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.

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.

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.

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.

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.

The mechanism behind enhanced reactivity of unsaturated phosphorus(v) electrophiles towards thiols.

Vinyl- and ethynyl phosphorus(v) electrophiles are a versatile class of thiol-reactive reagents suitable for cysteine-selective peptide and protein modifications, especially for the generation of antibody conjugates. Herein we investigated the reactivity of various P(v) reagents towards thiol addition. Complementing previous studies, we observed that the heteroatoms X (X = S, O, NH) as well as the vinyl- vs. ethynyl-substituent bound to phosphorus greatly influence the overall reactivity. These experimentally observed trends, as well as the high Z-selectivity for thiol additions to ethynyl derivatives, were further elucidated using DFT calculations. Hyperconjugation was a key means of stabilizing the intermediate generated upon the thiol addition, thus determining both the reactivity and stereoselectivity of unsaturated P(v) electrophiles. Specifically, the energetically low-lying σ antibonding orbital of the P-S bond more readily stabilizes the electron density from the lone pair (LP) of the generated carbanion, rendering the phosphonothiolates more reactive compared to the derivatives bearing oxygen and nitrogen. Our studies provide a detailed mechanistic picture for designing P(v)-based electrophiles with fine-tuned reactivity profiles.

Diethynyl Phosphinates for Cysteine-Selective Protein Labeling and Disulfide Rebridging.

Diethynyl phosphinates were developed as bisfunctional electrophiles for the site-selective modification of peptides, proteins and antibodies. One of their electron-deficient triple bonds reacts selectively with a thiol and positions an electrophilic moiety for a subsequent intra- or intermolecular reaction with another thiol. The obtained conjugates were found to be stable in human plasma and in the presence of small thiols. We further demonstrate that this method is suitable for the generation of functional protein conjugates for intracellular delivery. Finally, this reagent class was used to generate functional homogeneously rebridged antibodies that remain specific for their target. Their modular synthesis, thiol selectivity and conjugate stability make diethynyl phosphinates ideal candidates for protein conjugation for biological and pharmaceutical applications.

Discovery, X-ray structure and CPP-conjugation enabled uptake of p53/MDM2 macrocyclic peptide inhibitors.

Mouse double minute 2 homolog (MDM2, Hdm2) is an important negative regulator of the tumor suppressor p53. Using a mRNA based display technique to screen a library of >1012 in vitro-translated cyclic peptides, we have identified a macrocyclic ligand that shows picomolar potency on MDM2. X-Ray crystallography reveals a novel binding mode utilizing a unique pharmacophore to occupy the Phe/Trp/Leu pockets on MDM2. Conjugation of a cyclic cell-penetrating peptide (cCPP) to the initially non cell-permeable ligand enables cellular uptake and a pharmacodynamic response in SJSA-1 cells. The demonstrated enhanced intracellular availability of cyclic peptides that are identified by a display technology exemplifies a process for the application of intracellular tools for drug discovery projects.

Synthesis and Evaluation of Non-Hydrolyzable Phospho-Lysine Peptide Mimics.

The intrinsic lability of the phosphoramidate P-N bond in phosphorylated histidine (pHis), arginine (pHis) and lysine (pLys) residues is a significant challenge for the investigation of these post-translational modifications (PTMs), which gained attention rather recently. While stable mimics of pHis and pArg have contributed to study protein substrate interactions or to generate antibodies for enrichment as well as detection, no such analogue has been reported yet for pLys. This work reports the synthesis and evaluation of two pLys mimics, a phosphonate and a phosphate derivative, which can easily be incorporated into peptides using standard fluorenyl-methyloxycarbonyl- (Fmoc-)based solid-phase peptide synthesis (SPPS). In order to compare the biophysical properties of natural pLys with our synthetic mimics, the pKa values of pLys and analogues were determined in titration experiments applying nuclear magnetic resonance (NMR) spectroscopy in small model peptides. These results were used to compute electrostatic potential (ESP) surfaces obtained after molecular geometry optimization. These findings indicate the potential of the designed non-hydrolyzable, phosphonate-based mimic for pLys in various proteomic approaches.

Chemically Induced Vinylphosphonothiolate Electrophiles for Thiol-Thiol Bioconjugations.

Herein we introduce vinylphosphonothiolates as a new class of cysteine-selective electrophiles for protein labeling and the formation of stable protein-protein conjugates. We developed a straightforward synthetic route to convert nucleophilic thiols into electrophilic, thiol-selective vinylphosphonothiolates: In this protocol, intermediately formed disulfides can be chemoselectively substituted with vinylphosphonites under acidic conditions to yield the desired vinylphosphonothiolates. Notably, this reaction sequence enables the installation of vinylphosphonothiolate electrophiles directly on cysteine side chains within peptides and proteins. In addition to labeling the monoclonal antibody trastuzumab with excellent cysteine-selectivity, we applied our protocol for the site-specific conjugation of two proteins with unique cysteine residues yielding a nonhydrolyzable phosphonothiolate-linked diubiquitin and an ubiquitin-α-synuclein conjugate. The latter was recognized as a substrate in a subsequent enzymatic ubiquitination reaction.

N-Hydroxysuccinimide-Modified Ethynylphosphonamidates Enable the Synthesis of Configurationally Defined Protein Conjugates.

Herein, the application of N-hydroxysuccinimide-modified phosphonamidate building blocks for the incorporation of cysteine-selective ethynylphosphonamidates into lysine residues of proteins, followed by thiol addition with small molecules and proteins, is reported. It is demonstrated that the building blocks significantly lower undesired homo-crosslinking side products that can occur with commonly applied succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) under physiological pH. The previously demonstrated stability of the phosphonamidate moiety additionally solves the problem of premature maleimide hydrolysis, which can hamper the efficiency of subsequent thiol addition. Furthermore, a method to separate the phosphonamidate enantiomers to be able to synthesize protein conjugates in a defined configuration has been developed. Finally, the building blocks are applied to the construction of functional antibody-drug conjugates, analogously to FDA-approved, SMCC-linked Kadcyla, and to the synthesis of a functional antibody-protein conjugate.

One-Step Fluorescent Protein Labeling by Tubulin Tyrosine Ligase.

Tub-tag labeling, a novel chemoenzymatic protein functionalization method, facilitates one-step fluorescent labeling of functional biomolecules. The enzyme tubulin tyrosine ligase incorporates coumarin-amino acids to the terminal carboxylic acid of proteins containing a short peptidic recognition sequence called Tub-tag. Here we describe the one-step Tub-tag protein modification protocol in detail and explain its utilization to generate fluorescently labeled proteins for advanced applications in imaging and diagnostics.

Vinylphosphonites for Staudinger-induced chemoselective peptide cyclization and functionalization.

In this paper, we introduce vinylphosphonites for chemoselective Staudinger-phosphonite reactions (SPhR) with azides to form vinylphosphonamidates for the subsequent modification of cysteine residues in peptides and proteins. An electron-rich alkene is turned into an electron-deficient vinylphosphonamidate, thereby inducing electrophilic reactivity for a following thiol addition. We show that by varying the phosphonamidate ester substituent we can fine-tune the reactivity of the thiol addition and even control the functional properties of the final conjugate. Furthermore, we observed a drastic increase in thiol addition efficiency when the SPhR is carried out in the presence of a thiol substrate in a one-pot reaction. Hence, we utilize vinylphosphonites for the chemoselective intramolecular cyclization of peptides carrying an azide-containing amino acid and a cysteine in high yields. Our concept was demonstrated for the stapling of a cell-permeable peptidic inhibitor for protein-protein interaction (PPI) between BCL9 and beta-catenin, which is known to create a transcription factor complex playing a role in embryonic development and cancer origin, and for macrocyclization of cell-penetrating peptides (CPPs) to enhance the cellular uptake of proteins.

Ethynylphosphonamidates for the Rapid and Cysteine-Selective Generation of Efficacious Antibody-Drug Conjugates.

Requirements for novel bioconjugation reactions for the synthesis of antibody-drug conjugates (ADCs) are exceptionally high, since conjugation selectivity as well as the stability and hydrophobicity of linkers and payloads drastically influence the performance and safety profile of the final product. We report Cys-selective ethynylphosphonamidates as new reagents for the rapid generation of efficacious ADCs from native non-engineered monoclonal antibodies through a simple one-pot reduction and alkylation. Ethynylphosphonamidates can be easily substituted with hydrophilic residues, giving rise to electrophilic labeling reagents with tunable solubility properties. We demonstrate that ethynylphosphonamidate-linked ADCs have excellent properties for next-generation antibody therapeutics in terms of serum stability and in vivo antitumor activity.

Electron Transfer/Higher Energy Collisional Dissociation of Doubly Charged Peptide Ions: Identification of Labile Protein Phosphorylations.

In recent years, labile phosphorylation sites on arginine, histidine, cysteine, and lysine as well as pyrophosphorylation of serine and threonine have gained more attention in phosphoproteomic studies. However, the analysis of these delicate posttranslational modifications via tandem mass spectrometry remains a challenge. Common fragmentation techniques such as collision-induced dissociation (CID) and higher energy collisional dissociation (HCD) are limited due to extensive phosphate-related neutral loss. Electron transfer dissociation (ETD) has shown to preserve labile modifications, but is restricted to higher charge states, missing the most prevalent doubly charged peptides. Here, we report the ability of electron transfer/higher energy collisional dissociation (EThcD) to fragment doubly charged phosphorylated peptides without losing the labile modifications. Using synthetic peptides that contain phosphorylated arginine, histidine, cysteine, and lysine as well as pyrophosphorylated serine residues, we evaluated the optimal fragmentation conditions, demonstrating that EThcD is the method of choice for unambiguous assignment of tryptic, labile phosphorylated peptides. Graphical Abstract.

Cysteine-Selective Phosphonamidate Electrophiles for Modular Protein Bioconjugations.

We describe a new technique in protein synthesis that extends the existing repertoire of methods for protein modification: A chemoselective reaction that induces reactivity for a subsequent bioconjugation. An azide-modified building block reacts first with an ethynylphosphonite through a Staudinger-phosphonite reaction (SPhR) to give an ethynylphosphonamidate. The resulting electron-deficient triple bond subsequently undergoes a cysteine-selective reaction with proteins or antibodies. We demonstrate that ethynylphosphonamidates display excellent cysteine-selective reactivity combined with superior stability of the thiol adducts, when compared to classical maleimide linkages. This turns our technique into a versatile and powerful tool for the facile construction of stable functional protein conjugates.