L-type calcium channels expressed in the brain are heterogeneous. The predominant class of L-type calcium channels has a Ca V 1.2 pore-forming subunit. L-type calcium channels with a Ca V 1.3 pore-forming subunit are much less abundant, but have been implicated in the generation of mitochondrial oxidant stress underlying pathogenesis in Parkinson's disease. Thus, selectively antagonizing Ca V 1.3 L-type calcium channels could provide a means of diminishing cell loss in Parkinson's disease without producing side effects accompanying general antagonism of L-type calcium channels. However, there are no known selective antagonists of Ca V 1.3 L-type calcium channel. Here we report high-throughput screening of commercial and 'in-house' chemical libraries and modification of promising hits. Pyrimidine-2,4,6-triones were identified as a potential scaffold; structure-activity relationship-based modification of this scaffold led to 1-(3-chlorophenethyl)-3-cyclopentylpyrimidine-2,4,6-(1H,3H,5H)-trione (8), a potent and highly selective Ca V 1.3 L-type calcium channel antagonist. The biological relevance was confirmed by whole-cell patch-clamp electrophysiology. These studies describe the first highly selective Ca V 1.3 L-type calcium channel antagonist and point to a novel therapeutic strategy for Parkinson's disease.

Although it is known that the inflammatory response that results from disruption of epithelial barrier function after injury results in excessive scarring, the upstream signals remain unknown. It has also been observed that epithelial disruption results in reduced hydration status and that the use of occlusive dressings that prevent water loss from wounds decreases scar formation. We hypothesized that hydration status changes sodium homeostasis and induces sodium flux in keratinocytes, which result in activation of pathways responsible for keratinocyte-fibroblast signaling and ultimately lead to activation of fibroblasts. Here, we demonstrate that perturbations in epithelial barrier function lead to increased sodium flux in keratinocytes. We identified that sodium flux in keratinocytes is mediated by epithelial sodium channels (ENaCs) and causes increased secretion of proinflammatory cytokines, which activate fibroblast via the cyclooxygenase 2 (COX-2)/prostaglandin E2 (PGE2) pathway. Similar changes in signal transduction and sodium flux occur by increased sodium concentration, which simulates reduced hydration, in the media in epithelial cultures or human ex vivo skin cultures. Blockade of ENaC, prostaglandin synthesis, or PGE2 receptors all reduce markers of fibroblast activation and collagen synthesis. In addition, employing a validated in vivo excessive scar model in the rabbit ear, we demonstrate that utilization of either an ENaC blocker or a COX-2 inhibitor results in a marked reduction in scarring. Other experiments demonstrate that the activation of COX-2 in response to increased sodium flux is mediated through the PIK3/Akt pathway. Our results indicate that ENaC responds to small changes in sodium concentration with inflammatory mediators and suggest that the ENaC pathway is a potential target for a strategy to prevent fibrosis.

CaV1.3 L-type calcium channels (LTCCs) have been a potential target for Parkinson’s disease since calcium ion influx through the channel was implicated in the generation of mitochondrial oxidative stress, causing cell death in the dopaminergic neurons. Selective inhibition of CaV1.3 over other LTCC isoforms, especially CaV1.2, is critical to minimize potential side effects. We recently identified pyrimidinetriones (PYTs) as a CaV1.3-selective scaffold; here we report the structure-activity relationship of PYTs with both CaV1.3 and CaV1.2 LTCCs. By variation of the substituents on the cyclopentyl and arylalkyl groups of PYT, SAR studies allowed characterization of the CaV1.3 and CaV1.2 LTCCs binding sites. The SAR also identified four important moieties that either retain selectivity or enhance binding affinity. Our study represents a significant enhancement of the SAR of PYTs at CaV1.3 and CaV1.2 LTCCs and highlights several advances in the lead optimization and diversification of this family of compounds for drug development.

Ca 2+ channels with a Ca V 1.3 pore-forming α 1 subunit have been implicated in both neurodegenerative and neuropsychiatric disorders, motivating the development of selective and potent inhibitors of Ca V 1.3 vs Ca V 1.2 channels, the calcium channels implicated in hypertensive disorders. We have previously identified pyrimidine-2,4,6-triones (PYTs) that preferentially inhibit Ca V 1.3 channels, but the structural determinants of their interaction with the channel have not been identified, impeding their development into drugs. Using a combination of biochemical, computational, and molecular biological approaches, it was found that PYTs bind to the dihydropyridine (DHP) binding pocket of the Ca V 1.3 subunit, establishing them as negative allosteric modulators of channel gating. Site-directed mutagenesis, based on homology models of Ca V 1.3 and Ca V 1.2 channels, revealed that a single amino acid residue within the DHP binding pocket (M1078) is responsible for the selectivity of PYTs for Ca V 1.3 over Ca V 1.2. In addition to providing direction for chemical optimization, these results suggest that, like dihydropyridines, PYTs have pharmacological features that could make them of broad clinical utility.

Calcium influx elevates mitochondrial oxidant stress (mOS) in dorsal motor nucleus of the vagus (DMV) neurons that are prone to Lewy body pathologies in presymptomatic Parkinson's disease (PD) patients. In experimental PD models, treatment with isradipine, the dihydropyridine with the highest affinity to Cav1.3 channels, prevents subthreshold calcium influx via Cav1.3 channels into midbrain dopamine neurons and protects them from mOS. In DMV neurons, isradipine is also effective in reducing mOS despite overwhelming evidence that subthreshold calcium influx is negligible compared with spike-triggered influx. To solve this conundrum we combined slice electrophysiology, two-photon laser scanning microscopy, mRNA profiling, and computational modeling. We find that the unusually depolarized subthreshold voltage trajectory of DMV neurons is positioned between the relatively hyperpolarized activation curve of Cav1.3 channels and that of other high-voltage activated (HVA) calcium channels, thus creating a functional segregation between Cav1.3 and HVA calcium channels. The HVA channels flux the bulk of calcium during spikes but can only influence pacemaking through their coupling to calcium-activated potassium currents. In contrast, Cav1.3 currents, which we show to be more than an order-of-magnitude smaller than the HVA calcium currents, are able to introduce sufficient inward current to speed up firing. However, Kv4 channels that are constitutively open in the subthreshold range guarantee slow pacemaking, despite the depolarizing action of Cav1.3 and other pacemaking currents. We propose that the efficacy of isradipine in preventing mOS in DMV neurons arises from its mixed effect on Cav1.3 channels and on HVA Cav1.2 channels.