Fluorogenic cell surface glycan labelling with fluorescence molecular rotor dyes and nucleic acid stains.

We show that covalent labelling of sialic acids on live cell surfaces or mucin increases the fluorescence of the fluorescence molecular rotors (FMRs) CCVJ, Cy3 and thioazole orange, enabling wash-free imaging of cell surfaces. Dual labelling with an FMR and an environmentally insensitive dye allows detection of changes that occur, for example, when cross-linking is altered.

Rapid building block-economic synthesis of long, multi-O-GalNAcylated MUC5AC tandem repeat peptides.

The study of mucin function requires access to highly O-glycosylated peptides with multiple tandem repeats. Solid-phase synthesis would be a suitable method, however, the central problem in the synthesis of mucin glycopeptides is the need to use precious and potentially vulnerable glycoamino acid building blocks in excess. In this article, we report the development of a method based on SPPS and native chemical ligation/desulfurization chemistry that allows the rapid, reliable, and glyco-economical synthesis of long multi-O-GalNAcylated peptides. To facilitate access to the glycosyl donor required for the preparation of Fmoc-Ser/Thr(αAc3GalNAc)-OH we used an easily scalable azidophenylselenylation of galactal instead of azidonitration. The problem of low yield when coupling glycoamino acids in small excess was solved by carrying out the reactions in 2-MeTHF instead of DMF and using DIC/Oxyma. Remarkably, quantitative coupling was achieved within 10 minutes using only 1.5 equivalents of glycoamino acid. The method does not require (microwave) heating, thus avoiding side reactions such as acetyl transfer to the N-terminal amino acid. This method also improved the difficult coupling of glycoamino acid to the hydrazine-resin and furnished peptides carrying 10 GalNAc units in high purities (>95%) of crude products. Combined with a one-pot method involving native chemical ligation at a glycoamino acid junction and superfast desulfurization, the method yielded highly pure MUC5AC glycopeptides comprising 10 octapeptide tandem repeats with 20 α-O-linked GalNAc residues within a week.

Design and Evaluation of Phosphonamidate-Linked Exatecan Constructs for Highly Loaded, Stable, and Efficacious Antibody-Drug Conjugates.

Topoisomerase I (TOP1) Inhibitors constitute an emerging payload class to engineer antibody-drug conjugates (ADC) as next-generation biopharmaceutical for cancer treatment. Existing ADCs are using camptothecin payloads with lower potency and suffer from limited stability in circulation. With this study, we introduce a novel camptothecin-based linker-payload platform based on the highly potent camptothecin derivative exatecan. First, we describe general challenges that arise from the hydrophobic combination of exatecan and established dipeptidyl p-aminobenzyl-carbamate (PAB) cleavage sites such as reduced antibody conjugation yields and ADC aggregation. After evaluating several linker-payload structures, we identified ethynyl-phosphonamidates in combination with a discrete PEG24 chain to compensate for the hydrophobic PAB-exatecan moiety. Furthermore, we demonstrate that the identified linker-payload structure enables the construction of highly loaded DAR8 ADCs with excellent solubility properties. Head-to-head comparison with Enhertu, an approved camptothecin-based ADC, revealed improved target-mediated killing of tumor cells, excellent bystander killing, drastically improved linker stability in vitro and in vivo and superior in vivo efficacy over four tested dose levels in a xenograft model. Moreover, we show that ADCs based on the novel exatecan linker-payload platform exhibit antibody-like pharmacokinetic properties, even when the ADCs are highly loaded with eight drug molecules per antibody. This ADC platform constitutes a new and general solution to deliver TOP1 inhibitors with highest efficiency to the site of the tumor, independent of the antibody and its target, and is thereby broadly applicable to various cancer indications.

Cell-Surface-Retained Peptide Additives for the Cytosolic Delivery of Functional Proteins.

The delivery of functional proteins remains a major challenge in advancing biological and pharmaceutical sciences. Herein, we describe a powerful, simple, and highly effective strategy for the intracellular delivery of functional cargoes. Previously, we demonstrated that cell-penetrating peptide (CPP) additives equipped with electrophilic thiol-reactive moieties temporarily attach to the cellular membrane, thereby facilitating the cellular uptake of protein- and antibody-CPP cargoes through direct membrane transduction at low concentrations. Now, we hypothesize that CPP-additives with an increased retention on the cellular membrane will further enhance intracellular uptake. We discovered that adding a small hydrophobic peptide sequence to an arginine-rich electrophilic CPP-additive further improved the uptake of protein-CPP conjugates, whereas larger hydrophobic anchors showed increased cytotoxicity. Cell viability and membrane integrity measurements, structure-activity relationship studies, and quantitative evaluation of protein-CPP uptake revealed important design principles for cell-surface-retained CPP-additives. These investigations allowed us to identify a nontoxic, thiol-reactive CPP-additive containing the hydrophobic ILFF sequence, which can deliver fluorescent model proteins at low micromolar concentrations. This hydrophobic CPP-additive allowed the addition of protein cargoes for intracellular delivery after initial additive incubation. Time-lapse fluorescence microscopy and membrane tension analysis of cells treated with fluorescent ILFF-CPP-additives supported the claim of increased cell surface retention and suggested that the protein-CPP cargoes enter the cell through a mechanism involving lowered cell membrane tension. Finally, we demonstrated that our newly engineered hydrophobic CPP-additive enabled the uptake of a functional macrocyclic peptidic MDM2-inhibitor and a recombinant genome editing protein. This indicates that the developed hydrophobic CPP-additive holds promise as a tool to enhance the intracellular delivery of peptide and protein cargoes.

Small Molecules Targeting Human UDP-GlcNAc 2-Epimerase.

Uridine diphosphate N-acetylglucosamine 2-epimerase (GNE) is a key enzyme in the sialic acid biosynthesis pathway. Sialic acids are primarily terminal carbohydrates on glycans and play fundamental roles in health and disease. In search of effective GNE inhibitors not based on a carbohydrate scaffold, we performed a high-throughput screening campaign of 68,640 drug-like small molecules against recombinant GNE using a UDP detection assay. We validated nine of the primary actives with an orthogonal real-time NMR assay and verified their IC50 values in the low micromolar to nanomolar range manually. Stability and solubility studies revealed three compounds for further evaluation. Thermal shift assays, analytical size exclusion, and interferometric scattering microscopy demonstrated that the GNE inhibitors acted on the oligomeric state of the protein. Finally, hydrogen-deuterium exchange mass spectrometry (HDX-MS) revealed which sections of GNE were shifted upon the addition of the inhibitors. In summary, we have identified three small molecules as GNE inhibitors with high potency in vitro, which serve as promising candidates to modulate sialic acid biosynthesis in more complex systems.

Real-time monitoring of the sialic acid biosynthesis pathway by NMR.

Sialic acids are part of the outermost component of the glycocalyx of all vertebrates; as such, they are fundamental markers in physiological and pathological processes. In this study, we introduce a real-time assay to monitor individual enzymatic steps of sialic acid biosynthesis, either with recombinant enzymes, in particular using UDP-N-acetylglucosamine 2-epimerase (GNE) or N-acetylmannosamine kinase (MNK), or in cytosolic rat liver extract. Using state-of-the-art NMR techniques, we are able to follow the characteristic signal of the N-acetyl methyl group, which displays different chemical shifts for the biosynthesis intermediates UDP-N-acetylglucosamine, N-acetylmannosamine (and its 6-phosphate) and N-acetylneuraminic acid (and its 9-phosphate). Pseudo 2- and 3-D NMR demonstrated that in rat liver cytosolic extract, the phosphorylation reaction of MNK is exclusive for N-acetylmannosamine generated by GNE. Thus, we speculate that phosphorylation of this sugar from other sources (e.g. external application to cells) or N-acetylmannosamine derivatives often applied in metabolic glycoengineering is not conducted by MNK but by a yet unknown sugar kinase. Competition experiments with the most prevalent neutral carbohydrates demonstrated that of these, only N-acetylglucosamine slowed N-acetylmannosamine phosphorylation kinetics, suggesting an N-acetylglucosamine-preferring kinase as the acting enzyme.

Compact hydrophilic electrophiles enable highly efficacious high DAR ADCs with excellent in vivo PK profile.

The recent success of antibody-drug conjugates (ADC), exemplified by seven new FDA-approvals within three years, has led to increased attention for antibody based targeted therapeutics and fueled efforts to develop new drug-linker technologies for improved next generation ADCs. We present a highly efficient phosphonamidate-based conjugation handle that combines a discrete hydrophilic PEG-substituent, an established linker-payload and a cysteine-selective electrophile in one compact building block. This reactive entity provides homogeneous ADCs with a high drug-to-antibody ratio (DAR) of 8 in a one-pot reduction and alkylation protocol from non-engineered antibodies. The compact branched PEG-architecture introduces hydrophilicity without increasing the distance between antibody and payload, allowing the generation of the first homogeneous DAR 8 ADC from VC-PAB-MMAE without increased in vivo clearance rates. This high DAR ADC exhibits excellent in vivo stability and increased antitumor activity in tumour xenograft models relative to the established FDA approved VC-PAB-MMAE ADC Adcetris, clearly showing the benefit of the phosphonamidate based building-blocks as a general tool for the efficient and stable antibody-based delivery of highly hydrophobic linker-payload systems.

(3+2)-Cyclization Reactions of Unsaturated Phosphonites with Aldehydes and Thioketones.

By exploiting the unique reactivity of ethynyl-phosphonites we obtain novel P(V)-containing five-membered heterocycles via (3+2)-cyclization reactions with aldehydes or cycloaliphatic thioketones in satisfactory to excellent yields. Whereas reactions with thioketones to yield 1,3-thiaphospholes-3-oxides occur smoothly at room temperature with equimolar amounts of the starting materials in absence of any catalyst, the analogous conversions with aldehydes to generate 3-oxides of 1,3-oxaphospholes require addition of triethylamine as a base. We postulate a step-wise (3+2)-cyclization mechanism for the formation of the 1,3-thiaphosphole ring based on DFT quantum chemical calculations. With this study, we introduce new cyclization reactions originating from unsaturated phosphonites as central synthetic building blocks to yield previously inaccessible stable phosphorus-containing heterocycles with unexplored potential for the molecular sciences.

Glycation Interferes with the Activity of the Bi-Functional UDP-N-Acetylglucosamine 2-Epimerase/N-Acetyl-mannosamine Kinase (GNE).

Mutations in the gene coding for the bi-functional UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE), the key enzyme of the sialic acid biosynthesis, are responsible for autosomal-recessive GNE myopathy (GNEM). GNEM is an adult-onset disease with a yet unknown exact pathophysiology. Since the protein appears to work adequately for a certain period of time even though the mutation is already present, other effects appear to influence the onset and progression of the disease. In this study, we want to investigate whether the late onset of GNEM is based on an age-related effect, e.g., the accumulation of post-translational modifications (PTMs). Furthermore, we also want to investigate what effect on the enzyme activity such an accumulation would have. We will particularly focus on glycation, which is a PTM through non-enzymatic reactions between the carbonyl groups (e.g., of methylglyoxal (MGO) or glyoxal (GO)) with amino groups of proteins or other biomolecules. It is already known that the levels of both MGO and GO increase with age. For our investigations, we express each domain of the GNE separately, treat them with one of the glycation agents, and determine their activity. We demonstrate that the enzymatic activity of the N-acetylmannosamine kinase (GNE-kinase domain) decreases dramatically after glycation with MGO or GO-with a remaining activity of 13% ± 5% (5 mM MGO) and 22% ± 4% (5 mM GO). Whereas the activity of the UDP-N-acetylglucosamine 2-epimerase (GNE-epimerase domain) is only slightly reduced after glycation-with a remaining activity of 60% ± 8% (5 mM MGO) and 63% ± 5% (5 mM GO).

Targeting FLT3 with a new-generation antibody-drug conjugate in combination with kinase inhibitors for treatment of AML.

Fms-like tyrosine kinase 3 (FLT3) is often overexpressed or constitutively activated by internal tandem duplication (ITD) and tyrosine kinase domain (TKD) mutations in acute myeloid leukemia (AML). Despite the use of receptor tyrosine kinase inhibitors (TKI) in FLT3-ITD-positive AML, the prognosis of patients is still poor, and further improvement of therapy is required. Targeting FLT3 independent of mutations by antibody-drug conjugates (ADCs) is a promising strategy for AML therapy. Here, we report the development and preclinical characterization of a novel FLT3-targeting ADC, 20D9-ADC, which was generated by applying the innovative P5 conjugation technology. In vitro, 20D9-ADC mediated potent cytotoxicity to Ba/F3 cells expressing transgenic FLT3 or FLT3-ITD, to AML cell lines, and to FLT3-ITD-positive patient-derived xenograft AML cells. In vivo, 20D9-ADC treatment led to a significant tumor reduction and even durable complete remission in AML xenograft models. Furthermore, 20D9-ADC demonstrated no severe hematotoxicity in in vitro colony formation assays using concentrations that were cytotoxic in AML cell line treatment. The combination of 20D9-ADC with the TKI midostaurin showed strong synergy in vitro and in vivo, leading to reduction of aggressive AML cells below the detection limit. Our data indicate that targeting FLT3 with an advanced new-generation ADC is a promising and potent antileukemic strategy, especially when combined with FLT3-TKI in FLT3-ITD-positive AML.

Structure-Uptake Relationship Study of DABCYL Derivatives Linked to Cyclic Cell-Penetrating Peptides for Live-Cell Delivery of Synthetic Proteins.

Modifying cyclic cell-penetrating deca-arginine (cR10) peptides with 4-(4-dimethylaminophenylazo)benzoic acid (DABCYL) improves the uptake efficiency of synthetic ubiquitin (Ub) cargoes into living cells. To probe the role of the DABCYL moiety, we performed time-lapse microscopy and fluorescence lifetime imaging microscopy (FLIM) of fluorescent DABCYL-R10 to evaluate the impact on cell entry by the formation of nucleation zones. Furthermore, we performed a structure-uptake relationship study with 13 DABCYL derivatives coupled to CPP to examine their effect on the cell-uptake efficiency when conjugated to mono-Ub through disulfide linkages. Our results show that through structure variations of the DABCYL moiety alone we could reach, at nanomolar concentration, an additional threefold increase in the cytosolic delivery of Ub, which will enable studies on various intracellular processes related to Ub signaling.

Synthetic α-Helical Peptides as Potential Inhibitors of the ACE2 SARS-CoV-2 Interaction.

During viral cell entry, the spike protein of SARS-CoV-2 binds to the α1-helix motif of human angiotensin-converting enzyme 2 (ACE2). Thus, alpha-helical peptides mimicking this motif may serve as inhibitors of viral cell entry. For this purpose, we employed the rigidified diproline-derived module ProM-5 to induce α-helicity in short peptide sequences inspired by the ACE2 α1-helix. Starting with Ac-QAKTFLDKFNHEAEDLFYQ-NH2 as a relevant section of α1, a series of peptides, N-capped with either Ac-ßHAsp-[ProM-5] or Ac-ßHAsp-PP, were prepared and their α-helicities were investigated. While ProM-5 clearly showed a pronounced effect, an even increased degree of helicity (up to 63 %) was observed in sequences in which non-binding amino acids were replaced by alanine. The binding affinities of the peptides towards the spike protein, as determined by means of microscale thermophoresis (MST), revealed only a subtle influence of the α-helical content and, noteworthy, led to the identification of an Ac-ßHAsp-PP-capped peptide displaying a very strong binding affinity (KD =62 nM).

Modular solid-phase synthesis of electrophilic cysteine-selective ethynyl-phosphonamidate peptides.

We report an efficient method to install electrophilic cysteine-selective ethynyl-phosphonamidates on peptides during Fmoc-based solid phase peptide synthesis (SPPS). By performing Staudinger-phosphonite reactions between different solid supported azido-peptides and varying ethynylphosphonites, we obtained ethynyl-phosphonamidate containing peptidic compounds after acidic deprotection, including an electrophilic cell-penetrating peptide that showed high efficiency as an additive for cellular delivery of proteins.

DFT-Guided Discovery of Ethynyl-Triazolyl-Phosphinates as Modular Electrophiles for Chemoselective Cysteine Bioconjugation and Profiling.

We report the density functional theory (DFT) guided discovery of ethynyl-triazolyl-phosphinates (ETPs) as a new class of electrophilic warheads for cysteine selective bioconjugation. By using CuI -catalysed azide alkyne cycloaddition (CuAAC) in aqueous buffer, we were able to access a variety of functional electrophilic building blocks, including proteins, from diethynyl-phosphinate. ETP-reagents were used to obtain fluorescent peptide-conjugates for receptor labelling on live cells and a stable and a biologically active antibody-drug-conjugate. Moreover, we were able to incorporate ETP-electrophiles into an azide-containing ubiquitin under native conditions and demonstrate their potential in protein-protein conjugation. Finally, we showcase the excellent cysteine-selectivity of this new class of electrophile in mass spectrometry based, proteome-wide cysteine profiling, underscoring the applicability in homogeneous bioconjugation strategies to connect two complex biomolecules.

Design and Functional Analysis of Heterobifunctional Multivalent Phage Capsid Inhibitors Blocking the Entry of Influenza Virus.

Multiple conjugation of virus-binding ligands to multivalent carriers is a prominent strategy to construct highly affine virus binders for the inhibition of viral entry into host cells. In a previous study, we introduced rationally designed sialic acid conjugates of bacteriophages (Qß) that match the triangular binding site geometry on hemagglutinin spike proteins of influenza A virions, resulting in effective infection inhibition in vitro and in vivo. In this work, we demonstrate that even partially sialylated Qß conjugates retain the inhibitory effect despite reduced activity. These observations not only support the importance of trivalent binding events in preserving high affinity, as supported by computational modeling, but also allow us to construct heterobifunctional modalities. Capsids carrying two different sialic acid ligand-linker structures showed higher viral inhibition than their monofunctional counterparts. Furthermore, capsids carrying a fluorescent dye in addition to sialic acid ligands were used to track their interaction with cells. These findings support exploring broader applications as multivalent inhibitors in the future.

Atomistic insight into the essential binding event of ACE2-derived peptides to the SARS-CoV-2 spike protein.

The pathogenic agent of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters into human cells through the interaction between the receptor binding domain (RBD) of its spike glycoprotein and the angiotensin-converting enzyme 2 (ACE2) receptor. Efforts have been made towards finding antivirals that block this interaction, therefore preventing infection. Here, we determined the binding affinity of ACE2-derived peptides to the RBD of SARS-CoV-2 experimentally and performed MD simulations in order to understand key characteristics of their interaction. One of the peptides, p6, binds to the RBD of SARS-CoV-2 with nM affinity. Although the ACE2-derived peptides retain conformational flexibility when bound to SARS-CoV-2 RBD, we identified residues T27 and K353 as critical anchors mediating the interaction. New ACE2-derived peptides were developed based on the p6-RBD interface analysis and expecting the native conformation of the ACE2 to be maintained. Furthermore, we found a correlation between the helicity in trifluoroethanol and the binding affinity to RBD of the new peptides. Under the hypothesis that the conservation of peptide secondary structure is decisive to the binding affinity, we developed a cyclized version of p6 which had more helicity than p6 and approximately half of its KD value.

A silyl ether-protected building block for O-GlcNAcylated peptide synthesis to enable one-pot acidic deprotection.

In this report, we introduce a novel building block for Fmoc/tBu solid phase peptide synthesis (SPPS) of ß-linked O-GlcNAcylated peptides. This building block carries acid labile silyl ether protecting groups, which are fully removed under TFA-mediated peptide cleavage conditions from the resin, thus requiring fewer synthetic steps and no intermediate purification as compared to other acid or base labile protecting group strategies.

Targeted Subcellular Protein Delivery Using Cleavable Cyclic Cell-Penetrating Peptide-Conjugates.

Proteins conjugated to cyclic cell-penetrating peptides (cCPPs) haAbstractve been shown to be effectively taken up by living cells. Conjugation of proteins to cCPPs in a cleavable manner leads to localization in the cytosol and immediate bioavailability of the protein after uptake. Here we describe how mCherry, a fluorescent protein, can be targeted to a membrane-bound compartment, the nucleus, and a subcellular structure like the actin cytoskeleton after cCPP-mediated uptake into living cells. Targeting peptides are genetically fused to the mCherry protein sequence and the protein is conjugated to the cCPP via a cleavable disulfide bond. Localization of mCherry in the nucleus or the actin skeleton respectively can be observed by live cell confocal fluorescence microscopy.

Evaluation of Multivalent Sialylated Polyglycerols for Resistance Induction in and Broad Antiviral Activity against Influenza A Viruses.

The development of multivalent sialic acid-based inhibitors active against a variety of influenza A virus (IAV) strains has been hampered by high genetic and structural variability of the targeted viral hemagglutinin (HA). Here, we addressed this challenge by employing sialylated polyglycerols (PGs). Efficacy of prototypic PGs was restricted to a narrow spectrum of IAV strains. To understand this restriction, we selected IAV mutants resistant to a prototypic multivalent sialylated PG by serial passaging. Resistance mutations mapped to the receptor binding site of HA, which was accompanied by altered receptor binding profiles of mutant viruses as detected by glycan array analysis. Specifying the inhibitor functionalization to 2,6-α-sialyllactose (SL) and adjusting the linker yielded a rationally designed inhibitor covering an extended spectrum of inhibited IAV strains. These results highlight the importance of integrating virological data with chemical synthesis and structural data for the development of sialylated PGs toward broad anti-influenza compounds.