AUTACs: Cargo-Specific Degraders Using Selective Autophagy.

Protein silencing represents an essential tool in biomedical research. Targeted protein degradation (TPD) strategies exemplified by PROTACs are rapidly emerging as modalities in drug discovery. However, the scope of current TPD techniques is limited because many intracellular materials are not substrates of proteasomal clearance. Here, we described a novel targeted-clearance strategy (autophagy-targeting chimera [AUTAC]) that contains a degradation tag (guanine derivatives) and a warhead to provide target specificity. As expected from the substrate scope of autophagy, AUTAC degraded fragmented mitochondria as well as proteins. Mitochondria-targeted AUTAC accelerated both the removal of dysfunctional fragmented mitochondria and the biogenesis of functionally normal mitochondria in patient-derived fibroblast cells. Cytoprotective effects against acute mitochondrial injuries were also seen. Canonical autophagy is viewed as a nonselective bulk decomposition system, and none of the available autophagy-inducing agents exhibit useful cargo selectivity. With its target specificity, AUTAC provides a new modality for research on autophagy-based drugs.

Endogenous nitrated nucleotide is a key mediator of autophagy and innate defense against bacteria.

Autophagy is a cellular self-catabolic process wherein organelles, macromolecules, and invading microbes are sequestered in autophagosomes that fuse with lysosomes. In this study, we uncover the role of nitric oxide (NO) as a signaling molecule for autophagy induction via its downstream mediator, 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP). We found that 8-nitro-cGMP-induced autophagy is mediated by Lys63-linked polyubiquitination and that endogenous 8-nitro-cGMP promotes autophagic exclusion of invading group A Streptococcus (GAS) from cells. 8-nitro-cGMP can modify Cys residues by S-guanylation of proteins. We showed that intracellular GAS is modified with S-guanylation extensively in autophagosomes-like vacuoles, suggesting the role of S-guanylation as a marker for selective autophagic degradation. This finding is supported by the fact that S-guanylated bacteria were selectively marked with polyubiquitin, a known molecular tag for selective transport to autophagosomes. These results collectively indicate that 8-nitro-cGMP plays a crucial role in cytoprotection during bacterial infections or inflammations via autophagy upregulation.

Endothelial cells are intrinsically defective in xenophagy of Streptococcus pyogenes.

Group A Streptococcus (GAS) is deleterious pathogenic bacteria whose interaction with blood vessels leads to life-threatening bacteremia. Although xenophagy, a special form of autophagy, eliminates invading GAS in epithelial cells, we found that GAS could survive and multiply in endothelial cells. Endothelial cells were competent in starvation-induced autophagy, but failed to form double-membrane structures surrounding GAS, an essential step in xenophagy. This deficiency stemmed from reduced recruitment of ubiquitin and several core autophagy proteins in endothelial cells, as demonstrated by the fact that it could be rescued by exogenous coating of GAS with ubiquitin. The defect was associated with reduced NO-mediated ubiquitin signaling. Therefore, we propose that the lack of efficient clearance of GAS in endothelial cells is caused by their intrinsic inability to target GAS with ubiquitin to promote autophagosome biogenesis for xenophagy.

8-Nitro-cGMP: A Novel Protein-Reactive cNMP and Its Emerging Roles in Autophagy.

Nitric oxide (NO) raises the intracellular 3',5'-cyclic guanosine monophosphate (cGMP) level through the activation of soluble guanylate cyclase and, in the presence of reactive oxygen species (ROS), reacts with biomolecules to produce nitrated cGMP derivatives. 8-Nitro-cGMP was the first endogenous cGMP derivative discovered in mammalian cells (2007) and was later found in plant cells. Among the six nitrogen atoms in this molecule, the one in the nitro group (NO2) comes from NO. This chapter asserts that this newly found cGMP is undoubtedly one of the major physiological cNMPs. Multiple studies suggest that its intracellular abundance might exceed that of unmodified cGMP. The characteristic chemical feature of 8-nitro-cGMP is its ability to modify proteinous cysteine residues via a stable sulfide bond. In this posttranslational modification, the nitro group is detached from the guanine base. This modification, termed "protein S-guanylation," is known to regulate the physiological functions of several important proteins. Furthermore, 8-nitro-cGMP participates in the regulation of autophagy. For example, in antibacterial autophagy (xenophagy), S-guanylation accumulates around invading bacterial cells and functions as a "tag" for subsequent clearance of the organism via ubiquitin modifications. This finding suggests the existence of a system for recognizing the cGMP structure on proteins. Autophagy induction by 8-nitro-cGMP is mechanistically distinct from the well-described starvation-induced autophagy and is independent of the action of mTOR, the master regulator of canonical autophagy.

Novel blood-brain barrier-permeable spin probe for in vivo electron paramagnetic resonance imaging.

A novel blood-brain barrier (BBB)-permeable compound 10 was discovered, wherein the nitroxide moiety was linked to a nicotine acetylcholine receptor ligand. It was applied as a probe for electron paramagnetic resonance (EPR) imaging of the mouse brain. The results demonstrated that the newly synthesized compound 10 exhibited BBB permeability. These findings provide an essential discovery for in vivo EPR imaging.

Binding properties of antimicrobial agents to dipeptide terminal of lipid II using surface plasmon resonance.

We developed a surface plasmon resonance (SPR) assay to estimate the interactions of antimicrobial agents with the dipeptide terminal of lipid II (D-alanyl-D-alanine) and its analogous dipeptides (L-alanyl-L-alanine and D-alanyl-D-lactate) as ligands. The established SPR method showed the reproducible immobilization of ligands on sensor chip and analysis of binding kinetics of antimicrobial agents to ligands. The ligand-immobilized chip could be used repeatedly for at least 200 times for the binding assay of antimicrobial agents, indicating that the ligand-immobilized chip is sufficiently robust for the analysis of binding kinetics. In this SPR system, the selective and specific binding characteristics of vancomycin and its analogs to the ligands were estimated and the kinetic parameters were calculated. The kinetic parameters revealed that one of the remarkable binding characteristics was the specific interaction of vancomycin to only the D-alanyl-D-alanine ligand. In addition, the kinetic binding data of SPR showed close correlation with the antimicrobial activity. The SPR data of other antimicrobial agents (e.g., teicoplanin) to the ligands showed correlation with the antimicrobial activity on the basis of the therapeutic mechanism. Our SPR method could be a valuable tool for predicting the binding characteristics of antimicrobial agents to the dipeptide terminal of lipid II.

Binding properties of antimicrobial agents to lipid membranes using surface plasmon resonance.

In the present study, we examined the interaction of antimicrobial agents with four model lipid membranes that mimicked mammalian cell membranes and Gram-positive and -negative bacterial membranes and analyzed the binding kinetics using our surface plasmon resonance (SPR) technique. The selective and specific binding characteristics of antimicrobial agents to the lipid membranes were estimated, and the kinetic parameters were analyzed by application of a two-state reaction model. Reproducible analysis of binding kinetics was observed. Vancomyicn, teicoplanin, erythromycin, and linezolid showed little interaction with the four lipid membranes in the SPR system. On the other hand, vancomycin analogues showed interaction with the model lipid membranes in the SPR system. The selective and specific binding characteristics of vancomycin analogues to the lipid membranes are discussed based on data for in vitro antibacterial activities and our data on the binding affinity of the D-alanyl-D-alanine terminus of a pentapeptide cell wall obtained by SPR. The mechanism of antibacterial activity against Staphylococcus aureus and vancomycin-resistant enterococci could be evaluated using the binding affinity obtained with our SPR techniques. The results indicate that the SPR method could be widely applied to predict binding characteristics, such as selectivity and specificity, of many antimicrobial agents to lipid membranes.

Total synthesis of the antibiotic kendomycin: a macrocyclization using the Tsuji-Trost etherification.

A highly stereocontrolled, convergent total synthesis of kendomycin [(-)-TAN2162], an ansa-macrocyclic antibiotic, is reported. The key of the strategy is an unprecedented Tsuji-Trost macrocyclic etherification, followed by a transannular Claisen rearrangement to construct the 18-membered carbocyclic framework. The oxa-six- and five-membered rings were also stereoselectively constructed respectively by a cascade oxidative cyclization at an unfunctionalized benzylic position and using a one-pot epoxidation/5-exo-tet epoxide opening.

Simultaneous imaging of an enantiomer pair by electron paramagnetic resonance using isotopic nitrogen labeling.

This Article describes the simultaneous imaging of chiral nitroxyl radicals using electron paramagnetic resonance (EPR). Chiral nitroxyl radicals could be simultaneously visualized with the labeling of isotopic nitrogen. Chiral nitroxyl radicals, hydroxylmethyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl, were visualized using the method of simultaneous EPR imaging, which refers to the visualization of two kinds of molecules with unpaired electrons in a single image scan. EPR spectra of a racemic mixture of chiral nitroxyl radicals and those of the respective R and S configurations confirmed labeling by isotopic nitrogen. (1)H nuclear magnetic resonance (NMR) imaging and simultaneous imaging of solutions of chiral nitroxyl radicals were performed. The advantages and limitations of simultaneous imaging using EPR are also discussed. Simultaneous imaging with chiral-labeled nitroxyl radicals is a new application of EPR imaging and may be useful for biological studies involving biologically active chiral molecules.

Fluorescent probes for live cell imaging of endogenous Guanine nitration.

Seeing is believing: S-guanylation is a novel key mechanism by which signal transduction under oxidative stress is regulated. A chemical probe whose fluorescent intensity increases after the reaction with proteinous cysteine (S-guanylation) is described. The use of this probe revealed that S-guanylation products localized in lysosomes.

Staphylococcus aureus Penicillin-Binding Protein 2 Can Use Depsi-Lipid II Derived from Vancomycin-Resistant Strains for Cell Wall Synthesis.

Vancomycin-resistant Staphylococcus aureus (S. aureus) (VRSA) uses depsipeptide-containing modified cell-wall precursors for the biosynthesis of peptidoglycan. Transglycosylase is responsible for the polymerization of the peptidoglycan, and the penicillin-binding protein 2 (PBP2) plays a major role in the polymerization among several transglycosylases of wild-type S. aureus. However, it is unclear whether VRSA processes the depsipeptide-containing peptidoglycan precursor by using PBP2. Here, we describe the total synthesis of depsi-lipid I, a cell-wall precursor of VRSA. By using this chemistry, we prepared a depsi-lipid II analogue as substrate for a cell-free transglycosylation system. The reconstituted system revealed that the PBP2 of S. aureus is able to process a depsi-lipid II intermediate as efficiently as its normal substrate. Moreover, the system was successfully used to demonstrate the difference in the mode of action of the two antibiotics moenomycin and vancomycin.

C. elegans ATG-5 mutants associated with ataxia.

Intercellular cleaning via autophagy is crucial for maintaining cellular homeostasis, and impaired autophagy has been associated with the accumulation of protein aggregates that can contribute to neurological diseases. Specifically, the loss-of-function mutation in the human autophagy-related gene 5 (ATG5) at E122D has been linked to the pathogenesis of spinocerebellar ataxia in humans. In this study, we generated two homozygous C. elegans strains with mutations (E121D and E121A) at positions corresponding to the human ATG5 ataxia mutation to investigate the effects of ATG5 mutations on autophagy and motility. Our results showed that both mutants exhibited a reduction in autophagy activity and impaired motility, suggesting that the conserved mechanism of autophagy-mediated regulation of motility extends from C. elegans to humans.

Second-Generation AUTACs for Targeted Autophagic Degradation.

Targeted protein degradation via the ubiquitin-proteasome system has emerged as one of the most promising drug discovery modalities. Autophagy, another intracellular degradation system, can target a wide range of nonproteinous substrates as well as proteins, but its application to targeted degradation is still in its infancy. Our previous work revealed a relationship between guanine modification of cysteine residues on intracellular proteins and selective autophagy, resulting in the first autophagy-based degraders, autophagy-targeted chimeras (AUTACs). Based on the research background, all the reported AUTACs compounds contain cysteine as a substructure. Here, we examine the importance of this substructure by conducting SAR studies and report significant improvements in the degrader's activity by replacing cysteine with other moieties. Several derivatives showed sub-µM range degrading activity, demonstrating the increased practical value of AUTACs.

Elucidation of the active conformation of vancomycin dimers with antibacterial activity against vancomycin-resistant bacteria.

Covalently linked vancomycin dimers have attracted a great deal of attention among researchers because of their enhanced antibacterial activity against vancomycin-resistant strains. However, the lack of a clear insight into the mechanisms of action of these dimers hampers rational optimization of their antibacterial potency. Here, we describe the synthesis and antibacterial activity of novel vancomycin dimers with a constrained molecular conformation achieved by two tethers between vancomycin units. Conformational restriction is a useful strategy for studying the relationship between the molecular topology and biological activity of compounds. In this study, two vancomycin units were linked at three distinct positions of the glycopeptide (vancosamine residue (V), C terminus (C), and N terminus (N)) to form two types of novel vancomycin cyclic dimers. Active NC-VV-linked dimers with a stable conformation as indicated by molecular mechanics calculations selectively suppressed the peptidoglycan polymerization reaction of vancomycin-resistant Staphylococcus aureus in vitro. In addition, double-disk diffusion tests indicated that the antibacterial activity of these dimers against vancomycin-resistant enterococci might arise from the inhibition of enzymes responsible for peptidoglycan polymerization. These findings provide a new insight into the biological targets of vancomycin dimers and the conformational requirements for efficient antibacterial activity against vancomycin-resistant strains.

Pinnarine, another member of the halichlorine family. Isolation and preparation from pinnaic acid.

Pinnarine (1), a new macrocyclic alkaloid, was isolated from the black marine sponge Halichondria okadai. The structure was elucidated on the basis of 2D NMR and comparison with the spectra of the co-isolated known halichlorine. Further confirmation of the structure and the absolute configuration was validated by a synthetic method from authentic pinnaic acid and CD analysis. The isolation of pinnarine also suggested a biogenetic pathway from pinnaic acid to halichlorine.

Selective autophagy as the basis of autophagy-based degraders.

Degrader technologies, which enable the chemical knockdown of disease-causing proteins, are promising for drug discovery. After two decades of research, degraders using the ubiquitin-proteasome system (UPS) are currently in clinical trials. However, the UPS substrates are mainly limited to soluble proteins. Autophagy-targeting chimeras and autophagosome-tethering compounds are degraders that use autophagy, which has functions complementary to the UPS. They can degrade organelles and aggregate-prone proteins, making them promising treatments against age-related conditions such as mitochondrial dysfunction and neurodegenerative diseases. The molecular mechanism of selective autophagy is an ongoing research topic, which explains why autophagy-based degraders were not available until recently. In this review, we introduce four classifications of selective autophagy mechanisms to facilitate the understanding of the degrader design.

Mode of action of Van-M-02, a novel glycopeptide inhibitor of peptidoglycan synthesis, in vancomycin-resistant bacteria.

Van-M-02, a novel glycopeptide, was revealed to exert potent activities against Gram-positive bacteria, including vancomycin-resistant enterococci (VRE) and vancomycin-resistant Staphylococcus aureus (VRSA). A crude assay system was then used to study the mode of action of Van-M-02 as a peptidoglycan synthesis model of both vancomycin-susceptible and -resistant strains. The results suggested that Van-M-02 inhibits the synthesis of lipid intermediates irrespective of their termini. This inhibitory activity may contribute to the anti-VRE and anti-VRSA activities observed.

Regulation of redox signaling involving chemical conjugation of protein thiols by nitric oxide and electrophiles.

Nitric oxide (NO), a gaseous free radical that is biologically synthesized by nitric oxide synthases, participates in a critical fashion in the regulation of diverse physiological functions including vascular and neuronal signal transduction, host defense, and cell death regulation. This article reviews the chemical and biochemical mechanisms of protein thiol modifications caused by NO and by electrophiles derived from NO metabolism. The classical NO signaling pathway involves formation of the second messenger guanosine 3',5'-cyclic monophosphate (cGMP). Post-translational modifications of redox-sensitive protein thiols have also been shown to be important in this signaling pathway. For instance, redox-sensitive thiols are targets for NO conjugation and formation of S-nitrosothiols. Electrophiles generated by reactions of NO or reactive nitrogen oxide species and biomolecules (i.e., fatty acids) effect thiol conjugations through S-alkylation. Among this class of reactions, S-guanylation is particularly emphasized. S-Guanylation is a novel type of S-alkylation with nitrated cGMP that contributes to the cytoprotective effects of NO. Post-translational modifications of thiols affect protein structures and functions: allosteric effects may alter protein structure, modification of active centers of enzymes may suppress enzyme actions, and modifications may modulate protein-protein interactions. Better understanding of protein modification by NO-derived electrophiles and the molecular basis of NO signaling would be useful in the development of new diagnostic methods and treatment of diseases related to NO metabolism.

Targeting selective autophagy by AUTAC degraders.

Targeted degradation is a promising new modality in drug discovery that makes it possible to reduce intracellular protein levels with small molecules. It is a complementary approach to the conventional protein knockdown typically used in laboratories and may offer a way to approach the currently undruggable human proteome. Recently, the first autophagy-mediated degraders, called AUTACs, were developed based on observations in a xenophagy study.