Nucleic acid aptamers are synthetic, single-stranded oligonucleotides that bind targets with high affinity and specificity, enabling precise and programmable functional modulation. However, low biostability remains a key druggability bottleneck of therapeutic aptamers. In vivo enzymatic lability constitutes a primary pharmacokinetic barrier, impeding systemic exposure, tissue distribution, and sustained target occupancy, all critical determinants of in vivo efficacy. Consequently, rational chemical stabilization via enzyme-resistant modifications has emerged as a cornerstone strategy in aptamer drug development. In this review, we systematically categorize current stabilization approaches across two complementary design dimensions: local-level modifications (including terminal modification, sugar modification, backbone modification, and base modification) and global structural-level engineering (including Spiegelmers, cyclization modification, and multivalent assembly). Furthermore, we discuss persistent translational challenges to illuminate a coherent framework for designing aptamers with high biostability that achieve enhanced nuclease resistance while concurrently exhibiting favorable pharmacokinetics, high target binding affinity and specificity.

Macrophages play a critical role in sepsis, a life-threatening systemic inflammatory syndrome that necessitates urgent therapeutic intervention. Unfortunately, no approved drugs currently target this specific pathological mechanism. In this study, we identify Igalan, a natural compound that selectively disrupts macrophage-mediated inflammatory cascades in macrophages. Mechanistic studies demonstrate that Igalan alleviates oxidative stress and maintains mitochondrial integrity. Crucially, we reveal that Igalan covalently modifies the NLRP3 (NLR family pyrin domain containing 3) NACHT domain, thereby irreversibly suppressing inflammasome activation and subsequent pro-inflammatory signaling. In vivo studies demonstrate potent therapeutic effects of Igalan, mitigating systemic inflammation in LPS-challenged zebrafish and providing protection against pulmonary injury and intestinal barrier dysfunction in murine sepsis models. Collectively, our work establishes Igalan as a covalent NLRP3 inhibitor with translational potential for sepsis treatment.

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.

The human islet amyloid polypeptide (hIAPP) aggregates into amyloid fibrils that contribute to β-cell failure in type 2 diabetes. hIAPP is produced from a 67-residue precursor, proIAPP, but incomplete cleavage by prohormone convertase 2 (PC2) produces the 48-residue intermediate proIAPP(1-48), which accelerates amyloid formation in vivo. Here we show that proIAPP(1-48) assembles almost exclusively into a single fibril polymorph. Using cryo-electron microscopy we solved its structure at 3.5 Å resolution and uncovered a P-shaped, C2-symmetric dimer whose backbone and side-chain packing are nearly identical to the disease-associated TW2 polymorph propagated from pancreatic tissue, although with different helical symmetry. All eleven extra N-terminal residues remain disordered but create a weak density around His29. Based on time-averaged density derived from molecular dynamics (MD) simulations, we identified multiple hydrogen(H)-bonding interactions, which may contribute to stabilising the TW2-like fold and explain the peripheral cryo-EM density. These data establish a structural link between defective proIAPP processing and the polymorphic spectrum of islet amyloid and suggest a seeding pathway by which proIAPP(1-48) templates pathogenic architectures that fully processed hIAPP rarely adopts in vitro.

Development of chemically modified oligonucleotides, nucleic acid mimics, protein-based constructs, and other ligands - capable of sequence-unrestricted recognition of specific double-stranded (ds) DNA regions - is an area of research that continues to attract considerable attention. Efforts are fueled by the need for diagnostic agents, modulators of gene expression, and novel therapeutic modalities against genetic diseases. While pioneering approaches focused on accessing nucleotide-specific features from the grooves of DNA duplexes, recent developments have entailed strand-invading probes, i.e., probes capable of binding to DNA duplexes by breaking existing Watson-Crick base pairs and forming new, more stable base pairs. For the past twenty years, our laboratory has pursued the development of a type of dsDNA-targeting strand-invading probes, which we have named Invader probes. These double-stranded oligonucleotide probes feature intercalator-functionalized nucleotides that are specifically arranged to promote destabilization of the probe duplex, whereas individual strands exhibit very high affinity towards complementary DNA. This account details the discovery, principles, and applications of Invader probes.

Small molecule- and antibody-based approaches have shown tremendous success in both therapeutic and diagnostic applications. Aptamers, which are engineered nucleic acid ligands for proteins, have not found similar broad applicability, potentially due to their susceptibility to nuclease-mediated degradation and short engagement times of their targets. One approach to mitigate these issues is the use of covalent aptamers. Here, the aptamer sequence is functionalized with an electrophilic motif, combining the high specificity of aptamer-protein binding with the ability to form a permanent covalent bond at nucleophilic residues on the target protein. These electrophilic motifs can be either non-cleavable, allowing for the formation of aptamer-protein conjugates, or cleavable, allowing for transfer of a payload onto the target protein. The chemical structures of these motifs define their functions which range from protein detection to targeted protein degradation. The covalent bond formed between the electrophile and a nucleophilic amino acid sidechain at the protein surface dramatically increases the engagement time and duration of action of the functional moiety. In this review, we summarize efforts in establishing, understanding, and applying the chemistry of covalent aptamers.

Iterative polyketide synthases (iPKSs) rely on communication between acyl carrier protein (ACP) and acyltransferase (AT) domains to ensure efficient delivery of starter and extender substrates during biosynthesis. However, the molecular determinants governing the AT:ACP interface remain poorly understood. Here, we use the fungal highly reducing PKS, SimG, a component of the cyclosporin biosynthetic pathway, as a model system to dissect the AT:ACP interface. Using alanine scanning mutagenesis combined with a high-throughput intact protein mass spectrometry assay, we identified interface residues that affect AT:ACP interaction. These experimental constraints were used to guide docking and molecular dynamics simulations to produce a data-driven structural model of the SimG AT:ACP complex in a catalytically competent geometry. We also demonstrate that the SimG AT domain transacylates ACP domains from a range of fungal PKS architectural classes, highlighting significant interface plasticity. These insights advance our fundamental understanding of domain communication in these enigmatic megasynthases and provide a foundation for rational engineering to expand substrate scope towards novel polyketide scaffolds.

Treatment of acute promyelocytic leukemia (APL) remains challenged by toxicities associated with current regimens, highlighting the need for novel and safer therapeutic agents. Here, we identify Eupalinolide B (EB), a natural sesquiterpene lactone isolated from Eupatorium lindleyanum DC., as a potent anti-leukemic compound targeting the human APL-derived HL-60 cell line. Through integrated chemoproteomic profiling and functional validation, we demonstrate that EB covalently binds and inhibits 26S proteasome non-ATPase regulatory subunit 14 (PSMD14), a deubiquitinase enzyme (DUB) within the 19S proteasome regulatory particle. This inhibition disrupts PSMD14-mediated stabilization of key oncoproteins RAC-alpha serine/threonine-protein kinase 1 (AKT1) and cyclin-dependent kinase 4 (CDK4), promoting their proteasomal degradation. As a result, EB induces G2/M cell cycle arrest and apoptosis in leukemia cells. Both genetic knockdown and pharmacological inhibition of PSMD14 recapitulate EB's effects, confirming its essential role in leukemia cell survival and proliferation. Collectively, these findings uncover a previously unrecognized PSMD14-AKT1/CDK4 regulatory axis in leukemia and position EB as a promising chemical probe and lead compound for the development of targeted covalent inhibitors against oncogenic DUBs.

High-affinity inhibitors of specific receptors are a valuable tool to elucidate cellular signaling, control biological systems and develop therapeutic drugs. Here, we report mixed squaramide thioesters as a novel electrophilic handle, demonstrating an optimal balance between nucleophile accessibility and hydrolytic stability in aqueous environments. Density function theory calculations show that the energy of the lowest unoccupied molecular orbital (LUMO) of mixed squaramide thioesters is lower compared to mixed squaramide esters, rendering them more reactive to nucleophilic attack. Synthetic access to various lysophosphatidylglucoside (LysoPtdGlc) analogues incorporating mixed squaramide thioesters as phosphate bioisosteres was readily established by condensation of mixed squaramide esters with the corresponding thiols. Next, we characterised the inhibitory activity of these analogues in biological assays of axon growth cone chemotropism in cultured primary nociceptive neurons. These synthetic analogues induce both acute and sustained (more than 12 h) inhibition of the GPR55/LysoPtdGlc signaling. This inhibition resulted in sustained antagonistic attenuation of GPR55-mediated axon chemotropism while preserving growth cone sensitivity to other GPR55-independent chemotropic signaling molecules. Our findings demonstrate the potential of thiosquaramide-based phosphate bioisosteres as highly specific inhibitors with well controlled reactivity, expanding the repertoire of modulators for lipid-sensing GPCRs.