Antibiotics' resistance to pathogens is a major threat to public health and safety, increasing the risk of mortality, especially in hospital settings. This issue also includes preparedness to deal with bio-terrorism. Currently, the antibiotics market is dominated by small molecule classes, which all face increased drug resistance and require product differentiation. While this remains a primary focus in antibiotics development, new concepts for entirely new classes of substances for the treatment of bacteria, fungi, viruses and protozoa are urgently needed.
The objectives of the ET-PA project aimedare to develop an open, generic platform to enable the development of a new class of protein-antibiotics. The key technology (REPPs) is based on a principle that is proprietary to one of the participating small and medium-sized enterprises (SMEs, ), and consists of rationally modified, single-chain class II restriction enzymes (REs) fused to cell penetration peptide (PP) sequences that selectively allow microbial cell penetration.
RE's are homodimeric enzymes produced by bacteria to defend themselves against infection by bacterial viruses (phages), that cut bacterial DNA at specific sites. Bacteria that produce a particular RE are protected against them, but others that don't produce it would suffer extensive damage to their DNA, so that REs can act as effective protein antibiotics. However, this requires engineering the enzymes so that they can penetrate into susceptible pathogens, and be present in an active form even at low concentrations. This is why tThe ET-PA projectconsortium intendssought to fuseing an appropriate cell PP sequence to an engineered RE that includes both subunits in a single chain, so as to produce a "'REPP'" construct capable of microbial cell penetration and autonomous folding to an active unit within the cell .
Penetration peptides (PP) have been extensively studied for targeting biopharmaceuticals to eukaryotic cells, but less is known about penetration into bacterial cells. One of the principal aims of the ET-PA project is to identify such peptides. Excellent candidates include the antimicrobial peptides produced by the host-defence systems of animals and plants. Many of these are membrane active, acting either by damaging the bacterial membrane or translocating into bacteria to reach internal targets. By fusing these peptides to an engineered single-chain RE, they can carry it into cells as "cargo", allowing it to reach the bacterial DNA . This is a denaturing process, so the enzyme cannot pass as a dimer. The advantage of a single-chain REPP is that re-folding to the active enzyme within the cell occurs in single molecules even at very low concentrations.
The ET-PA concept thus uses a natural system that has evolved as the primary prokaryotic defence shield against horizontal DNA transfer, originally discovered for bacteriophages. Horizontal DNA transfer is also one of the main routes by which antibiotic resistance is selected. Moreover, some of the naturally occurring colicin proteins, produced by some bacteria to defend their ecological niches from rival strains, show similar features for an albeit narrower host range (e.g. E2, E7, E8, E9 colicins display DNAse activities as their action mechanism). The fact that these relatively large proteins internalize provides a strong support for the ET-PA concept. REPP constructs are expected to constitute highly efficient antibiotics by killing bacterial cells through DNA hydrolysis, and have the potential for a direct targeting of drug resistance.
The project brings together three SMEs and two RTD performers from four different European countries, which are leaders in their field (www.et-pa.org) . It will provide for a technology platform that allows the construction and screening of protein libraries. The RTD partners are contributing fundamental design features and will conduct all major molecule and equipment testing in approved research settings. This platform will be developed in particular for the design of Restriction Enzyme-Penetration Peptide (REPP) protein construct libraries in the multi-well format.
The ET-PA project has different layers, introduced to maximise success rates for a commercial outcome for each individual SME. A primary aim is the development of active REPP antibiotic lead substances, benefiting the SME that invented the system. Equally important is the setting up of a powerful and versatile platform that spaces beyond REPP research, as it would be applicable for bio-pharmaceutical screening-programs in general. Participation in the development of this platform by the other SMEs creates an added value for their existing individual proprietary technology portfolios and allows them to leverage their share in the growing proteomics and genomics R&D market.
The major ET-PAmilestone was to provide a clear proof of concept for the introduction of the class of REPP molecules as antibiotics. Lead substances for further preclinical development were expected. The ET-PA consortium has defined that for a successful targeting of prokaryotic pathogens by REPP antibiotics, these molecules must clearly accomplish four tasks cross the outer cell wall barriers of bacteria and bind to the membrane surface [these include the outer membrane, peptidoglycan (PG) layer and bio film penetration;