The adhesion domain of human CD2 bears a single N-linked carbohydrate. The solution structure of a fragment of CD2 containing the covalently bound high-mannose N-glycan [-(N-acetylglucosamine)2-(mannose)5-8] was solved by nuclear magnetic resonance. The stem and two of three branches of the carbohydrate structure are well defined and the mobility of proximal glycan residues is restricted. Mutagenesis of all residues in the vicinity of the glycan suggests that the glycan is not a component of the CD2-CD58 interface; rather, the carbohydrate stabilizes the protein fold by counterbalancing an unfavorable clustering of five positive charges centered about lysine-61 of CD2.

Thiolutin is a disulfide-containing antibiotic and anti-angiogenic compound produced by Streptomyces. Its biological targets are not known. We show that reduced thiolutin is a zinc-chelator that inhibits the JAB1/MPN/Mov34 (JAMM) domain-containing metalloprotease Rpn11, a de-ubiquinating enzyme of the 19S proteasome. Thiolutin also inhibits the JAMM metalloproteases Csn5, the deneddylase of the COP9 signalosome, Associated-molecule-with-the-SH3-Domain-of-STAM (AMSH), which regulates ubiquitin-dependent sorting of cell-surface receptors, and Brcc36, a K63-specific deubiquitnase of BRCC36-containing isopeptidase complex (BRISC) and BRCA1-BRCA2-containing complex (BRCC). We provide evidence that other dithiolopyrrolones also function as inhibitors of JAMM metalloproteases.

Dendrimers are macromolecular, nanoscale objects that are widely recognized as precise, mathematically defined, covalent core-shell assemblies. As such, they are composed of quantized numbers of atoms, monomers, and terminal functional groups relative to the respective shell levels (generations) surrounding their cores. Dendrimers have been referred to as molecular-level analogs of atoms. This perspective arises from their potential to function as precise macromolecular tectons (modules), suitable for the synthesis of structure-controlled complexity beyond dendrimers. We have termed this major new class of generic structures "megamers". Our group has now synthesized such "megamer complexity" in the form of both covalent and supra-macromolecular dendri-catenanes, dendri-macrocycles, dendri-clefts, and dendri-clusters. The covalent dendri-cluster subset of megamers has been coined "core-shell tecto(dendrimers)". New mathematically defined, covalent bonding rules for tecto(dendrimer) formation are consistent with sterically induced stoichiometry (SIS) predictions and have been verified experimentally.

O-linked β-N-acetyl-glucosamine (O-GlcNAc) is an essential and ubiquitous post-translational modification present in nucleic and cytoplasmic proteins of multicellular eukaryotes. The metabolic chemical probes such as GlcNAc or GalNAc analogues bearing ketone or azide handles, in conjunction with bioorthogonal reactions, provide a powerful approach for detecting and identifying this modification. However, these chemical probes either enter multiple glycosylation pathways or have low labeling efficiency. Therefore, selective and potent probes are needed to assess this modification. We report here the development of a novel probe, 1,3,6-tri-O-acetyl-2-azidoacetamido-2,4-dideoxy-d-glucopyranose (Ac4dGlcNAz), that can be processed by the GalNAc salvage pathway and transferred by O-GlcNAc transferase (OGT) to O-GlcNAc proteins. Due to the absence of a hydroxyl group at C4, this probe is less incorporated into α/β 4-GlcNAc or GalNAc containing glycoconjugates. Furthermore, the O-4dGlcNAz modification was resistant to the hydrolysis of O-GlcNAcase (OGA), which greatly enhanced the efficiency of incorporation for O-GlcNAcylation. Combined with a click reaction, Ac4dGlcNAz allowed the selective visualization of O-GlcNAc in cells and accurate identification of O-GlcNAc-modified proteins with LC-MS/MS. This probe represents a more potent and selective tool in tracking, capturing, and identifying O-GlcNAc-modified proteins in cells and cell lysates.

NA interference (RNAi) therapeutics use an endogenous mechanism whereby short interfering RNAs (siRNAs) direct the RNA-induced silencing complex (RISC) to sequence matched target transcripts for knockdown 1 . Both lipid nanoparticles and N-acetylgalactosamine (GalNAc) conjugates are clinically validated and approved delivery strategies for liver targets [2][3][4][5][6][7][8] . Building on nearly 2 decades of siRNA design and chemistry optimization [9][10][11][12] , we demonstrate here that, with suitable delivery solutions, the RNAi pathway can be harnessed in extrahepatic tissues, such as the central nervous system (CNS), eye and lung. Multiple CNS diseases, representing some of the highest unmet medical needs and greatest therapeutic challenges, have been associated with dominant gain-of-function mutations, making them suitable candidates for an RNAi-based silencing approach. As such, chemically modified siRNAs have demonstrated potent and sustained silencing in rodents and non-human primates (NHPs); however, using an invasive intracerebroventricular (ICV) administration approach 13 that is not suitable for repeated dosing in humans. Furthermore, technologies enabling siRNA delivery across the blood-brain barrier following a less challenging systemic administration are similarly being explored [14][15][16][17] , which are, however, still in early stages of discovery. In the eye, intravitreal (IVT) dosing of siRNAs has been evaluated in late-stage clinical studies, with few safety concerns, but did not advance further due to lack of efficacy 18 . Recently, the Coronavirus Disease 2019 (COVID-19) pandemic has highlighted the importance of optimizing siRNA delivery to the lung for the treatment of emergent viral respiratory diseases. Although earlier programs have already shown potential clinical benefits of siRNA-based therapeutics in the lung 19 , 2′-O-hexadecyl (C16) conjugates demonstrate enhanced delivery and siRNA uptake into the alveolar and bronchiolar epithelium. Taken together, this work highlights that the combination of a C16 lipophilic modification with our fully chemically modified, metabolically stable siRNAs achieves efficient delivery to the CNS, eye and lung, resulting in a robust and durable gene silencing in rodents and NHPs, with a favorable safety profile. We think that these advances have the potential to generate multiple candidates for investigating clinical safety and efficacy in humans. ResultsOptimization of the siRNA conjugate design. Lipophilic moieties represent one of the earliest approaches to improve cellular uptake and delivery of antisense oligonucleotides (ASOs) and siRNAs to the liver and various other organ systems 20 , including the CNS [21][22][23] . We reasoned that, by carefully optimizing the lipophilicity of chemically modified siRNAs, we could enhance intracellular delivery without compromising broad biodistribution, potency and safety. We used the 2′ position of the ribose sugar backbone to introduce