Periodic Reporting for period 1 - MIM (Molecular Imaging of Microglia)
Periodo di rendicontazione: 2020-05-01 al 2022-04-30
Neurodegenerative and neurological diseases, such as multiple sclerosis (MS), Alzheimer’s (AD) and Parkinson’s disease (PD) affect millions of people worldwide. MS is the most common neurological disorder among young adults and the number of affected people continues to grow. To date, already 700.000 people in Europe are affected. In order to guide drug development, a better understanding of the underlying mechanism is of utmost importance. However, diseases in the central nervous system (CNS) are complex pathologies, in which several factors and mechanisms are involved and contribute to the development and progression. A common hallmark for most of these diseases is neuroinflammation – an inflammatory response within the CNS caused by a disturbance in the brain from e.g. tissue damage or the formation of protein aggregates. As a result, microglia, the brain-specific immune cells become activated. The activation process of microglia is highly dynamic, covering a broad spectrum of activation phenotypes with pro-inflammatory neurotoxic microglia on one end and anti-inflammatory neuroprotective microglia on the other end of the activation spectrum. The exact role of the different phenotypes in neuroinflammation is still not fully clarified. The activation state is dependent on several factors and most likely it changes during disease progression. To better understand the role of activated microglia in the living brain, a molecular imaging technique is required. Positron Emission Tomography (PET) is such a technique. It makes use of radiolabeled molecules at tracer levels, so-called PET tracers, which target a specific protein that is representative for the biochemical process being studied. To date, the most often applied target to image activated microglia is the 18 kDa translocator protein (TSPO), a mitochondrial protein, overexpressed on activated microglia. Even though TSPO enables the detection of activated microglia, it cannot be used to discriminate between the pro-inflammatory and anti-inflammatory phenotype. In addition, a number of other drawbacks with PET tracers targeting TSPO have been reported, such as low brain uptake, high non-specific binding and a high level of variability of binding between patients due to polymorphism in the TSPO binding domain. Therefore, there is an immediate need to explore alternative targets, beyond TSPO, for selective PET imaging of a specific phenotype of activated microglia. PET tracers that can be used for imaging of either pro- or anti-inflammatory microglia, would allow for monitoring and measuring the dynamical changes in microglia activity in the onset and progression of neuroinflammatory diseases. This, in turn, can guide drug development and the identification of new treatment opportunities in such diseases.
The overall objective of MIM was to develop a novel BBB-penetrant PET tracer targeting the P2Y12R for imaging of anti-inflammatory microglia. In combination with the previously developed PET tracer for pro-inflammatory microglia, developed by the host institution, these PET tracers will allow for imaging of the dynamical changes of activated microglia.
The overall objective of MIM was to develop a novel BBB-penetrant PET tracer targeting the P2Y12R for imaging of anti-inflammatory microglia. In combination with the previously developed PET tracer for pro-inflammatory microglia, developed by the host institution, these PET tracers will allow for imaging of the dynamical changes of activated microglia.
By using an interdisciplinary approach spanning from computational design techniques through synthetic organic chemistry and in vitro assays to radiochemistry and in vivo evaluation studies we were able to investigate several potential P2Y12R PET tracers. In total, four different scaffolds were studied based on both leads from literature and novel structural frameworks found in a virtual screening campaign. So far, two second generation tracer candidates showed promising characteristics with regard to in silico prediction of BBB-permeability and affinity toward P2Y12R (Ki-values were 1.16 and 1.43 nM, respectively). These candidates were designed based on modifying a lead found in literature and the modifications were guided by molecular docking in a crystal structure of P2Y12R. Preliminary radiolabeling of these tracers have been carried out and further studies will elucidate the potential of these tracers in vivo in an animal model for MS. In order to find additional scaffolds for optimization toward PET tracer development, molecular modeling was carried out. From this, we established complementary pharmacophore models and a high-throughput docking protocol for virtual screening. Afterward, an in-house library of drug-like compounds and fragments were screened. The in silico hit list were filtered for structures with high probability for BBB-permeability (based on in silico prediction) and a set of representative compounds with favorable physicochemical properties were tested for P2Y12R affinity. After hit exploration, a new lead was identified based on a scaffold that has not yet been explored for P2Y12R antagonists. Future work will be directed toward screening a larger database to cover a greater chemical space, as well as optimizing the identified lead. In conclusion, we believe that the main goal of MIM could be fulfilled and the project resulted in several good starting points for future research and new collaborations were initiated.
The project has been presented at conferences for molecular imaging and radiopharmaceutical chemistry e.g. EMIM and iSRS. Moreover, two papers have been published in open access and more manuscripts are in preparation to be submitted.
The project has been presented at conferences for molecular imaging and radiopharmaceutical chemistry e.g. EMIM and iSRS. Moreover, two papers have been published in open access and more manuscripts are in preparation to be submitted.
A PET tracer targeting P2Y12R will provide detailed information regarding the dynamical changes in microglia activation and be an important milestone in the research regarding neuroinflammation. This can pave the way for improved diagnosis, staging and treatment monitoring of neurological and neurodegenerative diseases. These diseases affect millions of people worldwide and the number of affected people continues to dramatically increase. Consequently, these diseases comprise an enormous medical and socio-economic problem and research that will deliver new information for treatment, diagnosis and/or prognosis will have a huge societal impact. In addition, in MIM has trained a researcher in all aspects of PET tracer development and there is at the moment a shortage of properly trained PET scientists in Europe.