title: Functional Characterization of Human ProNGF and NGF Mutants: Identification of NGF P61SR100E as a "Painless" Lead Investigational Candidate for Therapeutic Applications.
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.
  
  
  OBJECTIVE
  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.
authors.affiliation: Neurotrophic Factors and Neurodegenerative Diseases Unit, European Brain Research Institute, "Rita Levi-Montalcini" Foundation, Rome, Italy; Neurobiology Laboratory of Biology, Scuola Normale Superiore, Pisa, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Malerba
authors.affiliation: Neurotrophic Factors and Neurodegenerative Diseases Unit, European Brain Research Institute, "Rita Levi-Montalcini" Foundation, Rome, Italy; Neurobiology Laboratory of Biology, Scuola Normale Superiore, Pisa, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Paoletti
authors.affiliation: Neurotrophic Factors and Neurodegenerative Diseases Unit, European Brain Research Institute, "Rita Levi-Montalcini" Foundation, Rome, Italy.
authors.forename: Bruno
authors.initials: B
authors.lastname: Bruni Ercole
authors.affiliation: Department of Health Sciences, Section of Clinical Pharmacology and Oncology, University of Florence, Florence, Italy.
authors.forename: Serena
authors.initials: S
authors.lastname: Materazzi
authors.affiliation: Department of Health Sciences, Section of Clinical Pharmacology and Oncology, University of Florence, Florence, Italy.
authors.forename: Romina
authors.initials: R
authors.lastname: Nassini
authors.affiliation: Department of Health Sciences, Section of Clinical Pharmacology and Oncology, University of Florence, Florence, Italy.
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Coppi
authors.affiliation: Department of Pharmacology, Chiesi Farmaceutici, Parma, Italy.
authors.forename: Riccardo
authors.initials: R
authors.lastname: Patacchini
authors.affiliation: Neurobiology Laboratory of Biology, Scuola Normale Superiore, Pisa, Italy.
authors.forename: Simona
authors.initials: S
authors.lastname: Capsoni
authors.affiliation: Istituto di Cristallografia, Consiglio Nazionale delle Ricerche, Area Science Park-Basovizza, Trieste, Italy.
authors.forename: Doriano
authors.initials: D
authors.lastname: Lamba
authors.affiliation: Neurotrophic Factors and Neurodegenerative Diseases Unit, European Brain Research Institute, "Rita Levi-Montalcini" Foundation, Rome, Italy; Neurobiology Laboratory of Biology, Scuola Normale Superiore, Pisa, Italy.
authors.forename: Antonino
authors.initials: A
authors.lastname: Cattaneo
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007700:Kinetics; D009154:Mutation; D020932:Nerve Growth Factor / Q000009:adverse effects* / Q000235:genetics* / Q000378:metabolism / Q000627:therapeutic use; D009836:Oligodendroglia / Q000166:cytology / Q000187:drug effects; D010146:Pain / Q000139:chemically induced*; D015202:Protein Engineering; D011498:Protein Precursors / Q000009:adverse effects* / Q000235:genetics* / Q000378:metabolism / Q000627:therapeutic use; D055550:Protein Stability; D059748:Proteolysis; D051381:Rats; D020800:Receptor, Nerve Growth Factor / Q000378:metabolism; D020917:Receptor, trkA / Q000378:metabolism; D013696:Temperature
chemical_list: C502240:NGF protein, human; D011498:Protein Precursors; D020800:Receptor, Nerve Growth Factor; D020932:Nerve Growth Factor; D020917:Receptor, trkA