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    • MK-8745 After initial validation of the ability of Ad Cre vi

    2021-12-01

    After initial validation of the ability of Ad5-Cre virus to express Cre recombinase protein in glial cells in vitro and in vivo, we employed this virus to induce site-specific deletion of our target GlyT1 in the thalamus, through injection of the cKO GlyT1 mice. This transgenic line was constructed with loxP sites flanking the GlyT1 gene, so that Cre-induced site-specific recombination would result in the production of a non-functional GlyT1 transporter. Comparison of GlyT1 binding level in the Ad5-Cre injected area with both the signal in the Ad5-GFP or vehicle injected area revealed a statistically significant reduction in the GlyT1 binding. The reduction of GlyT1 binding activity as measured by autoradiography was approximately 40% relative to vehicle. Since gene deletion is dependent upon viral infection and subsequent Cre expression, it is very likely that this modest, though significant, reduction could be explained by suboptimal levels of viral infection in the thalamus at the dose of vector used in this study (estimated to be approximately 50–60% of glial cells). It is therefore not unexpected to achieve a lower level of gene deletion using a viral approach in adult mice than what can be achieved utilizing a conventional germline deletion strategy. It is noted that in the recent report of Eulenburg et al. (2010), such a germline approach yielded a 60–70% reduction of GlyT1-specific uptake. A viral approach has the advantage of allowing one to be much more specific in the delivery of Cre protein and hence mediate deletion in specific, predetermined locations in the brain, critical where a gene has different functions in different MK-8745 regions. Furthermore, in some situations, it may be advantageous to reduce the level of expression of a target gene, rather than to ablate it. It is reasonable to assume that by increasing the dose of Ad-Cre virus injected, a higher percentage of cells would be infected, resulting in an increased number of cells undergoing gene deletion and thus exhibit a greater reduction in GlyT1 ligand binding. It is noted that, in our study, GlyT1 binding activity was not measured exclusively in the Ad5-Cre transduced area, due to the restricted area of viral vector diffusion, and therefore the actual level of GlyT1 suppression is likely to be underestimated. In conclusion, our study demonstrates the efficacy of using Ad5-Cre virus to mediate site-specific recombination of a target gene in the thalamic nuclei of conditional loxP transgenic mouse model, allowing the study of the functional consequence of GlyT1 reduction in specific brain nuclei. The thalamus plays a central role in filtering and relaying information between a variety of subcortical areas and the cerebral cortex. A defect in gating or filtering in this circuitry could lead to input overload seen in schizophrenia (Young et al., 2000). Thus, targeting the thalamus could shed some light over the role of GlyT1 in psychiatric disorders. Moreover, by modulating the area of viral vector diffusion, it will be possible to explore in more detail GlyT1 function in other distinct brain areas. Strategies such as this are potentially extremely useful in the study of genes of neurobiological interest.
    Scaffold hopping has emerged as an attractive approach to rapidly access new chemical space and enable fast-follower programs without the need for expensive and time-consuming HTS campaigns., , , As the negative symptom cluster in schizophrenia remains a critical unmet medical need,, , and GlyT1 inhibition has been shown to be affective toward negative symptoms in Phase II clicnial trials,, , , , , , we initiated scaffold hopping efforts to expediently develop novel GlyT1 inhibitors within a crowded intellectual property (IP) space. In a recent Letter, we reported on our preliminary scaffold hopping exercise () employing GlyT1 inhibitors from Merck and Pfizer, and , respectively, that generated a novel, patented series exemplified by . Notably, was a potent GlyT1 inhibitor with an exceptional DMPK profile, high CNS penetration and robust efficacy in preclinical models of schizophrenia.