Active Dimer Research Articles

Altering the bilayer motif in the ERBB2 HER2 TMD and ErbB HER TMD dimer causing in vitro and in vivo tumor suppression

AbstractIn silico, we modelled the possible docking conformation of human WNT16B and the human ERBB2 TMD homodimer, resulting in a mutant complex. The ribbon structure and the C-terminal, N-terminal, and GG4-like motif structures are similar in HER2 TMD and HER TMD: we modelled the possible docking conformation of WNT16B to the HER1 TMD (ErbB), which also resulted in a mutant complex. If a strong relationship exists between TMD mutations that improve the active dimer interface or stabilizing an activated conformation and the potency with which HER2 (and possibly also HER), then the dimerization part of the TMD seems to be the ideal reagent target. The agent we tested –4-(Furan-2-yl)hepta-1,6-dien-4-ol (AKOS004122375) – can connect directly into human ERBB2 TMD (HER2), to the ErbB TMD (HER1) dimer bilayer motif, and human WNT16B, ERBB2 TMD (HER2) and WNT16B ErbB TMD (HER1) mutant complex residues. We tested the agent ligand in vitro and in vivo in several tumor models, which highlighted that targeting the EGFR's TMD with an agent not only reduced treatment-induced metastasis but also radically decreased tumor growth. Because of the analogous structure of HER2 TMD and HER TMD, this dimerization motif targeting can also be successful in HER and HER2 EGFR signalling. In vitro, we reached an antiproliferation rate of 80%–94% in different tumor models, while in vivo we reached a rate of 35%–61% tumor suppression in different tumor models. The metastasis inhibition effect of the compound was between 82% and 87% in different tumor models. The referenced experiments took place in 2015 in Hungary.

Metal ion cofactors modulate integral enzyme activity by varying differential membrane curvature stress.

Metal ions are well-known cofactors of protein function and stability. In the case of the integral membrane enzyme OmpLA (outer membrane phospholipase A) the active dimer is stabilized by calcium ions. We studied the lipid hydrolysis kinetics of OmpLA in charge-neutral and charged membranes with symmetric or asymmetric transbilayer lipid distributions. In charge-neutral membranes, OmpLA was more active in symmetric bilayers due to the lower differential curvature stress between membrane leaflets. Strikingly, this behavior was completely reversed in charged bilayers. Measurements revealed intrinsic molecular shape changes in the charged lipids upon addition of calcium. This effectively reduces the differential curvature stress in charged asymmetric membranes leading to increased protein activity. This conclusion is further supported by similar effects observed upon the addition of sodium ions, which also alter the shape of the lipids, but do not specifically interact with the protein. Additional lipid-protein interactions likely contribute to this phenomenon. Our findings demonstrate that ion cofactors not only interact directly with membrane proteins but also modulate protein activity indirectly by altering the effective molecular shape of charged lipid species.

Role of TLR4 activation and signaling in bone remodeling, and afferent sprouting in serum transfer arthritis

BackgroundIn the murine K/BxN serum transfer rheumatoid arthritis (RA) model, tactile allodynia persists after resolution of inflammation in male and partially in female wild type (WT) mice, which is absent in Toll-like receptor (TLR)4 deficient animals. We assessed the role of TLR4 on allodynia, bone remodeling and afferent sprouting in this model of arthritis.MethodsK/BxN sera were injected into male and female mice with conditional or stable TLR4 deletion and controls. Paw swelling was scored and allodynia assessed by von Frey filaments. At day 28, synovial neural fibers were visualized with confocal microscopy and bone density assayed with microCT. Microglial activity and TLR4 dimerization in spinal cords were examined by immunofluorescence and flow cytometry.ResultsIn the synovium, K/BxN injected WT male and female mice showed robust increases in calcitonin gene related-peptide (CGRP+), tyrosine hydroxylase (TH)+ and GAP43+ nerve fibers. Trabecular bone density by microCT was significantly decreased in K/BxN WT female but not in WT male mice. The number of osteoclasts increased in both sexes of WT mice, but not in Tlr4-/- K/BxN mice. We used conditional strains with Cre drivers for monocytes/osteoclasts (lysozyme M), microglia (Tmem119 and Cx3CR1), astrocytes (GFAP) and sensory neurons (advillin) for Tlr4f/f disruption. All strains developed similar arthritis scores after K/BxN serum injection with the exception being the Tlr4Tmem119 mice which showed a reduction. Both sexes of Tlr4Lyz2, Tlr4Tmem119 and Tlr4Cx3cr1 mice displayed a partial reversal of the chronic pain phenotype but not in Tlr4Avil, and Tlr4Gfap mice. WT K/BxN male mice showed increases in spinal Iba1, but not GFAP, compared to Tlr4-/- male mice. To determine whether spinal TLR4 was indeed activated in the K/BxN mice, flow cytometry of lumbar spinal cords of WT K/BxN male mice was performed and revealed that TLR4 in microglia cells (CD11b+ /TMEM119+) demonstrated dimerization (e.g. activation) and a characteristic increase in lipid rafts.ConclusionThese results demonstrated a complex chronic allodynia phenotype associated with TLR4 in microglia and monocytic cell lineages, and a parallel spinal TLR4 activation. However, TLR4 is dispensable for the development of peripheral nerve sprouting in this model.

Structural basis of orientated asymmetry in a mGlu heterodimer

The structural basis for the allosteric interactions within G protein-coupled receptors (GPCRs) heterodimers remains largely unknown. The metabotropic glutamate (mGlu) receptors are complex dimeric GPCRs important for the fine tuning of many synapses. Heterodimeric mGlu receptors with specific allosteric properties have been identified in the brain. Here we report four cryo-electron microscopy structures of mGlu2-4 heterodimer in different states: an inactive state bound to antagonists, two intermediate states bound to either mGlu2 or mGlu4 agonist only and an active state bound to both glutamate and a mGlu4 positive allosteric modulator (PAM) in complex with Gi protein. In addition to revealing a unique PAM binding pocket among mGlu receptors, our data bring important information for the asymmetric activation of mGlu heterodimers. First, we show that agonist binding to a single subunit in the extracellular domain is not sufficient to stabilize an active dimer conformation. Single-molecule FRET data show that the monoliganded mGlu2-4 can be found in both intermediate states and an active one. Second, we provide a detailed view of the asymmetric interface in seven-transmembrane (7TM) domains and identified key residues within the mGlu2 7TM that limits its activation leaving mGlu4 as the only subunit activating G proteins.

Dynamics and Collective Behavior of Chemically Propelled Janus Sphere Dimers in Complex Solvents.

The propulsion mechanisms and collective dynamics of chemically powered Janus sphere dimers at the micro- and nanoscales, confined in a quasi-two-dimensional geometry, are investigated using a coarse-grained microscopic dynamical model. These active Janus dimers consist of two identical Janus spheres, featuring a catalytic cap on one hemisphere. The chemical reaction taking place on the catalytic surface generates asymmetric concentration gradients of product molecules around the Janus sphere, leading to the self-propulsion of the dimers. Depending on the dimer configuration, they exhibit various motion behaviors such as forward propulsion, rotation, and restricted stochastic motion. Due to chemotactic effects and self-diffusiophoretic forces, ensembles of dimers spontaneously form diverse structures, such as transient clusters, stable rotational ensembles, and antiparallel aligned doublets. This study demonstrates that the configurations of Janus sphere dimers significantly influence their self-propulsion and collective behaviors, providing crucial insights for the design and control of active micro- and nanoscale systems.

Downregulation of neuronal nitric oxide synthase (nNOS) within the paraventricular nucleus in Ins2Akita-type-1 diabetic mice contributes to sympatho-excitation

Activation of both renin-angiotensin system (RAS) and the sympathetic system is the primary etiologic event in developing cardiovascular complications in diabetes mellitus (DM). However, the precise mechanisms for sympathetic activation in DM have not been elucidated. Here we attempted to investigate diabetes-linked cardiovascular dysregulation due to angiotensin II (Ang II)-mediated reduction in neuronal nitric oxide (NO) synthase (nNOS) within the paraventricular neuleus (PVN). In the present study, we used Ins2+/−Akita (a spontaneous, insulin-dependent genetic diabetic non-obese murine model) and wild-type (WT) littermates mice as controls. At 14 weeks of age, we found the Akita mice had increased renal sympathetic nerve activity and elevated levels of plasma norepinephrine. There was decreased expression of nNOS protein (Akita 0.43 ± 0.11 vs. WT 0.75 ± 0.05, P < 0.05) in the PVN of Akita mice. Akita mice had increased expression of angiotensin-converting enzyme (ACE) (Akita 0.58 ± 0.05 vs. WT 0.34 ± 0.04, P < 0.05) and Ang II type 1 receptor (Akita 0.49 ± 0.03 vs. WT 0.29 ± 0.09, P < 0.05), decreased expressions of ACE2 (Akita 0.17 ± 0.05 vs. WT 0.27 ± 0.03, P < 0.05) and angiotensin (1–7) Mas receptor (Akita 0.46 ± 0.02 vs. WT 0.77 ± 0.07, P < 0.05). Futher, there were increased protein levels of protein inhibitor of nNOS (PIN) (Akita 1.75 ± 0.08 vs. WT 0.71 ± 0.09, P < 0.05) with concomitantly decreased catalytically active dimers of nNOS (Akita 0.11 ± 0.04 vs. WT 0.19 ± 0.02, P < 0.05) in the PVN in Akita mice. Our studies suggest that activation of the excitatory arm of RAS, leads to a decrease NO, causing an over-activation of the sympathetic drive in DM.

Response of chemically active dimer motor in phase-separating binary fluid mixture: Motilityregulation and self-aggregation.

The design of synthetic chemically powered nanomotors often considers the fuel and product to be miscible. The propulsion properties of such motors can be altered if the binary fluid consisting of fuel and product is phase separating. The dynamical properties of a dimer motor in a phase-separating binary mixture are discussed. Depending on the strength of phase separation and the activity of the dimer, the single-motor propulsion velocity either decreases or reverses its direction. The velocity reversal is shown to be related to the generated fluid flow around the motor. The collective dynamics of the motors in such phase-separating fluid results in the formation of self-assembled structures.

Understanding and simulating mechanochromism in dye-dispersed polymer blends: from atomistic insights to macroscopic properties.

In this work, we propose a computational protocol enabling the simulation of mechanochromic responses in dye-dispersed polymer blends. The main objective is the modeling of the molecular-level structural changes responsible for the modulation of the photophysical properties that lead to the mechanochromic phenomenon. In this demonstrative study, we focus on predicting the changes in optical absorption displayed by a model system consisting of a dimer of a tetraphenylethylene derivative dispersed in a polyethylene matrix. The blend is subjected to an external stimulus that causes a modulation of the polymer matrix density that translates, in turn, into the emergence of specific mechanical constraints on the optically active dimers. The accurate description of this phenomenon requires the reliable sampling of the dimer configurations induced by the interaction with the matrix under stress. These molecular geometries are associated with modified electronic structures that confer novel absorption responses to the dispersed dyes. In the present contribution, the sampling of these structures is achieved through classical molecular dynamics (MD) simulations including a model element to apply an anisotropic mechanical force. This element allows the microscopic modeling of the chains' and dyes' structural rearrangements under stress. After the sampling, we compare the results of two approaches for the prediction of the optical response: (i) the calculation of a mean response from a statistical average over quantum chemical calculations on the sampled MD structures and (ii) a prediction via a more expensive hybrid scheme allowing the relaxation of the sampled molecular geometries in the presence of the matrix constraints.

O-GlcNAcylation of enolase 1 serves as a dual regulator of aerobic glycolysis and immune evasion in colorectal cancer

Aerobic glycolysis and immune evasion are two key hallmarks of cancer. However, how these two features are mechanistically linked to promote tumor growth is not well understood. Here, we show that the glycolytic enzyme enolase-1 (ENO1) is dynamically modified with an O-linked β-N-acetylglucosamine (O-GlcNAcylation), and simultaneously regulates aerobic glycolysis and immune evasion via differential glycosylation. Glycosylation of threonine 19 (T19) on ENO1 promotes its glycolytic activity via the formation of active dimers. On the other hand, glycosylation of serine 249 (S249) on ENO1 inhibits its interaction with PD-L1, decreases association of PD-L1 with the E3 ligase STUB1, resulting in stabilization of PD-L1. Consequently, blockade of T19 glycosylation on ENO1 inhibits glycolysis, and decreases cell proliferation and tumor growth. Blockade of S249 glycosylation on ENO1 reduces PD-L1 expression and enhances T cell-mediated immunity against tumor cells. Notably, elimination of glycosylation at both sites synergizes with PD-L1 monoclonal antibody therapy to promote antitumor immune response. Clinically, ENO1 glycosylation levels are up-regulated and show a positive correlation with PD-L1 levels in human colorectal cancers. Thus, our findings provide a mechanistic understanding of how O-GlcNAcylation bridges aerobic glycolysis and immune evasion to promote tumor growth, suggesting effective therapeutic opportunities.

Protein disulfide isomerase plays a crucial role in mediating chemically-induced, glutathione depletion-associated hepatocyte injury in vitro and in vivo

Recently we have shown that protein disulfide isomerase (PDI or PDIA1) is involved in mediating chemically-induced, glutathione (GSH) depletion-associated ferroptotic cell death through NOS activation (dimerization) and NO accumulation. The present study aims to determine the role of PDI in mediating chemically-induced hepatocyte injury in vitro and in vivo and whether PDI inhibitors can effectively protect against chemically-induced hepatocyte injury. We show that during the development of erastin-induced ferroptotic cell death, accumulation of cellular NO, ROS and lipid-ROS follows a sequential order, i.e., cellular NO accumulation first, followed by accumulation of cellular ROS, and lastly cellular lipid-ROS. Cellular NO, ROS and lipid-ROS each play a crucial role in mediating erastin-induced ferroptosis in cultured hepatocytes. In addition, it is shown that PDI is an important upstream mediator of erastin-induced ferroptosis through PDI-mediated conversion of NOS monomer to its dimer, which then leads to accumulation of cellular NO, ROS and lipid-ROS, and ultimately ferroptotic cell death. Genetic manipulation of PDI expression or pharmacological inhibition of PDI function each can effectively abrogate erastin-induced ferroptosis. Lastly, evidence is presented to show that PDI is also involved in mediating acetaminophen-induced liver injury in vivo using both wild-type C57BL/6J mice and hepatocyte-specific PDI conditional knockout (PDIfl/fl Alb-cre) mice. Together, our work demonstrates that PDI is an important upstream mediator of chemically-induced, GSH depletion-associated hepatocyte ferroptosis, and inhibition of PDI can effectively prevent this injury.

Dimerization activates the Inversin complex in C. elegans.

Genetic, colocalization, and biochemical studies suggest that the ankyrin repeat-containing proteins Inversin (INVS) and ANKS6 function with the NEK8 kinase to control tissue patterning and maintain organ physiology. It is unknown whether these three proteins assemble into a static "Inversin complex" or one that adopts multiple bioactive forms. Through the characterization of hyperactive alleles in C. elegans, we discovered that the Inversin complex is activated by dimerization. Genome engineering of an RFP tag onto the nematode homologues of INVS (MLT-4) and NEK8 (NEKL-2) induced a gain-of-function, cyst-like phenotype that was suppressed by monomerization of the fluorescent tag. Stimulated dimerization of MLT-4 or NEKL-2 using optogenetics was sufficient to recapitulate the phenotype of a constitutively active Inversin complex. Further, dimerization of NEKL-2 bypassed a lethal MLT-4 mutant, demonstrating that the dimeric form is required for function. We propose that dynamic switching between at least two functionally distinct states - an active dimer and an inactive monomer - gates the output of the Inversin complex.

A phase 1/2, first-in-human, open-label, dose-escalation study of TAK-280, an investigational B7-H3 x CD3ε conditional bispecific redirected activation (COBRA) T-cell engager, in adult patients with unresectable, locally advanced, or metastatic solid tumors.

TPS2684 Background: TAK-280 is a novel COBRA T-cell engager that targets the B7 homolog 3 protein (B7-H3), an antigen highly expressed in a range of solid tumors including metastatic castration-resistant prostate cancer (mCRPC) and non–small cell lung cancer. In its prodrug form, TAK-280 binds to B7-H3, but not to CD3ε. Once in the protease-rich tumor microenvironment, TAK-280 undergoes protease-mediated activation through formation of CD3ε-binding active dimers, which stimulate CD3 T-cell activation and cytotoxic antitumor response against co-engaged B7-H3–expressing cells. In preclinical studies, TAK-280 demonstrated cleavage-dependent conditionality, engagement of tumor target antigen, and induction of T-cell mediated tumor killing. TAK-280 has the potential to target solid tumors that are not amenable to treatment with conventional first- or second-generation T-cell engagers, while limiting toxicity to normal tissues. Methods: This phase 1/2 study is enrolling adult patients with unresectable, locally advanced, or metastatic solid tumors described in the literature to have enhanced B7-H3 expression, who are ineligible or intolerant to standard therapies. Other eligibility criteria include Eastern Cooperative Oncology Group performance status ≤1 and measurable disease per RECIST v1.1. Key exclusion criteria include history of autoimmune disease, major surgery ≤8 weeks before first dose of TAK-280, and history of clinically significant cardiac or gastrointestinal disorders. During phase 1 dose escalation, TAK-280 is administered as an intravenous infusion, once weekly, in 28-day cycles at a pre-defined dose level range. Patients who do not experience dose-limiting or other unacceptable toxicities may receive up to 14 treatment cycles depending on response; patients are treated until disease progression, unacceptable toxicity, or withdrawal from study. The dose-escalation phase will determine two recommended doses for expansion to be assessed during the cohort-expansion phase. The primary endpoint is incidence of dose-limiting or other toxicities during dose escalation; secondary endpoints include pharmacokinetic parameters, response assessment per RECIST v1.1, overall response rate, duration of response, progression-free survival, overall survival, disease control rate, and prostate-specific antigen response in patients with mCRPC. Cytokine release syndrome is closely managed and reported per American Society for Transplantation and Cellular Therapy consensus grading. Approximately 182 patients are planned to be enrolled (42 in dose escalation and 100–142 in cohort-expansion); as of the data cutoff date of Jan 10, 2024, 18 patients have received the study drug. Clinical trial information: NCT05220098 .

Reconstitution and characterization of BRAF in complex with 14-3-3 and KRAS4B on nanodiscs.

RAF kinases are key components of the RAS-MAPK signaling pathway, which drives cell growth and is frequently overactivated in cancer. Upstream signaling activates the small GTPase RAS, which recruits RAF to the cell membrane, driving a transition of the latter from an auto-inhibited monomeric conformation to an active dimer. Despite recent progress, mechanistic details underlying RAF activation remain unclear, particularly the role of RAS and the membrane in mediating this conformational rearrangement of RAF together with 14-3-3 to permit RAF kinase domain dimerization. Here, we reconstituted an active complex of dimeric BRAF, a 14-3-3 dimer and two KRAS4B on a nanodisc bilayer and verified that its assembly is GTP-dependent. Biolayer interferometry (BLI) was used to compare the binding affinities of monomeric versus dimeric full-length BRAF:14-3-3 complexes for KRAS4B-conjugated nanodiscs (RAS-ND) and to investigate the effects of membrane lipid composition and spatial density of KRAS4B on binding. 1,2-Dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) and higher KRAS4B density enhanced the interaction of BRAF:14-3-3 with RAS-ND to different degrees depending on BRAF oligomeric state. We utilized our reconstituted system to dissect the effects of KRAS4B and the membrane on the kinase activity of monomeric and dimeric BRAF:14-3-3 complexes, finding that KRAS4B or nanodiscs alone were insufficient to stimulate activity, whereas RAS-ND increased activity of both states of BRAF. The reconstituted assembly of full-length BRAF with 14-3-3 and KRAS on a cell-free, defined lipid bilayer offers a more holistic biophysical perspective to probe regulation of this multimeric signaling complex at the membrane surface.

Bimetallic Metal Sites in Metal-Organic Frameworks Facilitate the Production of 1-Butene from Electrosynthesized Ethylene.

Converting CO2 to synthetic hydrocarbon fuels is of increasing interest. In light of progress in electrified CO2 to ethylene, we explored routes to dimerize to 1-butene, an olefin that can serve as a building block to ethylene longer-chain alkanes. With goal of selective and active dimerization, we investigate a series of metal-organic frameworks having bimetallic catalytic sites. We find that the tunable pore structure enables optimization of selectivity and that periodic pore channels enhance activity. In a tandem system for the conversion of CO2 to 1-C4H8, wherein the outlet cathodic gas from a CO2-to-C2H4 electrolyzer is fed directly (via a dehumidification stage) into the C2H4 dimerizer, we study the highest-performing MOF found herein: M' = Ru and M″ = Ni in the bimetallic two-dimensional M'2(OAc)4M″(CN)4 MOF. We report a 1-C4H8 production rate of 1.3 mol gcat-1 h-1 and a C2H4 conversion of 97%. From these experimental data, we project an estimated cradle-to-gate carbon intensity of -2.1 kg-CO2e/kg-1-C4H8 when CO2 is supplied from direct air capture and when the required energy is supplied by electricity having the carbon intensity of wind.

Regulation of RAF family kinases: new insights from recent structural and biochemical studies.

The RAF kinases are required for signal transduction through the RAS-RAF-MEK-ERK pathway, and their activity is frequently up-regulated in human cancer and the RASopathy developmental syndromes. Due to their complex activation process, developing drugs that effectively target RAF function has been a challenging endeavor, highlighting the need for a more detailed understanding of RAF regulation. This review will focus on recent structural and biochemical studies that have provided 'snapshots' into the RAF regulatory cycle, revealing structures of the autoinhibited BRAF monomer, active BRAF and CRAF homodimers, as well as HSP90/CDC37 chaperone complexes containing CRAF or BRAFV600E. In addition, we will describe the insights obtained regarding how BRAF transitions between its regulatory states and examine the roles that various BRAF domains and 14-3-3 dimers play in both maintaining BRAF as an autoinhibited monomer and in facilitating its transition to an active dimer. We will also address the function of the HSP90/CDC37 chaperone complex in stabilizing the protein levels of CRAF and certain oncogenic BRAF mutants, and in serving as a platform for RAF dephosphorylation mediated by the PP5 protein phosphatase. Finally, we will discuss the regulatory differences observed between BRAF and CRAF and how these differences impact the function of BRAF and CRAF as drivers of human disease.

Structural insights into the functional mechanism of the ubiquitin ligase E6AP

E6AP dysfunction is associated with Angelman syndrome and Autism spectrum disorder. Additionally, the host E6AP is hijacked by the high-risk HPV E6 to aberrantly ubiquitinate the tumor suppressor p53, which is linked with development of multiple types of cancer, including most cervical cancers. Here we show that E6AP and the E6AP/E6 complex exist, respectively, as a monomer and a dimer of the E6AP/E6 protomer. The short α1-helix of E6AP transforms into a longer helical structure when in complex with E6. The extended α1-helices of the dimer intersect symmetrically and contribute to the dimerization. The two protomers sway around the crossed region of the two α1-helices to promote the attachment and detachment of substrates to the catalytic C-lobe of E6AP, thus facilitating ubiquitin transfer. These findings, complemented by mutagenesis analysis, suggest that the α1-helix, through conformational transformations, controls the transition between the inactive monomer and the active dimer of E6AP.

Biophysical characterization of the CXC chemokine receptor 2 ligands.

The chemokines of the immune system act as first responders by operating as chemoattractants, directing immune cells to specific locations of inflamed tissues. This promiscuous network is comprised of 50 ligands and 18 receptors where the ligands may interact with the receptors in various oligomeric states i.e., monomers, homodimers, and heterodimers. Chemokine receptors are G-protein coupled receptors (GPCRs) present in the membrane of immune cells. The migration of immune cells occurs in response to a concentration gradient of the ligands. Chemotaxis of neutrophils is directed by CXC-ligand (CXCL) activation of the membrane bound CXC chemokine receptor 2 (CXCR2). CXCR2 plays an important role in human health and is linked to disorders such as autoimmune disorders, inflammation, and cancer. Yet, despite their important role, little is known about the biophysical characteristics controlling ligand:ligand and ligand:receptor interaction essential for biological activity. In this work, we study the homodimers of three of the CXCR2 cognate ligands, CXCL1, CXCL5, and CXCL8. The ligands share high structural integrity but a low sequence identity. We show that the sequence diversity has evolved different binding affinities and stabilities for the CXC-ligands resulting in diverse agonist/antagonist behavior. Furthermore, CXC-ligands fold through a three-state mechanism, populating a folded monomeric state before associating into an active dimer.

Scutellarin activates IDH1 to exert antitumor effects in hepatocellular carcinoma progression

Isochlorate dehydrogenase 1 (IDH1) is an important metabolic enzyme for the production of α-ketoglutarate (α-KG), which has antitumor effects and is considered to have potential antitumor effects. The activation of IDH1 as a pathway for the development of anticancer drugs has not been attempted. We demonstrated that IDH1 can limit glycolysis in hepatocellular carcinoma (HCC) cells to activate the tumor immune microenvironment. In addition, through proteomic microarray analysis, we identified a natural small molecule, scutellarin (Scu), which activates IDH1 and inhibits the growth of HCC cells. By selectively modifying Cys297, Scu promotes IDH1 active dimer formation and increases α-KG production, leading to ubiquitination and degradation of HIF1a. The loss of HIF1a further leads to the inhibition of glycolysis in HCC cells. The activation of IDH1 by Scu can significantly increase the level of α-KG in tumor tissue, downregulate the HIF1a signaling pathway, and activate the tumor immune microenvironment in vivo. This study demonstrated the inhibitory effect of IDH1–α-KG–HIF1a on the growth of HCC cells and evaluated the inhibitory effect of Scu, the first IDH1 small molecule agonist, which provides a reference for cancer immunotherapy involving activated IDH1.

Synthesis and antihepatoma activity of guaianolide dimers derived from lavandiolide I

Guaianolide dimers represent a unique class of natural products with anticancer activities, but their low content in plants has limited in-depth pharmacological studies. Lavandiolide I is a guaianolide dimer isolated from Artemisia species, and had been synthesized on a ten-gram scale in four steps with 60 % overall yield, which showed potent antihepatoma activity on the HepG2, Huh7, and SK-Hep-1 cell lines with IC50 values of 12.1, 18.4, and 17.6 µM, respectively. To explore more active dimers, 33 lavandiolide I derivatives were designed, synthesized, and evaluated for their inhibitory activity on human hepatoma cell lines. Among them, 10 derivatives were more active than lavandiolide I and sorafenib on the three cell lines. The primary structure–activity relationship concluded that the introduction of aldehyde, ester, azide, amide, carbamate and urea functional groups at C-14′ of the guaianolide dimer significantly enhanced the antihepatoma activity. Among these compounds, derivatives 25, 27, and 33 enhanced antihepatoma activity more than 1.2–5.8 folds than that of lavandiolide I, and demonstrated low toxicity to the human liver cell lines (THLE-2) and good safety profiles with selective index ranging from 1.3 to 3.4, while lavandiolide I was more toxic to THLE-2 cells. This work provides new insights into enhancing the antihepatoma efficacy and reducing the toxicity of sesquiterpenoid dimers.

Drug-resistant EGFR mutations promote lung cancer by stabilizing interfaces in ligand-free kinase-active EGFR oligomers

The Epidermal Growth Factor Receptor (EGFR) is frequently found to be mutated in non-small cell lung cancer. Oncogenic EGFR has been successfully targeted by tyrosine kinase inhibitors, but acquired drug resistance eventually overcomes the efficacy of these treatments. Attempts to surmount this therapeutic challenge are hindered by a poor understanding of how and why cancer mutations specifically amplify ligand-independent EGFR auto-phosphorylation signals to enhance cell survival and how this amplification is related to ligand-dependent cell proliferation. Here we show that drug-resistant EGFR mutations manipulate the assembly of ligand-free, kinase-active oligomers to promote and stabilize the assembly of oligomer-obligate active dimer sub-units and circumvent the need for ligand binding. We reveal the structure and assembly mechanisms of these ligand-free, kinase-active oligomers, uncovering oncogenic functions for hitherto orphan transmembrane and kinase interfaces, and for the ectodomain tethered conformation of EGFR. Importantly, we find that the active dimer sub-units within ligand-free oligomers are the high affinity binding sites competent to bind physiological ligand concentrations and thus drive tumor growth, revealing a link with tumor proliferation. Our findings provide a framework for future drug discovery directed at tackling oncogenic EGFR mutations by disabling oligomer-assembling interactions.