Evaluation of edonerpic maleate as a CRMP2 inhibitor for pain relief

ABSTRACT We have previously reported that the microtubule-associated collapsin response mediator protein 2 (CRMP2) is necessary for the expression of chronic pain. CRMP2 achieves this control of nociceptive signaling by virtue of its ability to regulate voltage-gated calcium and sodium channels. To date, however, no drugs exist that target CRMP2. Recently, the small molecule edonerpic maleate (1 -{3-[2-(1-benzothiophen-5-yl)ethoxy]propyl}azetidin-3-ol maleate), a candidate therapeutic for Alzheimer’s disease was reported to be a novel CRMP2 binding compound with the potential to decrease its phosphorylation level in cortical tissues in vivo. Here we sought to determine the mechanism of action of edonerpic maleate and test its possible effect in a rodent model of chronic pain. We observed: (i) no binding between human CRMP2 and edonerpic maleate; (ii) edonerpic maleate had no effect on CRMP2 expression and phosphorylation in dorsal root ganglion (DRG) neurons; (iii) edonerpic maleate-decreased calcium but increased sodium current density in DRG neurons; and (iv) edonerpic maleate was ineffective in reversing post-surgical allodynia in male and female mice. Thus, while CRMP2 inhibiting compounds remain a viable strategy for developing new mechanism-based pain inhibitors, edonerpic maleate is an unlikely candidate.

When searching for novel non-opioid therapeutic targets for chronic pain treatment, we identified the collapsin response mediator protein 2 (CRMP2) as necessary for mechanical allodynia [1]. CRMP2 is localized pre-synaptically in the dorsal horn of the spinal cord where dorsal root ganglia (DRG) sensory neurons synapse onto second-order neurons [2,3]. At this location, CRMP2 controls the presynaptic levels of both CaV2.2 and NaV1.7, two voltage gated ion channels essential for nociceptive signal transmission [3]. Actions of CRMP2 are tightly regulated by post-translational modifications [1]. In chronic neuropathic pain, we found that CRMP2 phosphorylation by cyclin-dependent kinase 5 (Cdk5) on Serine 522 (S522) was sufficient for mechanical allodynia [2] through the facilitation of CaV2.2 and NaV1.7 function [4][5][6][7][8]. By contrast, CRMP2 phosphorylation by the src tyrosineprotein kinase Fyn on tyrosine 32 (Y32) inhibits NaV1.7 [4]. Thus, CRMP2 phosphorylation could be leveraged for the identification of new therapeutics for pain management. Along these lines, a recent study reported the small molecule edonerpic maleate (T-817MA, 1 -{3- [2-(1-benzothiophen-5-yl)ethoxy] propyl}azetidin-3-ol maleate), a candidate therapeutic for Alzheimer's disease found to increase neurite outgrowth [9], as a novel CRMP2-binding compound with the potential to decrease its phosphorylation level in cortical tissues in vivo [10]. Here we sought to determine the mechanism of action of edonerpic maleate and its possible effect in a rodent model of chronic pain.
Edonerpic maleate was reported to bind to CRMP2 with a Kd of~735 µM by isothermal titration calorimetry (ITC) [10]. Given this lowaffinity binding, we first set out to confirm this through complementary biophysical approaches. We used saturation transfer difference nuclear magnetic resonance (STD-NMR) -a method for studying transient protein-ligand interactions in solutionto assess a direct binding between this small molecule and CRMP2. We tested three different ratios of CRMP2/edonerpic maleate: no binding was detected between CRMP2 and edonerpic maleate (Figure 1(a)). (S)-lacosamide, an inhibitor of CRMP2 phosphorylation [3,5,7], was used as a positive control at the same concentrations and showed binding to CRMP2 (not shown), consistent with our previously published finding [7]. Next, we used microscale thermophoresis (MST), a method for the biophysical analysis of biomolecular interactions, but were unable to detect any interaction between edonerpic maleate and CRMP2 with this method either (not shown). Thus, our results argue against a direct binding between CRMP2 and this small molecule. The differences between our results and previous findings may reflect differences of sensitivity between these methodologies as the Kd obtained by ITC required utilization of~10 fold more CRMP2 than our studies. Notwithstanding the incongruency between our findings and those of Abe et al. [10] we pursued further exploration of the biological effect of edonerpic maleate on CRMP2.
Despite the opposing effect on calcium and sodium channels, we next asked whether edonerpic maleate could be beneficial for pain. As calcium channels are known mediators of post-surgical allodynia [5,21], we evaluated the anti-allodynic potential of edonerpic maleate in a mouse model of post-surgical pain [27]. Female and male mice were subjected to a paw incision which resulted in allodynia at 24 h after injury and then treated intraperitoneally with edonerpic maleate (20 mg/kg, i.p.). Edonerpic maleate failed to reverse post-surgical allodynia in the 5-h window following injection (Figure 1(l-o)). These results show that although edonerpic maleate treatment results in inhibition of calcium currents in sensory neurons, it fails to reverse allodynia.
Together, these results show that edonerpic maleate is unlikely to be a CRMP2 targeting small molecule. We base this conclusion on (i) our inability to detect any binding between edonerpic maleate and CRMP2, and (ii) the observation that edonerpic maleate treatment of DRG sensory neurons had no effect on CRMP2 expression and phosphorylation. That edonerpic maleate decreased calcium but increased sodium current density in DRG neurons likely accounts for why the compound was ineffective in reversing post-surgical allodynia. In contrast, . Summary of the normalized (pA/pF) total calcium current (I Ca ) density versus voltage relationship (d) and peak total Ca 2+ current density at +10 mV (mean ± SEM) (E) from DRG sensory neurons treated as indicated (n=10 each). Boltzmann fits for normalized conductance G/G max voltage relations for voltage-dependent activation (f) and inactivation (g) of sensory neurons treated as indicated. Summary of the normalized (pA/pF) total sodium current (I Na ) density versus voltage relationship (h) and peak total Na 2+ current density at +10 mV (mean ± SEM) (I) from DRG sensory neurons treated as indicated (n=9 for 0.1%DMSO and n=10 for edonerpic maleate). Boltzmann fits for normalized conductance G/G max voltage relations for voltage-dependent activation (j) and inactivation (k) of sensory neurons treated as indicated. After a paw incision surgery, both male and female mice developed mechanical allodynia at 24 h. Paw withdrawal threshold of adult (L) female (n = 9) or (n) male (n=9) was measured after injection with edonerpic maleate (20 mg/kg, i.p.) or vehicle (0.1% DMSO in saline). Area under the curve was derived for (m) female or (o) male mice. Experimenter was blinded to the treatment condition. Exact p-values are indicated for each panel (Mann-Whitney test).
subverting CRMP2 phosphorylation with (S)lacosamide not only decreases calcium currents but is also able to reverse post-surgical allodynia, supporting our premise that CRMP2 inhibiting compounds could be useful for mitigating pain. However, edonerpic maleate does not meet the criteria as a CRMP2 inhibitor. Recently, edonerpic maleate showed no clinical efficacy in patients with mild to moderate Alzheimer's disease [28]. This clinical study with our pre-clinical results may indicate that the therapeutic actions of Edonerpic maleate could be specific to stroke.

Animals
As done previously [3], adult female Sprague Dawley rats (for electrophysiology experiments, Pathogen-free; 100 g; Envigo) or CD1 mice (19-22 g, Charles River) were housed in light and temperature-controlled conditions (12-h light/12-h dark cycle; lights on 07:00-19:00; 23 ± 3°C), fed ad libitum with standard rodent chow and water. All experiments and procedures were conducted in accordance with the regulations of the Institutional Animal Care and Use Committee of the University of Arizona's College of Medicine and the NIH-published Guide for Care and Use of Laboratory Animals', as well as the ethical regulations of the International Association for the Study of Pain. With regard to experimental design, behavioral experiments were done with random assignment of animals to both treatment and control conditions; experimenters were blinded to both experimental groups and treatments.
Saturation transfer difference nuclear magnetic resonance spectroscopy 1 D 1 H saturation transfer difference nuclear magnetic resonance (STD NMR) spectra with a spectral width of 12 ppm were collected for samples containing 500 or 100 µM endonerpic compound with either 1 or 5 µM CRMP2-His (1:100 dilution was always maintained) in PBS, 10% D 2 O. STD NMR spectra were collected with a spectral width of 12 ppm, 16 K data points, and 3 second repetition delay. A saturation of the protein was achieved by a 2 second train of selective 50 ms Gaussian pulses centered at 0.74 ppm (on-resonance) and 30 ppm (off resonance). A 20ms spin-lock was used to suppress the protein signal, followed by the double PFG spin echo to remove residual water signal. We acquired 512 scans per experiment. The on-resonance and offresonance spectra were acquired interleaved, and the difference spectrum was acquired by phase cycling. Spectra processing and analysis were performed with the VNMRJ 3.2 (Agilent Technologies, Santa Clara, CA) and MestReNova 7.1 (Mestrelab Research, S.L., Santiago de Compostela, Spain).

Preparation of acutely dissociated dorsal root ganglion neurons
Dorsal root ganglia from all levels were acutely dissociated using methods as described previously [4]. Rat DRG neurons were isolated from 100 g Sprague-Dawley rats using previously developed procedures [9]. In brief, removing dorsal skin and muscle and cutting the vertebral bone processes parallel to the dissection stage-exposed DRG. Dorsal root ganglia were then collected, trimmed at their roots, and enzymatically digested in 3 mL bicarbonate-free, serum-free, sterile DMEM (Cat# 11965, Thermo Fisher Scientific) solution containing neutral protease (3.125 mg.ml −1 , Cat#LS02104; Worthington, Lakewood, NJ) and collagenase type I (5 mg/mL, Cat# LS004194, Worthington, Lakewood, NJ) and incubated for 60 min at 37°C under gentile agitation. Dissociated DRG neurons (~1.5 × 10 6 ) were then gently centrifuged to collect cells and washed with DRG media DMEM containing 1% penicillin/streptomycin sulfate from 10,000 μg/mL stock, 30 ng/mL nerve growth factor, and 10% fetal bovine serum (Hyclone) before plating onto poly-D-lysineand laminin-coated glass 12-or 15-mm coverslips. Small diameter neurons were selected to target Aδ-and c-fiber nociceptive neurons. For rat DRG cultures, small cells were considered to be~< 30 μm. All cultures were used within 48 h.
Whole-cell patch recordings of Ca 2+ currents in acutely dissociated dorsal root ganglion neurons Recordings were obtained from acutely dissociated DRG neurons as described previously [10,11]. To isolate calcium currents, Na + and K + currents were blocked with 500 nM tetrodotoxin (TTX; Alomone Laboratories) and 30 mM tetraethylammonium chloride (TEA-Cl; Sigma). Extracellular recording solution (at~310 mOsm) consisted of the following (in mM): 110 N-methyl-D-glucamine (NMDG), 10 BaCl 2 , 30 TEA-Cl, 10 HEPES, 10 glucose, pH at 7.4, 0.001 TTX, 0.01 nifedipine. The intracellular recording solution (at~310 mOsm) consisted of the following (in mM): contained 150 CsCl 2 , 10 HEPES, 5 Mg-ATP, 5 BAPTA, pH at 7.4. Activation of I Ca was measured by using a holding voltage of −90 mV with voltage steps 200 ms in duration applied at 5-s intervals in +10 mV increments from −70 to +60 mV. Current density was calculated as peak I Ca /cell capacitance. Steady-state inactivation of I Ca was determined by applying an 800-ms conditioning prepulse (−100 to −20 mV in +10 mV increments) after which the voltage was stepped to −20 mV for 200 ms; a 15-s interval separated each acquisition.
Whole-cell voltage clamp recordings were performed at room temperature (RT) using an EPC 10 Amplifier-HEKA as previously described [6]. DRG neurons were subjected to current-density (I-V) and fast-inactivation voltage protocols as previously described [4,12]. In the I-V protocol, cells were held at a −80 mV holding potential before depolarization by 20-ms voltage steps from −70 to +60 mV in 5-mV increments. This allowed for collection of current density data to analyze activation of sodium channels as a function of current vs voltage and also peak current density, which was typically observed near~0 to 10 mV and normalized to cell capacitance (pF). In the fast-inactivation protocol, cells were held at a − 80 mV holding potential prior to hyperpolarizing and repolarizing pulses for 500 ms between −120 and −10 mV in 5 mV increments. This step conditioned various percentages of channels into fast-inactivated states so that a 0-mV test pulse for 20 ms could reveal relative fast inactivation normalized to maximum current. Firepolished recording pipettes, 2 to 5 MΩ resistance were used for all recordings. Whole-cell recordings were obtained with a HEKA EPC-10 USB (HEKA Instruments Inc., Bellmore, NY); data were acquired with a Patchmaster (HEKA) and analyzed with a Fitmaster (HEKA). Capacitive artifacts were fully compensated, and series resistance was compensated by~70%. Recordings made from cells with greater than a 5 mV shift in series resistance compensation error were excluded from analysis. All experiments were performed at room temperature (~23°C).
The Boltzmann relation was used to determine the voltage dependence for activation of I Ca and I Na wherein the conductance-voltage curve was fit by the equation G/G max = 1/[1 + exp (V 0.5 − V m )/k], where G is the conductance G = I/(V m −E Ca or E Na ), G max is the maximal conductance obtained from the Boltzmann fit under control conditions, V 0.5 is the voltage for half-maximal activation, V m is the membrane potential, and k is a slope factor. E Ca is the reversal potential for I Ca ; E Na is the reversal potential for I Na and was determined for each individual neuron. The values of I Ca and I Na around the reversal potential were fit with a linear regression line to establish the voltage at which the current was zero. The Boltzmann parameters were determined for each individual neuron and then used to calculate the mean ± S.E.M.

Surgeries and behavioral analysis
Mouse paw incision and pSNL surgeries were done following published methods [13]. Allodynia was tested as described previously [3,14]. Data was analyzed as described by Chaplan et al. [15] using the nonparametric method of Dixon.

Statistics
Statistical analyses were performed using GraphPad Prism 8 (GraphPad, La Jolla, CA). Data were sourced from a minimum of three independent biological replicates unless indicated otherwise. All data represent the mean ± S.E.M. The statistical significance of differences between groups was determined by non-parametric Student's t-test, analysis of variance (ANOVA) followed by post hoc comparisons (Tukey) using Prism 8. Statistical significance was set at p < 0.05.