AMFR dysfunction causes autosomal recessive spastic paraplegia in human that is amenable to statin treatment in a preclinical model.

Hereditary spastic paraplegias (HSP) are rare, inherited neurodegenerative or neurodevelopmental disorders that mainly present with lower limb spasticity and muscle weakness due to motor neuron dysfunction. Whole genome sequencing identified bi-allelic truncating variants in AMFR, encoding a RING-H2 finger E3 ubiquitin ligase anchored at the membrane of the endoplasmic reticulum (ER), in two previously genetically unexplained HSP-affected siblings. Subsequently, international collaboration recognized additional HSP-affected individuals with similar bi-allelic truncating AMFR variants, resulting in a cohort of 20 individuals from 8 unrelated, consanguineous families. Variants segregated with a phenotype of mainly pure but also complex HSP consisting of global developmental delay, mild intellectual disability, motor dysfunction, and progressive spasticity. Patient-derived fibroblasts, neural stem cells (NSCs), and in vivo zebrafish modeling were used to investigate pathomechanisms, including initial preclinical therapy assessment. The absence of AMFR disturbs lipid homeostasis, causing lipid droplet accumulation in NSCs and patient-derived fibroblasts which is rescued upon AMFR re-expression. Electron microscopy indicates ER morphology alterations in the absence of AMFR. Similar findings are seen in amfra-/- zebrafish larvae, in addition to altered touch-evoked escape response and defects in motor neuron branching, phenocopying the HSP observed in patients. Interestingly, administration of FDA-approved statins improves touch-evoked escape response and motor neuron branching defects in amfra-/- zebrafish larvae, suggesting potential therapeutic implications. Our genetic and functional studies identify bi-allelic truncating variants in AMFR as a cause of a novel autosomal recessive HSP by altering lipid metabolism, which may potentially be therapeutically modulated using precision medicine with statins.

Evaluation of Mean Percentage of Full-Length SMN Transcripts as a Molecular Biomarker of Spinal Muscular Atrophy.

The elevation of SMN transcript and protein level remains the principal aim of SMA therapy. Still, there is no standard molecular biomarker for the assessment of its efficacy. In the current study, we tested three methods of SMN transcript level measurement using real-time RT-PCR, quantitative fluorescent RT-PCR, and a semiquantitative RT-PCR gel densitometric assay. We examined several potential mRNA-based biomarkers and examined their sensitivity and reliability by comparing the obtained values in peripheral blood mononuclear cells of SMA patients, SMA carriers, and healthy individuals. We found that the mean percentage of full-length (FL-SMN) transcripts relative to the total sum of FL-SMN and exon 7-deleted (Δ7 SMN) transcripts detected by semiquantitative and quantitative fluorescence RT-PCR differed significantly between the three analyzed groups. The relevance of this biomarker was proven in an SMN2-targeting therapeutic experiment. We showed that the values of the biomarker changed significantly in SMA fibroblast cell cultures after treatment with therapeutic antisense oligonucleotides targeting the ISS-N1 site in intron 7 of the SMN2 gene. The obtained results indicate the convenience of using the mean percentage of FL-SMN transcripts determined by semiquantitative and quantitative fluorescence RT-PCR as a putative biomarker for the assessment of SMA therapy efficacy in vitro.

Comprehensive multi-omics integration identifies differentially active enhancers during human brain development with clinical relevance.

BACKGROUND: Non-coding regulatory elements (NCREs), such as enhancers, play a crucial role in gene regulation, and genetic aberrations in NCREs can lead to human disease, including brain disorders. The human brain is a complex organ that is susceptible to numerous disorders; many of these are caused by genetic changes, but a multitude remain currently unexplained. Understanding NCREs acting during brain development has the potential to shed light on previously unrecognized genetic causes of human brain disease. Despite immense community-wide efforts to understand the role of the non-coding genome and NCREs, annotating functional NCREs remains challenging. METHODS: Here we performed an integrative computational analysis of virtually all currently available epigenome data sets related to human fetal brain. RESULTS: Our in-depth analysis unravels 39,709 differentially active enhancers (DAEs) that show dynamic epigenomic rearrangement during early stages of human brain development, indicating likely biological function. Many of these DAEs are linked to clinically relevant genes, and functional validation of selected DAEs in cell models and zebrafish confirms their role in gene regulation. Compared to enhancers without dynamic epigenomic rearrangement, DAEs are subjected to higher sequence constraints in humans, have distinct sequence characteristics and are bound by a distinct transcription factor landscape. DAEs are enriched for GWAS loci for brain-related traits and for genetic variation found in individuals with neurodevelopmental disorders, including autism. CONCLUSION: This compendium of high-confidence enhancers will assist in deciphering the mechanism behind developmental genetics of human brain and will be relevant to uncover missing heritability in human genetic brain disorders.

Assessing In Vitro Resistance Development in Enterovirus A71 in the Context of Combination Antiviral Treatment.

There are currently no antivirals available to treat infection with enterovirus A71 (EV-A71) or any other enterovirus. The extensively studied capsid binders rapidly select for drug-resistant variants. We here explore whether the combination of two direct-acting enterovirus inhibitors with a different mechanism of action may delay or prevent resistance development to the capsid binders. To that end, the in vitro dynamics of resistance development to the capsid binder pirodavir was studied either alone or in combination with a viral 2C-targeting compound (SMSK_0213), a viral 3C-protease inhibitor (rupintrivir) or a viral RNA-dependent RNA polymerase inhibitor (7DMA). We demonstrate that combining pirodavir with either rupintrivir or 7DMA delays the development of resistance to pirodavir and that no resistance to the protease or polymerase inhibitor develops. The combination of pirodavir with the 2C inhibitor results in a double-resistant virus population, where only the minority carries the resistant mutation.

Comparative analysis of the molecular mechanism of resistance to vapendavir across a panel of picornavirus species.

Vapendavir is a rhino/enterovirus inhibitor that targets a hydrophobic pocket in the viral capsid preventing the virus from entering the cell. We set out to study and compare the molecular mechanisms of resistance to vapendavir among clinically relevant Picornavirus species. To this end in vitro resistance selection of drug-resistant isolates was applied in rhinovirus 2 and 14, enterovirus-D68 and Poliovirus 1 Sabin. Mutations in the drug-binding pocket in VP1 (C199R/Y in hRV14; I194F in PV1; M252L and A156T in EV-D68), typical for this class of compounds, were identified. Interestingly, we also observed mutations located outside the pocket (K167E in EV-D68 and G149C in hRV2) that contribute to the resistant phenotype. Remarkably, the G149C substitution rendered the replication of human rhinovirus 2 dependent on the presence of vapendavir. Our data suggest that the binding of vapendavir to the capsid of the G149C isolate may be required to stabilize the viral particle and to allow efficient dissemination of the virus. We observed the dependency of the G149C isolate on other compounds of this class, suggesting that this phenotype is common for capsid binders. In addition the VP1 region containing the G149C substitution has not been associated with antiviral resistance before. Our results demonstrate that the phenotype and genotype of clinically relevant vapendavir-resistant picornavirus species is more complex than generally believed.

Delineating the molecular and phenotypic spectrum of the SETD1B-related syndrome.

PURPOSE: Pathogenic variants in SETD1B have been associated with a syndromic neurodevelopmental disorder including intellectual disability, language delay, and seizures. To date, clinical features have been described for 11 patients with (likely) pathogenic SETD1B sequence variants. This study aims to further delineate the spectrum of the SETD1B-related syndrome based on characterizing an expanded patient cohort. METHODS: We perform an in-depth clinical characterization of a cohort of 36 unpublished individuals with SETD1B sequence variants, describing their molecular and phenotypic spectrum. Selected variants were functionally tested using in vitro and genome-wide methylation assays. RESULTS: Our data present evidence for a loss-of-function mechanism of SETD1B variants, resulting in a core clinical phenotype of global developmental delay, language delay including regression, intellectual disability, autism and other behavioral issues, and variable epilepsy phenotypes. Developmental delay appeared to precede seizure onset, suggesting SETD1B dysfunction impacts physiological neurodevelopment even in the absence of epileptic activity. Males are significantly overrepresented and more severely affected, and we speculate that sex-linked traits could affect susceptibility to penetrance and the clinical spectrum of SETD1B variants. CONCLUSION: Insights from this extensive cohort will facilitate the counseling regarding the molecular and phenotypic landscape of newly diagnosed patients with the SETD1B-related syndrome.

The non-coding genome in genetic brain disorders: new targets for therapy?

The non-coding genome, consisting of more than 98% of all genetic information in humans and once judged as 'Junk DNA', is increasingly moving into the spotlight in the field of human genetics. Non-coding regulatory elements (NCREs) are crucial to ensure correct spatio-temporal gene expression. Technological advancements have allowed to identify NCREs on a large scale, and mechanistic studies have helped to understand the biological mechanisms underlying their function. It is increasingly becoming clear that genetic alterations of NCREs can cause genetic disorders, including brain diseases. In this review, we concisely discuss mechanisms of gene regulation and how to investigate them, and give examples of non-coding alterations of NCREs that give rise to human brain disorders. The cross-talk between basic and clinical studies enhances the understanding of normal and pathological function of NCREs, allowing better interpretation of already existing and novel data. Improved functional annotation of NCREs will not only benefit diagnostics for patients, but might also lead to novel areas of investigations for targeted therapies, applicable to a wide panel of genetic disorders. The intrinsic complexity and precision of the gene regulation process can be turned to the advantage of highly specific treatments. We further discuss this exciting new field of 'enhancer therapy' based on recent examples.

Bi-allelic variants in HOPS complex subunit VPS41 cause cerebellar ataxia and abnormal membrane trafficking.

Membrane trafficking is a complex, essential process in eukaryotic cells responsible for protein transport and processing. Deficiencies in vacuolar protein sorting (VPS) proteins, key regulators of trafficking, cause abnormal intracellular segregation of macromolecules and organelles and are linked to human disease. VPS proteins function as part of complexes such as the homotypic fusion and vacuole protein sorting (HOPS) tethering complex, composed of VPS11, VPS16, VPS18, VPS33A, VPS39 and VPS41. The HOPS-specific subunit VPS41 has been reported to promote viability of dopaminergic neurons in Parkinson's disease but to date has not been linked to human disease. Here, we describe five unrelated families with nine affected individuals, all carrying homozygous variants in VPS41 that we show impact protein function. All affected individuals presented with a progressive neurodevelopmental disorder consisting of cognitive impairment, cerebellar atrophy/hypoplasia, motor dysfunction with ataxia and dystonia, and nystagmus. Zebrafish disease modelling supports the involvement of VPS41 dysfunction in the disorder, indicating lysosomal dysregulation throughout the brain and providing support for cerebellar and microglial abnormalities when vps41 was mutated. This provides the first example of human disease linked to the HOPS-specific subunit VPS41 and suggests the importance of HOPS complex activity for cerebellar function.

α-Ketoamides as Broad-Spectrum Inhibitors of Coronavirus and Enterovirus Replication: Structure-Based Design, Synthesis, and Activity Assessment.

The main protease of coronaviruses and the 3C protease of enteroviruses share a similar active-site architecture and a unique requirement for glutamine in the P1 position of the substrate. Because of their unique specificity and essential role in viral polyprotein processing, these proteases are suitable targets for the development of antiviral drugs. In order to obtain near-equipotent, broad-spectrum antivirals against alphacoronaviruses, betacoronaviruses, and enteroviruses, we pursued a structure-based design of peptidomimetic α-ketoamides as inhibitors of main and 3C proteases. Six crystal structures of protease-inhibitor complexes were determined as part of this study. Compounds synthesized were tested against the recombinant proteases as well as in viral replicons and virus-infected cell cultures; most of them were not cell-toxic. Optimization of the P2 substituent of the α-ketoamides proved crucial for achieving near-equipotency against the three virus genera. The best near-equipotent inhibitors, 11u (P2 = cyclopentylmethyl) and 11r (P2 = cyclohexylmethyl), display low-micromolar EC50 values against enteroviruses, alphacoronaviruses, and betacoronaviruses in cell cultures. In Huh7 cells, 11r exhibits three-digit picomolar activity against the Middle East Respiratory Syndrome coronavirus.

Multitarget CFTR Modulators Endowed with Multiple Beneficial Side Effects for Cystic Fibrosis Patients: Toward a Simplified Therapeutic Approach †.

Cystic fibrosis (CF) is a multiorgan disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR). In addition to respiratory impairment due to mucus accumulation, viruses and bacteria trigger acute pulmonary exacerbations, accelerating disease progression and mortality rate. Treatment complexity increases with patients' age, and simplifying the therapeutic regimen represents one of the key priorities in CF. We have recently reported the discovery of multitarget compounds able to "kill two birds with one stone" by targeting F508del-CFTR and PI4KIIIß and thus acting simultaneously as CFTR correctors and broad-spectrum enterovirus (EV) inhibitors. Starting from these preliminary results, we report herein a hit-to-lead optimization and multidimensional structure-activity relationship (SAR) study that led to compound 23a. This compound showed good antiviral and F508del-CFTR correction potency, additivity/synergy with lumacaftor, and a promising in vitro absorption, distribution, metabolism, and excretion (ADME) profile. It was well tolerated in vivo with no sign of acute toxicity and histological alterations in key biodistribution organs.

Viral engagement with host receptors blocked by a novel class of tryptophan dendrimers that targets the 5-fold-axis of the enterovirus-A71 capsid.

Enterovirus A71 (EV-A71) is a non-polio neurotropic enterovirus with pandemic potential. There are no antiviral agents approved to prevent or treat EV-A71 infections. We here report on the molecular mechanism by which a novel class of tryptophan dendrimers inhibits (at low nanomolar to high picomolar concentration) EV-A71 replication in vitro. A lead compound in the series (MADAL385) prevents binding and internalization of the virus but does not, unlike classical capsid binders, stabilize the particle. By means of resistance selection, reverse genetics and cryo-EM, we map the binding region of MADAL385 to the 5-fold vertex of the viral capsid and demonstrate that a single molecule binds to each vertex. By interacting with this region, MADAL385 prevents the interaction of the virus with its cellular receptors PSGL1 and heparan sulfate, thereby blocking the attachment of EV-A71 to the host cells.

Antiviral effects of selected nucleoside analogues against human parechoviruses A1 and A3.

Parechoviruses A (HPeV, Picornaviridae) are neglected human pathogens that cause sepsis-like illness and severe neurological complications in infants. There are no antivirals available for the treatment of HPeV infections. We here report on cell-based assays that allow for medium-throughput antiviral screening of compound libraries against HPeV. The nucleoside viral polymerase inhibitor 2'-C-methylcytidine was identified as being an in vitro replication inhibitor of HPeV1 and HPeV3 that can serve as a reference molecule for further antiviral studies.

Molecular Factors Involved in Spinal Muscular Atrophy Pathways as Possible Disease-modifying Candidates.

Spinal Muscular Atrophy (SMA) is a neuromuscular disorder caused by mutations in the SMN1 gene. Being a monogenic disease, it is characterized by high clinical heterogeneity. Variations in penetrance and severity of symptoms, as well as clinical discrepancies between affected family members can result from modifier genes influence on disease manifestation. SMN2 gene copy number is known to be the main phenotype modifier and there is growing evidence of additional factors contributing to SMA severity. Potential modifiers of spinal muscular atrophy can be found among the wide variety of different factors, such as multiple proteins interacting with SMN or promoting motor neuron survival, epigenetic modifications, transcriptional or splicing factors influencing SMN2 expression. Study of these factors enables to reveal mechanisms underlying SMA pathology and can have pronounced clinical application.

Replication of the Zika virus in different iPSC-derived neuronal cells and implications to assess efficacy of antivirals.

Infections with the Zika virus (ZIKV) are responsible for congenital abnormalities and neurological disorders. We here demonstrate that ZIKV productively infects three types of human iPSC (induced pluripotent stem cells)-derived cells from the neural lineage, i.e. cortical and motor neurons as well as astrocytes. ZIKV infection results in all three cell types in the production of infectious virus particles and induces cytopathic effects (CPE). In cortical and motor neurons, an Asian isolate (PRVABC59) produced roughly 10-fold more virus than the prototypic African strain (MR766 strain). Viral replication and CPE is efficiently inhibited by the nucleoside polymerase inhibitor 7-deaza-2'-C-methyladenosine (7DMA). However, ribavirin and favipiravir, two molecules that inhibit ZIKV replication in Vero cells, did not inhibit ZIKV replication in the neuronal cells. These results highlight the need to assess the potential antiviral activity of novel ZIKV inhibitors in stem cell derived neuronal cultures.

Discovery of Multitarget Agents Active as Broad-Spectrum Antivirals and Correctors of Cystic Fibrosis Transmembrane Conductance Regulator for Associated Pulmonary Diseases.

Enteroviruses (EVs) are among the most frequent infectious agents in humans worldwide and represent the leading cause of upper respiratory tract infections. No drugs for the treatment of EV infections are currently available. Recent studies have also linked EV infection with pulmonary exacerbations, especially in cystic fibrosis (CF) patients, and the importance of this link is probably underestimated. The aim of this work was to develop a new class of multitarget agents active both as broad-spectrum antivirals and as correctors of the F508del-cystic fibrosis transmembrane conductance regulator (CFTR) folding defect responsible for >90% of CF cases. We report herein the discovery of the first small molecules able to simultaneously act as correctors of the F508del-CFTR folding defect and as broad-spectrum antivirals against a panel of EVs representative of all major species.