Investigating SH-SY5Y Neuroblastoma Cell Surfaceome as a Model for Neuronal-Targeted Novel Therapeutic Modalities.

The SH-SY5Y neuroblastoma cells are a widely used in vitro model approximating neurons for testing the target engagement of therapeutics designed for neurodegenerative diseases and pain disorders. However, their potential as a model for receptor-mediated delivery and uptake of novel modalities, such as antibody-drug conjugates, remains understudied. Investigation of the SH-SY5Y cell surfaceome will aid in greater in vitro to in vivo correlation of delivery and uptake, thereby accelerating drug discovery. So far, the majority of studies have focused on total cell proteomics from undifferentiated and differentiated SH-SY5Y cells. While some studies have investigated the expression of specific proteins in neuroblastoma tissue, a global approach for comparison of neuroblastoma cell surfaceome to the brain and dorsal root ganglion (DRG) neurons remains uninvestigated. Furthermore, an isoform-specific evaluation of cell surface proteins expressed on neuroblastoma cells remains unexplored. In this study, we define a bioinformatic workflow for the identification of high-confidence surface proteins expressed on brain and DRG neurons using tissue proteomic and transcriptomic data. We then delineate the SH-SY5Y cell surfaceome by surface proteomics and show that it significantly overlaps with the human brain and DRG neuronal surface proteome. We find that, for 32% of common surface proteins, SH-SY5Y-specific major isoforms are alternatively spliced, maintaining their protein-coding ability, and are predicted to localize to the cell surface. Validation of these isoforms using surface proteomics confirms a SH-SY5Y-specific alternative NRCAM (neuron-glia related cell adhesion molecule) isoform, which is absent in typical brain neurons, but present in neuroblastomas, making it a receptor of interest for neuroblastoma-specific therapeutics.

Crystal structure of active CDK4-cyclin D and mechanistic basis for abemaciclib efficacy.

Despite the biological and therapeutic relevance of CDK4/6 for the treatment of HR+, HER2- advanced breast cancer, the detailed mode of action of CDK4/6 inhibitors is not completely understood. Of particular interest, phosphorylation of CDK4 at T172 (pT172) is critical for generating the active conformation, yet no such crystal structure has been reported to date. We describe here the x-ray structure of active CDK4-cyclin D3 bound to the CDK4/6 inhibitor abemaciclib and discuss the key aspects of the catalytically-competent complex. Furthermore, the effect of CDK4/6 inhibitors on CDK4 T172 phosphorylation has not been explored, despite its role as a potential biomarker of CDK4/6 inhibitor response. We show mechanistically that CDK4/6i stabilize primed (pT172) CDK4-cyclin D complex and selectively displace p21 in responsive tumor cells. Stabilization of active CDK4-cyclin D1 complex can lead to pathway reactivation following alternate dosing regimen. Consequently, sustained binding of abemaciclib to CDK4 leads to potent cell cycle inhibition in breast cancer cell lines and prevents rebound activation of downstream signaling. Overall, our study provides key insights demonstrating that prolonged treatment with CDK4/6 inhibitors and composition of the CDK4/6-cyclin D complex are both critical determinants of abemaciclib efficacy, with implications for this class of anticancer therapy.

Optimization of Metabolic Oligosaccharide Engineering with Ac4GalNAlk and Ac4GlcNAlk by an Engineered Pyrophosphorylase.

Metabolic oligosaccharide engineering (MOE) has fundamentally contributed to our understanding of protein glycosylation. Efficient MOE reagents are activated into nucleotide-sugars by cellular biosynthetic machineries, introduced into glycoproteins and traceable by bioorthogonal chemistry. Despite their widespread use, the metabolic fate of many MOE reagents is only beginning to be mapped. While metabolic interconnectivity can affect probe specificity, poor uptake by biosynthetic salvage pathways may impact probe sensitivity and trigger side reactions. Here, we use metabolic engineering to turn the weak alkyne-tagged MOE reagents Ac4GalNAlk and Ac4GlcNAlk into efficient chemical tools to probe protein glycosylation. We find that bypassing a metabolic bottleneck with an engineered version of the pyrophosphorylase AGX1 boosts nucleotide-sugar biosynthesis and increases bioorthogonal cell surface labeling by up to two orders of magnitude. A comparison with known azide-tagged MOE reagents reveals major differences in glycoprotein labeling, substantially expanding the toolbox of chemical glycobiology.

Metabolic precision labeling enables selective probing of O-linked N-acetylgalactosamine glycosylation.

Protein glycosylation events that happen early in the secretory pathway are often dysregulated during tumorigenesis. These events can be probed, in principle, by monosaccharides with bioorthogonal tags that would ideally be specific for distinct glycan subtypes. However, metabolic interconversion into other monosaccharides drastically reduces such specificity in the living cell. Here, we use a structure-based design process to develop the monosaccharide probe N-(S)-azidopropionylgalactosamine (GalNAzMe) that is specific for cancer-relevant Ser/Thr(O)-linked N-acetylgalactosamine (GalNAc) glycosylation. By virtue of a branched N-acylamide side chain, GalNAzMe is not interconverted by epimerization to the corresponding N-acetylglucosamine analog by the epimerase N-acetylgalactosamine-4-epimerase (GALE) like conventional GalNAc-based probes. GalNAzMe enters O-GalNAc glycosylation but does not enter other major cell surface glycan types including Asn(N)-linked glycans. We transfect cells with the engineered pyrophosphorylase mut-AGX1 to biosynthesize the nucleotide-sugar donor uridine diphosphate (UDP)-GalNAzMe from a sugar-1-phosphate precursor. Tagged with a bioorthogonal azide group, GalNAzMe serves as an O-glycan-specific reporter in superresolution microscopy, chemical glycoproteomics, a genome-wide CRISPR-knockout (CRISPR-KO) screen, and imaging of intestinal organoids. Additional ectopic expression of an engineered glycosyltransferase, "bump-and-hole" (BH)-GalNAc-T2, boosts labeling in a programmable fashion by increasing incorporation of GalNAzMe into the cell surface glycoproteome. Alleviating the need for GALE-KO cells in metabolic labeling experiments, GalNAzMe is a precision tool that allows a detailed view into the biology of a major type of cancer-relevant protein glycosylation.

Manno-epi-cyclophellitols Enable Activity-Based Protein Profiling of Human α-Mannosidases and Discovery of New Golgi Mannosidase II Inhibitors.

Golgi mannosidase II (GMII) catalyzes the sequential hydrolysis of two mannosyl residues from GlcNAcMan5GlcNAc2 to produce GlcNAcMan3GlcNAc2, the precursor for all complex N-glycans, including the branched N-glycans associated with cancer. Inhibitors of GMII are potential cancer therapeutics, but their usefulness is limited by off-target effects, which produce α-mannosidosis-like symptoms. Despite many structural and mechanistic studies of GMII, we still lack a potent and selective inhibitor of this enzyme. Here, we synthesized manno-epi-cyclophellitol epoxide and aziridines and demonstrate their covalent modification and time-dependent inhibition of GMII. Application of fluorescent manno-epi-cyclophellitol aziridine derivatives enabled activity-based protein profiling of α-mannosidases from both human cell lysate and mouse tissue extracts. Synthesized probes also facilitated a fluorescence polarization-based screen for dGMII inhibitors. We identified seven previously unknown inhibitors of GMII from a library of over 350 iminosugars and investigated their binding modalities through X-ray crystallography. Our results reveal previously unobserved inhibitor binding modes and promising scaffolds for the generation of selective GMII inhibitors.

Engineering Orthogonal Polypeptide GalNAc-Transferase and UDP-Sugar Pairs.

O-Linked α-N-acetylgalactosamine (O-GalNAc) glycans constitute a major part of the human glycome. They are difficult to study because of the complex interplay of 20 distinct glycosyltransferase isoenzymes that initiate this form of glycosylation, the polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts). Despite proven disease relevance, correlating the activity of individual GalNAc-Ts with biological function remains challenging due to a lack of tools to probe their substrate specificity in a complex biological environment. Here, we develop a "bump-hole" chemical reporter system for studying GalNAc-T activity in vitro. Individual GalNAc-Ts were rationally engineered to contain an enlarged active site (hole) and probed with a newly synthesized collection of 20 (bumped) uridine diphosphate N-acetylgalactosamine (UDP-GalNAc) analogs to identify enzyme-substrate pairs that retain peptide specificities but are otherwise completely orthogonal to native enzyme-substrate pairs. The approach was applicable to multiple GalNAc-T isoenzymes, including GalNAc-T1 and -T2 that prefer nonglycosylated peptide substrates and GalNAcT-10 that prefers a preglycosylated peptide substrate. A detailed investigation of enzyme kinetics and specificities revealed the robustness of the approach to faithfully report on GalNAc-T activity and paves the way for studying substrate specificities in living systems.

Synthesis and self-assembly of well-defined elastin-like polypeptide-poly(ethylene glycol) conjugates.

A series of stimulus-responsive elastin-like polypeptide-poly(ethylene glycol) (ELP-PEG) block copolymers was synthesized. The polymeric building blocks were conjugated via the efficient and specific strain-promoted alkyne-azide cycloaddition (SPAAC). For this purpose, ELP and PEG blocks were functionalized with azide and cyclooctyne moieties, respectively. Azides were introduced by applying a recently developed pH-controlled diazotransfer reaction on the primary amines present in ELP (N-terminus and lysine side chains). By varying pH, ELP-blocks with one or two azides were obtained, which subsequently allowed us to synthesize both ELP-PEG diblock copolymers and miktoarm star polymers. Triggering the phase transition of the ELP-block resulted in the formation of an amphiphilic block copolymer, which self-assembled into micelles. This is the first example of an ELP-containing hybrid block copolymer in which PEG as the hydrophilic corona-forming domain is combined with a stimulus-responsive ELP-block. The encapsulation of a hydrophobic fluorescent dye was shown to exemplify the potential of the micelles to serve as nanocarriers for hydrophobic drugs, with the PEG corona providing stealth and steric protection of encapsulated materials.

Nanobody-functionalized polymersomes for tumor-vessel targeting.

Targeted carrier systems (e.g., liposomes or nanoparticles) are used to specifically deliver drugs to a site of interest. Site-direction can be achieved by attachment of targeting molecules, such as peptides, DNA/RNA, or antibodies, to the surface of the carrier. Here, the formation of polymersomes with tumor-targeting potential is described. A single-domain antibody (A12) that specifically targets PlexinD1 (a transmembrane protein overexpressed in tumor vasculature) is equipped with an azide-functionality using expressed protein ligation. This azide-containing A12 can subsequently be attached to BCN-functionalized polymersomes using a strain-promoted azide alkyne cycloaddition, thereby forming polymersomes with tumor-targeting potential.

Detection of transglutaminase activity using click chemistry.

Transglutaminase 2 (TG2) is a Ca(2+)-dependent enzyme able to catalyze the formation of ε(γ-glutamyl)-lysine crosslinks between polypeptides, resulting in high molecular mass multimers. We have developed a bioorthogonal chemical method for the labeling of TG2 glutamine-donor proteins. As amine-donor substrates we used a set of azide- and alkyne-containing primary alkylamines that allow, after being crosslinked to glutamine-donor proteins, specific labeling of these proteins via the azide-alkyne cycloaddition. We demonstrate that these azide- and alkyne-functionalized TG2 substrates are cell permeable and suitable for specific labeling of TG2 glutamine-donor substrates in HeLa and Movas cells. Both the Cu(I)-catalyzed and strain promoted azide-alkyne cycloaddition proved applicable for subsequent derivatization of the TG2 substrate proteins with the desired probe. This new method for labeling TG2 substrate proteins introduces flexibility in the detection and/or purification of crosslinked proteins, allowing differential labeling of cellular proteins.