Dimerization of kringle 1 domain from hepatocyte growth factor/scatter factor provides a potent minimal MET receptor agonist

Abstract

Degenerative diseases of major internal epithelial organs such as liver, lung and kidney account for more than one third of mortality worldwide. The huge demand for drugs able to limit epithelial tissue degradation and eventually restore its functionality, place mimics of the hepatocyte growth factor/scatter factor (HGF/SF), the physiological ligand for the MET receptor tyrosine kinase, at the forefront of potential drug candidates. HGF/SF is a growth and motility factor with essential physiological roles in development and regeneration of epithelial organs. Unfortunately, HGF/SF itself is unsuitable for therapy because naturally the factor acts only locally as a morphogen and chemoattractant and has poor in vivo distribution and shelf life profile. We have therefore designed, produced, solved the crystal structure and characterized the biochemical and biological properties of K1K1, a new engineered fragment of HGF/SF for applications in tissue/organ regeneration. K1K1, a covalent dimer of the first kringle domain of HGF/SF, is recombinantly produced in bacterial cells, shows superior stability at physiological pH and ionic strength and is a potent receptor agonist as demonstrated in a wide range of biological assays with cells in culture and initial in vivo studies. K1K1 has broad potential in regenerative medicine with diseases such as acute liver failure, non-alcoholic steatohepatitis, chronic obstructive pulmonary disease and acute kidney injury.

A Novel HGF/SF Receptor (MET) Agonist Transiently Delays the Disease Progression in an Amyotrophic Lateral Sclerosis Mouse Model by Promoting Neuronal Survival and Dampening the Immune Dysregulation

“…Native HGF/SF, the MET-agonists NK1 and K1K1 were produced in highly optimized expression systems. NK1 is a natural splice-variant with reduced agonistic activity, while the design of recombinant K1K1 is based on extensive functional and structural studies performed in our laboratories in collaboration with colleagues at the Institute Pasteur in Lille, France [ 19 ] and is comprised of two linked HGF/SF kringle 1 domains providing two MET receptor binding sites [ 19 , 20 ] ( Figure S1 ). For therapeutic purposes, K1K1 has several advantages over HGF/SF, one of which is superior stability in buffered solutions such as phosphate buffer saline (PBS) as well as in mammalian cell culture medium.…”

“…Mouse kringle 1 domain differs from the human kringle 1 in only two amino acid positions: G134 (R134 in human) and N152 (S152 in human). The mouse protein was produced as described for human K1K1 in material and methods [ 19 ]. The mouse protein was tested in an MDCK assay and its biological activity was confirmed to be comparable to that of human K1K1 and mouse and human HGF/SF showing observable colony scattering down to the picomolar concentration and differing only by a half-log (3.16×) dilution ( Figure S3 ).…”

“…The natural splice-variant NK1 was produced recombinantly in the yeast expression system Pichia pastoris (Invitrogen, Carlsbad, CA, USA) [ 41 ]. The engineered recombinant K1K1 was produced as described in Leclercq et al, 2020 [ 19 ]. Briefly, K1K1 was produced in bacterial inclusion bodies using the E. coli strain BL21 (Invitrogen) giving yields of around 10 mg/litre.…”

“…We recently developed and tested in vitro stable and potent recombinant MET agonists based on HGF/SF. They include NK1, a small natural fragment of the growth factor consisting of the N and first kringle domain (K1) of HGF/SF and encompassing the high-affinity MET binding site [ 18 ] ( Supplementary Figure S1 ), and a novel dimeric form of the K1 domain, designated K1K1 [ 19 ]. After preliminary studies in vitro, we selected the most potent protein, K1K1, as the molecule of choice to assess the neuroprotective effect in in vitro and in vivo models of ALS.…”