Inhibition of soluble and membrane‐bound neutrophil elastase by inhibitor POL6014

Abstract

Neutrophil elastase (NE) is involved in the degradation of extracellular matrix molecules making it an attractive target for the development of anti‐inflammatory compounds in neutrophilic pulmonary diseases. It is mainly secreted extracellularly by activated neutrophils at inflammatory sites but a minor fraction is translocated to the membrane where it remains proteolytically active. We investigated the properties of the novel Protein Epitope Mimetic (PEM) POL6014, a medium sized fully synthetic macrocycle, towards soluble and membrane‐bound NE. Circulating human neutrophils from healthy volunteers were purified and used within 3 hours after activation from Calcium ionophore (A23187) as source of membrane bound NE. The enzymatic reaction using the Fluorescence Resonance Energy Transfer (FRET) Technology was started by addition of a specific human NE substrate (ABZ‐APEEIMRRQ‐EDDnp, 13 μM final) 5 minutes after incubation of POL6014 with human NE. We found that POL6014 inhibits membrane‐bound human NE and soluble human NE with similar Ki values in the sub‐nanomolar range (0.69 ± 0.31 nM (n=7) and 0.53 ± 0.37 nM (n=3), respectively). This means that POL6014 could inhibit human NE stoichiometrically in pathophysiological conditions so that no molar excess of inhibitor would be required to control NE activity. In addition we evaluated POL6014 activity on rat and non‐human primate (NHP) NE in order to qualify these species for in vivo testing. Lysates of circulating neutrophils from both species were used as source of NE. Species selective FRET substrates (ABZ‐QPMAVVQSVPQ‐Yno2 for rodent, ABZ‐APQQIMDDQ‐EDDnp for primate) were used to determine the inhibitory potency of POL6014. We found that POL6014 is also a potent low nanomolar inhibitor with IC50 = 1.0 ± 0.5 nM (n=4) against rat NE and IC50 = 2.2 ± 1.4 nM (n=3) against Cynomolgus (Macaca fascicularis) NHP NE, which makes these species appropriate to test the in vivo efficacy and toxicology of POL6014. In conclusion, POL6014 showed potent inhibition of soluble and membrane‐bound human NE and similar potency across human, rat and NHP.Support or Funding InformationThis work was supported by Polyphor Ltd.