Derivation and characterization of ubiquitin-specific protease 18 inhibitors

JCI Insight · 2026-05-19

Ubiquitin-Specific Protease 18 (USP18) is a deISGylation enzyme and antineoplastic target. To develop USP18 inhibitors, an enzymatically active human recombinant USP18 protein was engineered suitable for high-throughput screening of ~80,000 chemical compounds. Three of them substantially inhibited USP18 enzymatic activity with β-lapachone having prominent antineoplastic activity. Independent β-lapachone treatments of murine and human lung cancer cell lines statistically-significantly reduced proliferation and increased apoptosis. Gain of USP18 expression antagonized these effects. β-lapachone treatments statistically-significantly repressed lung cancer xenograft growth. β-lapachone increased reactive oxygen species (ROS), but antineoplastic effects occurred at dosages with negligible ROS production. ROS scavenger treatments did not rescue β-lapachone effects at these concentrations, consistent with an ROS-independent mechanism. Interferon-Stimulated Response Element (ISRE) reporter assays following β-lapachone treatment activated this reporter. USP18 co-transfection antagonized this activity. β-lapachone treatments increased global ISGylation. RNA sequencing of lung cancer cells engineered with or without enhanced USP18 expression showed specific pathways affected by β-lapachone treatment. Proteomic analysis of these treated cells revealed known and new ISGylated proteins. In silico modeling identified a unique USP18 pocket where these USP18 inhibitors bind. Engineered mutation of this pocket disrupted β-lapachone activity. Taken together, β-lapachone is an antineoplastic tool compound useful for USP18 inhibitor development.

Correction: BRAF inhibitors suppress apoptosis through off-target inhibition of JNK signaling

eLife · 2026-02-13

Abstract 1972 Leveraging Calmodulin's C-lobe to Design a Constitutively Active Elongation Factor-2 Kinase

Journal of Biological Chemistry · 2025-05-01

Eukaryotic elongation factor-2 kinase (eEF-2K) modulates elongation rates by phosphorylating translation factor eEF2, a GTPase that provides the energy for the translocation of ribosomes along mRNA. eEF-2K is regulated by diverse cellular cues, many of which sensitize it to the calcium-effector protein calmodulin (CaM), which binds and allosterically activates it in the presence of calcium. In the active complex, the C-terminal lobe (C-lobe) of CaM makes critical interactions with eEF-2K at the CaM targeting motif (CTM) and the kinase domain.

Phosphorylation of a conserved aspartate in the catalytic site of eukaryotic elongation factor 2 kinase

Protein Science · 2025-12-23 · 1 citations

Abstract Eukaryotic elongation factor 2 kinase (eEF‐2K) is a member of the α‐kinase family of atypical serine/threonine kinases. eEF‐2K, the only calmodulin‐activated α‐kinase, phosphorylates the ribosome‐associated GTPase, eukaryotic elongation factor 2 (eEF‐2), suppressing translational elongation. α‐kinases, including eEF‐2K, possess catalytic site geometries that are distinct from those of typical kinases, suggesting possible divergence in their phospho‐transfer mechanisms. Unlike typical protein kinases, where chemistry is known to proceed through a sequential mechanism involving a ternary kinase‐substrate‐ATP•Mg 2+ complex, the nature of the chemical step catalyzed by α‐kinases remains poorly defined. Here, multiple orthogonal lines of evidence, including a crystal structure and solution‐state mass spectrometry data, suggest phosphorylation of a catalytically essential aspartate residue (D284) at the eEF‐2K active site. Previous crystallographic evidence of the presence of a phospho‐aspartate at an equivalent position (D766) in the related Dictyostelium α‐kinase MHCK‐A strongly suggests that this species represents a conserved active‐site feature in α‐kinase family members, despite their disparate modes of activation. This observation, together with existing kinetics data on eEF‐2K, raises the possibility that phospho‐transfer chemistry in α‐kinases occurs via an ordered stepwise mechanism involving a phospho‐enzyme intermediate, contrasting with typical protein kinases.

Pro-metastatic collagen lysyl hydroxylase dimer assemblies stabilized by Fe2+-binding

UNC Libraries · 2025-08-15

Abstract 2069 Insights Into Mechanisms of the Activation and Activity of Eukaryotic Elongation Factor-2 Kinase

Journal of Biological Chemistry · 2025-05-01

In eukaryotes, the elongation factor-2 kinase (eEF2K) downregulates protein biosynthesis by phosphorylating residue T56 on the GTPase eEF2, putatively hindering its ability to engage the ribosome. eEF2K is a member of the atypical family of α-kinases whose activation, regulation, and catalytic mechanisms remain poorly understood. It is the only α-kinase that relies on calmodulin (CaM) for activation, which occurs through a unique mechanism distinct from other CaM-activated kinases. Upregulation of eEF2K activity involves binding calmodulin (CaM), autophosphorylation on a specific threonine residue (T348), and the intermolecular docking of this phospho-threonine into a conserved regulatory site.

Ser500 phosphorylation acts as a conformational switch to prime eEF-2K for activation

Journal of Biological Chemistry · 2025-12-22

Eukaryotic elongation factor-2 kinase (eEF-2K), a member of the -kinase family of atypical serine/threonine kinases, phosphorylates eEF-2 to slow ribosomal translocation and modulate translational elongation.eEF-2K activation requires Ca 2+ / calmodulin (CaM) and integrates upstream signals through specific sites within an intrinsically disordered regulatory loop (R-loop; 321-520) that links the -kinase core to a C-terminal domain.Unlike canonical CaM-dependent kinases that are activated by displacement of an autoinhibitory segment that occludes the active site, eEF-2K is activated by CaM-driven stabilization of an active state; Ca 2+ /CaM engagement triggers rapid autophosphorylation at T348, which is essential for full activity.Phosphorylation on S500, by eEF-2K or PKA, lowers the CaM requirement (20-fold) without increasing maximal catalytic turnover.Here we show that the phosphomimetic S500D markedly enhances CaM binding in the T348-phosphorylated enzyme.S500D also elevates CaM-independent ("intrinsic") activity even in the absence of phosphorylation at T348, although maximal activity requires modification at both sites.Hydrogen-deuterium exchange mass spectrometry reveals CaM-dependent conformational changes near S500, consistent with relief of inhibitory constraints.Deletion of residues near S500 mimics S500D, increasing intrinsic activity and CaM binding in vitro and enhancing eEF-2 phosphorylation in cells, supporting an inhibitory role for this segment.Prior studies have linked S500 phosphorylation to eEF-2K degradation, suggesting a dual regulatory role.We demonstrate that phosphorylation at T348 and S500 synergize to stabilize an active-like conformation and increase CaM responsiveness, effectively lowering the Ca 2+ / CaM threshold for eEF-2K activation and enabling the integration of Ca 2+ , cAMP/PKA, and metabolic cues.

Advancing the development of TRIP13 inhibitors: A high-throughput screening approach

SLAS DISCOVERY · 2025-04-12

<h2>Abstract</h2> TRIP13, a promising target for cancer therapy, has been identified as a key regulator of the mitotic checkpoint. Overexpression of TRIP13 is associated with poor clinical outcomes in various cancers. Inhibition of TRIP13 has the potential to address therapeutic challenges in cancer, particularly in therapy-resistant and Rb-deficient cancers. Despite the potential therapeutic benefits of TRIP13 inhibition, the development of TRIP13 inhibitors has been hindered by the lack of a robust high-throughput screening (HTS) assay. We developed a luminescence-based biochemical assay for TRIP13 activity to address this challenge using the ADP-Glo detection system. This assay offers high sensitivity, low background signal, and ease of automation, making it ideal for HTS applications. A pilot screen of kinase-focused inhibitors library and a large-scale screen of 4000 additional compounds demonstrated the assay's robust performance with a z'-factor exceeding 0.85 and a signal-to-background (S/B) ratio near 6. From the 50 initial hits, rigorous validation identified anlotinib as the most potent TRIP13 inhibitor with an IC₅₀ of 5 μM. A cellular thermal shift assay (CETSA) confirmed the direct binding of anlotinib to TRIP13, validating the potential of our biochemical assay for identifying novel TRIP13 inhibitors. Our study provides a valuable tool for discovering novel TRIP13 inhibitors and advances our understanding of the therapeutic potential of targeting TRIP13 in cancer.

Transcription factor EB (TFEB) activity increases resistance of TNBC stem cells to metabolic stress

Life Science Alliance · 2025-01-15 · 3 citations

Breast cancer stem cells (CSCs) are difficult to therapeutically target, but continued efforts are critical given their contribution to tumor heterogeneity and treatment resistance in triple-negative breast cancer. CSC properties are influenced by metabolic stress, but specific mechanisms are lacking for effective drug intervention. Our previous work on TFEB suggested a key function in CSC metabolism. Indeed, TFEB knockdown (KD) inhibited mammosphere formation in vitro and tumor initiation/growth in vivo. These phenotypic effects were accompanied by a decline in CD44 high /CD24 low cells. Glycolysis inhibitor 2-deoxy-D-glucose (2-DG) induced TFEB nuclear translocation, indicative of TFEB transcriptional activity. TFEB KD blunted, whereas TFEB (S142A) augmented 2-DG–driven unfolded protein response (UPR) mediators, notably BiP/HSPA5 and CHOP. Like TFEB KD, silencing BiP/HSPA5 inhibited CSC self-renewal, suggesting that TFEB augments UPR-related survival. Further studies showed that TFEB KD attenuated 2-DG–directed autophagy, suggesting a mechanism whereby TFEB protects CSCs against 2-DG–induced stress. Our data indicate that TFEB modulates CSC metabolic stress response via autophagy and UPR. These findings reveal the novel role of TFEB in regulating CSCs during metabolic stress in triple-negative breast cancer.

Abstract P3-01-23: MELK controls stromal components through fibronectin modulation in highly aggressive breast cancers

Clinical Cancer Research · 2025-06-13

Abstract Background: Triple-negative breast cancer (TNBC) and inflammatory breast cancer (IBC) have limited therapeutic options due to advanced stage at diagnosis, high rate of metastasis, and lack of actionable targets. In a previous study, we showed that MELK is a potential target in highly aggressive breast cancers; a driver of cell stemness, and promotes epithelial-mesenchymal transition and metastasis in a TNBC xenograft model. Fibronectin (FN1) produced by mesenchymal cells has been implicated in breast cancer metastasis, and previous studies have shown that FN1 expression is higher in TNBCs than in HER2-positive or hormone-receptor-positive breast cancers. Studies have also shown that an increase in FN1 can lead to the formation of extracellular fibronectin fibrils, which promote directional cancer cell motility. We previously showed that inhibition of MELK using siRNA against MELK and using a MELK inhibitor reduced the expression of FN1 in vitro and in a xenograft mouse model, suggesting that MELK kinase activity may be important for maintaining FN1 levels in TNBC. We hypothesized that MELK promotes the expression of FN1, which is involved in reorganization of the extracellular matrix. Methods and results: To determine if MELK promotes transition to mesenchymal-like stromal components, we used qPCR to evaluate the expression of FN1 in MDA-MB-231 clones with CRISPR-based MELK knockout. The clones C3 and C28 showed 0.54-fold ± 0.03-fold (p ≤ 0.01) and 0.76-fold ± 0.01-fold (p ≤ 0.001) reduction in expression of FN1 compared to Cas9 control cells. To determine if MELK promotes the motility and invasion of TNBC through FN1, we performed a migration assay by plating MELK-knockdown and MELK-inhibitor-treated TNBC/IBC cells in FN1-coated migration chambers. When MELK was knocked down using three siRNAs against MELK in MDA-MB-231 cells, the relative percentage of migration was reduced by 69.01% ± 0.03, 44.53% ± 0.05, and 11.80 ± 0.10, respectively, in the MELK-knockdown cells compared to the control cells transfected with non-targeting siRNA (p ≤ 0.001). Similarly, treatment of MDA-MB-231 and SUM149 cells with the MELK inhibitor 30e reduced the relative percentage migration by 30.45% ± 0.10 and 20.00% ± 0.14 compared to the respective DMSO-treated controls (p ≤ 0.05) for both comparisons. To evaluate the effect of MELK on stromal organization, we used the 3D Spheroid BME Invasion Assay to measure the invasion of MDA-MB-231 cells in the presence of FN1 and MELK-In-30e. The presence of FN1 enhanced the invasion of these cells compared to the control cells, and inhibition of MELK with MELK-In-30e reduced the invasion of these cells compared to the DMSO-treated control cells. To pave the path for clinical translation of MELK inhibitors, we evaluated three MELK inhibitors (MELK-In-17, MELK-In-30e, and OTS167) to find the inhibitor with the highest efficacy and lowest toxicity alone or in combination with paclitaxel in the murine TNBC 4T1 mouse model. All the treated groups exhibited a reduction in tumor growth compared to the vehicle-treated group. The MELK–In–30–treated group exhibited significant inhibition in tumor growth compared to the OTS167-treated group (p=0.002). The combinations of MELK inhibitors with paclitaxel did not show any synergism with respect to tumor growth inhibition. Conclusions and Future Directions: Our results showed that MELK promotes FN1 expression, which may be involved in stromal reorganization and contribute to tumor progression in TNBC. Currently, we are evaluating MELK inhibitors with other rational combination therapies in a triple-negative IBC xenograft model. Citation Format: Mohd Mughees, Alex Tan, Jian Wang, Savitri Krishnamurthy, Senthil Damodaran, Debu Tripathy, Wendy A. Woodward, Kevin Dalby, Chandra Bartholomeusz. MELK controls stromal components through fibronectin modulation in highly aggressive breast cancers [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P3-01-23.