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Abstract: Protein–DNA interfaces are central to genome maintenance but remain challenging targets for small-molecule discovery because they are often broad, polar, solvent-exposed, and lack classical druggable pockets. Here, we establish a structure-guided strategy to chemically target such interfaces by combining the fragment-based identification of DNA-mimetic binders with the covalent capture of a proximal nucleophilic residue. Using the ssDNA-binding groove of RAD52 as a representative model system, an X-ray fragment screen identified carboxylate-containing fragments that recapitulate key phosphate backbone interactions of ssDNA within the conserved DNA-binding groove. Structural analysis revealed a recurrent 2:1 fragment-binding mode that enabled fragment linking and preorganization, yielding reversible inhibitors with nanomolar biochemical activity. Subsequent exploitation of C64, located adjacent to the bound ligand, converted this reversible scaffold into potent covalent inhibitors. Optimization led to the identification of FORX-864, a beyond-rule-of-5 covalent RAD52 chemical probe with low nanomolar ssDNA-displacement activity, efficient covalent target engagement, favorable in vitro properties, and submicromolar inhibition of RAD52-dependent single-strand annealing in cells. This work provides a chemical strategy for targeting DNA-binding proteins through DNA-mimetic fragment recognition, structure-guided preorganization, and covalent stabilization while delivering validated probes to interrogate RAD52 biology.

Open Access

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Abstract: The Fc region of immunoglobulin E (IgE-Fc) is an important therapeutic target due to the antibody’s critical role in allergic disorders through interactions with high-affinity (FcεRI) and low-affinity (CD23) receptors. IgE-Fc is known to be conformationally flexible, undergoing large-scale structural changes that modulate receptor binding and downstream biological functions. The mechanistic basis for how ligands induce these allosteric changes is not fully understood. In this study, we investigate structure–function relationships of IgE by characterizing four closely related anti-IgE-Fc Fab fragments with highly conserved sequences that recognize a shared epitope at the base of the Cε2 domain. Despite their sequence similarity, these Fabs exhibit markedly different binding characteristics, stoichiometries, and functional activities. Furthermore, the crystal structures of these four Fab complexes reveal that the bound IgE-Fc adopts a wide range of conformations. These structural differences directly dictate the functional outcomes, ranging from conformations that facilitate FcεRI binding to those that prevent or destabilize receptor interaction. Our findings demonstrate that highly similar antibodies binding to the same epitope on a protein that exhibits significant conformational plasticity can elicit distinct and divergent functional outcomes. This work challenges the conventional antibody discovery paradigm, i.e., “same epitope, same functional outcome,” and highlights the importance of accounting for both the target’s structural flexibility and the resulting functional diversity within antibody selection strategies. This is especially critical when targeting dynamic proteins where allosteric mechanisms are fundamental to function.

Open Access

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Abstract: Structure-guided exploitation of symmetry within multimeric protein interfaces offers an opportunity to convert partial ligand-binding poses into higher-affinity modulators. Here, we report a class of symmetrical “Z-shaped” allosteric activators of human liver pyruvate kinase (PKL), generated by symmetry-guided fusion of binding poses identified in inhibitor-bound crystal structures. The resulting ligands span the inter-subunit pocket at the tetrameric dimer–dimer interface and display substantially enhanced activity, with several analogues showing nanomolar activation potency. In HepG2 PKM2-knockout cells, the compounds increased PKL activity, maximal mitochondrial respiratory capacity and glycolytic proton efflux without altering PKL expression, Ser113 phosphorylation-dependent localisation, cell proliferation or triglyceride accumulation under the conditions examined. These findings establish symmetry-guided ligand fusion as a productive design strategy in this PKL system and provide structurally defined chemical tools for investigating the metabolic consequences of direct PKL activation.

Open Access

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Abstract: Odorant-binding proteins (OBPs) in mosquitoes play central roles in chemosensory perception and are attractive targets for vector control strategies. However, some OBPs are also expressed in non-olfactory tissues, suggesting a pleiotropic function that has not been sufficiently investigated. The crystal structures of the pleiotropic AgamOBP9 of the Anopheles gambiae mosquito were determined in complex with three plant-derived bioactive molecules: the phenylpropanoids n-butyl cinnamate and methyl eugenol (ME), as well as the monoterpene p-menthane-3, 8-diol (PMD). Fluorescence competitive binding assays and binding free-energy calculations identified that n-butyl cinnamate and ME bind with Ki values in the micromolar range, in contrast to the weak affinity observed for PMD. Structural analysis revealed an extended internal cavity comprising two distinct ligand-binding regions. One region, located at the bottom of the cavity, accommodated all investigated ligands without undergoing significant conformational changes, suggesting a structurally preordered binding pocket. The second region, situated at the cavity entrance, binds MPD and PEG, both used as crystallization agents, and may therefore serve as a recognition site for molecules with diverse chemical features. In the AgamOBP9–butyl cinnamate–MPD complex, MPD binding to the entrance-site induces rearrangements of surrounding residues, including Arg8, Arg15, Tyr32, Lys33, and Trp35. These interactions appear to promote the convergence of helices α1, α2, and the α2-α3 connecting loop toward a more “closed” protein conformation. The conformational flexibility of AgamOBP9 at the entrance region was further supported by differential scanning calorimetry and molecular dynamics simulations, which suggested the presence of two independent thermodynamic domains within the protein. Structural superposition with the homologous AaegOBP22–linoleic acid complex indicated that the AgamOBP9 cavity could accommodate fatty acids or other long-chain molecules spanning both binding regions, implying potential functions beyond conventional olfactory signaling. These findings provide new insights into the molecular basis of ligand recognition by AgamOBP9, which may guide the discovery of novel OBP9-targeting ligands.

Open Access

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Abstract: P450 BM3 is a natural fusion protein with a P450 domain fused to its necessary redox partner and capable of binding a range of fatty acids at high catalytic rates. The flexibility of the active site and catalytic efficiency have led to extensive research aimed at modifying substrate and product profiles for its utilization as a biocatalyst. The introduction of a double mutation (DM) at the base of the active site (A82F/F87V) allows the binding of a broad range of structurally diverse small molecules, including pharmaceutical drugs. Herein, we describe screening with an FDA-approved drug compound library, and the results exhibit the promiscuous nature of the DM BM3 variant. Of the 978 compounds screened; 59% of the library elicited UV–vis spectral shifts indicative of binding with a range of structurally diverse drugs. The interaction of the DM BM3 variant with a range of structurally diverse drugs, with a particular focus on the antidiabetic glitazone (thiazolidinedione) class, was investigated to observe ligand binding modes and metabolite production. BM3-derived metabolites were successfully produced and structurally elucidated for several structurally diverse drugs in the steroid, glitazone, meglitinide, terpene lactone, retinoid, and fibrate classes. The range of modifications performed includes some that are human P450-derived metabolites. Structural elucidation of the troglitazone-bound DM variant P450 domain shows an unusually close crystallographic interaction between the substrate and heme cofactor. We report here the use of the DM BM3 variant as a model for the main human metabolizing P450 enzymes CYP1A1, CYP2B6, CYP2C8, CYP2C9, and CYP3A4, as well as a biotechnological tool for the production of metabolites with diverse functionality.