Christoph Nitsche

L -DOPA-containing peptides enable intrinsic, chelator-free Ga( iii ) binding under mild aqueous conditions. Orthogonal Ga( iii )/Bi( iii ) coordination within a single peptide scaffold is demonstrated.

The transformation of cysteine residues into more reactive species is a powerful entry point to post-translational protein modification and the synthesis of complex bioactive peptides, yet existing approaches often suffer from limited selectivity and undesired side reactions. Here we report iodomethylboronic acid (IMBA) as a small alkylating agent that selectively converts cysteine residues in peptides and proteins into dimethylsulfonium species via rapid protodeboronation. The resulting S,S-dimethylsulfonium-modified cysteine can be stabilized at low temperature. Subsequent β-elimination furnishes dehydroalanine under mild and biocompatible conditions. Multiple cysteine residues can be simultaneously modified. The overall transformation generates only iodide, boric acid and dimethyl sulfide as byproducts. Full conversion of cysteine is achieved with low equivalents of IMBA reagent, enabling direct downstream diversification of the resulting dehydroalanine with nucleophiles and eliminating the need for conventional two-step workflows.

Affibodies are remarkably stable three‐helix bundle proteins that can be engineered to selectively bind target proteins. When combined with radioactive metals, they serve as imaging agents or cancer therapeutics, depending on the metal used. Traditionally, this involves bifunctional linkers that attach large chelators to the affibody via reactive groups. Here, we present an alternative approach that eliminates the need for such linkers by burying the metal within the core of the affibody, surrounded by its three helices. A simple engineered triple cysteine motif, with one cysteine in each helix, stably binds Bi(III), Pb(II), In(III), and Ga(III), which are commonly used in imaging and radiotherapy. Quantitative metal uptake is instantaneous at room temperature and physiological pH, and all metal‐affibody complexes remain fully intact for one week at 4 °C. All retain their metal cargo when challenged with cellular concentrations of glutathione, while only the bismuth‐affibody complex withstands a challenge with 100 equivalents of strong chelators, even over two weeks. We demonstrate that, a bismuth‑loaded affibody retains binding affinity to the HER2 receptor comparable to the wildtype affibody, while selectively binding and retaining ²¹³ Bi, a promising radioisotope for targeted alpha therapy, thereby enabling targeting of HER2‑overexpressing SKBR3 cancer cells.

Affibodies are remarkably stable three‐helix bundle proteins that can be engineered to selectively bind target proteins. When combined with radioactive metals, they serve as imaging agents or cancer therapeutics, depending on the metal used. Traditionally, this involves bifunctional linkers that attach large chelators to the affibody via reactive groups. Here, we present an alternative approach that eliminates the need for such linkers by burying the metal within the core of the affibody, surrounded by its three helices. A simple engineered triple cysteine motif, with one cysteine in each helix, stably binds Bi(III), Pb(II), In(III), and Ga(III), which are commonly used in imaging and radiotherapy. Quantitative metal uptake is instantaneous at room temperature and physiological pH, and all metal‐affibody complexes remain fully intact for one week at 4 °C. All retain their metal cargo when challenged with cellular concentrations of glutathione, while only the bismuth‐affibody complex withstands a challenge with 100 equivalents of strong chelators, even over two weeks. We demonstrate that, a bismuth‑loaded affibody retains binding affinity to the HER2 receptor comparable to the wildtype affibody, while selectively binding and retaining ²¹³ Bi, a promising radioisotope for targeted alpha therapy, thereby enabling targeting of HER2‑overexpressing SKBR3 cancer cells.

Cell-penetrating bismuth peptides are visualised inside cells by combined X-ray fluorescence (XFM) and optical microscopy. This multimodal approach enables direct tracking of metal-based peptides, offering new insight into bioinorganic therapeutics.

Heparanase is the sole enzyme responsible for breaking down heparan sulfate within the extracellular matrix, and its overexpression is linked to human diseases. Despite heparanase being a promising drug target, most efforts have focused on substrate mimetics, which have failed clinical trials, highlighting the need for new inhibitor scaffolds. Here, we employed fragment-based drug design to explore a novel chemical space to develop small molecule inhibitors of heparanase. We used a crystallographic and computational approach to identify 31 fragments that bind heparanase; five of these inhibited heparanase in the micromolar range. One of these fragments underwent two cycles of fragment growing, which resulted in a compound with a 7-fold increased potency compared to the initial hit. The results from our fragment screen unveil untapped chemical space for heparanase inhibition, paving the way for the development of potent drug leads with the potential to transform the treatment of heparanase-related diseases.

Affibodies are remarkably stable three-helix bundle proteins that can be engineered to selectively bind target proteins. When combined with radioactive metals, they serve as imaging agents or cancer therapeutics, depending on the metal used. Traditionally, this involves bifunctional linkers that attach large chelators to the affibody via reactive groups. Here, we present an alternative approach that eliminates the need for such linkers by burying the metal within the core of the affibody, surrounded by its three helices. A simple engineered triple cysteine motif, with one cysteine in each helix, stably binds Bi(III), Pb(II), In(III) and Ga(III), which are commonly used in imaging and radiotherapy. Quantitative metal uptake is instantaneous at room temperature and physiological pH, and all metal-affibody complexes remain fully intact for one week at 4 °C. All retain their metal cargo when challenged with cellular concentrations of glutathione, while only the bismuth-affibody complex withstands a challenge with 100 equivalents of strong chelators, even over two weeks. We demonstrate selective uptake and retention of ²¹³ Bi, a promising isotope for targeted alpha therapy.

Proteins and peptides underpin essential biological functions and technological applications, from targeting disease-relevant interactions to providing broad enzymatic activities. However, engineering molecules with desired properties remains difficult, owing to complex sequence-structure-function relationships and the lack of data on specific systems. Experimental selection strategies, including directed evolution, phage display, and mRNA display, address this challenge by leveraging high diversity libraries and iterative enrichment under defined selection pressures. This allows for the identification of candidates without requiring extensive prior knowledge, and can generate extensive datasets for use in machine learning. While many selection systems exist, comparisons across different selection approaches are hindered by the lack of a unifying analytical framework. Here, we present a set of broadly applicable analyses for assessing selection dynamics in multi-round or multi-condition experiments, ranging from position level analysis of sequence properties to full sequence space mappings through protein language model embeddings. Using the toolset to analyze a variety of different datasets in parallel, we explore the potential effects of diversity, coverage, and reproducibility, offering generalizable insights to guide experimental design, interpretation, and troubleshooting across protein and peptide discovery platforms.

No specific vaccines or therapeutics are currently available for the prevention or treatment of Zika virus infections. The viral protease NS2B‐NS3 is essential for the replication of Zika and other orthoflaviviruses, making it a target for antiviral drug development. Traditional discovery of competitive inhibitors has relied on substrate recognition sequences, typically yielding multibasic peptides. Herein, a de novo strategy is presented for identifying competitive inhibitors using peptide phage display in combination with Bi(III)‐mediated in situ formation of bicyclic peptides. In an initial screening, phages displaying a library of randomized peptide‐bismuth bicycles are eluted by interrupting the phage‐target interactions at low pH. This approach yields a small number of peptides biased toward the active site, characterized by dibasic motifs, but only one low‐ranking sequence shows modest inhibitory activity. To enhance specificity, a second screening campaign employs competitive phage elution using the dibasic boronate inhibitor CN‐714 that covalently binds to the catalytically active serine residue S135 of NS2B‐NS3. This strategy enriches a larger pool of competitive inhibitors sharing the characteristic dibasic substrate recognition motif. The most potent peptide‐bismuth bicycle identified and synthesized features a completely novel sequence, exhibits an inhibition constant of 3.9 µM and displays remarkable proteolytic stability over 24 h.

Bismuth peptides and proteins are emerging as versatile tools for medicinal chemistry and chemical biology. Bismuth(III) binds three cysteine residues in peptides and proteins with remarkable selectivity. While the thermodynamic stability of these bismuth complexes is outstanding, their kinetic lability imposes limitations. Introducing bismuth selenopeptides, we demonstrate that selenocysteine binds bismuth with substantially higher kinetic stability than cysteine. This effect was quantified by directly comparing a peptide containing three cysteine residues with an identical peptide containing three selenocysteines. Bismuth selenopeptides are not only inert to strong chelators such as ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA) but also to the natural metal‐binding protein transferrin present in human plasma. We further demonstrate biological utility by developing bismuth selenopeptides that selectively bind and inhibit unrelated target proteins. To extend this concept to a more complex system, we investigated the human epidermal growth factor (EGF), a small protein comprising three disulfide bonds. We established that precisely two bismuth atoms bind to the six cysteine or selenocysteine residues in EGF and seleno‐EGF, respectively. In the presence of EDTA, bismuth seleno‐EGF remains fully intact, unlike its cysteine‐based analog, consistent with observations from smaller peptides. Structural models confirm full preservation of the native EGF fold.