Functional Characterization of Human ProNGF and NGF Mutants: Identification of NGF P61SR100E as a "Painless" Lead Investigational Candidate for Therapeutic Applications

PLoS One. 2015 Sep 15;10(9):e0136425. doi: 10.1371/journal.pone.0136425. eCollection 2015.

Abstract

Background: Nerve Growth Factor (NGF) holds a great therapeutic promise for Alzheimer's disease, diabetic neuropathies, ophthalmic diseases, dermatological ulcers. However, the necessity for systemic delivery has hampered the clinical applications of NGF due to its potent pro-nociceptive action. A "painless" human NGF (hNGF R100E) mutant has been engineered. It has equal neurotrophic potency to hNGF but a lower nociceptive activity. We previously described and characterized the neurotrophic and nociceptive properties also of the hNGF P61S and P61SR100E mutants, selectively detectable against wild type hNGF. However, the reduced pain-sensitizing potency of the "painless" hNGF mutants has not been quantified.

Objectives and results: Aiming at the therapeutic application of the "painless" hNGF mutants, we report on the comparative functional characterization of the precursor and mature forms of the mutants hNGF R100E and hNGF P61SR100E as therapeutic candidates, also in comparison to wild type hNGF and to hNGF P61S. The mutants were assessed by a number of biochemical, biophysical methods and assayed by cellular assays. Moreover, a highly sensitive ELISA for the detection of the P61S-tagged mutants in biological samples has been developed. Finally, we explored the pro-nociceptive effects elicited by hNGF mutants in vivo, demonstrating an expanded therapeutic window with a ten-fold increase in potency.

Conclusions: This structure-activity relationship study has led to validate the concept of developing painless NGF as a therapeutic, targeting the NGF receptor system and supporting the choice of hNGF P61S R100E as the best candidate to advance in clinical development. Moreover, this study contributes to the identification of the molecular determinants modulating the properties of the hNGF "painless" mutants.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Differentiation / drug effects
  • Cell Line, Tumor
  • Cell Proliferation / drug effects
  • Escherichia coli / genetics
  • Humans
  • Kinetics
  • Mutation*
  • Nerve Growth Factor / adverse effects*
  • Nerve Growth Factor / genetics*
  • Nerve Growth Factor / metabolism
  • Nerve Growth Factor / therapeutic use
  • Oligodendroglia / cytology
  • Oligodendroglia / drug effects
  • Pain / chemically induced*
  • Protein Engineering*
  • Protein Precursors / adverse effects*
  • Protein Precursors / genetics*
  • Protein Precursors / metabolism
  • Protein Precursors / therapeutic use
  • Protein Stability
  • Proteolysis
  • Rats
  • Receptor, Nerve Growth Factor / metabolism
  • Receptor, trkA / metabolism
  • Temperature

Substances

  • NGF protein, human
  • Protein Precursors
  • Receptor, Nerve Growth Factor
  • Nerve Growth Factor
  • Receptor, trkA

Grant support

This work was supported by the following grants: MIUR, project PRIN (#2010N8PBAA_006) to AC (http://prin.miur.it/); ADDF (grant #20120601) to AC (https://www.alzdiscovery.org/); and European Union, project “PAINCAGE” (GA n. 603191) to AC (http://www.paincage.eu/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.