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Chemical probes and chemogenomic compounds are valuable tools to link gene to phenotype, explore human biology, and uncover novel targets for precision medicine. The mission of the Target 2035 initiative is to discover chemical tools for all human proteins by the year 2035. Here, we draw a landscape of the current chemical coverage of human biological pathways. Although available chemical tools target only 3% of the human proteome, they already cover 53% of human biological pathways and represent a versatile toolkit to dissect a vast portion of human biology. Pathways targeted by existing drugs may be enriched in unknown but valid drug targets and could be prioritized in future Target 2035 efforts., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Peptides are used for diagnostics, therapeutics, and as antimicrobial agents. Most peptides are produced by chemical synthesis, but recombinant production has recently become an attractive alternative due to the advantages of high titers, less toxic waste and correct folding of tertiary structure. Somatostatin-28 is a peptide hormone that regulates the endocrine system, cell proliferation and inhibits the release of numerous secondary hormones in human body. It is composed of 28 amino acids and has one disulfide bond, which makes it to an optimal model peptide for a whole downstream purification process. We produced the peptide in the periplasm of E. coli using the CASPON™ technology, an affinity fusion technology system that enables high soluble expression of recombinant proteins and cleaves the fusion tag with a circularly permuted human caspase-2. Furthermore, purification of the products is straight forward using an established platform process. Two different case studies for downstream purification are presented, starting with either hydrochloric acid or polyethyleneimine as an extraction aid. After release of affinity-tagged somatostatin-28 out of E. coli's periplasm, several purification steps were performed, delivering a pure peptide solution after the final polishing step. The process was monitored by reversed-phase high-performance liquid chromatography as well as mass spectrometry to determine the yield and correct disulfide bond formation. Monitoring of impurities like host cell proteins, DNA and endotoxins after each downstream unit confirmed effective removal for both purification pathways., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Mutations in the Kirsten rat sarcoma viral oncogene homolog (KRAS) protein are highly prevalent in cancer. However, small-molecule concepts that address oncogenic KRAS alleles remain elusive beyond replacing glycine at position 12 with cysteine (G12C), which is clinically drugged through covalent inhibitors. Guided by biophysical and structural studies of ternary complexes, we designed a heterobifunctional small molecule that potently degrades 13 out of 17 of the most prevalent oncogenic KRAS alleles. Compared with inhibition, KRAS degradation results in more profound and sustained pathway modulation across a broad range of KRAS mutant cell lines, killing cancer cells while sparing models without genetic KRAS aberrations. Pharmacological degradation of oncogenic KRAS was tolerated and led to tumor regression in vivo. Together, these findings unveil a new path toward addressing KRAS-driven cancers with small-molecule degraders.

p53 is known as the guardian of the genome and is one of the most important tumor-suppressors. It is inactivated in most tumors, either via tumor protein p53 (TP53) gene mutation or copy number amplification of key negative regulators, e.g., mouse double minute 2 (MDM2). Compounds that bind to the MDM2 protein and disrupt its interaction with p53 restore p53 tumor suppressor activity, thereby promoting cell cycle arrest and apoptosis. Previous clinical experience with MDM2-p53 protein-protein interaction antagonists (MDM2-p53 antagonists) have demonstrated that thrombocytopenia and neutropenia represent on-target dose-limiting toxicities that might restrict their therapeutic utility. Dosing less frequently, while maintaining efficacious exposure, represents an approach to mitigate toxicity and improve the therapeutic window of MDM2-p53 antagonists. However, to achieve this, a molecule possessing excellent potency and ideal pharmacokinetic properties is required. Here, we present the discovery and characterization of brigimadlin (BI 907828), a novel, investigational spiro-oxindole MDM2-p53 antagonist. Brigimadlin exhibited high bioavailability and exposure, as well as dose-linear pharmacokinetics in preclinical models. Brigimadlin treatment restored p53 activity and led to apoptosis induction in preclinical models of TP53 wild-type, MDM2-amplified cancer. Oral administration of brigimadlin in an intermittent dosing schedule induced potent tumor growth inhibition in several TP53 wild-type, MDM2-amplified xenograft models. Exploratory clinical pharmacokinetic studies (NCT03449381) showed high systemic exposure and a long plasma elimination half-life in cancer patients who received oral brigimadlin. These findings support the continued clinical evaluation of brigimadlin in patients with MDM2-amplified cancers, such as dedifferentiated liposarcoma.

Mutations in HER2 occur in 2-4% of non-small cell lung cancer (NSCLC) and confer poor prognosis. ERBB-targeting tyrosine kinase inhibitors, approved for treating other HER2-dependent cancers, are ineffective in HER2 mutant NSCLC due to dose-limiting toxicities or suboptimal potency. We report the discovery of zongertinib (BI 1810631), a covalent HER2 inhibitor. Zongertinib potently and selectively blocks HER2, while sparing EGFR, and inhibits the growth of cells dependent on HER2 oncogenic driver events, including HER2-dependent human cancer cells resistant to trastuzumab deruxtecan. Zongertinib displays potent anti-tumor activity in HER2-dependent human NSCLC xenograft models and enhances the activities of antibody-drug conjugates and KRASG12C inhibitors, without causing obvious toxicities. The preclinical efficacy of zongertinib translates in objective responses in patients with HER2-dependent tumors, including cholangiocarcinoma (SDC4-NRG1 fusion) and breast cancer (V777L HER2 mutation) thus supporting the ongoing clinical development of zongertinib.

In this study, we explore the possibility of inferring characteristics of the tumor immune microenvironment from the blood. Specifically, we investigate two datasets of patients with head and neck squamous cell carcinoma with matched single-cell RNA sequencing (scRNA-seq) from peripheral blood mononuclear cells (PBMCs) and tumor tissues. Our analysis shows that the immune cell fractions and gene expression profiles of various immune cells within the tumor microenvironment can be inferred from the matched PBMC scRNA-seq data. We find that the established exhausted T-cell signature can be predicted from the blood and serve as a valuable prognostic blood biomarker of immunotherapy response. Additionally, our study reveals that the inferred ratio between tumor memory B- and regulatory T-cell fractions is predictive of immunotherapy response and is superior to the well-established cytolytic and exhausted T-cell signatures. These results highlight the promising potential of PBMC scRNA-seq in cancer immunotherapy and warrant, and will hopefully facilitate, further investigations on a larger scale. The code for predicting tumor immune microenvironment from PBMC scRNA-seq, TIMEP, is provided, offering other researchers the opportunity to investigate its prospective applications in various other indications., Significance: Our work offers a new and promising paradigm in liquid biopsies to unlock the power of blood single-cell transcriptomics in cancer immunotherapy., (©2024 The Authors; Published by the American Association for Cancer Research.)

Combination approaches are needed to strengthen and extend the clinical response to KRAS G12C inhibitors (KRAS G12C i). Here, we assessed the antitumor responses of KRAS G12C mutant lung and colorectal cancer models to combination treatment with a SOS1 inhibitor (SOS1i), BI-3406, plus the KRAS G12C inhibitor, adagrasib. We found that responses to BI-3406 plus adagrasib were stronger than to adagrasib alone, comparable to adagrasib with SHP2 (SHP2i) or EGFR inhibitors and correlated with stronger suppression of RAS-MAPK signaling. BI-3406 plus adagrasib treatment also delayed the emergence of acquired resistance and elicited antitumor responses from adagrasib-resistant models. Resistance to KRAS G12C i seemed to be driven by upregulation of MRAS activity, which both SOS1i and SHP2i were found to potently inhibit. Knockdown of SHOC2, a MRAS complex partner, partially restored response to KRAS G12C i treatment. These results suggest KRAS G12C plus SOS1i to be a promising strategy for treating both KRAS G12C i naive and relapsed KRAS G12C -mutant tumors., (© 2024. The Author(s).)

Although γδ T cells are known to participate in immune dysregulation in solid tumors, their relevance to human microsatellite-stable (MSS) colorectal cancer (CRC) is still undefined. Here, using integrated gene expression analysis and T cell receptor sequencing, we characterized γδ T cells in MSS CRC, with a focus on Vδ1 + T cells. We identified Vδ1 + T cells with shared motifs in the third complementarity-determining region of the δ-chain, reflective of antigen recognition. Changes in gene and protein expression levels suggested a dysfunctional effector state of Vδ1 + T cells in MSS CRC, distinct from Vδ1 + T cells in microsatellite-instable (MSI). Interaction analysis highlighted an immunosuppressive role of fibroblasts in the dysregulation of Vδ1 + T cells in MSS CRC via the TIGIT-NECTIN2 axis. Blocking this pathway with a TIGIT antibody partially restored cytotoxicity of the dysfunctional Vδ1 phenotype. These results define an operative pathway in γδ T cells in MSS CRC., (© 2024. The Author(s).)

Fragment-based drug design is heavily dependent on the optimization of initial low-affinity binders. Herein we introduce an approach that uses selective labeling of methyl groups in leucine and isoleucine side chains to directly probe methyl-π contacts, one of the most prominent forms of interaction between proteins and small molecules. Using simple NMR chemical shift perturbation experiments with selected BRD4-BD1 binders, we find good agreement with a commonly used model of the ring-current effect as well as the overall interaction geometries extracted from the Protein Data Bank. By combining both interaction geometries and chemical shift calculations as fit quality criteria, we can position dummy aromatic rings into an AlphaFold model of the protein of interest. The proposed method can therefore provide medicinal chemists with important information about binding geometries of small molecules in fast and iterative matter, even in the absence of high-resolution experimental structures.

During neural tube (NT) development, the notochord induces an organizer, the floorplate, which secretes Sonic Hedgehog (SHH) to pattern neural progenitors. Conversely, NT organoids (NTOs) from embryonic stem cells (ESCs) spontaneously form floorplates without the notochord, demonstrating that stem cells can self-organize without embryonic inducers. Here, we investigated floorplate self-organization in clonal mouse NTOs. Expression of the floorplate marker FOXA2 was initially spatially scattered before resolving into multiple clusters, which underwent competition and sorting, resulting in a stable "winning" floorplate. We identified that BMP signaling governed long-range cluster competition. FOXA2 + clusters expressed BMP4, suppressing FOXA2 in receiving cells while simultaneously expressing the BMP-inhibitor NOGGIN, promoting cluster persistence. Noggin mutation perturbed floorplate formation in NTOs and in the NT in vivo at mid/hindbrain regions, demonstrating how the floorplate can form autonomously without the notochord. Identifying the pathways governing organizer self-organization is critical for harnessing the developmental plasticity of stem cells in tissue engineering., Competing Interests: Declaration of interests A.H. and R.A.N. are full-time employees of Boehringer Ingelheim RCV., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Identifying promising chemical starting points for small molecule inhibitors of active, GTP-loaded KRAS "on" remains of great importance to clinical oncology and represents a significant challenge in medicinal chemistry. Here, we describe broadly applicable learnings from a KRAS hit finding campaign: While we initially identified KRAS inhibitors in a biochemical high-throughput screen, we later discovered that compound potencies were all but assay artifacts linked to metal salts interfering with KRAS AlphaScreen assay technology. The source of the apparent biochemical KRAS inhibition was ultimately traced to unavoidable palladium impurities from chemical synthesis. This discovery led to the development of a Metal Ion Interference Set (MIIS) for up-front assay development and testing. Profiling of the MIIS across 74 assays revealed a reduced interference liability of label-free biophysical assays and, as a result, provided general estimates for luminescence- and fluorescence-based assay susceptibility to metal salt interference.

The WWE domain is a relatively under-researched domain found in twelve human proteins and characterized by a conserved tryptophan-tryptophan-glutamate (WWE) sequence motif. Six of these WWE domain-containing proteins also contain domains with E3 ubiquitin ligase activity. The general recognition of poly-ADP-ribosylated substrates by WWE domains suggests a potential avenue for development of Proteolysis-Targeting Chimeras (PROTACs). Here, we present novel crystal structures of the HUWE1, TRIP12, and DTX1 WWE domains in complex with PAR building blocks and their analogs, thus enabling a comprehensive analysis of the PAR binding site structural diversity. Furthermore, we introduce a versatile toolbox of biophysical and biochemical assays for the discovery and characterization of novel WWE domain binders, including fluorescence polarization-based PAR binding and displacement assays, 15 N-NMR-based binding affinity assays and 19 F-NMR-based competition assays. Through these assays, we have characterized the binding of monomeric iso-ADP-ribose (iso-ADPr) and its nucleotide analogs with the aforementioned WWE proteins. Finally, we have utilized the assay toolbox to screen a small molecule fragment library leading to the successful discovery of novel ligands targeting the HUWE1 WWE domain., (© 2024. The Author(s).)

Targeted protein degradation has recently emerged as a novel option in drug discovery. Natural protein half-life is expected to affect the efficacy of degrading agents, but to what extent it influences target protein degradation has not been systematically explored. Using simple mathematical modeling of protein degradation, we find that the natural half-life of a target protein has a dramatic effect on the level of protein degradation induced by a degrader agent which can pose significant hurdles to screening efforts. Moreover, we show that upon screening for degraders of short-lived proteins, agents that stall protein synthesis, such as GSPT1 degraders and generally cytotoxic compounds, deceptively appear as protein-degrading agents. This is exemplified by the disappearance of short-lived proteins such as MCL1 and MDM2 upon GSPT1 degradation and upon treatment with cytotoxic agents such as doxorubicin. These findings have implications for target selection as well as for the type of control experiments required to conclude that a novel agent works as a bona fide targeted protein degrader.

KRAS inhibitors have demonstrated exciting preclinical and clinical responses, although resistance occurs rapidly. Here, we investigate the effects of KRAS-targeting therapies on the tumor microenvironment using a library of KrasG12D, p53-mutant, murine pancreatic ductal adenocarcinoma-derived cell lines (KPCY) to leverage immune-oncology combination strategies for long-term tumor efficacy. Our findings show that SOS1 and MEK inhibitors (SOS1i+MEKi) suppressed tumor growth in syngeneic models and increased intratumoral CD8+ T cells without durable responses. Single-cell RNA sequencing revealed an increase in inflammatory cancer-associated fibroblasts (iCAF), M2 macrophages, and a decreased dendritic cell (DC) quality that ultimately resulted in a highly immunosuppressive microenvironment driven by IL6+ iCAFs. Agonist CD40 treatment was effective to revert macrophage polarization and overcome the lack of mature antigen-presenting DCs after SOS1i+MEKi therapy. Treatment increased the overall survival of KPCY tumor-bearing mice. The addition of checkpoint blockade to SOS1i+MEKi combination resulted in tumor-free mice with established immune memory. Our data suggest that KRAS inhibition affects myeloid cell maturation and highlights the need for combining KRAS cancer-targeted therapy with myeloid activation to enhance and prolong antitumor effects., Significance: Combination of SOS1 and MEK inhibitors increase T cell infiltration while blunting pro-immune myeloid cell maturation and highlights the need for combining KRAS cancer-targeted therapy with myeloid activation to enhance and prolong anti-tumor effects., (© 2024 The Authors; Published by the American Association for Cancer Research.)

We describe the single-step formation of complex tetracyclic fused scaffolds enabled by (3 + 2) cycloaddition of azomethine ylides. Various indoles, N-protecting groups, and amino acids are well tolerated. The products are obtained in a catalyst-free manner with moderate to excellent yield and high diastereoselectivity. Representing a new scaffold that is not yet found in nature, the construction of pyrrolidine-fused cyclohepta-, azepino-, or oxepinoindoles could be found valuable in the synthesis of new pseudo-natural products.

Background: Recombinant peptide production in Escherichia coli provides a sustainable alternative to environmentally harmful and size-limited chemical synthesis. However, in-vivo production of disulfide-bonded peptides at high yields remains challenging, due to degradation by host proteases/peptidases and the necessity of translocation into the periplasmic space for disulfide bond formation., Results: In this study, we established an expression system for efficient and soluble production of disulfide-bonded peptides in the periplasm of E. coli. We chose model peptides with varying complexity (size, structure, number of disulfide bonds), namely parathyroid hormone 1-84, somatostatin 1-28, plectasin, and bovine pancreatic trypsin inhibitor (aprotinin). All peptides were expressed without and with the N-terminal, low molecular weight CASPON™ tag (4.1 kDa), with the expression cassette being integrated into the host genome. During BioLector™ cultivations at microliter scale, we found that most of our model peptides can only be sufficiently expressed in combination with the CASPON™ tag, otherwise expression was only weak or undetectable on SDS-PAGE. Undesired degradation by host proteases/peptidases was evident even with the CASPON™ tag. Therefore, we investigated whether degradation happened before or after translocation by expressing the peptides in combination with either a co- or post-translational signal sequence. Our results suggest that degradation predominantly happened after the translocation, as degradation fragments appeared to be identical independent of the signal sequence, and expression was not enhanced with the co-translational signal sequence. Lastly, we expressed all CASPON™-tagged peptides in two industry-relevant host strains during C-limited fed-batch cultivations in bioreactors. We found that the process performance was highly dependent on the peptide-host-combination. The titers that were reached varied between 0.6-2.6 g L -1 , and exceeded previously published data in E. coli. Moreover, all peptides were shown by mass spectrometry to be expressed to completion, including full formation of disulfide bonds., Conclusion: In this work, we demonstrated the potential of the CASPON™ technology as a highly efficient platform for the production of soluble peptides in the periplasm of E. coli. The titers we show here are unprecedented whenever parathyroid hormone, somatostatin, plectasin or bovine pancreatic trypsin inhibitor were produced in E. coli, thus making our proposed upstream platform favorable over previously published approaches and chemical synthesis., (© 2024. The Author(s).)

Transcription factors are generally considered challenging, if not "undruggable", targets but they promise new therapeutic options due to their fundamental involvement in many diseases. In this study, we aim to assess the ligandability of the C-terminal Rel-homology domain of nuclear factor of activated T cells 1 (NFAT1), a TF implicated in T-cell regulation. Using a combination of experimental and computational approaches, we demonstrate that small molecule fragments can indeed bind to this protein domain. The newly identified binder is the first small molecule binder to NFAT1 validated with biophysical methods and an elucidated binding mode by X-ray crystallography. The reported eutomer/distomer pair provides a strong basis for potential exploration of higher potency binders on the path toward degrader or glue modalities., (© 2024 The Authors. Archiv der Pharmazie published by Wiley‐VCH GmbH on behalf of Deutsche Pharmazeutische Gesellschaft.)

Transcription factors (TFs) are important mediators of aberrant transcriptional programs in cancer cells. In this study, we focus on TF activity (TFa) as a biomarker for cell-line-selective anti-proliferative effects, in that high TFa predicts sensitivity to loss of function of a given gene (i.e., genetic dependencies [GDs]). Our linear-regression-based framework identifies 3,047 pan-cancer and 3,952 cancer-type-specific candidate TFa-GD associations from cell line data, which are then cross-examined for impact on survival in patient cohorts. One of the most prominent biomarkers is TEAD1 activity, whose associations with its predicted GDs are validated through experimental evidence as proof of concept. Overall, these TFa-GD associations represent an attractive resource for identifying innovative, biomarker-driven hypotheses for drug discovery programs in oncology., Competing Interests: Declaration of interests All authors, except A.S.B., M.G.R., M.M.R., J.A.R., R.S., and L.B. are full-time employees of Boehringer Ingelheim RCV. V.T. is now a full-time employee of Exscientia GmbH., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Small cell lung cancer (SCLC) is the most aggressive lung cancer entity with an extremely limited therapeutic outcome. Most patients are diagnosed at an extensive stage. However, the molecular mechanisms driving SCLC invasion and metastasis remain largely elusive. We used an autochthonous SCLC mouse model and matched samples from patients with primary and metastatic SCLC to investigate the molecular characteristics of tumor metastasis. We demonstrate that tumor cell invasion and liver metastasis in SCLC are triggered by an Angiopoietin-2 (ANG-2)/Integrin β-1-dependent pathway in tumor cells, mediated by focal adhesion kinase/Src kinase signaling. Strikingly, CRISPR-Cas9 KO of Integrin β-1 or blocking Integrin β-1 signaling by an anti-ANG-2 treatment abrogates liver metastasis formation in vivo. Interestingly, analysis of a unique collection of matched samples from patients with primary and metastatic SCLC confirmed a strong increase of Integrin β-1 in liver metastasis in comparison with the primary tumor. We further show that ANG-2 blockade combined with PD-1-targeted by anti-PD-1 treatment displays synergistic treatment effects in SCLC. Together, our data demonstrate a fundamental role of ANG-2/Integrin β-1 signaling in SCLC cells for tumor cell invasion and liver metastasis and provide a potentially new effective treatment strategy for patients with SCLC.

The Wnt receptor ROR1 has generated increased interest as a cancer therapeutic target. Research on several therapeutic approaches involving this receptor is ongoing; however, ROR1 tissue expression remains understudied. We performed an immunohistochemistry analysis of ROR1 protein expression in a large cohort of multiple tumor and histologic types. We analyzed 12 anonymized multi-tumor tissue microarrays (TMAs), including mesothelioma, esophageal and upper gastrointestinal carcinomas, and uterine endometrioid carcinoma, among other tumor types. Additionally, we studied 5 different sarcoma types of TMAs and 6 patient-derived xenografts (PDX) TMAs developed from 19 different anatomic sites and tumor histologic types. A total of 1142 patient cases from different histologic types and 140 PDXs placed in TMAs were evaluated. Pathologists assessed the percentage of tumor cells in each case that were positive for ROR1 and the intensity of staining. For determining the prevalence of staining for each tumor type, a case was considered positive if >1% of its tumor cells showed ROR1 staining. Our immunohistochemistry assays revealed a heterogeneous ROR1 expression profile. A high prevalence of ROR1 expression was found in mesothelioma (84.6%), liposarcoma (36.1%), gastrointestinal stromal tumors (33.3%), and uterine endometrioid carcinoma (28.9%). Other histologic types such as breast, lung, renal cell, hepatocellular, urothelial carcinoma, and colon carcinomas; glioblastoma; cholangiocarcinoma; and leiomyosarcoma showed less ROR1 overall expression, ranging between 0.9 and 13%. No ROR1 expression was seen in mesenchymal chondrosarcoma, rhabdomyosarcoma, or gastric adenocarcinoma cases. Overall, ROR1 expression was relatively infrequent and low in most tumor types investigated; however, ROR1 expression was infrequent but high in selected tumor types, such as gastroesophageal GIST, suggesting that ROR1 prescreening may be preferable for those indications. Further, mesothelioma exhibited frequent and high levels of ROR1 expression, which represents a previously unrecognized therapeutic opportunity. These findings can contribute to the development of ROR1-targeted therapies.

Biochemical and cell-based assays are essential to discovering and optimizing efficacious and safe drugs, agrochemicals and cosmetics. However, false assay readouts stemming from colloidal aggregation, chemical reactivity, chelation, light signal attenuation and emission, membrane disruption, and other interference mechanisms remain a considerable challenge in screening synthetic compounds and natural products. To address assay interference, a range of powerful experimental approaches are available and in silico methods are now gaining traction. This Review begins with an overview of the scope and limitations of experimental approaches for tackling assay interference. It then focuses on theoretical methods, discusses strategies for their integration with experimental approaches, and provides recommendations for best practices. The Review closes with a summary of the critical facts and an outlook on potential future developments., (© 2024. Springer Nature Limited.)

The 20S U5 small nuclear ribonucleoprotein particle (snRNP) is a 17-subunit RNA-protein complex and a precursor of the U4/U6.U5 tri-snRNP, the major building block of the precatalytic spliceosome. CD2BP2 is a hallmark protein of the 20S U5 snRNP, absent from the mature tri-snRNP. Here we report a high-resolution cryogenic electron microscopy structure of the 20S U5 snRNP, shedding light on the mutually exclusive interfaces utilized during tri-snRNP assembly and the role of the CD2BP2 in facilitating this process., (© 2024. The Author(s).)

Purpose: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) serve as the standard first-line therapy for EGFR-mutated non-small cell lung cancer (NSCLC). Despite the sustained clinical benefits achieved through optimal EGFR-TKI treatments, including the third-generation EGFR-TKI osimertinib, resistance inevitably develops. Currently, there are no targeted therapeutic options available postprogression on osimertinib. Here, we assessed the preclinical efficacy of BI-4732, a novel fourth-generation EGFR-TKI, using patient-derived preclinical models reflecting various clinical scenarios., Experimental Design: The antitumor activity of BI-4732 was evaluated using Ba/F3 cells and patient-derived cell/organoid/xenograft models with diverse EGFR mutations. Intracranial antitumor activity of BI-4732 was evaluated in a brain-metastasis mouse model., Results: We demonstrated the remarkable antitumor efficacy of BI-4732 as a single agent in various patient-derived models with EGFR_C797S-mediated osimertinib resistance. Moreover, BI-4732 exhibited activity comparable to osimertinib in inhibiting EGFR-activating (E19del and L858R) and T790M mutations. In a combination treatment strategy with osimertinib, BI-4732 exhibited a synergistic effect at significantly lower concentrations than those used in monotherapy. Importantly, BI-4732 displayed potent antitumor activity in an intracranial model, with low efflux at the blood-brain barrier., Conclusions: Our findings highlight the potential of BI-4732, a selective EGFR-TKI with high blood-brain barrier penetration, targeting a broad range of EGFR mutations, including C797S, warranting clinical development., (©2024 American Association for Cancer Research.)

In this study, we introduce a convenient Heck vinylation protocol that eliminates the requirement for ethylene gas as a coupling partner. In contrast to traditional methodologies, quaternary ammonium salts can serve as solid olefin precursors under ambient atmosphere conditions. The practicality of this method, distinguished by its convenience and safety in a one-pot reaction, renders it appealing for applications in research and discovery context.

In-vitro to in-vivo correlations (IVIVC), relating in-vitro parameters like IC50 to in-vivo drug exposure in plasma and tumour growth, are widely used in oncology for experimental design and dose decisions. However, they lack a deeper understanding of the underlying mechanisms. Our paper therefore focuses on linking empirical IVIVC relations for small-molecule kinase inhibitors with a semi-mechanistic tumour-growth model. We develop an approach incorporating parameters like the compound's peak-trough ratio (PTR), Hill coefficient of in-vitro dose-response curves, and xenograft-specific properties. This leads to formulas for determining efficacious doses for tumor stasis under linear pharmacokinetics equivalent to traditional empirical IVIVC relations, but enabling more systematic analysis. Our findings reveal that in-vivo xenograft-specific parameters, specifically the growth rate (g) and decay rate (d), along with the average exposure, are generally more significant determinants of tumor stasis and effective dose than the compound's peak-trough ratio. However, as the Hill coefficient increases, the dependency of tumor stasis on the PTR becomes more pronounced, indicating that the compound is more influenced by its maximum or trough values rather than the average exposure. Furthermore, we discuss the translation of our method to predict population dose ranges in clinical studies and propose a resistance mechanism that solely relies on specific in-vivo xenograft parameters instead of IC50 exposure coverage. In summary, our study aims to provide a more mechanistic understanding of IVIVC relations, emphasizing the importance of xenograft-specific parameters and PTR on tumor stasis., (© 2023. The Author(s).)

Skeletal muscle adaptation to external stimuli, such as regeneration following injury and hypertrophy in response to resistance exercise, are blunted with advanced age. The accumulation of senescent cells, along with defects in myogenic progenitor cell (MPC) proliferation, have been strongly linked as contributing factors to age-associated impairment in muscle adaptation. p53 plays an integral role in all these processes, as upregulation of p53 causes apoptosis in senescent cells and prevents mitotic catastrophe in MPCs from old mice. The goal of this study was to determine if a novel pharmaceutical agent (BI01), which functions by upregulating p53 through inhibition of binding to MDM2, the primary p53 regulatory protein, improves muscle regeneration and hypertrophy in old mice. BI01 effectively reduced the number of senescent cells in vitro but had no effect on MPC survival or proliferation at a comparable dose. Following repeated oral gavage with 2 mg/kg of BI01 (OS) or vehicle (OV), old mice (24 months) underwent unilateral BaCl 2 injury in the tibialis anterior (TA) muscle, with PBS injections serving as controls. After 7 days, satellite cell number was higher in the TA of OS compared to OV mice, as was the expression of genes involved in ATP production. By 35 days, old mice treated with BI01 displayed reduced senescent cell burden, enhanced regeneration (higher muscle mass and fiber cross-sectional area) and restoration of muscle function relative to OV mice. To examine the impact of 2 mg/kg BI01 on muscle hypertrophy, the plantaris muscle was subjected to 28 days of mechanical overload (MOV) in OS and OV mice. In response to MOV, OS mice had larger plantaris muscles and muscle fibers than OV mice, particularly type 2b + x fibers, associated with reduced senescent cells. Together our data show that BI01 is an effective senolytic agent that may also augment muscle metabolism to enhance muscle regeneration and hypertrophy in old mice., (© 2023. The Author(s).)

Cell disintegration and protein extraction are crucial steps in downstream process development for biopharmaceuticals produced in E. coli. In this study, we explored the extraction mechanism of polyethyleneimine (PEI) at the cellular level and characterized the floc network that is formed upon PEI addition by Focused Beam Reflectance Measurement and Dispersion Analyzer. PEI disintegrates the cells by detachment of the outer membrane allowing protein to diffuse into the interspace of the flocs. Protein release into the supernatant occurs by diffusion out of the floc network. We could show that the type and concentrations of PEIs with varying molecular weight determines the floc properties and thus the extraction efficiency. We could demonstrate why optimal conditions, using 70 kDa PEI at 0.25 g/g cell dry mass, lead to efficient extraction while at suboptimal conditions extraction is almost negligible. Our findings provide valuable insights into the relationship between floc properties and PEI-driven protein extraction, with potential applications in bioprocessing and biotechnology., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Hepatocellular carcinoma (HCC) and solid cancers with liver metastases are indications with high unmet medical need. Interleukin-12 (IL-12) is a proinflammatory cytokine with substantial anti-tumor properties, but its therapeutic potential has not been realized due to severe toxicity. Here, we show that orthotopic liver tumors in mice can be treated by targeting hepatocytes via systemic delivery of adeno-associated virus (AAV) vectors carrying the murine IL-12 gene. Controlled cytokine production was achieved in vivo by using the tetracycline-inducible K19 riboswitch. AAV-mediated expression of IL-12 led to STAT4 phosphorylation, interferon-γ (IFNγ) production, infiltration of T cells and, ultimately, tumor regression. By detailed analyses of efficacy and tolerability in healthy and tumor-bearing animals, we could define a safe and efficacious vector dose. As a potential clinical candidate, we characterized vectors carrying the human IL-12 (huIL-12) gene. In mice, bioactive human IL-12 was expressed in a vector dose-dependent manner and could be induced by tetracycline, suggesting tissue-specific AAV vectors with riboswitch-controlled expression of highly potent proinflammatory cytokines as an attractive approach for vector-based cancer immunotherapy., Competing Interests: All authors are employees of Boehringer Ingelheim., (Copyright © 2024 Düchs, Kratzer, Vieyra-Garcia, Strobel, Schönberger, Groß, Aljayyoussi, Gupta, Lang, Klein, Morilla, Hopf, Park, Kreuz, Klugmann and Igney.)

Precise information regarding the interaction between proteins and ligands at molecular resolution is crucial for effectively guiding the optimization process from initial hits to lead compounds in early stages of drug development. In this study, we introduce a novel aliphatic side chain isotope-labeling scheme to directly probe interactions between ligands and aliphatic sidechains using NMR techniques. To demonstrate the applicability of this method, we selected a set of Brd4-BD1 binders and analyzed 1 H chemical shift perturbation resulting from CH-π interaction of H β -Val and H γ -Leu as CH donors with corresponding ligand aromatic moieties as π acceptors., (© 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Cross-presentation by type 1 DCs (cDC1) is critical to induce and sustain antitumoral CD8 T cell responses to model antigens, in various tumor settings. However, the impact of cross-presenting cDC1 and the potential of DC-based therapies in tumors carrying varied levels of bona-fide neoantigens (neoAgs) remain unclear. Here we develop a hypermutated model of non-small cell lung cancer in female mice, encoding genuine MHC-I neoepitopes to study neoAgs-specific CD8 T cell responses in spontaneous settings and upon Flt3L + αCD40 (DC-therapy). We find that cDC1 are required to generate broad CD8 responses against a range of diverse neoAgs. DC-therapy promotes immunogenicity of weaker neoAgs and strongly inhibits the growth of high tumor-mutational burden (TMB) tumors. In contrast, low TMB tumors respond poorly to DC-therapy, generating mild CD8 T cell responses that are not sufficient to block progression. scRNA transcriptional analysis, immune profiling and functional assays unveil the changes induced by DC-therapy in lung tissues, which comprise accumulation of cDC1 with increased immunostimulatory properties and less exhausted effector CD8 T cells. We conclude that boosting cDC1 activity is critical to broaden the diversity of anti-tumoral CD8 T cell responses and to leverage neoAgs content for therapeutic advantage., (© 2024. The Author(s).)

Antimicrobial resistance is a global health threat that requires the development of new treatment concepts. These should not only overcome existing resistance but be designed to slow down the emergence of new resistance mechanisms. Targeted protein degradation, whereby a drug redirects cellular proteolytic machinery towards degrading a specific target, is an emerging concept in drug discovery. We are extending this concept by developing proteolysis targeting chimeras active in bacteria (BacPROTACs) that bind to ClpC1, a component of the mycobacterial protein degradation machinery. The anti-Mycobacterium tuberculosis (Mtb) BacPROTACs are derived from cyclomarins which, when dimerized, generate compounds that recruit and degrade ClpC1. The resulting Homo-BacPROTACs reduce levels of endogenous ClpC1 in Mycobacterium smegmatis and display minimum inhibitory concentrations in the low micro- to nanomolar range in mycobacterial strains, including multiple drug-resistant Mtb isolates. The compounds also kill Mtb residing in macrophages. Thus, Homo-BacPROTACs that degrade ClpC1 represent a different strategy for targeting Mtb and overcoming drug resistance., (© 2024. The Author(s).)

In this study, we present the synthesis and incorporation of a metabolic isoleucine precursor compound for selective methylene labeling. The utility of this novel α-ketoacid isotopologue is shown by incorporation into the protein Brd4-BD1, which regulates gene expression by binding to acetylated histones. High quality single quantum 13 C- 1  H-HSQC were obtained, as well as triple quantum HTQC spectra, which are superior in terms of significantly increased 13 C-T 2 times. Additionally, large chemical shift perturbations upon ligand binding were observed. Our study thus proves the great sensitivity of this precursor as a reporter for side-chain dynamic studies and for investigations of CH-π interactions in protein-ligand complexes., (© 2023. The Author(s).)

Herein we report a method for the synthesis of indazoles from readily available 2-aminomethyl-phenylamines via N-N bond-forming oxidative cyclization. Inspired by indazole formation initially observed as a side product by N. Coskun et al. we developed a robust protocol to access indazoles in all three tautomeric forms. The method selectively gives access to various 2-substituted 2 H -indazoles which are frequently used in drug design, and we also demonstrated its applicability to less studied 3 H -indazoles.

Background: Chemotherapy-induced peripheral neuropathy (CIPN) represents a major unmet medical need that currently has no preventive and/or curative treatment. This is, among others, driven by a poor understanding of the contributive role of drug transport across biological barriers to target-site exposure., Methods: Here, we systematically investigated the transport of 11 small-molecule drugs, both, associated and not with CIPN development, at conventional (dorsal root ganglia, sciatic nerve) and non-conventional (brain, spinal cord, skeletal muscle) CIPN sites. We developed a Combinatory Mapping Approach for CIPN, CMA-CIPN, combining in vivo and in vitro elements., Results: Using CMA-CIPN, we determined the unbound tissue-to-plasma concentration ratio (K p,uu ) and the unbound intracellular-to-extracellular concentration ratio (K p,uu,cell ), to quantitatively assess the extent of unbound drug transport across endothelial interfaces and parenchymal cellular barriers of investigated CIPN-sites, respectively, in a rat model. The analysis revealed that unique pharmacokinetic characteristics underly time-dependent accumulation of the CIPN-positive drugs paclitaxel and vincristine at conventional (dorsal root ganglia and sciatic nerve) and non-conventional (skeletal muscle) CIPN sites. Investigated CIPN-positive drugs displayed intracellular accumulation contrary to CIPN-negative drugs nilotinib and methotrexate, which lacked this feature in all investigated tissues., Conclusions: Hence, high unbound drug intracellular and extracellular exposure at target sites, driven by an interplay of drug transport across the endothelial and parenchymal cellular barriers, is a predisposing factor to CIPN development for CIPN-positive drugs. Critical drug-specific features of unbound drug disposition at various CIPN- sites provide invaluable insights into understanding the pharmacological/toxicological effects at the target-sites which will inform new strategies for monitoring and treatment of CIPN., (© 2024. The Author(s).)

Background: Preclinical and early clinical data suggest that the irreversible ErbB family blocker afatinib may be effective in urothelial cancers harbouring ERBB mutations., Methods: This open-label, phase II, single-arm trial (LUX-Bladder 1, NCT02780687) assessed the efficacy and safety of second-line afatinib 40 mg/d in patients with metastatic urothelial carcinoma with ERBB1-3 alterations. The primary endpoint was 6-month progression-free survival rate (PFS6) (cohort A); other endpoints included ORR, PFS, OS, DCR and safety (cohorts A and B). Cohort A was planned to have two stages: stage 2 enrolment was based on observed antitumour activity., Results: Thirty-four patients were enroled into cohort A and eight into cohort B. In cohorts A/B, PFS6 was 11.8%/12.5%, ORR was 5.9%/12.5%, DCR was 50.0%/25.0%, median PFS was 9.8/7.8 weeks and median OS was 30.1/29.6 weeks. Three patients (two ERBB2-amplified [cohort A]; one EGFR-amplified [cohort B]) achieved partial responses. Stage 2 for cohort A did not proceed. All patients experienced adverse events (AEs), most commonly (any/grade 3) diarrhoea (76.2%/9.5%). Two patients (4.8%) discontinued due to AEs and one fatal AE was observed (acute coronary syndrome; not considered treatment-related)., Conclusions: An exploratory biomarker analysis suggested that basal-squamous tumours and ERBB2 amplification were associated with superior response to afatinib., Clinical Trial Registration: NCT02780687., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: Immunotherapy based on checkpoint inhibitors is highly effective in mismatch repair deficient (MMRd) colorectal cancer (CRC). These tumors carry a high number of mutations, which are predicted to translate into a wide array of neoepitopes; however, a systematic classification of the neoantigen repertoire in MMRd CRC is lacking. Mass spectrometry peptidomics has demonstrated the existence of MHC class I associated peptides (MAPs) originating from non-coding DNA regions. Based on these premises we investigated DNA genomic regions responsible for generating MMRd-induced peptides., Methods: We exploited mouse CRC models in which the MMR gene Mlh1 was genetically inactivated. Isogenic cell lines CT26 Mlh1 +/+ and Mlh1 -/- were inoculated in immunocompromised and immunocompetent mice. Whole genome and RNA sequencing data were generated from samples obtained before and after injection in murine hosts. First, peptide databases were built from transcriptomes of isogenic cell lines. We then compiled a database of peptides lost after tumor cells injection in immunocompetent mice, likely due to immune editing. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and matched next-generation sequencing databases were employed to identify the DNA regions from which the immune-targeted MAPs originated. Finally, we adopted in vitro T cell assays to verify whether MAP-specific T cells were part of the in vivo immune response against Mlh1 -/- cells., Results: Whole genome sequencing analyses revealed an unbalanced distribution of immune edited alterations across the genome in Mlh1 -/- cells grown in immunocompetent mice. Specifically, untranslated (UTR) and coding regions exhibited the largest fraction of mutations leading to highly immunogenic peptides. Moreover, the integrated computational and LC-MS/MS analyses revealed that MAPs originate mainly from atypical translational events in both Mlh1 +/+ and Mlh1 -/- tumor cells. In addition, mutated MAPs-derived from UTRs and out-of-frame translation of coding regions-were highly enriched in Mlh1 -/- cells. The MAPs trigger T-cell activation in mice primed with Mlh1 -/- cells., Conclusions: Our results suggest that-in comparison to MMR proficient CRC-MMRd tumors generate a significantly higher number of non-canonical mutated peptides able to elicit T cell responses. These results reveal the importance of evaluating the diversity of neoepitope repertoire in MMRd tumors., (© 2024. The Author(s).)

Background: Escherichia coli is a cost-effective expression system for production of antibody fragments like Fabs. Various yield improvement strategies have been applied, however, Fabs remain challenging to produce. This study aimed to characterize the gene expression response of commonly used E. coli strains BL21(DE3) and HMS174(DE3) to periplasmic Fab expression using RNA sequencing (RNA-seq). Two Fabs, Fabx and FTN2, fused to a post-translational translocation signal sequence, were produced in carbon-limited fed-batch cultivations., Results: Production of Fabx impeded cell growth substantially stronger than FTN2 and yields of both Fabs differed considerably. The most noticeable, common changes in Fab-producing cells suggested by our RNA-seq data concern the cell envelope. The Cpx and Psp stress responses, both connected to inner membrane integrity, were activated, presumably by recombinant protein aggregation and impairment of the Sec translocon. The data additionally suggest changes in lipopolysaccharide synthesis, adjustment of membrane permeability, and peptidoglycan maturation and remodeling. Moreover, all Fab-producing strains showed depletion of Mg 2+ , indicated by activation of the PhoQP two-component signal transduction system during the early stage and sulfur and phosphate starvation during the later stage of the process. Furthermore, our data revealed ribosome stalling, caused by the Fabx amino acid sequence, as a contributor to low Fabx yields. Increased Fabx yields were obtained by a site-specific amino acid exchange replacing the stalling sequence. Contrary to expectations, cell growth was not impacted by presence or removal of the stalling sequence. Considering ribosome rescue is a conserved mechanism, the substantial differences observed in gene expression between BL21(DE3) and HMS174(DE3) in response to ribosome stalling on the recombinant mRNA were surprising., Conclusions: Through characterization of the gene expression response to Fab production under industrially relevant cultivation conditions, we identified potential cell engineering targets. Thereby, we hope to enable rational approaches to improve cell fitness and Fab yields. Furthermore, we highlight ribosome stalling caused by the amino acid sequence of the recombinant protein as a possible challenge during recombinant protein production., (© 2024. The Author(s).)

The availability of high-resolution 3D structural information is crucial for investigating guest-host systems across a wide range of fields. In the context of drug discovery, the information is routinely used to establish and validate structure-activity relationships, grow initial hits from screening campaigns, and to guide molecular docking. For the generation of protein-ligand complex structural information, X-ray crystallography is the experimental method of choice, however, with limited information on protein flexibility. An experimentally verified structural model of the binding interface in the native solution-state would support medicinal chemists in their molecular design decisions. Here we demonstrate that protein-bound ligand 1 H NMR chemical shifts are highly sensitive and accurate probes for the immediate chemical environment of protein-ligand interfaces. By comparing the experimental ligand 1 H chemical shift values with those computed from the X-ray structure using quantum mechanics methodology, we identify significant disagreements for parts of the ligand between the two experimental techniques. We show that quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) ensembles can be used to refine initial X-ray co-crystal structures resulting in a better agreement with experimental 1 H ligand chemical shift values. Overall, our findings highlight the usefulness of ligand 1 H NMR chemical shift information in combination with a QM/MM MD workflow for generating protein-ligand ensembles that accurately reproduce solution structural data., (© 2023 The Authors. ChemPhysChem published by Wiley-VCH GmbH.)

In this study, a one-pot synthesis via photoinduced C(sp 2 )-C(sp 3 ) coupling followed by amide formation to access proteolysis targeting chimeras (PROTACs) was developed. The described protocol was studied on cereblon (CRBN)-based E3-ligase binders and (+)-JQ-1, a bromodomain inhibitor, to generate BET (bromodomain and extra terminal domain) targeting protein degraders. The generated PROTACs were profiled in vitro and tested for their degradation ability with several potent candidates identified. Upfront, the individual reactions of the one-pot transformation were carefully optimized for CRBN binder functionalization and multiple heterobifunctional linker moieties were designed and synthesized. Separate scopes detailing the C(sp 2 )-C(sp 3 ) coupling and one-pot PROTAC synthesis are described in this report as well as a library miniaturization study showing the high-throughput compatibility. Overall, the developed protocol provides rapid access to PROTACs in a single process, thereby allowing efficient generation of CRBN-based PROTAC libraries.

Intracellular bacteria produce antigens, which serve as potent activators of γδ T cells. Phosphoantigens are presented via a complex of butyrophilins (BTN) to signal infection to human Vγ9+Vδ2+ T cells. Here, we established an in vitro system allowing for studies of Vγ9+Vδ2+ T cell activity in coculture with epithelial cells infected with the intracellular bacterial pathogen Listeria monocytogenes. We report that the Vγ9+Vδ2+ T cells efficiently control L. monocytogenes growth in such cultures. This effector function requires the expression of members of the BTN3A family on epithelial cells. Specifically, we observed a BTN3A1-independent BTN3A3 activity to present antigen to Vγ9+Vδ2+ T cells. Since BTN3A1 is the only BTN3A associated with phosphoantigen presentation, our study suggests that BTN3A3 may present different classes of antigens to mediate Vγ9+Vδ2+ T cell effector function against L. monocytogenes-infected epithelia., (© 2023. The Author(s).)

The effect of combination therapies in many cancers has often been shown to be superior to that of monotherapies. This success is commonly attributed to drug synergies. Combinations of two (or more) drugs in xenograft tumor growth inhibition (TGI) studies are typically designed at fixed doses for each compound. The available methods for assessing synergy in such study designs are based on combination indices (CI) and model-based analyses. The former methods are suitable for screening exercises but are difficult to verify in in vivo studies, while the latter incorporate drug synergy in semi-mechanistic frameworks describing disease progression and drug action but are unsuitable for screening. In the current study, we proposed the empirical radius additivity (Rad-add) score, a novel CI for synergy detection in fixed-dose xenograft TGI combination studies. The Rad-add score approximates model-based analysis performed using the semi-mechanistic constant-radius growth TGI model. The Rad-add score was compared with response additivity, defined as the addition of the two response values, and the bliss independence model in combination studies derived from the Novartis PDX dataset. The results showed that the bliss independence and response additivity models predicted synergistic interactions with high and low probabilities, respectively. The Rad-add score predicted synergistic probabilities that appeared to be between those predicted with response additivity and the Bliss model. We believe that the Rad-add score is particularly suitable for assessing synergy in the context of xenograft combination TGI studies, as it combines the advantages of CI approaches suitable for screening exercises with those of semi-mechanistic TGI models based on a mechanistic understanding of tumor growth., Competing Interests: Authors NM, JD, and HM were employed by Seda Pharmaceutical Developments Services, Unit D, Cheadle Royal Business Park. Authors LT and HH were employed by Boehringer Ingelheim RCV GmbH & Co KG., (Copyright © 2023 Melillo, Dickinson, Tan, Mistry and Huber.)

Background: This multicentre, open-label, Phase Ib/II trial evaluated the insulin-like growth factor (IGF) 1/2 neutralising antibody xentuzumab plus enzalutamide in metastatic castrate-resistant prostate cancer (mCRPC)., Methods: The trial included Phase Ib escalation and expansion parts and a randomised Phase II part versus enzalutamide alone. Primary endpoints in the Phase Ib escalation, Phase Ib expansion and Phase II parts were maximum tolerated dose (MTD), prostate-specific antigen response and investigator-assessed progression-free survival (PFS), respectively. Patients in the Phase Ib escalation and Phase II parts had progressed on/after docetaxel/abiraterone., Results: In the Phase Ib escalation (n = 10), no dose-limiting toxicities were reported, and xentuzumab 1000 mg weekly plus enzalutamide 160 mg daily (Xe1000 + En160) was defined as the MTD and recommended Phase 2 dose. In the Phase Ib expansion (n = 24), median PFS was 8.2 months, and one patient had a confirmed, long-term response. In Phase II (n = 86), median PFS for the Xe1000 + En160 and En160 arms was 7.4 and 6.2 months, respectively. Subgroup analysis suggested trends towards benefit with Xe1000 + En160 in patients whose tumours had high levels of IGF1 mRNA or PTEN protein. Overall, the combination was well tolerated., Conclusions: Xentuzumab plus enzalutamide was tolerable but lacked antitumour activity in unselected patients with mCRPC., Clinical Trial Registration: EudraCT number 2013-004011-41., (© 2023. The Author(s).)

Purpose: Since glioma therapy is currently still limited until today, new treatment options for this heterogeneous group of tumours are of great interest. Eukaryotic initiation factors (eIFs) are altered in various cancer entities, including gliomas. The purpose of our study was to evaluate the potential of eIFs as novel targets in glioma treatment., Methods: We evaluated eIF protein expression and regulation in 22 glioblastoma patient-derived xenografts (GBM PDX) after treatment with established cytostatics and with regards to mutation profile analyses of GBM PDX., Results: We observed decreased expression of several eIFs upon temozolomide (TMZ) treatment independent from the phosphatidylinositol 3-kinase (PI3K)/ AKT/ mammalian target of the rapamycin (mTOR) signalling pathway. These effects of TMZ treatment were not present in TMZ-resistant PDX. Combination therapy of regorafenib and TMZ re- established the eIF/AKT/mTOR axis., Conclusion: Our study provides novel insights into chemotherapeutic effects on eIF regulation in gliomas and suggests that eIFs are interesting candidates for future research to improve glioma therapy., (© 2023. The Author(s).)

Glial cells have been proposed as a source of neural progenitors, but the mechanisms underpinning the neurogenic potential of adult glia are not known. Using single cell transcriptomic profiling, we show that enteric glial cells represent a cell state attained by autonomic neural crest cells as they transition along a linear differentiation trajectory that allows them to retain neurogenic potential while acquiring mature glial functions. Key neurogenic loci in early enteric nervous system progenitors remain in open chromatin configuration in mature enteric glia, thus facilitating neuronal differentiation under appropriate conditions. Molecular profiling and gene targeting of enteric glial cells in a cell culture model of enteric neurogenesis and a gut injury model demonstrate that neuronal differentiation of glia is driven by transcriptional programs employed in vivo by early progenitors. Our work provides mechanistic insight into the regulatory landscape underpinning the development of intestinal neural circuits and generates a platform for advancing glial cells as therapeutic agents for the treatment of neural deficits., (© 2023. Springer Nature Limited.)

Immobilized metal affinity chromatography (IMAC) is a powerful technique for capture and purification of relevant biopharmaceuticals in complex biological matrices. However, protein recovery can be drastically compromised due to surface induced spreading and unfolding of the analyte, leading to fouling of the stationary phase. Here, we report on the kinetics of irreversible adsorption of a protease on an IMAC resin in a time span ranging from minutes to several hours. This trend correlated with the thermal data measured by nano differential scanning calorimetry, and showed a time-dependent change in protein unfolding temperature. Our results highlight that 'soft' proteins show a strong time dependent increase in irreversible adsorption. Furthermore, commonly used co-solvents for preservation of the native protein conformation are tested for their ability to reduce fouling. Thermal data suggests that the amino acid l-arginine is beneficial in preventing unfolding, which was confirmed in batch adsorption experiments. The choice of counter-ions has to be considered when using this amino acid. These results show that l-arginine sulfate decelerates the irreversible adsorption kinetics of proteins on the IMAC stationary phase to a greater extent than l-arginine chloride., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)