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Novel COVID-19 therapeutics are urgently needed. We generated a phage-displayed human antibody VH domain library from which we identified a high-affinity VH binder ab8. Bivalent VH, VH-Fc ab8, bound with high avidity to membrane-associated S glycoprotein and to mutants found in patients. It potently neutralized mouse-adapted SARS-CoV-2 in wild-type mice at a dose as low as 2 mg/kg and exhibited high prophylactic and therapeutic efficacy in a hamster model of SARS-CoV-2 infection, possibly enhanced by its relatively small size. Electron microscopy combined with scanning mutagenesis identified ab8 interactions with all three S protomers and showed how ab8 neutralized the virus by directly interfering with ACE2 binding. VH-Fc ab8 did not aggregate and did not bind to 5,300 human membrane-associated proteins. The potent neutralization activity of VH-Fc ab8 combined with good developability properties and cross-reactivity to SARS-CoV-2 mutants provide a strong rationale for its evaluation as a COVID-19 therapeutic.
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Infecciones por Coronavirus/tratamiento farmacológico , Cadenas Pesadas de Inmunoglobulina/administración & dosificación , Región Variable de Inmunoglobulina/administración & dosificación , Biblioteca de Péptidos , Neumonía Viral/tratamiento farmacológico , Enzima Convertidora de Angiotensina 2 , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/ultraestructura , Anticuerpos Antivirales/administración & dosificación , Anticuerpos Antivirales/química , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/ultraestructura , Afinidad de Anticuerpos , COVID-19 , Cricetinae , Femenino , Humanos , Fragmentos Fc de Inmunoglobulinas/inmunología , Cadenas Pesadas de Inmunoglobulina/química , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas Pesadas de Inmunoglobulina/ultraestructura , Región Variable de Inmunoglobulina/química , Región Variable de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/ultraestructura , Ratones , Ratones Endogámicos BALB C , Mutación , Pandemias , Peptidil-Dipeptidasa A/metabolismo , Dominios Proteicos , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Glicoproteína de la Espiga del Coronavirus/ultraestructura , Tratamiento Farmacológico de COVID-19RESUMEN
As important π-skeletons, benzosiloles often possess unique electronic and optical properties and have been widely used in semiconductor materials. Therefore, great attention has been drawn to the area of developing novel synthetic methods for various benzosiloles. However, the synthesis of enantioenriched silicon-stereogenic benzosiloles is still at an early stage and remains to be explored. Herein, we performed systematic density functional theory studies on the recently reported nickel-catalyzed asymmetric synthesis of silicon-stereogenic benosiloles, which was enabled by an enantioselective desymmetrization of (2-alkenyl)aryl-substituted silacyclobutanes. Our computational study shows that the reaction mechanism involves ligand exchange, oxidative addition, alkene insertion, and hydrogen-transfer coupled reductive-demetalation steps. The proposed transmetalation and ß-hydride elimination mechanism was not found, which might be due to the unfavorable ring strain of the multicyclic intermediates. The novel hydrogen-transfer coupled reductive-demetalation mechanism was shown to be reasonable for the generation of the silicon-stereogenic benzosilole. Noncovalent interactions (including C-H···π and hydrogen bonding) in the rate-determining alkene insertion transition state account for the origins of the enantioselectivity. Our computational study sheds light on the detailed reaction mechanism and also provides insights for the development of novel approaches for synthesis of high-value silicon-stereogenic compounds.
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Compare with transient expression, stable cell lines generally have higher productivity and better quality for protein expression. However, selection of stable cell line is time-consuming and laborious. Here we describe an optimized selection method to achieve high-efficient stable cell pools with Expi293F suspension cells. This method only takes 2-3 weeks to generate stable cell pools with 2- to 10-fold higher productivity than transient gene expression (TGE). In fed-batch culture with Yeastolate, >1 g/L yield was achieved with our KTN0239-IgG stable cell pool in shaker flasks. This method can be also applied to efficiently display proteins on the cell surface.
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Proteínas , Proteómica , Cricetinae , Animales , Cricetulus , Células CHO , Proteínas RecombinantesRESUMEN
Effective therapies are urgently needed for the SARS-CoV-2/COVID-19 pandemic. We identified panels of fully human monoclonal antibodies (mAbs) from large phage-displayed Fab, scFv, and VH libraries by panning against the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) glycoprotein. A high-affinity Fab was selected from one of the libraries and converted to a full-size antibody, IgG1 ab1, which competed with human ACE2 for binding to RBD. It potently neutralized replication-competent SARS-CoV-2 but not SARS-CoV, as measured by two different tissue culture assays, as well as a replication-competent mouse ACE2-adapted SARS-CoV-2 in BALB/c mice and native virus in hACE2-expressing transgenic mice showing activity at the lowest tested dose of 2 mg/kg. IgG1 ab1 also exhibited high prophylactic and therapeutic efficacy in a hamster model of SARS-CoV-2 infection. The mechanism of neutralization is by competition with ACE2 but could involve antibody-dependent cellular cytotoxicity (ADCC) as IgG1 ab1 had ADCC activity in vitro. The ab1 sequence has a relatively low number of somatic mutations, indicating that ab1-like antibodies could be quickly elicited during natural SARS-CoV-2 infection or by RBD-based vaccines. IgG1 ab1 did not aggregate, did not exhibit other developability liabilities, and did not bind to any of the 5,300 human membrane-associated proteins tested. These results suggest that IgG1 ab1 has potential for therapy and prophylaxis of SARS-CoV-2 infections. The rapid identification (within 6 d of availability of antigen for panning) of potent mAbs shows the value of large antibody libraries for response to public health threats from emerging microbes.
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Prueba Serológica para COVID-19/métodos , Vacunas contra la COVID-19/inmunología , COVID-19/terapia , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos , Prueba Serológica para COVID-19/normas , Vacunas contra la COVID-19/normas , Chlorocebus aethiops , Cricetinae , Femenino , Humanos , Inmunización Pasiva/métodos , Inmunización Pasiva/normas , Inmunogenicidad Vacunal , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Ratones , Ratones Endogámicos BALB C , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/inmunología , Células Vero , Sueroterapia para COVID-19RESUMEN
The Biltz synthesis establishes straightforward access to 5,5-disubstituted (thio)hydantoins by combining a 1,2-diketone and a (thio)urea. Its appealing features include inherent atom and step economy together with the potential to generate structurally diverse products. However, control of the stereochemistry of this reaction has proven to be a daunting challenge. Herein, we describe the first example of enantioselective catalytic Biltz synthesis which affords more than 40 thiohydantoins with high stereo- and regio-control, irrespective of the symmetry of thiourea structure. A one pot synthesis of corresponding hydantoins is also documented. Remarkably, experimental studies and DFT calculations establish the reaction pathway and origin of stereoselectivity.
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Glutamine synthetase (GS), a central nitrogen metabolic enzyme, plays important roles in the nitrogen regulation network and secondary metabolism in fungi. However, the mechanisms by which external nitrogen sources regulate fungal GS activity have not been determined. Here, we found that GS activity was inhibited under nitrate conditions in Ganoderma lucidum. By constructing gs-silenced strains and adding 1 mM GS inhibitor to inhibit GS activity, we found that a decrease in GS activity led to a decrease in ganoderic acid biosynthesis. The transcription of gs increased approximately five fold under nitrate conditions compared with that under ammonia. Electrophoretic mobility shift and yeast one-hybrid assay showed that gs was transcriptionally regulated by AreA. Although both gs expression and GS protein content increased under nitrate conditions, the GS activity still decreased. Treatment of recombinant GS with SIN-1 (protein nitration donor) resulted in a strengthened nitration accompanied by a 71% decrease in recombinant GS activity. Furthermore, intracellular GS could be nitrated from mycelia cultivated under nitrate conditions. These results indicated that GS activity could be inhibited by NO-mediated protein nitration. Our findings provide the first insight into the role of transcriptional and posttranslational regulation of GS activity in regulating secondary metabolism in fungi.
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Regulación Fúngica de la Expresión Génica , Glutamato-Amoníaco Ligasa/genética , Glutamato-Amoníaco Ligasa/metabolismo , Reishi/metabolismo , Triterpenos/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Micelio/metabolismo , Nitratos/metabolismo , Nitrógeno/metabolismo , Procesamiento Proteico-Postraduccional , Reishi/genética , Metabolismo SecundarioRESUMEN
Neutrophil elastase (NE) is a serine protease released during neutrophil maturation. High levels of NE are related to lung tissue damage and poor prognosis in cancer; thus, NE is a potential target for therapeutic immunotherapy for multiple lung diseases and cancers. Here, we isolate and characterize two high-affinity, specific, and noncompetitive anti-NE antibodies Fab 1C10 and VH 1D1.43 from two large phage-displayed human Fab and VH libraries. After fusion with human IgG1 Fc, both of them (VH-Fc 1D1.43 and IgG1 1C10) inhibit NE enzymatic activity with VH-Fc 1D1.43 showing comparable inhibitory effects to that of the small molecule NE inhibitor SPCK and IgG1 1C10 exhibiting even higher (2.6-fold) activity than SPCK. Their epitopes, as mapped by peptide arrays combined with structural modeling, indicate different mechanisms for blocking NE activity. Both VH-Fc and IgG1 antibodies block NE uptake by cancer cells and fibroblast differentiation. VH-Fc 1D1.43 and IgG1 1C10 are promising for the antibody-based immunotherapy of cancer and inflammatory diseases.
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Inflamación/tratamiento farmacológico , Elastasa de Leucocito/inmunología , Neoplasias/tratamiento farmacológico , Proteínas Inhibidoras de Proteinasas Secretoras/uso terapéutico , Células Cultivadas , Mapeo Epitopo , Humanos , Dominios de Inmunoglobulinas/fisiología , Fragmentos de Inmunoglobulinas/química , Fragmentos de Inmunoglobulinas/farmacología , Fragmentos de Inmunoglobulinas/uso terapéutico , Inmunoterapia/métodos , Inflamación/inmunología , Elastasa de Leucocito/antagonistas & inhibidores , Masculino , Modelos Moleculares , Terapia Molecular Dirigida , Neoplasias/inmunología , Células PC-3 , Estructura Secundaria de Proteína , Proteínas Inhibidoras de Proteinasas Secretoras/química , Proteínas Inhibidoras de Proteinasas Secretoras/farmacologíaRESUMEN
Inbred mouse models are widely used to study age-related hearing loss (AHL). Many genes associated with AHL have been mapped in a variety of strains. However, little is known about gene variants that have the converse function-protective genes that confer strong resistance to hearing loss. Previously, we reported that C57BL/6J (B6) and DBA/2J (D2) strains share a common hearing loss allele in Cdh23. The cadherin 23 (Cdh23) gene is a key contributor to early-onset hearing loss in humans. In this study, we tested hearing across a large family of 54 BXD strains generated from B6 to D2 crosses. Five of 54 strains maintain the normal threshold (20 dB SPL) even at 2 years old-an age at which both parental strains are essentially deaf. Further analyses revealed an age-related hearing protection (ahp) locus on chromosome 16 (Chr 16) at 57~76 Mb with a maximum LOD of 5.7. A small number of BXD strains at 2 years with good hearing correspond roughly to the percentage of humans who have good hearing at 90 years old. Further studies to define candidate genes in the ahp locus and related molecular mechanisms involved in age-related resilience or resistance to AHL are warranted.
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Alelos , Umbral Auditivo/fisiología , Cadherinas/genética , Cromosomas de los Mamíferos , Potenciales Evocados Auditivos del Tronco Encefálico/genética , Pérdida Auditiva/genética , Audición/fisiología , Animales , Predisposición Genética a la Enfermedad , Genotipo , Ratones , FenotipoRESUMEN
Nitrogen metabolism repression (NMR) has been well studied in filamentous fungi, but the molecular mechanism of its effects on fungal secondary metabolism has been generally unexplored. Ganoderic acid (GA) biosynthesis in Ganoderma lucidum differs between ammonia and nitrate nitrogen sources. To explain the functions of NMR in secondary metabolism, AreA, which is a core transcription factor of NMR, was characterized in G. lucidum. The transcription level of AreA was dramatically increased (approximately 4.5-folds), with the nitrate as the sole nitrogen source, compared with that with ammonia as the source. In addition, the expression of related genes involved in NMR was changed (upregulated of MeaB and downregulated of Nmr and GlnA) when AreA was knockdown. Yeast one-hybrid and electrophoretic mobility shift assay results showed that AreA could directly bind to the promoter of fps (encoding farnesyl-diphosphate synthase) to activate its expression. However, GA biosynthesis was increased (27% in the ammonia source and 77% in the nitrate source) in AreAi mutant strains versus that in control strains. These results showed that another important factor must participate in regulating GA biosynthesis other than the direct activation of AreA. Furthermore, we found that the content of nitric oxide (NO) was increased approximately 2.7-folds in the nitrate source compared with that in the ammonia. By adding the NO donor (SNP) or scavenger (cPTIO) and using NR-silenced or NR-overexpressed strains, we found that there was a negative correlation between the NO contents and GA biosynthesis. NO generated by nitrate reductase (NR) during the nitrogen utilization burst and could negatively influence GA biosynthesis. As a global transcription factor, AreA could also regulate the expression of NR. Our studies provide novel insight into the dual functions of AreA in GA biosynthesis during nitrogen assimilation.
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Proteínas Fúngicas/metabolismo , Reishi/genética , Reishi/metabolismo , Factores de Transcripción/metabolismo , Triterpenos/metabolismo , Proteínas Fúngicas/genética , Técnicas de Silenciamiento del Gen , Óxido Nítrico/metabolismo , Saccharomyces cerevisiae/genética , Factores de Transcripción/genéticaAsunto(s)
Carcinoma de Células Renales , Inflamación , Neoplasias Renales , Humanos , Carcinoma de Células Renales/patología , Carcinoma de Células Renales/cirugía , Neoplasias Renales/patología , Inflamación/patología , Pronóstico , Clasificación del Tumor , Coagulación Sanguínea , Femenino , MasculinoAsunto(s)
Carcinoma de Células Renales , Neoplasias Renales , Humanos , Carcinoma de Células Renales/cirugía , Carcinoma de Células Renales/patología , Riñón/cirugía , Riñón/patología , Neoplasias Renales/cirugía , Neoplasias Renales/patología , Grasa Intraabdominal/patología , Complicaciones Posoperatorias/etiología , Complicaciones Posoperatorias/patologíaRESUMEN
Ganoderma lucidum, which contains many pharmacologically active compounds, is regarded as a traditional medicinal fungus. Nevertheless, the scarcity of basic research limits the commercial value and utilization of G. lucidum. As a class of highly conserved, phosphopeptide-binding proteins present in all eukaryotes, 14-3-3 proteins play vital roles in controlling multiple physiological processes, including signal transduction, primary metabolism, and stress responses. However, knowledge of the roles of 14-3-3 proteins in Basidiomycetes is sparse. In this article, two homologs of 14-3-3 proteins, encoded by the two distinct genes GlBmh1 and GlBmh2, were distinguished in G. lucidum. We found that GlBmh1 and GlBmh2 were expressed at various developmental stages, including in vegetative mycelium cultivated on solid medium and in primordia and fruiting bodies. Moreover, we constructed GlBmh1 single-silenced strains, GlBmh2 single-silenced strains, and 14-3-3 double-silenced mutants for further study. When GlBmh1 and GlBmh2 were inhibited by RNA interference, the growth rate of mycelia was decreased, and the distance between the aerial hyphal branches was reduced; responses to various abiotic stresses such as oxidants and cell wall and osmotic stressors were also changed. Furthermore, the contents of secondary metabolite ganoderic acids (GAs) were increased after GlBmh1 and GlBmh2 were simultaneously silenced. Taken together, we provide evidence that implicates potential roles for the two 14-3-3 proteins in affecting growth and GA biosynthesis, thereby providing new insights into the basic functions of 14-3-3 proteins in G. lucidum.
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Proteínas 14-3-3/metabolismo , Proteínas Fúngicas/metabolismo , Hifa/crecimiento & desarrollo , Reishi/crecimiento & desarrollo , Reishi/fisiología , Estrés Fisiológico , Triterpenos/metabolismo , Proteínas 14-3-3/genética , Proteínas Fúngicas/genética , Perfilación de la Expresión Génica , Silenciador del Gen , Reishi/genéticaRESUMEN
With the recent development of single B-cell cloning techniques, an increasing number of human immunodeficiency virus type 1 (HIV-1)-specific broadly neutralizing antibodies have been isolated since 2009. However, knowledge regarding HIV-1-specific B cells in vivo is limited. In this study, an HIV-1-specific B-cell line was established using healthy PBMC donors by the highly efficient Epstein-Barr virus transformation method to generate immortalized human naïve B-cell libraries. The enrichment of HIV-1 envelope-specific B cells was observed after four rounds of cell panning with the HIV-1 envelope glycoprotein. An HIV-1 envelope-specific stable B-cell line (LCL-P4) was generated. Although this cell line acquired a lymphoblastic phenotype, no expression was observed for activation-induced cytidine deaminase, an enzyme responsible for initiating somatic hypermutation and class switch recombination in B cells. This study describes a method that enables fast isolation of HIV-1-specific B cells, and this approach may extend to isolating other B-cell-specific antigens for further experiments.
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Anticuerpos Neutralizantes/metabolismo , Linfocitos B/inmunología , Anticuerpos Anti-VIH/metabolismo , VIH-1/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Transformación Celular Viral , Herpesvirus Humano 4/genética , HumanosRESUMEN
The alternative oxidase (AOX), which forms a branch of the mitochondrial respiratory electron transport pathway, functions to sustain electron flux and alleviate reactive oxygen species (ROS) production. In this article, a homologous AOX gene was identified in Ganoderma lucidum. The coding sequence of the AOX gene in G. lucidum contains 1038 nucleotides and encodes a protein of 39.48 kDa. RNA interference (RNAi) was used to study the function of AOX in G. lucidum, and two silenced strains (AOXi6 and AOXi21) were obtained, showing significant decreases of approximately 60 and 50â%, respectively, in alternative pathway respiratory efficiency compared to WT. The content of ganoderic acid (GA) in the mutant strains AOXi6 and AOXi21 showed significant increases of approximately 42 and 44â%, respectively, compared to WT. Elevated contents of intermediate metabolites in GA biosynthesis and elevated transcription levels of corresponding genes were also observed in the mutant strains AOXi6 and AOXi21. In addition, the intracellular ROS content in strains AOXi6 and AOXi21 was significantly increased, by approximately 1.75- and 1.93-fold, respectively, compared with WT. Furthermore, adding N-acetyl-l-cysteine (NAC), a ROS scavenger, significantly depressed the intracellular ROS content and GA accumulation in AOX-silenced strains. These results indicate that AOX affects GA biosynthesis by regulating intracellular ROS levels. Our research revealed the important role of AOX in the secondary metabolism of G. lucidum.
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Proteínas Mitocondriales/metabolismo , Oxidorreductasas/metabolismo , Proteínas de Plantas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Reishi/metabolismo , Triterpenos/metabolismo , Secuencia de Aminoácidos , Clonación Molecular , Biología Computacional/métodos , Citocromos/metabolismo , Expresión Génica , Silenciador del Gen , Redes y Vías Metabólicas , Proteínas Mitocondriales/genética , Modelos Biológicos , Estrés Oxidativo , Oxidorreductasas/genética , Filogenia , Proteínas de Plantas/genética , Interferencia de ARN , Reishi/clasificación , Reishi/genética , Análisis de Secuencia de ADNRESUMEN
The mitogen-activated protein kinases (MAPKs) are crucial signaling instruments in eukaryotes that play key roles in regulating fungal growth, development, and secondary metabolism and in adapting to the environment. In this study, we characterized an Slt2-type MAPK in Ganoderma lucidum, GlSlt2, which was transcriptionally induced during the primordium and fruiting body stages. RNA interference was used to examine the function of GlSlt2. Knockdown of GlSlt2 caused defects in growth and increased hyphal branching as well as hypersensitivity to cell wall-disturbing substances. Consistently, the chitin and ß-1,3-d-glucan contents and the expression of cell wall biosynthesis genes were decreased and down-regulated, respectively, in GlSlt2 knockdown strains compared with those in the wild type (WT). In addition, no primordium or fruiting body could be observed in GlSlt2 knockdown strains. Furthermore, the intracellular reactive oxygen species (ROS) content and ganoderic acid biosynthesis also decreased in GlSlt2 knockdown strains. Addition of H2O2 could recover the decreased ganoderic acid content in GlSlt2 knockdown strains, indicating that GlSlt2 might regulate ganoderic acid biosynthesis via the intracellular ROS level. Overall, GlSlt2 is involved in hyphal growth, fruiting body development, cell wall integrity, oxidative stress and ganoderic acid biosynthesis in G. lucidum.
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Cuerpos Fructíferos de los Hongos/crecimiento & desarrollo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Reishi/enzimología , Reishi/crecimiento & desarrollo , Triterpenos/metabolismo , Pared Celular/fisiología , Quitina/metabolismo , Clonación Molecular , Técnicas de Silenciamiento del Gen , Peróxido de Hidrógeno/farmacología , Estrés Oxidativo , Proteoglicanos , Interferencia de ARN , Especies Reactivas de Oxígeno/metabolismo , Reishi/efectos de los fármacos , beta-Glucanos/metabolismoRESUMEN
Federated learning has recently been applied to recommendation systems to protect user privacy. In federated learning settings, recommendation systems can train recommendation models by collecting the intermediate parameters instead of the real user data, which greatly enhances user privacy. In addition, federated recommendation systems (FedRSs) can cooperate with other data platforms to improve recommendation performance while meeting the regulation and privacy constraints. However, FedRSs face many new challenges such as privacy, security, heterogeneity, and communication costs. While significant research has been conducted in these areas, gaps in the surveying literature still exist. In this article, we: 1) summarize some common privacy mechanisms used in FedRSs and discuss the advantages and limitations of each mechanism; 2) review several novel attacks and defenses against security; 3) summarize some approaches to address heterogeneity and communication costs problems; 4) introduce some realistic applications and public benchmark datasets for FedRSs; and 5) present some prospective research directions in the future. This article can guide researchers and practitioners understand the research progress in these areas.
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A novel photocatalytic palladium-induced 6-endo-selective alkyl Heck reaction of unactivated alkyl iodides and alkyl bromides has been described. This strategy facilitates the gentle and efficient synthesis of a variety of 5-phenyl-1,2,3,6-tetrahydropyridine derivatives. It demonstrates a broad substrate tolerance and excellent 6-endo selectivity. Unlike the high-temperature requirements of traditional alkyl Heck reactions, this transformation efficiently proceeds at room temperature and shows significant promise for industrial-scale applications. Mechanistic investigations reveal that this alkyl Heck reaction proceeds via a hybrid palladium-radical process.
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The anaplastic lymphoma kinase (ALK, CD247) is a potential target for antibody-based therapy. However, no antibody-based therapeutics targeting ALK have entered clinical trials, necessitating the development of novel antibodies with unique therapeutic merits. Single-domain antibodies (sdAb) bear therapeutic advantages compared to the full-length antibody including deeper tumor penetration, cost-effective production and fast washout from normal tissues. In this study, we identified a human immunoglobulin heavy chain variable domain (VH domain) (VH20) from an in-house phage library. VH20 exhibits good developability and high specificity with no off-target binding to ~6000 human membrane proteins. VH20 efficiently bound to the glycine-rich region of ALK with an EC50 of 0.4 nM and a KD of 6.54 nM. Both VH20-based bispecific T cell engager (TCE) and chimeric antigen receptor T cells (CAR Ts) exhibited potent cytolytic activity to ALK-expressing tumor cells in an ALK-dependent manner. VH20 CAR Ts specifically secreted proinflammatory cytokines including IL-2, TNFα and IFNγ after incubation with ALK-positive cells. To our knowledge, this is the first reported human single-domain antibody against ALK. Our in vitro characterization data indicate that VH20 could be a promising ALK-targeting sdAb with potential applications in ALK-expressing tumors, including neuroblastoma (NBL) and non-small cell lung cancer.