Design, synthesis and biological evaluation of novel bioactive molecules for the treatment of protozoan and human diseases

DESIGN, SYNTHESIS AND BIOLOGICAL EVALUATION OF NOVEL PSEUDOPEPTIDES AS INHIBITORS OF RHODESAIN OF T. brucei rhodesiense Human African Trypanosomiasis (HAT) is caused by a protozoan of Trypanosoma genus. There are two forms of HAT, the chronic form caused by Trypanosoma brucei gambiense and the acute form caused by Trypanosoma brucei rhodesiense. This infection is transmitted to humans by the bite of a fly belonging to Glossina genus (tsetse fly). There is an urgent need to find new targets for HAT treatment because current therapy shows many problems. In this research area, rhodesain, a cysteine protease that plays a key role in Trypanosoma brucei rhodesiense life cycle, can be considered a promising target for HAT treatment. Our research team has been involved in the last decade into the development of rhodesain inhibitors as novel agents for HAT treatment. In particular, I focused my research on the development of peptidomimetics or pseudopeptides as inhibitors of rhodesain. Starting from the structure of the reversible rhodesain inhibitors 1a-c, we designed a new series of peptidomimetics 2a-g maintaining the benzodiazepine scaffold as a β-turn mimetic. We introduced a characteristic peptide sequence for rhodesain inhibition, i.e. Phe-HomoPhe P2-P1, finally the 3-bromoisoxazoline warhead was replaced with a vinyl ester moiety, able to react as Michael acceptor, while at the P1’ site we selected a panel of aliphatic or aromatic nuclei to evaluate the size of the corresponding S1’ pocket. Moreover, starting from the potent irreversible rhodesain inhibitor 3, we designed a new series of dipeptide nitriles 4-5 a-f , showing a Phe or a Leu residue at the P2 site, strongly preferred by rhodesain. The amino group of the P2 substituent was protected with a series of variously decorated substituents, spanning into the P3 region, with the aim to optimize the interactions with the S3 pocket. While the hPhe residue at the P1 site was kept unchanged, due to the strong affinity for rhodesain S1 pocket; on the contrary, the vinyl ketone warhead was replaced with a nitrile group, to explore its reactivity towards the catalytic cysteine residue. All the new synthesized inhibitors were tested against rhodesain to evaluate their inhibitory properties. Their selectivity profile for the target protease was also evaluated by testing inhibitors against the cathepsin L, a human cysteine protease belonging to papain family, like rhodesain. The most active compounds were also tested against Trypanosoma brucei brucei to evaluate their antitrypanosomal activity. Docking studies on the most active inhibitors will allow us to clarify the binding mode of the new class of inhibitors. During my PhD work I considered the possibility to combine different inhibitors, e.g. a natural product and a synthetized inhibitor, to evaluate the combination effect on the rhodesain inhibition. The first study has been based on the combination of curcumin and RK-52, which showed an impressive potency (k2nd = 67 × 106 M−1 min−1) and a picomolar binding affinity of 38 pM against rhodesain. Curcumin is a natural product obtained from Curcuma longa L. endowed with many biological properties, more recently curcumin was shown to inhibit rhodesain of T. b. rhodesiense with an IC50 value of 7.75 µM. In the present work, we designed drug-combination studies at five selected combined doses by Chou and Talalay method for the evaluation of the potential synergistic or additive effects for the inhibition of rhodesain. Another combination study that has been carried out is based on the combination of quercetin and PS-1. The first one is a polyphenolic compound with a well-known antiparasitic activity. The second one is a rhodesain inhibitor with a Ki in a picomolar range. Also in this case, we evaluated by Chou and Talalay method if the combination PS-1 and quercetin could exert a synergistic or additive effects for the inhibition of the trypanosomal protease. In conclusion, the combination study let us to identify if the combination of two drugs induces an antagonist, additive or synergic effect. If an additive or synergic effect occurs, this could reduce some problems related to the toxicity because in this way the two inhibitors could be administer at a lower dose.