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

<div><p>Background</p><p>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.</p><p>Objectives and Results</p><p>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 <i>in vivo</i>, demonstrating an expanded therapeutic window with a ten-fold increase in potency.</p><p>Conclusions</p><p>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.</p></div

Chemical denaturation.

<p>Guanidinium Chloride (Gdm-Cl) denaturation profile of WT (blue), P61S (green), P61SR100E (red), R100E (purple) hNGF (panel A), hproNGF (Panel B). The fraction of folded protein is plotted as a function of Gdm-Cl concentration. The inset in Panel A shows an alternative normalization, obtained to consider the anomalous behaviour of the mutants that show an increase in the emission fluorescence intensity at low Gdm-Cl concentrations (0–1.5M). The data were normalized assuming that the mutants, unlike hNGF WT, are in a fully folded state (Fraction folded = 1) at 0.5M Gdm-Cl (instead of that at 0M Gdm-Cl) and allow to better compare all the proteins at a glance.</p

Painful effect induced by hNGF WT or mutants.

<p>Pooled data of the mechanical allodynic response (A) and the thermal (hot) hyperalgesic response (B) evoked by intraplantar (i.pl.) injection (20 μl) of hNGF WT, hNGF R100E, hNGF P61S, hNGF P61S R100E or their vehicle (Veh, isotonic saline), measured 5 hours post-treatment. Data are mean ± sem of at least n = 4 mice per group; *P<0.05 vs. Veh or hNGF WT 0.1 μg or hNGF P61S 0.1 μg; #P<0.05 vs. hNGF WT 1 μg or hNGF P61S 0.1 μg; §P<0.05 vs. hNGF WT 10 μg or hNGF P61S 10 μg. One-way ANOVA followed by Bonferroni post-test.</p

hNGF WT and mutants bioactivity on <i>in vitro</i> differentiation of purified primary rat OPCs.

<p>Panel A: Immunolabelling of primary purified OPC cultures grown for 24 hours in control conditions or in the presence of hNGF WT or its mutants (R100E, P61S, P61S R100E; all compounds are 150 ng/ml). Cell nuclei are marked with DAPI (blue). Scale bar: 50 μm. hNGF increases the percentage of undifferentiated NG2+ cells (left panels: in red) and decreases the percentage of O4+ pre-oligodendrocytes (right panels: in red), indicating that NGF inhibits OPC differentiation. The same effect is observed when cells were growth in the presence of the hNGF mutant P61S but not of R100E nor P61S R100E. Panel B. Quantification of the percentage NG2+ (upper panel) and O4+ (lower panel) OPCs in all different experimental conditions. Three coverslips per experiment were performed in each experimental group. Ten random microscopic fields (20x) per coverslip were evaluated. Experiments were run in triplicate. *P<0.05 vs CTL, #P<0.05 vs hNGF, One-way ANOVA followed by Newman-Keuls post-test. Panel C. None of the compounds tested (all 150 ng/ml; 24 hours exposure) induced toxicity in rat OPC cultures.</p

Behavioral results.

<p>A. Nine hole peg test (NHPT) score: left hand in pianists was significantly faster than in controls (p = 0.001). In the control group the time required to perform NHPT was shorter for the right than the left hand (p = 0.003). B Finger tapping (FT) scores: a significant effect of “side” factor was observed (F_1,22 = 100.6, p<0.0001) being the right hand faster than the left hand. A trend was observed for the “group” comparison (F_1,22 = 3.8, p = 0.06) and the “side” x “group” interaction was not significant (F_1,22 = 0.18, p = 0.6). C. NHPT asymmetry index (AI) score was significantly lower in pianist than in controls indicating more symmetric motor performance in pianists (p = 0.005) D. FT AI was significantly lower in pianists than in controls (p = 0.01).</p

ELISA assay to measure hNGF P61S tagged mutants.

<p>Panel A. Standard calibration curve, obtained with hNGF P61S (red diamonds) or hNGF P61SR100E (orange squares). Panel B. Calibration curves carried out with hNGF P61S (red diamonds), rat NGF (green squares), mouse NGF (yellow circles) and hNGF WT (blue triangles). The experimental points are the mean values of the different experiments, the error bars represent the standard deviations.</p

Cortical motor representation of the hand muscles (mean of APB, ADM and ECR) over the dominant (LH) and non-dominant (RH) hemisphere in pianists and controls (mean and standard error).

<p><b>A.</b> Map_area in the dominant hemisphere of the control group was significantly larger compared with their non-dominant hemisphere (** p = 0.001) and with the dominant hemisphere of pianists (*p = 0.029). <b>B</b>. Example of cortical motor mapping of ADM in a pianist and a control naïve subject. MEPs amplitudes higher than 50 mV were interpolated and projected on an average brain cortical surface reconstruction using Curry software V4.6. The interhemispheric asymmetry in map_area, with larger representation of the dominant hemisphere, is of note only in the naïve subject.</p

Kinetics of proteolytic cleavage of proNGF WT and mutants.

<p>Representative SDS-PAGE of hproNGF WT (panel A), hproNGF P61S (panel B), hproNGF P61SR100E (panel C), hproNGF R100E (panel D) digested by trypsin. The lanes correspond to aliquots of the reaction mixtures, taken at time 0 (immediately after trypsin addition) and after 0.5, 1, 1.5, 2, 3, 4, 6, 20 hrs. The loading position of the molecular weight marker is indicated by M.</p

Inflection points extrapolated by the chemical denaturation profiles of human NGF and proNGF Wild Type and mutants.

<p>Inflection points extrapolated by the chemical denaturation profiles of human NGF and proNGF Wild Type and mutants.</p

<i>In vitro</i> stability by TF1 Proliferation assay read-out.

<p>The fold changes of ΔC50 values for all the destabilizing treatments (temperature 4°C-22°C, freeze-thaw cycles 5–12 and lyophilization) carried out on hNGF WT (blue), hNGF P61S (red), hNGF P61SR100E (yellow) and hNGF R100E (green) is shown. The reference curves exhibits a ΔC50 value equal to 0, so that ΔC50 values higher than 0 indicate that the stability of the NGF sample tested was affected. The hNGF R100E fold change in ΔC50 value corresponding to the incubation at 4°C for 2 weeks is missing (arrow). The experimental points corresponding to this treatment could not be interpolated because they did not fit with the theoretical curve. This behavior indicates a strong destabilization of the protein (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136425#pone.0136425.s002" target="_blank">S2 Fig</a>). Asterisks represent the samples with a p<0.05 when compared to hNGF WT. Hashes represent the samples with a p<0.05 when compared to hNGF P61S. Circles represent the samples with a p<0.05 when compared to hNGF P61SR100E.</p