Novel peptides of the venom of the Hymenoptera (wasps and ants)
Isolation, structure elucidation, synthesis and biological properties

Insect antimicrobial peptides have been found mainly within the haemolymph and in the venom. The venom of the hymenoptera is a complex mixture of low molecular weight chemicals, high molecular weight proteins and small to medium size peptides which acting together produce various biological effects. Stinging with the venom causes severe prolonged pain, inflammation, local tissue damage, and may result even in the death of large vertebrate. The social wasps are using venom for the self-defense against the predators. The venom of solitary wasps paralyzes insects or spiders and their larvae feed on the paralyzed pray. The peptide constituents of wasp venom studied up to now were identified as cationic peptides featuring amphipathic alpha-helical conformation permitting them to interact specifically with the anionic components of the cell or bacterial membranes by the way which triggers the cascade of reactions leading to biological responses, such as killing of bacteria, viruses and fungi, mast cell degranulation, lysis of the cells, increase of the chemotaxis of neutrophiles, activation of leucocytes in the inflammation site etc. These peptides are represented by tetradecapeptide amides of mastoparan group, chemotactic peptides (dodeca- or tridecapeptide amides) and neurotoxic kinins (related to nonapeptide bradykinin). Kinins were studied as a research tool to elucidate the action mechanisms of ion channels or characterizing receptor functions, and some of them are of clinical interest for neurological disorders. Large number of wasp venom peptides display only partial or no structural homology with the peptides of the groups mentioned above. Sometimes, their primary structures are to certain degree homologous to mastoparans, chemotactic peptides or kinins, but they do not posses appropriate biological activity. Few of them also contain intramolecular disulfide bridge in the molecule. Ponericins, the AMPs isolated from the venom gland of ant subfamily Ponerinae, represent abundant group of 24 to 30 amino acid long peptides.

Scientists estimate that there are more than 300 000 species of Hymenoptera in the world; however the peptides from the venom of only tens of wasp species and only a few ant species have been characterized chemically.

Since there are only few published reports about the peptide components and biological properties of wasp and ant venom of the species inhabiting the European continent, we have been initiating the study of the chemistry of the venom peptides isolated from the species of Hymenoptera collected in the area of the Czech Republic. Hymenoptera used for our study have been classified with the help of entomologists from Charles University, Prague.

The venom reservoirs of wasps or ants are obtained by dissection, venom is extracted and the peptides are isolated by RP-HPLC. The primary structures of peptides are determined with the help of mass spectrometry, enzymatic digestions and Edman degradation. Several peptides of mastoparan group and their analogues were already synthesized by the method of solid phase peptide synthesis providing a material for biological assays. In the antimicrobial test using the disc diffusion method carried on Petri dishes, some of these synthetic peptides exhibited strong antimicrobial activities against Bacillus subtilis and Escherichia coli in addition to the mast cell degranulation activity.

At the moment, biological testing of the isolated substances and their synthetic analogues consists primarily from the assay of antimicrobial activities using different gram positive and gram negative bacteria, hemolytic test using rat red blood cells, mast cell degranulating activity using rat mast cells and kinin activity (rat uterus). We intend to introduce also the test for the chemotactic activity.

Inhibitors of microbial enzymes

Another part of our research is focused on potential inhibitors of microbial enzymes, which catalyze metabolic processes leading specifically to pathogenic bacteria. Since there are no similar pathways in mammals, the inhibitors could provide selective toxicity against bacteria with no effect on humans.

During this program we are synthesizing and testing new derivatives of selected amino acids with aim (i) to study the role of microbial enzymes in vivo, (ii) to study the interaction of new compounds with the active site of enzymes and (iii) to test possible application of new amino acid derivatives as antibiotics and antifungal drugs.

The new potential inhibitors are designed as the selected protected amino acids derivatives containing bonds resistant against enzymatic degradation. The inhibition of the enzymes function should cause an interruption of pathways leading to development of bacteria. Thus the discovery of powerful inhibitors of enzymatic degradation might lead to a class of new antibiotics.

Since the synthetic alterations of parent molecules are based on principal methodology of organic, amino acid and peptide chemistries, a relatively easy preparation of majority of the analogues designed is supposed. It concerns, e.g. derivatives of α,α-diaminocarboxylic acids. However, the synthesis of diamino-dicarboxylic acid derivatives is a more challenging task and we are developing new synthetic approaches in this field.

Synthesized compounds have been tested for their inhibition potency towards available bacterial enzymes in the collaborating laboratory of Prof. Richard Holz, Utah State University (USA).

Antimicrobial peptides for treatment of osteomyelitis

Osteomyelitis, the infection of the bones is a refractory disease which may lead to amputation or even death. Despite the improving of prevention, operating technique, postoperative care and better availability of antibiotics, bone infection remains one of the most serious complications in orthopedics and traumatology. The main reason is growing resistance of causative microorganisms such as Staphylococcus aureus, Enterobacter species, Streptococcus species and Pseudomonas aeruginosa against conventional antibiotics.

Currently the treatment of osteomyelitis is based on antimicrobial therapy (traditional antibiotics) combined with surgical treatment and adjuvant therapy. A key problem is the treatment of the "dead space" after debridement and removal of sequester formation which is colonized by bacterial biofilm. One of the ways of increasing the therapeutic potential is filling the cavity in the infected bone with local carriers loaded with antibiotics. Local application of antimicrobial peptide (AMP) released from local carrier to the site of the bone infection seems to be very promising therapeutic method for treatment of osteomyelitis caused by resistant pathogens. Our former discoveries of potent AMP challenge us to apply synthetically available AMP for eradication of the bone infections. We will prepare series of analogs of native AMP named LL-III, select the most promising one and incorporate it into various local carriers and test it against bone infection in the in vitro experiments and in animal models. The design of this analog will aim to the improvement of its antimicrobial potency against bacteria as well as those in biofilm and to elimination of its side effects. This will include chemical modifications of native peptide structure resulting to the change of the desired parameters such as net charge, helicity, hydrophobicity, amphipathicity and stability against degradation in biological fluids. The development of the analog of interest will involve flexible and economic synthetic route. In parallel we will search for novel AMP from natural sources aiming to identify another lead compound of promising antimicrobial activity. The final goal of this proposal is to find out suitable combination of selected AMP with a local carrier as a safe formula for the treatment of osteomyelitis. Hopefully, some of our compounds will lead to the promising formula that can enter into preclinical and clinical development.