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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.

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).)

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.

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.

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.