Iterative computational design and crystallographic screening identifies potent inhibitors targeting the Nsp3 macrodomain of SARS-CoV-2.

The nonstructural protein 3 (NSP3) of the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) contains a conserved macrodomain enzyme (Mac1) that is critical for pathogenesis and lethality. While small-molecule inhibitors of Mac1 have great therapeutic potential, at the outset of the COVID-19 pandemic, there were no well-validated inhibitors for this protein nor, indeed, the macrodomain enzyme family, making this target a pharmacological orphan. Here, we report the structure-based discovery and development of several different chemical scaffolds exhibiting low- to sub-micromolar affinity for Mac1 through iterations of computer-aided design, structural characterization by ultra-high-resolution protein crystallography, and binding evaluation. Potent scaffolds were designed with in silico fragment linkage and by ultra-large library docking of over 450 million molecules. Both techniques leverage the computational exploration of tangible chemical space and are applicable to other pharmacological orphans. Overall, 160 ligands in 119 different scaffolds were discovered, and 153 Mac1-ligand complex crystal structures were determined, typically to 1 Å resolution or better. Our analyses discovered selective and cell-permeable molecules, unexpected ligand-mediated conformational changes within the active site, and key inhibitor motifs that will template future drug development against Mac1.

Biallelic PRMT7 pathogenic variants are associated with a recognizable syndromic neurodevelopmental disorder with short stature, obesity, and craniofacial and digital abnormalities.

PURPOSE: Protein arginine methyltransferase 7 (PRMT7) is a member of a family of enzymes that catalyzes the methylation of arginine residues on several protein substrates. Biallelic pathogenic PRMT7 variants have previously been associated with a syndromic neurodevelopmental disorder characterized by short stature, brachydactyly, intellectual developmental disability, and seizures. To our knowledge, no comprehensive study describes the detailed clinical characteristics of this syndrome. Thus, we aim to delineate the phenotypic spectrum of PRMT7-related disorder. METHODS: We assembled a cohort of 51 affected individuals from 39 different families, gathering clinical information from 36 newly described affected individuals and reviewing data of 15 individuals from the literature. RESULTS: The main clinical characteristics of the PRMT7-related syndrome are short stature, mild to severe developmental delay/intellectual disability, hypotonia, brachydactyly, and distinct facial morphology, including bifrontal narrowing, prominent supraorbital ridges, sparse eyebrows, short nose with full/broad nasal tip, thin upper lip, full and everted lower lip, and a prominent or squared-off jaw. Additional variable findings include seizures, obesity, nonspecific magnetic resonance imaging abnormalities, eye abnormalities (i.e., strabismus or nystagmus), and hearing loss. CONCLUSION: This study further delineates and expands the molecular, phenotypic spectrum and natural history of PRMT7-related syndrome characterized by a neurodevelopmental disorder with skeletal, growth, and endocrine abnormalities.

Iterative computational design and crystallographic screening identifies potent inhibitors targeting the Nsp3 Macrodomain of SARS-CoV-2.

The nonstructural protein 3 (NSP3) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contains a conserved macrodomain enzyme (Mac1) that is critical for pathogenesis and lethality. While small molecule inhibitors of Mac1 have great therapeutic potential, at the outset of the COVID-19 pandemic there were no well-validated inhibitors for this protein nor, indeed, the macrodomain enzyme family, making this target a pharmacological orphan. Here, we report the structure-based discovery and development of several different chemical scaffolds exhibiting low- to sub-micromolar affinity for Mac1 through iterations of computer-aided design, structural characterization by ultra-high resolution protein crystallography, and binding evaluation. Potent scaffolds were designed with in silico fragment linkage and by ultra-large library docking of over 450 million molecules. Both techniques leverage the computational exploration of tangible chemical space and are applicable to other pharmacological orphans. Overall, 160 ligands in 119 different scaffolds were discovered, and 152 Mac1-ligand complex crystal structures were determined, typically to 1 Å resolution or better. Our analyses discovered selective and cell-permeable molecules, unexpected ligand-mediated protein dynamics within the active site, and key inhibitor motifs that will template future drug development against Mac1.

Venus: Elucidating the Impact of Amino Acid Variants on Protein Function Beyond Structure Destabilisation.

Exploring the functional effect of a non-synonymous coding variant at the protein level requires multiple pieces of information to be interpreted appropriately. This is particularly important when embarking on the study of a potentially pathogenic variant linked to a rare or monogenic disease. Whereas accurate protein stability predictions alone are generally informative, other effects, such as disruption of post-translational modifications or weakened ligand binding, may also contribute to the disease phenotype. Furthermore, consideration of nearby variants that are found in the healthy population may strengthen or refute a given mechanistic hypothesis. Whilst there are several bioinformatics tools available that score a genetic variant in terms of deleteriousness, there is no single tool that assembles multiple effects of a variant on the encoded protein, beyond structural stability, and presents them on the structure for inspection. Venus is a web application which, given a protein substitution, rapidly estimates the predicted effect on protein stability of the variant, flags if the variant affects a post-translational modification site, a predicted linear motif or known annotation, and determines the effect on protein stability of variants which affect nearby residues and have been identified in healthy populations. Venus is built upon Michelanglo and the results can be exported to it, allowing them to be annotated and shared with other researchers. Venus is freely accessible at https://venus.cmd.ox.ac.uk and its source code is openly available at https://github.com/CMD-Oxford/Michelanglo-and-Venus.

Recurrent de novo missense variants in GNB2 can cause syndromic intellectual disability.

PURPOSE: Binding proteins (G-proteins) mediate signalling pathways involved in diverse cellular functions and comprise Gα and Gßγ units. Human diseases have been reported for all five Gß proteins. A de novo missense variant in GNB2 was recently reported in one individual with developmental delay/intellectual disability (DD/ID) and dysmorphism. We aim to confirm GNB2 as a neurodevelopmental disease gene, and elucidate the GNB2-associated neurodevelopmental phenotype in a patient cohort. METHODS: We discovered a GNB2 variant in the index case via exome sequencing and sought individuals with GNB2 variants via international data-sharing initiatives. In silico modelling of the variants was assessed, along with multiple lines of evidence in keeping with American College of Medical Genetics and Genomics guidelines for interpretation of sequence variants. RESULTS: We identified 12 unrelated individuals with five de novo missense variants in GNB2, four of which are recurrent: p.(Ala73Thr), p.(Gly77Arg), p.(Lys89Glu) and p.(Lys89Thr). All individuals have DD/ID with variable dysmorphism and extraneurologic features. The variants are located at the universally conserved shared interface with the Gα subunit, which modelling suggests weaken this interaction. CONCLUSION: Missense variants in GNB2 cause a congenital neurodevelopmental disorder with variable syndromic features, broadening the spectrum of multisystem phenotypes associated with variants in genes encoding G-proteins.

DogCatcher allows loop-friendly protein-protein ligation.

There are many efficient ways to connect proteins at termini. However, connecting at a loop is difficult because of lower flexibility and variable environment. Here, we have developed DogCatcher, a protein that forms a spontaneous isopeptide bond with DogTag peptide. DogTag/DogCatcher was generated initially by splitting a Streptococcus pneumoniae adhesin. We optimized DogTag/DogCatcher through rational design and evolution, increasing reaction rate by 250-fold and establishing millimolar solubility of DogCatcher. When fused to a protein terminus, DogTag/DogCatcher reacts slower than SpyTag003/SpyCatcher003. However, inserted in loops of a fluorescent protein or enzyme, DogTag reacts much faster than SpyTag003. Like many membrane proteins, the ion channel TRPC5 has no surface-exposed termini. DogTag in a TRPC5 extracellular loop allowed normal calcium flux and specific covalent labeling on cells in 1 min. DogTag/DogCatcher reacts under diverse conditions, at nanomolar concentrations, and to 98% conversion. Loop-friendly ligation should expand the toolbox for creating protein architectures.

Biallelic variants in SLC38A3 encoding a glutamine transporter cause epileptic encephalopathy.

The solute carrier (SLC) superfamily encompasses >400 transmembrane transporters involved in the exchange of amino acids, nutrients, ions, metals, neurotransmitters and metabolites across biological membranes. SLCs are highly expressed in the mammalian brain; defects in nearly 100 unique SLC-encoding genes (OMIM: https://www.omim.org) are associated with rare Mendelian disorders including developmental and epileptic encephalopathy and severe neurodevelopmental disorders. Exome sequencing and family-based rare variant analyses on a cohort with neurodevelopmental disorders identified two siblings with developmental and epileptic encephalopathy and a shared deleterious homozygous splicing variant in SLC38A3. The gene encodes SNAT3, a sodium-coupled neutral amino acid transporter and a principal transporter of the amino acids asparagine, histidine, and glutamine, the latter being the precursor for the neurotransmitters GABA and glutamate. Additional subjects with a similar developmental and epileptic encephalopathy phenotype and biallelic predicted-damaging SLC38A3 variants were ascertained through GeneMatcher and collaborations with research and clinical molecular diagnostic laboratories. Untargeted metabolomic analysis was performed to identify novel metabolic biomarkers. Ten individuals from seven unrelated families from six different countries with deleterious biallelic variants in SLC38A3 were identified. Global developmental delay, intellectual disability, hypotonia, and absent speech were common features while microcephaly, epilepsy, and visual impairment were present in the majority. Epilepsy was drug-resistant in half. Metabolomic analysis revealed perturbations of glutamate, histidine, and nitrogen metabolism in plasma, urine, and CSF of selected subjects, potentially representing biomarkers of disease. Our data support the contention that SLC38A3 is a novel disease gene for developmental and epileptic encephalopathy and illuminate the likely pathophysiology of the disease as perturbations in glutamine homeostasis.

Expanding the phenotypic spectrum of BCS1L-related mitochondrial disease.

OBJECTIVE: To delineate the full phenotypic spectrum of BCS1L-related disease, provide better understanding of the genotype-phenotype correlations and identify reliable prognostic disease markers. METHODS: We performed a retrospective multinational cohort study of previously unpublished patients followed in 15 centres from 10 countries. Patients with confirmed biallelic pathogenic BCS1L variants were considered eligible. Clinical, laboratory, neuroimaging and genetic data were analysed. Patients were stratified into different groups based on the age of disease onset, whether homozygous or compound heterozygous for the c.232A>G (p.Ser78Gly) variant, and those with other pathogenic BCS1L variants. RESULTS: Thirty-three patients were included. We found that growth failure, lactic acidosis, tubulopathy, hepatopathy and early death were more frequent in those with disease onset within the first month of life. In those with onset after 1 month, neurological features including movement disorders and seizures were more frequent. Novel phenotypes, particularly involving movement disorder, were identified in this group. The presence of the c.232A>G (p.Ser78Gly) variant was associated with significantly worse survival and exclusively found in those with disease onset within the first month of life, whilst other pathogenic BCS1L variants were more frequent in those with later symptom onset. INTERPRETATION: The phenotypic spectrum of BCS1L-related disease comprises a continuum of clinical features rather than a set of separate syndromic clinical identities. Age of onset defines BCS1L-related disease clinically and early presentation is associated with poor prognosis. Genotype correlates with phenotype in the presence of the c.232A>G (p.Ser78Gly) variant.

Biallelic PI4KA variants cause neurological, intestinal and immunological disease.

Phosphatidylinositol 4-kinase IIIα (PI4KIIIα/PI4KA/OMIM:600286) is a lipid kinase generating phosphatidylinositol 4-phosphate (PI4P), a membrane phospholipid with critical roles in the physiology of multiple cell types. PI4KIIIα's role in PI4P generation requires its assembly into a heterotetrameric complex with EFR3, TTC7 and FAM126. Sequence alterations in two of these molecular partners, TTC7 (encoded by TTC7A or TCC7B) and FAM126, have been associated with a heterogeneous group of either neurological (FAM126A) or intestinal and immunological (TTC7A) conditions. Here we show that biallelic PI4KA sequence alterations in humans are associated with neurological disease, in particular hypomyelinating leukodystrophy. In addition, affected individuals may present with inflammatory bowel disease, multiple intestinal atresia and combined immunodeficiency. Our cellular, biochemical and structural modelling studies indicate that PI4KA-associated phenotypical outcomes probably stem from impairment of PI4KIIIα-TTC7-FAM126's organ-specific functions, due to defective catalytic activity or altered intra-complex functional interactions. Together, these data define PI4KA gene alteration as a cause of a variable phenotypical spectrum and provide fundamental new insight into the combinatorial biology of the PI4KIIIα-FAM126-TTC7-EFR3 molecular complex.

A human importin-ß-related disorder: Syndromic thoracic aortic aneurysm caused by bi-allelic loss-of-function variants in IPO8.

Importin 8, encoded by IPO8, is a ubiquitously expressed member of the importin-ß protein family that translocates cargo molecules such as proteins, RNAs, and ribonucleoprotein complexes into the nucleus in a RanGTP-dependent manner. Current knowledge of the cargoes of importin 8 is limited, but TGF-ß signaling components such as SMAD1-4 have been suggested to be among them. Here, we report that bi-allelic loss-of-function variants in IPO8 cause a syndromic form of thoracic aortic aneurysm (TAA) with clinical overlap with Loeys-Dietz and Shprintzen-Goldberg syndromes. Seven individuals from six unrelated families showed a consistent phenotype with early-onset TAA, motor developmental delay, connective tissue findings, and craniofacial dysmorphic features. A C57BL/6N Ipo8 knockout mouse model recapitulates TAA development from 8-12 weeks onward in both sexes but most prominently shows ascending aorta dilatation with a propensity for dissection in males. Compliance assays suggest augmented passive stiffness of the ascending aorta in male Ipo8-/- mice throughout life. Immunohistological investigation of mutant aortic walls reveals elastic fiber disorganization and fragmentation along with a signature of increased TGF-ß signaling, as evidenced by nuclear pSmad2 accumulation. RT-qPCR assays of the aortic wall in male Ipo8-/- mice demonstrate decreased Smad6/7 and increased Mmp2 and Ccn2 (Ctgf) expression, reinforcing a role for dysregulation of the TGF-ß signaling pathway in TAA development. Because importin 8 is the most downstream TGF-ß-related effector implicated in TAA pathogenesis so far, it offers opportunities for future mechanistic studies and represents a candidate drug target for TAA.

Non-coding region variants upstream of MEF2C cause severe developmental disorder through three distinct loss-of-function mechanisms.

Clinical genetic testing of protein-coding regions identifies a likely causative variant in only around half of developmental disorder (DD) cases. The contribution of regulatory variation in non-coding regions to rare disease, including DD, remains very poorly understood. We screened 9,858 probands from the Deciphering Developmental Disorders (DDD) study for de novo mutations in the 5' untranslated regions (5' UTRs) of genes within which variants have previously been shown to cause DD through a dominant haploinsufficient mechanism. We identified four single-nucleotide variants and two copy-number variants upstream of MEF2C in a total of ten individual probands. We developed multiple bespoke and orthogonal experimental approaches to demonstrate that these variants cause DD through three distinct loss-of-function mechanisms, disrupting transcription, translation, and/or protein function. These non-coding region variants represent 23% of likely diagnoses identified in MEF2C in the DDD cohort, but these would all be missed in standard clinical genetics approaches. Nonetheless, these variants are readily detectable in exome sequence data, with 30.7% of 5' UTR bases across all genes well covered in the DDD dataset. Our analyses show that non-coding variants upstream of genes within which coding variants are known to cause DD are an important cause of severe disease and demonstrate that analyzing 5' UTRs can increase diagnostic yield. We also show how non-coding variants can help inform both the disease-causing mechanism underlying protein-coding variants and dosage tolerance of the gene.

Fragment binding to the Nsp3 macrodomain of SARS-CoV-2 identified through crystallographic screening and computational docking.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) macrodomain within the nonstructural protein 3 counteracts host-mediated antiviral adenosine diphosphate-ribosylation signaling. This enzyme is a promising antiviral target because catalytic mutations render viruses nonpathogenic. Here, we report a massive crystallographic screening and computational docking effort, identifying new chemical matter primarily targeting the active site of the macrodomain. Crystallographic screening of 2533 diverse fragments resulted in 214 unique macrodomain-binders. An additional 60 molecules were selected from docking more than 20 million fragments, of which 20 were crystallographically confirmed. X-ray data collection to ultra-high resolution and at physiological temperature enabled assessment of the conformational heterogeneity around the active site. Several fragment hits were confirmed by solution binding using three biophysical techniques (differential scanning fluorimetry, homogeneous time-resolved fluorescence, and isothermal titration calorimetry). The 234 fragment structures explore a wide range of chemotypes and provide starting points for development of potent SARS-CoV-2 macrodomain inhibitors.

Pervasive cooperative mutational effects on multiple catalytic enzyme traits emerge via long-range conformational dynamics.

Multidimensional fitness landscapes provide insights into the molecular basis of laboratory and natural evolution. To date, such efforts usually focus on limited protein families and a single enzyme trait, with little concern about the relationship between protein epistasis and conformational dynamics. Here, we report a multiparametric fitness landscape for a cytochrome P450 monooxygenase that was engineered for the regio- and stereoselective hydroxylation of a steroid. We develop a computational program to automatically quantify non-additive effects among all possible mutational pathways, finding pervasive cooperative signs and magnitude epistasis on multiple catalytic traits. By using quantum mechanics and molecular dynamics simulations, we show that these effects are modulated by long-range interactions in loops, helices and ß-strands that gate the substrate access channel allowing for optimal catalysis. Our work highlights the importance of conformational dynamics on epistasis in an enzyme involved in secondary metabolism and offers insights for engineering P450s.

An ancestral 10-bp repeat expansion in VWA1 causes recessive hereditary motor neuropathy.

The extracellular matrix comprises a network of macromolecules such as collagens, proteoglycans and glycoproteins. VWA1 (von Willebrand factor A domain containing 1) encodes a component of the extracellular matrix that interacts with perlecan/collagen VI, appears to be involved in stabilizing extracellular matrix structures, and demonstrates high expression levels in tibial nerve. Vwa1-deficient mice manifest with abnormal peripheral nerve structure/function; however, VWA1 variants have not previously been associated with human disease. By interrogating the genome sequences of 74 180 individuals from the 100K Genomes Project in combination with international gene-matching efforts and targeted sequencing, we identified 17 individuals from 15 families with an autosomal-recessive, non-length dependent, hereditary motor neuropathy and rare biallelic variants in VWA1. A single disease-associated allele p.(G25Rfs*74), a 10-bp repeat expansion, was observed in 14/15 families and was homozygous in 10/15. Given an allele frequency in European populations approaching 1/1000, the seven unrelated homozygote individuals ascertained from the 100K Genomes Project represents a substantial enrichment above expected. Haplotype analysis identified a shared 220 kb region suggesting that this founder mutation arose >7000 years ago. A wide age-range of patients (6-83 years) helped delineate the clinical phenotype over time. The commonest disease presentation in the cohort was an early-onset (mean 2.0 ± 1.4 years) non-length-dependent axonal hereditary motor neuropathy, confirmed on electrophysiology, which will have to be differentiated from other predominantly or pure motor neuropathies and neuronopathies. Because of slow disease progression, ambulation was largely preserved. Neurophysiology, muscle histopathology, and muscle MRI findings typically revealed clear neurogenic changes with single isolated cases displaying additional myopathic process. We speculate that a few findings of myopathic changes might be secondary to chronic denervation rather than indicating an additional myopathic disease process. Duplex reverse transcription polymerase chain reaction and immunoblotting using patient fibroblasts revealed that the founder allele results in partial nonsense mediated decay and an absence of detectable protein. CRISPR and morpholino vwa1 modelling in zebrafish demonstrated reductions in motor neuron axonal growth, synaptic formation in the skeletal muscles and locomotive behaviour. In summary, we estimate that biallelic variants in VWA1 may be responsible for up to 1% of unexplained hereditary motor neuropathy cases in Europeans. The detailed clinical characterization provided here will facilitate targeted testing on suitable patient cohorts. This novel disease gene may have previously evaded detection because of high GC content, consequential low coverage and computational difficulties associated with robustly detecting repeat-expansions. Reviewing previously unsolved exomes using lower QC filters may generate further diagnoses.

Fragment Binding to the Nsp3 Macrodomain of SARS-CoV-2 Identified Through Crystallographic Screening and Computational Docking.

The SARS-CoV-2 macrodomain (Mac1) within the non-structural protein 3 (Nsp3) counteracts host-mediated antiviral ADP-ribosylation signalling. This enzyme is a promising antiviral target because catalytic mutations render viruses non-pathogenic. Here, we report a massive crystallographic screening and computational docking effort, identifying new chemical matter primarily targeting the active site of the macrodomain. Crystallographic screening of diverse fragment libraries resulted in 214 unique macrodomain-binding fragments, out of 2,683 screened. An additional 60 molecules were selected from docking over 20 million fragments, of which 20 were crystallographically confirmed. X-ray data collection to ultra-high resolution and at physiological temperature enabled assessment of the conformational heterogeneity around the active site. Several crystallographic and docking fragment hits were validated for solution binding using three biophysical techniques (DSF, HTRF, ITC). Overall, the 234 fragment structures presented explore a wide range of chemotypes and provide starting points for development of potent SARS-CoV-2 macrodomain inhibitors.

MichelaNglo: sculpting protein views on web pages without coding.

MOTIVATION: The sharing of macromolecular structural information online by scientists is predominantly performed via 2D static images, since the embedding of interactive 3D structures in webpages is non-trivial. Whilst the technologies to do so exist, they are often only implementable with significant web coding experience. RESULTS: Michelaɴɢʟo is an accessible and open-source web-based application that supports the generation, customization and sharing of interactive 3D macromolecular visualizations for digital media without requiring programming skills. A PyMOL file, PDB file, PDB identifier code or protein/gene name can be provided to form the basis of visualizations using the NGL JavaScript library. Hyperlinks that control the view can be added to text within the page. Protein-coding variants can be highlighted to support interpretation of their potential functional consequences. The resulting visualizations and text can be customized and shared, as well as embedded within existing websites by following instructions and using a self-contained download. Michelaɴɢʟo allows researchers to move away from static images and instead engage, describe and explain their protein to a wider audience in a more interactive fashion. AVAILABILITY AND IMPLEMENTATION: Michelaɴɢʟo is hosted at michelanglo.sgc.ox.ac.uk. The Python code is freely available at https://github.com/thesgc/MichelaNGLo, along with documentations about its implementation.

Identification of Circulating Genomic and Metabolic Biomarkers in Intrahepatic Cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is an aggressive cancer arising from the bile ducts with a need for earlier diagnosis and a greater range of treatment options. KRAS/NRAS mutations are common in ICC tumours and 6-32% of patients also have isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) gene mutations associated with metabolic changes. This feasibility study investigated sequencing circulating tumour DNA (ctDNA) combined with metabolite profiling of plasma as a method for biomarker discovery in ICC patients. Plasma was collected from four ICC patients receiving radio-embolisation and healthy controls at multiple time points. ctDNA was sequenced using Ampliseq cancer hotspot panel-v2 on Ion Torrent PGM for single nucleotide variants (SNV) detection and with Illumina whole genome sequencing for copy number variants (CNV) and further targeted examination for SNVs. Untargeted analysis of metabolites from patient and control plasma was performed using liquid chromatography coupled with high-resolution tandem mass spectrometry (LC-MS/MS). Metabolite identification was performed using multi-parameter comparisons with analysis of authentic standards, and univariate statistical analysis was performed to identify differences in metabolite abundance between patient and control samples. Recurrent somatic SNVs and CNVs were identified in ctDNA from three out of four patients that included both NRAS and IDH1 mutations linked to ICC. Plasma metabolite analysis revealed biomarker metabolites associated with ICC and in particular 2-hydroxyglutarate (2-HG) levels were elevated in both samples from the only patient showing a variant allele in IDH1. A reduction in the number of CNVs was observed with treatment. This study demonstrates that ctDNA and metabolite levels can be identified and correlated in ICC patient blood samples and differentiated from healthy controls. We conclude that combining genomic and metabolic analysis of plasma offers an effective approach to biomarker identification with potential for disease stratification and early detection studies.