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Novel Atg4B-inhibitors and dual [Atg4B-carbonic anhydrase] inhibitors for interfering with cytoprotective mechanisms of cancer cells in the acidic tumor micro-environment.

Periodic Reporting for period 1 - ONCOPHAGY (Novel Atg4B-inhibitors and dual [Atg4B-carbonic anhydrase]inhibitors for interfering with cytoprotective mechanisms of cancercells in the acidic tumor micro-environment.)

Período documentado: 2017-05-01 hasta 2019-04-30

Cancer cells are remarkably capable of surviving the harsh conditions present in tumors. In this framework, two metabolic adaptations of cancer cells have received significant attention: (1) the presence of chronic autophagy and (2) the strongly increased production of carbonic anhydrases (CAs). Autophagy is a process with which cells recycle old proteins and other life-sustaining building blocks. Autophagy allows them to limit the exchange of materials with their environment and this confers a protective effect, for example against chemotherapy and other cancer drugs. CAs are enzymes that are directly involved in maintaining a neutral pH inside the cells. They protect cancer cells against the tumor microenvironment, which is typically acidic.

The project wants to block these protective processes in cancer cells and, in this way, aims at increasing cellular stress and making cancer cells more sensitive to therapy. Two strategies are followed. 1) Designing,making and testing molecules that block autophagy in cancer cells. These molecules are designed to disrupt the activity of a key player in autophagy: the protein Atg4B. 2) Design, make and test hybrid molecules, consisting of the Atg4B targeting moiety linked to a CA targeting moiety. The combined blocking of autophagy and CAs can be expected to be particularly effective at making cancer cells vulnerable. In the project, the newly prepared molecules are first extensively tested on cancer cells and normal cells. Molecules that combine the highest potency against cancer cells and a lack of toxicity against normal cells, are subsequently investigated in a battery of in vitro tests to predict behaviour in a living organism (e.g. stability in blood, resistance against liver metabolism). Only after maximal in vitro characterization, the single most promising molecule is allowed for testing in tumour-bearing mice.

Cancer is the most common cause of death and morbidity in Europe, after cardiovascular diseases. In spite of continuous advance in the treatment of the disease, challenges to make cancer a 100% curable disease remain immense. The societal relevance of this project therefore is primarily related to the fact that it contributes to improvement of cancer therapy. In addition, autophagy and CAs are also emerging pharmacological targets in other areas (neurodegeneration, cardiovascular disease).

The scientific innovativity of the project can be demonstrated using the following characteristics: 1) It delivers novel, specific autophagy inhibitors that target Atg4B. The relevance of these compounds is clear when taking into account both the currently unmet demand for reliable, specific autophagy inhibitors and the status of Atg4B as a daunting target. 2) To the best of our knowledge, no reports exist on the combined targeting of autophagy and CAs as an anti-tumor strategy. 3) The in vivo study allows to determine the translational potential of the compounds in the framework of cancer therapy and these data will directly support the economic valorization strategy of the work.
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.
The project has delivered the following results that go significantly beyond the state-of-the art:

1) A set of novel Atg4B-targeting autophagy blockers and a large portfolio of associated biological data. These compounds prepared in this project are more potent and have higher plasma stability than the molecules reported in literature.

2) A set of hybrid [Atg4B-CA] targeting molecules. To the best of our knowledge, such hybrid molecules represent a radically novel approach in cancer drug development.

We are confident that the delivered compounds have significantly higher potential for full development into approved drugs than the currently available molecules form other parties. They can therefore contribute to the delivery of new and better cancer treatments and this is the main reason for their societal impact. A main requirement to reach this impact is that the compounds should attract sufficient exploitation interest from industrial parties. To maximize chances of economic exploitation, an exploitation strategy is already operationaland all team members are committed to make this plan successfull.
Unpublished, non-confidential project results.