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Structure of the human C9orf72-SMCR8 complex reveals a multivalent protein interaction architecture.
No full textA major cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) spectrum disorder is the hexanucleotide G4C2 repeat expansion in the first intron of the C9orf72 gene. Many underlying mechanisms lead to manifestation of disease that include toxic gain of function by repeat G4C2 RNAs, dipeptide repeat proteins, and a reduction of the C9orf72 gene product. The C9orf72 protein interacts with SMCR8 and WDR41 to form a trimeric complex and regulates multiple cellular pathways including autophagy. Here, we report the structure of the C9orf72-SMCR8 complex at 3.8 Å resolution using single-particle cryo-electron microscopy (cryo-EM). The structure reveals 2 distinct dimerization interfaces between C9orf72 and SMCR8 that involves an extensive network of interactions. Homology between C9orf72-SMCR8 and Folliculin-Folliculin Interacting Protein 2 (FLCN-FNIP2), a GTPase activating protein (GAP) complex, enabled identification of a key residue within the active site of SMCR8. Further structural analysis suggested that a coiled-coil region within the uDenn domain of SMCR8 could act as an interaction platform for other coiled-coil proteins, and its deletion reduced the interaction of the C9orf72-SMCR8 complex with FIP200 upon starvation. In summary, this study contributes toward our understanding of the biological function of the C9orf72-SMCR8 complex
CRISPR/Cas9-engineered inducible gametocyte producer lines as a valuable tool for Plasmodium falciparum malaria transmission research.
No full textThe malaria parasite Plasmodium falciparum replicates inside erythrocytes in the blood of infected humans. During each replication cycle, a small proportion of parasites commits to sexual development and differentiates into gametocytes, which are essential for parasite transmission via the mosquito vector. Detailed molecular investigation of gametocyte biology and transmission has been hampered by difficulties in generating large numbers of these highly specialised cells. Here, we engineer P. falciparum NF54 inducible gametocyte producer (iGP) lines for the routine mass production of synchronous gametocytes via conditional overexpression of the sexual commitment factor GDV1. NF54/iGP lines consistently achieve sexual commitment rates of 75% and produce viable gametocytes that are transmissible by mosquitoes. We also demonstrate that further genetic engineering of NF54/iGP parasites is a valuable tool for the targeted exploration of gametocyte biology. In summary, we believe the iGP approach developed here will greatly expedite basic and applied malaria transmission stage research
Orally administered branaplam does not impact neurogenesis in juvenile mice, rats, and dogs
No full textBranaplam is a therapeutic agent currently in clinical development for the treatment of infants with type 1 spinal muscular atrophy (SMA). Since preclinical studies showed that branaplam had cell-cycle arrest effects; we sought to determine whether branaplam may affect postnatal cerebellar development and brain neurogenesis. Here, we describe a novel approach for developmental neurotoxicity testing (DNT) of a central nervous system (CNS) active drug . The effects of orally administered branaplam were evaluated in the SMA neonatal mouse model (SMN∆7), and in juvenile Wistar Hanover rats and Beagle dogs. Histopathological examination and complementary immunohistochemical studies focused on areas of neurogenesis in the cerebellum (mice, rats, and dogs), and the subventricular zone of the striatum and dentate gyrus (rats and dogs) using antibodies directed against Ki67, phosphorylated histone H3, cleaved caspase-3, and glial fibrillary acidic protein. Additionally, image analysis based quantification of calbindin-D28k and Ki67 was performed in rats and dogs. The patterns of cell proliferation and apoptosis, and neural migration and innervation in the cerebellum and other brain regions of active adult neurogenesis did not differ between branaplam- and control-treated animals. Quantitative image analysis did not reveal any changes in calbindin-D28k and Ki67 expression in rats and dogs. The data show that orally administered branaplam has no impact on neurogenesis in juvenile animals. Application of selected immunohistochemical stainings in combination with quantitative image analysis on a few critical areas of postnatal CNS development offer a reliable approach to assess DNT of CNS-active drug candidates in juvenile animal toxicity studies
Analyzing patient trajectories with artificial intelligence
No full textIn digital medicine, patient data typically record health events over time (eg, through electronic health records, wearables, or other sensing technologies) and thus form unique patient trajectories. Patient trajectories are highly predictive of the future course of diseases and therefore facilitate effective care. However, digital medicine often uses only limited patient data, consisting of health events from only a single or small number of time points while ignoring additional information encoded in patient trajectories. To analyze such rich longitudinal data, new artificial intelligence (AI) solutions are needed. In this paper, we provide an overview of the recent efforts to develop trajectory-aware AI solutions and provide suggestions for future directions. Specifically, we examine the implications for developing disease models from patient trajectories along the typical workflow in AI: problem definition, data processing, modeling, evaluation, and interpretation. We conclude with a discussion of how such AI solutions will allow the field to build robust models for personalized risk scoring, subtyping, and disease pathway discovery
Split chloramphenicol acetyl-transferase assay reveals self-ubiquitylation-dependent regulation of UBE3B
No full textSplit reporter protein-based genetic section systems are widely used to identify and
characterize protein-protein interactions (PPI). Assembly of split markers that
antagonize toxins, rather than required for synthesis of missing essential metabolites,
facilitate the seeding of high density of cells and selective growth. Here we present adeveloped split chloramphenicol acetyltransferase (split-CAT) -based genetic selection
system. The N-terminus fragment of CAT is fused downstream of the protein of interest
and the C-terminus fragment is tethered upstream of a postulated protein partner. We
demonstrate the system's advantages for the study of PPIs. Moreover, we show that
co-expression of a functional ubiquitylation cascade where the target and ubiquitin are
tethered to the split-CAT fragments results in ubiquitylation-dependent growth on
selective media. The fact that proteins do not have to be purified from bacteria and the
high sensitivity of the split-CAT reporter, enable the detection of challenging protein
cascades and post-translation modifications. In addition, we demonstrate that the split-
CAT system responds to small molecule inhibitors and molecular glues (GLUTACs).
The absence of ubiquitylation-dependent degradation and deubiquitylation in E. coli
significantly simplify the interpretation of the results. We demonstrate that the spit-CAT
system provides a readout for the known self-ubiquitylation-dependent inactivation of
NEDD4. Subsequently, we harnessed the system to explore if UBE3B, a HECT ligase
not belonging to the Nedd4 subfamily, is also regulated by self-ubiquitylation. We found
that self-ubiquitylation of UBE3B at residue K665 inactivates the enzyme in the E. coli
system and in mammalian cells due to its oligomerization
Achiral SFC for Purification of Pharmaceuticals
No full textWith large quantities of small scale (<1000mg) separations typically being needed to be purified and evaluated for discovery in-vitro and in-vivo testing regimes, a chromatographic purification technique needs to be easily integrated into discovery chemistry laboratories and highly reproducible. HPLC has been generally accepted as the main technique of choice for analysis and purification, utilizing either normal phase or reverse phase conditions, with reverse phase being the generally accepted as a high throughput approach to purify small scale drug discovery products At Novartis, the integration of supercritical fluid chromatography (SFC) has several advantages such as faster run times due to lower viscosity, short equilibration times, reduced solvent consumption and, in preparative applications, fast solvent removal. SFC has rapidly become a very attractive alternative to Normal Phase and Reverse Phase purification for achiral samples and this short article highlights the benefits of this approach
Addition of iptacopan, an oral factor B inhibitor, to eculizumab in patients with paroxysmal nocturnal haemoglobinuria and active haemolysis: an open-label, single-arm, phase 2, proof-of-concept trial
No full textBackground: The haematological benefit of standard-of-care anti-C5 treatment for haemolytic paroxysmal nocturnal haemoglobinuria is limited by residual intravascular haemolysis or emerging C3-mediated extravascular haemolysis.
Therefore, the aim of this phase 2 study was to assess the safety, tolerability, pharmacokinetics and pharmacodynamics,
and activity of the new complement factor B inhibitor, iptacopan, in patients with paroxysmal nocturnal haemoglobinuria
who have active haemolysis despite anti-C5 therapy.
Methods: In this multicentre, open-label, single-arm, phase 2 trial, we enrolled adult patients (aged 18–80 years) with
paroxysmal nocturnal haemoglobinuria who showed signs of active haemolysis despite receiving eculizumab
treatment. Patients were enrolled at Federico II University Hospital (Naples, Italy), Hôpital Saint-Louis (Paris, France),
and University Hospital Essen (Essen, Germany). For enrolment, patients were required to show lactate dehydrogenase
more than 1·5-times the upper limit of normal and a paroxysmal nocturnal haemoglobinuria type 3 erythrocyte or
granulocyte clone size of 10% or greater. Patients with bone marrow failure, on systemic steroid or immunosuppressive
drugs, or with severe comorbidities were excluded from the study. Iptacopan was given orally as an add-on therapy at
a dose of 200 mg twice daily. The primary endpoint was the effect of iptacopan on the reduction of chronic residual
intravascular haemolysis measured as change in lactate dehydrogenase from baseline value to week 13. At 13 weeks,
patients could opt into a long-term study extension (ongoing), allowing for modifications of standard treatment. This
trial is registered at ClinicialTrials.gov, NCT03439839.
Findings: Between May 31, 2018, and April 9, 2019, ten patients had twice daily 200 mg iptacopan. Iptacopan resulted in
marked reduction of lactate dehydrogenase from baseline versus at week 13 (mean 539 IU/L [SD 263] vs 235 IU/L
[44], change from baseline –309·2 IU/L [SD 265·5], 90% CI –473·77 to –144·68, p=0·0081), associated with significant
improvement of haemoglobin concentrations (mean 97·7 g/L [SD 10·5] vs 129·5 g/L [18·3] change from baseline
31·9 g/L [14·5], 90% CI 23·42–40·28, p<0·0001). All biomarkers of haemolysis improved on iptacopan treatment.
Observed haematological benefits were maintained longer than the 13-week study period, throughout the study extension,
including seven patients who stopped concomitant standard-of-care treatment and continued iptacopan as monotherapy.
There were no deaths or treatment-related serious adverse events during the study period. Of three non-related serious
adverse events, two occurred in the same patient (one during run-in and before exposure to iptacopan).
Interpretation: Iptacopan at a chronic dose of 200 mg twice daily was well tolerated without any major drug-related
safety findings and shows lactate dehydrogenase reduction and haemoglobin normalisation in most patients with
paroxysmal nocturnal haemoglobinuria at week 13 and beyond, even in monotherapy. On the basis of these data,
iptacopan will be tested as monotherapy in pivotal trials investigating its haematological benefit in a broader paroxysmal
nocturnal haemoglobinuria population.
Funding: Novartis Institutes for Biomedical Research
Multi-Species Phenotypic Screening across Disease Models of Mucolipidosis Type IV
No full textInvertebrate model organisms (the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster) are valuable tools to bridge the gap between traditional in vitro discovery and preclinical animal models. Invertebrate model organisms are poised to serve as better disease models than 2D cellular monocultures for drug discovery, as well as easier and more cost-effective to scale up than 3D organoids/assembloids or co-cultures. A strength of model organisms is the opportunity to probe conserved biology such as lysosomal function and autophagy in a physiological setting. However, invertebrate models are not without pharmacokinetic and pharmacodynamic challenges, such as poor tissue penetration and confidence in a compound’s mechanism of action. To confront those challenges, we took advantage of the Novartis mechanism-of-action box (MoA Box), a compound library of well-annotated and drug-like chemical probes. Curious as to how the MoA Box, comprised of chemical probes optimized for mammalian targets, would fare in an invertebrate setting we screened the MoA Box across three different models of the lysosomal storage disease mucolipidosis Type IV (MLIV). MLIV is caused by mutations in the lysosomal transient receptor potential ion channel mucolipin-1 (TRPML1) resulting in hyper-acidic lysosomes and dysregulated autophagy. The overlap of screening hits from worm, fly, and patient fibroblast screens identified cyclin-dependent kinase (CDK) inhibition as an evolutionarily conserved disease modifier and potential drug repurposing strategy
Greening up nitro group reductions. 1-Pot sequences and applications to syntheses of pharmaceutical intermediates
No full textCommercially available Pd/C was used as a catalyst with only 0.4 mol % Pd loading. The reaction can be performed using either silane as transfer hydrogenating agent or simply hydrogen balloon (~1 atm pressure). With this technology, a series of nitro compounds were reduced to the desired amines in high chemical yields. Both the catalyst and surfactant were recycled several times without loss of reactivity