Engineering hepatocyte growth factor fragments with high stability and activity as Met receptor agonists and antagonists

Abstract

The Met receptor tyrosine kinase and its ligand hepatocyte growth factor (HGF) play an important role in mediating both tumor progression and tissue regeneration. The N-terminal and first Kringle domains (NK1) of HGF comprise a naturally occurring splice variant that retains the ability to activate the Met receptor. However, NK1 is a weak agonist and is relatively unstable, limiting its therapeutic potential. Here, we engineered NK1 mutants with improved biochemical and biophysical properties that function as Met receptor agonists or antagonists. We first engineered NK1 for increased stability and recombinant expression yield using directed evolution. The NK1 variants isolated from our library screens acted as weak Met receptor antagonists due to a mutation at the NK1 homodimerization interface. We introduced point mutations that restored this NK1 homodimerization interface to create an agonistic ligand, or that further disrupted this interface to create more effective antagonists. The rationally engineered antagonists exhibited melting temperatures up to approximately 64°C, a 15°C improvement over antagonists derived from wild-type NK1, and approximately 40-fold improvement in expression yield. Next, we created disulfide-linked NK1 homodimers through introduction of an N-terminal cysteine residue. These covalent dimers exhibited nearly an order of magnitude improved agonistic activity compared to wild-type NK1, approaching the activity of full-length HGF. Moreover, covalent NK1 dimers formed from agonistic or antagonistic monomeric subunits elicited similar activity, further signifying that NK1 dimerization mediates agonistic activity. These engineered NK1 proteins are promising candidates for therapeutic development and will be useful tools for further exploring determinants of Met receptor activation.

Degradable Acetalated Dextran Microparticles for Tunable Release of an Engineered Hepatocyte Growth Factor Fragment

“…Specifically, we were able to investigate how delivery rate impacted the efficacy of a known cardioprotective growth factor, HGF. Despite the benefits of HGF therapy, including increased angiogenesis 6, 21–27 , reduced apoptosis 6, 22–26, 28 , and reduced fibrosis 6, 21–22, 24, 26–28 , the use of recombinant HGF is limited by poor stability and high cost and thus a more cost effective and practical alternative is to deliver HGF-f, which has shown enhanced stability and expression yield, comparable c-met activation compared to recombinant HGF 12 , and efficacy post-MI when delivered in a biomaterial 13 . In all measured outcomes it was shown that delivering HGF-f over a three-day time frame was optimal.…”

An allosteric switch for pro-HGF/Met signaling using zymogen activator peptides

“…Until now, pharmacological stimulation of Met receptor signaling has involved some form of recombinant HGF or alternative direct receptor agonists [4][5][6][7][8] . Using ZAPtides, we have validated a new mechanism for reversible, allosteric activation of the pro-HGF ligand, which may provide greater control over Met stimulation for tissue repair applications.…”

Rational and Combinatorial Methods to Create Designer Protein Interfaces

“…In this example, mutants with increased thermal stability were also displayed at higher levels on the yeast cell surface and exhibited significantly higher soluble expression yields. 193 The yeast surface display system has also proven to be robust for engineering proteins with therapeutic potential. 194 The human proteins EGF and IL-2 have been engineered for 30-and 150-fold increased binding affinity for their receptors, respectively.…”