Improving Bicyclic Peptide Phage Display and Development of Sortase A Inhibitors

Bicyclic peptide ligands are promising molecules for the development of new therapeutics. They combine advantages from large protein therapeutics and small molecule drugs. Large combinatorial libraries of bicyclic peptides can be generated and screened by phage display using a recently developed strategy. Potent and selective bicyclic peptide inhibitors against several therapeutic targets have already been developed and their therapeutic potential is currently being evaluated in animal models. One aim of my thesis was the exploration of ring size diversity in bicyclic peptides. I generated a set of libraries of the format Cys-(Xaa)m-Cys-(Xaa)n-Cys, where 'm' and 'n' = 3, 4, 5 or 6, and performed affinity selections against the serine protease urokinase-type plasminogen activator. Bicyclic peptide inhibitors from virtually all ring size combinations were isolated, suggesting that many peptide formats can be accommodated in the active site of this enzyme. Moreover, they showed a large variety of consensus sequences and several of the identified consensus sequences were exclusively found in bicyclic peptides having specific ring size combinations. Having available multiple leads isolated from such bicyclic peptide libraries with variable ring sizes could be a great asset for the generation of high affinity binders. Additionally, other targets may have preferences for specific peptide constraints and the availability of these libraries increases the chances to isolate high affinity binders to any desired target. A second goal of my thesis was to apply high throughput sequencing technologies to phage display selections of bicyclic peptides, in order to identify a larger number of specific target-binding sequences and motifs. I developed a procedure to efficiently compare the sequences of large numbers of phage-selected peptides to identify target-binding peptide motifs based on abundance and sequence similarity. Applying this approach to phage isolated in selections against five different protein targets, I was able to identify rare target-binding peptide motifs and could more precisely define groups and sub-groups of consensus sequences. This information is valuable to choose peptide leads for drug development and facilitates the identification of epitopes. Moreover, binding motifs could be identified after a single round of phage panning. The final aim of my thesis was to discover bicyclic peptides that could be used as new antibiotics. Towards this end, I combined the newly generated variable ring size libraries and high-throughput sequencing procedures. I focused on the development of inhibitors of Staphylococcus aureus sortase A, an antivirulence target for which no potent and specific inhibitors have been reported. For the isolation of bicyclic peptide inhibitors to this target, the ring size diversity of the libraries turned out to be key. Inhibitors all shared the same motif in a loop of 5 residues. Further characterization of their effects on S. aureus showed that they could inhibit sortase-mediated incorporation of external substrates on the staphylococcal cell wall. However, they were not sufficiently potent to compete with the native substrates of the enzyme. More potent inhibitors are needed to effectively inhibit sortase A on S. aureus cells, and the bicyclic peptide inhibitors isolated constitute promising leads for the development of future antisortase therapeutics.

Development of potent and selective S. aureus sortase A inhibitors Based on Peptide Macrocycles

Davide Bertoldo, Christian Heinis, Inmaculada Rentero Rebollo

Sortases are transpeptidase enzymes that anchor surface proteins, including virulence factors, to the cell wall of Gram-positive bacteria, and they are potential targets for the development of anti-infective agents. While several large compound libraries were searched by high-throughput screening, no high-affinity inhibitors of sortases could be developed to date. Here, we applied phage display to screen billions of peptide macrocycles against sortase A (SrtA) of Staphylococcus aureus (S. aureus). We were able to identify potent and selective inhibitors of SrtA that blocked SrtA-mediated anchoring of synthetic substrates to the surface of live S. aureus cells. A region present in all inhibitory peptides (Leu-Pro-Pro) resembled the natural substrates of SrtA (Leu-Pro-Xaa-Thr-Gly), suggesting that the macrocycles bind to the enzyme's active site and that they form similar molecular contacts as natural substrates. The evolved peptide macrocycles may be used as lead structures for the development of potent peptidomimetic SrtA inhibitors.

2016

Phage display libraries of differently sized bicyclic peptides

Alessandro Angelini, Christian Heinis, Inmaculada Rentero Rebollo

Phage selections with combinatorial libraries of uniformly sized bicyclic peptides have recently yielded potent and selective binders of several protein targets. In this work we varied in a combinatorial fashion the ring sizes of bicyclic peptides in phage libraries, expecting that they would yield binders with higher affinities and/or more diverse binding motifs that could be affinity matured. 14 new phage peptide libraries of the format Cys-(Xaa)(m)-Cys-(Xaa)(n)-Cys (Xaa are random amino acids, m and n = 3, 4, 5 or 6) were generated and cyclized with tris-(bromomethyl) benzene. Affinity selections against the tumor-associated serine protease urokinase-type plasminogen activator yielded bicyclic peptide inhibitors with a large variety of consensus sequences. Several of the identified consensus sequences were exclusively found in bicyclic peptides having defined ring size combinations. Some of these peptides may bind in orientations that allow affinity maturation of non-conserved regions, while others do not. Having available multiple leads isolated from such bicyclic peptide libraries with variable ring sizes could therefore be a great asset for the generation of high affinity binders.

Royal Soc Chemistry2013

A novel cell-based sensor detecting the activity of individual basic proprotein convertases

Daniel Constam, Stefan Kunz

The basic proprotein convertases (PCs) furin, PC1/3, PC2, PC5/6, PACE4, PC4, and PC7 are promising drug targets for human diseases. However, developing selective inhibitors remains challenging due to overlapping substrate recognition motifs and limited structural information. Classical drug screening approaches for basic PC inhibitors involve homogeneous biochemical assays using soluble recombinant enzymes combined with fluorogenic substrate peptides that may not accurately recapitulate the complex cellular context of the basic PC-substrate interaction. Herein we report basic PC sensor (BPCS), a novel cell-based molecular sensor that allows rapid screening of candidate inhibitors and their selectivity toward individual basic PCs within mammalian cells. BPCS consists of Gaussia luciferase linked to a sortilin-1 membrane anchor via a cleavage motif that allows efficient release of luciferase specifically if individual basic PCs are provided in the same membrane. Screening of selected candidate peptidomimetic inhibitors revealed that BPCS can readily distinguish between general and selective PC inhibitors in a high-throughput screening format. The robust and cost-effective assay format of BPCS makes it suitable to identify novel specific small-molecule inhibitors against basic PCs for therapeutic application. Its cell-based nature will allow screening for drug targets in addition to the catalytically active mature enzyme, including maturation, transport, and cellular factors that modulate the enzyme's activity. This broadened 'target range' will enhance the likelihood to identify novel small-molecule compounds that inhibit basic PCs in a direct or indirect manner and represents a conceptual advantage.

WILEY2019