Despite the widespread success of transition-metal-catalysed cross-coupling methodologies, considerable limitations still exist in reactions at sp
-hybridized carbon atoms, with most approaches ...relying on prefunctionalized alkylmetal or bromide coupling partners
. Although the use of native functional groups (for example, carboxylic acids, alkenes and alcohols) has improved the overall efficiency of such transformations by expanding the range of potential feedstocks
, the direct functionalization of carbon-hydrogen (C-H) bonds-the most abundant moiety in organic molecules-represents a more ideal approach to molecular construction. In recent years, an impressive range of reactions that form C(sp
)-heteroatom bonds from strong C-H bonds has been reported
. Additionally, valuable technologies have been developed for the formation of carbon-carbon bonds from the corresponding C(sp
)-H bonds via substrate-directed transition-metal C-H insertion
, undirected C-H insertion by captodative rhodium carbenoid complexes
, or hydrogen atom transfer from weak, hydridic C-H bonds by electrophilic open-shell species
. Despite these advances, a mild and general platform for the coupling of strong, neutral C(sp
)-H bonds with aryl electrophiles has not been realized. Here we describe a protocol for the direct C(sp
) arylation of a diverse set of aliphatic, C-H bond-containing organic frameworks through the combination of light-driven, polyoxometalate-facilitated hydrogen atom transfer and nickel catalysis. This dual-catalytic manifold enables the generation of carbon-centred radicals from strong, neutral C-H bonds, which thereafter act as nucleophiles in nickel-mediated cross-coupling with aryl bromides to afford C(sp
)-C(sp
) cross-coupled products. This technology enables unprecedented, single-step access to a broad array of complex, medicinally relevant molecules directly from natural products and chemical feedstocks through functionalization at sites that are unreactive under traditional methods.
Establishment of a proper chromatin landscape is central to genome function. Here, we explain H3 variant distribution by specific targeting and dynamics of deposition involving the CAF-1 and HIRA ...histone chaperones. Impairing replicative H3.1 incorporation via CAF-1 enables an alternative H3.3 deposition at replication sites via HIRA. Conversely, the H3.3 incorporation throughout the cell cycle via HIRA cannot be replaced by H3.1. ChIP-seq analyses reveal correlation between HIRA-dependent H3.3 accumulation and RNA pol II at transcription sites and specific regulatory elements, further supported by their biochemical association. The HIRA complex shows unique DNA binding properties, and depletion of HIRA increases DNA sensitivity to nucleases. We propose that protective nucleosome gap filling of naked DNA by HIRA leads to a broad distribution of H3.3, and HIRA association with Pol II ensures local H3.3 enrichment at specific sites. We discuss the importance of this H3.3 deposition as a salvage pathway to maintain chromatin integrity.
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► Failure in H3.1 deposition at replication sites permits replacement by H3.3 via HIRA ► RNA pol II presence correlates with H3.3 enrichment at genes and associates with HIRA ► DNA binding ability of HIRA reveals a gap-filling mechanism for H3.3 deposition ► HIRA-dependent H3.3 deposition as a salvage pathway to maintain chromatin integrity
The introduction of a trifluoromethyl (CF
) group can dramatically improve a compound's biological properties. Despite the well-established importance of trifluoromethylated compounds, general ...methods for the trifluoromethylation of alkyl C-H bonds remain elusive. Here we report the development of a dual-catalytic C(sp
)-H trifluoromethylation through the merger of light-driven, decatungstate-catalysed hydrogen atom transfer and copper catalysis. This metallaphotoredox methodology enables the direct conversion of both strong aliphatic and benzylic C-H bonds into the corresponding C(sp
)-CF
products in a single step using a bench-stable, commercially available trifluoromethylation reagent. The reaction requires only a single equivalent of substrate and proceeds with excellent selectivity for positions distal to unprotected amines. To demonstrate the utility of this new methodology for late-stage functionalization, we have directly derivatized a broad range of approved drugs and natural products to generate valuable trifluoromethylated analogues. Preliminary mechanistic experiments reveal that a 'Cu-CF
' species is formed during this process and the critical C(sp
)-CF
bond-forming step involves the copper catalyst.
The gene encoding ARID1A, a chromatin remodeler, shows one of the highest mutation rates across many cancer types. Notably, ARID1A is mutated in over 50% of ovarian clear cell carcinomas, which ...currently have no effective therapy. To date, clinically applicable targeted cancer therapy based on ARID1A mutational status has not been described. Here we show that inhibition of the EZH2 methyltransferase acts in a synthetic lethal manner in ARID1A-mutated ovarian cancer cells and that ARID1A mutational status correlated with response to the EZH2 inhibitor. We identified PIK3IP1 as a direct target of ARID1A and EZH2 that is upregulated by EZH2 inhibition and contributed to the observed synthetic lethality by inhibiting PI3K-AKT signaling. Importantly, EZH2 inhibition caused regression of ARID1A-mutated ovarian tumors in vivo. To our knowledge, this is the first data set to demonstrate a synthetic lethality between ARID1A mutation and EZH2 inhibition. Our data indicate that pharmacological inhibition of EZH2 represents a novel treatment strategy for cancers involving ARID1A mutations.
High-throughput experimentation (HTE) has revolutionized the pharmaceutical industry, most notably allowing for rapid screening of compound libraries against therapeutic targets. The past decade has ...also witnessed the extension of HTE principles toward the realm of small-molecule process chemistry. Today, most major pharmaceutical companies have created dedicated HTE groups within their process development teams, invested in automation technology to accelerate screening, or both. The industry’s commitment to accelerating process development has led to rapid innovations in the HTE space. This review will deliver an overview of the latest best practices currently taking place within our teams in process chemistry by sharing frequently studied transformations, our perspective for the next several years in the field, and manual and automated tools to enable experimentation. A series of case studies are presented to exemplify state-of-the-art workflows developed within our laboratories.
The identification of the human homologue of the yeast CST in 2009 posed a new challenge in our understanding of the mechanism of telomere capping in higher eukaryotes. The high-resolution structure ...of the human Stn1-Ten1 (hStn1-Ten1) complex presented here reveals that hStn1 consists of an OB domain and tandem C-terminal wHTH motifs, while hTen1 consists of a single OB fold. Contacts between the OB domains facilitate formation of a complex that is strikingly similar to the replication protein A (RPA) and yeast Stn1-Ten1 (Ten1) complexes. The hStn1-Ten1 complex exhibits non-specific single-stranded DNA activity that is primarily dependent on hStn1. Cells expressing hStn1 mutants defective for dimerization with hTen1 display elongated telomeres and telomere defects associated with telomere uncapping, suggesting that the telomeric function of hCST is hTen1 dependent. Taken together the data presented here show that the structure of the hStn1-Ten1 subcomplex is conserved across species. Cell based assays indicate that hTen1 is critical for the telomeric function of hCST, both in telomere protection and downregulation of telomerase function.
Mutations in epigenetic pathways are common oncogenic drivers. Histones, the fundamental substrates for chromatin-modifying and remodelling enzymes, are mutated in tumours including gliomas, ...sarcomas, head and neck cancers, and carcinosarcomas. Classical 'oncohistone' mutations occur in the N-terminal tail of histone H3 and affect the function of polycomb repressor complexes 1 and 2 (PRC1 and PRC2). However, the prevalence and function of histone mutations in other tumour contexts is unknown. Here we show that somatic histone mutations occur in approximately 4% (at a conservative estimate) of diverse tumour types and in crucial regions of histone proteins. Mutations occur in all four core histones, in both the N-terminal tails and globular histone fold domains, and at or near residues that contain important post-translational modifications. Many globular domain mutations are homologous to yeast mutants that abrogate the need for SWI/SNF function, occur in the key regulatory 'acidic patch' of histones H2A and H2B, or are predicted to disrupt the H2B-H4 interface. The histone mutation dataset and the hypotheses presented here on the effect of the mutations on important chromatin functions should serve as a resource and starting point for the chromatin and cancer biology fields in exploring an expanding role of histone mutations in cancer.
There is an urgent need for antivirals to treat the newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To identify new candidates, we screen a repurposing library of ∼3,000 ...drugs. Screening in Vero cells finds few antivirals, while screening in human Huh7.5 cells validates 23 diverse antiviral drugs. Extending our studies to lung epithelial cells, we find that there are major differences in drug sensitivity and entry pathways used by SARS-CoV-2 in these cells. Entry in lung epithelial Calu-3 cells is pH independent and requires TMPRSS2, while entry in Vero and Huh7.5 cells requires low pH and triggering by acid-dependent endosomal proteases. Moreover, we find nine drugs are antiviral in respiratory cells, seven of which have been used in humans, and three are US Food and Drug Administration (FDA) approved, including cyclosporine. We find that the antiviral activity of cyclosporine is targeting Cyclophilin rather than calcineurin, revealing essential host targets that have the potential for rapid clinical implementation.
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•3,000 compounds screened in two cell types against SARS-CoV-2•Entry pathways are distinct in hepatocyte Huh7.5 and respiratory Calu-3 cells•Only nine compounds that are active in Huh7.5 cells are active in Calu-3 cells•Cyclosporin and cyclophilin inhibitors block SARS-CoV-2 infection in diverse cells
There is an urgent need for antivirals to treat the newly emerged SARS-CoV-2. Dittmar et al. find nine host-directed drugs are antiviral in respiratory cells, seven of which have been given to humans, and three are FDA approved. We show host targets that have the potential for rapid clinical implementation.
Administering intramammary antimicrobials to all mammary quarters of dairy cows at drying-off i.e., blanket dry cow therapy (BDCT) has been a mainstay of mastitis prevention and control. However, as ...udder health has considerably improved over recent decades with reductions in intramammary infection prevalence at drying-off and the introduction of teat sealants, BDCT may no longer be necessary on all dairy farms, thereby supporting antimicrobial stewardship efforts. This narrative review summarizes available literature regarding current dry cow therapy practices and associated impacts of selective dry cow therapy (SDCT) on udder health, milk production, economics, antimicrobial use, and antimicrobial resistance. Various methods to identify infections at drying-off that could benefit from antimicrobial treatment are described for selecting cows or mammary quarters for treatment, including utilizing somatic cell count thresholds, pathogen identification, previous clinical mastitis history, or a combination of criteria. Selection methods may be enacted at the herd, cow, or quarter levels. Producers' and veterinarians' motivations for antimicrobial use are discussed. Based on review findings, SDCT can be adopted without negative consequences for udder health and milk production, and concurrent teat sealant use is recommended, especially in udder quarters receiving no intramammary antimicrobials. Furthermore, herd selection should be considered for SDCT implementation in addition to cow or quarter selection, as BDCT may still be temporarily necessary in some herds for optimal mastitis control. Costs and benefits of SDCT vary among herds, whereas impacts on antimicrobial resistance remain unclear. In summary, SDCT is a viable management option for maintaining udder health and milk production while improving antimicrobial stewardship in the dairy industry.
Accumulation of senescent cells during aging contributes to chronic inflammation and age-related diseases. While senescence is associated with profound alterations of the epigenome, a systematic view ...of epigenetic factors in regulating senescence is lacking. Here, we curated a library of short hairpin RNAs for targeted silencing of all known epigenetic proteins and performed a high-throughput screen to identify key candidates whose downregulation can delay replicative senescence of primary human cells. This screen identified multiple new players including the histone acetyltransferase p300 that was found to be a primary driver of the senescent phenotype. p300, but not the paralogous CBP, induces a dynamic hyper-acetylated chromatin state and promotes the formation of active enhancer elements in the non-coding genome, leading to a senescence-specific gene expression program. Our work illustrates a causal role of histone acetyltransferases and acetylation in senescence and suggests p300 as a potential therapeutic target for senescence and age-related diseases.
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•High-throughput screening identifies key epigenetic proteins driving senescence•Depletion of the histone acetyltransferase p300 delays senescence•p300 induces the formation of de novo super enhancers in senescence•Depletion of p300 suppresses senescence-related gene expression
Epigenetic dysregulation is a hallmark of senescence and aging. In this article, Sen et al. have identified the histone acetyltransferase p300 as a key protein regulating senescence from a high-throughput screen. p300 induces formation of new super enhancers that drive senescence-related gene expression. p300 is an attractive candidate for anti-aging therapy.
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