Novartis Institutes for BioMedical Research

Introduction/Aims Studies have demonstrated the potential of muscle MRIs to measure disease progression in ALS. However, the responsiveness and utility of quantitative muscle MRIs in an ALS clinical trial remain unknown. This study aimed to determine the responsiveness of quantitative muscle MRIs to measure disease progression in ALS. Methods Longitudinal quantitative muscle MRIs were obtained in an ALS study that delivered human neural progenitor cells to the spinal cord (NCT02943850). Participants underwent MRIs at baseline, 1, 3, 6, 9, and 12 months. MRI measures included fat fraction (ff), water T 2 ( T 2m ), cross‐sectional area (CSA), and remaining muscle area (RMA). Non‐MRI measures included strength via Accurate Test of Limb Isometric Strength (ATLIS) and the ALSFRS‐R. Standardized response means (SRM) were calculated at 1, 3, 6, and 12 months. Results Significant increases in muscle FF and decreases in CSA and RMA were seen as early as 1 month from baseline. At 6 months, the most responsive measures were muscle FF (SRM thigh = 1.85, SRM calf = 1.39), T 2m (SRM thigh = 1.2, SRM calf = 1.71), CSA (SRM thigh = −1.58, SRM calf = −1.14), RMA (SRM thigh = −1.77, SRM calf = −1.28), and strength tested via ATLIS (SRM knee extension = −1.79, SRM knee flexion = −1.3). The ALSFRS‐R was the least responsive at 6 months (SRM = −0.85). Muscle FF and T 2m correlated with ALSFRS‐R leg subscores and MRI measures demonstrated varying degrees of correlation with strength. Discussion High responsiveness and low variability make quantitative muscle MRI a novel and complementary outcome measure for ALS clinical trials.

We describe the identification of a candidate positron emission tomography (PET) imaging agent for the NLRP3 protein. NLRP3 plays a critical role in the immune system and has proven a difficult target for the development of imaging agents due to its low and cell‐specific expression profile. A recently described series of pyridazine‐based inhibitors, with improved permeability and brain‐penetration properties, was used as a starting point for the development of a suitable PET imaging agent. Optimization of affinity, non‐specific binding and pharmacokinetic properties led to the identification of aminopyridazine (R)‐2‐(6‐((1‐cyclopropylpiperidin‐3‐yl)amino)pyridazin‐3‐yl)‐5‐fluoro‐3‐methylphenol (17 b), which meets the preclinical profile of a successful imaging agent, and whose tritiated version demonstrated excellent specificity in a radioligand saturation binding assay, confirming its imaging potential.¹⁸F labeling led to [¹⁸F]NP3‐627, the proposed PET imaging agent.

Controlled modifications of amino acids are an indispensable tool for advancing fundamental and translational research based on peptides and proteins. Yet, we still lack methods to chemically modify each naturally occurring amino acid sidechain. To help address this gap, we show that N,α‐diaryl oxaziridines expand the scope of bioconjugation methods to chemically modify cysteine, methionine, and tryptophan residues with evidence for additional tyrosine labelling in a proteomic context. Conjugation primarily at tryptophan sites can be accessed by selective cleavage of modifications at other sidechains. The N,α‐diaryl oxaziridine reagents are accessed through photoisomerization of nitrones, which serve as photocaged reagents, thus providing an additional level of control over reactivity. Initial guiding principles for the design of nitrone reagents are developed by exploring the impact of structure on UV/Vis absorption, photoisomerization, and reactivity. We identify a nitrone structure that maximizes photoisomerization efficiency, the aqueous stability of the oxaziridine, the extent of amino acid modification, and the stability of the resulting amino acid conjugates. We then translate nitrone reagents to modify proteins in aqueous conditions. Finally, we use nitrones to profile reactive residues across the proteome of a mammalian cell line and find that they expand the proteome coverage.

Objectives To develop a protocol for largescale analysis of synovial fluid proteins, for the identification of biological networks associated with subtypes of osteoarthritis. Methods Synovial Fluid To detect molecular Endotypes by Unbiased Proteomics in Osteoarthritis (STEpUP OA) is an international consortium utilising clinical data (capturing pain, radiographic severity and demographic features) and knee synovial fluid from 17 participating cohorts. 1746 samples from 1650 individuals comprising OA, joint injury, healthy and inflammatory arthritis controls, divided into discovery (n = 1045) and replication (n = 701) datasets, were analysed by SomaScan Discovery Plex V4.1 (>7000 SOMAmers/proteins). An optimised approach to standardisation was developed. Technical confounders and batch-effects were identified and adjusted for. Poorly performing SOMAmers and samples were excluded. Variance in the data was determined by principal component (PC) analysis. Results A synovial fluid standardised protocol was optimised that had good reliability (<20% co-efficient of variation for >80% of SOMAmers in pooled samples) and overall good correlation with immunoassay. 1720 samples and >6290 SOMAmers met inclusion criteria. 48% of data variance (PC1) was strongly correlated with individual SOMAmer signal intensities, particularly with low abundance proteins (median correlation coefficient 0.70), and was enriched for nuclear and non-secreted proteins. We concluded that this component was predominantly intracellular proteins, and could be adjusted for using an ‘intracellular protein score’ (IPS). PC2 (7% variance) was attributable to processing batch and was batch-corrected by ComBat. Lesser effects were attributed to other technical confounders. Data visualisation revealed clustering of injury and OA cases in overlapping but distinguishable areas of high-dimensional proteomic space. Conclusions We have developed a robust method for analysing synovial fluid protein, creating a molecular and clinical dataset of unprecedented scale to explore potential patient subtypes and the molecular pathogenesis of OA. Such methodology underpins the development of new approaches to tackle this disease which remains a huge societal challenge.

Loss of cytosolic actin filaments upon TORC2 inhibition triggers chromosome fragmentation in yeast, which results from altered base excision repair of Zeocin-induced lesions. To find the link between TORC2 kinase and this yeast chromosome shattering (YCS) we performed phosphoproteomics. YCS-relevant phospho-targets included plasma membrane-associated regulators of actin polymerization, such as Las17, the yeast Wiscott-Aldrich Syndrome protein. Induced degradation of Las17 was sufficient to trigger YCS in presence of Zeocin, bypassing TORC2 inhibition. In yeast, Las17 does not act directly at damage, but instead its loss, like TORC2 inhibition, raises nuclear actin levels. Nuclear actin, in complex with Arp4, forms an essential subunit of several nucleosome remodeler complexes, including INO80C, which facilitates DNA polymerase elongation. Here we show that the genetic ablation of INO80C activity leads to partial YCS resistance, suggesting that elevated levels of nuclear G-actin may stimulate INO80C to increase DNA polymerase processivity and convert single-strand lesions into double-strand breaks.

Combinational therapies provoking cell death are of major interest in oncology. Combining TORC2 kinase inhibition with the radiomimetic drug Zeocin results in a rapid accumulation of double-strand breaks (DSB) in the budding yeast genome. This lethal Yeast Chromosome Shattering (YCS) requires conserved enzymes of base excision repair. YCS can be attenuated by eliminating three N-glycosylases or endonucleases Apn1/Apn2 and Rad1, which act to convert oxidized bases into abasic sites and single-strand nicks. Adjacent lesions must be repaired in a step-wise fashion to avoid generating DSBs. Artificially increasing nuclear actin by destabilizing cytoplasmic actin filaments or by expressing a nuclear export-deficient actin interferes with this step-wise repair and generates DSBs, while mutants that impair DNA polymerase processivity reduce them. Repair factors that bind actin include Apn1, RFA and the actin-dependent chromatin remodeler INO80C. During YCS, increased INO80C activity could enhance both DNA polymerase processivity and repair factor access to convert clustered lesions into DSBs.

Introduction: Pelacarsen, a hepatocyte-directed, N-acetyl galactosamine (GalNAc 3 )–conjugated antisense oligonucleotide, reduces plasma lipoprotein(a) levels by inhibiting apolipoprotein(a) translation. Pelacarsen uptake is mediated by GalNAc 3 binding to the hepatocyte-specific asialoglycoprotein receptor. Hypothesis: It is unknown whether hepatic impairment (HI) impacts pelacarsen uptake and systemic exposure. Aim: This single-dose, open-label, parallel-group, Phase 1 study (NCT05026996) assessed the pharmacokinetics (PK), safety, and tolerability of a single 80 mg subcutaneous dose of pelacarsen in participants with mild HI compared to healthy controls (normal hepatic function). Methods: Eight adults with prior liver cirrhosis and mild HI (Child-Pugh Class A) were matched for sex, age, and body weight with nine healthy controls. PK parameters (C max , AUC 0-72, AUC last , and AUC inf ) were determined using non-compartmental methods. Log-transformed PK parameters were analyzed using a statistical model with group and matching covariates as fixed effects. Least-square geometric means for each group and geometric mean ratios between participants with mild HI and healthy controls were extracted. Safety was also assessed. Results: Pelacarsen C max , AUC last, and AUC inf were, on average, 7%, 37%, and 50% higher, respectively, in participants with mild HI versus matched controls. All 90% confidence intervals around the HI versus healthy control geometric mean ratios included 1 ( Table ). The ranges of all PK parameters and estimated half-lives were similar between groups. In participants with mild HI, pelacarsen exposure approached the same level as controls after eight hours post-dose ( Figure ). No serious adverse events occurred. Conclusion(s): In participants with mild HI, pelacarsen was well tolerated. Mild HI had no significant effect on pelacarsen C max . The non-statistically significant transient increase in AUC was within the exposure range tested in the first-in-human study.

Introduction: Patients with Duchenne muscular dystrophy (DMD) universally develop dilated cardiomyopathy. Characteristic left ventricular (LV) fibrosis and decreased LV systolic function can increase the likelihood of fatal ventricular arrhythmias (VA). However, the benefits of ICD therapy in DMD remain uncertain, and there are no established VA risk stratification methods to guide clinical decision making. Here, we developed a digital twin approach that uses standard-of-care late-gadolinium enhanced cardiac magnetic resonance (LGE CMR) imaging alone to non-invasively screen for VA risk in DMD. Methods: Thirty-nine DMD patients from three centers with LGE CMR and Ziopatch monitoring were included, with fast nonsustained VT (NSVT; >170 BPM) as the primary clinical outcome (positive control). From short-axis 2D LGE CMR, we constructed 3D computational models of the LV with patient-specific fibrosis distribution (Fig A). These models were separated into positive and negative groups by performing unsupervised clustering on regional substrate percentages (Fig B). DMD-specific EP properties were then assigned to the 3D models, and a rapid pacing protocol was used to assess in silico VT inducibility (Fig C). Patients were predicted to have VA if they tested positive with both clustering and simulation methods. Tests were performed blinded to clinical outcome, cohort positivity rate, and covariates such as ejection fraction. Results: Five patients were observed to have fast NSVT. Our approach achieved 80% (4/5) sensitivity, 79.4% (27/34) specificity, and 79.5% (31/39) accuracy in predicting these events. The sole false negative patient had lower LGE CMR resolution relative to the rest of the cohort (p < 0.01). While extensive LGE was present in all patients, the positive cluster showed higher inferolateral, apical, and total fibrosis burden (p < 0.01) compared to the negative cluster. Without using simulation, specificity and accuracy decreased to 58.8% and 64.1%. Conclusion: LGE-derived digital twin models show strong potential for screening for VA risk in DMD patients. Future application of these models may help define optimal indications for ICD implantation in DMD.

Lipid nanoparticles (LNPs) have emerged as the leading nonviral nucleic acid (NA) delivery system, gaining widespread attention for their use in COVID‐19 vaccines. They are recognized for their efficient NA encapsulation, modifiability, and scalable production. However, LNPs face efficacy and potency limitations due to suboptimal intracellular processing, with endosomal escape efficiencies (ESE) below 2.5%. Additionally, up to 70% of NPs undergo recycling and exocytosis after cellular uptake. In contrast, cell‐derived vesicles offer biocompatibility and high‐delivery efficacy but are challenging to load with exogenous NAs and to manufacture at large‐scale. To leverage the strengths of both systems, a hybrid system is designed by combining cell‐derived vesicles, such as nano plasma membrane vesicles (nPMVs), with LNPs through microfluidic mixing and subsequent dialysis. These hybrids demonstrate up to tenfold increase in ESE and an 18‐fold rise in reporter gene expression in vitro and in vivo in zebrafish larvae (ZFL) and mice, compared to traditional LNPs. These improvements are linked to their unique physico‐chemical properties, composition, and morphology. By incorporating cell‐derived vesicles, this strategy streamlines the development process, significantly enhancing the efficacy and potency of gene delivery systems without the need for extensive screening.

Embedded within the field of drug metabolism and pharmacokinetics (DMPK), biotransformation is a discipline that studies the origins, disposition, and structural identity of metabolites to provide a comprehensive safety assessment, including the assessment of exposure coverage in toxicological species. Spanning discovery and development, metabolite identification (metID) scientists employ various strategies and tools to address stage-specific questions aimed at guiding the maturation of early chemical matter into drug candidates. During this process, the identity of major (and minor) circulating human metabolites is ascertained to comply with the regulatory requirements such as the Metabolites in Safety Testing (MIST) guidance. Through the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ), the “Translatability of MetID In Vitro Systems Working Group” was created within the Translational and ADME Sciences Leadership Group. The remit of this group was to objectively determine how accurate commonly employed in vitro systems have been with respect to prediction of circulating human metabolites, both qualitatively and quantitatively. A survey composed of 34 questions was conducted across 26 pharmaceutical companies to obtain a foundational understanding of current metID practices, preclinically and clinically, as well as to provide perspective on how successful these practices have been at predicting circulating human metabolites. The results of this survey are presented as an initial snapshot of current industry-based metID practices, including our perspective on how a harmonized framework for the conduct of in vitro metID studies could be established. Future perspectives from current practices to emerging advances with greater translational capability are also provided.

Cancer progression and response to therapy are inextricably reliant on the coevolution of a supportive tissue microenvironment. This is particularly evident in pancreatic ductal adenocarcinoma, a tumor type characterized by expansive and heterogeneous stroma. Herein, we employed single-cell RNA sequencing and spatial transcriptomics of normal, inflamed, and malignant pancreatic tissues to contextualize stromal dynamics associated with disease and treatment status, identifying temporal and spatial trajectories of fibroblast differentiation. Using analytical tools to infer cellular communication, together with a newly developed assay to annotate genomic alterations in cancer cells, we additionally explored the complex intercellular networks underlying tissue circuitry, highlighting a fibroblast-centric interactome that grows in strength and complexity in the context of malignant transformation. Our study yields new insights on the stromal remodeling events favoring the development of a tumor-supportive microenvironment and provides a powerful resource for the exploration of novel points of therapeutic intervention in pancreatic ductal adenocarcinoma. Significance Pancreatic cancer remains a high unmet medical need. Understanding the interactions between stroma and cancer cells in this disease may unveil new opportunities for therapeutic intervention.

Necroptosis is a regulated form of cell death that has been observed in Alzheimer’s disease (AD) along with the classical pathological hallmark lesions of amyloid plaques and Tau neurofibrillary tangles. To understand the neurodegenerative process in AD, we studied the role of necroptosis in mouse models and primary mouse neurons. Using immunohistochemistry, we demonstrated activated necroptosis-related proteins in transgenic mice developing Tau pathology and in primary neurons from amyloid precursor protein (APP)–Tau double transgenic mice treated with phosphorylated Tau seeds derived from a patient with AD but not in APP transgenic mice that only exhibited β-amyloid deposits. Necroptosis proteins in granulovacuolar degeneration (GVD) bodies were associated with neuronal loss in mouse brain regions also known to be vulnerable to GVD in the human AD brain. Necroptosis inhibitors lowered the percentage of neurons showing GVD and reduced neuronal loss, both in transgenic mice and in primary mouse neurons. This suggests that a GVD-associated form of necroptosis that we refer to as “GVD-necroptosis” may represent a delayed form of necroptosis in AD. We propose that inhibition of necroptosis could rescue this type of neuronal death in AD.

RAS proteins control cell proliferation and activating mutations are collectively the most frequent oncogenic event observed in cancer patients, justifying investments into multiple drug discovery efforts. While RAS-directed therapeutic agents targeting either the inactive GDP-bound or the active GTP-bound state have entered the clinic, invariably resistance is observed. Mutations at drug binding sites represent a common resistance mechanism indicating the need to discover new targetable pockets in RAS. Such efforts are hindered by the small globular size of the protein, for long considered undruggable. Here we perform macrocyclic peptides mRNA and nanobody yeast display screens and discover a new targetable ligand-induced pocket in RAS. In vitro and cellular experiments with the KM12 and KM12-AM nanobodies show RAS inhibition via displacement of cRAF, by affecting their protein-protein interaction via the less studied cRAF CRD domain. Further, we provide orthogonal functional validation for the new binding pocket via mutagenesis experiments. Notably, the newly discovered RAS-targeting approach enable simultaneous targeting of both GTP-bound active and GDP-bound inactive states and leaves the SwII pocket unaltered, opening possibilities of combinatorial approaches with clinically approved SwII pocket inhibitors.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates plasma low‐density lipoprotein cholesterol (LDL−C) levels by promoting hepatic LDL receptor (LDL−R) degradation. We previously identified and optimized 13‐mer cyclic peptides that bind to a novel, induced‐fit pocket adjacent to the binding interface of PCSK9 and LDL−R and effectively disrupted the PCSK9/LDL−R protein‐protein interaction (PPI) both in vitro and in vivo. However this series of large cyclic peptides required charged groups for function and lacked oral bioavailability in rodents. We describe herein multiple structure‐based modifications to these original peptides to yield truncated, neutral molecules with full PPI function in both biochemical and cellular assays. In parallel, new mRNA‐peptide display screens identified non‐functional 8‐ and 9‐mer compounds which ligand the induced‐fit pocket in a distinct manner. Taken together, these studies indicate multiple directions to reduce the size and complexity of this peptide class toward a true small molecule oral agent.

Plasma membrane integrity is vital not only for cell survival but also nearly all aspects of cell functioning. Mechanical stress can cause plasma membrane damage, but it is not known whether there are large molecules (proteins) that control plasma membrane integrity. Here we constructed a 384-well cellular stretch system that delivers precise, reproducible mechanical strain to adherent cells. Using the system, we screened 10,843 siRNAs targeting 2,726 multi-pass transmembrane proteins for stretch-induced membrane permeability changes. The screen identified NINJ1, a protein recently proposed to regulate pyroptosis and other lytic cell death, as the top hit. We demonstrate that NINJ1 is a critical regulator for mechanical force-induced plasma membrane rupture (PMR), without the need of stimulating any cell death programs. Low NINJ1 expression renders the membrane more resistant to stretching, while high expression of NINJ1 lowers the threshold of PMR under mechanical strain. NINJ1 level on the plasma membrane is inversely correlated to tension required to rupture the membrane. In the pyroptosis context, NINJ1 on its own is not sufficient to fully rupture the membrane, and additional mechanical stress is required for full PMR. Our work establishes that NINJ1 functions as a bona fide determinant of membrane biomechanical properties. Our study also suggests that PMR across tissues of distinct mechanical environments is subjected to fine tuning by differences in NINJ1 expression and external mechanical forces.

Diarrhoeal disease caused by Cryptosporidium is a major cause of morbidity and mortality in young and malnourished children from low- and middle-income countries, with no vaccine or effective treatment. Here we describe the discovery of EDI048, a Cryptosporidium PI(4)K inhibitor, designed to be active at the infection site in the gastrointestinal tract and undergo rapid metabolism in the liver. By using mutational analysis and crystal structure, we show that EDI048 binds to highly conserved amino acid residues in the ATP-binding site. EDI048 is orally efficacious in an immunocompromised mouse model despite negligible circulating concentrations, thus demonstrating that gastrointestinal exposure is necessary and sufficient for efficacy. In neonatal calves, a clinical model of cryptosporidiosis, EDI048 treatment resulted in rapid resolution of diarrhoea and significant reduction in faecal oocyst shedding. Safety and pharmacological studies demonstrated predictable metabolism and low systemic exposure of EDI048, providing a substantial safety margin required for a paediatric indication. EDI048 is a promising clinical candidate for the treatment of life-threatening paediatric cryptosporidiosis.