Streamlined design and production of bioactive variants of recombinant human keratinocyte growth factor to improve acidic pH stability; promising therapeutic candidates for wound treatment

Keratinocyte Growth Factor (KGF) plays a crucial role in promoting epithelial cell proliferation, making it a valuable candidate for wound healing therapies. However, its instability in acidic environments limits its effectiveness in early-stage wound care. This study addresses the instability of recombinant human Keratinocyte Growth Factor (rhKGF) in acidic environments. Using pH molecular dynamics (pH-MD) simulations, we previously investigated the influence of pH on rhKGF stability and identified positively charged residues as contributors to its instability. Building on these findings, we employed a rational design strategy to lower KGF's isoelectric point (pI) by introducing targeted mutations. This led to the development of 15 mutants which of them two mutants, K76E and K126E, were selected based on molecular dynamics simulations, and docking studies. Further experimental evaluations including secondary structure analysis, thermal stability, acidic pH stability, and mitogenic activity were performed on these two mutants. According to the results, both mutants retained similar structural characteristics and potency, and showed lower thermal stability, but modest acidic pH stability improvements compared to native rhKGF. K126E exhibited enhanced stability over extended periods in acidic environments, indicating its potential as a viable alternative for topical applications. Our findings highlight the efficacy of targeted mutations in acidic pH stability enhancement and underscore the role of electrostatic interactions in KGF stability. Future research should focus on targeting other hotspots for mutants, further computational analysis, and optimizing formulations that leverage these mutants for enhanced therapeutic efficacy in wound care applications.