Selective degradation of multimeric proteins by TRIM21-based molecular glue and PROTAC degraders.

Targeted protein degradation (TPD) utilizes molecular glues or proteolysis-targeting chimeras (PROTACs) to eliminate disease-causing proteins by promoting their interaction with E3 ubiquitin ligases. Current TPD approaches are limited by reliance on a small number of constitutively active E3 ubiquitin ligases. Here, we report that (S)-ACE-OH, a metabolite of the antipsychotic drug acepromazine, acts as a molecular glue to induce an interaction between the E3 ubiquitin ligase TRIM21 and the nucleoporin NUP98, leading to the degradation of nuclear pore proteins and disruption of nucleocytoplasmic trafficking. Functionalization of acepromazine into PROTACs enabled selective degradation of multimeric proteins, such as those within biomolecular condensates, while sparing monomeric proteins. This selectivity is consistent with the requirement of substrate-induced clustering for TRIM21 activation. As aberrant protein assemblies cause diseases such as autoimmunity, neurodegeneration, and cancer, our findings highlight the potential of TRIM21-based multimer-selective degraders as a strategy to tackle the direct causes of these diseases.

Shigella evades pyroptosis by arginine ADP-riboxanation of caspase-11.

Mouse caspase-11 and human caspase-4 and caspase-5 recognize cytosolic lipopolysaccharide (LPS) to induce pyroptosis by cleaving the pore-forming protein GSDMD1-5. This non-canonical inflammasome defends against Gram-negative bacteria6,7. Shigella flexneri, which causes bacillary dysentery, lives freely within the host cytosol where these caspases reside. However, the role of caspase-11-mediated pyroptosis in S. flexneri infection is unknown. Here we show that caspase-11 did not protect mice from S. flexneri infection, in contrast to infection with another cytosolic bacterium, Burkholderia thailandensis8. S. flexneri evaded pyroptosis mediated by caspase-11 or caspase 4 (hereafter referred to as caspase-11/4) using a type III secretion system (T3SS) effector, OspC3. OspC3, but not its paralogues OspC1 and 2, covalently modified caspase-11/4; although it used the NAD+ donor, this modification was not ADP-ribosylation. Biochemical dissections uncovered an ADP-riboxanation modification on Arg314 and Arg310 in caspase-4 and caspase-11, respectively. The enzymatic activity was shared by OspC1 and 2, whose ankyrin-repeat domains, unlike that of OspC3, could not recognize caspase-11/4. ADP-riboxanation of the arginine blocked autoprocessing of caspase-4/11 as well as their recognition and cleavage of GSDMD. ADP-riboxanation of caspase-11 paralysed pyroptosis-mediated defence in Shigella-infected mice and mutation of ospC3 stimulated caspase-11- and GSDMD-dependent anti-Shigella humoral immunity, generating a vaccine-like protective effect. Our study establishes ADP-riboxanation of arginine as a bacterial virulence mechanism that prevents LPS-induced pyroptosis.

Enantioselective Decarboxylative C(sp3)-C(sp3) Cross-Coupling of Aliphatic Redox-Active Esters with gem-Borazirconocene Alkanes.

Asymmetric decarboxylative cross-couplings of carboxylic acids are powerful methods for synthesizing chiral building blocks essential in medicinal chemistry and material science. Despite their potential, creating versatile chiral alkylboron derivatives through asymmetric decarboxylative C(sp3)-C(sp3) cross-coupling from readily available primary aliphatic acids and mild organometallic reagents remains challenging. In this study, we present a visible light-induced Ni-catalyzed enantioconvergent C(sp3)-C(sp3) cross-coupling of unactivated primary aliphatic acid NHPI esters with gem-borazirconocene alkanes, producing a diverse array of valuable chiral alkylboron building blocks. The method boasts a broad substrate scope, high functional group tolerance, and the ability for late-stage modification of complex drug molecules and natural products with high enantioselectivity, showcasing its synthetic potential. Mechanistic investigations suggest a nickel-catalyzed enantioconvergent radical cross-coupling pathway, wherein the primary radical from a redox-active ester is generated through single-electron reduction with ZrIII species. This represents an unprecedented example of enantioselective radical C(sp3)-C(sp3) cross-coupling in the absence of photocatalysts.

Genetically encoded tags for direct synthesis of EM-visible gold nanoparticles in cells.

Genetically encoded tags for single-molecule imaging in electron microscopy (EM) are long-awaited. Here, we report an approach for directly synthesizing EM-visible gold nanoparticles (AuNPs) on cysteine-rich tags for single-molecule visualization in cells. We first uncovered an auto-nucleation suppression mechanism that allows specific synthesis of AuNPs on isolated tags. Next, we exploited this mechanism to develop approaches for single-molecule detection of proteins in prokaryotic cells and achieved an unprecedented labeling efficiency. We then expanded it to more complicated eukaryotic cells and successfully detected the proteins targeted to various organelles, including the membranes of endoplasmic reticulum (ER) and nuclear envelope, ER lumen, nuclear pores, spindle pole bodies and mitochondrial matrices. We further implemented cysteine-rich tag-antibody fusion proteins as new immuno-EM probes. Thus, our approaches should allow biologists to address a wide range of biological questions at the single-molecule level in cellular ultrastructural contexts.

Author Correction: Genetically encoded tags for direct synthesis of EM-visible gold nanoparticles in cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Oxidative Coupling Approach to Sarpagine Alkaloids: Total Synthesis of (-)-Trinervine, Vellosimine, (+)-Normacusine†B, and (-)-Alstomutinine†C.

Sarpagine alkaloids are bioactive indole natural products that contain a highly rigid indole-fused 1-azabicyclo[2.2.2]octane, more than 100 members of which have been identified. Herein, a detailed examination of the intramolecular oxidative coupling between a ketone and a Weinreb amide for assembling the complex 1-azabicyclo[2.2.2]octane core structure of sarpagine family alkaloids is described. Precise late-stage manipulations of the ketone and Weinreb amide enable the divergent syntheses of (-)-trinervine, (+)-vellosimine, (+)-normacusine B, and (-)-alstomutinine C. Other notable transformations of the synthesis featured an aza-Achmatowicz/indole cyclization cascade to generate the azabicyclo[3.3.1]nonane structure, a regioselective elimination reaction to form the ethylidene motif embedded in the (+)-vellosimine and (+)-normacusine B structures, and a diastereoselective indole oxidative rearrangement to form the spirooxindole structure in (-)-alstomutinine C.

Flavin-Dependent Monooxygenase-Mediated 1,2-Oxazine Construction via Meisenheimer Rearrangement in the Biosynthesis of Paeciloxazine.

The [1,2]-Meisenheimer rearrangement is well known as the [1,2]-migration of an O-substituted hydroxylamine from a tertiary amine N-oxide, and it is frequently employed in organic synthesis to enforce adjacent carbon oxidation or install a 1,2-oxazine core, which is a prevalent structural feature and pharmacophore of many bioactive natural products. Although the [1,2]-Meisenheimer rearrangement was proposed to occur in the biosynthesis of a number of 1,2-oxazine-containing natural products, it has never been proved biosynthetically. Here, we identified the biosynthetic gene cluster of an insecticidal natural product, paeciloxazine (1), from Penicillium janthinellum and characterized a flavin-dependent monooxygenase, PaxA, as the first example that mediates the formation of a 1,2-oxazine moiety via Meisenheimer rearrangement. In vitro biochemical assays, site-directed mutations, docking and molecular dynamics simulations, and density functional theory calculations support the mechanism that PaxA first catalyzes N-oxidation to form an N-oxide intermediate, which undergoes [1,2]-Meisenheimer rearrangement with the assistance of an amino acid with proton transfer property. This study expands the repertoire of rearrangement reactions during the biosynthesis of natural products and provides a new strategy for discovering natural products with N-O tethers by genome mining.

BAFinder: A Software for Unknown Bile Acid Identification Using Accurate Mass LC-MS/MS in Positive and Negative Modes.

Most LC-MS based bile acid analyses target common bile acids. The identification of unknown bile acids remains challenging in untargeted experiments. Here, a software named BAFinder was developed to improve the identification of unknown bile acids from accurate mass LC-MS/MS data in both the positive and negative ESI modes. A wide variety of bile acid structures were covered in BAFinder, including oxidized bile acids and sugar conjugates that were often ignored. The annotation of unknown bile acids was based on a thorough investigation of MS/MS fragmentation patterns of 84 bile acid reference standards in both modes. Specifically, BAFinder took the peak alignment result and MS/MS spectra, grouped candidate features in positive and negative modes, searched their representative MS/MS spectra against a MS/MS library, and used characteristic product ions and neutral losses to annotate bile acids not covered in the library. Finally, the number of hydroxyl groups and double bonds, conjugation, and isomer information of bile acids were reported with four different levels of annotation confidence. The use of BAFinder was demonstrated through successful application to the analysis of human plasma and urine samples, in which a total of 112 and 244 bile acids were annotated and 75 and 111 of them were confirmed with standards or synthesized compounds, respectively. The software is freely available at https://bafinder.github.io/.

Asymmetric Total Syntheses of Strychnos Alkaloids via Selective Fischer Indolization.

The complex structures and important biological functions of Strychnos alkaloids have attracted a great deal of attention from synthetic chemists. Herein, we describe the concise asymmetric total syntheses of the Strychnos alkaloids, (-)-dehydrotubifoline, (-)-tubifoline, and (-)-tubifolidine, as well as the formal total synthesis of (-)-strychnine. Our strategy features the construction of the common tetracyclic pyrrolo[2,3-d]carbazole structure using regioselective Fischer indolization on unsymmetrical cyclic ketones and late-stage functionalization for divergent synthesis. We developed a stepwise Fischer indolization featuring selective formation of enol triflate to solve the challenging regioselectivity problem, leading to the common tetracyclic ring skeleton in these Strychnos alkaloids. The regioselectivity of Fischer indolization on unsymmetrical cyclic ketones was studied on the basis of different types of ring systems and supported by density functional theory calculations. Overall, our success in the construction of this tetracyclic ring secured the syntheses of Strychnos alkaloids and may provide a general method for the total syntheses of various alkaloids containing this skeleton.

A small molecule protects mitochondrial integrity by inhibiting mTOR activity.

Apoptosis activation by cytochrome c release from mitochondria to cytosol is a normal cellular response to mitochondrial damage. Using cellular apoptosis assay, we have found small-molecule apoptosis inhibitors that protect cells from mitochondrial damage. Previously, we reported the discovery of a small molecule, Compound A, which blocks dopaminergic neuron death in a rat model of Parkinson's disease through targeting succinate dehydrogenase subunit B (SDHB) of complex II to protect the integrity of the mitochondrial respiratory chain. Here, we report a small molecule, Compound R6, which saves cells from apoptosis via mammalian target of rapamycin (mTOR)-mediated induction of autophagy. Additionally, we show that Compound R6 protects mitochondrial integrity and respiration after induction of the intrinsic apoptosis pathway. Encouragingly, and supporting the potential further application of Compound R6 as a tool for basic and medicinal research, a pharmacokinetics (PK) profiling study showed that Compound R6 is metabolically stable and can pass the blood-brain barrier. Moreover, Compound R6 accumulates in the brain of test animals via intravenous and intraperitoneal administration. Finally, we found that Compound R6 confers significant neuroprotective effects on a rat cerebral ischemia/reperfusion model, demonstrating its potential as a promising drug candidate for neurodegenerative diseases.

Expeditious and Efficient ortho-Selective Trifluoromethane-sulfonylation of Arylhydroxylamines.

A metal- and oxidant-free, practical and efficient method for the synthesis of highly versatile and synthetically useful ortho-trifluoromethanesulfonylated anilines from arylhydroxylamines and trifluoromethanesulfinic chloride was developed. This rapid transformation proceeded smoothly with good yields and excellent ortho-selectivity in the absence of any metals or ligands. Mechanistically, the reaction comprised a noncanonical O-trifluoromethanesulfinylation of the arylhydroxylamine, and the subsequent [2,3]-sigmatropic rearrangement to afford ortho-trifluoromethanesulfonylated aniline derivatives. The practical application of this reaction was demonstrated by further conversion into a series of functional molecules under different reaction conditions.

A hybridization-chain-reaction-based method for amplifying immunosignals.

Immunosignal hybridization chain reaction (isHCR) combines antibody-antigen interactions with hybridization chain reaction (HCR) technology, which results in amplification of immunofluorescence signals by up to two to three orders of magnitude with low background. isHCR's highly modular and easily adaptable design enables the technique to be applied broadly, and we further optimized its use in multiplexed imaging and in state-of-the-art tissue expansion and clearing techniques.

A thiazole-derived oridonin analogue exhibits antitumor activity by directly and allosterically inhibiting STAT3.

Constitutive activation of signal transducer and activator of transcription 3 (STAT3) occurs in ∼70% of human cancers, and STAT3 is regarded as one of the most promising targets for cancer therapy. However, specific direct STAT3 inhibitors remain to be developed. Oridonin is an ent-kaurane plant-derived diterpenoid with anti-cancer and anti-inflammatory activities. Here, using an array of cell-based and biochemical approaches, including cell proliferation and apoptosis assays, pulldown and reporter gene assays, site-directed mutagenesis, and molecular dynamics analyses, we report that a thiazole-derived oridonin analogue, CYD0618, potently and directly inhibits STAT3. We found that CYD0618 covalently binds to Cys-542 in STAT3 and suppresses its activity through an allosteric effect, effectively reducing STAT3 dimerization and nuclear translocation, as well as decreasing expression of STAT3-targeted oncogenes. Remarkably, CYD0618 not only strongly inhibited growth of multiple cancer cell lines that harbor constitutive STAT3 activation, but it also suppressed in vivo tumor growth via STAT3 inhibition. Taken together, our findings suggest Cys-542 as a druggable site for selectively inhibiting STAT3 and indicate that CYD0618 represents a promising lead compound for developing therapeutic agents against STAT3-driven diseases.

Chemoselective Cross-Coupling of gem-Borazirconocene Alkanes with Aryl Halides.

The direct and chemoselective conversion of the carbon-metal bond of gem-dimetallic reagents enables rapid and sequential formation of multiple carbon-carbon and carbon-heteroatom bonds, thus representing a powerful method for efficiently increasing structural complexity. Herein, we report a visible-light-induced, nickel-catalyzed, chemoselective cross-coupling reaction between gem-borazirconocene alkanes and diverse aryl halides, affording a wide range of alkyl Bpin derivatives in high yields with excellent regioselectivity. This practical method features attractively simple reaction conditions and a broad substrate scope. Additionally, we systematically investigated a Bpin-directed chain walking process underlying the regioselectivity of alkylzirconocenes, thus uncovering the mechanism of the remote functionalization of internal olefins achieved with our method. Finally, DFT calculations indicate that the high regioselectivity of this reaction originates from the directing effect of the Bpin group.

A novel inhibitor of pyruvate dehydrogenase kinase stimulates myocardial carbohydrate oxidation in diet-induced obesity.

The pyruvate dehydrogenase complex (PDC) is a key control point of energy metabolism and is subject to regulation by multiple mechanisms, including posttranslational phosphorylation by pyruvate dehydrogenase kinase (PDK). Pharmacological modulation of PDC activity could provide a new treatment for diabetic cardiomyopathy, as dysregulated substrate selection is concomitant with decreased heart function. Dichloroacetate (DCA), a classic PDK inhibitor, has been used to treat diabetic cardiomyopathy, but the lack of specificity and side effects of DCA indicate a more specific inhibitor of PDK is needed. This study was designed to determine the effects of a novel and highly selective PDK inhibitor, 2((2,4-dihydroxyphenyl)sulfonyl) isoindoline-4,6-diol (designated PS10), on pyruvate oxidation in diet-induced obese (DIO) mouse hearts compared with DCA-treated hearts. Four groups of mice were studied: lean control, DIO, DIO + DCA, and DIO + PS10. Both DCA and PS10 improved glucose tolerance in the intact animal. Pyruvate metabolism was studied in perfused hearts supplied with physiological mixtures of long chain fatty acids, lactate, and pyruvate. Analysis was performed using conventional 1H and 13C isotopomer methods in combination with hyperpolarized [1-13C]pyruvate in the same hearts. PS10 and DCA both stimulated flux through PDC as measured by the appearance of hyperpolarized [13C]bicarbonate. DCA but not PS10 increased hyperpolarized [1-13C]lactate production. Total carbohydrate oxidation was reduced in DIO mouse hearts but increased by DCA and PS10, the latter doing so without increasing lactate production. The present results suggest that PS10 is a more suitable PDK inhibitor for treatment of diabetic cardiomyopathy.

Total Synthesis of (-)-Alstofolinine†A through a Furan Oxidation/Rearrangement and Indole Nucleophilic Cyclization Cascade.

A reaction cascade of aza-Achmatowicz rearrangement followed by indole nucleophilic cyclization was developed to generate the common indole-fused azabicyclo[3.3.1]nonane core of the macroline family alkaloids. The key to the success of the strategy relies on the careful manipulation of protecting groups and judicious selection of chemoselective furan oxidation conditions. The synthetic utility was further demonstrated on the asymmetric total synthesis of (-)-alstofolinine A.

Development of the Vinylogous Pictet-Spengler Cyclization and Total Synthesis of (±)-Lundurine†A.

A novel vinylogous Pictet-Spengler cyclization has been developed for the generation of indole-annulated medium-sized rings. The method enables the synthesis of tetrahydroazocinoindoles with a fully substituted carbon center, a prevalent structural motif in many biologically active alkaloids. The strategy has been applied to the total synthesis of (±)-lundurine A.

Structure-based design and mechanisms of allosteric inhibitors for mitochondrial branched-chain α-ketoacid dehydrogenase kinase.

The branched-chain amino acids (BCAAs) leucine, isoleucine, and valine are elevated in maple syrup urine disease, heart failure, obesity, and type 2 diabetes. BCAA homeostasis is controlled by the mitochondrial branched-chain α-ketoacid dehydrogenase complex (BCKDC), which is negatively regulated by the specific BCKD kinase (BDK). Here, we used structure-based design to develop a BDK inhibitor, (S)-α-chloro-phenylpropionic acid [(S)-CPP]. Crystal structures of the BDK-(S)-CPP complex show that (S)-CPP binds to a unique allosteric site in the N-terminal domain, triggering helix movements in BDK. These conformational changes are communicated to the lipoyl-binding pocket, which nullifies BDK activity by blocking its binding to the BCKDC core. Administration of (S)-CPP to mice leads to the full activation and dephosphorylation of BCKDC with significant reduction in plasma BCAA concentrations. The results buttress the concept of targeting mitochondrial BDK as a pharmacological approach to mitigate BCAA accumulation in metabolic diseases and heart failure.

Structure-guided development of specific pyruvate dehydrogenase kinase inhibitors targeting the ATP-binding pocket.

Pyruvate dehydrogenase kinase isoforms (PDKs 1-4) negatively regulate activity of the mitochondrial pyruvate dehydrogenase complex by reversible phosphorylation. PDK isoforms are up-regulated in obesity, diabetes, heart failure, and cancer and are potential therapeutic targets for these important human diseases. Here, we employed a structure-guided design to convert a known Hsp90 inhibitor to a series of highly specific PDK inhibitors, based on structural conservation in the ATP-binding pocket. The key step involved the substitution of a carbonyl group in the parent compound with a sulfonyl in the PDK inhibitors. The final compound of this series, 2-[(2,4-dihydroxyphenyl)sulfonyl]isoindoline-4,6-diol, designated PS10, inhibits all four PDK isoforms with IC50 = 0.8 µM for PDK2. The administration of PS10 (70 mg/kg) to diet-induced obese mice significantly augments pyruvate dehydrogenase complex activity with reduced phosphorylation in different tissues. Prolonged PS10 treatments result in improved glucose tolerance and notably lessened hepatic steatosis in the mouse model. The results support the pharmacological approach of targeting PDK to control both glucose and fat levels in obesity and type 2 diabetes.

Benzothiophene carboxylate derivatives as novel allosteric inhibitors of branched-chain α-ketoacid dehydrogenase kinase.

The mitochondrial branched-chain α-ketoacid dehydrogenase complex (BCKDC) is negatively regulated by reversible phosphorylation.BCKDC kinase (BDK) inhibitors that augment BCKDC flux have been shown to reduce branched-chain amino acid (BCAA) concentrations in vivo. In the present study, we employed high-throughput screens to identify compound 3,6- dichlorobenzo[b]thiophene-2-carboxylic acid (BT2) as a novel BDK inhibitor (IC(50) = 3.19 µM). BT2 binds to the same site in BDK as other known allosteric BDK inhibitors, including (S)-α-cholorophenylproprionate ((S)-CPP). BT2 binding to BDK triggers helix movements in the N-terminal domain, resulting in the dissociation of BDK from the BCKDC accompanied by accelerated degradation of the released kinase in vivo. BT2 shows excellent pharmacokinetics (terminal T(1/2) = 730 min) and metabolic stability (no degradation in 240 min), which are significantly better than those of (S)-CPP. BT2, its analog 3-chloro-6-fluorobenzo[ b]thiophene-2-carboxylic acid (BT2F), and a prodrug of BT2 (i.e. N-(4-acetamido-1,2,5-oxadiazol-3-yl)-3,6-dichlorobenzo[ b]thiophene-2-carboxamide (BT3)) significantly increase residual BCKDC activity in cultured cells and primary hepatocytes from patients and a mouse model of maple syrup urine disease. Administration of BT2 at 20 mg/kg/day to wild-type mice for 1 week leads to nearly complete dephosphorylation and maximal activation of BCKDC in heart, muscle, kidneys, and liver with reduction in plasma BCAA concentrations. The availability of benzothiophene carboxylate derivatives as stable BDK inhibitors may prove useful for the treatment of metabolic disease caused by elevated BCAA concentrations.