Dual-specificity RNA aptamers: A sweet new tool for studying O-GlcNAc biology.

Zhu and Hart1 use dual-specificity RNA aptamers to recruit cellular O-GlcNAc transferase (OGT) and induce O-GlcNAc on target proteins like ß-catenin, revealing that O-GlcNAc stabilizes ß-catenin and enhances its transcriptional activity.

The E3 ligase adapter cereblon targets the C-terminal cyclic imide degron.

The ubiquitin E3 ligase substrate adapter cereblon (CRBN) is a target of thalidomide and lenalidomide1, therapeutic agents used in the treatment of haematopoietic malignancies2-4 and as ligands for targeted protein degradation5-7. These agents are proposed to mimic a naturally occurring degron; however, the structural motif recognized by the thalidomide-binding domain of CRBN remains unknown. Here we report that C-terminal cyclic imides, post-translational modifications that arise from intramolecular cyclization of glutamine or asparagine residues, are physiological degrons on substrates for CRBN. Dipeptides bearing the C-terminal cyclic imide degron substitute for thalidomide when embedded within bifunctional chemical degraders. Addition of the degron to the C terminus of proteins induces CRBN-dependent ubiquitination and degradation in vitro and in cells. C-terminal cyclic imides form adventitiously on physiologically relevant timescales throughout the human proteome to afford a degron that is endogenously recognized and removed by CRBN. The discovery of the C-terminal cyclic imide degron defines a regulatory process that may affect the physiological function and therapeutic engagement of CRBN.

Photo-affinity and Metabolic Labeling Probes Based on the Opioid Alkaloids.

The opioids are powerful analgesics yet possess contingencies that can lead to opioid-use disorder. Chemical probes derived from the opioid alkaloids can provide deeper insight into the molecular interactions in a cellular context. Here, we designed and developed photo-click morphine (PCM-2) as a photo-affinity probe based on morphine and dialkynyl-acetyl morphine (DAAM) as a metabolic acetate reporter based on heroin. Application of these probes to SH-SY5Y, HEK293T, and U2OS cells revealed that PCM-2 and DAAM primarily localize to the lysosome amongst other locations inside the cell by confocal microscopy and chemical proteomics. Interaction site identification by mass spectrometry revealed the mitochondrial phosphate carrier protein, solute carrier family 25 member 3, SLC25A3, and histone H2B as acylation targets of DAAM. These data illustrate the utility of chemical probes to measure localization and protein interactions in a cellular context and will inform the design of probes based on the opioids in the future.

Community evaluation of glycoproteomics informatics solutions reveals high-performance search strategies for serum glycopeptide analysis.

Glycoproteomics is a powerful yet analytically challenging research tool. Software packages aiding the interpretation of complex glycopeptide tandem mass spectra have appeared, but their relative performance remains untested. Conducted through the HUPO Human Glycoproteomics Initiative, this community study, comprising both developers and users of glycoproteomics software, evaluates solutions for system-wide glycopeptide analysis. The same mass spectrometrybased glycoproteomics datasets from human serum were shared with participants and the relative team performance for N- and O-glycopeptide data analysis was comprehensively established by orthogonal performance tests. Although the results were variable, several high-performance glycoproteomics informatics strategies were identified. Deep analysis of the data revealed key performance-associated search parameters and led to recommendations for improved 'high-coverage' and 'high-accuracy' glycoproteomics search solutions. This study concludes that diverse software packages for comprehensive glycopeptide data analysis exist, points to several high-performance search strategies and specifies key variables that will guide future software developments and assist informatics decision-making in glycoproteomics.

Chemical tools for the opioids.

The opioids are potent and widely used pain management medicines despite also possessing severe liabilities that have fueled the opioid crisis. The pharmacological properties of the opioids primarily derive from agonism or antagonism of the opioid receptors, but additional effects may arise from specific compounds, opioid receptors, or independent targets. The study of the opioids, their receptors, and the development of remediation strategies has benefitted from derivatization of the opioids as chemical tools. While these studies have primarily focused on the opioids in the context of the opioid receptors, these chemical tools may also play a role in delineating mechanisms that are independent of the opioid receptors. In this review, we describe recent advances in the development and applications of opioid derivatives as chemical tools and highlight opportunities for the future.

A Versatile Isocyanate-Mediated Strategy for Appending Chemical Tags onto Drug-Like Small Molecules.

Target identification studies are a major hurdle in probe and drug discovery pipelines due to the need to chemically modify small molecules of interest, which can be time intensive and have low throughput. Here, we describe a versatile and scalable method for attaching chemical moieties to a small molecule, isocyanate-mediated chemical tagging (IMCT). By preparation of a template resin with an isocyanate capture group and a cleavable linker, nucleophilic groups on small molecules can be modified with an enforced one-to-one stoichiometry. We demonstrate a small molecule substrate scope that includes primary and secondary amines, thiols, phenols, benzyl alcohols, and primary alcohols. Cheminformatic analyses predict that IMCT is reactive with more than 25% of lead-like compounds in publicly available databases. To demonstrate that the method can produce biologically active molecules, we generated FKBP12 photoaffinity labeling (PAL) compounds with a wide range of affinities and showed that purified and crude cleavage products can bind to and label FKBP12. This method could be used to rapidly modify small molecules for many applications, including the synthesis of PAL probes, fluorescence polarization probes, pull-down probes, and degraders.

Target protein deglycosylation in living cells by a nanobody-fused split O-GlcNAcase.

O-linked N-acetylglucosamine (O-GlcNAc) is an essential and dynamic post-translational modification that is presented on thousands of nucleocytoplasmic proteins. Interrogating the role of O-GlcNAc on a single target protein is crucial, yet challenging to perform in cells. Herein, we developed a nanobody-fused split O-GlcNAcase (OGA) as an O-GlcNAc eraser for selective deglycosylation of a target protein in cells. After systematic cellular optimization, we identified a split OGA with reduced inherent deglycosidase activity that selectively removed O-GlcNAc from the desired target protein when directed by a nanobody. We demonstrate the generality of the nanobody-fused split OGA using four nanobodies against five target proteins and use the system to study the impact of O-GlcNAc on the transcription factors c-Jun and c-Fos. The nanobody-directed O-GlcNAc eraser provides a new strategy for the functional evaluation and engineering of O-GlcNAc via the selective removal of O-GlcNAc from individual proteins directly in cells.

The schizophrenia-associated variant in SLC39A8 alters protein glycosylation in the mouse brain.

A missense mutation (A391T) in SLC39A8 is strongly associated with schizophrenia in genomic studies, though the molecular connection to the brain is unknown. Human carriers of A391T have reduced serum manganese, altered plasma glycosylation, and brain MRI changes consistent with altered metal transport. Here, using a knock-in mouse model homozygous for A391T, we show that the schizophrenia-associated variant changes protein glycosylation in the brain. Glycosylation of Asn residues in glycoproteins (N-glycosylation) was most significantly impaired, with effects differing between regions. RNAseq analysis showed negligible regional variation, consistent with changes in the activity of glycosylation enzymes rather than gene expression. Finally, nearly one-third of detected glycoproteins were differentially N-glycosylated in the cortex, including members of several pathways previously implicated in schizophrenia, such as cell adhesion molecules and neurotransmitter receptors that are expressed across all cell types. These findings provide a mechanistic link between a risk allele and potentially reversible biochemical changes in the brain, furthering our molecular understanding of the pathophysiology of schizophrenia and a novel opportunity for therapeutic development.

Methods to characterize and discover molecular degraders in cells.

Cells use many post-translational modifications (PTMs) to tailor proteins and transduce cellular signals. Recent years have witnessed the rapid growth of small molecule and enzymatic strategies to purposely manipulate one particular PTM, ubiquitination, on desired target proteins in cells. These approaches typically act by induced proximity between an E3 ligase and a target protein resulting in ubiquitination and degradation of the substrate in cells. In this review, we cover recent approaches to study molecular degraders and discover their induced substrates in vitro and in live cells. Methods that have been adapted and applied to the development of molecular degraders are described, including global proteomics, affinity-purification, chemical proteomics and enzymatic strategies. Extension of these strategies to edit additional PTMs in cells is also discussed. This review is intended to assist researchers who are interested in editing PTMs with new modalities to select suitable method(s) and guide their studies.

Design and Evaluation of a Cyclobutane Diazirine Alkyne Tag for Photoaffinity Labeling in Cells.

Alkyl diazirines are frequently used in photoaffinity labeling to map small molecule-protein interactions in target identification studies. However, the alkyl diazirines can preferentially label acidic amino acids and acidic protein surfaces in a pH-dependent manner, presumably via a reactive alkyl diazo intermediate. Here, we explore the use of ring strain to alter these reactivity preferences and report the development of a cyclobutane diazirine photoaffinity tag with reduced pH-dependent reactivity, termed PALBOX. We show that PALBOX possesses differential reactivity profiles as compared to other diazirine tags in vitro and is readily incorporated into small molecules to profile their binding interactions in cells. Using a set of small molecule fragments and ligands, we show that photoaffinity probes equipped with PALBOX can label the known protein targets in cells with reduced labeling of known alkyl diazirine off-targets. Finally, we demonstrate that ligands equipped with PALBOX can accurately map small molecule-protein binding sites. Thus, PALBOX is a versatile diazirine-based photoaffinity tag for use in the development of chemical probes for photoaffinity labeling experiments, including the study of small molecule-protein interactions.

Development of Photolenalidomide for Cellular Target Identification.

The thalidomide analogue lenalidomide (Len) is a clinical therapeutic that alters the substrate engagement of cereblon (CRBN), a substrate receptor for the CRL4 E3 ubiquitin ligase. Here, we report the development of photolenalidomide (pLen), a Len probe with a photoaffinity label and enrichment handle, designed for target identification by chemical proteomics. pLen preserves the substrate degradation profile, phenotypic antiproliferative and immunomodulatory properties of Len, and enhances interactions with the thalidomide-binding domain of CRBN, as revealed by binding site mapping and molecular modeling. Using pLen, we captured the known targets IKZF1 and CRBN from multiple myeloma MM.1S cells and further identified a new target, eukaryotic translation initiation factor 3 subunit i (eIF3i), from HEK293T cells. eIF3i is directly labeled by pLen and forms a ternary complex with CRBN in the presence of Len across several epithelial cell lines but is itself not ubiquitylated or degraded. These data point to the existence of a broader array of targets induced by ligands to CRBN that may or may not be degraded, which can be identified by the highly translatable application of pLen to additional biological systems.

Labeling Preferences of Diazirines with Protein Biomolecules.

Diazirines are widely used in photoaffinity labeling (PAL) to trap noncovalent interactions with biomolecules. However, design and interpretation of PAL experiments is challenging without a molecular understanding of the reactivity of diazirines with protein biomolecules. Herein, we report a systematic evaluation of the labeling preferences of alkyl and aryl diazirines with individual amino acids, single proteins, and in the whole cell proteome. We find that alkyl diazirines exhibit preferential labeling of acidic amino acids in a pH-dependent manner that is characteristic of a reactive alkyl diazo intermediate, while the aryl-fluorodiazirine labeling pattern reflects reaction primarily through a carbene intermediate. From a survey of 32 alkyl diazirine probes, we use this reactivity profile to rationalize why alkyl diazirine probes preferentially enrich highly acidic proteins or those embedded in membranes and why probes with a net positive charge tend to produce higher labeling yields in cells and in vitro. These results indicate that alkyl diazirines are an especially effective chemistry for surveying the membrane proteome and will facilitate design and interpretation of biomolecular labeling experiments with diazirines.

Writing and erasing O-GlcNAc from target proteins in cells.

O-linked N-acetylglucosamine (O-GlcNAc) is a widespread reversible modification on nucleocytoplasmic proteins that plays an important role in many biochemical processes and is highly relevant to numerous human diseases. The O-GlcNAc modification has diverse functional impacts on individual proteins and glycosites, and methods for editing this modification on substrates are essential to decipher these functions. Herein, we review recent progress in developing methods for O-GlcNAc regulation, with a focus on methods for editing O-GlcNAc with protein- and site-selectivity in cells. The applications, advantages, and limitations of currently available strategies for writing and erasing O-GlcNAc and future directions are also discussed. These emerging approaches to manipulate O-GlcNAc on a target protein in cells will greatly accelerate the development of functional studies and enable therapeutic interventions in the O-GlcNAc field.

Truncation of the TPR domain of OGT alters substrate and glycosite selection.

O-GlcNAc transferase (OGT) is an essential enzyme that installs O-linked N-acetylglucosamine (O-GlcNAc) to thousands of protein substrates. OGT and its isoforms select from these substrates through the tetratricopeptide repeat (TPR) domain, yet the impact of truncations to the TPR domain on substrate and glycosite selection is unresolved. Here, we report the effects of iterative truncations to the TPR domain of OGT on substrate and glycosite selection with the model protein GFP-JunB and the surrounding O-GlcNAc proteome in U2OS cells. Iterative truncation of the TPR domain of OGT maintains glycosyltransferase activity but alters subcellular localization of OGT in cells. The glycoproteome and glycosites modified by four OGT TPR isoforms were examined on the whole proteome and a single target protein, GFP-JunB. We found the greatest changes in O-GlcNAc on proteins associated with mRNA splicing processes and that the first four TPRs of the canonical nucleocytoplasmic OGT had the broadest substrate scope. Subsequent glycosite analysis revealed that alteration to the last four TPRs corresponded to the greatest shift in the resulting O-GlcNAc consensus sequence. This dataset provides a foundation to analyze how perturbations to the TPR domain and expression of OGT isoforms affect the glycosylation of substrates, which will be critical for future efforts in protein engineering of OGT, the biology of OGT isoforms, and diseases associated with the TPR domain of OGT.

Enantioselective Synthesis and Biological Evaluation of Sanglifehrin†A and B and Analogs.

Sanglifehrin A and B are immunosuppressive macrocyclic natural products endowed with and differentiated by a unique spirocyclic lactam. Herein, we report an enantioselective total synthesis and biological evaluation of sanglifehrin A and B and analogs. Access to the spirocyclic lactam was achieved through convergent assembly of a key pyranone intermediate followed by a stereo-controlled spirocyclization. The 22-membered macrocyclic core was synthesized by ring-closing metathesis in the presence of 2,6-bis(trifluoromethyl) benzeneboronic acid (BFBB). The spirocyclic lactam and macrocycle fragments were united by a Stille coupling to furnish sanglifehrin A and B. Additional sanglifehrin B analogs with variation at the C40 position were additionally prepared. Biological evaluation revealed that the 2-CF3 analog of sanglifehrin B exhibited higher anti-proliferative activity than the natural products sanglifehrin A and B in Jurkat cells. Both natural products induced higher-order homodimerization of cyclophilin A (CypA), but only sanglifehrin A promoted CypA complexation with inosine-5'-monophosphate dehydrogenase 2 (IMPDH2). The synthesis reported herein will enable further evaluation of the spirolactam and its contribution to sanglifehrin-dependent immunosuppressive activity.

Mapping and Quantification of Over 2000 O-linked Glycopeptides in Activated Human T Cells with Isotope-Targeted Glycoproteomics (Isotag).

Post-translational modifications (PTMs) on proteins often function to regulate signaling cascades, with the activation of T cells during an adaptive immune response being a classic example. Mounting evidence indicates that the modification of proteins by O-linked N-acetylglucosamine (O-GlcNAc), the only mammalian glycan found on nuclear and cytoplasmic proteins, helps regulate T cell activation. Yet, a mechanistic understanding of how O-GlcNAc functions in T cell activation remains elusive, partly because of the difficulties in mapping and quantifying O-GlcNAc sites. Thus, to advance insight into the role of O-GlcNAc in T cell activation, we performed glycosite mapping studies via direct glycopeptide measurement on resting and activated primary human T cells with a technique termed Isotope Targeted Glycoproteomics. This approach led to the identification of 2219 intact O-linked glycopeptides across 1045 glycoproteins. A significant proportion (>45%) of the identified O-GlcNAc sites lie near or coincide with a known phosphorylation site, supporting the potential for PTM crosstalk. Consistent with other studies, we find that O-GlcNAc sites in T cells lack a strict consensus sequence. To validate our results, we employed gel shift assays based on conjugating mass tags to O-GlcNAc groups. Notably, we observed that the transcription factors c-JUN and JUNB show higher levels of O-GlcNAc glycosylation and higher levels of expression in activated T cells. Overall, our findings provide a quantitative characterization of O-GlcNAc glycoproteins and their corresponding modification sites in primary human T cells, which will facilitate mechanistic studies into the function of O-GlcNAc in T cell activation.

A Binding Site Hotspot Map of the FKBP12-Rapamycin-FRB Ternary Complex by Photoaffinity Labeling and Mass Spectrometry-Based Proteomics.

Structural characterization of small molecule binding site hotspots within the global proteome is uniquely enabled by photoaffinity labeling (PAL) coupled with chemical enrichment and unbiased analysis by mass spectrometry (MS). MS-based binding site maps provide structural resolution of interaction sites in conjunction with identification of target proteins. However, binding site hotspot mapping has been confined to relatively simple small molecules to date; extension to more complex compounds would enable the structural definition of new binding modes in the proteome. Here, we extend PAL and MS methods to derive a binding site hotspot map for the immunosuppressant rapamycin, a complex macrocyclic natural product that forms a ternary complex with the proteins FKBP12 and FRB. Photo-rapamycin was developed as a diazirine-based PAL probe for rapamycin, and the FKBP12-photo-rapamycin-FRB ternary complex formed readily in vitro. Photoirradiation, digestion, and MS analysis of the ternary complex revealed a McLafferty rearrangement product of photo-rapamycin conjugated to specific surfaces on FKBP12 and FRB. Molecular modeling based on the binding site map revealed two distinct conformations of complex-bound photo-rapamycin, providing a 5.0 Å distance constraint between the conjugated residues and the diazirine carbon and a 9.0 Å labeling radius for the diazirine upon photoactivation. These measurements may be broadly useful in the interpretation of binding site measurements from PAL. Thus, in characterizing the ternary complex of photo-rapamycin by MS, we applied binding site hotspot mapping to a macrocyclic natural product and extracted precise structural measurements for interpretation of PAL products that may enable the discovery of new binding sites in the "undruggable" proteome.

Aspartate Residues Far from the Active Site Drive O-GlcNAc Transferase Substrate Selection.

O-GlcNAc is an abundant post-translational modification found on nuclear and cytoplasmic proteins in all metazoans. This modification regulates a wide variety of cellular processes, and elevated O-GlcNAc levels have been implicated in cancer progression. A single essential enzyme, O-GlcNAc transferase (OGT), is responsible for all nucleocytoplasmic O-GlcNAcylation. Understanding how this enzyme chooses its substrates is critical for understanding, and potentially manipulating, its functions. Here we use protein microarray technology and proteome-wide glycosylation profiling to show that conserved aspartate residues in the tetratricopeptide repeat (TPR) lumen of OGT drive substrate selection. Changing these residues to alanines alters substrate selectivity and unexpectedly increases rates of protein glycosylation. Our findings support a model where sites of glycosylation for many OGT substrates are determined by TPR domain contacts to substrate side chains five to fifteen residues C-terminal to the glycosite. In addition to guiding design of inhibitors that target OGT's TPR domain, this information will inform efforts to engineer substrates to explore biological functions.

Structural basis for DNA cleavage by the potent antiproliferative agent (-)-lomaiviticin A.

(-)-Lomaiviticin A (1) is a complex antiproliferative metabolite that inhibits the growth of many cultured cancer cell lines at low nanomolar-picomolar concentrations. (-)-Lomaiviticin A (1) possesses a C2-symmetric structure that contains two unusual diazotetrahydrobenzo[b]fluorene (diazofluorene) functional groups. Nucleophilic activation of each diazofluorene within 1 produces vinyl radical intermediates that affect hydrogen atom abstraction from DNA, leading to the formation of DNA double-strand breaks (DSBs). Certain DNA DSB repair-deficient cell lines are sensitized toward 1, and 1 is under evaluation in preclinical models of these tumor types. However, the mode of binding of 1 to DNA had not been determined. Here we elucidate the structure of a 1:1 complex between 1 and the duplex d(GCTATAGC)2 by NMR spectroscopy and computational modeling. Unexpectedly, we show that both diazofluorene residues of 1 penetrate the duplex. This binding disrupts base pairing leading to ejection of the central AT bases, while placing the proreactive centers of 1 in close proximity to each strand. DNA binding may also enhance the reactivity of 1 toward nucleophilic activation through steric compression and conformational restriction (an example of shape-dependent catalysis). This study provides a structural basis for the DNA cleavage activity of 1, will guide the design of synthetic DNA-activated DNA cleavage agents, and underscores the utility of natural products to reveal novel modes of small molecule-DNA association.