Biology subjects

Singh, S.

Publications and source records attributed to Singh, S..

At least 163 records · Page 9Linked to original sources

bioRxiv2025

A bioinformatic workflow to facilitate the study of less-understood proteins: the case of SH2D2A

Adaptor proteins are key regulators of immune signalling but are challenging to study due to redundant pathways that obscure clear functional "anchor" phenotypes. Making use of public single-cell RNA sequencing (scRNA-seq) datasets, we theorised that anchors could be identified by working in the reverse direction: starting from the transcriptomic level and working "bottom-up" to identify anchoring phenomena at the cell surface. We have produced a prototype workflow for this method using the case of the lymphocyte-enriched adaptor protein SH2D2A, whose function remains uncertain. Using our bottom-up analysis, we have identified a link between SH2D2A and T cell-T cell (T-T) synapses enriched for ITGB2. The functional implications of this link are currently being explored, but further application of this bioinformatic approach to less-understood proteins may prove useful in understanding the minutiae of immune cell signalling, with implications for immunotherapy.

molecular biology↗

bioRxiv2025

Monoclonal antibody to Filarial serpin Wb123 impedes the urokinase plasminogen activator receptor-mediated Alternative activation of macrophages

Macrophages play a multifaceted role in mounting an immune response against pathogens through phagocytosis, antigen presentation and cytokine production. Activated macrophages are critical for the initiation, maintenance, and resolution of inflammation. They can either be classically activated (Th1 response), which promotes inflammation or alternatively activated (Th2 response), which resolves inflammation. The alternate activation of macrophages, along with diminished T cell response, is thought to be an important immune evasion strategy employed by the filarial parasites. However, the mechanism behind filaria-induced alternate activation remains unclear. In this study, by using in-silico approach we identified filarial serpins which are highly expressed in the infective L3 larval stages. We characterized the filarial serine protease inhibitor, Wb123 and established its role in alternate activation. We observed that Wb123 impairs nitric oxide (NO) and reactive oxygen species (ROS) expression, which plays a crucial role in mounting a strong immune response. Surprisingly, we also observed elevated IL6 expression and phosphorylation of STAT3, indicating IL-6-dependent alternate activation. Our findings indicate that the urokinase plasminogen activator receptor (uPAR) is essential for Wb123-induced alternate activation. Furthermore, we demonstrated that the monoclonal antibody MAbG8 impedes the Wb123-induced alternate activation, as shown by reduced CD163 expression along with increased ROS and uPA expression in response to lipopolysaccharide (LPS) and interferon-gamma (IFN-{gamma}). Overall, our study identifies the filarial serpin Wb123 and highlights its potent role in alternative activation. This interaction reveals, for the first time a mechanism exploited by filarial parasites to evade a strong pro-inflammatory immune response.

cell biology↗

bioRxiv2025

Neuronal Silencing and Protection in a Mouse Model of Demyelination

Damage to the myelin sheath that protects axons in the central nervous system is a hallmark pathology of demyelinating diseases like multiple sclerosis. Cuprizone-induced demyelination in mice is a common model for studying demyelination and remyelination. However, the relationship between myelin damage and recovery and neuronal function remains poorly understood. By monitoring hippocampal myelination and neuronal activity in the same mice, we assessed longitudinal changes following cuprizone consumption and remyelination treatment. Upon cuprizone consumption, a rapid decline in neuronal activity preceded slower demyelination. Female mice showed a more intense early decrease in brain activity and a stronger correlation with levels of myelin loss. Remyelination treatment led to increased myelination levels and recovery of neuronal activity compared to vehicle treatment. Changes in single-neuron firing rates during treatment were proportional to the pre-cuprizone firing rate in the same cells, highlighting a potential linkage between the status of myelin recovery and cellular activity.

neuroscience↗

bioRxiv2025

Critical Evaluation of Sphingolipids Detection by MALDI-MSI

The increasing interest in the role of sphingolipids in (patho)physiology has led to the demand for visualization of these lipids within tissue samples (both from animal models and patient specimens) using techniques such as matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). While increasingly adopted, detection of sphingolipids with MALDI-MSI is challenging due to: i) the significant structural variations of sphingolipid molecules, ii) the potential breakdown of the more complex molecules into structurally simpler species which may confound the analysis, and iii) the great difference in levels among sphingolipid classes and subspecies, with the low-abundant ones often being close to the detection limit. In this study, we adopted a multi-pronged approach to establish a robust pipeline for the detection of sphingolipids by MALDI-MSI and to establish best practices and limitations of this technology. First, we evaluated the more commonly adopted methods [2,5-Dihydroxyacetophenon (DHA) or 2,5-Dihydroxybenzoic acid (DHB) matrix in positive ion mode and 1,5-Diaminonaphthalene (DAN) matrix in negative ion mode] using MALDI-MS on reference standards. These standards were used at ratios similar to their relative levels in biological samples to evaluate signal artifacts originating from fragmentation of more complex sphingolipids and impacting low level species. Next, by applying the most appropriate protocol for each sphingolipid class, MALDI-MSI signals were validated in cell culture by modulating specific sphingolipid species using sphingolipid enzymes and inhibitors. Finally, the optimized parameters were utilized on breast cancer tissue from the PyMT mouse model. We report the optimal signal for sphingomyelin (SM) and, for the first time, Sph in DHB positive ion mode (in cells and PyMT tissue), and the validated detection of ceramides and glycosphingolipids in DAN negative ion mode. We document the extensive fragmentation of SM into sphingosine-1-phosphate (S1P) and even more so into ceramide-1-phosphate (C1P) using DAN in negative ion mode and its effect in generating an artifactual C1P tissue signal; we also report the lack of detectable signal for S1P and C1P in biological samples (cells and tissue) using the more suitable DHB positive ion mode protocol.

cell biology↗

bioRxiv2025

"Decoding Mobile Genetic Elements, Virulence Factors, and Antibiotic Resistance Genes Driving Biofilm Formation in MRSA via Network Analysis"

This study aimed to analyze the genome of the Methicillin-resistant Staphylococcus aureus (MRSA) strain d1418m22 through whole-genome sequencing (WGS) and comprehensive bioinformatics analysis with six references strains. De novo assembly resulted in 61 scaffolds with a total genome size of 2.78 Mb. Functional annotation revealed 2,625 predicted genes, including those involved in metabolism, cellular processes, and virulence. Comparative genomic analysis identified 20 antibiotic resistance genes, including those conferring resistance to beta-lactams, fluoroquinolones, and aminoglycosides. In addition, 21 virulence factors (VFs) were identified, including Panton-Valentine leukocidin (PVL) and various enterotoxins. The presence of staphylococcal cassette chromosome mec (SCCmec) type IVa and mobile genetic elements (MGEs), such as prophages and transposons, underscored the role of horizontal gene transfer (HGT) in the evolution and transmission of resistance and virulence factors. Interaction network analysis identified key hub genes involved in various cellular processes, including biofilm formation. This comprehensive genomic analysis provides valuable insights into the genetic makeup of MRSA strain d1418m22, contributing to a better understanding of its pathogenicity and potential public health implications. Six reference MRSA strains were isolated from (MRSA-AMRF4 and MRSA-AMRF5) eye infections, (MRSA and MRSA-15) wound pus, (VMRSA-WC071, and VMRSA-WC081) urine samples. The analysis identified resistance genes, virulence factors, GC content, ANI values, and SCCmec elements, which were found to be similar to those present in the resistant MRSA d1418m22 strain genome.

genomics↗

bioRxiv2025

Cell Painting for cytotoxicity and mode-of-action analysis in primary human hepatocytes

High-throughput, human-relevant approaches for predicting chemical toxicity are urgently needed for better decision-making in human health. Here, we apply image-based profiling (the Cell Painting assay) and two cytotoxicity assays (metabolic and membrane damage readouts) to primary human hepatocytes after exposure to eight concentrations of 1085 compounds that include pharmaceuticals, pesticides, and industrial chemicals with known liver toxicity-related outcomes. Three computational methods (CellProfiler, a Cell Painting-specific convolutional neural network, and a pretrained vision transformer) were compared to extract morphology features from single cells or entire images. We used these morphology features to predict activity in the measured cytotoxicity assays, as well as in 412 curated ToxCast assays that span cytotoxicity, cell-based, and cell-free categories. We found that the morphological profiles detect compound bioactivity at lower concentrations than standard cytotoxicity assays. In supervised analyses, they predict cytotoxicity and targeted cell-based assay readouts, but not cell-free assay readouts. We also found that the various feature extraction methods performed relatively similarly and that filtering out non-bioactive or cytotoxic concentrations did not boost supervised assay prediction performance for any assay endpoint category, although it did have a large influence on unsupervised cluster analysis. We envision that image-based profiling could serve as a key component of modern safety assessment.

pharmacology and toxicology↗

bioRxiv2025

Understanding RNA Chaperone Activity of ProQ Protein

RNA-binding proteins (RBPs) are crucial in recognizing and binding small RNAs that modulate gene expression. RBPs often act as RNA chaperones, facilitating the sRNA-mRNA duplex formation. One of the most studied sRNA chaperones is Hfq, the post-transcriptional gene regulator in bacteria. Recently, a new RNA-chaperone protein, ProQ, has been discovered, which binds to a novel sRNA RaiZ, derived from the 3-end of mRNA encoding ribosome-inactivating protein, and promotes its base pairing with the ribosome binding site of hupA mRNA, leading to the repression of HU- protein synthesis. Despite these studies, the RNA-binding mechanism in ProQ has not been well explored in detail to date. In this work, we studied the binding of raiZ RNA to ProQ using atomistic long-timescale molecular dynamics and on-the-fly probability-based enhanced sampling (OPES) simulations. The raiZ RNA binds to ProQs concave surface, mostly decorated with polar and positively charged residues like Arginine and Lysine, exerting electrostatic stabilization to the RNA. In-silico mutations of these crucial residues lead to a significant loss of the intermolecular interactions, resulting in the detachment of the RNA from the protein. The free energy surface obtained from the OPES simulation helps identify stable raiZ-bound ProQ structures as well as intermediate states. The pertinent atomistic insights into the protein-RNA interactions and deciphering the binding mechanism of the raiZ RNA to ProQ enrich our knowledge about ProQ and its RNA chaperone activity.

biophysics↗

bioRxiv2025

A mechanistic understanding of the effect of Staphylococcus aureus VraS histidine kinase single point mutation on antibiotic resistance

Bacterial genomic mutations in Staphylococcus aureus (S. aureus) have been detected in isolated resistant clinical strains, yet their mechanistic effect on the development of antimicrobial resistance remains unclear. The resistance-associated regulatory systems acquire adaptive mutations under stress conditions that may lead to a gain of function effect and contribute to the resistance phenotype. Here, we investigate the effect of a single-point mutation (T331I) in VraS histidine kinase, part of the VraSR two-component system in S. aureus. VraSR senses and responds to environmental stress signals by upregulating gene expression for cell wall synthesis. A combination of enzyme kinetics, microbiological, and transcriptomic analysis revealed the mechanistic effect of the mutation on VraS and S. aureus. Michaelis Mentens kinetics show that the VraS mutation caused an increase in the autophosphorylation rate of VraS and enhanced its catalytic efficiency. The introduction of the mutation through recombineering coupled with CRISPR-Cas9 counterselection to the Newman strain wild-type (WT) genome doubled the minimum inhibitory concentration of three cell wall-targeting antibiotics. The mutation caused an enhanced S. aureus growth rate at sub-lethal doses of the antibiotics, confirming the causative effect of mutation on bacterial persistence. Transcriptomic analysis showed a genome-wide alteration in gene expression levels and protein-protein interaction network of the mutant compared to the WT strain after exposure to vancomycin. The results suggest that vraS mutation causes several mechanistic changes at the protein and cellular levels that favor bacterial survival under antibiotic stress and cause the mutation-harboring strains to become the dominant population during infection. ImportanceRising antimicrobial resistance (AMR) is a global health problem. Mutations in the two- component system have been linked to drug- resistance in Staphylococcus aureus, yet the exact mechanism through which these mutations work is understudied. We investigated the T331I mutation in the vraS gene linked to sensing and responding to cell wall stress. The mutation caused changes at the protein level by increasing the catalytic efficiency of VraS kinase activity. Introducing the mutation to the genome of an S. aureus strain resulted in changes in the phenotypic antibiotic susceptibility, growth kinetics, and genome-wide transcriptomic alterations. By a combination of enzyme kinetics, microbiological, and transcriptomic approaches, we highlight how small genetic changes can significantly impact bacterial physiology and survival under antibiotic stress. Understanding the mechanistic basis of antibiotic resistance is crucial to guide the development of novel therapeutic agents to combat AMR.

microbiology↗

bioRxiv2024

The RAB27A effector SYTL5 regulates mitophagy and mitochondrial metabolism

SYTL5 is a member of the Synaptotagmin-Like (SYTL) protein family that differs from the Synaptotagmin family by having a unique N-terminal Synaptotagmin homology domain that directly interacts with the small GTPase RAB27A. Several SYTL protein family members have been implicated in plasma membrane transport and exocytosis, but the specific function of SYTL5 remains unknown. We here show that SYTL5 is a RAB27A effector and that both proteins localise to mitochondria and vesicles containing mitochondrial material. Mitochondrial recruitment of SYTL5 depends on its interaction with functional RAB27A. We demonstrate that SYTL5-RAB27A positive vesicles containing mitochondrial material, autophagy proteins and LAMP1 form during hypoxia and that depletion of SYTL5 and RAB27A reduces mitophagy under hypoxia mimicking conditions, indicating a role for these proteins in mitophagy. Indeed, we find that SYTL5 interacts with proteins involved in vesicle-mediated transport and cellular response to stress and that its depletion compromises mitochondrial respiration and increases glucose uptake. Intriguingly, SYTL5 expression is significantly reduced in tumours of the adrenal gland, and correlates positively with survival for patients with adrenocortical carcinoma.

cell biology↗

bioRxiv2024

Evaluation of Immunopharmacological Efficacy of Live Leishmania donovani Overexpressing Ld_ζ1domain for Protection Against Experimental Human Visceral Leishmaniasis

ObjectiveTo evaluate the efficacy and immunogenicity of the zeta domain over-expressing Leishmania donovani (Ld_{zeta}1domain) as a vaccination candidate against visceral leishmaniasis (VL). MethodsIn this study, Leishmania overexpressor Ld_{zeta}1domain (OE) were transformed by electroporation using a GFP-tagged Ld_{zeta}1domain recombinant plasmid. The resulting overexpressing cells were analysed in vitro to assess their growth dynamics and infectivity. We also investigated the immune-protective potential of these overexpressor in a mouse model challenged with Leishmania donovani. The immune response, including Th1 and Th2 pathways, was thoroughly characterized using RT-PCR and ELISA assays. In addition, the study conducted a thorough evaluation of the mouses spleen and liver parasites, as well as quantitative evaluation of tissue pathological changes. ResultsLd_{zeta}1 domain (OE) parasites exhibited significantly lower viability and replication rates than WT parasites, and in vivo studies showed that mice immunized with the Ld_{zeta}1(OE) domain had lower parasite numbers than mice infected with LdWT. Spleen and liver showed significant histological changes suggestive of protection. Parasite burden in the spleen and liver of vaccinated mice were significantly reduced. The immune response showed increased IFN-{gamma} levels and lower IL-10 production, resulting in a greater IFN-{gamma}/IL-10 ratio, indicating parasite elimination. The vaccination also caused a significant IgG humoral response and increased nitric oxide production in immunized mice. ConclusionOur findings demonstrated that overexpressing the zeta toxin resulted in controlled parasite attenuation, lowering pathogenicity while retaining immunogenic features. Our work established the zeta over-expressors protective efficacy, immunogenicity, and proliferation in response to a Leishmania challenge in vitro and in vivo. This preliminary prototype study suggested that Ld_{zeta}1domain (OE) parasites may be suitable for developing an attenuated vaccine against leishmaniasis. Graphical representation O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=88 SRC="FIGDIR/small/630021v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@32dfc0org.highwire.dtl.DTLVardef@1ffe89dorg.highwire.dtl.DTLVardef@d9feorg.highwire.dtl.DTLVardef@1e7bae7_HPS_FORMAT_FIGEXP M_FIG C_FIG A schematic representation on the protective effectiveness, immunogenicity, and proliferation of the zeta over-expressor in response to Leishmania challenge in vitro and in vivo model.

pathology↗

bioRxiv2024

Chemical degradation identified Human p38-MAPK as a host target to combat parasitic infections

The interplay between host and parasite determines parasite burden and disease outcome. Parasite exploits host signaling pathways like p38-MAPK for its survival and pathogenesis. NR-7h, a proteolysis-targeting chimera (PROTAC) targeting human p38-MAPK was used to assess p38-MAPKs role in Leishmania donovani and Plasmodium falciparum infection in their respective hosts. NR-7h degraded host p38-MAPK in a time- and dose-dependent manner. Degradation of host p38-MAPK by NR-7h reduced parasite load in host cells dose-dependently, implicating the role of p38-MAPK in parasite survival. The modulation of cytokine profiling and oxidative burst upon NR-7h mediated degradation of host p38-MAPK was further correlated with parasite death. The synergistic effect of host p38-MAPK degradation by NR-7h with Amphotericin B enhances the efficacy of parasite-directed therapy. This study underscores the importance of host p38-MAPK for L. donovani and P. falciparum progression and highlights NR-7hs potential in antiparasitic therapy by targeting this pathway.

microbiology↗

bioRxiv2024

A comprehensive spatiotemporal map of dystrophin isoform expression in the developing and adult human brain

Mutations in the dystrophin gene (DMD) cause the severe muscle-wasting disease Duchenne Muscular Dystrophy (DMD). Additionally, there is a high incidence of intellectual disability and neurobehavioural comorbidities in individuals with DMD. Similar behavioural abnormalities are found in mdx dystrophic mouse models. Unlike muscle, several dystrophin isoforms are expressed in the human brain, but a detailed map of regional and cellular localisation of dystrophin isoforms is missing. This is crucial in understanding the neuropathology of DMD individuals, and for evaluating the translatability of pre-clinical findings in DMD mouse models receiving genetic therapy interventions. Here, we provide a comprehensive dystrophin expression profile in human brains from early development to adulthood. We reveal expression of dp427p2, dp427c, dp427m and dp40 isoforms in embryonic brains, not previously reported. Dp427p2 and dp140 were greatly downregulated in adult brains, although the latter continued to be expressed across several regions. Importantly, we demonstrate for the first-time expression of DMD transcripts in human motor neurons and co-expression of different dystrophin isoforms within single neurons in both developing and adult brains. Finally, we show localisation of DMD transcripts with GAD1+ GABAergic-associated transcripts in neurons including cerebellar Purkinje cells and interneurons, as well as in the majority of neocortical and hippocampal SLC17A7+ glutamatergic neurones, suggesting a role for dystrophin in signalling at the neuronal inhibitory and excitatory synapses. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=178 HEIGHT=200 SRC="FIGDIR/small/629620v1_ufig1.gif" ALT="Figure 1"> View larger version (70K): org.highwire.dtl.DTLVardef@1dda96corg.highwire.dtl.DTLVardef@19e133corg.highwire.dtl.DTLVardef@100e8c5org.highwire.dtl.DTLVardef@b1ef77_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2024

Cell-type specific autophagy in human leukocytes

Autophagy is a naturally conserved mechanism crucial for degrading and recycling damaged organelles and proteins to support cell survival. This process slows biological ageing and age-related disease in preclinical models. However, there has been little translation of autophagy to the clinic, and we have identified a lack of measurement tools for physiological human autophagy as a barrier. To address this, we have previously developed a direct measurement tool for autophagy in pooled human peripheral blood mononuclear cells (PBMCs) in the context of whole blood. In order to better understand how autophagy behaves and changes in humans, we measured human autophagic flux using flow cytometry in 19 cell sub-populations in whole blood to retain physiological flux. Autophagic flux was different between different cell types, being highest in B lymphocytes and lowest in T lymphocytes and monocytes. Autophagic flux also varied with sex, being higher in monocytes in females compared with males. In keeping with previous observations in humans, autophagy also increased with ageing at sub-population levels. Importantly, we found that only monocytes - specifically, non-classical monocytes - displayed increased autophagic flux following amino acid withdrawal, underscoring the importance of population selection for measurement of autophagic flux during nutrient restriction studies in humans. Collectively, these data show PBMC population level analysis improves sensitivity of human autophagic flux measurement.

cell biology↗

bioRxiv2024

Fluorescence based live cell imaging identifies exon 14 skipped hepatocyte growth factor receptor (MET) degraders

Despite ongoing efforts to employ structure-based methods to discover targeted protein degraders (TPD), the prevailing strategy continues to be the synthesis of a focused set of heterobifunctional compounds and screen them for target protein degradation. Here we used a fluorescence based live cell imaging screen to identify degraders that target exon 14 skipped hepatocyte growth factor receptor (MET). MET is a known oncogenic driver. MET exon 14 skipping mutations (METex14{Delta}) are found in lung cancers and result in the loss of a degron that is required for E3-ligase recognition and subsequent ubiquitination, prolonging the half-life and oncogenicity of MET. Since proteolysis targeting chimeras (PROTACs) are heterobifunctional molecules that promote target degradation by the proteosome, we sought to restore degradation of MET lost with METex14{Delta} using a MET-targeting PROTAC. We generated a library of sixty PROTACs of which 37 used the MET inhibitor capmatinib as the protein of interest targeting ligand. We screened this PROTAC library for targeted degradation of METex14{Delta}-GFP using live cell imaging. We benchmarked out MET-targeting PROTACs to that of a previously reported MET-targeting PROTAC, SJF8240. Curve fitting live cell imaging data affords determination of time required to degrade 50% of the target protein (DT50), which was used in determining structure activity relationships. A promising candidate, 48-284, identified from the screen, exhibited classic PROTAC characteristics, was > 15-fold more potent than SJF8240, had fewer off targets compared to SJF8240, and degraded MET in multiple cell lines.

cancer biology↗

bioRxiv2024

Id1 Promotes Clonal Hematopoiesis in Mice with Tet2 Loss of Function

Hematopoietic malignancies emerge through the gradual acquisition of genetic mutations within hematopoietic stem and progenitor cells (HSPCs). Mutations that occur early in disease progression impart a selective growth advantage to HSPCs, which allows them to expand and contribute to a substantial percentage of mature blood cells. This increased expansion is termed clonal hematopoiesis (CH) and is a preleukemic phase associated with an increased risk of developing leukemia. Inhibitor of DNA binding 1 (ID1) protein is a transcriptional regulator of proliferation/differentiation of neuronal, muscle, hematopoietic and other cells, and is frequently overexpressed in cancer. Id1 is expressed at low levels in normal HSCs and is induced by growth factors and other mediators of inflammatory stress and promotes HSPC proliferation in vitro and in vivo. Since chronic inflammation is associated with the progression of hematopoietic malignancies, reducing Id1 expression during CH may be therapeutic. Mutations in TET2 are frequently observed in patients with CH, and Tet2-/-mice develop CH that progress to hematopoietic malignancies. Id1 is upregulated in murine Tet2-/- HSPCs and in AML, CMML and MDS patient samples with TET2 mutations. Genetic ablation of Id1 in Tet2-/- HSPCs reduces HSPC expansion/self-renewal/CH, extramedullary hematopoiesis, myeloid skewing, genetic instability and delays the onset of disease. Mechanistically, p16 expression, senescence and apoptosis were increased and proliferation decreased in Tet2-/-; Id1-/- HSPCs. Thus, ID1 may represent a potential therapeutic target to reduce CH, hematopoietic hyperplasia, and delay the onset of disease. One Sentence SummaryGenetic ablation of Id1 in Tet2-/- mice rescues clonal hematopoiesis by increasing CDKI expression, apoptosis, senescence, and differentiation, and reducing cell growth.

cancer biology↗

bioRxiv2024

Prospective evaluation of structure-based simulations reveal their ability to predict the impact of kinase mutations on inhibitor binding

Small molecule kinase inhibitors are critical in the modern treatment of cancers, evidenced by the existence of over 80 FDA-approved small-molecule kinase inhibitors. Unfortunately, intrinsic or acquired resistance, often causing therapy discontinuation, is frequently caused by mutations in the kinase therapeutic target. The advent of clinical tumor sequencing has opened additional opportunities for precision oncology to improve patient outcomes by pairing optimal therapies with tumor mutation profiles. However, modern precision oncology efforts are hindered by lack of sufficient biochemical or clinical evidence to classify each mutation as resistant or sensitive to existing inhibitors. Structure-based methods show promising accuracy in retrospective benchmarks at predicting whether a kinase mutation will perturb inhibitor binding, but comparisons are made by pooling disparate experimental measurements across different conditions. We present the first prospective benchmark of structure-based approaches on a blinded dataset of in-cell kinase inhibitor affinities to Abl kinase mutants using a NanoBRET reporter assay. We compare NanoBRET results to structure-based methods and their ability to estimate the impact of mutations on inhibitor binding (measured as {Delta}{Delta}G). Comparing physics-based simulations, Rosetta, and previous machine learning models, we find that structure-based methods accurately classify kinase mutations as inhibitor-resistant or inhibitor-sensitizing, and each approach has a similar degree of accuracy. We show that physics-based simulations are best suited to estimate {Delta}{Delta}G of mutations that are distal to the kinase active site. To probe modes of failure, we retrospectively investigate two clinically significant mutations poorly predicted by our methods, T315A and L298F, and find that starting configurations and protonation states significantly alter the accuracy of our predictions. Our experimental and computational measurements provide a benchmark for estimating the impact of mutations on inhibitor binding affinity for future methods and structure-based models. These structure-based methods have potential utility in identifying optimal therapies for tumor-specific mutations, predicting resistance mutations in the absence of clinical data, and identifying potential sensitizing mutations to established inhibitors.

biophysics↗

bioRxiv2024

Combining NeuroPainting with transcriptomics reveals cell-type-specific morphological and molecular signatures of the 22q11.2 deletion

Neuropsychiatric conditions pose substantial challenges for therapeutic development due to their complex and poorly understood underlying mechanisms. High-throughput, unbiased phenotypic assays present a promising path for advancing therapeutic discovery, especially within disease-relevant neural tissues. Here, we introduce NeuroPainting, a novel adaptation of the Cell Painting assay, optimized for high-dimensional morphological phenotyping of neural cell types, including neurons, neuronal progenitor cells, and astrocytes derived from human stem cells. Using NeuroPainting, we quantified cell structure and organelle behavior across various brain cell types, creating a public dataset of over 4,000 cellular traits. This extensive dataset not only sets a new benchmark for phenotypic screening in neuropsychiatric research but also serves as a gold standard for the research community, enabling comparisons and validation of results. We then applied NeuroPainting to identify morphological signatures associated with the 22q11.2 deletion, a major genetic risk factor for schizophrenia. We observed profound cell-type-specific effects of the 22q11.2 deletion, with significant alterations in mitochondrial structure, endoplasmic reticulum organization, and cytoskeletal dynamics, particularly in astrocytes. Transcriptomic analysis revealed reduced expression of cell adhesion genes in 22q11.2 deletion astrocytes, consistent with recent post-mortem findings. Integrating the RNA sequencing data and morphological profiles uncovered a novel biological link between altered expression of specific cell adhesion molecules and observed changes in mitochondrial morphology in 22q11.2 deletion astrocytes. These findings underscore the power of combined phenomic and transcriptomic analyses to reveal mechanistic insights associated with human genetic variants of neuropsychiatric conditions.

neuroscience↗

bioRxiv2024

Lipid composition differentiates ferroptosis sensitivity between in vitro and in vivo systems

Ferroptosis is a regulated non-apoptotic cell death process characterized by iron-dependent lipid peroxidation. This process has recently emerged as a promising approach for cancer therapy. Peroxidation of polyunsaturated fatty acid-containing phospholipids (PUFA-PLs) is necessary for the execution of ferroptosis. Ferroptosis is normally suppressed by glutathione peroxidase 4 (GPX4), which reduces lipid hydroperoxides to lipid alcohols. Some evidence indicates that GPX4 may be a useful target for drug development, yet factors that govern GPX4 inhibitor sensitivity in vivo are poorly understood. We find that pharmacological and genetic loss of GPX4 function was sufficient to induce ferroptosis in multiple adherent ("2D") cancer cell cultures. However, reducing GPX4 protein levels did not affect tumor xenograft growth when these cells were implanted in mice. Furthermore, sensitivity to GPX4 inhibition was markedly reduced when cells were cultured as spheroids ("3D"). Mechanistically, growth in 3D versus 2D conditions reduced the abundance of PUFA-PLs. 3D culture conditions upregulated the monounsaturated fatty acid (MUFA) biosynthetic gene stearoyl-CoA desaturase (SCD). SCD-derived MUFAs appear to protect against ferroptosis in 3D conditions by displacing PUFAs from phospholipids. Various structurally related long chain MUFAs can inhibit ferroptosis through this PUFA-displacement mechanism. These findings suggest that growth-condition-dependent lipidome remodeling is an important mechanism governing GPX4 inhibitor effects. This resistance mechanism may specifically limit GPX4 inhibitor effectiveness in vivo.

cancer biology↗