Technology Platform

 

Inhibitors of protein-protein interactions

In drug discovery there is a high interest in protein-protein interactions as they represent appealing drug targets. PROCOMCURE has established procedures to obtain peptide-ligands derived from interacting proteins, described in a PROCOMCURE patent of which the key claims describe the production, modification, and use of the peptides blocking protein-protein interactions (cf. WO2009027495). PROCOMCURE uses libraries of all protein-encoding open reading frames to obtain peptides derived from any one partner in a protein-protein interaction that block this very protein-protein interaction. Following mutagenesis, peptides can be selected in vivo for high-affinity binding to a defined target-protein. PROCOMCURE uses optimized automated screening protocols and proprietary screening libraries, namely peptides derived from ORFeomes (all protein encoding Open Reading Frames of an Organism).
This is detailed below.
 

Use of ORFeome resources

Omics technologies can take full advantage of the information found in a genome sequence. Fulfilling the genomics-promise requires libraries composed of all protein-encoding open reading frames (ORFs) cloned in highly flexible vectors. Using novel technologies for cloning and expressing entire proteomes, PROCOMCURE has achieved the creation of such ORFeome resources for Staphylococcus aureus [1], Candida albicans, Homo sapiens, Escherichia coli, Chlamydia pneumoniae and Chlamydia trachomatis. PROCOMCURE produced pooled, screening-ready libraries by merging ORFeomes with a streamlined and easily executable single-tube design, and found that the complexity of the gene population is maintained once an entry resource has been successfully established, and that no apparent size-selection bias or loss of large inserts takes place.

Thus collections of full-length genes have been established. They are used in identifying inhibitors [2]. Peptides of any one partner in a protein-protein interaction (ORFeomers) are obtained from the ORFeomes. These protein-binding peptides block the protein-protein interaction, and by mutagenesis and selection are improved for binding to the target-protein. Once a series of inhibitory peptide ligands are identified, thousands of close derivates of these first hits are arrayed on chips to identify peptides with improved specificity and selectivity in binding. PROCOMCURE has established peptide-fusion & cyclization methods to derive from the peptides the scaffolds for inhibitors of protein-protein interactions. Function assays and quantitative protein-protein interaction assays are used to select inhibitors.
 

Applications in antibacterial research

For one of the full gene collections, the ORFeome of Staphylococcus aureus, we converted the gene collection into a unique strain collection. For this purpose we produced a series of novel S. aureus and E. coli shuttle plasmids, which allow the introduction, stable maintenance, and tagged expression of a certain gene in S. aureus. In addition, a series of rolling circle and theta-mode amplification vectors were made, each equipped with different resistance genes for selection and with constitutive weak or moderate or strong as well as inducible expression-promoters.

The strains can be used as follows:

    • For a new antibacterial substance the MIC can be systematically measured in the background of 3000 MRSA, linezolid-resistant S. aureus or vancomycin-resistant S. aureus strains overexpressing a certain gene
    • Antibacterials towards which resistance has developed can be screened for re-sensibilizing bacterial factors
    • Hits from high-throughput screening of compound libraries can be selected for specific and unspecific inhibitors
    • Of special interest is a strain collections, which consists of 500 MRSA strains expressing the full complements of S. aureus essential genes and all known antibiotics targets

 

PROCOMCURE would welcome opportunities to collaborate with partners interested in mode of action (MOA) studies for bacterial inhibitors or with partners interested in protein targeting ligand discovery.

[1] Brander et al, BMC Genomics, 2008, 9, 321; Rid et al, Assays Drug Dev Technol, 2012, May 23
[2] Published in a patent application as EP2034020, Method for manufacturing a modified peptide