Periodic Reporting for period 1 - SECRET-DOCK (Transient inactivation of hypothalamic hormone release to prevent post-traumatic stress sensitization)
Periodo di rendicontazione: 2022-09-01 al 2024-02-29
Stress is the foremost consequence of human life, and became a pressing societal burden through the many sensory and societal pressures that have evolved during the past decades. Severe stress induces maladaptive changes in the brain that clinically manifest as post-traumatic stress disorder (PTSD). Despite ~4% of the population presenting PTSD, only symptomatic therapies are available. In the ’SECRET-CELLS’ ERC award, we have identified a multimodal neurocircuit that induces brain-wide sensitization to stress through the sequential recruitment of corticotropin releasing hormone (CRH)-containing neuroendocrine command cells in the hypothalamus and cortically-projecting norepinephrinergic neurons. Particularly, we developed mouse models to interrogate molecular underpinnings of fast, pulsatile hormone release from the hypothalamus into the blood stream. By using unbiased proteomics in combination with reverse single-cell transcriptomics to identify protein interactors, we have mapped probable druggable targets of an intracellular signaling cascade to inhibit the initiation and/or execution of hormone exocytosis. The 'SECRET-DOCK' project was aimed at evaluating the hypothesis that the inhibition of protein-protein interactions at hormone release sites could lead to the transient and reversible cessation of CRh release and peripheral sensitisation to stress.
We have first tested biochemical parameters (dynamics, stoichiometry) of direct protein-protein interactions by using recombinant proteins in vitro. Subsequently, we used X-ray crystallography to define the structure of druggable candidates in complex, with particular focus on their interaction sites/surfaces. Thereafter, a high-throughput screen was established to identify inhibitors. Templates were then used for small-molecule inhibitor discovery, in silico optimization, and the synthesis of derivatives. Finally, the candidates’ pharmacology and cytotoxicity were profiled in cells in vitro.
We discovered novel protein-protein interactions that participate in the control of vesicle entry into and recycling within release terminals of hypothalamic neurons. Using a reverse template-inhibition screen, we have identified original compounds and their derivatives (179 in total), which significantly inhibited protein-protein interactions of biological relevance. Thereafter, we have synthesised 81 analogues of 4 lead compounds for structure-based optimisation. Finally, we have performed in-cell evaluation, including the assessment of general cytotoxicity and biological activity, on three cell lines (derived from human, rat and mouse, and with different cellular origins and characteristics), and scheduled hit-characterisation for the top 2 candidates. Thus, we have succeeded in 1) producing a novel biological theory, 2) defining the strucutural basis of this theory, 3) using the foreground knowledge to develop a screening platform, and 4) identifying, selecting, optimising, and testing for basic safety the lead compounds that could be amenable to reversibly inhibiting protein interactions, thus acutely reducing hormone release. Demonstration of biological activity, specificity for the site of action, and tolerability in model organisms will be necessary for IPR protection, investor identification, and commercialisation.