Abstract
242217
Introduction: Glutamate, the primary excitatory neurotransmitter in the vertebrate CNS, modulates over 50% of excitatory synaptic transmission through its interaction with ionotropic receptors (iGluRs) or metabotropic glutamate receptors (mGluRs). Categorized into three groups (I, II, and III) with eight subtypes (mGluR1-8), mGluRs display diversity in sequence homologies, pharmacological properties, and signal transduction pathways. Group III mGluRs (mGluR4, 6, and 8) are predominantly expressed in the presynaptic terminals of basal ganglia (BG) circuitry. In Parkinson's disease (PD), dopamine depletion induces hyperactivity in this circuitry, underscoring the importance of Group III mGluRs, especially mGluR4, as critical therapeutic targets for non-dopaminergic interventions. We pioneered on development of mGluR4 PET radioligands by designing and characterizing several mGluR4 tracers as positive allosteric modulators (PAMs) for mGluR4. One such tracer, initially named [11C]mG4P012 and later renamed [11C]PXT012253 by Prexton Therapeutics, underwent evaluation in a clinical trial (NCT03826134) as a PET tracer for mGluR4. Since PET imaging may face resolution limitations (mm), a fluorescent ligand may provide high spatial and temporal resolution, easy operation, compatibility with several equipment, and high throughput in drug screening. Conjugating a fluorophore to an mGluR allosteric modulator poses significant challenges, as even a minor modification by a single atom can lead to substantial activity loss. In this report, we introduce a novel molecule highly specific to mGluR4, showing a strong fluorescent turn-on effect in cell culture, enabling fluorescent in vitro assays and PET imaging could also be achieved by the same molecule.
Methods: mG4PFL01, designed and synthesized based on known PAM ligands, underwent evaluation in HEK293 cells overexpressing all mGluR subtypes using confocal imaging. An mGluR4 PAM ligand 1 was used to replace this fluorescent ligand for potential drug screening. The PAM activity was assessed in the overexpressed cells using cAMP assays. Fluorescent response was verified using both a UV-vis spectrometer and a fluorometer, and imaging was captured under a confocal microscope. Autoradiography of a derivative was conducted on sagittal rat brain sections at a concentration of 1.0 µCi/mL in PBS. Immunohistochemical staining utilized an mGluR4 selective antibody.
Results: mG4PFL01 exhibits a significant fluorescent turn-on effect upon binding to mGluR4, displaying high signal and good subtype selectivity to mGluR4 while it generates minimal signal for other subtypes of mGluRs. With a PAM activity of <100 nM, the fluorescent signal is interchangeable with both the mGluR4 antibody and the PAM ligand 1, indicating its suitability for high-throughput screening (HTS) and new drug applications. Derivatives from the same chemotype demonstrate high signals in the cerebellum, aligning with immunohistochemical staining, affirming the promise of this chemotype as mGluR4 ligands for both in vitro and in vivo imaging.
Conclusions: The mGluR4 fluorescent turn-on probe, mG4PFL01, was deliberately designed and synthesized, demonstrating a highly effective fluorescent turn-on effect towards mGluR4. This probe can be replaced by the mGluR4 antibody and a known PAM ligand, making it suitable for HTS in new drug discovery. The radioligand of the same chemotype exhibits high specificity on rat brain sections, presenting a promising chemotype for in vivo imaging. With methyl groups in its composition, mG4PFL01 can be readily developed as PET C-11 probes, and we plan to evaluate its PET version both in vitro and in vivo. This molecule holds the potential to advance mGluR4 drug development and disease imaging across micro to macro scales.
Acknowledgments: The studies were supported by NIH R01NS100164 and NIH/NIA K25AG061282.
References:
• J. Med. Chem. 2014, 57, 9130−91381,
• J. Med. Chem. 2020, 63,3381-3389.
In this issue
Jump to section
Related Articles
Cited By...
- No citing articles found.