Insect peptides are key elements of the innate immunity against bacteria and fungi. Thesemolecules offer remarkable properties: high efficacy, a low probability of resistance, limited toxicity, andimmunogenicity. In this context, we are investigating several classes of peptides, and we have beensuccessful in identifying biologically important classes of peptides and small molecules that will providea stream of drug candidates for treating severe, life-threatening, hospital-acquired infections and otherpathologies of high medical need. Recently, we have isolated a new class of antifungal peptides from thecoleopteran
Acrocinus longimanus. Three homologous peptides, Alo-1, Alo-2, and Alo-3, with sequenceidentity above 80% and active against the
Candida glabrata yeast strain were identified. Alo-3 displayedthe highest activity against
Candida glabrata and was thus chosen for structure determination using NMRspectroscopy and molecular modeling. Alo-3 contains six cysteine residues forming three disulfide bridges.The pairing of the cysteines was assessed using ambiguous disulfide restraints within the ARIA software,allowing us to establish that Alo-3 belongs to the inhibitor cystine-knot family. It exhibits all the structuralfeatures characteristic of the knottin fold, namely, a triple-stranded antiparallel
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-sheet with a long flexibleloop connecting the first strand to the second strand and a series of turns. To our knowledge, Alo-3 is thefirst peptide from insects with antimicrobial activity adopting the knottin fold. Alo-3 shows a level ofactivity significantly higher against
C. glabrata than Alo-1 or Alo-2. It has no negatively charged residuesand displays on its surface a cationic pole that may account for its antifungal activity. This finding isvalidated by the comparison of the structure of Alo-3 with the structure of other structurally related peptidesfrom other sources also showing antifungal activity.