Structure-Based Optimization of Pyrazinamide-Containing Macrocyclic Derivatives as Fms-like Tyrosine Kinase 3 (FLT3) Inhibitors to Overcome Clinical Mutations.

Secondary mutations in Fms-like tyrosine kinase 3-tyrosine kinase domain (FLT3-TKD) (e.g., D835Y and F691L) have become a major on-target resistance mechanism of FLT3 inhibitors, which present a significant clinical challenge. To date, no effective drugs have been approved to simultaneously overcome clinical resistance caused by these two mutants. Thus, a series of pyrazinamide macrocyclic compounds were first designed and evaluated to overcome the secondary mutations of FLT3. The representative 8v exhibited potent inhibitory activities against FLT3D835Y and FLT3D835Y/F691L with IC50 values of 1.5 and 9.7 nM, respectively. 8v also strongly suppressed the proliferation against Ba/F3 cells transfected with FLT3-ITD, FLT3-ITD-D835Y, FLT3-ITD-F691L, FLT3-ITD-D835Y-F691L, and MV4-11 acute myeloid leukemia (AML) cell lines with IC50 values of 12.2, 10.5, 24.6, 16.9, and 6.8 nM, respectively. Furthermore, 8v demonstrated ideal anticancer efficacy in a Ba/F3-FLT3-ITD-D835Y xenograft model. The results suggested that 8v can serve as a promising macrocycle-based FLT3 inhibitor for the treatment of AML.

Kinase Inhibition via Small Molecule-Induced Intramolecular Protein Cross-Linking.

Remarkable progress has been made in the development of cysteine-targeted covalent inhibitors. In kinase drug discovery, covalent inhibitors capable of targeting other nucleophilic residues (i.e. lysine, or K) has emerged in recent years. Besides a highly conserved catalytic lysine, almost all human protein kinases possess an equally conserved glutamate/aspartate (e.g. E/D) that forms a K-E/D salt bridge within the enzyme active-site. Electrophilic ynamides were previously used as effective peptide coupling reagents and to develop E/D-targeting covalent protein inhibitors/probes. In the present study, we report the first ynamide-based small-molecule inhibitors capable of inducing intramolecular cross-linking of various protein kinases, leading to subsequent irreversible inhibition of kinase activity. Our strategy took advantage of the close distance between the highly conserved catalytic K and E/D residues in a targeted kinase, thus providing a conceptually general approach to achieve irreversible kinase inhibition with high specificity and desirable cellular potency. Finally, this ynamide-facilitated, ligand-induced mechanism leading to intramolecular kinase cross-linking and inhibition was unequivocally established by using recombinant ABL kinase as a representative.

Bacterial host adaptation through sequence and structural variations of a single type III effector gene.

Molecular mechanisms underlying quantitative variations of pathogenicity remain elusive. Here, we identified the Xanthomonas campestris XopJ6 effector that triggers disease resistance in cauliflower and Arabidopsis thaliana. XopJ6 is a close homolog of the Ralstoniapseudosolanacearum PopP2 YopJ family acetyltransferase. XopJ6 is recognized by the RRS1-R/RPS4 NLR pair that integrates a WRKY decoy domain mimicking effector targets. We identified a XopJ6 natural variant carrying a single residue substitution in XopJ6 WRKY-binding site that disrupts interaction with WRKY proteins. This mutation allows XopJ6 to evade immune perception while retaining some XopJ6 virulence functions. Interestingly, xopJ6 resides in a Tn3-family transposon likely contributing to xopJ6 copy number variation (CNV). Using synthetic biology, we demonstrate that xopJ6 CNV tunes pathogen virulence on Arabidopsis through gene dosage-mediated modulation of xopJ6 expression. Together, our findings highlight how sequence and structural genetic variations restricted at a particular effector gene contribute to bacterial host adaptation.

Discovery of 6-Formylpyridyl Urea Derivatives as Potent Reversible-Covalent Fibroblast Growth Factor Receptor 4 Inhibitors with Improved Anti-Hepatocellular Carcinoma Activity.

Fibroblast growth factor receptor 4 (FGFR4) has been considered as a potential anticancer target due to FGF19/FGFR4 mediated aberrant signaling in hepatocellular carcinoma (HCC). Several FGFR4 inhibitors have been reported, but none have gained approval. Herein, a series of 5-formyl-pyrrolo[3,2-b]pyridine-3-carboxamides and a series of 6-formylpyridyl ureas were characterized as selective reversible-covalent FGFR4 inhibitors. The representative 6-formylpyridyl urea 8z exhibited excellent potency against FGFR4WT, FGFR4V550L, and FGFR4V550M with IC50 values of 16.3, 12.6, and 57.3 nM, respectively. It also potently suppressed proliferation of Ba/F3 cells driven by FGFR4WT, FGFR4V550L, and FGFR4V550M, and FGFR4-dependent Hep3B and Huh7 HCC cells, with IC50 values of 1.2, 13.5, 64.5, 15.0, and 20.4 nM, respectively. Furthermore, 8z displayed desirable microsomal stability and significant in vivo efficacy in the Huh7 HCC cancer xenograft model in nude mice. The study provides a promising new lead for anticancer drug discovery directed toward overcoming FGFR4 gatekeeper mutation mediated resistance in HCC patients.

Global Reactivity Profiling of the Catalytic Lysine in Human Kinome for Covalent Inhibitor Development.

Advances in targeted covalent inhibitors (TCIs) have been made by using lysine-reactive chemistries. Few aminophiles possessing balanced reactivity/stability for the development of cell-active TCIs are however available. We report herein lysine-reactive activity-based probes (ABPs; 2-14) based on the chemistry of aryl fluorosulfates (ArOSO2 F) capable of global reactivity profiling of the catalytic lysine in human kinome from mammalian cells. We concurrently developed reversible covalent ABPs (15/16) by installing salicylaldehydes (SA) onto a promiscuous kinase-binding scaffold. The stability and amine reactivity of these probes exhibited a broad range of tunability. X-ray crystallography and mass spectrometry (MS) confirmed the successful covalent engagement between ArOSO2 F on 9 and the catalytic lysine of SRC kinase. Chemoproteomic studies enabled the profiling of >300 endogenous kinases, thus providing a global landscape of ligandable catalytic lysines of the kinome. By further introducing these aminophiles into VX-680 (a noncovalent inhibitor of AURKA kinase), we generated novel lysine-reactive TCIs that exhibited excellent in vitro potency and reasonable cellular activities with prolonged residence time. Our work serves as a general guide for the development of lysine-reactive ArOSO2 F-based TCIs.

Novel Covalent Probe Selectively Targeting Glutathione Peroxidase 4 In Vivo: Potential Applications in Pancreatic Cancer Therapy.

Glutathione peroxidase 4 (GPX4) emerges as a promising target for the treatment of therapy-resistant cancer through ferroptosis. Thus, there is a broad interest in the development of GPX4 inhibitors. However, a majority of reported GPX4 inhibitors utilize chloroacetamide as a reactive electrophilic warhead, and the selectivity and pharmacokinetic properties still need to be improved. Herein, we developed a compound library based on a novel electrophilic warhead, the sulfonyl ynamide, and executed phenotypic screening against pancreatic cancer cell lines. Notably, one compound A16 exhibiting potent cell toxicity was identified. Further chemical proteomics investigations have demonstrated that A16 specifically targets GPX4 under both in situ and in vivo conditions, inducing ferroptosis. Importantly, A16 exhibited superior selectivity and potency compared to reported GPX4 inhibitors, ML210 and ML162. This provides the structural diversity of tool probes for unraveling the fundamental biology of GPX4 and exploring the therapeutic potential of pancreatic cancer via ferroptosis induction.

5mC modification orchestrates choriogenesis and fertilization by preventing prolonged ftz-f1 expression.

DNA methylation at the fifth position of cytosine (5-methylcytosine, 5mC) is a crucial epigenetic modification for regulating gene expression, but little is known about how it regulates gene expression in insects. Here, we pursue the detailed molecular mechanism by which DNMT1-mediated 5mC maintenance regulates female reproduction in the German cockroach, Blattella germanica. Our results show that Dnmt1 knockdown decreases the level of 5mC in the ovary, upregulating numerous genes during choriogenesis, especially the transcription factor ftz-f1. The hypomethylation at the ftz-f1 promoter region increases and prolongs ftz-f1 expression in ovarian follicle cells during choriogenesis, which consequently causes aberrantly high levels of 20-hydroxyecdysone and excessively upregulates the extracellular matrix remodeling gene Mmp1. These changes further impair choriogenesis and disrupt fertilization by causing anoikis of the follicle cells, a shortage of chorion proteins, and malformation of the sponge-like bodies. This study significantly advances our understanding of how DNA 5mC modification regulates female reproduction in insects.

Exploration of tricyclic heterocycles as core structures for RIOK2 inhibitors.

Right open reading frame kinase 2 (RIOK2) is an atypical kinase and has been proved to be involved in multiple human cancers including non-small cell lung cancer (NSCLC), acute myeloid leukemia (AML), glioblastoma and anemia. Although tremendous efforts have been devoted to the studies of RIOK2, its biological functions remain poorly understood. It is highly important to develop potent and selective RIOK2 inhibitors as potential research tools to elucidate its functions and as drug candidates for further therapies. We have previously identified a highly potent and selective RIOK2 inhibitor (CQ211). To confirm the importance of the "V-shaped" structure of CQ211 for binding with RIOK2, a variety of tricyclic compounds with different core structures instead of the [1,2,3]triazolo[4,5-c]quinolin-4-one core of CQ211 were designed, synthesized, and the binding affinities of these tricyclic heterocycles with RIOK2 were also evaluated.

N-N-Bridged Polynuclear POM-Based Coordination Polymers Based on a V-Type Ligand: Proton Conduction and Magnetism.

The construction of polyoxometalate (POM)-based coordination polymers, in the presence of a nitrogen heterocyclic ligand, is intriguing due to the potential for obtaining diverse structures. These structures exhibit extensive application possibilities in the fields of proton conductivity and magnetism. Herein, four new POM-based polynuclear coordination polymers with the formulas of {[Fe2(btb)3(H2O)2(SiW12O40)]·3H2O}n (1), {[Cd2(btb)2(H2O)6(HPMoVI10MoV2O40)]·2H2O}n (2), {[Co3(OH)2(btb)2(H2O)5(HPMoVI10MoV2O40)]·7H2O}n (3), and {[Cu3(OH)(btb)2(H2O)(HP2Mo5O23)]·6H2O}n (4) have been prepared using the V-type 1,3-bis(4H-1,2,4-triazole-4-yl)benzene (btb) ligand. Compounds 1 and 2 feature similar two-dimensional (2D) structures, derived from the binuclear Fe2N6 and Cd2N4 subunits connected by tridentate btb ligands. Meanwhile, in compound 3, hexanuclear Co6(OH)4 units are bound by quadridentate btb ligands forming a 2D layer with the same 4-c sql topology simplification as compounds 1 and 2. In compound 1, Keggin-type polyoxoanions are monodentate-coordinated to metal ions and suspended on the 2D structure, while, in compounds 2 and 3, they act as discrete counterions residing in the interstitial spaces between two adjacent layers, thereby extending the 2D structures into 3D structures through hydrogen bonding interactions. In compound 4, trinuclear Cu3(OH) subunits are further constructed into a 3D framework through cooperation with four tridentate and quadridentate btb ligands as well as Strandberg-type anions. Furthermore, the proton conduction of the four compounds has been investigated. They display high proton conductivities at 358 K and 98% RH with powdered samples, which are 1.26 × 10-3, 1.24 × 10-3, 3.24 × 10-4, and 2.57 × 10-4 S cm-1, respectively. Interestingly, by mixing with Nafion, the composite membranes of compounds 2 and 4 exhibit enhanced proton conductivities, measuring at 4.87 × 10-2 and 1.28 × 10-2 S cm-1, respectively, at 358 K and 98% RH, which suggests excellent potential for applications. In addition, compounds 1, 3, and 4 display antiferromagnetic behaviors due to similar magnetic interactions. This work can provide research insights into the assembly of 2D POM-based coordination polymers with nitrogen heterocyclic ligands and Keggin-type POMs and further promote their research progress in proton conduction.

Characteristics of fish community structure and environmental driving factors in Taihu Lake during the first year of fishing ban.

Taihu Lake has officially implemented the full fishing ban policy since October 1, 2020. We investigated fish community of Taihu Lake in the four seasons of 2020. A total of 42 fish species were collected, belonging to 6 orders, 7 families, and 33 genera. The first five dominant species ranked by the index of relative importance were Coilia nasus, Toxabramis swinhonis, Hypophthalmichthys molitrix, Hypophthalmichthys nobilis, and Salangichthys tangkahkeii. The number of C. nasus accounted for 85.1% of the total number of catches. According to the distributional characteristics of cyanobacterial blooms and aquatic plants, Taihu Lake could be divided into the northern, central, and eastern regions. There was no significant difference in catch per unit effort (CPUE) among different lake regions, but Shannon diversity index and Pielou evenness index in the eastern region was greater than in the other two regions. The CPUE, Shannon diversity index, and Pielou evenness index were significantly different among the four seasons, with the lowest CPUE in autumn and higher diversity in autumn and winter than in spring and summer. Electrical conductivity, water depth, chloride, and transparency were the main environmental factors driving the seasonal variations of fish community in Taihu Lake, while electrical conductivity, dissolved oxygen, total alkalinity, and transparency were key variables driving the spatial patterns. The results could be used as the baseline data for fish community studies in Taihu Lake after the fishing ban.

Structure-Based Optimization of the Third Generation Type II Macrocycle TRK Inhibitors with Improved Activity against Solvent-Front, xDFG, and Gatekeeper Mutations.

The solvent-front (SF), gatekeeper, and xDFG motif mutations of tropomyosin receptor kinase (TRK) mediating acquired resistance of larotrectinib and entrectinib represent an unmet clinical need. To date, no effective drugs are being approved to overcome these mutants. Thus, a series of macrocycle compounds were designed and synthesized as new type II TRK inhibitors to combat clinically relevant mutations. The representative compound 10g exhibited excellent potency against wide type TRKA/C, TRKAG595R, TRKAG667C, and TRKAF589L with IC50 values of 5.21, 4.51, 6.77, 1.42, and 6.13 nM, respectively, and a good kinome selectivity against 378 kinases. 10g also strongly suppressed the proliferation of Ba/F3 cells transfected with SF, GK, xDFG, and others (Val to Met) single mutants with IC50 values of 1.43-47.56 nM. Moreover, 10g demonstrated ideal antitumor efficacy in both BaF3-CD74-NTRK1G595R and BaF3-CD74-NTRK1G667C xenograft models. The study provides a promising lead compound for pan-anticancer drug discovery.

Characterization of two O-methyltransferases involved in the biosynthesis of O-methylated catechins in tea plant.

Tea is known for having a high catechin content, with the main component being (-)-epigallocatechin gallate (EGCG), which has significant bioactivities, including potential anti-cancer and anti-inflammatory activity. The poor intestinal stability and permeability of EGCG, however, undermine these health-improving benefits. O-methylated EGCG derivatives, found in a few tea cultivars in low levels, have attracted considerable interest due to their increased bioavailability. Here, we identify two O-methyltransferases from tea plant: CsFAOMT1 that has a specific O-methyltransferase activity on the 3''-position of EGCG to generate EGCG3''Me, and CsFAOMT2 that predominantly catalyzes the formation of EGCG4″Me. In different tea tissues and germplasms, the transcript levels of CsFAOMT1 and CsFAOMT2 are strongly correlated with the amounts of EGCG3''Me and EGCG4''Me, respectively. Furthermore, the crystal structures of CsFAOMT1 and CsFAOMT2 reveal the key residues necessary for 3''- and 4''-O-methylation. These findings may provide guidance for the future development of tea cultivars with high O-methylated catechin content.

Simultaneous Covalent Modification of K-Ras(G12D) and K-Ras(G12C) with Tunable Oxirane Electrophiles.

Owing to their remarkable pharmaceutical properties compared to those of noncovalent inhibitors, the development of targeted covalent inhibitors (TCIs) has emerged as a powerful method for cancer treatment. The K-Ras mutant, which is prevalent in multiple cancers, has been confirmed to be a crucial drug target in the treatment of various malignancies. However, although the K-Ras(G12D) mutation is present in up to 33% of K-Ras mutations, no covalent inhibitors targeting K-Ras(G12D) have been developed to date. The relatively weak nucleophilicity of the acquired aspartic acid (12D) residue in K-Ras may be the reason for this. Herein, we present the first compound capable of covalently engaging both K-Ras(G12D) and K-Ras(G12C) mutants. Proteome profiling revealed that this compound effectively conjugates with G12C and G12D residues, modulating the protein functions in situ. These findings offer a unique pathway for the development of novel dual covalent inhibitors.

Multitarget inhibitors/probes that target LRRK2 and AURORA A kinases noncovalently and covalently.

Herein, we report a salicylaldehyde-based, reversible covalent inhibitor (A2) that possesses moderate cellular activity against AURKA with a prolonged residence time and shows significant non-covalent inhibition towards LRRK2. Our results indicated that this multitarget kinase inhibitor may be used as the starting point for future development of more potent, selective and dual-targeting covalent kinase inhibitors against AURKA and LRRK2 for mitophagy.

lncRNA ASBEL and lncRNA Erbb4-IR reduce chemoresistance against gemcitabine and cisplatin in stage IV lung squamous cell carcinoma via the microRNA-21/LZTFL1 axis.

Drug resistance is a major cause of treatment failure and post-treatment disease progression in patients with cancer. This study aimed to investigate the mechanisms of chemoresistance to gemcitabine (GEM) plus cisplatin (cis-diamminedichloroplatinum, DDP) combination therapy in stage IV lung squamous cell carcinoma (LSCC). It also examined the functional role of lncRNA ASBEL and lncRNA Erbb4-IR in the malignant progression of LSCC. The expression of lncRNA ASBEL, lncRNA Erbb4-IR, miR-21, and LZTFL1 mRNA was examined in human stage IV LSCC tissues and adjacent normal tissues, human LSCC cells and normal human bronchial epithelial cells using qRT-PCR. Furthermore, LZTFL1 protein levels were also examined using western blots. Cell proliferation, cell migration and invasion, and cell cycle progression and apoptosis were evaluated in vitro using the CCK-8, transwell, and flow cytometry assays, respectively. Based on the treatment response, LSCC tissues were classified as GEM-, DDP-, and GEM+DDP-sensitive/resistant. The MTT assay was performed to assess the chemoresistance of human LSCC cells to GEM, DDP, and GEM+DDP following transfection experiments. The results showed that lncRNA ASBEL, lncRNA Erbb4-IR, and LZTFL1 were down-regulated in human LSCC tissues and cells, whereas miR-21 was up-regulated. In stage IV human LSCC tissues, miR-21 levels were negatively correlated with those of lncRNA ASBEL, lncRNA Erbb4-IR, and LZTFL1 mRNA. The overexpression of lncRNA ASBEL and lncRNA Erbb4-IR inhibited cell proliferation, migration, and invasion. It also blocked cell cycle entry and accelerated apoptosis. These effects were mediated by the miR-21/LZTFL1 axis and reduced chemoresistance to GEM+DDP combination therapy in stage IV human LSCC. These findings indicate that lncRNA ASBEL and lncRNA Erbb4-IR function as tumor suppressors in stage IV LSCC and attenuate chemoresistance to GEM+DDP combination therapy via the miR-21/LZTFL1 axis. Hence, lncRNA ASBEL, lncRNA Erbb4-IR, and LZTFL1 may be targeted to enhance the efficacy of GEM+DDP combination chemotherapy against LSCC.

Midbrain dopamine oxidation links ubiquitination of glutathione peroxidase 4 to ferroptosis of dopaminergic neurons.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the gradual loss of midbrain dopaminergic neurons in association with aggregation of α-synuclein. Oxidative damage has been widely implicated in this disease, though the mechanisms involved remain elusive. Here, we demonstrated that preferential accumulation of peroxidized phospholipids and loss of the antioxidant enzyme glutathione peroxidase 4 (GPX4) were responsible for vulnerability of midbrain dopaminergic neurons and progressive motor dysfunctions in a mouse model of PD. We also established a mechanism wherein iron-induced dopamine oxidation modified GPX4, thereby rendering it amenable to degradation via the ubiquitin-proteasome pathway. In conclusion, this study unraveled what we believe to be a novel pathway for dopaminergic neuron degeneration during PD pathogenesis, driven by dopamine-induced loss of antioxidant GPX4 activity.

Ynamide Coupling Reagent for the Chemical Cross-Linking of Proteins in Live Cells.

Chemical cross-linking of proteins coupled with mass spectrometry analysis (CXMS) is a powerful method for the study of protein structure and protein-protein interactions (PPIs). However, the chemical probes used in the CXMS are limited to bidentate reactive warheads, and the available zero-length cross-linkers are restricted to 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM). To alleviate this issue, an efficient coupling reagent, sulfonyl ynamide, was developed as a new zero-length cross-linker that can connect high-abundance carboxyl residues (D/E) with lysine (K) to form amide bonds in the absence of any catalyst. Significant improvement in the cross-linking efficiency and specificity in comparison with traditional EDC/NHS was achieved with model proteins, which includes inter- and intramolecular conjugations. The cross-linked structures were validated by X-ray crystallography. Importantly, this coupling reagent can be successfully used to capture interacting proteins in the whole proteome and can be a useful reagent for probing potential protein-protein interactions in situ.

Molecular insights into the catalytic mechanism of plasticizer degradation by a monoalkyl phthalate hydrolase.

Phthalate acid esters (PAEs), a group of xenobiotic compounds used extensively as plasticizers, have attracted increasing concern for adverse effects to human health and the environment. Microbial degradation relying on PAE hydrolases is a promising treatment. However, only a limited number of PAE hydrolases were characterized to date. Here we report the structures of MehpH, a monoalkyl phthalate (MBP) hydrolase that catalyzes the reaction of MBP to phthalic acid and the corresponding alcohol, in apo and ligand-bound form. The structures reveal a positively-charged catalytic center, complementary to the negatively-charged carboxyl group on MBP, and a penetrating tunnel that serves as exit of alcohol. The study provides a first glimpse into the enzyme-substrate binding model for PAE hydrolases, leading strong support to the development of better enzymes in the future.

Hot Deformation Behavior and Processing Map Considering Strengthening Effect for Al-10.0Zn-3.0Mg-2.8Cu Alloy.

In this paper, a hot processing map that takes into the strengthening effect into account is optimized for the Al-10.0Zn-3.0Mg-2.8Cu alloy, mainly considering the crushing and dissolving behavior of the insoluble phase. The hot deformation experiments were performed by compression testing with strain rates ranging from 0.001 to 1 s-1 and the temperature ranging from 380 to 460 °C. The hot processing map was established at the strain of 0.9. It exhibits that the appropriate hot processing region is located at the temperature from 431 to 456 °C and its strain rate is within 0.004-0.108 s-1. The recrystallization mechanisms and insoluble phase evolution were demonstrated using the real-time EBSD-EDS detection technology for this alloy. It is verified that the work hardening can also be consumed by the coarse insoluble phase refinement with the strain rate increasing from 0.001 to 0.1 s-1, besides the traditional recovery and recrystallization, but the effect of the insoluble phase crushing was weakened when strain rate increased over 0.1 s-1. Better refinement of the insoluble phase was around strain rate in 0.1 s-1, which exhibits adequate dissolving during the solid solution treatment, leading to excellent aging strengthen effects. Finally, the hot processing region was further optimized, so that the strain rate approaches 0.1 s-1 instead of 0.004-0.108 s-1. This will provide a theoretical support for the subsequent deformation of the Al-10.0Zn-3.0Mg-2.8Cu alloy and its' engineering application in aerospace, defense and military fields.