On 2017 Feb 13, Massimiliano Tognolini commented:

None

On 2014 Dec 13, Massimiliano Tognolini commented:

• CPD1 is a not reliable pharmacological tool as it spontaneously forms a mixture of polymers, with a molecular weight of 40 kDa, which are likely responsible for its biological activity (Baell and Holloway, 2010; Zhu et al., 2013). Conversely pure, freshly synthesized CPD1 is inactive (Noberini et al., 2011) on EphA4 receptor, consequently data reported in Figure 2F, showing that the inhibition of EphA4 by CPD1 prevents the Eph-mediated neurotransmission impairment, should be carefully considered.

• A not-reported concentration of biotinylated rhynchophylline (and not rhynchophylline alone), co-precipitates with the chimeric protein composed by the Fc region of immunoglobulin and the extracellular domain of EphA4. As a signal can be detected also for the control (Figure 4A, lane 1 column 1) the interpretation of this data is unclear.

• Rhynchophylline inhibition of EphA4 phosphorylation in rat neurons was claimed. However, the poor reduction of EphA4 phosphorylation reported in Figures 4B and 4C (20-25% of inhibition), with an unknown concentration of rhynchophylline, is not supported by a cell-viability study nor by an inhibition assay on the isolated kinase domain of EphA4. Without these data, the mechanism of action of rhynchophylline is not proved.

• The indication of concentration is essential (fig 4A, 4B, 4C). The evaluation of Rhynchophylline Ki, Kd or IC50 is crucial.

• Considering the large number of pharmacological activities attributed to Rhynchophylline, i.e. interference with : i) platelet aggregation (Jin et al., 1991), ii) 5HT and dopamine release (Shi et al., 1993), iii) calcium channels (Wang et al., 1994; Shimada et al., 1999), apoptosis (Shi and Kenneth, 2002), iv) NMDA receptors (Kang et al., 2002), v) voltage-gated channel (Chou et al., 2009), vi) NF-kappaB and AP-1(Hsieh et al., 2009), NR2B expression (Zhou et al., 2010), is rather complex demonstrating that this compound can act on a single target.

• In our hands, when tested in our binding study (Giorgio, Plos one, 2011 and Tognolini, ACS chem neurosci, 2014) rhynchophylline does not interfere with Eph-ephrin interaction.

Thank you for your attention.

On 2014 Dec 13, Massimiliano Tognolini commented:

• CPD1 is a not reliable pharmacological tool as it spontaneously forms a mixture of polymers, with a molecular weight of 40 kDa, which are likely responsible for its biological activity (Baell and Holloway, 2010; Zhu et al., 2013). Conversely pure, freshly synthesized CPD1 is inactive (Noberini et al., 2011) on EphA4 receptor, consequently data reported in Figure 2F, showing that the inhibition of EphA4 by CPD1 prevents the Eph-mediated neurotransmission impairment, should be carefully considered.

• A not-reported concentration of biotinylated rhynchophylline (and not rhynchophylline alone), co-precipitates with the chimeric protein composed by the Fc region of immunoglobulin and the extracellular domain of EphA4. As a signal can be detected also for the control (Figure 4A, lane 1 column 1) the interpretation of this data is unclear.

• Rhynchophylline inhibition of EphA4 phosphorylation in rat neurons was claimed. However, the poor reduction of EphA4 phosphorylation reported in Figures 4B and 4C (20-25% of inhibition), with an unknown concentration of rhynchophylline, is not supported by a cell-viability study nor by an inhibition assay on the isolated kinase domain of EphA4. Without these data, the mechanism of action of rhynchophylline is not proved.

• The indication of concentration is essential (fig 4A, 4B, 4C). The evaluation of Rhynchophylline Ki, Kd or IC50 is crucial.

• Considering the large number of pharmacological activities attributed to Rhynchophylline, i.e. interference with : i) platelet aggregation (Jin et al., 1991), ii) 5HT and dopamine release (Shi et al., 1993), iii) calcium channels (Wang et al., 1994; Shimada et al., 1999), apoptosis (Shi and Kenneth, 2002), iv) NMDA receptors (Kang et al., 2002), v) voltage-gated channel (Chou et al., 2009), vi) NF-kappaB and AP-1(Hsieh et al., 2009), NR2B expression (Zhou et al., 2010), is rather complex demonstrating that this compound can act on a single target.

• In our hands, when tested in our binding study (Giorgio, Plos one, 2011 and Tognolini, ACS chem neurosci, 2014) rhynchophylline does not interfere with Eph-ephrin interaction.

Thank you for your attention.

On 2017 Feb 13, Massimiliano Tognolini commented:

None