Novel Influenza Peptide Inhibitors by Directed Evolution – FluPept

Influenza is a common respiratory disease that affects 10-30% of the population each year and kills 290-650,000 people annually worldwide. Antiviral drugs complement the annual global vaccine strategy, notably when vaccines match poorly with the epidemic strains. They become the first line of defence during influenza pandemics when at least 6 months are required to produce a vaccine. A major limitation of existing anti-influenza drugs is the frequent development of viral resistance due to the high mutation rate of the viral RNA polymerase. Therefore, there is an ongoing and urgent need for new drugs for use alone or in combination with existing treatments.
Our FluPept project will develop the proof-of-concept as well as novel lead drug candidates that target two protein complexes essential for influenza viral replication: the PA-PB1 sub-complex of the viral RNA polymerase that catalyses replication and transcription of the viral genome, and the host spliceosome-associated RED-SMU1 complex required for viral mRNA processing. The two target complexes assemble via protein-protein interactions (PPIs) mediated by short, highly conserved motifs of PB1/RED that bind their partner domains of PA/SMU1.
Guided by our extensive functional knowledge of the two complexes and their 3D structures, we will perform directed evolution by phage display to evolve high affinity peptide mimics of these natural helical interaction motifs that will competitively inhibit complex assembly. These hits will be assessed initially in cell-based assays by intracellular expression of EGFP-peptide fusions, then as chemically synthesised Tat cell penetrating peptide (CPP) fusions that cross the cell membrane. Structural, biophysical and cellular studies on these hits will guide further rounds of in vitro evolution. In addition, peptide modifications including alternative CPP motifs, helix-stabilising cross-links and protease-resisting D-amino acid incorporation will be included in the subsequent phase of hit-to-lead optimisation.
Our preliminary data suggest that we can develop lead peptides that inhibit viral replication at sub-micromolar concentrations in cell-based assays. Viral resistance will be assessed by performing serial passaging of different viral strains in the presence of the lead peptides and monitoring for the selection of escape mutations. The large surface of these conserved PPI interfaces (compared to small-molecule binding sites), the structural similarity of our inhibitors to the natural peptide ligands, and the host origin of the RED-SMU1 target lead us to expect that they will exhibit low susceptibility to viral resistance.
The originality and innovation in our proposal lie in the combination of i) our choice of targets: the PA-PB1 dimer and, in line with the emerging concept of host-directed therapy, the RED-SMU1 host factor; and ii) our choice of a peptide-based antiviral strategy, enabled by structure-guided phage display with huge but structurally focused libraries of >10^9 individual members, as well as through incorporation of CPP technology and chemistry-facilitated optimisation steps.
The overall vision of FluPept is to develop novel peptide-based drugs with low levels of drug-resistance that can be applied to virus-infected epithelial cells of the upper respiratory tract using inhaler or spray-based technologies. The workflow presented will advance this vision towards first preclinical tests in a mouse model; this will open the way for clinical trials, in line with an increasing number of peptide-based therapeutics. Importantly, such peptide-based drugs will have orthogonal mechanisms of action compared to existing anti-influenza drugs and will thus offer the potential to significantly increase the therapeutic armoury for this important disease.