Search

Ionic detergents enable applications and cause harm in biospheres due to cell toxicity. The utility of covalent combinations between ionic and non-ionic detergent headgroups in modulating cell toxicity remains speculative due to the yet rarely explored synthesis. We close this gap and establish the modular synthesis of ionic/non-ionic hybrid detergents. We restructure a combinatorial methallyl dichloride one-pot coupling into a two-step coupling, which reduces by-products, improves product yields, and enables the gram-scale preparation of asymmetric, cationic/non-ionic and anionic/non-ionic hybrid detergents. Our modular synthesis delivers new modalities for the design of ionic detergents, including an unprecedented scaling of properties that determine applications, such as charge, critical micelle concentration, solubilizing properties, hard water tolerance, and cell compatibility. We uncover that shielding the charge in ionic headgroups can switch the detergent species that is toxic to cells from monomers to mixtures of monomers and micellar assemblies. Establishing the chemistry of ionic/non-ionic hybrid detergents provides a missing evolutionary link in the structural comparison of ionic and non-ionic detergents, enables an easy synthesis access to yet unexplored chemical spaces of asymmetric hybrid materials, and delivers new modalities for designing the toxicity of supramolecular nanomaterials., (© 2024 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Akt kinase is vital in cell growth, survival, metabolism, and migration. Dysregulation of Akt signaling is implicated in cancer and metabolic disorders. In the context of cancer, overactive Akt promotes cell survival and proliferation. This has spurred extensive research into developing Akt inhibitors as potential therapeutic agents to disrupt aberrant Akt signaling. Akt inhibitors are classified into three main types: ATP-competitive, allosteric, and covalent-allosteric inhibitors (CAAIs). ATP-competitive inhibitors compete with ATP for binding to Akt, allosteric inhibitors interact with the Pleckstrin homology (PH) domain, and covalent-allosteric inhibitors form covalent bonds, making them more potent and selective. Notably, capivasertib (AZD5363), a potent ATP-competitive Akt inhibitor, received FDA approval in November 2023 for use in combination with the estrogen receptor degrader fulvestrant to treat breast cancer. Challenges remain, including improving selectivity, identifying biomarkers to tailor treatments, and enhancing therapeutic efficacy while minimizing adverse effects. Particularly covalent-allosteric inhibitors hold promise for future more effective and personalized treatments.

The serine/threonine kinase Akt1 is part of the PI3 K/Akt pathway and plays a key role in the regulation of various cellular processes such as cell growth, proliferation, and apoptosis. Here, we analyzed the elasticity between the two domains of the kinase Akt1, connected by a flexible linker, recording a wide variety of distance restraints by electron paramagnetic resonance (EPR) spectroscopy. We studied full length Akt1 and the influence of the cancer-associated mutation E17K. The conformational landscape in the presence of different modulators, like different types of inhibitors and membranes was presented, revealing a tuned flexibility between the two domains, dependent on the bound molecule., (© 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Ten-eleven translocation dioxygenases (TETs) are the erasers of 5-methylcytosine (mC), the central epigenetic regulator of mammalian DNA. TETs convert mC to three oxidized derivatives with unique physicochemical properties and inherent regulatory potential, and it initializes active demethylation by the base excision repair pathway. Potent small molecule inhibitors would be useful tools to study TET functions by conditional control. To facilitate the discovery of such tools, we here report a high-throughput screening pipeline and its application to screen and validate 31.5k compounds for inhibition of TET2. Using a homogenous fluorescence assay, we discover a novel quinoline-based scaffold that we further validate with an orthogonal semi-high throughput MALDI-MS assay for direct monitoring of substrate turnover. Structure-activity relationship (SAR) studies involving >20 derivatives of this scaffold led to the identification of optimized inhibitors, and together with computational studies suggested a plausible model for its mode of action., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Misregulation and mutations of the transcription factor Nrf2 are involved in the development of a variety of human diseases. In this study, we employed the technology of stapled peptides to address a protein-DNA-complex and designed a set of Nrf2-based derivatives. Varying the length and position of the hydrocarbon staple, we chose the best peptide for further evaluation in both fixed and living cells. Peptide 4 revealed significant enrichment within the nucleus compared to its linear counterpart 5, indicating potent binding to DNA. Our studies suggest that these molecules offer an interesting strategy to target activated Nrf2 in cancer cells., Competing Interests: The authors have declared that no competing interests exist.

Akt is a protein kinase that has been implicated in the progression of cancerous tumours. A number of covalent allosteric Akt inhibitors are known, and based on these scaffolds, a small library of novel potential covalent allosteric imidazopyridine-based inhibitors was designed. The envisaged compounds were synthesised, with click chemistry enabling a modular approach to a number of the target compounds. The binding modes, potencies and antiproliferative activities of these synthesised compounds were explored, thereby furthering the structure activity relationship knowledge of this class of Akt inhibitors. Three novel covalent inhibitors were identified, exhibiting moderate activity against Akt1 and various cancer cell lines, potentially paving the way for future covalent allosteric inhibitors with improved properties., (© 2022 The Authors. ChemMedChem published by Wiley-VCH GmbH.)

Despite the clinical efficacy of epidermal growth factor receptor (EGFR) inhibitors, a subset of patients with non-small cell lung cancer displays insertion mutations in exon20 in EGFR and Her2 with limited treatment options. Here, we present the development and characterization of the novel covalent inhibitors LDC8201 and LDC0496 based on a 1 H -pyrrolo[2,3- b ]pyridine scaffold. They exhibited intense inhibitory potency toward EGFR and Her2 exon20 insertion mutations as well as selectivity over wild type EGFR and within the kinome. Complex crystal structures with the inhibitors and biochemical and cellular on-target activity document their favorable binding characteristics. Ultimately, we observed tumor shrinkage in mice engrafted with patient-derived EGFR-H773_V774insNPH mutant cells during treatment with LDC8201. Together, these results highlight the potential of covalent pyrrolopyridines as inhibitors to target exon20 insertion mutations.

The protein kinase Akt plays a pivotal role in cellular processes. However, its isoforms' distinct functions have not been resolved to date, mainly due to the lack of suitable biochemical and cellular tools. Against this background, we present the development of an isoform-dependent Ba/F3 model system to translate biochemical results on isoform specificity to the cellular level. Our cellular model system complemented by protein X-ray crystallography and structure-based ligand design results in covalent-allosteric Akt inhibitors with unique selectivity profiles. In a first proof-of-concept, the developed molecules allow studies on isoform-selective effects of Akt inhibition in cancer cells. Thus, this study will pave the way to resolve isoform-selective roles in health and disease and foster the development of next-generation therapeutics with superior on-target properties., (© 2021. The Author(s).)

Mutated or amplified Her2 serves as a driver of non-small cell lung cancer or mediates resistance toward the inhibition of its family member epidermal growth factor receptor with small-molecule inhibitors. To date, small-molecule inhibitors targeting Her2 which can be used in clinical routine are lacking, and therefore, the development of novel inhibitors was undertaken. In this study, the well-established pyrrolopyrimidine scaffold was modified with structural motifs identified from a screening campaign with more than 1600 compounds, which were applied against wild-type Her2 and its mutant variant Her2-A775_G776insYVMA. The resulting inhibitors were designed to covalently target a reactive cysteine in the binding site of Her2 and were further optimized by means of structure-based drug design utilizing a set of obtained complex crystal structures. In addition, the analysis of binding kinetics and absorption, distribution, metabolism, and excretion parameters as well as mass spectrometry experiments and western blot analysis substantiated our approach.

The conformational dynamics of a kinase's activation loop have been challenging to assess due to the activation loop's intrinsic flexibility. To directly probe the conformational equilibrium of the activation loop of mitogen-activated protein kinase p38α, we present an approach based on site-directed spin labeling, electron paramagnetic resonance (EPR) distance restraints, and multilateration. We demonstrate that the activation loop of apo p38α resides in a highly flexible equilibrium state and we reveal that binding of small molecules significantly alters this equilibrium and the populated sub-states.

Isoforms of protein kinase Akt are involved in essential processes including cell proliferation, survival, and metabolism. However, their individual roles in health and disease have not been thoroughly evaluated. Thus, there is an urgent need for perturbation studies, preferably mediated by highly selective bioactive small molecules. Herein, we present a structure-guided approach for the design of structurally diverse and pharmacologically beneficial covalent-allosteric modifiers, which enabled an investigation of the isoform-specific preferences and the important residues within the allosteric site of the different isoforms. The biochemical, cellular, and structural evaluations revealed interactions responsible for the selective binding profiles. The isoform-selective covalent-allosteric Akt inhibitors that emerged from this approach showed a conclusive structure-activity relationship and broke ground in the development of selective probes to delineate the isoform-specific functions of Akt kinases., (© 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

In photopharmacology, photoswitchable compounds including azobenzene or other diarylazo moieties exhibit bioactivity against a target protein typically in the slender E-configuration, whereas the rather bulky Z-configuration usually is pharmacologically less potent. Herein we report the design, synthesis and photochemical/inhibitory characterization of new photoswitchable kinase inhibitors targeting p38α MAPK and CK1δ. A well characterized inhibitor scaffold was used to attach arylazo- and diazocine moieties. When the isolated isomers, or the photostationary state (PSS) of isomers, were tested in commonly used in vitro kinase assays, however, only small differences in activity were observed. X-ray analyses of ligand-bound p38α MAPK and CK1δ complexes revealed dynamic conformational adaptations of the protein with respect to both isomers. More importantly, irreversible reduction of the azo group to the corresponding hydrazine was observed. Independent experiments revealed that reducing agents such as DTT (dithiothreitol) and GSH (glutathione) that are typically used for protein stabilization in biological assays were responsible. Two further sources of error are the concentration dependence of the E-Z-switching efficiency and artefacts due to incomplete exclusion of light during testing. Our findings may also apply to a number of previously investigated azobenzene-based photoswitchable inhibitors.

Aberrations within the PI3K/AKT signaling axis are frequently observed in numerous cancer types, highlighting the relevance of these pathways in cancer physiology and pathology. However, therapeutic interventions employing AKT inhibitors often suffer from limitations associated with target selectivity, efficacy, or dose-limiting effects. Here we present the first crystal structure of autoinhibited AKT1 in complex with the covalent-allosteric inhibitor borussertib, providing critical insights into the structural basis of AKT1 inhibition by this unique class of compounds. Comprehensive biological and preclinical evaluation of borussertib in cancer-related model systems demonstrated a strong antiproliferative activity in cancer cell lines harboring genetic alterations within the PTEN, PI3K, and RAS signaling pathways. Furthermore, borussertib displayed antitumor activity in combination with the MEK inhibitor trametinib in patient-derived xenograft models of mutant KRAS pancreatic and colon cancer. SIGNIFICANCE: Borussertib, a first-in-class covalent-allosteric AKT inhibitor, displays antitumor activity in combination with the MEK inhibitor trametinib in patient-derived xenograft models and provides a starting point for further pharmacokinetic/dynamic optimization., (©2019 American Association for Cancer Research.)

The Ser/Thr kinase Akt (Protein Kinase B/PKB) is a master switch in cellular signal transduction pathways. Its downstream signaling influences cell proliferation, cell growth, and apoptosis, rendering Akt a prominent drug target. The unique activation mechanism of Akt involves a change of the relative orientation of its N-terminal pleckstrin homology (PH) and the kinase domain and makes this kinase suitable for highly specific allosteric modulation. Here we present a unique set of crystal structures of covalent-allosteric interdomain inhibitors in complex with full-length Akt and report the structure-based design, synthesis, biological and pharmacological evaluation of a focused library of these innovative inhibitors.

Conformational transitions in protein kinases are crucial for the biological function of these enzymes. Here, we characterize and assess conformational equilibria of the activation loop and the effect of small molecule inhibitors in the MAP kinase p38α. Our work experimentally revealed the existence of a two-state equilibrium for p38α while the addition of inhibitors shifts the equilibrium between these two states.

The identification of compounds for dissecting biological functions and the development of novel drug molecules are central tasks that often require screening campaigns. However, the required architecture is cost- and time-intensive. Herein we describe the devices and technologies that comprise a Robotics-Assisted Screening Platform for Efficient Ligand Discovery (RASPELD), which we set up in an academic laboratory. RASPELD provides semi-automated high-end screening, and it can be maintained by graduate students. We demonstrate its successful application in biochemical and cellular screens for the identification and validation of bioactive chemical entities as candidate cancer-relevant inhibitors. Specifically, we examined the interaction between a transcription factor, Nrf2, and its key regulator, Keap1. We also examined drug-resistant mutants of the epidermal growth factor receptor (EGFR). Screening campaigns with more than 30 000 compounds were performed in a reasonable period of time. We identified the molecule RSL6586 as a starting point for hit optimization, which is currently ongoing., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Transcription factors (TFs) constitute a diverse class of sequence-specific DNA-binding proteins, which are key to the modulation of gene expression. TFs have been associated with human diseases, including cancer, Alzheimer's and other neurodegenerative diseases, which makes this class of proteins attractive targets for chemical biology and medicinal chemistry research. Since TFs lack a common binding site or structural similarity, the development of small molecules to efficiently modulate TF biology in cells and in vivo is a challenging task. This review highlights various strategies that are currently being explored for the identification and development of modulators of Myc, p53, Stat, Nrf2, CREB, ER, AR, HIF, NF-κB, and BET proteins.

A chemical genetic approach is presented to covalently target a unique lipid binding pocket in the protein kinase p38α, whose function is not yet known. Based on a series of cocrystal structures, a library of 2-arylquinazolines that were decorated with electrophiles were designed and synthesized to covalently target tailored p38α mutants containing artificially introduced cysteine residues. Matching protein-ligand pairs were identified by MS analysis and further validated by MS/MS studies and protein crystallography. The covalent ligands that emerged from this approach showed excellent selectivity towards a single p38α mutant and will be applicable as suitable probes in future studies of biological systems to dissect the function of the lipid pocket by means of pharmacological perturbations., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Targeting and stabilizing distinct kinase conformations is an instrumental strategy for dissecting conformation-dependent signaling of protein kinases. Herein the structure-based design, synthesis, and evaluation of pleckstrin homology (PH) domain-dependent covalent-allosteric inhibitors (CAIs) of the kinase Akt is reported. These inhibitors bind covalently to a distinct cysteine of the kinase and thereby stabilize the inactive kinase conformation. These modulators exhibit high potency and selectivity, and represent an innovative approach for chemical biology and medicinal chemistry research., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Autism spectrum disorders (ASD) are associated with differences in sensory sensitivity and affective response to sensory stimuli, the neural basis of which is still largely unknown. We used psychophysics and functional magnetic resonance imaging (fMRI) to investigate responses to somatosensory stimulation with three textured surfaces that spanned a range of roughness and pleasantness in a sample of adults with ASD and a control group. While psychophysical ratings of roughness and pleasantness were largely similar across the two groups, the ASD group gave pleasant and unpleasant textures more extreme average ratings than did controls. In addition, their ratings for a neutral texture were more variable than controls, indicating they are less consistent in evaluating a stimulus that is affectively ambiguous. Changes in brain blood oxygenation level-dependent (BOLD) signal in response to stimulation with these textures differed substantially between the groups, with the ASD group exhibiting diminished responses compared to the control group, particularly for pleasant and neutral textures. For the most unpleasant texture, the ASD group exhibited greater BOLD response than controls in affective somatosensory processing areas such as the posterior cingulate cortex and the insula. The amplitude of response in the insula in response to the unpleasant texture was positively correlated with social impairment as measured by the Autism Diagnostic Interview-Revised (ADI-R). These results suggest that people with ASD tend to show diminished response to pleasant and neutral stimuli, and exaggerated limbic responses to unpleasant stimuli, which may contribute to diminished social reward associated with touch, perpetuating social withdrawal, and aberrant social development., (© 2012 International Society for Autism Research, Wiley Periodicals, Inc.)

Vancomycin resistance in Enterococcus gallinarum results from the production of UDP-MurNAc-pentapeptide[D-Ser]. VanT, a membrane-bound serine racemase, is one of three proteins essential for this resistance. To investigate the selectivity of racemization of L-Ser or L-Ala by VanT, a strain of Escherichia coli TKL-10 that requires D-Ala for growth at 42 degrees C was used as host for transformation experiments using plasmids containing the full-length vanT from Ent. gallinarum or the alanine racemase gene (alr) of Bacillus stearothermophilus: both plasmids were able to complement E. coli TKL-10 at 42 degrees C. No alanine or serine racemase activities were detected in the host strain E. coli TKL-10 grown at 30, 34 or 37 degrees C. Serine and alanine racemase activities were found almost exclusively (96%) in the membrane fraction of E. coli TKL-10/pCA4(vanT): the alanine racemase activity of VanT was 14% of the serine racemase activity in both E. coli TKL-10/pCA4(vanT) and E. coli XL-1 Blue/pCA4(vanT). Alanine racemase activity was present mainly (95%) in the cytoplasmic fraction of E. coli TKL-10/pJW40(alr), with a trace (1.6%) of serine racemase activity. Additionally, DNA encoding the soluble domain of VanT was cloned and expressed in E. coli M15 as a His-tagged polypeptide and purified: this polypeptide also exhibited both serine and alanine racemase activities; the latter was approximately 18% of the serine racemase activity, similar to that of the full-length, membrane-bound enzyme. N-terminal sequencing of the purified His-tagged polypeptide revealed a single amino acid sequence, indicating that the formation of heterodimers between subunits of His-tagged C-VanT and endogenous alanine racemases from E. coli was unlikely. The authors conclude that the membrane-bound serine racemase VanT also has alanine racemase activity but is able to racemize serine more efficiently than alanine, and that the cytoplasmic domain is responsible for the racemase activity.