Changing the backbone connectivity of proteins can impart useful new traits while maintaining essential structural and functional features. In design of artificial proteomimetic agents, backbone modification is usually isolated to sites that are solvent-exposed in the folded state, as similar changes at buried residues can alter the fold. Recent work has shown that core backbone modification without structural perturbation is possible; however, the modifications in that study were consistently destabilizing and made in a prototype of exceptionally high conformational stability. Here, we report efforts to broaden the scope and improve the efficacy of core backbone engineering by applying it to the C-terminal subdomain of villin headpiece. A series of variants are prepared in which different artificial residue types are incorporated at core positions throughout the sequence, including a crucial aromatic triad. Impacts on folding energetics are quantified by biophysical methods, and high-resolution structures of several variants determined by NMR. We go on to construct a variant with ∼40% of its core modified that adopts a fold identical to the prototype while showing enhanced thermodynamic stability.

As the global fight against antimicrobial resistance in bacteria becomes increasingly pressing, new tool compounds are needed to study and evaluate novel therapeutic targets. Here, cysteine-directed fragment-based drug discovery is coupled with high throughput chemistry direct-to-biology screening to target the catalytic cysteine of a family of bacterial effector proteins, the novel E3 ligases (NELs) from Salmonella and Shigella. These effector E3 ligases are attractive as potential drug targets because they are delivered into host cells during infection, have no human homologues and disrupt host immune response to infection. We successfully identify hit compounds against the SspH subfamily of NELs from Salmonella and show that these proteins are inhibited by compound treatment, representing an exciting starting point for development into specific and potent tool compounds.

The growing need for effective HBV treatments and lead compounds with novel mechanisms prompted us to explore synthetic strategies for generating skeletally diverse alkaloidal Michael acceptors. Our approach uniquely embeds Michael acceptors directly within multicyclic alkaloid-inspired frameworks, exploiting the azepinoindole scaffold—a privileged structure in indole alkaloids. A single-step assembly between the versatile intermediate 13 with methyl propiolate 14 or its derivatives enabled the rapid and divergent synthesis of six alkaloidal Michael acceptors (15–20). This strategy facilitated systematic diversification of three-dimensional functional group arrangements and precise tuning of the electronic and steric properties of the embedded α,β-unsaturated carbonyl moieties. The optimal hit 15 inhibited hepatitis B surface antigen (HBsAg) production with an IC50 of 2.48 μM and significantly reduced levels of covalently closed circular DNA (cccDNA), the master template of HBV. Unlike existing nucleoside/nucleotide-based anti-HBV drugs that primarily inhibit reverse transcription, the alkaloidal Michael acceptor 15 suppressed both cccDNA and relaxed circular DNA (rcDNA) levels, suggesting a potential pathway toward a functional HBV cure. Our study also streamlined the target identification by leveraging the covalent binding properties of the Michael acceptors and the operational simplicity of biotin- or fluorescent-tag attachment via a pre-installed alkyne moiety. Competitive pull-down experiments identified several potential target proteins, involving DNA polymerase epsilon subunit 3 (POLE3). Notably, the alkaloidal Michael acceptor 15 was demonstrated to covalently modify Cys51 in POLE3, providing new insights into virus–host interactions and opening novel avenues for targeted anti-HBV therapies. This approach represents a significant advance beyond traditional screening methods and underscores the potential of skeletally diverse alkaloidal Michael acceptors in antiviral drug development.

This article profiles the early career researchers whose work features in the RSC Chemical Biology Emerging Investigators Collection 2025.

d-Luciferin (d-LH2) is the most used substrate for beetle luciferases in various bioluminescence applications. Here, we successfully synthesized six d-LH2 analogues including 5′,7′-dimethoxy-d-LH2 and 7′-methylnaphthol-d-LH2 as novel compounds. We also developed a continuous one-pot green synthesis method to improve yields of luciferins from condensation of quinone and d-Cys (63-fold greater than the previous report). The novel d-LH2 analogues were tested with five luciferases (Fluc, SLR, Eluc, Pmluc-WT, and Pmluc-N230S), and all the compounds emitted bioluminescence at wavelengths longer than that of d-LH2 (>80 nm). The reaction of SLR with 5′,7′-dimethoxy-d-LH2 gave the longest red-shifted bioluminescence at 663 nm. Remarkably, the reactions of 5′-methyl-d-LH2 emit longer wavelengths and brighter light than those of d-LH2 in all tested luciferases, except for Eluc. Interestingly, the novel red-shifted 5′,7′-dimethyl-d-LH2 also provided prolonged bioluminescence with a rate of light decay slower than that of d-LH2. We further demonstrated applications of 5′-methyl-d-LH2 and 5′,7′-dimethyl-d-LH2 in mammalian cell lines expressing Fluc, SLR, and Pmluc-N230S. 5′-Methyl-d-LH2 provided about 11.2-fold greater sensitivity to detect Fluc in the HEK293T crude lysate than d-LH2, achieving the detection with a lower number of cell lines. The red-shifted 5′,7′-dimethyl-d-LH2 also exhibits high sensitivity when using a red light filter to monitor live cell bioluminescence. These d-LH2 analogues, 5′-methyl-d-LH2 and 5′,7′-dimethyl-d-LH2, are promising substrates for future cell-based assays and real-time monitoring applications.

Sialic acid mimetics (SAMs) are chemically modified derivatives of sialic acids that can act as metabolic inhibitors or as sugar donors for sialyltransferases. This makes SAMs highly useful research tools to study and manipulate the biosynthesis of sialic acid-carrying glycans (sialoglycans). Moreover, SAMs that inhibit aberrant sialylation in cancer cells are emerging as potential therapeutics. Despite the wide use of SAMs, many aspects regarding their cellular uptake and metabolic fate are unknown. Here, we investigated the metabolic fate of an inhibitory SAM (P-SiaFNEtoc) and an incorporative SAM (P-SiaNPoc) in various mammalian cell lines. Using kinetic experiments and read-outs based on sialic acid-binding lectins, click chemistry, and nucleotide sugar analysis, we monitored the key steps of cellular SAM utilization. We found differences in the metabolism of SAMs that determine their potency in different mammalian cell lines. By identifying a murine macrophage cell line that is insensitive to SAMs, we have identified esterase activity as a bottleneck for the cellular utilization of SAMs. This study contributes to the understanding of the mechanisms underlying SAMs utilization in mammalian cell lines and provide relevant considerations for the future chemical design of SAMs and their application in mammalian systems.

Abnormal glycosylation has been long considered a hallmark of cancer progression. Carbohydrate-binding proteins, also known as lectins, offer a unique way to target glycosylation changes in malignant cells. The present study repurposes SadP, a monomeric lectin from Streptococcus suis, to target globotriaosylceramide (Gb3), a glycosphingolipid overly abundant in many cancer types including Burkitt's lymphoma. The lectibody was designed as a fusion protein by linking the SadP to the scFv UCHT1 anti-CD3 antibody resulting in a bispecific T cell engager (BiTE)-like protein referred to as lectibody. Protein expression was carried out in Escherichia coli and the resulting lectibody was purified using affinity and size exclusion chromatography. The lectibody was tested for its specificity in binding Gb3-positive cancer cells by flow cytometry. T-cell-mediated cytotoxicity was measured in a bioluminescence-based cytotoxicity assay, and T-cell activation was assessed by evaluating CD69 and CD71 expression on PBMCs, incubated with target cells and the lectibody. The present study demonstrates that the monomeric and monovalent SadP-scFv UCHT1 lectibody can redirect T cell cytotoxicity towards Gb3+ Burkitt's lymphoma cells, resulting in a dose-dependent target cell lysis up to 65% in vitro at a concentration of 10 nM. In the same experimental setting, negative control cells characterized by a low or absent Gb3 content remained unaffected. Lectibody-induced T cell activation resulted in a significant increase in CD69 and CD71 surface expression in PBMCs incubated with SadP-scFv UCHT1 and Gb3 positive cancer cells. This study highlights the potential of lectins in immunotherapy for the treatment and eradication of malignant cells. The SadP-based lectibody demonstrates improved efficacy and yield when compared to the previously engineered StxB-scFv UCHT1 lectibody, therefore opening the possibility for its use in an in vivo model.

Fluorescent probes for measuring the structure, dynamics, cellular localization, and biochemistry of DNA and RNA are useful for determining the regulatory mechanisms of gene expression. Intrinsically fluorescent, Watson-Crick-capable nucleobase analogues are especially powerful because they can precisely probe desired loci while minimally perturbing native nucleic acid function. Here, we study the fluorescent responses of the tricyclic pyrimidine analogue DEAtC to base pairing with adenine, guanine, and a set of noncanonical nucleobases in duplex DNA oligonucleotides. We find that single-stranded oligonucleotides containing one DEAtC exhibit up to a fivefold fluorescence increase upon hybrid duplex formation and base pairing with G, and a lesser degree of fluorescence turn-on when base pairing with inosine. Other purine nucleobases do not induce significant fluorescence turn-on. Solvent kinetic isotope effect measurements, excitation-emission matrix (EEM) analysis, and spectral comparisons indicate that fluorescence turn-on originates from base pairing-templated tautomerism. The non-emissive T-like form predominates in the single strand and in duplexes paired with A, whereas the emissive C-like tautomer is selectively stabilized upon duplex formation when paired with G. Density functional theory (DFT) calculations further support this tautomeric control model. Although base stacking influences overall brightness, it does not alter the mechanism or specificity of fluorescence turn-on. Modulation of emission through tautomeric control offers a powerful strategy for designing nucleobase analogues with base pairing-specific fluorescence responses.