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

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.

Small Molecule Based Approaches to Inhibit Macrophage Migration Inhibitory Factor (MIF) Activities and Elucidate its Role in Health and Disease

Hajer Ouertatani-Sakouhi

Macrophage migration inhibitory factor (MIF) is a major mediator in innate immunity and inflammation and a potential therapeutic target in multiple inflammatory, infectious and autoimmune diseases including cancer. Current therapeutic strategies for targeting MIF focus on modulating its biological activities using anti-MIF neutralizing antibodies or developing inhibitors of its tautomerase activity. Although the identity of its natural substrate remains unknown, several small molecule inhibitors have been reported to be effective inhibitors of MIF tautomerase activity in vitro. All these inhibitors were identified by rational design and structure-activity studies based on the structure of the catalytic site and/or non physiologic substrates of MIF. The lack of suitable high-throughput screening (HTS) assays has hindered the screening of chemical libraries and discovery of more diverse inhibitors. Motivated by the goal of discovering diverse classes of MIF inhibitors that modulate MIF activity specific targeting of its biochemical, conformation and quaternary structure properties, both activity based endpoint and kinetic HTS assays were developed, optimized and performed on diverse set of libraries. Using the endpoint assay, out of 15440 compounds screened, twelve novel inhibitors of MIF's catalytic activity (∼ 0.1% hit rate) with IC50s in the range of 1.5 to 15.5 µM were identified and validated. The interaction site and mechanism of action of all these inhibitors were defined using structure activity studies and a battery of biochemical and biophysical methods, including mass spectrometry, light scattering, and NMR. The effect on MIF biological activities was also examined on MIF-mediated glucocorticoid overriding and MIF-induced Akt phosphorylation. Using the kinetic-based activity assay, we screened 80,000 small molecules and identified and validated thirteen novel inhibitors of MIF catalytic activity with inhibition constant (Ki,app) values ranging from 0.5 to 13 µM. According to the structure and potency some of these molecules could be of great therapeutic potential. The MIF inhibitors emerging from these studies could be divided into four classes: 1) molecules that covalently modify the catalytic site at the N-terminal proline residue, Pro1 which could be classified into 5 groups; 2) a novel class of catalytic site inhibitors, 3) a class of trimer destabilizing inhibitors. These findings demonstrate the potential of HTS assays as attractive means to identify novel classes of MIF inhibitors as lead drug candidates and/or chemical tools for elucidating the biochemical and structural bases underlying the multifunctionality of MIF's in health and disease. Having these molecules in hand, will advance our understanding of protein-protein interactions mechanism for MIF oligomerization and may help to elucidate the role of MIF enzymatic activity in health and disease.

EPFL2010

Improving Bicyclic Peptide Phage Display and Development of Sortase A Inhibitors

Inmaculada Rentero Rebollo

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.

EPFL2014

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.