(117d) A High-Throughput Activity Screen for Reprogramming Proteases (HARP)

Protease dysregulation is a hallmark of many human diseases^1–3. Due to this, controlling proteolytic activity is the goal of many therapeutics. Protease-targeting therapeutics are primarily small molecules discovered via binding-based high-throughput screens, often resulting in molecules with an active site bias⁴. Targeting protease active sites with small molecules remains challenging and often lacks enzyme selectivity due to the highly similar active topologies of related proteases². Additionally, active site binding primarily results in enzyme inhibition. This approach limits the scope of current therapeutic approaches and can negatively affect proteolytic involvement in homeostatic pathways. Efforts have been made to discover distal regulators that modulate protease activity beyond inhibition, but these sites are challenging to find, and the screens require extensive structural analysis⁵. A robust functional screen to isolate protease modulators as a direct result of functional outcomes is needed to address these challenges.

We have developed the High-Throughput (HT) Activity screen for Reprogramming Proteases (HARP) system, a functional HT screening platform that links protease activity and modulation to observable fluorescent phenotypes on the yeast surface. HARP allows one to (1) modulate the activity of an exogenous protease using protein-based binders, (2) select protease modulators from large ligand libraries in a high-throughput fashion, (3) relate modulation phenotypes directly to the physicochemical properties of the ligand, and (4) map the binding epitopes of the modulator to infer their mechanism of action. We have used HARP to (1) optimize the activity of human and viral proteases in yeast and (2) isolate protease inhibitory nanobodies (Nbs) from large synthetic libraries.

HARP includes three gene cassettes, one containing an active protease sequence, one containing the protease’s target substrate(s), and one containing an Nb modulator sequence (Figure 1A). Using the system to screen human proteases active in yeast is achieved by selecting a high anti-FLAG, low anti-HA phenotype using a two-color stain, as the HA tag is lost when the substrate is cleaved. If one wants to screen for an inhibitory effect of a protease: Nb interaction, selection of the fluorescent phenotype high anti-FLAG and anti-HA signals is used. This methodology can be used to observe and induce modulation beyond inhibition, for instance, by reprogramming substrate selectivity and activating proteases on non-naïve substrates. We have observed significant activity of PLpro and 3Cl, the two main proteases involved in SARS-CoV-2 disease pathology, and MMP8, a protease associated with cancer tumor proliferation. Using previously published nanobodies, we have also used HARP to inhibit PLpro⁶ and MMP8⁷(Figure 1B).

This system can be expanded to discover modulators by introducing an Nb modulator library and sorting cell populations for a desired modulatory phenotype using fluorescence-activated cell sorting (FACS). In a proof-of-concept, we show that we can recover an MMP8 specific nanobody inhibitor from a large population of non-binders and non-inhibitors. We aim to show early results of isolating Nbs from a large synthetic Nb library that are highly selective against disease-related proteases (i.e., BACE1, IDE, MMP8). Additionally, we are further expanding HARP to discover substrate-selective modulators by incorporating multi-display systems.

The use of HARP to screen and discover protease modulators that reprogram activity with increased specificity and selectivity opens many doors for advancement in protease targeting, therapeutic development, and distal site regulation. Using a functional screen as the basis for modulator discovery allows us to select modulatory outcomes such as activation and enhancement that are not discoverable in current systems. As we explore Nb-based modulation and isolation from large Nb libraries, we aim to discover modulators with favorable kinetics and potencies that rival current modulators. With each new application of this system, we look forward to tuning the HARP and finding new avenues for controlling proteases.

References: (1) Hampel, H., et. al., Biol. Psychiatry, (2021). (2) Coussens, L.M., et.al., Science, (2002). (3) López-Otín, C., et. al., J. Biol. Chem., (2008). (4) Schmidt, M.F., et. al., Trends Biotechnol., (2009). (5) Maianti, J.P., et. al., Nat. Chem. Biol., (2019). (6) Armstrong, L.A., PLOS ONE, (2021). (7) Demeestere, D., et. al., Mol. Ther., (2016).