Research comprised the molecular design of new Atg4B targeting autophagy inhibitors. Next, chemical strategies to prepare these compounds were conceived and then experimentally elaborated. 20 Chemically distinct, potential autophagy inhibitors were delivered in this way. All these products and the chemical intermediates en route to these products were characterized using NMR spectroscopy, chromatographic techniques and mass spectrometry. Next, they were progressed to the biological investigation level. The latter involved two types of cellular assays that were applied to all new molecules. In the so-called 'cyto-id' experiment, the compounds' potency to reduce autophagy was quantified by counting autophagosomes via flow cytometry. In addition, complementary experiments were carried out, to quantify cellular levels of LC3-II and p62. The best compounds identified in these experiments were progressed to in vitro characterization of biopharmaceutical parameters: solubility, lipohilicity, plasma stability and microsomal stability. Especially plasma stability is a known liability of published compounds. Overall, obtained results at this stage pointed out that the best novel molecules had significantly improved autophagy blocking potency (3-fold lower autophagosome numbers at 10 µM) than the best published reference. In addition, plasma stability was also increased for the most potent molecule (>4-fold increase of stability half-life) (Figure 1). Finally, a selected Atg4B inhibitor was then evaluated in vivo, in tumour bearing mice. Gratifyingly, obtained data demonstrated that autophagy inhibition indeed increased the sensitivity of tumours to chemotherapy and targeted cancer therapy (Figure 1). Next, the Fellow delivered 4 novel hybrid [Atg4B-CA] blocking molecules, based on the best compounds that he had prepared earlier and based on CA-inhibitors that have been published in literature. The biological evaluation of these compounds is currently ongoing.
So far, the Fellow has published one peer-reviewed publication on his work (Tanç et al. Bioorg. Chem. 2019, 163-168). This paper does not yet cover the most potent molecules he has identified. It was decided to not disclose these compounds yet in order to conserve their potential for patenting. Based on the full evaluation data set of the structurally derived hybrid molecules, a decision on patent coverage will be taken during Q4 2019. Only after that date and after patent submission, the best compounds will be published in the peer-reviewed literature. The patenting issue is important because patenting is prerequisite to support industrial exploitation of the results. In this framework, an exploitation strategy for the Fellow's results has already been designed and the Fellow has actively participated to these talks. One of the clinical co-PIs of the project (Prof. Marc Peeters) is presenting non-confidential project data to the pharmaceutical companies of which he is an Advisory Board member to already raise industrial project interest. Next to peer-reviewed dissemination, the Fellow has also presented his work via poster presentation at several conferences, both to other scientists and to the general public.
