A Technology for Developing Synbodies with Antibacterial Activity

Abstract

The rise in antibiotic resistance has led to an increased research focus on discovery of new antibacterial candidates. While broad-spectrum antibiotics are widely pursued, there is evidence that resistance arises in part from the wide spread use of these antibiotics. Our group has developed a system to produce protein affinity agents, called synbodies, which have high affinity and specificity for their target. In this report, we describe the adaptation of this system to produce new antibacterial candidates towards a target bacterium. The system functions by screening target bacteria against an array of 10,000 random sequence peptides and, using a combination of membrane labeling and intracellular dyes, we identified peptides with target specific binding or killing functions. Binding and lytic peptides were identified in this manner and in vitro tests confirmed the activity of the lead peptides. A peptide with antibacterial activity was linked to a peptide specifically binding Staphylococcus aureus to create a synbody with increased antibacterial activity. Subsequent tests showed that this peptide could block S. aureus induced killing of HEK293 cells in a co-culture experiment. These results demonstrate the feasibility of using the synbody system to discover new antibacterial candidate agents.

Scalable high-density peptide arrays for comprehensive health monitoring

“…Peptide arrays have been used to assay kinase activity and inhibitors 23,31 , map monoclonal antibodies 32 , identify autoantigens 33 , even bind proteins 34 and cells 35 . The synthesis fidelity and peptide density of these arrays make it feasible to tile the entire human proteome or the proteomes of most human pathogens for studies of autoimmunity or vaccine development 23,26,36 .…”

“…The synthesis fidelity and peptide density of these arrays make it feasible to tile the entire human proteome or the proteomes of most human pathogens for studies of autoimmunity or vaccine development 23,26,36 . Random-sequence arrays have been the starting point for developing synthetic antibodies 34,35,37,38 using branched peptides, and a screening system for identifying antimicrobial peptides 35 . We expect this platform will find many other uses.…”

Targeted treatment for bacterial infections: prospects for pathogen-specific antibiotics coupled with rapid diagnostics

“…164 This strategy has been employed in cancer therapy with several FDA-approved antibody-drug conjugates [brentuximab vedotin (Adcetris), trastuzumab emtansine (Kadcyla)]. 165 Examples of targeting domains include peptides, 166,167 antibodies, 168 and phage. 169 The conjugates are intended to bind specifically to the pathogen of interest and thus create a locally high concentration of the antibiotic that is sufficient to kill the target.…”

Conjugation Approach To Produce a Staphylococcus aureus Synbody with Activity in Serum

“…The plate was incubated for the indicated times at 37°C with shaking and aliquots were Hemolytic Assay. We adapted our previously published hemolytic assay 39 for use with ASU007 and ASU008. Briefly, blood from female BALB/C mice was collected, centrifuged at 300 g for 5 min, the sera was discarded, red blood cells were rinsed with 1×PBS (v/v − RBC: PBS), and centrifuged at 900 g for 15 min.…”

“…33 Our group recently developed a specific bivalent peptide, called a synbody, that inhibited the growth of Staphylococcus aureus. 39 The synbody ASU001 was constructed from a 20-amino-acid (aa) peptide that specifically bound S. aureus linked to a second 20-aa peptide that inhibited S. aureus growth. The resulting synbody had a narrow spectrum of antibacterial activity with an MIC of 14 μM for S. aureus.…”