Pharmacological Profile of the Novel Antiepileptic Drug Candidate Padsevonil: Interactions with Synaptic Vesicle 2 Proteins and the GABAA Receptor.

Padsevonil is an antiepileptic drug (AED) candidate synthesized in a medicinal chemistry program initiated to rationally design compounds with high affinity for synaptic vesicle 2 (SV2) proteins and low-to-moderate affinity for the benzodiazepine binding site on GABAA receptors. The pharmacological profile of padsevonil was characterized in binding and electrophysiological experiments. At recombinant SV2 proteins, padsevonil's affinity for SV2A was greater than that of levetiracetam and brivaracetam (pKi 8.5, 5.2, and 6.6, respectively). Unlike the latter AEDs, both selective SV2A ligands, padsevonil also displayed high affinity for the SV2B and SV2C isoforms (pKi 7.9 and 8.5, respectively). Padsevonil's interaction with SV2A differed from that of levetiracetam and brivaracetam; it exhibited slower binding kinetics: dissociation t 1/2 30 minutes from the human protein at 37°C compared with <0.5 minute for levetiracetam and brivaracetam. In addition, its binding was not potentiated by the allosteric modulator UCB1244283. At recombinant GABAA receptors, padsevonil displayed low to moderate affinity (pIC50≤6.1) for the benzodiazepine site, and in electrophysiological studies, its relative efficacy compared with zolpidem (full-agonist reference drug) was 40%, indicating partial agonist properties. In in vivo (mice) receptor occupancy studies, padsevonil exhibited SV2A occupancy at low ED50 (0.2 mg/kg) and benzodiazepine site occupancy at higher doses (ED50 36 mg/kg), supporting in vitro results. Padsevonil's selectivity for its intended targets was confirmed in profiling studies, where it lacked significant effects on a wide variety of ion channels, receptors, transporters, and enzymes. Padsevonil is a first-in-class AED candidate with a unique target profile allowing for presynaptic and postsynaptic activity. SIGNIFICANCE STATEMENT: Padsevonil is an antiepileptic drug candidate developed as a single molecular entity interacting with both presynaptic and postsynaptic targets. Results of in vitro and in vivo radioligand binding assays confirmed this target profile: padsevonil displayed nanomolar affinity for the three synaptic vesicle 2 protein isoforms (SV2A, B, and C) and micromolar affinity for the benzodiazepine binding site on GABAA receptors. Furthermore, padsevonil showed greater affinity for and slower binding kinetics at SV2A than the selective SV2A ligands, levetiracetam, and brivaracetam.

Tissue-Specific Ferritin- and GFP-Based Genetic Vectors Visualize Neurons by MRI in the Intact and Post-Ischemic Rat Brain.

(1) Background: Neurogenesis is considered to be a potential brain repair mechanism and is enhanced in stroke. It is difficult to reconstruct the neurogenesis process only from the histological sections taken from different animals at different stages of brain damage and restoration. Study of neurogenesis would greatly benefit from development of tissue-specific visualization probes. (2) Purpose: The study aimed to explore if overexpression of ferritin, a nontoxic iron-binding protein, under a doublecortin promoter can be used for non-invasive visualization of neurogenesis using magnetic resonance imaging (MRI). (3) Methods: Ferritin heavy chain (FerrH) was expressed in the adeno-associated viral backbone (AAV) under the doublecortin promoter (pDCX), specific for young neurons, in the viral construct AAV-pDCX-FerrH. Expression of the enhanced green fluorescent protein (eGFP) was used as an expression control (AAV-pDCX-eGFP). The viral vectors or phosphate-buffered saline (PBS) were injected intracerebrally into 18 adult male Sprague-Dawley rats. Three days before injection, rats underwent transient middle-cerebral-artery occlusion or sham operation. Animals were subjected to In vivo MRI study before surgery and on days 7, 14, 21, and 28 days after injection using a Bruker BioSpec 11.7 T scanner. Brain sections obtained on day 28 after injection were immunostained for ferritin, young (DCX) and mature (NeuN) neurons, and activated microglia/macrophages (CD68). Additionally, RT-PCR was performed to confirm ferritin expression. (4) Results: T2* images in post-ischemic brains of animals injected with AAV-pDCX-FerrH showed two distinct zones of MRI signal hypointensity in the ipsilesioned hemisphere starting from 14 days after viral injection-in the ischemic lesion and near the lateral ventricle and subventricular zone (SVZ). In sham-operated animals, only one zone of hypointensity near the lateral ventricle and SVZ was revealed. Immunochemistry showed that ferritin-expressing cells in ischemic lesions were macrophages (88.1%), while ferritin-expressing cells near the lateral ventricle in animals both after ischemia and sham operation were mostly mature (55.7% and 61.8%, respectively) and young (30.6% and 7.1%, respectively) neurons. RT-PCR confirmed upregulated expression of ferritin in the caudoputamen and corpus callosum. Surprisingly, in animals injected with AAV-pDCX-eGFP we similarly observed two zones of hypointensity on T2* images. Cellular studies also showed the presence of mature (81.5%) and young neurons (6.1%) near the lateral ventricle in both postischemic and sham-operated animals, while macrophages in ischemic lesions were ferritin-positive (98.2%). (5) Conclusion: Ferritin overexpression induced by injection of AAV-pDCX-FerrH was detected by MRI using T2*-weighted images, which was confirmed by immunochemistry showing ferritin in young and mature neurons. Expression of eGFP also caused a comparable reduced MR signal intensity in T2*-weighted images. Additional studies are needed to investigate the potential and tissue-specific features of the use of eGFP and ferritin expression in MRI studies.

Synaptic vesicle glycoprotein 2A modulates vesicular release and calcium channel function at peripheral sympathetic synapses.

Synaptic vesicle glycoprotein (SV)2A is a transmembrane protein found in secretory vesicles and is critical for Ca(2+) -dependent exocytosis in central neurons, although its mechanism of action remains uncertain. Previous studies have proposed, variously, a role of SV2 in the maintenance and formation of the readily releasable pool (RRP) or in the regulation of Ca(2+) responsiveness of primed vesicles. Such previous studies have typically used genetic approaches to ablate SV2 levels; here, we used a strategy involving small interference RNA (siRNA) injection to knockdown solely presynaptic SV2A levels in rat superior cervical ganglion (SCG) neuron synapses. Moreover, we investigated the effects of SV2A knockdown on voltage-dependent Ca(2+) channel (VDCC) function in SCG neurons. Thus, we extended the studies of SV2A mechanisms by investigating the effects on vesicular transmitter release and VDCC function in peripheral sympathetic neurons. We first demonstrated an siRNA-mediated SV2A knockdown. We showed that this SV2A knockdown markedly affected presynaptic function, causing an attenuated RRP size, increased paired-pulse depression and delayed RRP recovery after stimulus-dependent depletion. We further demonstrated that the SV2A-siRNA-mediated effects on vesicular release were accompanied by a reduction in VDCC current density in isolated SCG neurons. Together, our data showed that SV2A is required for correct transmitter release at sympathetic neurons. Mechanistically, we demonstrated that presynaptic SV2A: (i) acted to direct normal synaptic transmission by maintaining RRP size, (ii) had a facilitatory role in recovery from synaptic depression, and that (iii) SV2A deficits were associated with aberrant Ca(2+) current density, which may contribute to the secretory phenotype in sympathetic peripheral neurons.

Novel Green Fluorescent Polyamines to Analyze ATP13A2 and ATP13A3 Activity in the Mammalian Polyamine Transport System.

Cells acquire polyamines putrescine (PUT), spermidine (SPD) and spermine (SPM) via the complementary actions of polyamine uptake and synthesis pathways. The endosomal P5B-type ATPases ATP13A2 and ATP13A3 emerge as major determinants of mammalian polyamine uptake. Our biochemical evidence shows that fluorescently labeled polyamines are genuine substrates of ATP13A2. They can be used to measure polyamine uptake in ATP13A2- and ATP13A3-dependent cell models resembling radiolabeled polyamine uptake. We further report that ATP13A3 enables faster and stronger cellular polyamine uptake than does ATP13A2. We also compared the uptake of new green fluorescent PUT, SPD and SPM analogs using different coupling strategies (amide, triazole or isothiocyanate) and fluorophores (symmetrical BODIPY, BODIPY-FL and FITC). ATP13A2 promotes the uptake of various SPD and SPM analogs, whereas ATP13A3 mainly stimulates the uptake of PUT and SPD conjugates. However, the polyamine linker and coupling position on the fluorophore impacts the transport capacity, whereas replacing the fluorophore affects polyamine selectivity. The highest uptake in ATP13A2 or ATP13A3 cells is observed with BODIPY-FL-amide conjugated to SPD, whereas BODIPY-PUT analogs are specifically taken up via ATP13A3. We found that P5B-type ATPase isoforms transport fluorescently labeled polyamine analogs with a distinct structure-activity relationship (SAR), suggesting that isoform-specific polyamine probes can be designed.

Inhibition of FK506 binding proteins reduces alpha-synuclein aggregation and Parkinson's disease-like pathology.

alpha-Synuclein (alpha-SYN) is a key player in the pathogenesis of Parkinson's disease (PD). In pathological conditions, the protein is present in a fibrillar, aggregated form inside cytoplasmic inclusions called Lewy bodies. Members of the FK506 binding protein (FKBP) family are peptidyl-prolyl isomerases that were shown recently to accelerate the aggregation of alpha-SYN in vitro. We now established a neuronal cell culture model for synucleinopathy based on oxidative stress-induced alpha-SYN aggregation and apoptosis. Using high-content analysis, we examined the role of FKBPs in aggregation and apoptotic cell death. FK506, a specific inhibitor of this family of proteins, inhibited alpha-SYN aggregation and neuronal cell death in this synucleinopathy model dose dependently. Knockdown of FKBP12 or FKBP52 reduced the number of alpha-SYN aggregates and protected against cell death, whereas overexpression of FKBP12 or FKBP52 accelerated both aggregation of alpha-SYN and cell death. Thus, FK506 likely targets FKBP members in the cell culture model. Furthermore, oral administration of FK506 after viral vector-mediated overexpression of alpha-SYN in adult mouse brain significantly reduced alpha-SYN aggregate formation and neuronal cell death. Our data explain previously described neuroregenerative and neuroprotective effects of immunophilin ligands and validate FKBPs as a novel drug target for the causative treatment of PD.

Insight into the mode of action of the LRRK2 Y1699C pathogenic mutant.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most prevalent known cause of autosomal dominant Parkinson's disease. The LRRK2 gene encodes a Roco protein featuring a Ras of complex proteins (ROC) GTPase and a kinase domain linked by the C-terminal of ROC (COR) domain. Here, we explored the effects of the Y1699C pathogenic LRRK2 mutation in the COR domain on GTPase activity and interactions within the catalytic core of LRRK2. We observed a decrease in GTPase activity for LRRK2 Y1699C comparable to the decrease observed for the R1441C pathogenic mutant and the T1348N dysfunctional mutant. To study the underlying mechanism, we explored the dimerization in the catalytic core of LRRK2. ROC-COR dimerization was significantly weakened by the Y1699C or R1441C/G mutation. Using a competition assay, we demonstrated that the intra-molecular ROC : COR interaction is favoured over ROC : ROC dimerization. Interestingly, the intra-molecular ROC : COR interaction was strengthened by the Y1699C mutation. This is supported by a 3D homology model of the ROC-COR tandem of LRRK2, showing that Y1699 is positioned at the intra-molecular ROC : COR interface. In conclusion, our data provides mechanistic insight into the mode of action of the Y1699C LRRK2 mutant: the Y1699C substitution, situated at the intra-molecular ROC : COR interface, strengthens the intra-molecular ROC : COR interaction, thereby locally weakening the dimerization of LRRK2 at the ROC-COR tandem domain resulting in decreased GTPase activity.

Exploring the interaction of SV2A with racetams using homology modelling, molecular dynamics and site-directed mutagenesis.

The putative Major Facilitator Superfamily (MFS) transporter, SV2A, is the target for levetiracetam (LEV), which is a successful anti-epileptic drug. Furthermore, SV2A knock out mice display a severe seizure phenotype and die after a few weeks. Despite this, the mode of action of LEV is not known at the molecular level. It would be extremely desirable to understand this more fully in order to aid the design of improved anti-epileptic compounds. Since there is no structure for SV2A, homology modelling can provide insight into the ligand-binding site. However, it is not a trivial process to build such models, since SV2A has low sequence identity to those MFS transporters whose structures are known. A further level of complexity is added by the fact that it is not known which conformational state of the receptor LEV binds to, as multiple conformational states have been inferred by tomography and ligand binding assays or indeed, if binding is exclusive to a single state. Here, we explore models of both the inward and outward facing conformational states of SV2A (according to the alternating access mechanism for MFS transporters). We use a sequence conservation analysis to help guide the homology modelling process and generate the models, which we assess further with Molecular Dynamics (MD). By comparing the MD results in conjunction with docking and simulation of a LEV-analogue used in radioligand binding assays, we were able to suggest further residues that line the binding pocket. These were confirmed experimentally. In particular, mutation of D670 leads to a complete loss of binding. The results shed light on the way LEV analogues may interact with SV2A and may help with the on-going design of improved anti-epileptic compounds.

The Parkinson disease-associated leucine-rich repeat kinase 2 (LRRK2) is a dimer that undergoes intramolecular autophosphorylation.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are a common cause of familial and apparently sporadic Parkinson disease. LRRK2 is a multidomain protein kinase with autophosphorylation activity. It has previously been shown that the kinase activity of LRRK2 is required for neuronal toxicity, suggesting that understanding the mechanism of kinase activation and regulation may be important for the development of specific kinase inhibitors for Parkinson disease treatment. Here, we show that LRRK2 predominantly exists as a dimer under native conditions, a state that appears to be stabilized by multiple domain-domain interactions. Furthermore, an intact C terminus, but not N terminus, is required for autophosphorylation activity. We identify two residues in the activation loop that contribute to the regulation of LRRK2 autophosphorylation. Finally, we demonstrate that LRRK2 undergoes intramolecular autophosphorylation. Together, these results provide insight into the mechanism and regulation of LRRK2 kinase activity.