Human PET imaging using the second-generation TSPO radiotracer [(11)C]DPA-713 revealed a strong trend towards reduced TSPO binding in the middle frontal gyrus of patients with recent-onset schizophrenia, who were previously shown to display increased levels of inflammatory cytokines in peripheral and central tissues. Together, our findings challenge the common assumption that central low-grade inflammation in schizophrenia is mirrored by increased TSPO expression or ligand binding. Our study further underscores the need to interpret altered TSPO binding in schizophrenia with caution, especially when measures of TSPO are not complemented with other markers of inflammation. Unless more selective microglial markers are available for PET imaging, quantification of cytokines and other inflammatory biomarkers, along with their molecular signaling pathways, may be more accurate in attempts to characterize inflammatory profiles in schizophrenia and other mental disorders that lack robust reactive gliosis.Molecular Psychiatry advance online publication, 17 January 2017; doi:10.1038/mp.2016.248.
Pub.: 18 Jan '17, Pinned: 29 Aug '17
Abstract: Activation of the innate immune system plays a significant role in pathologies of the central nervous system (CNS). In order to follow disease progression and evaluate effectiveness of potential treatments involved in neuroinflammation, it is important to track neuroinflammatory markers in vivo longitudinally. The translocator protein (TSPO) is used as a target to image neuroinflammation as its expression is upregulated in reactive glial cells during CNS pathologies. However, it remains unclear in which microglial phenotypes TSPO levels are upregulated, as microglia can display a plethora of activation states that can be protective or detrimental to the CNS.We assessed the levels of TSPO transcripts in cultured microglia that were polarized into pro- and anti-inflammatory states in vitro and in the brain of mice in which an anti-inflammatory environment was induced in vivo. In addition, we used a mouse model of peroxisomal multifunctional protein-2 (MFP2) deficiency that exhibits widespread neuroinflammation despite no neuronal loss and monitored TSPO expression by immunohistochemistry and by imaging using the TSPO radiotracer [(18)F]DPA-714.TSPO expression was selectively increased in so-called classically activated or M1 microglia but not in alternatively activated or M2 microglia in vitro. In agreement, TSPO transcript levels were not induced in an anti-inflammatory brain environment. We found that both transcript and protein levels of TSPO are significantly increased in the brain of Mfp2 (-/-) compared to those of the control mice and TSPO immunoreactivity colocalized predominantly with microglia in Mfp2 (-/-) brain. In vitro and ex vivo autoradiography in Mfp2 (-/-) mice using the TSPO radiotracer [(18)F]DPA-714 confirmed increased expression of TSPO. These data demonstrate that TSPO imaging reveals microgliosis in non-neurodegenerative brain pathologies.We show that induced TSPO expression marks a pro-inflammatory brain environment that is not necessarily accompanied by neuronal loss.
Pub.: 12 Jul '17, Pinned: 29 Aug '17
Abstract: The 18kDa translocator protein (TSPO) is a target for novel glioblastoma therapies due to its upregulation in this cancer and relatively low levels of expression in the healthy cortex. The pyrazolo[1,5-a] pyrimidine acetamides, exemplified by DPA-713 and DPA-714, are a class of high affinity TSPO ligands with selectivity over the central benzodiazepine receptor. In this study we have explored the potential anti-glioblastoma activity of a library of DPA-713 and DPA-714 analogues, and investigated the effect of amending the alkyl ether chain on TSPO affinity and functional potential. All ligands demonstrated nanomolar affinity for TSPO, but showed diverse functional activity, for example DPA-713 and DPA-714 did not affect the proliferation or viability of human T98G glioblstoma cells, while the hexyl ether and benzyl ether derivatives decreased proliferation of T98G cells without affecting proliferation in human fetal glial SVGp12 cells. These ligands also induced apoptosis and dissipated T98G mitochondrial membrane potential. This suggests that the nature of the alkyl ether chain of pyrazolo[1,5-a] pyrimidine acetamides has little influence on TSPO affinity but is important for functional activity of this class of TSPO ligands.
Pub.: 24 Sep '16, Pinned: 29 Aug '17
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