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Metabolic networks are interconnected and influence diverse cellular processes. The protein-metabolite interactions that mediate these networks are frequently low affinity and challenging to systematically discover. We developed mass spectrometry integrated with equilibrium dialysis for the discovery of allostery systematically (MIDAS) to identify such interactions. Analysis of 33 enzymes from human carbohydrate metabolism identified 830 protein-metabolite interactions, including known regulators, substrates, and products as well as previously unreported interactions. We functionally validated a subset of interactions, including the isoform-specific inhibition of lactate dehydrogenase by long-chain acyl–coenzyme A. Cell treatment with fatty acids caused a loss of pyruvate-lactate interconversion dependent on lactate dehydrogenase isoform expression. These protein-metabolite interactions may contribute to the dynamic, tissue-specific metabolic flexibility that enables growth and survival in an ever-changing nutrient environment.

The PA28 family proteasome activators play important roles in regulating proteasome activities. Though the three paralogs (PA28α, PA28β, and PA28γ) are similar in terms of primary sequence, they show significant differences in expression pattern, cellular localization and most importantly, biological functions. While PA28αβ is responsible for promoting peptidase activity of proteasome to facilitate MHC-I antigen processing, but unable to promote protein degradation, PA28γ is well-known to not only promote peptidase activity but also proteolytic activity of proteasome. However, why this paralog has the unique function remains elusive. Previous structural studies have mainly focused on mammalian PA28α, PA28β and PA28αβ heptamers, while structural studies on mammalian PA28γ of atomic resolution are still absent to date. In the present work, we determined the Cryo-EM structure of the human PA28γ heptamer at atomic resolution, revealing interesting unique structural features that may hint our understanding the functional mechanisms of this proteasome activator.

Supplemental Figures 1 - 7. Figure S1. Sequence tracings from WZ4002 resistant Ba/F3 cells demonstrating EGFR L718Q, C797S and L844V mutations. Figure S2. EGFR Del19 Ba/F3 cells alone or expressing EGFR L718Q were treated with EGF. Figure S3. Structure of TX 2-30. Figure S4. Impact of EGFR L718Q and L844V on sensitivity to WZ4002 and quinazoline based EGFR inhibitors Figure S5. Impact of EGFR Q791R mutation. Figure S6. EGFR L718Q and L844V cause resistance in trans in EGFR mutant NSCLC cell lines. Figure S7. Evaluation of cetuximab on EGFR dimerization and growth in EGFR C797S expressing cells.

CCR Translation for This Article from EGFR Exon 19 Insertions: A New Family of Sensitizing EGFR Mutations in Lung Adenocarcinoma

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Supplemental Figure legends. This file contains the detailed descriptions of the supplementaal figures.

Supplemental Tables 1-4. Table S1. Secondary EGFR mutations recovered in the presence of WZ4002. Table S2. Efficacy (IC50 values) of EGFR kinase inhibitors in EGFR mutant Ba/F3 cells Table S3. Efficacy (IC50 values) of covalent EGFR kinase inhibitors in EGFR mutant Ba/F3 cells or lung cancer cell lines. Table S4. Frequency and type of EGFR secondary mutation following ENU mutagenesis screen using irreversible pyrimidine EGFR inhibitors.

Purpose: Epidermal growth factor receptor (EGFR) genotyping is now standard in the management of advanced lung adenocarcinoma, as this biomarker predicts marked benefit from treatment with EGFR tyrosine kinase inhibitors (TKI). EGFR exon 19 insertions are a poorly described family of EGFR mutations, and their association with EGFR-TKI sensitivity in lung adenocarcinoma is uncertain.Experimental Design: Patients with lung cancers harboring EGFR exon 19 insertions were studied. The predicted effects of the insertions on the structure of the EGFR protein were examined, and EGFR exon 19 insertions were introduced into Ba/F3 cells to assess oncogenicity and in vitro sensitivity to EGFR-TKIs. In patients receiving TKI, response magnitude was assessed with serial computed tomographic (CT) measurement.Results: Twelve tumors harboring EGFR exon 19 insertions were identified; patients were predominately female (92%) and never-smokers (75%). The 11 specimens available for full sequencing all showed an 18-bp insertion that resulted in the substitution of a Pro for Leu at residue 747. The mutant EGFR transformed the Ba/F3 cells, which were then sensitive to EGFR-TKI. Six patients with measurable disease received TKI and five had a response on serial CT.Conclusions:EGFR exon 19 insertions are a newly appreciated family of EGFR-TKI–sensitizing mutations, and patients with tumors harboring these mutations should be treated with EGFR-TKI. While these mutations may be missed through the use of some mutation-specific assays, the addition of PCR product size analysis to multigene assays allows sensitive detection of both exon 19 insertion and deletion mutations. Clin Cancer Res; 18(6); 1790–7. ©2011 AACR.

1,3-β-Glucan is the major component of the fungal cell wall and is synthesized by 1,3-β-glucan synthase located in the plasma membrane, which is a molecular target of anti-fungal drugs echinocandins and the triterpenoid ibrexafungerp. In this study, we report the 3.0-Å resolution cryo-EM structure ofSaccharomyces cerevisiae1,3-β-glucan synthase, Fks1. The structure reveals a central catalytic region adopting a cellulose synthase fold with a cytosolic conserved GT-A type glycosyltransferase domain and a closed transmembrane glucan-transporting channel. We found that two extracellular disulfide bonds are crucial for Fks1 enzymatic activity. Structural comparison between Fks1 and cellulose synthases and structure-guided mutagenesis studies provided novel insights into the molecular mechanisms of the fungal 1,3-β-glucan synthase.

[Image: see text] A series of brigatinib derivatives were designed and synthesized as new potent and selective EGFR(T790M/C797S) inhibitors. One of the most potent and selective compounds 18k strongly suppressed the EGFR(L858R/T790M/C797S) and EGFR(19Del/T790M/C797S) kinases with IC(50) values of 0.7 and 3.6 nM, respectively, which were over 54-fold more potent than the lead compound. 18k also demonstrated promising EGFR(T790M/C797S) mutant selectivity, and was 94-fold less potent against the wild type EGFR. A cocrystal structure of EGFR(T790M/C797S) with a close derivative 18f was solved to provide insight on the inhibitor’s binding mode. Moreover, compound 18k was orally bioavailable and demonstrated highly desirable PK properties, making it a promising lead compound for further structural optimization.

Upon binding to DNA breaks, poly(ADP-ribose) polymerase 1 (PARP1) ADP-ribosylates itself and other factors to initiate DNA repair. Serine is the major residue for ADP-ribosylation upon DNA damage, which strictly depends on HPF1. Here, we report the crystal structures of human HPF1/PARP1-CAT ΔHD complex at 1.98 Å resolution, and mouse and human HPF1 at 1.71 Å and 1.57 Å resolution, respectively. Our structures and mutagenesis data confirm that the structural insights obtained in a recent HPF1/PARP2 study by Suskiewicz et al. apply to PARP1. Moreover, we quantitatively characterize the key residues necessary for HPF1/PARP1 binding. Our data show that through salt-bridging to Glu284/Asp286, Arg239 positions Glu284 to catalyze serine ADP-ribosylation, maintains the local conformation of HPF1 to limit PARP1 automodification, and facilitates HPF1/PARP1 binding by neutralizing the negative charge of Glu284. These findings, along with the high-resolution structural data, may facilitate drug discovery targeting PARP1., Once DNA breaks occur, poly(ADP-ribose) polymerase 1 (PARP1) ADP-ribosylates itself and other DNA repair factors to initiate the repair process. Here, the authors resolve the crystal structures of mouse and human HPF1, and human HPF1/PARP1 complex proving insights into PARP1 regulation.

Schaftoside and isoschaftoside are bioactive natural products widely distributed in higher plants including cereal crops and medicinal herbs. Their biosynthesis may be related with plant defense. However, little is known on the glycosylation biosynthetic pathway of these flavonoid di-C-glycosides with different sugar residues. Herein, we report that the biosynthesis of (iso)schaftosides is sequentially catalyzed by two C-glycosyltransferases (CGTs), i.e., CGTa for C-glucosylation of the 2-hydroxyflavanone aglycone and CGTb for C-arabinosylation of the mono-C-glucoside. The two enzymes of the same plant exhibit high homology but remarkably different sugar acceptor and donor selectivities. A total of 14 CGTa and CGTb enzymes were cloned and characterized from seven dicot and monocot plants, including Scutellaria baicalensis, Glycyrrhiza uralensis, Oryza sativa ssp. japonica, and Zea mays, and the in vivo functions for three enzymes were verified by RNA interference and overexpression. Through transcriptome analysis, we found homologous genes in 119 other plants, indicating this pathway is general for the biosynthesis of (iso)schaftosides. Furthermore, we resolved the crystal structures of five CGTs and realized the functional switch of SbCGTb to SbCGTa by structural analysis and mutagenesis of key amino acids. The CGT enzymes discovered in this paper allow efficient synthesis of (iso)schaftosides, and the general glycosylation pathway presents a platform to study the chemical defense mechanisms of higher plants.

Extensive structure-activity relationships (SARs) study of JND3229 was conducted to yield a series of new reversible 2-oxo-3,4-dihydropyrimido[4,5-d]pyrimidine privileged scaffold as EGFRC797S inhibitors. One of the most potent compound 6i potently suppressed EGFRL858R/T790M/C797S kinase with an IC50 value of 3.1 nmol/L, and inhibited the proliferation of BaF3 cells harboring EGFRL858R/T790M/C797S and EGFR19D/T790M/C797S mutants with IC50 values of 290 nmol/L and 316 nmol/L, respectively. Further, 6i dose-dependently induced suppression of the phosphorylation of EGFRL858R/T790M/C797S and EGFR19D/T790M/C797S in BaF3 cells. Compound 6i may serve as a promising lead compound for further drug discovery overcoming the acquired resistance of non-small cell lung cancer (NSCLC) patients.

A highly efficient di-C-glycosyltransferase GgCGT was discovered from the medicinal plant Glycyrrhiza glabra. GgCGT catalyzes a two-step di-C-glycosylation of flopropione-containing substrates with conversion rates of >98%. To elucidate the catalytic mechanisms of GgCGT, we solved its crystal structures in complex with UDP-Glc, UDP-Gal, UDP/phloretin, and UDP/nothofagin, respectively. Structural analysis revealed that the sugar donor selectivity was controlled by the hydrogen-bond interactions of sugar hydroxyl groups with D390 and other key residues. The di-C-glycosylation capability of GgCGT was attributed to a spacious substrate-binding tunnel, and the G389K mutation could switch di- to mono-C-glycosylation. GgCGT is the first di-C-glycosyltransferase with a crystal structure, and the first C-glycosyltransferase with a complex structure containing a sugar acceptor. This work could benefit the development of efficient biocatalysts to synthesize C-glycosides with medicinal potential.

More than 60% of nonsmall cell lung cancer (NSCLC) patients show a positive response to the first ALK inhibitor, crizotinib, which has been used as the standard treatment for newly diagnosed patients with ALK rearrangement. However, most patients inevitably develop crizotinib resistance due to acquired secondary mutations in the ALK kinase domain, such as the gatekeeper mutation L1196M and the most refractory mutation, G1202R. Here, we develop XMU‐MP‐5 as a new‐generation ALK inhibitor to overcome crizotinib resistance mutations, including L1196M and G1202R. XMU‐MP‐5 blocks ALK signaling pathways and inhibits the proliferation of cells harboring either wild‐type or mutant EML4‐ALK in vitro and suppresses tumor growth in xenograft mouse models in vivo. Structural analysis provides insights into the mode of action of XMU‐MP‐5. In addition, XMU‐MP‐5 induces significant regression of lung tumors in two genetically engineered mouse (GEM) models, further demonstrating its pharmacological efficacy and potential for clinical application. These preclinical data support XMU‐MP‐5 as a novel selective ALK inhibitor with high potency and selectivity. XMU‐MP‐5 holds great promise as a new therapeutic against clinically relevant secondary ALK mutations., Despite the clinical success of ALK inhibitors in NSCLC, multiple drug‐resistant mutations in ALK are inevitably reported. XMU‐MP‐5 overcomes resistance to first and second generation ALK inhibitors in vitro and in vivo, thus holds great promise for the therapeutic use against ALK‐positive NSCLC.

Background and purpose Bruton's tyrosine kinase (BTK) plays a key role in B-cell receptor signalling by regulating cell proliferation and survival in various B-cell malignancies. Covalent low-MW BTK kinase inhibitors have shown impressive clinical efficacy in B-cell malignancies. However, the mutant BtkC481S poses a major challenge in the management of B-cell malignancies by disrupting the formation of the covalent bond between BTK and irreversible inhibitors, such as ibrutinib. The present studies were designed to develop novel BTK inhibitors targeting ibrutinib-resistant BtkC481S mutation. Experimental approach BTK-Ba/F3, BTK(C481S)-Ba/F3 cells, and human malignant B-cells JeKo-1, Ramos, and NALM-6 were used to evaluate cellular potency of BTK inhibitors. The in vitro pharmacological efficacy and compound selectivity were assayed via cell viability, colony formation, and BTK-mediated signalling. A tumour xenograft model with BTK-Ba/F3, Ramos and BTK(C481S)-Ba/F3 cells in Nu/nu BALB/c mice was used to assess in vivo efficacy of XMU-MP-3. Key results XMU-MP-3 is one of a group of low MW compounds that are potent non-covalent BTK inhibitors. XMU-MP-3 inhibited both BTK and the acquired mutant BTKC481S, in vitro and in vivo. Further computational modelling, site-directed mutagenesis analysis, and structure-activity relationships studies indicated that XMU-MP-3 displayed a typical Type-II inhibitor binding mode. Conclusion and implications XMU-MP-3 directly targets the BTK signalling pathway in B-cell lymphoma. These findings establish XMU-MP-3 as a novel inhibitor of BTK, which could serve as both a tool compound and a lead for further drug development in BTK relevant B-cell malignancies, especially those with the acquired ibrutinib-resistant C481S mutation.

Tertiary EGFRC797S mutation induced resistance against osimertinib (1) is an emerging "unmet clinical need" for non-small-cell lung cancer (NSCLC) patients. A series of 5-methylpyrimidopyridone derivatives were designed and synthesized as new selective EGFRL858R/T790M/C797S inhibitors. A representative compound, 8r-B, exhibited an IC50 of 27.5 nM against the EGFRL858R/T790M/C797S mutant, while being a significantly less potent for EGFRWT (IC50 > 1.0 μM). Cocrystallographic structure determination and computational investigation were conducted to elucidate its target selectivity.

ZAK is a new promising target for discovery of drugs with activity against antihypertrophic cardiomyopathy (HCM). A series of 1,2,3-triazole benzenesulfonamides were designed and synthesized as selective ZAK inhibitors. One of these compounds, 6p binds tightly to ZAK protein (Kd = 8.0 nM) and potently suppresses the kinase function of ZAK with single-digit nM (IC50 = 4.0 nM) and exhibits excellent selectivity in a KINOMEscan screening platform against a panel of 403 wild-type kinases. This compound dose dependently blocks p38/GATA-4 and JNK/c-Jun signaling and demonstrates promising in vivo anti-HCM efficacy upon oral administration in a spontaneous hypertensive rat (SHR) model. Compound 6p may serve as a lead compound for new anti-HCM drug discovery.

The exosome functions in the degradation of diverse RNA species, yet how it is negatively regulated remains largely unknown. Here, we show that NRDE2 forms a 1:1 complex with MTR4, a nuclear exosome cofactor critical for exosome recruitment, via a conserved MTR4-interacting domain (MID). Unexpectedly, NRDE2 mainly localizes in nuclear speckles, where it inhibits MTR4 recruitment and RNA degradation, and thereby ensures efficient mRNA nuclear export. Structural and biochemical data revealed that NRDE2 interacts with MTR4's key residues, locks MTR4 in a closed conformation, and inhibits MTR4 interaction with the exosome as well as proteins important for MTR4 recruitment, such as the cap-binding complex (CBC) and ZFC3H1. Functionally, MID deletion results in the loss of self-renewal of mouse embryonic stem cells. Together, our data pinpoint NRDE2 as a nuclear exosome negative regulator that ensures mRNA stability and nuclear export.

Schaftoside and isoschaftoside are bioactive natural products widely distributed in higher plants including cereal crops and medicinal herbs. Their biosynthesis may be related with plant defense. However, little is known on the glycosylation biosynthetic pathway of these flavonoid di-C-glycosides with different sugar residues. Herein, we report that the biosynthesis of (iso)schaftosides is sequentially catalyzed by two C-glycosyltransferases (CGTs), i.e., CGTa for C-glucosylation of the 2-hydroxyflavanone aglycone and CGTb for C-arabinosylation of the mono-C-glucoside. The two enzymes of the same plant exhibit high homology but remarkably different sugar acceptor and donor selectivities. A total of 14 CGTa and CGTb enzymes were cloned and characterized from seven dicot and monocot plants, including Scutellaria baicalensis, Glycyrrhiza uralensis, Oryza sativa ssp. japonica, and Zea mays, and the in vivo functions for three enzymes were verified by RNA interference and overexpression. Through transcriptome analysis, we found homologous genes in 119 other plants, indicating this pathway is general for the biosynthesis of (iso)schaftosides. Furthermore, we resolved the crystal structures of five CGTs and realized the functional switch of SbCGTb to SbCGTa by structural analysis and mutagenesis of key amino acids. The CGT enzymes discovered in this paper allow efficient synthesis of (iso)schaftosides, and the general glycosylation pathway presents a platform to study the chemical defense mechanisms of higher plants.

Upon binding to DNA breaks, poly(ADP-ribose) polymerase 1 (PARP1) ADP-ribosylates itself and other factors to initiate DNA repair. Serine is the major residue for ADP-ribosylation upon DNA damage, which strictly depends on HPF1. Here we report the crystal structures of human HPF1/PARP1-CAT ΔHD complex at 1.98 Å resolution and mouse and human HPF1 at 1.71 Å and 1.57 Å resolution, respectively. These structures and mutagenesis data confirm that the structural insights obtained in the HPF1/PARP2 study apply to PARP1. Moreover, we quantitatively characterize the key residues for HPF1/PARP1 binding. This information clarifies the confusion regarding HPF1/PARP1 assembly and can direct following functional studies. Our data show that through salt-bridging to Glu284/Asp286, Arg239 positions Glu284 for catalyzing serine ADP-ribosylation, maintains the local conformation of HPF1 to limit PARP1 automodification and facilitates HPF1/PARP1 binding through neutralizing the negative charge of Glu284. These findings and the high-resolution structural data may facilitate drug discovery targeting PARP1.

Macrolactins (MLNs) are a class of important antimacular degeneration and antitumor agents. Malonylated/succinylated MLNs are even more important due to their efficacy in overcoming multi-drug-resistant bacteria. However, which enzyme catalyzes this reaction remains enigmatic. Herein, we deciphered a β-lactamase homologue BmmI to be responsible for this step. BmmI could specifically attach C3-C5 alkyl acid thioesters onto 7-OH of MLN A and also exhibits substrate promiscuity toward acyl acceptors with different scaffolds. The crystal structure of BmmI covalently linked to the succinyl group and systematic mutagenesis highlighted the role of oxyanion holelike geometry in the recognition of carboxyl-terminated acyl donors. The engineering of this geometry expanded its substrate scope, with the R166A/G/Q variants recognizing up to C12 alkyl acid thioester. The structure of BmmI with acyl acceptor MLN A revealed the importance of Arg292 in the recognition of macrolide substrates. Moreover, the mechanism of the BmmI-catalyzed acyltransfer reaction was established, unmasking the deft role of Lys76 in governing acyl donors as well as catalysis. Our studies uncover the delicate mechanism underlying the substrate selectivity of acyltransferases, which would guide rational enzyme engineering for drug development.

AZD9291 (Osimertinib) is highly effective in treating EGFR-mutated non-small-cell lung cancers (NSCLCs) with T790M-mediated drug resistance. Despite the remarkable success of AZD9291, its binding pose with EGFR T790M remains unclear. Here, we report unbiased, atomic-level molecular dynamics (MD) simulations in which spontaneous association of AZD9291 with EGFR kinases having WT and T790M mutant gatekeepers was observed. Simulation-generated structural models suggest that the binding pose of AZD9291 with T790M differs from its binding pose with the WT, and that AZD9291 interacts extensively with the gatekeeper residue (Met 790) in T790M but not with Thr 790 in the WT, which explains why AZD9291 binds T790M with higher affinity. The MD simulation-generated models were confirmed by experimentally determined EGFR/T790M complex crystal structures. This work may facilitate the rational design of drugs that can overcome resistance mutations to AZD9291, and more generally it suggests the potential of using unbiased MD simulation to elucidate small-molecule binding poses.

Microtubule inhibitors as chemotherapeutic drugs are widely used for cancer treatment. However, the development of multidrug resistance (MDR) in cancer is a major challenge for microtubule inhibitors in their clinical implementation. From a high-throughput drug screen using cells transformed by oncogenic RAS, we identify a lead heteroaryl amide compound that blocks cell proliferation. Analysis of the structure-activity relationship indicated that this series of scaffolds (exemplified by MP-HJ-1b) represents a potent inhibitor of tumor cell growth. MP-HJ-1b showed activities against a panel of more than 1,000 human cancer cell lines with a wide variety of tissue origins. This compound depolymerized microtubules and affected spindle formation. It also induced the spike-like conformation of microtubules in vitro and in vivo, which is different from typical microtubule modulators. Structural analysis revealed that this series of compounds bound the colchicine pocket at the intra-dimer interface, although mostly not overlapping with colchicine binding. MP-HJ-1b displayed favorable pharmacological properties for overcoming tumor MDR, both in vitro and in vivo. Taken together, our data reveal a novel scaffold represented by MP-HJ-1b that can be developed as a cancer therapeutic against tumors with MDR. Significance: Paclitaxel is a widely used chemotherapeutic drug in patients with multiple types of cancer. However, resistance to paclitaxel is a challenge. This study describes a novel class of microtubule inhibitors with the ability to circumvent multidrug resistance across multiple tumor cell lines. Cancer Res; 78(20); 5949–57. ©2018 AACR.

Through exploration of the non-highly conserved allosteric hydrophobic pocket generated by DFG-out shifting in the inactive conformation, we discovered a highly selective type II PDGFRα kinase inhibitor 15i (CHMFL-PDGFRα-159), which exhibited strong potency against purified PDGFRα (IC50: 132 nM) but not structurally similar PDGFRβ, ABL, c-KIT and VEGFR2 kinases. In addition, it displayed a high selectivity profile (S score (10) = 0.02) at the concentration of 1 μM among 468 kinases/mutants in the KINOMEscan profiling. X-ray crystal structure of 15i in complex with PDGFRα revealed a distinct binding feature in the allosteric hydrophobic pocket which might help to expand the diversity of type II kinase inhibitors. Compound 15i potently inhibited the proliferation of PDGFRα driving Chronic Eosinophilic Leukemia (CEL) cell line EOL-1 through strong blockage of PDGFRα mediated signaling pathways, arresting cell cycle progression, and induction of apoptosis. Furthermore, compound 15i effectively suppressed the EOL-1 tumor progression in the xenograft model and increased the survival rate in the engraftment tumor model.

RCC2 (also known as TD60) is a highly conserved protein involved in prognosis in colorectal cancer. However, its relationship with tumor development is less understood. Here we demonstrate a signaling pathway defining regulation of RCC2 and its functions in tumor progression. We report that p53 is a transcriptional regulator of RCC2 that acts through its binding to a palindromic motif in the RCC2 promoter. RCC2 physically interacts and deactivates a small GTPase Rac1 that is known to be involved in metastasis. We solved a high-resolution crystal structure of RCC2 and revealed one RCC1-like domain with a unique β-hairpin that is requisite for RCC2 interaction with Rac1. p53 or RCC2 deficiency leads to activation of Rac1 and deterioration of extracellular matrix sensing (haptotaxis) of surface-bound gradients. Ectopic expression of RCC2 restores directional migration in p53-null cells. Our results demonstrate that p53 and RCC2 signaling is important for regulation of cell migration and suppression of metastasis. We propose that the p53/RCC2/Rac1 axis is a potential target for cancer therapy.

A series of N-(3-((1H-pyrazolo[3,4-b]pyridin-5-yl)ethynyl)benzenesulfonamides were designed as the first class of highly selective ZAK inhibitors. The representative compound 3h strongly inhibits the kinase activity of ZAK with an IC50 of 3.3 nM and dose-dependently suppresses the activation of ZAK downstream signals in vitro and in vivo, while it is significantly less potent for the majority of 403 nonmutated kinases evaluated. Compound 3h also exhibits orally therapeutic effects on cardiac hypertrophy in a spontaneous hypertensive rat model.

// Chen Hu 1, 2, * , Aoli Wang 1, 2, * , Hong Wu 1, 3, * , Ziping Qi 1, 3, * , Xixiang Li 1, 3, * , Xiao-E Yan 4, * , Cheng Chen 1, 2 , Kailin Yu 1, 2 , Fengming Zou 1, 3 , Wenchao Wang 1, 3 , Wei Wang 1, 3 , Jiaxin Wu 1, 2 , Juan Liu 1, 2 , Beilei Wang 1, 2 , Li Wang 1, 2 , Tao Ren 5 , Shanchun Zhang 3, 6 , Cai-Hong Yun 4 , Jing Liu 1, 3 , Qingsong Liu 1, 2, 3, 5 1 High Magnetic Field Laboratory, Chinese Academy of Sciences, Hefei, Anhui 230031, P. R. China 2 University of Science and Technology of China, Hefei, Anhui 230036, P. R. China 3 CHMFL-HCMTC Target Therapy Joint Laboratory, Hefei, Anhui 230031, P. R. China 4 Institute of Systems Biomedicine, Department of Biophysics, Beijing Key Laboratory of Tumor Systems Biology and Center for Molecular and Translational Medicine, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191, P.R. China 5 Precision Targeted Therapy Discovery Center, Institute of Technology Innovation, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, Anhui 230088, P. R. China 6 Hefei Cosource Medicine Technology Co. LTD., Hefei, Anhui 230031, P. R. China * These authors have contributed equally to this work Correspondence to: Cai-Hong Yun, email: yunch@hsc.pku.edu.cn Jing Liu, email: jingliu@hmfl.ac.cn Qingsong Liu, email: qsliu97@hmfl.ac.cn Keywords: EGFR, EGFRT790M, NSCLC, kinase inhibitors, drug resistance Received: December 09, 2016 Accepted: January 23, 2017 Published: February 17, 2017 ABSTRACT EGFR T790M mutation accounts for about 40-55% drug resistance for the first generation EGFR kinase inhibitors in the NSCLC. Starting from ibrutinib, a highly potent irreversible BTK kinase inhibitor, which was also found to be moderately active to EGFR T790M mutant, we discovered a highly potent irreversible EGFR inhibitor CHMFL-EGFR-26, which is selectively potent against EGFR mutants including L858R, del19, and L858R/T790M. It displayed proper selectivity window between the EGFR mutants and the wide-type. CHMFL-EGFR-26 exhibited good selectivity profile among 468 kinases/mutants tested (S score (1)=0.02). In addition, X-ray crystallography revealed a distinct “DFG-in” and “cHelix-out” inactive binding mode between CHMFL-EGFR-26 and EGFR T790M protein. The compound showed highly potent anti-proliferative efficacy against EGFR mutant but not wide-type NSCLC cell lines through effective inhibition of the EGFR mediated signaling pathway, induction of apoptosis and arresting of cell cycle progression. CHMFL-EGFR-26 bore acceptable pharmacokinetic properties and demonstrated dose-dependent tumor growth suppression in the H1975 (EGFR L858R/T790M) and PC-9 (EGFR del19) inoculated xenograft mouse models. Currently CHMFL-EGFR-26 is undergoing extensive pre-clinical evaluation for the clinical trial purpose.

Structural optimization of pyrido[2,3-d]pyrimidin-7-ones was conducted to yield a series of new selective EGFRT790M inhibitors with improved pharmacokinetic properties. One of the most promising compound 9s potently suppressed EGFRL858R/T790M kinase and inhibited the proliferation of H1975 cells with IC50 values of 2.0 nM and 40 nM, respectively. The compound dose-dependently induced reduction of the phosphorylation of EGFR and downstream activation of ERK in NCIH1975 cells. It also exhibited moderate plasma exposure after oral administration and an oral bioavailability value of 16%. Compound 9s may serve as a promising lead compound for further drug discovery overcoming the acquired resistance of non-small cell lung cancer (NSCLC) patients.

[Image: see text] Allosteric kinase inhibitors represent a promising new therapeutic strategy for targeting kinases harboring oncogenic driver mutations in cancers. Here, we report the discovery, optimization, and structural characterization of allosteric mutant-selective EGFR inhibitors comprising a 5,10-dihydro-11H-dibenzo[b,e][1,4]diazepin-11-one scaffold. Our structure-based medicinal chemistry effort yielded an inhibitor (3) of the EGFR(L858R/T790M) and EGFR(L858R/T790M/C797S) mutants with an IC(50) of ∼10 nM and high selectivity, as assessed by kinome profiling. Further efforts to develop allosteric dibenzodiazepinone inhibitors may serve as the basis for new therapeutic options for targeting drug-resistant EGFR mutations.

Tertiary EGFR

Herein, the catalytic promiscuity of TcCGT1, a new C-glycosyltransferase (CGT) from the medicinal plant Trollius chinensis is explored. TcCGT1 could efficiently and regio-specifically catalyze the 8-C-glycosylation of 36 flavones and other flavonoids and could also catalyze the O-glycosylation of diverse phenolics. The crystal structure of TcCGT1 in complex with uridine diphosphate was determined at 1.85 A resolution. Molecular docking revealed a new model for the catalytic mechanism of TcCGT1, which is initiated by the spontaneous deprotonation of the substrate. The spacious binding pocket explains the substrate promiscuity, and the binding pose of the substrate determines C- or O-glycosylation activity. Site-directed mutagenesis at two residues (I94E and G284K) switched C- to O-glycosylation. TcCGT1 is the first plant CGT with a crystal structure and the first flavone 8-C-glycosyltransferase described. This provides a basis for designing efficient glycosylation biocatalysts.

Platelet-derived growth factor receptor α (PDGFRA) is a Type III receptor tyrosine kinase, and this kinase is a target for treatment of gastrointestinal stromal tumors (GIST) as it is frequently mutated in these cancers. Most of the mutations that cause constitutive activation of PDGFRA occur in either the activation loop (A-loop) or in the juxtamembrane (JM) domain, such as the mutations D842V or V561D respectively. Treatment of PDGFRA-mutated GIST with imatinib is successful in some cases, but the D842V mutation is imatinib-resistant. To better understand the mechanism of PDGFRA drug-resistance, we have determined the crystal structure of the PDGFRA kinase domain in the auto-inhibited form, and studied the kinetics of the D842V mutation. Auto-inhibited PDGFRA is stabilized by the JM domain, which inserts into the active site of the kinase. The conserved residue Asp842 makes extensive contacts with several A-loop residues to maintain PDGFRA in the "DFG out" conformation, which stabilizes the kinase in the inactive state and facilitates the binding of imatinib. The D842V mutation would therefore be expected to activate the kinase and hinder the binding of drug through destabilizing the "DFG out" conformation. Furthermore, our kinetic data show that drug resistance in the D842V mutation may also in part result from its increased affinity for ATP. The PDGFRA kinase domain structure we report in this study has potential to facilitate development of new agents which can inhibit this kinase, including both its activating and drug-resistant mutations.

// Su Hang 1, 2 , Agnes F.Y. Tiwari 1 , Hextan Y.S. Ngan 3 , Yim-Ling Yip 4 , Annie L.M. Cheung 4 , Sai Wah Tsao 4 , Wen Deng 1 1 School of Nursing, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, SAR, China 2 College of Forensic Sciences, Xi’an Jiaotong University Health Science Center, Xi’an, Shanxi Province, P.R. China 3 Department of Obstetrics and Gynaecology, The University of Hong Kong, Hong Kong, SAR, China 4 School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, SAR, China Correspondence to: Wen Deng, email: wdeng@hku.hk Keywords: cervical epithelial cells, immortalization, p16 INK4a , telomerase, HPV-negative Received: March 13, 2016 Accepted: May 29, 2016 Published: June 17, 2016 ABSTRACT Cervical epithelial cell immortalization with defined genetic factors without viral oncogenes has never been reported. Here we report that HPV-negative cervical epithelial cells failed to be immortalized by telomerase activation or the combination of p53 knockdown and telomerase activation. Under those conditions, p16 INK4a expression was always elevated during the late stage of limited cell lifespan, suggesting that cervical epithelial cells possess an intrinsic property of uniquely stringent activation of p16 INK4a , which may offer an explanation for the rarity of HPV-negative cervical cancer. Combining p16 INK4a knockdown with telomerase activation resulted in efficient immortalization of HPV-negative cervical epithelial cells under ordinary culture conditions. Compared with the HPV16-E6E7-immortalized cell lines derived from the same primary cell sources, the novel HPV-negative immortalized cell lines had lower degrees of chromosomal instability, maintained more sensitive p53/p21 response to DNA damage, exhibited more stringent G2 checkpoint function, and were more resistant to replication-stress-induced genomic instability. The newly immortalized HPV-negative cervical epithelial cell lines were non-tumorigenic in nude mice. The cell lines can be used not only as much-needed HPV-negative non-malignant cell models but also as starting models that can be genetically manipulated in a stepwise fashion to investigate the roles of defined genetic alterations in the development of HPV-negative cervical cancer.

Bruton's tyrosine kinase (BTK) plays a key role in B-cell receptor signalling by regulating cell proliferation and survival in various B-cell malignancies. Covalent low-MW BTK kinase inhibitors have shown impressive clinical efficacy in B-cell malignancies. However, the mutant BtkBTK-Ba/F3, BTK(C481S)-Ba/F3 cells, and human malignant B-cells JeKo-1, Ramos, and NALM-6 were used to evaluate cellular potency of BTK inhibitors. The in vitro pharmacological efficacy and compound selectivity were assayed via cell viability, colony formation, and BTK-mediated signalling. A tumour xenograft model with BTK-Ba/F3, Ramos and BTK(C481S)-Ba/F3 cells in Nu/nu BALB/c mice was used to assess in vivo efficacy of XMU-MP-3.XMU-MP-3 is one of a group of low MW compounds that are potent non-covalent BTK inhibitors. XMU-MP-3 inhibited both BTK and the acquired mutant BTKC481S, in vitro and in vivo. Further computational modelling, site-directed mutagenesis analysis, and structure-activity relationships studies indicated that XMU-MP-3 displayed a typical Type-II inhibitor binding mode.XMU-MP-3 directly targets the BTK signalling pathway in B-cell lymphoma. These findings establish XMU-MP-3 as a novel inhibitor of BTK, which could serve as both a tool compound and a lead for further drug development in BTK relevant B-cell malignancies, especially those with the acquired ibrutinib-resistant C481S mutation.

[Image: see text] EGFR(C797S) mutation inducing resistance against third generation EGFR inhibitor drugs is an emerging “unmet clinical need” for nonsmall cell lung cancer patients. The pyrimidopyrimidinone derivative JND3229 was identified as a new highly potent EGFR(C797S) inhibitor with single digit nM potency. It also exhibited good in vitro and in vivo monodrug anticancer efficacy in a xenograft mouse model of BaF3/EGFR(19D/T790M/C797S) cells. A high-resolution X-ray crystallographic structure was also determined to elucidate the interactions between JND3229 and EGFR(T790M/C797S). Our study provides an important structural and chemical basis for future development of new generation EGFR(C797S) inhibitors as anticancer drugs.

Some mutations of isocitrate dehydrogenase 1 and 2 observed in multiple kinds of malignant tumors can lead to a neomorphic enzyme activity that converts alpha-ketoglutarate (α-KG) to 2-hydroxyglutarate (2-HG). As an oncometabolite, 2-HG can cause epigenetic changes and impair cell differentiation. Inhibiting the activity of isocitrate dehydrogenase mutants (mIDH) is considered to be an effective therapy for the treatment of mIDH positive cancers, including glioma and acute myeloid leukemia (AML). The presently disclosed allosteric inhibitors work only on one of the mIDH1 and mIDH2, and it is shown that mIDH1 and mIDH2 have different allosteric inhibition pockets. However, AG-881 from Agios Pharmaceuticals was found to be a pan-IDH inhibitor against both mIDH1 and mIDH2, and is undergoing Phase I clinical trials for tumors with an IDH1 and/or IDH2 mutation. To understand the binding mode of AG-881 to mIDHs, we solved the crystal structures of IDH1-R132H/NADPH/AG-881 and IDH2-R140Q/NADPH/AG-881 complexes, and acquired the IC50 values of AG-881 for IDH1-R132H and IDH2-R140Q homodimers after different pre-incubation times. Our data show that AG-881 binds IDH1-R132H and IDH2-R140Q in the same allosteric pockets and that the subtle difference in the pockets of these two proteins may contribute to their remarkably different inhibitory kinetics by AG-881. The structural pharmacological data provided in this report may benefit the future development of pan-IDH inhibitors targeting mIDH1 and mIDH2.

Lung cancer is the leading cause of cancer deaths. Epidermal growth factor receptor (EGFR) kinase domain mutations are a common cause of non-small cell lung cancers (NSCLCs), a major subtype of lung cancers. Patients harboring most of these mutations respond well to the anti-EGFR tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib initially, but soon develop resistance to them in about half of the cases due to the emergence of the gatekeeper mutation T790M. The third-generation TKIs such as AZD9291, HM61713, CO-1686 and WZ4002 can overcome T790M through covalent binding to the EGFR kinase through Cys 797, but ultimately lose their efficacy upon emergence of the C797S mutation that abolishes the covalent bonding. Therefore to develop new TKIs to overcome EGFR drug-resistant mutants harboring T790M/C797S is urgently demanded. EAI001 and EAI045 are a new type of EGFR TKIs that bind to EGFR reversibly and not relying on Cys 797. EAI045 in combination with cetuximab is effective in mouse models of lung cancer driven by EGFR L858R/T790M and L858R/T790M/C797S. Here we report the crystal structure of EGFR T790M/C797S/V948R in complex with EAI045, and compare it to EGFR T790M/V948R in complex with EAI001. The complex structure reveals why EAI045 binds tighter to EGFR than does EAI001, and why EAI001 and EAI045 prefer binding to EGFR T790M. The knowledge may facilitate future drug development studies targeting this very important cancer target.

Diabetes-induced endothelial cell (EC) dysfunction and neovascularization impairment constitute vascular complications with limited treatment regimens. Transcription factor FOXO1 is a key angiogenic regulator and plays a pathologic role in progression of diabetes. The present study was designed to determine the involvement of FOXO1 in impaired EC function and post-ischemic neovascularization in diabetes and investigate underlying mechanisms. We found that FOXO1-selective inhibitor AS1842856 improved blood flow recovery and capillary density in ischemic hindlimb, and rescued the delay of wound closure with a concomitant augmentation of mean perfusion rate in diabetic mice. In vitro, treatment with AS1842856 or FOXO1 siRNA abrogated high glucose-induced apoptosis and ameliorated capillary tube formation in human umbilical vein endothelial cells (HUVECs). FOXO1 inhibition relieved alterations in mitochondrial networks and significantly suppressed the overproduction of mitochondrial reactive oxygen species (mtROS) induced by high glucose in ECs. Expression of dynamin-related protein-1 (Drp1) and phosphorylation at Ser616, a protein required for mitochondrial fission, were enhanced by hyperglycemia, which could be neutralized by FOXO1 inhibition. Moreover, the transcription of Rho-associated coiled-coil containing protein kinase 1 (ROCK1), which phosphorylates Drp1 at Ser616, was shown by luciferase assay to be directly regulated by FOXO1. These findings suggested that FOXO1 is critical to preserve mitochondrial quantity and function in ECs, and FOXO1 may serve as a therapeutic target for microvascular complications of diabetes.

Aberrant activation of the EGF receptor (EGFR) contributes to many human cancers by activating the Ras-MAPK pathway and other pathways. EGFR signaling is augmented by Src-family kinases, but the mechanism is poorly understood. Here, we show that human EGFR preferentially phosphorylates peptide substrates that are primed by a prior phosphorylation. Using peptides based on the sequence of the adaptor protein Shc1, we show that Src mediates the priming phosphorylation, thus promoting subsequent phosphorylation by EGFR. Importantly, the doubly phosphorylated Shc1 peptide binds more tightly than singly phosphorylated peptide to the Ras activator Grb2; this binding is a key step in activating the Ras-MAPK pathway. Finally, a crystal structure of EGFR in complex with a primed Shc1 peptide reveals the structural basis for EGFR substrate specificity. These results provide a molecular explanation for the integration of Src and EGFR signaling with downstream effectors such as Ras.

// Hong Wu 1, 2, * , Aoli Wang 1, 2, * , Wei Zhang 3, * , Beilei Wang 1, * , Cheng Chen 1, * , Wenchao Wang 1 , Chen Hu 1 , Zi Ye 4 , Zheng Zhao 1 , Li Wang 1 , Xixiang Li 1 , Kailin Yu 1 , Juan Liu 1 , Jiaxin Wu 1, 2 , Xiao-E Yan 5 , Peng Zhao 5 , Jinhua Wang 6 , Chu Wang 4 , Ellen L. Weisberg 7 , Nathanael S. Gray 6 , Cai-Hong Yun 5 , Jing Liu 1 , Liang Chen 3 , Qingsong Liu 1, 2, 8 1 High Magnetic Field Laboratory, Chinese Academy of Sciences, Hefei 230031, Anhui, P. R. China 2 University of Science and Technology of China, Hefei 230036, Anhui, P. R. China 3 Collaborative Innovation Center of Cancer Medicine, National Institute of Biological Sciences, Beijing, Beijing 102206, P.R. China 4 Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, College of Chemistry and Molecular Engineering, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, P.R. China 5 Institute of Systems Biomedicine, Department of Biophysics, Beijing Key Laboratory of Tumor Systems Biology and Center for Molecular and Translational Medicine, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191, P.R. China 6 Department of Cancer Biology, Dana-Farber Cancer Institute, and Department of Biological Chemistry & Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA 7 Department of Medical Oncology, Dana Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA 8 Hefei Science Center, Chinese Academy of Sciences, Hefei 230031, Anhui, P. R. China * These authors have contributed equally to this work Correspondence to: Jing Liu, e-mail: jingliu@hmfl.ac.cn Liang Chen, e-mail: chenliang@nibs.ac.cn Qingsong Liu, e-mail: qsliu97@hmfl.ac.cn Keywords: ibrutinib, NSCLC, EGFR mutation, drug resistance, drug combination Received: June 24, 2015 Accepted: August 24, 2015 Published: September 05, 2015 ABSTRACT Through comprehensive comparison study, we found that ibrutinib, a clinically approved covalent BTK kinase inhibitor, was highly active against EGFR (L858R, del19) mutant driven NSCLC cells, but moderately active to the T790M ‘gatekeeper’ mutant cells and not active to wild-type EGFR NSCLC cells. Ibrutinib strongly affected EGFR mediated signaling pathways and induced apoptosis and cell cycle arrest (G0/G1) in mutant EGFR but not wt EGFR cells. However, ibrutinib only slowed down tumor progression in PC-9 and H1975 xenograft models. MEK kinase inhibitor, GSK1120212, could potentiate ibrutinib’s effect against the EGFR (L858R/T790M) mutation in vitro but not in vivo . These results suggest that special drug administration might be required to achieve best clinical response in the ongoing phase I/II clinical trial with ibrutinib for NSCLC.

Thrombin is an effector enzyme for hemostasis and thrombosis; however, endogenous regulators of thrombin remain elusive. Cartilage oligomeric matrix protein (COMP), a matricellular protein also known as thrombospondin-5, is essential for maintaining vascular homeostasis. Here, we asked whether COMP is involved in the process of blood coagulation. COMP deficiency shortened tail-bleeding and clotting time and accelerated ferric-chloride-induced thrombosis in mice. The absence of COMP had no effect on platelet count. In contrast, COMP specifically inhibited thrombin-induced platelet aggregation, activation, and retraction and the thrombin-mediated cleavage of fibrinogen. Furthermore, surface plasmon resonance analysis revealed direct thrombin-COMP binding (KD = 1.38 ± 0.24 μM). In particular, blockage of thrombin exosites with compounds specific for exosite I (hirudin and HD1 aptamer) or exosite II (heparin and HD22 aptamer) impaired the COMP-thrombin interaction, indicating a 2-site binding mechanism. Additionally, epidermal growth factor-like repeats (amino acids 84-261) were identified as a COMP binding site for thrombin. Moreover, COMP was expressed in and secreted by platelets. Using bone marrow transplantation and platelet transfusion to create chimeric mice, platelet-derived but not vessel-wall-derived COMP was demonstrated to inhibit coagulation. Taken together, COMP is an endogenous thrombin inhibitor and negative regulator of hemostasis and thrombosis.

P53-binding protein 1 (53BP1) plays critical roles in DNA double strand break (DSB) repair by promoting non-homologous end joining (NHEJ), and loss of 53BP1 abolishes PARPi sensitivity in BRCA1-deficient cells by restoring homologous recombination (HR). 53BP1 is one of the proteins initially recruited to sites of DSBs via recognition of H4K20me2 through the Tudor-UDR domain and H2AK15ub through the UDR motif. Although extensive studies have been conducted, it remains unclear how the post-translational modification of 53BP1 affects DSB repair pathway choice. Here, we identified 53BP1 as an acetylated protein and determined that acetylation of 53BP1 inhibit NHEJ and promote HR by negatively regulating 53BP1 recruitment to DSBs. Mechanistically, CBP-mediated acetylation of K1626/1628 in the UDR motif disrupted the interaction between 53BP1 and nucleosomes, subsequently blocking the recruitment of 53BP1 and its downstream factors PTIP and RIF1 to DSBs. Hyperacetylation of 53BP1, similar to depletion of 53BP1, restored PARPi resistance in BRCA1-deficient cells. Interestingly, 53BP1 acetylation was tightly regulated by HDAC2 to maintain balance between the HR and NHEJ pathways. Together, our results demonstrate that the acetylation status of 53BP1 plays a key role in its recruitment to DSBs and reveal how specific 53BP1 modification modulates the choice of DNA repair pathway.

Treatment of non-small-cell lung cancers (NSCLCs) harboring primary EGFR oncogenic mutations such as L858R and exon 19 deletion delE746_A750 (Del-19) using gefitinib/erlotinib ultimately fails due to the emergence of T790M mutation. Though WZ4002/CO-1686/AZD9291 are effective in overcoming EGFR T790M by targeting Cys797 via covalent bonding, their efficacy is again limited due to the emergence of C797S mutation. New agents effectively inhibiting EGFR T790M without covalent linkage through Cys 797 may solve this problem. We presented here crystal structures of EGFR activating/drug-resistant mutants in complex with a panel of reversible inhibitors along with mutagenesis and enzyme kinetic data. These data revealed a previously un-described hydrophobic clamp structure in the EGFR kinase which may be exploited to facilitate development of next generation drugs targeting EGFR T790M with or without concomitant C797S. Interestingly, mutations in the hydrophobic clamp that hinder drug binding often also weaken ATP binding and/or abolish kinase activity, thus do not readily result in resistance to the drugs.

Drug-resistance is a major challenge in targeted therapy of EGFR mutated non-small cell lung cancers (NSCLCs). The third-generation irreversible inhibitors such as AZD9291, CO-1686 and WZ4002 can overcome EGFR T790M drug-resistance mutant through covalent binding through Cys 797, but ultimately lose their efficacy upon emergence of the new mutation C797S. To develop new reversible inhibitors not relying on covalent binding through Cys 797 is therefore urgently demanded. Go6976 is a staurosporine-like reversible inhibitor targeting T790M while sparing the wild-type EGFR. In the present work, we reported the complex crystal structures of EGFR T790M/C797S + Go6976 and T790M + Go6976, along with enzyme kinetic data of EGFR wild-type, T790M and T790M/C797S. These data showed that the C797S mutation does not significantly alter the structure and function of the EGFR kinase, but increases the local hydrophilicity around residue 797. The complex crystal structures also elucidated the detailed binding mode of Go6976 to EGFR and explained why this compound prefers binding to T790M mutant. These structural pharmacological data would facilitate future drug development studies.