Cav1.3-selective L-type calcium channel blockers

Ion channels are protein molecules in the membrane of cells, which, upon opening, allow a well- orchestrated influx of ions into cells. The activity of ion channels is essential for the normal functioning of living organisms. Many ion channels are selective for certain ions, such as sodium, potassium or calcium. Voltage-gated ion channels with selectivity for calcium (so-called voltage-gated calcium channels, VGCCs) open in response to electrical excitation, like in heart muscle, vascular smooth muscle, neurons and hormone-secreting cells. Therefore, they play a key role for many physiological processes. D rugs which inhibit VGCCs in the cardiovascular system are being used successfully used clinically for the treatment of hypertension since decades. In previous FWF-funded projects we showed that blood pressure-lowering is due to the inhibition of the Cav1.2 type of VGCCs. More recent data of our and other research groups now also point to a therapeutic potential of the Cav1.3 type, for example for the treatment of Parkinson Disease, treatment-resistant forms of hypertension and chronic muscle spasms after spinal cord injury. Therefore, it is the aim of this project to discover potent and selective inhibitors of Cav1.3 VGCCs. We will express Cav1.3 and other VGCC types in mammalian cells in culture to measure the inhibition of channel activity by chemical compounds using standard electrophysiological methods (whole-cell patch-clamp technique). Compounds with potential Cav1.3-selectivity are already available for further pharmacological characterization. In parallel we employ computer-based methods (ligand- and structure-based molecular modeling) to identify novel chemical structures with Cav1.3-selectivity based on our already developed computer models of these channels. By comparing predicted with the measured pharmacological properties the predictive capacity of these computer models can be constantly refined. This project has the potential to discover chemical lead structures with the desired pharmacological properties which could serve as a further basis for preclinical studies with the aim to test their potential for further clinical development.

We pursued several therapeutically relevant questions concerning the pharmacology of Cav1.3 L-type Ca2+-channels: 1. can symptoms in patients with a neurodevelopmental syndrome caused by gain-of-function mutations in Cav1.3 be ameliorated by inhibiting hyperactive channels by treatment with already licensed Ca2+-channel blockers (CCBs); 2. are mutated channels still sensitive to these blockers and, 3. is it possible to discover Cav1.3-selective inhibitors since available CCBs also potently inhibit Cav1.2 channels which can cause hypotension. We took advantage of a previously generated mouse model (FWF project P27809) carrying the pathogenic mutation A749G and found behavioral abnormalities corresponding to the phenotype in the A749G-patient (autism-like behaviors and novelty-induced hyperactivity). We developed a novel drug application protocol (including a separate pharmacokinetic study) for the oral administration of the CCB isradipine but did not find an effect on the pathological hyperactivity in these mice at therapeutic plasma levels. Likewise, in a patient with another pathogenic mutation no clinically meaningful improvement was observed with CCB treatment. This may be explained by our answer to question #2. We discovered that the previously observed higher sensitivity of several pathogenic mutants (like A749G) is only observed at negative holding potentials in standard electrophysiological protocols. However, apparent isradipine sensitivity of both wildtype and mutant channels (shown by us for A749T and L271H) increases at more positive holding potentials, since the drug has about 100-fold higher affinity for inactivated states. Notably, IC50-values are only in the therapeutic, low nanomolar concentration range when about 50% of channel are inactivated. This predicts that therapeutic concentrations of isradipine would inhibit wildtype and mutant Cav1.3 channels only in subsets of neurons operating at more depolarized membrane potentials. This may be insufficient for a symptomatic effect in our mouse model and in humans (e.g. on hyperactivity or self-injurious behaviors). We therefore also aimed at the discovery of state-independent Cav1.3-selective CCBs, which could also be beneficial for treating patients with CACNA1D mutations but also a number of other human diseases, including spasticity after spinal trauma, treatment resistant hypertension and Parkinsons disease. Using in silico-guided drug discovery and electrophysiological screening employing a previously generated stable Cav1.2- and Cav1.3-expressing HEK293-cells, we have already identified two lead compounds with 10-fold selectivity for Cav1.3. In addition, we have also tested >20 compounds structurally related to the about 10-fold Cav1.3-selective compound B. This study has already shown that none of the analogues is more potent or selective than compound B. Interestingly, the potency of compound B seems to differ in different Cav1.3 splice variants. We have also shown that the natural compound, claimed to be a Cav1.3-selective inhibitor, is a low potency and unselective inhibitor.