This file contains details of the BE sgRNA library

This file contain information on the Cell lines and organoids used in this study

KEAP1 is the key regulator of the NRF2-mediated cytoprotective response and a target for pathologies involving oxidative stress. Compounds that covalently modify KEAP1 to activate NRF2 are clinically validated; however, their chemical reactivity may drive increased off–target activity. A more selective approach involves inhibition of the KEAP1-NRF2 protein–protein interaction as exemplified by our previous bis-aryl lead KI-696 (4). We now describe the lead optimization of the bis-aryl series that focused on increasing the population of the bioactive conformation of the free ligand to drive potency while optimizing overall physicochemical properties. This resulted in the discovery of GSK3227634 (5), an ultrahigh-affinity noncovalent inhibitor of KEAP1-NRF2 (surface plasmon resonance pK d = 10.9) and the first NRF2 activator to demonstrate target engagement and efficacy in preclinical models of oxidative stress via direct delivery to the lung. Compound 5 therefore represents a novel potential agent to treat lung diseases involving oxidative stress, such as chronic obstructive pulmonary disease.

Human plasma proteomics is critical for biomedical research but challenged by an extraordinary dynamic range. We evaluated six mass spectrometry workflows, including bead-based enrichment (Proteograph XT, P2, Mag-Net), depletion of the 14 most abundant proteins, and neat plasma analysis. Enrichment methods achieved superior proteome coverage, identifying up to 4600 proteins, but exhibited acute susceptibility to preanalytical bias from cellular contaminants and vesicles in plasma. Increased centrifugation stringency before proteomics analysis drastically decreased protein identifications. Crucially, the abundance of the vast majority of secreted proteins was not significantly affected by centrifugation for all tested workflows, suggesting that their quantification is little influenced by contaminating cells or vesicles, whereas depleted proteins are enriched for cellular components like cytoskeleton organization and platelet activation. Our results underscore that nanoparticle-based methods allow robust and deep measurement of the secreted plasma proteome, providing additional insights by reporting the contributions of proteins of cellular origin when applying the common practice of 2000 × g centrifugation prior to analysis.

Supplementary Figure 1. Generating base editor-expressing MSI cell lines for screening. Supplementary Figure 2. Quality control for base-editor screens in MSI cell lines. Supplementary Figure 3. Discovery and characterization of novel inhibitors of WRN helicase. Supplementary Figure 4. Correlation of WRN inhibitor sensitivity with genetic and molecular markers in MSI models. Supplementary Figure 5. Comparative analysis of DNA damage in MSI and MSS cells treated with WRN inhibitors and genetic inactivation. Supplementary Figure 6: Effects of GSK_WRN4 on MSI and MSS colorectal cancer xenografts.

This table lists the results from Base editing screen

Kinobead assay (affinity enrichment chemoproteomics)

Supplementary Materials and Methods

Data-independent acquisition (DIA) on ion mobility mass spectrometers enables deep proteome coverage and high data completeness in large-scale proteomics studies. For advanced acquisition schemes such as parallel accumulation serial fragmentation-based DIA (diaPASEF) stability of ion mobility (1/K0) over time is crucial for consistent data quality. We found that minor changes in environmental air pressure systematically affect the vacuum pressure in the TIMS analyzer, causing ion mobility shifts. By comparing experimental ion mobilities with historical weather data, we attributed observed drifts to fluctuations in the ground air pressure. Moderate air pressure changes of e.g. fifteen mbar induce ion mobility shifts of 0.025 Vs/cm2. These drifts negatively impact peptide quantification across consecutively acquired samples due to drift-dependent abundance changes and increased missing values for ions located at the boundaries of diaPASEF isolation windows, which cannot be corrected by postprocessing. To address this, we applied an in-batch mobility autocalibration feature on a run-wise basis, leading to full elimination of ion mobility drifts.

Several transcription inhibitors have been developed as cancer therapies. However, they show modest clinical activity, highlighting that our understanding of the cellular response to transcriptional inhibition remains incomplete. Here we report that potent inhibitors of transcription not only impact mRNA output but also markedly impair mRNA transcript localization and nuclear export. We demonstrate that retention of newly transcribed mRNA in nuclear speckles is an adaptive response to chemically distinct transcriptional inhibitors. Retained transcripts are fully processed and accumulate in proportion to the expression level of the genes from which they emanate. The TREX mRNA export complex plays an integral role in directing nascent transcripts to nuclear speckles where they are bound to NXF1, protected from degradation, and poised for rapid export following re-initiation of transcription. Our findings provide new insights into the crosstalk between transcription and mRNA export with important implications for drugs aiming to inhibit transcription for therapeutic gain.

Niraparib matrix (affinity enrichment chemoproteomics)

Niraparib specific modulation of expression levels of proteins involved in cholesterol biosynthesis pathway in cancer cell lines (proteomics)

The first step in bottom-up proteomics is the assignment of measured fragmentation mass spectra to peptide sequences, also known as peptide spectrum matches. In recent years novel algorithms have pushed the assignment to new heights; unfortunately, different algorithms come with different strengths and weaknesses and choosing the appropriate algorithm poses a challenge for the user. Here we introduce PeptideForest, a semisupervised machine learning approach that integrates the assignments of multiple algorithms to train a random forest classifier to alleviate that issue. Additionally, PeptideForest increases the number of peptide-to-spectrum matches that exhibit a q-value lower than 1% by 25.2 ± 1.6% compared to MS-GF+ data on samples containing mixed HEK and Escherichia coli proteomes. However, an increase in quantity does not necessarily reflect an increase in quality and this is why we devised a novel approach to determine the quality of the assigned spectra through TMT quantification of samples with known ground truths. Thereby, we could show that the increase in PSMs below 1% q-value does not come with a decrease in quantification quality and as such PeptideForest offers a possibility to gain deeper insights into bottom-up proteomics. PeptideForest has been integrated into our pipeline framework Ursgal and can therefore be combined with a wide array of algorithms.

Therapeutics promoting the endogenous production of IL-10 have the potential to restore homeostasis in inflammatory disorders such as inflammatory bowel disease (IBD). Here we describe the identification of a series of IL-10 upregulators based on a pyrimidyl-piperidine scaffold through a high throughput phenotypic CD4+ T-cell multiplex assay. In vitro optimization of the initial hit yielded a lead with good potency and an in vitro clearance profile, compound 3-7, which additionally demonstrated efficacy in a murine endotoxin challenge PK-PD mechanistic model. Target deconvolution efforts identified compound 3-7 as a highly selective CDK8/19 inhibitor, and crystallographic studies unveiled its binding mode to the CDK8/Cyclin-C complex, characterized by an unusual water-mediated hydrogen bond to the kinase hinge region.

The efficacy and safety of kinase inhibitor drugs are largely influenced by their selectivity. Available profiling technologies are primarily based on over-expressed or endogenously expressed kinases in cell extracts. We compared kinase capture with the cell penetrant covalent probe XO44 to three derivatives and found that replacing the alkyne handle with a trans-cyclooctene group allowed the development of a more robust kinase capture and enrichment protocol. An intracellular chemoproteomics target profiling and engagement assay was devised by optimizing probe concentration and incubation time and using an isobaric mass tag-based strategy for relative quantification. Comparing intracellular kinase profiles of the marketed drug Dasatinib and the tool compound Dinaciclib with the lysate-based kinobeads assay revealed excellent agreement in rank-order of binding. Dinaciclib showed a systematic shift to higher IC50s suggesting that intracellular co-substrate concentrations, cell penetration of the compound as well as kinase localization and complexes in live cells influence target profiles. Further, we show that sepiapterin reductase SPR and Multidrug Resistance Protein 1 ABCC1 are off-targets of kinase inhibitor scaffolds with potential implications on efficacy and safety.

Selectivity profiling and Niraparib-LSS interaction