Protein tyrosine phosphatases (PTPs) represent an important pharmacological target and subject of study. Although a number of broad-spectrum electrophilic, phosphotyrosine-mimicking probes have been developed to covalently capture the catalytic site of these enzymes, there is still a high demand for PTP probes with high target selectivity that are accessible in a synthetically straightforward way. Unsaturated phosphorus (V) (P(V)) compounds have recently emerged as powerful cysteine-selective bioconjugation reagents (P5-labeling). Herein, we introduce ethynyl-substituted aryl phosphonamidic and phosphonic acids as phosphotyrosine mimics, which serve as active-site-directed, covalent probes for tyrosine phosphatases. We show that these P(V) electrophiles can be readily incorporated into a peptide sequence, allowing proximity-enabled reactivity and selective targeting of the catalytic cysteine residue of an interacting phosphatase, as exemplified for PTP1B, a protein tyrosine phosphatase that acts as a key negative regulator of insulin signaling. Both ethynyl phosphonamidic acid and ethynyl phosphonic acid show no reactivity towards nontarget cysteine residues, though the phosphonamidic acid probe was notably less reactive toward its intended target. Proteomics experiments in human cell lysates demonstrated that the phosphonic acid probe selectively enriches its interacting phosphatase in the human proteome. Our study highlights a versatile strategy to obtain remarkably precise peptide-based PTP probes, thereby enabling the characterization of phosphatase interactions with high specificity.

Recent advances in cell-penetrating peptide (CPP)-mediated intracellular protein delivery emphasized the critical role of sustained membrane association in enhancing delivery efficiency. Here, we report cell-surface-reactive, polyfluoroalkyl-tagged polyarginine peptides with varying fluorine content as CPP-additives that significantly enhance protein delivery in living cells. At low micromolar concentrations (2.5 µM), CPP-additives containing 11-13 fluorine atoms enhanced intracellular protein delivery over 2-fold relative to a tagless control without observable cytotoxicity. Live-cell time-lapse fluorescence imaging revealed that a CPP-additive with 13 fluorine atoms showed prolonged membrane association (>5 min) relative to a tagless control and facilitated rapid protein internalization within 10 min. Remarkably, surface-enhanced infrared absorption spectroscopy (SEIRAS) with POPC membranes showed that fluorous CPP-additives initially interacted with the lipid bilayer predominantly as aggregates but subsequently inserted into the membrane interior as monomers without fluorous tag-tag association. Complementary molecular dynamics simulations of the initial membrane-association step provided atomistic insight, showing partial lipid insertion of a monomeric CPP-additive with 13 fluorine atoms while no insertion was observed for a tagless control within the same time scale. Collectively, our findings establish polyfluoroalkyl-tagged CPP-additives as potent, non-cytotoxic vectors for intracellular protein delivery and provide mechanistic detail regarding the molecular basis of their lipid bilayer interactions.

Covalent inhibitors and chemical probes targeting ligandable cysteine residues have emerged as powerful tools for drug discovery and proteomics. In this study, we introduce vinyl phosphonamidates (VPAs) as a novel class of latent cysteine electrophiles and assess their reactivity, selectivity, and potential for developing covalent inhibitors. Compared to well-established chloroacetamide and acrylamide electrophiles, VPAs exhibit a significantly lower intrinsic reactivity toward the model thiol glutathione. Moreover, VPA-derived covalent fragments displayed only very limited nonspecific reactivity in human cell lysate. Encouraged by these results, we developed VPA-functionalized derivatives of the FDA-approved covalent inhibitors Afatinib and Ibrutinib and evaluated their ability to engage the target protein by gel-based and mass spectrometry-based activity-based protein profiling (ABPP). Compared to commonly employed Michael acceptor-based electrophilic groups, VPA-functionalized drug ligands displayed significantly less off-targets while maintaining inhibitor efficiency. Furthermore, we leveraged the modular nature and accessibility of VPAs to develop a bifunctional proteolysis targeting chimera (PROTAC) for targeted protein degradation. The demonstrated selectivity and modularity, as exemplified by the incorporation of various ligands on the phosphorus O-substituent, of the vinyl phosphonamidate group as a cysteine-directed electrophile highlight its ability to expand the chemical space in the development of covalent inhibitors with a favorable proteome-wide reactivity profile.

Brentuximab and derivatives show high affinity binding to CD30 and strongly internalize into CD30-positive cells

Providing immediate access for functional proteins inside living cells would unlock unprecedented control over cellular processes; however, commonly used endocytic delivery suffers from endosomal trapping and degradation. One of the most powerful non-endosomal delivery methods uses cell surface anchored cell penetrating peptide (CPP)-additives that allow proteins to enter cells directly. Nevertheless, the underlying molecular mechanism involved in direct entry via crossing the cell membrane (protein translocation through the cell) and the major driving forces remain controversially discussed. Here, we provide a stepwise molecular picture on how CPP-additives enable uptake of protein cargoes through direct membrane translocation. CPP-additives accumulate on the cell surface in nucleation zones, locally hyperpolarizing the membrane, and induce transient water pores that allow selective CPP-protein entry without compromising membrane integrity. These fundamental mechanistic insights provide a firm basis for rationally optimizing delivery strategies using highly cationic CPPs, ultimately resulting in innovative and smart protein delivery strategies to advance therapeutic protein applications.

ZUSAMMENFASSUNG Der aktuelle Stand der Forschung zu zellpenetrierenden Peptiden und ihrem Einfluss auf intrazelluläre Proteinaufnahme unterstreicht die entscheidende Rolle einer anhaltenden Membranassoziation für eine hohe Aufnahmeeffizienz. Diese Studie zeigt, wie zelloberflächenreaktive und polyfluoralkylierte Polyarginin‐Peptide mit unterschiedlichem Fluorgehalt als CPP‐Additive die Proteinaufnahme in lebende Säugerzellen signifikant verbessern. Bei niedrigen mikromolaren Konzentrationen (2.5 µM) verbesserten CPP‐Additive mit 11 bis 13 Fluoratomen die intrazelluläre Proteinaufnahme um mehr als das Zweifache im Vergleich zu einer unmarkierten Kontrolle. Hierbei wurde keine Zytotoxizität beobachtet. Zeitaufgelöste Fluoreszenzmikroskopie mit lebenden Zellen zeigte, dass ein CPP‐Additiv mit 13 Fluoratomen im Vergleich zu einem unmarkierten CPP‐Additiv eine längere Membranassoziation (> 5 Minuten) aufwies und gleichzeitig eine schnelle Proteininternalisierung ermöglichte (innerhalb von 10 Minuten). Bemerkenswerterweise zeigten oberflächenverstärkte Infrarotabsorptionsspektroskopie (SEIRAS) Experimente mit POPC‐Modellmembranen, dass fluorhaltige CPP‐Additive zunächst überwiegend als Aggregate mit der Lipid‐Doppelschicht interagierten und anschließend als Monomere und ohne intermolekulare Fluor‐Fluor Wechselwirkungen in das Membraninnere eindrangen. Ergänzende Molekulardynamiksimulationen der initialen Membranassoziation offenbarten die Membranverankerung eines CPP‐Additivs mit 13 Fluoratomen, während für ein unmarkiertes CPP‐Additiv innerhalb desselben Zeitraums keine Membranverankerung beobachtet wurde. Insgesamt zeigen unsere Ergebnisse, dass polyfluoralkylierte CPP‐Additive effiziente und zellverträgliche Hilfsmittel für eine effiziente intrazelluläre Proteinaufnahme sind. Gleichzeitig liefern wir mechanistische Einblicke in die molekularen Grundlagen der Membraninteraktion.

Intracellular delivery of functional proteins is emerging as a powerful strategy to interrogate and control cellular pathways with high spatial and temporal precision. Recent advances in chemical biology now enable the design of cell-permeable proteins through precise chemical and biochemical modification, bringing the field closer to achieving intracellularly targeted biologics as a "new class of drugs". By overcoming the limitations of genetic manipulation, researchers create solutions for basic research and medicine. In this review, we outline the key motivations that drive intracellular protein delivery. We highlight central mechanistic paradigms for protein entry, important parameters influencing protein delivery, and summarize analytical tools to assess successful delivery. We outline different chemical and biochemical conceptional approaches that resulted in breakthrough studies to achieve functional protein transport. Finally, we discuss ongoing efforts, highlighting the challenges for future research on protein delivery.

TUB-010 is a next-generation antibody-drug conjugate (ADC) targeting CD30 expressed on various hematopoietic malignancies such as Hodgkin lymphoma. Among the therapeutic options for patients with relapsed and refractory CD30-positive cancers is brentuximab vedotin (Adcetris), a monomethyl auristatin E (MMAE)-delivering anti-CD30 ADC with a mean drug-to-antibody ratio of 4. Adcetris exhibits a high response rate at the cost of significant toxicities, likely driven by the payload MMAE and the instability of the maleimide conjugation chemistry. TUB-010 uses the same antibody and payload as Adcetris but is based on the Tub-tag conjugation strategy, which stably attaches MMAE to the hydrophilic Tub-tag peptides on the light chains via chemoenzymatic conjugation. This new technology enables the generation of a homogeneous and site-specific drug-to-antibody ratio 2 ADC with unique biophysical properties. TUB-010 demonstrates similar binding and lysosomal release characteristics as Adcetris, which translates into comparable in vitro cytotoxicity on CD30-positive cell lines when normalized to the MMAE concentration. Importantly, TUB-010 exhibits higher stability with negligible premature deconjugation in circulation and reduced aggregation, as well as lower nonspecific cytotoxicity on target-negative cells compared with Adcetris. As a consequence, TUB-010 induces superior tumor control compared with Adcetris when dosed at equal MMAE concentrations in vivo and also lower toxicity and higher tolerability in rodents and nonhuman primates. Taken together, TUB-010 is a novel, potential best-in-class anti-CD30 ADC with improved biophysical properties designed to deliver MMAE with higher precision and a wider therapeutic window than Adcetris using Tub-tag technology. Therefore, TUB-010 may increase the clinical benefit of anti-CD30 ADC therapies.

Opioid receptors (ORs) orchestrate pain relief, reward, and dependence, yet their signaling arises from diverse cell types and subcellular compartments that cannot be selectively interrogated with existing pharmacological or genetic approaches. Single-domain antibodies, or nanobodies (Nbs), can probe receptor states, but their potential as tools for controlling native receptor signaling at the system level has remained unexplored. Here, we engineer a suite of high-affinity intracellular Nbs that bind active ORs through structure-guided evolution and in silico design. Iterative optimization yields Nb64, a potent inhibitor that rapidly suppresses transducer engagement, receptor internalization, and downstream signaling, including endogenous pathways in neuronal cells. Organelle targeting highlights Nb64's capacity to control OR activity with subcellular precision, while bio-reversible cell-penetrating peptide (CPP) conjugation enables non-genetic cytosolic delivery. Cell-type-specific expression of Nb64 in VTA interneurons attenuates fentanyl-levoked dopamine release and behavioral responses in mice, demonstrating targeted control of opioid actions in vivo. Nb64 provides a versatile strategy for dissecting OR biology and establishes a generalizable framework for precision inhibition of native GPCR signaling in vivo.

Canonical protein phosphorylation patterns are a thoroughly studied post-translational modification (PTM) driving distinct regulatory mechanisms in both prokaryotes, and eukaryotes. In contrast, the identification and investigation of essential components that regulate non-canonical phosphorylation has received considerably less attention, although these PTMs are associated with important functions. One notable example is arginine phosphorylation which modulates processes such as protein degradation, transcriptional regulation and spore germination in bacteria. Herein we introduce the first in class covalent activity-based probes to study phosphoarginine-phosphatases. We identify unsaturated phosphonamidic acids as bespoke electrophilic phosphoarginine (pArg) mimics, which allowed to uncover a series of unprecedented pArg-phosphatases, which in part had been previously annotated as low molecular weight tyrosine-phosphatases across phylogenetically distinct microbial species. This work, which serves as the first example of proteome-wide activity-based profiling of pArg phosphatases will help inform the development of new therapeutic modalities and expand our understanding of bacterial signal transduction. ### Competing Interest Statement The authors have declared no competing interest. Deutsche Forschungsgemeinschaft, 392923329, HA 4468/10-1 Leibniz Association, https://ror.org/01n6r0e97, SAW-2018-FMP-4-P5label European Union, breakingBAC 101096911

Elucidating protein-protein interactions plays a crucial part in understanding disease mechanisms and advancing pharmacological research. Photocatalytic proximity labeling using antibody-catalyst conjugates enables the highly target-specific analysis of protein-protein interactions on the cell surface without altering the native cellular state through genetic manipulation. Here, we describe an extension of the deazaflavin-diazirine energy-transfer (DarT) labeling platform through the development and evaluation of trastuzumab-deazaflavin (Tra-dFl) conjugates for mapping the extracellular microenvironment of human epidermal growth factor receptor 2 (HER2). Four Tra-dFl conjugates were synthesized via azide-DBCO click chemistry, varying in PEG linker size and catalyst loading: Tra-PEG0-dFl, Tra-PEG6-dFl, Tra-PEG12-dFl, and Tra-bis-dFl, exhibiting a branched linker for dual attachment. Imaging and proteomic pulldown experiments revealed that linker size influences biotinylation efficiency and proteomic enrichment, resulting in Tra-PEG12-dFl emerging as the most effective construct, enabling the enrichment of cancer-associated cell surface proteins in HER2-positive SK-BR-3 cells. Evaluation of catalyst valency using a branched linker resulted in fewer enriched proteins, suggesting that linker architecture is a more critical parameter for conjugate performance than increased catalyst loading. Together, these findings provide guidelines for antibody-based deazaflavin conjugates and expand the applicability of DarT labeling for target-directed surfaceome mapping.

Chemoselective or bioorthogonal modification reactions resulted in several breakthrough studies that enabled the incorporation of functional modules into proteins and antibodies for basic and translational research. In 2010, we published a paper in Chemical Science, which described a chemoselective method for synthesizing branched PEGylated peptides and proteins using the Staudinger-phosphite reaction (R. Serwa, P. Majkut, B. Horstmann, J. M. Swiecicki, M. Gerrits, E. Krause and C. P. R. Hackenberger, Chem. Sci., 2010, 1, 596-602, https://doi.org/10.1039/C0SC00324G). We discuss subsequent studies in using the protocol for the intracellular stabilization of peptides and the development of the P5-labeling platform, which we currently use in the generation of antibody-drug-conjugates (ADCs) as next-generation biopharmaceuticals in clinical studies, for which a first proof-of-concept study was also published in Chemical Science (P. Ochtrop, J. Jahzerah, P. Machui, I. Mai, D. Schumacher, J. Helma, M. A. Kasper and C. P. R. Hackenberger, Chem. Sci., 2023, 14, 2259-2266, https://doi.org/10.1039/D2SC05678J).

Despite recent advances in targeted drug delivery, approved Antibody-Drug-Conjugates (ADCs) are still limited by the delivery of a restricted set of payloads with limited modes of action (MOA). Versatile linkers, applicable to functional groups prevalent across diverse pharmacophores are needed to expand this space. We present phosphoramidate-based self-immolative linker-units that facilitate stable attachment in serum and traceless drug release in the target cell of aliphatic and aromatic alcohols. Studies with camptothecins show that stability and release are tunable and that various intracellular trigger events can be exploited to ensure traceless drug delivery. Superior stability, in vivo efficacy, and pharmacokinetics (PK) compared to approved camptothecin ADCs are demonstrated. Moreover, we report targeted delivery of 10 different hydroxy-containing cytotoxins with different intracellular MOAs. In vivo studies with gemcitabine show excellent PK and efficacy, unlocking gemcitabine’s full potential and illustrating the ability of the phosphoramidate-based linker system to expand the payload space for ADCs. Approved antibody–drug conjugates (ADCs) remain constrained by a limited repertoire of payloads with restricted modes of action. Here, the authors present phosphoramidate-based self-immolative linker units that facilitate stable attachment in serum and traceless drug release in the target cell from aliphatic and aromatic alcohols with various modes of action.

Nanobodies are emerging as attractive biopharmaceuticals due to their small size, stability and target specificity. However, their therapeutic use has largely been restricted to extracellular targets because of a lack of efficient delivery methods. This limitation is particularly relevant for diseases caused by dysfunctional intracellular proteins, such as cystic fibrosis. Here we show that cell-permeable nanobodies can modulate an intracellular disease-relevant target: the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel carrying the common F508del mutation. By combining a CFTR-binding nanobody with cell-penetrating peptides, we achieved intracellular delivery in cystic fibrosis bronchial epithelial cells. The delivered nanobody stabilizes misfolded F508del-CFTR, promotes its maturation and trafficking to the apical membrane and restores chloride channel activity. Moreover, the cell-permeable nanobody enhances the efficacy of approved CFTR modulator drug combination in primary airway epithelial cultures from patients with cystic fibrosis. These findings establish cell-permeable nanobodies as promising biopharmaceuticals for intracellular protein targeting and therapeutic modulation.

Refractory disease and relapse are major challenges in acute myeloid leukemia (AML) therapy attributed to survival of leukemic stem cells (LSC). To target LSCs, antibody-drug conjugates (ADCs) provide an elegant solution, combining the specificity of antibodies with highly potent payloads. We aimed to investigate if FLT3-20D9h3-ADCs delivering either the DNA-alkylator duocarmycin (DUBA) or the microtubule-toxin monomethyl auristatin F (MMAF) can eradicate quiescent LSCs. We show here that DUBA more potently kills cell-cycle arrested AML cells compared to microtubule-targeting auristatins. Due to limited stability of 20D9h3-DUBA ADC in vivo, we analyzed both ADCs in advanced in vitro stem cell assays. 20D9h3-DUBA successfully eliminated leukemic progenitors in vitro in colony-forming unit and long-term culture initiating cell assays, both in patient cells and in patient-derived xenograft (PDX) cells. Further, it completely prevented engraftment of AML PDX leukemia-initiating cells in NSG mice. 20D9h3-MMAF had a similar effect in engraftment assays, but a less prominent effect in colony assays. Both ADCs did not affect healthy stem and progenitor cells at comparable doses providing the rationale for FLT3 as therapeutic LSC target. Collectively, we show that FLT3-directed ADCs with DUBA or MMAF have potent activity against AML LSCs and represent promising candidates for further clinical development.

Proteinbasierte Werkzeuge gewinnen zunehmend an Bedeutung als innovative Ansätze zur gezielten Beeinflussung biologischer Signalwege in der Molekularbiologie und Medizin. Im Vergleich zu klassischen niedermolekularen Wirkstoffen bieten sie Vorteile durch ihre strukturelle Diversität und die Fähigkeit, bislang schwer zugängliche zelluläre Zielstrukturen zu adressieren. Allerdings durchdringen die meisten Proteine die Lipiddoppelschicht von Säugerzellen nicht und sind daher auf extrazelluläre Strukturen beschränkt. Trotz jüngster Fortschritte stellt der intrazelluläre Transport funktioneller Proteine in humane Zellen weiterhin eine erhebliche Herausforderung dar. In der vorliegenden Studie präsentieren wir eine bioreversible Modifikationsstrategie primärer Amine mithilfe kurzer, Arginin‐haltiger Peptide (bezeichnet als BioRAM), welche die zytosolische Aufnahme genetisch nicht veränderter Proteine ermöglicht. Die Biokonjugationsstrategie wurde dahingehend optimiert, eine schnelle intrazelluläre Abspaltung und vollständige Wiederherstellung der nativen Proteinfunktion zu gewährleisten. In Kombination mit unserem zuvor etablierten zellpenetrierenden Peptid‐Additivprotokoll demonstrieren wir einen effizienten intrazellulären Transport von fluoreszierenden Proteinen und funktioneller RNase A, die in der Zelle zu physiologisch relevanten Effekten führen. Darüber hinaus zeigen wir die hohe Leistungsfähigkeit von BioRAM auch in Anwesenheit von Serum, was das Anwendungsspektrum funktioneller Proteine im intrazellulären Kontext deutlich erweitert.

UDP-GlcNAc 2-epimerase/N-acetylmannosamine kinase (GNE/MNK) is the rate-limiting enzyme in sialic acid biosynthesis and a promising therapeutic target. We applied interferometric scattering microscopy (iSCAM) to investigate GNE oligomerization and its modulation by three small-molecule inhibitors (C5, C13, C15). Substrate binding (UDP-GlcNAc) stabilized tetramer formation by increasing dimer-dimer affinity 120-fold. All inhibitors destabilized tetramers in a concentration-dependent manner, with IC50 values in the low micromolar range. Using a modified Cheng-Prusoff equation, IC50 values were converted into Ki values. Schild analysis was applied to estimate an apparent KB,app value and assess cooperative inhibition effects. Molecular docking confirmed competitive binding for all inhibitors and helped rationalize observed potency trends. While iSCAM has previously been used to study protein assembly, our work demonstrates its applicability for the label-free, quantitative characterization of small-molecule inhibitors affecting protein oligomerization. These findings provide a foundation for further mechanistic studies and underscore the potential of iSCAM in drug-target interaction profiling. ### Competing Interest Statement The authors have declared no competing interest. Federal Ministry of Education and Research, https://ror.org/04pz7b180, 13XP511 Deutsche Forschungsgemeinschaft, https://ror.org/018mejw64, 431232613

Zusammenfassung Proteinbasierte Werkzeuge gewinnen zunehmend an Bedeutung als innovative Ansätze zur gezielten Beeinflussung biologischer Signalwege in der Molekularbiologie und Medizin. Im Vergleich zu klassischen niedermolekularen Wirkstoffen bieten sie Vorteile durch ihre strukturelle Diversität und die Fähigkeit, bislang schwer zugängliche zelluläre Zielstrukturen zu adressieren. Allerdings durchdringen die meisten Proteine die Lipiddoppelschicht von Säugerzellen nicht und sind daher auf extrazelluläre Strukturen beschränkt. Trotz jüngster Fortschritte stellt der intrazelluläre Transport funktioneller Proteine in humane Zellen weiterhin eine erhebliche Herausforderung dar. In der vorliegenden Studie präsentieren wir eine bioreversible Modifikationsstrategie primärer Amine mithilfe kurzer, Arginin‐haltiger Peptide (bezeichnet als BioRAM), welche die zytosolische Aufnahme genetisch nicht veränderter Proteine ermöglicht. Die Biokonjugationsstrategie wurde dahingehend optimiert, eine schnelle intrazelluläre Abspaltung und vollständige Wiederherstellung der nativen Proteinfunktion zu gewährleisten. In Kombination mit unserem zuvor etablierten zellpenetrierenden Peptid‐Additivprotokoll demonstrieren wir einen effizienten intrazellulären Transport von fluoreszierenden Proteinen und funktioneller RNase A, die in der Zelle zu physiologisch relevanten Effekten führen. Darüber hinaus zeigen wir die hohe Leistungsfähigkeit von BioRAM auch in Anwesenheit von Serum, was das Anwendungsspektrum funktioneller Proteine im intrazellulären Kontext deutlich erweitert.