Resistance to chemotherapy represents the major cause of treatment failure in cancer patients. Similarly, an increased bacterial resistance to conventional antibiotics is reported worldwide and represents an urgent public health threat. In the search for new safe and effective drugs, increasing attention is devoted to biologically active compounds derived from non-traditional and unexplored sources, in order to obtain innovative therapeutic solutions.

Antimicrobial peptides (AMPs) are small molecules produced by all organisms and are attractive candidates for the design of new antimicrobial agents because of their efficacy and a low propensity for the development of resistance. Some of the cationic AMPs which are toxic to bacteria but not to normal mammalian cells, exhibit a broad spectrum of cytotoxic activity against cancer cells being able to avoid the shortcomings of conventional chemotherapy, and are indicated as Anti Cancer Peptides (ACPs), but their mechanisms of actions are poorly understood. Hypothesis Biologically active peptides can represent useful alternatives being characterized by high therapeutic efficacy, a low probability of resistance emerging in target cells, and limited side effects. To this aim, the exploitation of new compounds and the identification of their mechanisms of action is essential for further development. Although antimicrobial peptides are produced by all organisms, insects represent the richest source of these molecules.

The larval stages of Hermetia illucens, an insect known as the black soldier fly, produce several AMPs reported to be active on bacterial strains pathogenic for humans. Thus, H. illucens is an excellent model system for the identification of new AMPs and ACPs. The present research project proposes the production, by chemical synthesis and recombinant methodologies, the characterization of the mechanism of action and the evaluation of antimicrobial and antitumor activity of different peptides derived from the insect H. illucens.

These activities will contribute to identify molecules with novel mechanisms of action potentially lacking toxicity to healthy cells and refractory to current resistance mechanisms. Experimental Design H. illucens larval and adult transcriptomes are already available and analyzed with the identification of potential AMP/ACP and two of them have been already synthesized and are available for subsequent analyses.

The already available transcriptomes will be further analyzed to identify other potential AMP/ACPs. In the meantime, peptides will be extracted from the soluble fraction of H. illucens larvae and adults and from larvae following immunization with bacteria. Extracted samples will be analysed by LC-MS/MS to identify AMPs specifically expressed and their variations in different conditions. Moreover, new transcriptomes will be built from immunized larvae and analyzed. Cross-analysis of transcriptomic and proteomic data will allow to identify the most promising AMP/ACP which will be then produced by chemical synthesis or recombinant methodology. Purified AMPs will be tested on a unique library of bacterial strains, most of them resistant to traditional antibiotics. MIC and MBC as well as their potential synergistic activity with conventional antibiotics will be defined for each putative AMP together with the possibility to encapsulate them in drug delivery systems to increase their activity. In parallel, AMPs effects will be evaluated on bacterial adhesion and biofilm formation in static or dynamic growth conditions. Their enzymatic proteolytic and degradation activity on biofilms will be also assessed. Effects of putative ACPs will be evaluated on the in vitro phenotype of cancer and cancer stem cells isolated from different human tumors and the molecular underlying mechanisms will be analyzed. Effects of ACPs will be also tested on the release as well as the cargo of exosomes released from both cancer and non-transformed cells. Finally, they will be tested in vivo in a model of tumor xenografts in mice. Functional proteomics approaches will be used to investigate AMP/ACP mechanisms of action and for the identification of their specific interactors in protein extracts from AMPs-treated bacteria and ACPs-treated cancer cells, respectively. Proteins differentially expressed in cells treated with ACPs will be also investigated.

This research project will use an integrated approach comprising in-silico screening as well as fine molecular, functional and pharmacological characterizations and a series of in vitro/in vivo tests, to identify novel insect-derived antimicrobial and/or antitumor peptides. The results obtained might allow the identification of previously unexplored mechanisms of action, which may foster the development of new, valid therapeutic strategies as well as the design of new drugs helpful in overriding resistance to currently available antibiotics and/or antineoplastic drugs. 

Partner:

1. Università Cattolica del Sacro Cuore (Coordinator);
2. Università degli Studi della Basilicata; 
3. Università degli Studi di Napoli Federico II;
4. Università degli Studi di Roma "La Sapienza";
5. Università degli Studi di Catania.