Heterovalent Inhibitors of CARbohydrate-processing Enzymes (Sialidases) – HICARE

The success of the HICARE project will be based on the association of different specialists at the chemistry-biology interface. The team is formed of partners working in complementary domains of research and well identified in their respective fields of competences. The project is well-balanced between the three main task that are 1) the chemical synthesis of the multi- and heterovalent ligands (Partner1 – P1); 2) the measurement of their inhibitory activities and selectivities among the tested sialidases (Partner2 – P2); 3) the determination of the binding modes (Partner3 – P3).
The compounds are designed starting from reported characteristics of the targeted SA. A first set of bivalent ligands have been designed to interact in a (hetero) chelate binding mode with the sialidase. A chelate binding mode may operate if the distance between binding epitopes can span the distance between the catalytic site and CBM. The linker length has been varied and adjusted to give the maximal effect. To further increase the affinity, additional ligands of the CBM or of catalytic domains will be grafted on polymer scaffolds. The future class of molecules will be composed of heterovalent ligands randomly presented on a biologically compatible polymer scaffold . This should allow the formation of large aggregates with membrane-bound sialidases that are highly dense and mobiles on the cell surface, or released extracellularly. This aggregative process allow the tight binding of the polymeric inhibitor on the parasite or bacterial surface. To probe the validity of the concept, we selected therapeutically relevant exo-sialidases from T. cruzi, A. fumigatus, V. cholera and S. pneumoniae. They are representative of the structural variety founded in GH33 family in terms of additional carbohydrate binding sites besides the catalytic site . All the structures of these sialidases have been solved and are available from the protein data bank.

We have designed non-hydrolysable multivalent thio-sialosides as probes and inhibitors of biologically relevant sialidases. A polymeric compoundwas by far the most active showing unprecedented levels of inhibitory capacity and binding affinity against the biologically relevant TcTS and NanA sialidases. Each thiosialoside ligand attached to the polymer backbone surpassed the inhibitory capacity of the corresponding monovalent reference by more than three orders of magnitude. Thus, polymeric thiosialosides and sialidases can reach the high level of inhibition previously reported for glycopolymers and lectins. This is relevant considering the difficulty in designing potent sialidases and trans-sialidases inhibitors. Importantly, the expression of the NanA (from S. pneumoniae) truncated domains NanA-C (catalytic) and NanA-L (lectinic) allowed a better understanding of the respective functions of the catalytic and lectinic domains of the pathogenic sialidases. The lectinic domain improves NanA binding to a sialylated SPR surface by more than two orders of magnitude . The affinity profile for the thiosialylated BSA was nearly identical for NanA and NanA-L, and two-orders of magnitudes lower for NanA-C. Thus, the binding to a sialylated surface is almost exclusively promoted by the lectin domain. The polymer exerted very high inhibition of the enzymatic activity (on both NanA and NanA-C ). This suggests that the activity of sialidases lacking a lectin domain can be efficiently inhibited by polymeric thiosialosides .
The design of polymeric thiosialosides led to highly potent sialidase inhibitors with high sygnergistic multivalent effects. Binding affinities were significantly increased with sialidase lectin domains, but strong inhibition of the enzymatic activity was observed on the catalytic domain alone.

The design of polymeric thiosialosides led to highly potent sialidase inhibitors with high sygnergistic multivalent effects. Binding affinities were significantly increased with sialidase lectin domains, but strong inhibition of the enzymatic activity was observed on the catalytic domain. The design of multivalent glycosidase modulators is a promising emerging field as illustrated by the continuous reports of potent glycosidase activators or inhibitors based on clusterized iminosugars or sugar substrates. This concept is now extended to the inhibition of bacterial and pathogenic sialidases for which transition-state inhibitors fail to reach the submicromolar level.
We are now designing polymeric compounds with more potent substrates in order to potentially provide compounds with outstanding inhibory potency for bacterial and parasitic sialidases

The first results have been published in a high impact journal (Chem. Eur. J IF = 5) as a hot paper.
Multivalent thiosialosides and their synergistic interaction with pathogenic sialidases. Brissonnet, Y. ; Assailly, C.; Saumonneau, A.; Bouckaert, J. ; Maillasson, M. ; Petitot, C. ; Roubinet, B. ; Didak, B. ; Landemarre, L. ; Bridot, C. ; Blossey, R. ; Deniaud, D.; Yan, X.; Bernard, J.; Tellier, C.; Grandjean, C.; Daligault, F.; Gouin, S.G.* Chem.Eur.J. 2019, 25, 2358-2365.

Carbohydrate-hydrolyzing enzymes (glycosidases) are involved in a host of biological processes and are therapeutic targets. During recent decades, tremendous efforts have been dedicated to designing potent and selective glycosidase inhibitors. Potential candidates often lack glycosidase selectivity and the resulting non- specific inhibition generally leads to severe side-effects. Limiting selectivity issues due to unwanted inhibition of related glycosidases is a challenge not achieved with the first generation of inhibitors. The aim of this project is based on an unprecedented approach for reaching high affinities and selectivities for carbohydrate-processing-enzymes with a special focus on sialidases. Many sialidases of biological interest possess carbohydrate-binding modules (CBM) in close proximity of the catalytic site (CAT). They functions as anchoring points to the targeted glycans, increasing the catalytic efficiency of the system. Sialidases with both CAT and CBM are particularly promising therapeutic targets common to a tremendous number of pathogenic bacteria and protozoan. Their extracellular expression should facilitate drug accessibility and will impair several internal resistance mechanisms of bacteria such as chemical drug modification, reduced intracellular accumulation and increased active efflux. Importantly, the human sialidases (Neu1, Neu2, Neu3) that are implied in important physiological functions, do not express CBM, and that should limits the potential side-effects of the heterovalent inhibitors of carbohydrate-hydrolyzing enzymes (HICARE) proposed here. The inhibitors should considerably increase the affinity and selectivity for the targets through multivalent and avidity effects. As far as we know, this concept of heterovalent targeting has never been applied to parasitic or microbial sialidases. The bifunctional compounds bearing one or several ligands of the two domains separated with appropriate spacers should strongly and selectively interact with the enzymes through specific (hetero) multivalent effects including chelate binding modes, aggregative process and bind and recapture mechanisms. The HICARE project include i) the chemical synthesis of the heterovalent ligands guided by computational methods, ii) the production of recombinant sialidase from pathogens in their full and truncated version and the assessment of the compounds inhibitory activities against the sialidases, iii) the determination of the multivalent binding modes by analytical techniques such as microcalorimetry or surface plasmon resonance. These three tasks will be conducted by a tryptic network of chemist-biochemist- biophysicians, experts in their respective fields. If successful, the HICARE project will be highly stimulating for the scientific community working in this topic considering the huge pharmaceutical potential in pathologies such as African and American trypanosomiases (T. brucei and T. cruzi), bacterial infections of the intestinal tract (C. perfringens or V. cholera), bacterial infections in cystic fibrosis ( A. fumigatus).