In detail

Bruxelles Woluwe


Tryptophan catabolism is an important mechanism of peripheral immune tolerance contributing to tumoral immune resistance, and indoleamine 2,3-dioxygenases (IDO and TDO) inhibition is a promising strategy for anticancer drug development. IDO and TDO are unrelated heme-containing enzymes catalyzing the oxidative cleavage of the indole ring of L-tryptophan (L-Trp), the first and rate-limiting step along the kynurenine pathway. The implication of IDO in the phenomenon of tumoral immune resistance is the focus of intense researches and the enzyme is now recognized as a validated target for anti-cancer therapy. Therefore, a number of groups, including us, are actively searching for novel original IDO inhibitors. In contrast, the effect of TDO expression on the immune response has only been recently investigated in detail. Indeed, we showed in collaboration with the group of Prof Van den Eynde that TDO was effectively overexpressed by a number of human tumors and that this expression prevented rejection of tumor cells. We designed a novel TDO inhibitor and proved, in a preclinical model, the concept that TDO inhibition promotes tumoral immune rejection. Interestingly, blocking both TDO and IDO to improve the efficacy of cancer immunotherapy would be complementary: in a series of 104 human tumor lines of various histological types, we showed that 20 tumors expressed only TDO, 17 only IDO and 16 expressed both enzymes. Therefore, targeting both IDO and TDO would allow reaching 51% of tumors instead of either 32% or 35% with a compound inhibiting IDO or TDO alone, respectively. The design of IDO, TDO or dual IDO/TDO inhibitors is thus of major importance. Interestingly, our recent works (PhD thesis of Arina Kozlova) led to the discovery of new TDO inhibitors. These data (still unpublished) are very encouraging to pursue the search for new anticancer agents targeting Trp catabolism.

Besides Trp catabolism, Arginine (L-Arg) catabolism by Arginase 1 (Arg1) is another mechanism contributing to tumoral immune resistance. In recent works (PhD thesis of Julien Prévost and Juhans Dechenne), we have identified novel boronic acids compounds as promising Arginase inhibitors.


The last ten years have witnessed an increased regain of interest for tumor metabolism. Recent advances in this field have shed light on how tumors fuel rapid growth by preferentially engaging biosynthetic pathways. Although cellular metabolic pathways are rich pickings for drug targets, pinpointing enzymes that critically contribute to tumor metabolism is key to establish a therapeutic window since most of metabolic enzymes also play important roles in normal tissues.

1. PHGDH (3-phosphoglycerate dehydrogenase) and PSAT1 (phosphoserine aminotransferase-1) represent ideal targets for new anticancer strategies. These enzymes catalyze the first and second steps in the serine biosynthetic pathway, respectively. This pathway diverts a relatively small fraction of 3-phosphoglycerate from glycolysis to generate serine as well as equimolar amounts of NADH and α-ketoglutarate (αKG).

In this project (PhD thesis of Quentin Spillier), our aim is to understand the role of the serine pathway in tumor progression and in particular to develop pharmacological tools to evaluate the extent of tumor addiction to this metabolic path and their therapeutic potential by exploring potential side effects on healthy tissues. To this end, novel innovative pharmacological inhibitors of PHGDH the first enzyme of the serine pathway, were designed and chemically synthesized.

A screening campaign of an in-house library of compounds was performed and led to the serendipitous discovery of two original series of very active PHGDH inhibitors: the first are derived from the FDA-approved drug Disulfiram, whereas the second is characterized by an original α-ketothioamide scaffold. Interestingly, the use of chemical biology methodologies such as cross-linking experiments and an original photoactivatable diazirine chemical probe (unpublished results) with mass spectrometry experiments led us to hypothesize that each series would act through a covalent allosteric mechanism, probably involving the ACT-regulatory domain, hence promoting disruption of the PHGDH tetramer. Although repurpos ing of Disulfiram is suggested and PHGDH inhibition could account for its anticancer activity, its direct optimization is not our intend here, but these preliminary results inspire the design and discovery of more specific covalent modifiers.

2. Tumor cells are also characterized by a remarkable metabolic plasticity allowing them to survive and proliferate in hypoxic and extracellular acidic environments. In tumor cells, this plasticity allows the coexistence and coordination of several metabolic phenotypes, leading to an optimal use or resources. Hypoxic cells uses glucose that is metabolized by anaerobic glycolysis. Lactate is secreted and diffuses, and can be subsequently used by oxygenated tumor cells as a preferred energetic source to glucose. The lactate oxidative pathway requires the entrance of lactate in oxidative cells via a process that is mainly facilitated by the Monocarboxylate Transporter MCT1 and the oxidation of lactate to pyruvate by the lactate dehydrogenase B (LDHB). The pyruvate can then fueled the Krebs cycle and NADH uses the malate-aspartate shuttle to directly fuel the mitochondrial respiration chain. The oxidative use of lactate in the oxygenated tumor compartment therefore optimizes the availability of glucose for cells of the hypoxic compartment, thus constituting a unique metabolic cooperation. If the use of lactate by oxidative cells is a proven fact, the advantage it gives them remains largely unknown. A first series of studies showed that lactate can act as a proangiogenic agent. This signaling activity also depends on the oxidation of lactate to pyruvate by LDHB, allowing pyruvate to inhibit enzymes of the prolylhydroxylase family and activate the hypoxia-inducible transcription factor factor-1 (HIF-1) independently of hypoxia. In addition, a recent collaborative led with the team of P. Sonveaux (IREC) has shown that the oxidative use of lactate promotes autophagy, ie, a process of degradation and recycling of proteins and organelles requiring formation of specialized structures, autophagosomes, and their fusion with lysosomes. To promote autophagy, LDHB physically interacts with VATPase, a proton pump located on the surface of lysosomes, which it feeds with the protons produced during the lactate + NAD+ -->/<--pyruvate + NADH + H+ reaction. This observation seems important to us as autophagy participates in tumor progression by recycling damaged proteins and organelles when cancer cells are exposed to oxidative stress, and because it provides cells with energy substrates under metabolic stress conditions.

All these observations suggest that LDHB may be a new target in cancer therapy. However, there is currently no specific inhibitor of this enzyme, and the consequences of systemic inhibition of LDHB activity remain largely unknown.

In this project (PhD thesis of Léopold Thabault, Chiara Brustenga & Perrine Savoyen), our aim is thus to develop and validate a peptide inhibitor and a non-peptide inhibitor to selectively inhibit tetramerization of LDHB. Our strategy will involve the use of Protein-Protein Interaction Inhibitor (PPI) identification methods that is, a highly multidisciplinary approach involving molecular modeling studies (identification of "Hot Spots"), biochemical studies (in vitro and in vivo inhibition of LDHB tetramerization, selectivity study) and biophysical studies (nuclear magnetic resonance analysis of ligand-LDHB interaction). To achieve the goal of a selective inhibition of LDHB, we will use an innovative strategy targeting the tetramerization site of LDHB rather than the active site of the enzyme. So far, our pivotal collaborative works led to (a) the delineation of hot spots at the LDH tetramerization site, (b) the design and synthesis of original (stapled) peptides capable of preventing LDH self-association and/or disrupting a preformed LDH tetramer, and (c) the development of some chemical biology tools to interrogate LDH tetramerization using NMR spectroscopy (STD and WaterLogSy experiments), thermal shift, microscale thermophoresis, and fluorescence spectroscopy experiments.

Ferroptosis, first coined in 2012, is a regulated cell death (RCD) characterized by iron-dependent accumulation of lipid hydroperoxides associated with an insufficient capacity to eliminate these oxidation products. A recent report uncovered acyl-CoA synthetase long-chain 4 (ACSL4) as a critical contributor to ferroptosis execution. Therefore, ACSL4 inhibitors are emerging as attractive anti-ferroptotic agents. The goal of our research program is to develop novel ACSL4 inhibitors to help establish the potential link between ACSL4, ferroptosis and NDDs. On a longer-term perspective, it should constitute a strong basis for the development of first-in-class drugs for the treatment of NDDs.

The present project grounds on important preliminary findings generated in our lab (PhD thesis of Romain Marteau). A screening of the Selective Optimization of Side Activity (SOSA) library against ACSL4 was undertaken by TSA. Typically, this approach starts with the screening of a set of limited and structurally-diverse drug-like compounds known to possess good bioavailability and safety in humans. So far, we identified three series of molecules that stabilize the folded state of ACSL4 and we validated these hits in our optimized enzymatic assay. The identified micromolar-range inhibitors of ACSL4 represent original starting points for our lead discovery program.

As the phenomenon of antibiotic resistance is dramatically increasing these days, the search for new therapeutic targets less vulnerable to these resistance mechanisms appears as a real need. The cell wall of bacteria and the enzymes that are involved in its synthesis are prime targets for many antibiotics, which inhibit the late stages of peptidoglycan biosynthetic pathway. But the resistance phenomena have revealed the high flexibility in this assembly pathway, and the need to target other enzymes acting on earlier steps of peptidoglycan synthesis. D-alanyl-D-alanine ligase (Ddl) is of particular interest as it utilizes a substrate (D-alanine) which is specific for bacterial peptidoglycan biosynthesis and essential for bacterial growth.

In this work (PhD thesis of Alice Ameryckx), we aim at designing novel DD-ligases inhibitors. Previous works in our group have highlighted a novel hit (S89) characterized with thiosemicarbazide motif. First, analogues of S89 were synthesized. Indeed, the thiosemicarbazide family is very promising due to its low half maximal effective concentration (EC50) and its good antibacterial activity. These compounds will be evaluated on recombinant protein Ddl-His6 produced and purified in our group. This study will provide initial structure-activity relationships (SAR) and thus help understanding the structure requirements to achieve a high DD-ligases inhibition. Then, novel hits will be identified through a fragment-based strategy. To this end, an in-house library of 280 diverse fragments will be first assessed. Finally, the more potent fragments will undergo a structure guided optimization to design potent DD-ligases inhibitors.

Many diseases such as cancer (solid tumors), ischemia, stroke or infection lead to the development of local hypoxia and acidosis. Acidosis results from enhanced glycolytic flux which produces lactate and H+ ions (but also in tumors from hydration of CO2 which represents another source of H+ ions). Hence, extracellular acidity might serve as a general marker for detecting and targeting ischemic tissues and tumors. However, since the bulk extracellular pH in these diseased tissues is only 0.5–0.8 pH units lower than the extracellular pH in healthy tissue, this strategy remains particularly challenging. Several pH-sensitive imaging and drug delivery systems have actually been envisioned as diagnostic or therapeutic modalities specifically triggered by the acidic tumor microenvironment. Among these are the pH Low Insertion Peptides (pHLIP) family derived from the bacteriorhodopsin C helix. This family represents a unique class of water-soluble membrane polypeptides which were found to insert across a membrane to form a stable transmembrane α-helix.

In the last years, pHLIP’s were investigated in various fields and for instance combined with fluorescent dyes in order to target different disorders. In vivo studies were performed to target tumors, ischemic myocardium, the sites of inflammatory arthritis and infections. More recently, the very first examples of pHLIP linked to chemotherapeutic agents were published: paclitaxel, doxorubicine and monomethyl auristatin F.

In our research project (Phd thesis of Marine Deskeuvre) we are studying, developing and applying the pHLIP technology in two promising fields of cancer therapy: tumor lipid metabolism and the response of T cells to tumor microenvironment acidification.

Sarcomas, neuroblastomas and brain tumors frequently activate an alternative and telomerase-independent mechanism of telomere maintenance, dubbed ALT, based on homologous recombination events between telomeric sequences. Being absent from normal cells, the ALT mechanism offers new interesting perspectives for specific and targeted anticancer therapy. However, “druggable” ALT-specific targets are still awaiting identification.

TSPYL5 is suggested as a possible ALT target candidate. Recent discoveries indicated that TSPYL5 depletion induces ALT+ cell death without impacting normal or telomerase-expressing cells. Cell death results from strong DNA damage activation in response to telomere deprotection due to the proteasomal degradation of POT1 telomeric protein. Our current hypothesis is that, through its competitive binding to USP7 deubiquitinase, TSPYL5 inhibits the recruitment of USP7 to telomeres and the subsequent degradation of POT1.

To give a better understanding this ALT+ mechanism, we will focus, in collaboration with the team of Anabelle Decottignies, on the TSPYL5-USP7 complex. Biophysical and biochemical experiments will be undertaken to identify the hot spots of this interaction and to discover small molecules capable of disrupting the TSPYL5-USP7 interaction. These pharmacological tools will help establishing the proof-of-concept that TSPYL5-USP7 disruption can exert anticancer activity in ALT+ tumour cells.