Pan-KRAS inhibitor disables oncogenic signalling and tumour growth.

KRAS is one of the most commonly mutated proteins in cancer, and efforts to directly inhibit its function have been continuing for decades. The most successful of these has been the development of covalent allele-specific inhibitors that trap KRAS G12C in its inactive conformation and suppress tumour growth in patients1-7. Whether inactive-state selective inhibition can be used to therapeutically target non-G12C KRAS mutants remains under investigation. Here we report the discovery and characterization of a non-covalent inhibitor that binds preferentially and with high affinity to the inactive state of KRAS while sparing NRAS and HRAS. Although limited to only a few amino acids, the evolutionary divergence in the GTPase domain of RAS isoforms was sufficient to impart orthosteric and allosteric constraints for KRAS selectivity. The inhibitor blocked nucleotide exchange to prevent the activation of wild-type KRAS and a broad range of KRAS mutants, including G12A/C/D/F/V/S, G13C/D, V14I, L19F, Q22K, D33E, Q61H, K117N and A146V/T. Inhibition of downstream signalling and proliferation was restricted to cancer cells harbouring mutant KRAS, and drug treatment suppressed KRAS mutant tumour growth in mice, without having a detrimental effect on animal weight. Our study suggests that most KRAS oncoproteins cycle between an active state and an inactive state in cancer cells and are dependent on nucleotide exchange for activation. Pan-KRAS inhibitors, such as the one described here, have broad therapeutic implications and merit clinical investigation in patients with KRAS-driven cancers.

Diverse alterations associated with resistance to KRAS(G12C) inhibition.

Inactive state-selective KRAS(G12C) inhibitors1-8 demonstrate a 30-40% response rate and result in approximately 6-month median progression-free survival in patients with lung cancer9. The genetic basis for resistance to these first-in-class mutant GTPase inhibitors remains under investigation. Here we evaluated matched pre-treatment and post-treatment specimens from 43 patients treated with the KRAS(G12C) inhibitor sotorasib. Multiple treatment-emergent alterations were observed across 27 patients, including alterations in KRAS, NRAS, BRAF, EGFR, FGFR2, MYC and other genes. In preclinical patient-derived xenograft and cell line models, resistance to KRAS(G12C) inhibition was associated with low allele frequency hotspot mutations in KRAS(G12V or G13D), NRAS(Q61K or G13R), MRAS(Q71R) and/or BRAF(G596R), mirroring observations in patients. Single-cell sequencing in an isogenic lineage identified secondary RAS and/or BRAF mutations in the same cells as KRAS(G12C), where they bypassed inhibition without affecting target inactivation. Genetic or pharmacological targeting of ERK signalling intermediates enhanced the antiproliferative effect of G12C inhibitor treatment in models with acquired RAS or BRAF mutations. Our study thus suggests a heterogenous pattern of resistance with multiple subclonal events emerging during G12C inhibitor treatment. A subset of patients in our cohort acquired oncogenic KRAS, NRAS or BRAF mutations, and resistance in this setting may be delayed by co-targeting of ERK signalling intermediates. These findings merit broader evaluation in prospective clinical trials.

Rapid non-uniform adaptation to conformation-specific KRAS(G12C) inhibition.

KRAS GTPases are activated in one-third of cancers, and KRAS(G12C) is one of the most common activating alterations in lung adenocarcinoma1,2. KRAS(G12C) inhibitors3,4 are in phase-I clinical trials and early data show partial responses in nearly half of patients with lung cancer. How cancer cells bypass inhibition to prevent maximal response to therapy is not understood. Because KRAS(G12C) cycles between an active and inactive conformation4-6, and the inhibitors bind only to the latter, we tested whether isogenic cell populations respond in a non-uniform manner by studying the effect of treatment at a single-cell resolution. Here we report that, shortly after treatment, some cancer cells are sequestered in a quiescent state with low KRAS activity, whereas others bypass this effect to resume proliferation. This rapid divergent response occurs because some quiescent cells produce new KRAS(G12C) in response to suppressed mitogen-activated protein kinase output. New KRAS(G12C) is maintained in its active, drug-insensitive state by epidermal growth factor receptor and aurora kinase signalling. Cells without these adaptive changes-or cells in which these changes are pharmacologically inhibited-remain sensitive to drug treatment, because new KRAS(G12C) is either not available or exists in its inactive, drug-sensitive state. The direct targeting of KRAS oncoproteins has been a longstanding objective in precision oncology. Our study uncovers a flexible non-uniform fitness mechanism that enables groups of cells within a population to rapidly bypass the effect of treatment. This adaptive process must be overcome if we are to achieve complete and durable responses in the clinic.

Factors associated with clopidogrel resistance and clinical outcomes in ischemic cerebrovascular disease: A retrospective study.

OBJECTIVE: Clopidogrel resistance may lead to the recurrence of cerebrovascular diseases. We aimed to identify potential factors associated with clopidogrel resistance and evaluate the clinical outcomes of the patients. MATERIALS AND METHODS: In this retrospective study, patients with ischemic cerebrovascular disease treated with clopidogrel were included and classified into 2 groups according to the adenosine diphosphate (ADP)-induced platelet aggregation. Patients with the ADP inhibition rate of <30 % were included in clopidogrel resistance group, otherwise were included in clopidogrel sensitive group. CYP2C19 genotype and other clinical data were analyzed to identify factors and clinical features in the multivariate analysis. The outcomes were vascular events in 6 months. RESULTS: In total, 139 patients were enrolled with 81 (58.27 %) in clopidogrel sensitive group and 58 (41.73 %) in clopidogrel resistance group. Female and CYP2C19 *2*3 carrying were risk factors for clopidogrel resistance, and female was an independent risk factor (OR 2.481, 95 % CI 1.066-5.771, P=0.035). The clopidogrel resistance group showed a higher use rate of argatroban (P=0.030) and a lower arachidonic acid-induced inhibition of platelet aggregation (P=0.036). Clopidogrel resistance was related to the progressing stroke (HR 3.521, 95 % CI 1.352-9.170, P=0.010), but had no influence on the bleeding events (P>0.05). CONCLUSIONS: The risk of clopidogrel resistance increased significantly in female patients. Patients with clopidogrel resistance may have an increased incidence of stroke progression in the acute phase.

Synthesis of Polycyclic 3,3'-Biindoles via AgOTf-Catalyzed Nucleophilic Addition and Cycloisomerization.

A method for the rapid synthesis of polycyclic 3,3'-biindole derivatives has been developed through AgOTf-catalyzed nucleophilic addition and cycloisomerization processes. The cascade reaction employs readily accessible indoles and their N-2-formylphenyl derivatives and provides functionalized polycyclic 3,3'-biindoles in moderate to good yields under mild conditions. This reaction is highly efficient and takes only several minutes (∼5 min). Notably, the method is also highlighted by a Selectfluor-mediated oxidation reaction that quickly generates the oxindole derivatives.

Electrochemical Cyclization of Alkynyl Enaminones: Controllable Synthesis of Indeno[1,2-c]pyrroles or Indanones.

We report an electrochemical intramolecular [3 + 2] cyclization of alkynyl enaminones in a user-friendly undivided cell under constant current conditions without an oxidant and catalyst, and indeno[1,2-c]pyrrole derivatives could be obtained in good to excellent yields. Notably, preliminary substituent-controlled selective transformation is also achieved under electrocatalysis alone, and indeno[1,2-c]pyrrole (R4 ≠ H) or indanone derivatives (R4 = H) could be prepared directly under electrocatalysis without adding a base and heating process.

Transcriptome Analysis Reveals Contrasting Plant Responses of Sorghum bicolor upon Colonization by Two Formae Speciales of Sporisorium reilianum.

The biotrophic fungus Sporisorium reilianum exists in two host-adapted formae speciales that cause head smut in maize (S. reilianum f. sp. zeae; SRZ) and sorghum (S. reilianum f. sp. reilianum; SRS). In sorghum, the spread of SRZ is limited to the leaves. To understand the plant responses to each forma specialis, we determined the transcriptome of sorghum leaves inoculated either with SRS or SRZ. Fungal inoculation led to gene expression rather than suppression in sorghum. SRZ induced a much greater number of genes than SRS. Each forma specialis induced a distinct set of plant genes. The SRZ-induced genes were involved in plant defense mainly at the plasma membrane and were associated with the Molecular Function Gene Ontology terms chitin binding, abscisic acid binding, protein phosphatase inhibitor activity, terpene synthase activity, chitinase activity, transmembrane transporter activity and signaling receptor activity. Specifically, we found an upregulation of the genes involved in phospholipid degradation and sphingolipid biosynthesis, suggesting that the lipid content of the plant plasma membrane may contribute to preventing the systemic spread of SRZ. In contrast, the colonization of sorghum with SRS increased the expression of the genes involved in the detoxification of cellular oxidants and in the unfolded protein response at the endoplasmic reticulum, as well as of the genes modifying the cuticle wax and lipid composition through the generation of alkanes and phytosterols. These results identified plant compartments that may have a function in resistance against SRZ (plasma membrane) and susceptibility towards SRS (endoplasmic reticulum) that need more attention in the future.

Transient Stabilization Effect of CO2 in the Electrochemical Hydrogenation of Azo Compounds and the Reductive Coupling of α-Ketoesters.

The carbon dioxide (CO2 ) capture and utilization has attracted a great attention in organic synthesis. Herein, an unpresented transient stabilization effect (TSE) of CO2 is disclosed and well applied to the electrochemical hydrogenation of azo compounds to hydrazine derivatives. Mechanistic experiments and computational studies imply that CO2 can capture azo radical anion intermediates to protect the hydrogenation from potential degradation reactions, and is finally released through decarboxylation. The promotion effect of CO2 was further demonstrated to work in the preliminary study of electrochemical reductive coupling of α-ketoesters to vicinal diol derivatives. For the electrochemical reductive reactions mentioned above, CO2 is indispensable. The presented results shed light on a different usage of CO2 and could inspire novel experimental design by using CO2 as a transient protecting group.

CF3CO2H-Catalyzed Synthesis of 3-Alkynylpyrrole Derivatives and Their Controlled Reduction.

A transition-metal-free methodology employing nitroenynes and enaminones has been developed to access 3-alkynylpyrrole derivatives. This mild cyclization reaction might proceed through the nucleophilic addition, intramolecular cyclization, and the subsequent elimination processes. The protocol features a broad substrate scope, good selectivity, and functional group tolerance. Notably, the advantage of this method is also highlighted by the controlled reduction to generate alkenyl- or alkylpyrrole derivatives in good to excellent yields.

Catalyst-Free and Transition-Metal-Free Approach to 1,2-Diketones via Aerobic Alkyne Oxidation.

A catalyst-free and transition-metal-free method for the synthesis of 1,2-diketones from aerobic alkyne oxidation was reported. The oxidation of various internal alkynes, especially more challenging aryl-alkyl acetylenes, proceeded smoothly with inexpensive, easily handled, and commercially available potassium persulfate and an ambient air balloon, achieving the corresponding 1,2-diketones with up to 85% yields. Meanwhile, mechanistic studies indicated a radical process, and the two oxygen atoms in the 1,2-diketons were most likely from persulfate salts and molecular oxygen, respectively, rather than water.

Hydrosilane-Assisted Synthesis of Urea Derivatives from CO2 and Amines.

A methodology employing CO2, amines, and phenylsilane was discussed to access aryl- or alkyl-substituted urea derivatives. This procedure was characterized by adopting hydrosilane to promote the formation of ureas directly, without the need to prepare silylamines in advance. Control reactions suggested that FeCl3 was a favorable additive for the generation of ureas, and this 1,5,7-triazabicyclo[4.4.0]dec-5-ene-catalyzed reaction might proceed through nucleophilic addition, silicon migration, and the subsequent formal substitution of silylcarbamate.

Mechanism and Origin of Ligand-Controlled Chemo- and Regioselectivities in Palladium-Catalyzed Methoxycarbonylation of Alkynes.

Pd-catalyzed alkoxycarbonylation of alkynes provided a redox-neutral method to selectively access branched/linear α,ß-unsaturated monoesters and 1,4-dicarboxylic acid diesters. Herein, a systematic computational study was performed to elucidate the mechanism and origin of ligand-controlled chemo- and regioselectivities. It is found that the catalytic cycle, including hydrometallation, carbon monoxide insertion, and methanolysis, is more likely than that involving palladium alkoxycarbonyl intermediates. Both hydrometallation and methanolysis stages are important to determine the chemo- and regioselectivities. Hydrometallation proceeds via anti-Markovnikov-selective migratory insertion or Markovnikov-selective ligand-participated electrophilic addition. A flexible bidentate phosphine ligand slows down migratory insertion due to the stronger trans effect of the CO ligand but accelerates the ligand-participated electrophilic addition by adopting better orbital orientations. On the other hand, a ligand-participated mechanism and an unrevealed mechanism involving ketene intermediates can promote methanolysis, whereas ligands with large bite angles or bulky substituents are detrimental to methanolysis. On the basis of these mechanistic foundations, the influence of the flexibility, basicity, bite angle, and steric hindrance of ligands on chemo- and regioselectivities was clarified. The present study provided more universal and deeper mechanistic insights into Pd-catalyzed alkoxycarbonylation reactions and shed light on the superior regulation performance of the bifunctional pyridyl-containing phosphine ligands.

One-Pot Methylenation-Cyclization Employing Two Molecules of CO2 with Arylamines and Enaminones.

One-pot methylenation-cyclization employing two molecules of CO2 with enaminones and primary aromatic amines was discussed for the first time to access cyclized products. This 1,5,7-triazabicyclo[4.4.0]dec-5-ene and ZnCl2-catalyzed procedure was characterized by the selective conversion of two molecules of CO2 into methylene groups in a multicomponent cyclization reaction. According to the computational study and control experiments, the reaction might proceed through the generation of bis(silyl)acetal and condensation of arylamine and aza-Diels-Alder processes. Moreover, the resulting products will probably be potential organic building blocks with adjustable photophysical properties.

Recent developments of nanoenzyme-based colorimetric sensors for heavy metal detection and the interaction mechanism.

Heavy metal contamination has posed a great threat to human survival and social development. For this, a series of nanoenzyme-based colorimetric sensors, e.g., metal nanoparticles, metal oxides, metal sulfides, graphene-based nanomaterials, G-quadruplex and so on, were developed for the rapid and efficient detection of toxic heavy metal ions, whose detection limit for heavy metal ions could be as low as the nmol L-1 level. The recognition mechanism was based on the catalysis and signal amplification of nanozymes, a new type of nanomaterial possessing specific catalytic activity towards certain chemical reactions such as the oxidation of colorless TMB to blue oxTMB. In this work, we are trying to present readers with a better understanding of this important colorimetric sensing material by illustrating its application in the detection of heavy metal ions using metal nanoparticles, metal oxides, metal sulfides, graphene-based nanomaterials, G-quadruplex, etc. respectively.

Synthesis of indoline-fused eight-membered azaheterocycles through Zn-catalyzed dearomatization of indoles and subsequent base-promoted C-C activation.

A cascade reaction involving the Zn-catalyzed dearomatization of indoles, base-promoted ring-expansion and intramolecular SNAr reaction has been developed. This process realized a novel, atom economical and efficient synthesis of indoline-fused eight-membered azaheterocycles in a one pot manner.

Synthesis of fused-tetrahydropyrimidines: one-pot methylenation-cyclization utilizing two molecules of CO2.

A methylenation-cyclization reaction, employing cyclic enaminones with primary aromatic amines and two molecules of CO2, furnishing fused-tetrahydropyrimidines, is discussed. In this Cs2CO3 and ZnI2 catalyzed one-pot two-step procedure, two molecules of CO2 were selectively converted to methylene groups. The multi-component reaction might proceed through the formation of bis(silyl)acetal which was followed by condensation and further aza-Diels-Alder reaction. Hydroquinazoline, hydrocyclopenta[d]pyrimidine and hydroindeno[1,2-d]pyrimidine derivatives could be prepared with CO2 as the C1 source, effectively.

TBAF-Catalyzed O-Nucleophilic Cyclization of Enaminones: A Process for the Synthesis of Dihydroisobenzofuran Derivatives.

A novel methodology for the stereoselective synthesis of dihydroisobenzofuran derivatives is described in this paper. The procedure was realized by the bifunctional TBAF catalyzed selective O-nucleophilic cyclization of enaminone with intramolecular alkyne under mild and non-metal-mediated conditions. The results of control experiments suggested that the cation-π interaction and basicity, offered by TBAF, might be indispensable for the isomerization of enaminone and the formation of carbon-oxygen bond.

Synthesis of Polycyclic Benzo[b]indolo[3,2,1-de]acridines via Sequential Allenylation, Diels-Alder Cyclization, and Hydrogen Migration Reaction.

A novel methodology for stereoselective synthesis of benzo[b]indolo[3,2,1-de]acridines through the tandem reaction of propargylic compounds with organoboron is described, and only one diastereoisomer was obtained. The sequential procedure was triggered by Pd(0)-catalyzed allenylation of propargyl carbonate. Then, Diels-Alder cyclization and hydrogen migration processes proceeded successively to furnish the polycyclic target molecules. Control reactions suggested the base (Cs2CO3) was indispensable for the hydrogen migration.

Selective synthesis of pyrrolo[1,2-a]azepines or 4,6-dicarbonyl indoles via tandem reactions of alkynones with pyrrole derivatives.

Novel methodologies for the selective synthesis of pyrrolo[1,2-a]azepines or 4,6-dicarbonyl indoles starting from pyrrole derivatives and alkynones are described. When reactions were carried out with 1,2,4-trisubstituted N-propargyl pyrroles using a ZnI2 catalyst, pyrrolo[1,2-a]azepines were obtained. Whereas 4,6-dicarbonyl indoles were produced selectively with 1,2-disubstituted pyrroles in the presence of silica gel. The reaction outcomes depend on the substituent pattern of the substrates and the nature of the catalysts chosen. Control reactions suggested that the formation of a conjugated enamine intermediate was crucial for both the processes. With easily accessible starting materials, inexpensive catalysts and an easy-to-handle procedure, this reaction has the potential to become a general protocol for the synthesis of pyrrolo[1,2-a]azepines or indoles.

Hippo Component TAZ Functions as a Co-repressor and Negatively Regulates ΔNp63 Transcription through TEA Domain (TEAD) Transcription Factor.

Transcriptional co-activator with a PDZ binding domain (TAZ) is a WW domain-containing transcriptional co-activator and a core component of an emerging Hippo signaling pathway that regulates organ size, tumorigenesis, metastasis, and drug resistance. TAZ regulates these biological functions by up-regulating downstream cellular genes through transactivation of transcription factors such as TEAD and TTF1. To understand the molecular mechanisms underlying TAZ-induced tumorigenesis, we have recently performed a gene expression profile analysis by overexpressing TAZ in mammary cells. In addition to the TAZ-up-regulated genes that were confirmed in our previous studies, we identified a large number of cellular genes that were down-regulated by TAZ. In this study, we have confirmed these down-regulated genes (including cytokines, chemokines, and p53 gene family members) as bona fide downstream transcriptional targets of TAZ. By using human breast and lung epithelial cells, we have further characterized ΔNp63, a p53 gene family member, and shown that TAZ suppresses ΔNp63 mRNA, protein expression, and promoter activity through interaction with the transcription factor TEAD. We also show that TEAD can inhibit ΔNp63 promoter activity and that TAZ can directly interact with ΔNp63 promoter-containing TEAD binding sites. Finally, we provide functional evidence that down-regulation of ΔNp63 by TAZ may play a role in regulating cell migration. Altogether, this study provides novel evidence that the Hippo component TAZ can function as a co-repressor and regulate biological functions by negatively regulating downstream cellular genes.