FINAL REPORT: Low-throughput evaluation of novel, selective CaV2.1 inhibitors in animal models of Familial Hemiplegic Migraine: modulation of excitatory neurotransmission and analysis of potential therapeutic value (2017)

Summary

Gain-of function mutations (those that promote new or enhanced activity) in the calcium channel (Ca_V2.1) cause multiple neurological diskh778kh778673orders, including both familial (FHM) and sporadic hemiplegic migraine. They produce an imbalance between excitatory and inhibitory neurotransmission at cortical synapses due to a specific enhancement of the excitatory function. This leads to hyper-excitation of neurons in the cerebral cortex that initiates cortical spreading depression and can also trigger the headache phase itself. Pharmacological evidence suggests that reduction of Ca_V2.1 activity has therapeutic potential in the treatment of hemiplegic migraine (HM) and the relief of common migraine. At present, the Ca_V2.1-selective inhibitors available are peptide toxins, which are not suitable therapeutic tools due to their undesirable side effects.

In a previous project funded by the Migraine Research Foundation, we identified 6 new classes of small organic molecules with higher selectivity for Ca_V2.1 inhibition as potential targets from which to develop therapeutic tools. In particular, one of them showed promise at reducing the gain-of-function induced by the pathogenic mutation.

For this grant, we tested these novel inhibitors to see if the reduction of excessive Ca_V2.1 activity was sufficient to prevent the hyper-excitability observed in HM. Our analysis showed that two selective Cav2.1 inhibitors diminished the excitatory synaptic function at the cortical neuronal network. These novel molecules modulated the Ca_V2.1 channel activity and reduced the pathological gain-of-function effect. They could contribute to the development of a new, effective and safe treatment for both hemiplegic and common migraine.

Hypothesis vs. Findings

For this grant, we tested six novel inhibitors to see if the reduction of excessive Ca_V2.1 activity was sufficient to prevent the hyper-excitability observed in HM. We evaluated the effect of the Ca_V2.1 inhibitors on excitability of cortical neurons obtained from both healthy wild-type and FHM model mice. Although our studies with FHM mice are not yet conclusive due to their reduced breeding performance, our analysis showed two selective Cav2.1 inhibitors that diminished by 25-45% the excitatory synaptic function at the cortical neuronal network. These novel molecules modulated the Ca_V2.1 channel activity and reduced the pathological gain-of-function effect.

Unanswered Questions

Our results highlight two novel, specific Ca_V2.1 inhibiOOOtors that reduce hyper-excitability in cortical neurons which might prevent the cortical spreading depression that triggers hemiplegic migraine. However, further validation of the inhibitors’ action is required by using other FHM animal models. We observed that FHM mice present serious breeding difficulties which greatly slows experimentation.

The reduction of cortical hyper-excitability by Ca_V2.1 inhibitors was observed in healthy wild-type mouse neurons after suppression of inhibitory neurotransmission. However, under FHM pathological conditions, those inhibitory inputs are present. As Ca_V2.1 is responsible for the release of both excitatory and inhibitory neurotransmitters, we must verify the effect of Ca_V2.1 inhibitors separately on both excitatory and inhibitory synapsis and in a situation closer to the pathological condition. Thus, further in vitro and in vivo research is required to characterize the pharmacological action of these novel Ca_V2.1 inhibitors to validate their therapeutic potential.

What This Research Means to You

The Migraine Research Foundation Grant allowed us to demonstrate the efficiency of novel, specific Ca_V2.1 inhibitors in reducing hyper-excitability in in vitro cortical neural networks obtained from mice. We will continue working on this research, which we hope will lead to a translational impact on hemiplegic migraine patients. Furthermore, since similar Ca_V2.1 malfunctions leading to hemiplegic migraine are also found in other neurological disorders, these compounds may help disorders beyond hemiplegic migraine and common migraine.

FINAL REPORT: Identification of novel, selective voltage-gated Ca_V2.1 calcium channel inhibitors which reverse the gain of channel function produced by Hemiplegic Migraine CACNA1A mutations (2014)

Summary

Human mutations in the P/Q-type calcium channel (CaV2.1) cause multiple neurological disorders, including Hemiplegic Migraine. These mutations induce a gain of CaV2.1 channel function that produces the hyper-excitation of neurons in the cerebral cortex to favor initiation and propagation of cortical spreading depression (CSD).

CSD is a key process in the origin of migraine: it is the physiological substrate of the migraine aura, and it has also been proposed as a trigger of the headache phase itself. Accordingly, there is evidence suggesting that reduction of CaV2.1 activity can provide a new therapeutic approach for the treatment of Hemiplegic Migraine and possibly, common migraine. Currently the only truly CaV2.1-selective inhibitors are peptide toxins, which are not suitable therapeutic tools, as their mode of inhibition can give rise to undesirable side effects.

Using computer simulations, we evaluated the potential of ~3.5 million compounds taken from several commercial databases to interact with the CaV2.1 channel. From this analysis we found ~17,500 materials that were positive (based on pre-determined criteria). A further refinement based on chemical treatability, availability, price and selectivity for CaV2.1 reduced the number to ~150 compounds, from which we finally evaluated 80 in functional electrophysiological studies. Based on this functional analysis, we identified 19 new compounds capable of selectively inhibiting CaV2.1 channel activity.

Our results suggest the identification of 6 structurally distinct and novel classes of small organic molecules with higher selectivity for CaV2.1 inhibition as prospective hits from which to develop Hemiplegic Migraine therapeutic tools in the future. One in particular shows inhibitory action on the Familial Hemiplegic Migraine mutant CaV2.1 disease relevant channel at concentrations which don’t affect the function of the “healthy” channel.

Hypothesis vs. Findings

We validated our hypothesis and achieved our main goal: the identification of novel potent and selective inhibitors of CaV2.1 capable of preventing the excessive activity of CaV2.1 channels produced by human mutations leading to Hemiplegic Migraine, with minor side effects. Additionally, we have functionally characterized a CaV2.1 modulatory mechanism that emerges as a novel potential therapeutic target for migraine in general.

Unanswered Questions

We focused our functional studies on one of the aspects of CaV2.1 channel activity: the opening of the channel in response to physiological electrical stimulation, which is the main property altered by mutations causing Hemiplegic Migraine. Further studies will be required to evaluate whether these compounds also alter other properties regulating CaV2.1 activity.

More importantly, future work is required to evaluate the consequences of CaV2.1 inhibition by these new compounds (and novel derivatives) on the release of neurotransmitters and neuronal hyper-excitation on Familial Hemiplegic Migraine animal models, in order to validate their therapeutic potential in preclinical studies.

What This Research Means to You

Our results provide novel molecular insights in the pharmacological modulation of CaV2.1 channels and form the basis for the refined development of small organic molecules with potential therapeutic use in Hemiplegic Migraine, and perhaps common migraine.