4. DISCUSSION
Our data suggest that riluzole is the first member of an entirely new
class of sodium channel inhibitor compounds. Its primary binding site is
the well-characterized ”local anesthetic receptor,” with F1579 as the
key residue. However, the location of the bound ligand is different from
that of local anesthetics, and the effect exerted by it is also
radically different. As for the location, in silico docking
revealed that unlike other sodium channel inhibitor molecules, riluzole
was preferentially located within the fenestrations, and tended to avoid
the central part of the pore or the vicinity of the selectivity filter,
where it could interfere with conduction. Localization within the
fenestration has been previously observed in molecular dynamics
simulations investigating the general anesthetic isoflurane (Raju et
al., 2013) and propofol (Wang et al., 2018), as well as the local
anesthetic benzocaine (Boiteux et al., 2014; Buyan et al., 2018; Martin
& Corry, 2014) and lidocaine (neutral form) (Nguyen et al., 2019), but
it has never been found to be the dominant position. The unusual
location predicted by in silico docking is supported by the way riluzole
acted in experiments. It stabilized inactivated conformation without
interference with conduction. Inactivated state stabilization affected
both the equilibrium and the recovery rate, as evidenced by the shift of
the SSI and the RFI curves, respectively (Fig. 1; see
also Fig. 1 in (Lukacs et al., 2018)).
The non-instantaneous onset (as seen in the SDO protocol) and
the delayed recovery (see the RFI protocol) together ensure
that effective inhibition is confined within a strict temporal window.
Single pulse-evoked currents or synaptic activity-evoked action
potentials are ”missed” by riluzole because inhibition becomes effective
only after the channel has reached inactivated conformation, and the
conformation of the ligand-channel complex has been stabilized. After
this, the inhibition is effective, but only for a limited time – even
in the case of the non-therapeutic concentration of 100 µM it was only
effective for approximately 10-20 ms. At therapeutic concentrations the
effect of riluzole is no more than a fine modulation: the refractory
time after an action potential is prolonged by a few milliseconds, and a
small fraction of the channel population is kept in inactivated
conformation. The exact extent of prolongation and the magnitude of the
fraction kept in inactivated conformation is dependent on the membrane
potential during the interspike interval. This temporal window of
effectiveness is the basis of both persistent (or ”late”) sodium current
selectivity and the low pass filtering effect. The contribution of the
persistent component of sodium currents (INaP) is
significant during gradual depolarizations preceding action potentials,
especially during burst firing (Del Negro et al., 2010; Taddese & Bean,
2002). In these cases, there is enough time for the onset of inhibition,
and the inhibition remains effective until the membrane becomes
hyperpolarized for a sufficient time.
An upregulation of the persistent sodium current has been shown to be
involved in a number of pathologies, including epilepsies (Oyrer et al.,
2018; Stafstrom, 2007), cardiac pathologies (Antzelevitch et al., 2014;
Chadda et al., 2017; Makielski, 2016), neurodegenerative disorders (van
Zundert et al., 2012; Waxman, 2008), pain disorders (Lampert et al.,
2006; Misawa et al., 2009; Xie et al., 2011), even cancer metastasis
(Djamgoz & Onkal, 2013), and type II diabetes (Rizzetto et al., 2015).
Selective inhibitors of INaP, including riluzole, have
been found to be effective in cardiac diseases (Antzelevitch et al.,
2014; Belardinelli et al., 2013; Karagueuzian et al., 2017), epilepsies
(Anderson et al., 2014), pain syndromes (Xie et al., 2011), and
neurodegenerative diseases. Riluzole was especially effective in
preventing damage in spinal cord and peripheral nerve injury(Fehlings et
al., 2016; Ghayour et al., 2017; Gloviczki et al., 2017).
Riluzole represents a new class among INaP selective
inhibitors because the non-blocking modulation mechanism endows it with
an ”ultrafast” offset kinetics, which, however, is not based on actual
dissociation. A similar mechanism has been assumed for inhibition by
riluzole in a recent computational study (Phillips & Rubin, 2019). To
illustrate how this compares to offset kinetics of other well-known
sodium channel inhibitors, we re-plotted the data from a comparative
study of nine inhibitor compounds (El-Bizri et al., 2018), which
included six antiarrhythmic drugs, and the three best known
INaP selective inhibitors. We supplemented these data
with our results on the effects of riluzole (Fig. 8). Binding rate
constants were plotted against unbinding rate constants. The latter were
calculated from the time constants of the offset
(koff = 1 / τoff ), then the
former was calculated from koff and theIC50 value (kon =
koff / IC50 ). We calculatedkon and koff values the
same way for riluzole using time constants of both the fast and slow
recovery processes (τfast = 2.25 ms, and
τslow = 823 ms), and the IC50value 3.98 µM. As for the slow recovery process (which reflects true
dissociation) riluzole did not differ from the other compounds, but when
we used data from fast recovery process, it was in a completely
different range. It seems that by staying in the fenestration, riluzole
can ”pretend” to have been dissociated, which practically increases its
speed 366-fold. This allows it to be effective in frequency ranges that
are unavailable for conventional drugs. If there was genuine
dissociation during the fast recovery process, the value ofkon would be 112
s-1µM-1. This would require an onset
time constant of 0.086 ms at 100 µM concentration (from the equationτon = 1/(cc *kon +koff )), which is clearly much faster than the
experimentally observed onset.
In summary, our data indicate that riluzole is unique in being able to
combine fast onset kinetics with high affinity to the conventional local
anesthetic binding site of sodium channels. This high-affinity binding
occurs only in depolarized (open or inactivated) conformations. The
binding itself does not prevent conduction, only slightly alters the
gating of channels, making them more likely to stay in inactivated
conformation. This produces a preferential inhibition of i)
INaP, ii) cells with depolarized membrane potential, and
iii) cells firing at high frequencies. The combination of increased
persistent component, compromised ability to keep resting membrane
potential, and abnormally high firing frequency almost always signifies
pathology, which is the reason why riluzole can selectively inhibit this
type of pathological activity. The basis of this selectivity is the
non-blocking modulation mechanism. We anticipate that compounds of this
class (fast-acting non-blocking modulators) may be favorable for the
treatment of a number of pathologies, such as neuropathic pain
syndromes, certain neuromuscular diseases, epilepsies, and cardiac
fibrillations, which are characterized by an enlarged persistent sodium
current and high-frequency firing.