Fast Substructure Search in Combinatorial Library Spaces.

We present an efficient algorithm for substructure search in combinatorial libraries defined by synthons, i.e., substructures with connection points. Our method improves on existing approaches by introducing powerful heuristics and fast fingerprint screening to quickly eliminate branches of nonmatching combinations of synthons. With this, we achieve typical response times of a few seconds on a standard desktop computer for searches in large combinatorial libraries like the Enamine REAL Space. We published the Java source as part of the OpenChemLib under the BSD license, and we implemented tools to enable substructure search in custom combinatorial libraries.

Fully Automated Creation of Virtual Chemical Fragment Spaces Using the Open-Source Library OpenChemLib.

Synthetically accessible chemical spaces provide a valuable source to search for small-molecule analogues or new starting points in drug discovery projects. Having a toolbox at hand that can automatically create searchable representations of such spaces using reaction definitions and building blocks as inputs is a prerequisite to put this approach into practice. Herein, we present a tool kit to create such virtual chemical spaces. It is part of the OpenChemLib, an open-source Cheminformatics tool kit. Furthermore, we demonstrate the creation of a several billion molecules large chemical space from commercial building blocks and a list of common organic chemistry reactions.

Gene expression analysis in epileptic hippocampi reveals a promoter haplotype conferring reduced aldehyde dehydrogenase 5a1 expression and responsiveness.

Increasing evidence indicates the pathogenetic relevance of regulatory genomic motifs for variability in the manifestation of brain disorders. In this context, cis-regulatory effects of single nucleotide polymorphisms (SNPs) on gene expression can contribute to changing transcript levels of excitability-relevant molecules and episodic seizure manifestation in epilepsy. Biopsy specimens of patients undergoing epilepsy surgery for seizure relief provide unique insights into the impact of promoter SNPs on corresponding mRNA expression. Here, we have scrutinized whether two linked regulatory SNPs (rs2744575; 4779C > G and rs4646830; 4854C > G) located in the aldehyde dehydrogenase 5a1 (succinic semialdehyde dehydrogenase; ALDH5A1) gene promoter are associated with expression of corresponding mRNAs in epileptic hippocampi (n = 43). The minor ALDH5A1-GG haplotype associates with significantly lower ALDH5A1 transcript abundance. Complementary in vitro analyses in neural cell cultures confirm this difference and further reveal a significantly constricted range for the minor ALDH5A1 haplotype of promoter activity regulation through the key epileptogenesis transcription factor Egr1 (early growth response 1). The present data suggest systematic analyses in human hippocampal tissue as a useful approach to unravel the impact of epilepsy candidate SNPs on associated gene expression. Aberrant ALDH5A1 promoter regulation in functional terms can contribute to impaired γ-aminobutyric acid homeostasis and thereby network excitability and seizure propensity.

Tautobase: An Open Tautomer Database.

There has been decades of research on determining and predicting acid dissociation constants (pKa) and the tautomer ratios both experimentally and theoretically. However, the lack of an extensive publicly available database of measured tautomeric ratios in water and nonaqueous solvents poses a challenge for the researchers interested in theoretical studies related to tautomers. Hereby, we present Tautobase, to date and to the best of our knowledge, the first extensive open-source tautomer database of measured and estimated tautomer ratios mainly in water, containing 1680 unique tautomer pairs.

No evidence for a BRD2 promoter hypermethylation in blood leukocytes of Europeans with juvenile myoclonic epilepsy.

Juvenile myoclonic epilepsy (JME) is a common syndrome of genetic generalized epilepsies (GGEs). Linkage and association studies suggest that the gene encoding the bromodomain-containing protein 2 (BRD2) may increase risk of JME. The present methylation and association study followed up a recent report highlighting that the BRD2 promoter CpG island (CpG76) is differentially hypermethylated in lymphoblastoid cells from Caucasian patients with JME compared to patients with other GGE subtypes and unaffected relatives. In contrast, we found a uniform low average percentage of methylation (<4.5%) for 13 CpG76-CpGs in whole blood cells from 782 unrelated European Caucasians, including 116 JME patients, 196 patients with genetic absence epilepsies, and 470 control subjects. We also failed to confirm an allelic association of the BRD2 promoter single nucleotide polymorphism (SNP) rs3918149 with JME (Armitage trend test, P = 0.98), and we did not detect a substantial impact of SNP rs3918149 on CpG76 methylation in either 116 JME patients (methylation quantitative trait loci [meQTL], P = 0.29) or 470 German control subjects (meQTL, P = 0.55). Our results do not support the previous observation that a high DNA methylation level of the BRD2 promoter CpG76 island is a prevalent epigenetic motif associated with JME in Caucasians.

Burden analysis of rare microdeletions suggests a strong impact of neurodevelopmental genes in genetic generalised epilepsies.

Genetic generalised epilepsy (GGE) is the most common form of genetic epilepsy, accounting for 20% of all epilepsies. Genomic copy number variations (CNVs) constitute important genetic risk factors of common GGE syndromes. In our present genome-wide burden analysis, large (≥ 400 kb) and rare (< 1%) autosomal microdeletions with high calling confidence (≥ 200 markers) were assessed by the Affymetrix SNP 6.0 array in European case-control cohorts of 1,366 GGE patients and 5,234 ancestry-matched controls. We aimed to: 1) assess the microdeletion burden in common GGE syndromes, 2) estimate the relative contribution of recurrent microdeletions at genomic rearrangement hotspots and non-recurrent microdeletions, and 3) identify potential candidate genes for GGE. We found a significant excess of microdeletions in 7.3% of GGE patients compared to 4.0% in controls (P = 1.8 x 10-7; OR = 1.9). Recurrent microdeletions at seven known genomic hotspots accounted for 36.9% of all microdeletions identified in the GGE cohort and showed a 7.5-fold increased burden (P = 2.6 x 10-17) relative to controls. Microdeletions affecting either a gene previously implicated in neurodevelopmental disorders (P = 8.0 x 10-18, OR = 4.6) or an evolutionarily conserved brain-expressed gene related to autism spectrum disorder (P = 1.3 x 10-12, OR = 4.1) were significantly enriched in the GGE patients. Microdeletions found only in GGE patients harboured a high proportion of genes previously associated with epilepsy and neuropsychiatric disorders (NRXN1, RBFOX1, PCDH7, KCNA2, EPM2A, RORB, PLCB1). Our results demonstrate that the significantly increased burden of large and rare microdeletions in GGE patients is largely confined to recurrent hotspot microdeletions and microdeletions affecting neurodevelopmental genes, suggesting a strong impact of fundamental neurodevelopmental processes in the pathogenesis of common GGE syndromes.

The Screening Compound Collection: A Key Asset for Drug Discovery.

In this case study on an essential instrument of modern drug discovery, we summarize our successful efforts in the last four years toward enhancing the Actelion screening compound collection. A key organizational step was the establishment of the Compound Library Committee (CLC) in September 2013. This cross-functional team consisting of computational scientists, medicinal chemists and a biologist was endowed with a significant annual budget for regular new compound purchases. Based on an initial library analysis performed in 2013, the CLC developed a New Library Strategy. The established continuous library turn-over mode, and the screening library size of 300'000 compounds were maintained, while the structural library quality was increased. This was achieved by shifting the selection criteria from 'druglike' to 'leadlike' structures, enriching for non-flat structures, aiming for compound novelty, and increasing the ratio of higher cost 'Premium Compounds'. Novel chemical space was gained by adding natural compounds, macrocycles, designed and focused libraries to the collection, and through mutual exchanges of proprietary compounds with agrochemical companies. A comparative analysis in 2016 provided evidence for the positive impact of these measures. Screening the improved library has provided several highly promising hits, including a macrocyclic compound, that are currently followed up in different Hit-to-Lead and Lead Optimization programs. It is important to state that the goal of the CLC was not to achieve higher HTS hit rates, but to increase the chances of identified hits to serve as the basis of successful early drug discovery programs. The experience gathered so far legitimates the New Library Strategy.

16p11.2 600 kb Duplications confer risk for typical and atypical Rolandic epilepsy.

Rolandic epilepsy (RE) is the most common idiopathic focal childhood epilepsy. Its molecular basis is largely unknown and a complex genetic etiology is assumed in the majority of affected individuals. The present study tested whether six large recurrent copy number variants at 1q21, 15q11.2, 15q13.3, 16p11.2, 16p13.11 and 22q11.2 previously associated with neurodevelopmental disorders also increase risk of RE. Our association analyses revealed a significant excess of the 600 kb genomic duplication at the 16p11.2 locus (chr16: 29.5-30.1 Mb) in 393 unrelated patients with typical (n = 339) and atypical (ARE; n = 54) RE compared with the prevalence in 65,046 European population controls (5/393 cases versus 32/65,046 controls; Fisher's exact test P = 2.83 × 10(-6), odds ratio = 26.2, 95% confidence interval: 7.9-68.2). In contrast, the 16p11.2 duplication was not detected in 1738 European epilepsy patients with either temporal lobe epilepsy (n = 330) and genetic generalized epilepsies (n = 1408), suggesting a selective enrichment of the 16p11.2 duplication in idiopathic focal childhood epilepsies (Fisher's exact test P = 2.1 × 10(-4)). In a subsequent screen among children carrying the 16p11.2 600 kb rearrangement we identified three patients with RE-spectrum epilepsies in 117 duplication carriers (2.6%) but none in 202 carriers of the reciprocal deletion. Our results suggest that the 16p11.2 duplication represents a significant genetic risk factor for typical and atypical RE.

Rare variants in γ-aminobutyric acid type A receptor genes in rolandic epilepsy and related syndromes.

OBJECTIVE: To test whether mutations in γ-aminobutyric acid type A receptor (GABAA -R) subunit genes contribute to the etiology of rolandic epilepsy (RE) or its atypical variants (ARE). METHODS: We performed exome sequencing to compare the frequency of variants in 18 GABAA -R genes in 204 European patients with RE/ARE versus 728 platform-matched controls. Identified GABRG2 variants were functionally assessed for protein stability, trafficking, postsynaptic clustering, and receptor function. RESULTS: Of 18 screened GABAA -R genes, we detected an enrichment of rare variants in the GABRG2 gene in RE/ARE patients (5 of 204, 2.45%) in comparison to controls (1 of 723, 0.14%; odds ratio = 18.07, 95% confidence interval = 2.01-855.07, p = 0.0024, pcorr = 0.043). We identified a GABRG2 splice variant (c.549-3T>G) in 2 unrelated patients as well as 3 nonsynonymous variations in this gene (p.G257R, p.R323Q, p.I389V). Functional assessment showed reduced surface expression of p.G257R and decreased GABA-evoked currents for p.R323Q. The p.G257R mutation displayed diminished levels of palmitoylation, a post-translational modification crucial for trafficking of proteins to the cell membrane. Enzymatically raised palmitoylation levels restored the surface expression of the p.G257R variant γ2 subunit. INTERPRETATION: The statistical association and the functional evidence suggest that mutations of the GABRG2 gene may increase the risk of RE/ARE. Restoring the impaired membrane trafficking of some GABRG2 mutations by enhancing palmitoylation might be an interesting therapeutic approach to reverse the pathogenic effect of such mutants.

Nocturnal frontal lobe epilepsy caused by a mutation in the GATOR1 complex gene NPRL3.

Mutations in NPRL3, one of three genes that encode proteins of the mTORC1-regulating GATOR1 complex, have recently been reported to cause cortical dysplasia with focal epilepsy. We have now analyzed a multiplex epilepsy family by whole exome sequencing and identified a frameshift mutation (NM_001077350.2; c.1522delG; p.E508Rfs*46) within exon 13 of NPRL3. This truncating mutation causes an epilepsy phenotype characterized by early childhood onset of mainly nocturnal frontal lobe epilepsy. The penetrance in our family was low (three affected out of six mutation carriers), compared to families with either ion channel- or DEPDC5-associated familial nocturnal frontal lobe epilepsy. The absence of apparent structural brain abnormalities suggests that mutations in NPRL3 are not necessarily associated with focal cortical dysplasia but might be able to cause epilepsy by different, yet unknown pathomechanisms.

CHD2 variants are a risk factor for photosensitivity in epilepsy.

Photosensitivity is a heritable abnormal cortical response to flickering light, manifesting as particular electroencephalographic changes, with or without seizures. Photosensitivity is prominent in a very rare epileptic encephalopathy due to de novo CHD2 mutations, but is also seen in epileptic encephalopathies due to other gene mutations. We determined whether CHD2 variation underlies photosensitivity in common epilepsies, specific photosensitive epilepsies and individuals with photosensitivity without seizures. We studied 580 individuals with epilepsy and either photosensitive seizures or abnormal photoparoxysmal response on electroencephalography, or both, and 55 individuals with photoparoxysmal response but no seizures. We compared CHD2 sequence data to publicly available data from 34 427 individuals, not enriched for epilepsy. We investigated the role of unique variants seen only once in the entire data set. We sought CHD2 variants in 238 exomes from familial genetic generalized epilepsies, and in other public exome data sets. We identified 11 unique variants in the 580 individuals with photosensitive epilepsies and 128 unique variants in the 34 427 controls: unique CHD2 variation is over-represented in cases overall (P = 2.17 × 10(-5)). Among epilepsy syndromes, there was over-representation of unique CHD2 variants (3/36 cases) in the archetypal photosensitive epilepsy syndrome, eyelid myoclonia with absences (P = 3.50 × 10(-4)). CHD2 variation was not over-represented in photoparoxysmal response without seizures. Zebrafish larvae with chd2 knockdown were tested for photosensitivity. Chd2 knockdown markedly enhanced mild innate zebrafish larval photosensitivity. CHD2 mutation is the first identified cause of the archetypal generalized photosensitive epilepsy syndrome, eyelid myoclonia with absences. Unique CHD2 variants are also associated with photosensitivity in common epilepsies. CHD2 does not encode an ion channel, opening new avenues for research into human cortical excitability.

Impairment of ceramide synthesis causes a novel progressive myoclonus epilepsy.

OBJECTIVE: Alterations of sphingolipid metabolism are implicated in the pathogenesis of many neurodegenerative disorders. METHODS: We identified a homozygous nonsynonymous mutation in CERS1, the gene encoding ceramide synthase 1, in 4 siblings affected by a progressive disorder with myoclonic epilepsy and dementia. CerS1, a transmembrane protein of the endoplasmic reticulum (ER), catalyzes the biosynthesis of C18-ceramides. RESULTS: We demonstrated that the mutation decreases C18-ceramide levels. In addition, we showed that downregulation of CerS1 in a neuroblastoma cell line triggers ER stress response and induces proapoptotic pathways. INTERPRETATION: This study demonstrates that impairment of ceramide biosynthesis underlies neurodegeneration in humans.

DEPDC5 mutations in genetic focal epilepsies of childhood.

Recent studies reported DEPDC5 loss-of-function mutations in different focal epilepsy syndromes. Here we identified 1 predicted truncation and 2 missense mutations in 3 children with rolandic epilepsy (3 of 207). In addition, we identified 3 families with unclassified focal childhood epilepsies carrying predicted truncating DEPDC5 mutations (3 of 82). The detected variants were all novel, inherited, and present in all tested affected (n=11) and in 7 unaffected family members, indicating low penetrance. Our findings extend the phenotypic spectrum associated with mutations in DEPDC5 and suggest that rolandic epilepsy, albeit rarely, and other nonlesional childhood epilepsies are among the associated syndromes.

Extending the phenotypic spectrum of RBFOX1 deletions: Sporadic focal epilepsy.

Partial deletions of the RBFOX1 gene encoding the neuronal splicing regulator have been reported in a range of neurodevelopmental diseases including idiopathic/genetic generalized epilepsy (IGE/GGE), childhood focal epilepsy, and self-limited childhood benign epilepsy with centrotemporal spikes (BECTS, rolandic epilepsy), and autism. The protein regulates alternative splicing of many neuronal transcripts involved in the homeostatic control of neuronal excitability. Herein, we examined whether structural deletions affecting RBFOX1 exons confer susceptibility to common forms of juvenile and adult focal epilepsy syndromes. We screened 807 unrelated patients with sporadic focal epilepsy, and we identified seven hemizygous exonic RBFOX1 deletions in patients with sporadic focal epilepsy (0.9%) in comparison to one deletion found in 1,502 controls. The phenotypes of the patients carrying RBFOX1 deletions comprise magnetic resonance imaging (MRI)-negative epilepsy of unknown etiology with frontal and temporal origin (n = 5) and two patients with temporal lobe epilepsy with hippocampal sclerosis. The epilepsies were largely pharmacoresistant but not associated with intellectual disability. Our study extends the phenotypic spectrum of RBFOX1 deletions as a risk factor for focal epilepsy and suggests that exonic RBFOX1 deletions are involved in the broad spectrum of focal and generalized epilepsies.

DataWarrior: an open-source program for chemistry aware data visualization and analysis.

Drug discovery projects in the pharmaceutical industry accumulate thousands of chemical structures and ten-thousands of data points from a dozen or more biological and pharmacological assays. A sufficient interpretation of the data requires understanding, which molecular families are present, which structural motifs correlate with measured properties, and which tiny structural changes cause large property changes. Data visualization and analysis software with sufficient chemical intelligence to support chemists in this task is rare. In an attempt to contribute to filling the gap, we released our in-house developed chemistry aware data analysis program DataWarrior for free public use. This paper gives an overview of DataWarrior's functionality and architecture. Exemplarily, a new unsupervised, 2-dimensional scaling algorithm is presented, which employs vector-based or nonvector-based descriptors to visualize the chemical or pharmacophore space of even large data sets. DataWarrior uses this method to interactively explore chemical space, activity landscapes, and activity cliffs.

Exonic microdeletions of the gephyrin gene impair GABAergic synaptic inhibition in patients with idiopathic generalized epilepsy.

Gephyrin is a postsynaptic scaffolding protein, essential for the clustering of glycine and γ-aminobutyric acid type-A receptors (GABAARs) at inhibitory synapses. An impairment of GABAergic synaptic inhibition represents a key pathway of epileptogenesis. Recently, exonic microdeletions in the gephyrin (GPHN) gene have been associated with neurodevelopmental disorders including autism spectrum disorder, schizophrenia and epileptic seizures. Here we report the identification of novel exonic GPHN microdeletions in two patients with idiopathic generalized epilepsy (IGE), representing the most common group of genetically determined epilepsies. The identified GPHN microdeletions involve exons 5-9 (Δ5-9) and 2-3 (Δ2-3), both affecting the gephyrin G-domain. Molecular characterization of the GPHN Δ5-9 variant demonstrated that it perturbs the clustering of regular gephyrin at inhibitory synapses in cultured mouse hippocampal neurons in a dominant-negative manner, resulting in a significant loss of γ2-subunit containing GABAARs. GPHN Δ2-3 causes a frameshift resulting in a premature stop codon (p.V22Gfs*7) leading to haplo-insufficiency of the gene. Our results demonstrate that structural exonic microdeletions affecting the GPHN gene constitute a rare genetic risk factor for IGE and other neuropsychiatric disorders by an impairment of the GABAergic inhibitory synaptic transmission.

A common SCN1A splice-site polymorphism modifies the effect of carbamazepine on cortical excitability--a pharmacogenetic transcranial magnetic stimulation study.

OBJECTIVE: SCN1A encodes the alpha subunit of the voltage-gated sodium channel and plays a crucial role in several epilepsy syndromes. The common SCN1A splice-site polymorphism rs3812718 (IVS5N+5 G>A) might contribute to the pathophysiology underlying genetic generalized epilepsies and is associated with electrophysiologic properties of the channel and the effect of sodium-channel blocking antiepileptic drugs. We assessed the effects of the rs3812718 genotype on cortical excitability at baseline and after administration of carbamazepine in order to investigate the mechanism of this association. METHODS: Paired-pulse transcranial magnetic stimulation (TMS) was applied in 92 healthy volunteers with the homozygous genotypes AA or GG of rs3812718 at baseline and after application of 400 mg of carbamazepine or placebo in a double-blind, randomized, crossover design. Resting motor threshold (RMT), short interval intracortical inhibition (SICI), intracortical facilitation (ICF), and cortical silent period (CSP) were determined. RESULTS: At baseline there was no significant difference in any TMS parameter. Genotype GG was associated with a higher carbamazepine-induced increase in CSP duration as compared to AA (multivariate analysis of covariance [MANCOVA], p = 0.013). An expected significant increase in RMT was genotype independent. SIGNIFICANCE: We found that the rs3812718 genotype modifies the effect of carbamazepine on CSP duration (mainly reflecting modulation of γ-aminobutyric acid (GABA)ergic inhibition), but not on RMT (mainly reflecting modulation of voltage-gated sodium channels). This provides evidence that rs3812718 affects the pharmacoresponse to carbamazepine via an effect on GABAergic cortical interneurons. Our results also confirm that TMS is useful to investigate the effect of genetic variants on cortical excitability and pharmacoresponse.

Analysis of ELP4, SRPX2, and interacting genes in typical and atypical rolandic epilepsy.

Rolandic epilepsy (RE) and its atypical variants (atypical rolandic epilepsy, ARE) along the spectrum of epilepsy-aphasia disorders are characterized by a strong but largely unknown genetic basis. Two genes with a putative (ELP4) or a proven (SRPX2) function in neuronal migration were postulated to confer susceptibility to parts of the disease spectrum: the ELP4 gene to centrotemporal spikes and SRPX2 to ARE. To reexamine these findings, we investigated a cohort of 280 patients of European ancestry with RE/ARE for the etiological contribution of these genes and their close interaction partners. We performed next-generation sequencing and single-nucleotide polymorphism (SNP)-array based genotyping to screen for sequence and structural variants. In comparison to European controls we could not detect an enrichment of rare deleterious variants of ELP4, SRPX2, or their interaction partners in affected individuals. The previously described functional p.N327S variant in the X chromosomal SRPX2 gene was detected in two affected individuals (0.81%) and also in controls (0.26%), with some preponderance of male patients. We did not detect an association of SNPs in the ELP4 gene with centrotemporal spikes as previously reported. In conclusion our data do not support a major role of ELP4 and SRPX2 in the etiology of RE/ARE.

Regulation of ClC-2 gating by intracellular ATP.

ClC-2 is a voltage-dependent chloride channel that activates slowly at voltages negative to the chloride reversal potential. Adenosine triphosphate (ATP) and other nucleotides have been shown to bind to carboxy-terminal cystathionine-ß-synthase (CBS) domains of ClC-2, but the functional consequences of binding are not sufficiently understood. We here studied the effect of nucleotides on channel gating using single-channel and whole-cell patch clamp recordings on transfected mammalian cells. ATP slowed down macroscopic activation and deactivation time courses in a dose-dependent manner. Removal of the complete carboxy-terminus abolishes the effect of ATP, suggesting that CBS domains are necessary for ATP regulation of ClC-2 gating. Single-channel recordings identified long-lasting closed states of ATP-bound channels as basis of this gating deceleration. ClC-2 channel dimers exhibit two largely independent protopores that are opened and closed individually as well as by a common gating process. A seven-state model of common gating with altered voltage dependencies of opening and closing transitions for ATP-bound states correctly describes the effects of ATP on macroscopic and microscopic ClC-2 currents. To test for a potential pathophysiological impact of ClC-2 regulation by ATP, we studied ClC-2 channels carrying naturally occurring sequence variants found in patients with idiopathic generalized epilepsy, G715E, R577Q, and R653T. All naturally occurring sequence variants accelerate common gating in the presence but not in the absence of ATP. We propose that ClC-2 uses ATP as a co-factor to slow down common gating for sufficient electrical stability of neurons under physiological conditions.