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A unified drug discovery platform for protein misfolding diseases

Periodic Reporting for period 2 - ProMiDis (A unified drug discovery platform for protein misfolding diseases)

Reporting period: 2020-09-01 to 2022-02-28

It is widely recognized that a variety of major diseases, such as Alzheimer’s disease, Huntington’s disease, systemic amyloidosis, cystic fibrosis, type 2 diabetes etc., are characterized by a common molecular origin: the misfolding of specific proteins. These disorders have been termed protein misfolding diseases (PMDs) and the vast majority of them remain incurable. In ProMiDis, I am developing a unified approach for the discovery of potential therapeutics against PMDs. More specifically, I am generating engineered bacterial cells that function as a broadly applicable discovery platform for compounds that rescue the misfolding of PMD-associated proteins (MisPs). These compounds are being selected from libraries of drug-like molecules biosynthesized in engineered bacteria using a technology that allows the facile production of billions of different test molecules. These libraries are then screened in the same bacterial cells that produce them and the rare molecules that rescue MisP misfolding effectively are selected using an ultrahigh-throughput genetic screen. The effect of the selected molecules on MisP folding is then evaluated by biochemical and biophysical methods, while their ability to inhibit MisP-induced pathogenicity is tested in appropriate mammalian cell assays and in established animal models of the associated PMD. The molecules that rescue the misfolding of the target MisPs and antagonize their associated pathogenicity both in vitro and in vivo, will become drug candidates against the corresponding diseases. This procedure is being applied for different MisPs to identify potential therapeutics for at least four major PMDs: Huntington’s disease, cardiotoxic light chain amyloidosis, dialysis-related amyloidosis and retinitis pigmentosa. Successful realization of ProMiDis will provide invaluable therapeutic leads against major diseases and a unified framework for anti-PMD drug discovery.
We have generated engineered bacteria with the ability to biosynthesize billions of different test molecules. These molecules belong to a certain class of compounds called cyclic peptides. We have developed a technology that allows us to produce cyclic peptides that contain not only naturally looking, but also multiple non-naturally-looking chemical entities. In this manner, we can expand significantly both the number of cyclic peptides that we are studying, but also their chemical complexity. When combined, these two characteristics increase significantly the chances of identifying molecules with the desired properties. Up to now, we have used these modified microbes to look for molecules that correct the problematic shapes of seven different proteins related to serious human diseases. We have already identified hits for five of them and we are currently validating these hits in the bacteria and it the test tube.
We have recently reported the largest screen of small molecule–like molecular entities described to date with the ability to perform direct functional screening for aggregation-inhibitory activity, which can go beyond simple detection of binding to the target protein. Currently, we are in the process of expanding these capabilities and limits even further. Furthermore, we have developed a technology that allows the simultaneous incorporation of multiple non-natural amino acids in combinatorial peptide/protein libraries during the screening process in living cells. This will significantly expand the levels of molecular and chemical diversity which can be achieved in directed peptide/protein evolution experiments performed in living cells. Based on the technologies, which are being developed as part of ProMiDis, we have established the spin-off company ResQ Biotech (www.resqbiotech.com). Recently, Nature has selected ResQ Biotech in the framework of the 2020 The Spinoff Prize among the most exciting science-based companies to have emerged from academic labs in the past three years [https:// www.nature.com/articles/d41586-020-01904-6].
During the second period of the project, our goal is to validate the hit molecules that we have acquired in established cellular and small animal models of the target PMDs. Ultimately, our goal is to identify potential therapeutics for at least four major PMDs. Even more importantly, by the end of this project we want to demonstrate that the approach developed in the framework of ProMiDis is a platform technology, which can be readily applicable to potentially any type of disease caused by protein misfolding.