1194 Citations
Gram-negative bacteria pose a threat to global healthcare mainly because their outer membrane OM provides an intrinsic barrier to many antimicrobials Key to this barrier function is the asymmetric structure of the OM with phospholipids constituting the inner leaflet and lipopolysaccharides the outer leaflet Although the mechanism of phospholipid transport between the inner membrane IM and OM remains poorly understood recent studies implicate TamB YhdP and YdbH as functionally redundant proteins mediating this process in Escherichia coli Accordingly collective loss of these three paralogs is lethal and any one of them is sufficient for growth YdbH is anchored to the ... More
Gram-negative bacteria pose a threat to global healthcare mainly because their outer membrane (OM) provides an intrinsic barrier to many antimicrobials. Key to this barrier function is the asymmetric structure of the OM, with phospholipids constituting the inner leaflet and lipopolysaccharides the outer leaflet. Although the mechanism of phospholipid transport between the inner membrane (IM) and OM remains poorly understood, recent studies implicate TamB, YhdP, and YdbH as functionally redundant proteins mediating this process in Escherichia coli. Accordingly, collective loss of these three paralogs is lethal and any one of them is sufficient for growth. YdbH is anchored to the IM and its periplasmic repeating β-sheet groove domain interacts with the OM lipoprotein YnbE via β-strand augmentation to form an intermembrane bridge. Additionally, YnbE multimerizes, and the periplasmic protein YdbL is proposed to modulate YnbE multimerization to facilitate its stacking on the C-terminus of YdbH. Here, we demonstrate that excess YdbL specifically inhibits the function of the YdbH-YnbE complex since overexpression of ydbL causes lethality in the ΔyhdP ΔtamB double mutant but the presence of both ydbH and ynbE in trans abrogates this lethality. We resolve high-resolution structural data for YdbL and ascertain its interaction site with the YnbE C-terminal α-helix, with residues mediating this interface highly conserved and critical for YdbL function. Finally, we show that YdbL is protected from degradation by the protease DegP when complexed with YnbE. Overall, our data supports a model in which YdbL ensures proper YdbH-YnbE intermembrane bridge formation by directly interacting with YnbE. Less
Interactions between plants and pathogens drive long-term co-evolution through cycles of effector diversification and immune recognition Effectors play central roles in this molecular interplay and zinc-binding folds have been identified in a subset of pathogen effectors yet the contribution of zinc coordination to effector stability and immune recognition remains unclear Here we investigated the structure and recognition of the AvrSr effector from the wheat stem rust fungus Puccinia graminis f sp tritici Pgt Structural and biochemical analyses show that AvrSr adopts a fold containing two zinc-binding sites Transient expression assays in Nicotiana benthamiana show that AvrSr directly interacts with Sr ... More
Interactions between plants and pathogens drive long-term co-evolution through cycles of effector diversification and immune recognition. Effectors play central roles in this molecular interplay, and zinc-binding folds have been identified in a subset of pathogen effectors, yet the contribution of zinc coordination to effector stability and immune recognition remains unclear.
Here, we investigated the structure and recognition of the AvrSr33 effector from the wheat stem rust fungus (Puccinia graminis f. sp. tritici, Pgt). Structural and biochemical analyses show that AvrSr33 adopts a fold containing two zinc-binding sites.
Transient expression assays in Nicotiana benthamiana show that AvrSr33 directly interacts with Sr33, and comparison of recognised and non-recognised AvrSr33 variants identifies a polymorphic loop associated with recognition. This loop is positioned adjacent to one of the zinc-binding sites with its orientation constrained by zinc coordination. Reciprocal mutations of key surface residues within this region alter recognition, whereas mutation of the zinc-binding site abolishes recognition.
Our data suggest that zinc coordination in AvrSr33 provides a structurally constrained scaffold that supports the surface features associated with Sr33 recognition. These findings provide a mechanistic framework for understanding how zinc coordination contributes to effector recognition and may influence the evolutionary trajectories of pathogen effectors. Less
Here, we investigated the structure and recognition of the AvrSr33 effector from the wheat stem rust fungus (Puccinia graminis f. sp. tritici, Pgt). Structural and biochemical analyses show that AvrSr33 adopts a fold containing two zinc-binding sites.
Transient expression assays in Nicotiana benthamiana show that AvrSr33 directly interacts with Sr33, and comparison of recognised and non-recognised AvrSr33 variants identifies a polymorphic loop associated with recognition. This loop is positioned adjacent to one of the zinc-binding sites with its orientation constrained by zinc coordination. Reciprocal mutations of key surface residues within this region alter recognition, whereas mutation of the zinc-binding site abolishes recognition.
Our data suggest that zinc coordination in AvrSr33 provides a structurally constrained scaffold that supports the surface features associated with Sr33 recognition. These findings provide a mechanistic framework for understanding how zinc coordination contributes to effector recognition and may influence the evolutionary trajectories of pathogen effectors. Less
Parasite-derived homologs of the cytokine macrophage migration inhibitory factor MIF function as virulent factors during parasitic infection Recent evidence suggests MIF-like products from multicellular helminthic species can be targeted to ameliorate parasite burden Here we identify a broad contingent of hypothetical MIF-like proteins from genomic helminth repositories and perform structure analysis to validate conserved homology Further we employ a diverse subset of MIF-specific assays to establish cross-species functionality of helminth MIF proteins including native enzymatic activity binding to cognate receptor CD direct interactions with human MIF and signaling through chemokine receptors CXCR and CXCR We demonstrate that MIF-like proteins retain ... More
Parasite-derived homologs of the cytokine macrophage migration inhibitory factor (MIF) function as virulent factors during parasitic infection. Recent evidence suggests MIF-like products from multicellular helminthic species can be targeted to ameliorate parasite burden. Here, we identify a broad contingent of hypothetical MIF-like proteins from genomic helminth repositories and perform structure analysis to validate conserved homology. Further, we employ a diverse subset of MIF-specific assays to establish cross-species functionality of helminth MIF proteins, including native enzymatic activity, binding to cognate receptor CD74, direct interactions with human MIF, and signaling through chemokine receptors CXCR2 and CXCR4. We demonstrate that MIF-like proteins retain a preserved architecture but are capable of diverse physiological outcomes due to small changes in key components of the conserved MIF structure. This work simultaneously provides a mechanistic understanding of helminth virulence upon infection, while broadly examining the potential to neutralize MIF-like proteins for protection against various pathological species. Less
Mycobacterium tuberculosis Mtb encodes a huge repertoire of toxin antitoxin TA systems many of which remain uncharacterized Here we report the crystal structures of the VapC toxin and VapBC TA complex at a resolution of and respectively We show that VapC adopts a unique open dimeric conformation and inhibits mycobacterial growth by cleaving tRNA at the variable or anticodon loop region Structure reveals that VapB adopts a distinct structural architecture and binds VapC with a stoichiometry respectively Interestingly binding of ssDNA activates VapB peptidase domain resulting in auto-cleavage of VapB N-terminal domain which is critical for VapBC complex formation and ... More
Mycobacterium tuberculosis (Mtb) encodes a huge repertoire of toxin–antitoxin (TA) systems, many of which remain uncharacterized. Here, we report the crystal structures of the VapC52 toxin and VapBC52 TA complex at a resolution of 2.6 and 3.2 Å, respectively. We show that VapC52 adopts a unique open dimeric conformation and inhibits mycobacterial growth by cleaving tRNA at the variable or anticodon loop region. Structure reveals that VapB52 adopts a distinct structural architecture and binds VapC52 with a 1:2 stoichiometry, respectively. Interestingly, binding of ssDNA activates VapB52 peptidase domain, resulting in auto-cleavage of VapB52 N-terminal domain which is critical for VapBC complex formation and neutralization. In addition to VapB52, co-expression of several other non-cognate VapB antitoxins abrogates the growth inhibition associated with VapC52 overexpression in Mycobacterium smegmatis (Msm) suggesting crosstalk among VapBC TA systems. Further, we demonstrate that the vapBC52 locus is dispensable for in vitro growth but essential for Mtb intracellular growth in macrophages and guinea pigs. Notably, VapC52 also cleaves mycobacteriophage D29 encoded tRNAs and confers resistance to phage infection in Msm. Taken together, we show that VapBC52 adopts a unique structural architecture, plays role in pathogenesis, and is possibly involved in mycobacterial antiphage defense mechanisms. Less
Pyrophosphate-dependent phosphofructokinases provide an alternative route through central carbon metabolism by using inorganic pyrophosphate rather than ATP to phosphorylate fructose- -phosphate These enzymes are absent from mammals but occur in several anaerobic and parasitic organisms where they may support metabolic flexibility and represent potential therapeutic vulnerabilities Here we report three crystal structures of Trichomonas vaginalis PPi-dependent phosphofructokinase TvPPi-PFK a cytosolic enzyme that contributes substantially to parasite glycolytic flux The structures show that TvPPi-PFK adopts the conserved two-domain PFK fold assembles as a tetramer and preserves the canonical PPi MgPPi -binding motifs required for pyrophosphate-dependent catalysis Mass photometry confirms that TvPPi-PFK ... More
Pyrophosphate-dependent phosphofructokinases provide an alternative route through central carbon metabolism by using inorganic pyrophosphate rather than ATP to phosphorylate fructose-6-phosphate. These enzymes are absent from mammals but occur in several anaerobic and parasitic organisms, where they may support metabolic flexibility and represent potential therapeutic vulnerabilities. Here, we report three crystal structures of Trichomonas vaginalis PPi-dependent phosphofructokinase 1 (TvPPi-PFK), a cytosolic enzyme that contributes substantially to parasite glycolytic flux. The structures show that TvPPi-PFK adopts the conserved two-domain PFK fold, assembles as a tetramer, and preserves the canonical PPi/MgPPi²⁻-binding motifs required for pyrophosphate-dependent catalysis. Mass photometry confirms that TvPPi-PFK is predominantly tetrameric in solution and shows that nucleotide- and Mg²⁺/PPi-containing conditions further stabilize the tetrameric assembly. Unexpectedly, the ligand-bound structures reveal ATP, AMP, and sugar-phosphate ligands at a recurring dimer-interface site that is spatially distinct from the canonical PPi/MgPPi²⁻ catalytic donor-binding pocket. This non-canonical interface is more accessible in TvPPi-PFK than in available PPi-PFK structures, suggesting a previously unrecognized ligand-recognition feature. These findings expand the structural landscape of the PFK superfamily and provide a framework for testing how nucleotide and sugar-phosphate binding may influence TvPPi-PFK function, regulation, and future antitrichomonal drug-discovery efforts. Less
Chemical topology has emerged as a unique dimension in protein engineering motivating the pursuit of topologically nontrivial protein architectures for functional advantages such as enhanced stability and rich dynamics However the structural diversity of artificial mechanically interlocked proteins remains limited Here we report the computational design and cellular synthesis of a pair of topological isomers via symmetric assembly of orthogonal entangling motifs By fusing two C symmetric entangling motifs i e p dim and HP in specific arrangements we programmed the formation of multiple crossings which upon cyclization yielded a protein Solomon link and a protein three-twist knot The fusion ... More
Chemical topology has emerged as a unique dimension in protein engineering, motivating the pursuit of topologically nontrivial protein architectures for functional advantages, such as enhanced stability and rich dynamics. However, the structural diversity of artificial mechanically interlocked proteins remains limited. Here, we report the computational design and cellular synthesis of a pair of topological isomers via symmetric assembly of orthogonal entangling motifs. By fusing two C2 symmetric entangling motifs, i.e., p53dim and HP0242, in specific arrangements, we programmed the formation of multiple crossings, which upon cyclization yielded a protein Solomon link and a protein three-twist knot. The fusion patterns and linker lengths were systematically optimized to direct the formation of the intended topologies. Their successful cellular synthesis was validated through biophysical and structural analyses, including sodium dodecyl sulfate-polyacrylamide gel electrophoresis, size exclusion chromatography, and liquid chromatography-mass spectrometry. Notably, we report the crystal structure of an artificial protein three-twist knot. Both the Solomon link and the three-twist knot displayed increased structural compactness and stability relative to their controls with lower topological complexity (e.g., Hopf link, trefoil knot, and linear forms), as evidenced by their superior thermal stability and resistance to chemical denaturation. This modular design strategy provides a rational and extensible route to diverse mechanically interlocked proteins and could be generalized to access even more complex architectures, such as protein chainmail-like nanocages and woven protein frameworks. Less
EV-A has been responsible for recent severe HFMD outbreaks We report structures for potently neutralizing human anti EV-A monoclonal antibody Fabs alone and complexed with virus Most recognize the native antigenic state with epitopes that span interfaces together covering of the capsid surface The majority of bind the canyon while the others cluster around the icosahedral two- and threefold axes Blocking SCARB receptor binding likely contributes to neutralization for all and a subset induces empty particles A predominant gene family IGHV - does not dictate a common binding pose Long CDR-H loops are frequently key to binding especially at the ... More
EV-A71 has been responsible for recent severe HFMD outbreaks. We report structures for 12 potently neutralizing human anti–EV-A71 monoclonal antibody Fabs, alone and complexed with virus. Most recognize the native antigenic state with epitopes that span interfaces, together covering 85% of the capsid surface. The majority (8 of 12) bind the canyon, while the others cluster around the icosahedral two- and threefold axes. Blocking SCARB2 receptor binding likely contributes to neutralization for all, and a subset induces empty particles. A predominant gene family (IGHV4-39) does not dictate a common binding pose. Long CDR-H3 loops are frequently key to binding, especially at the canyon, suggesting that antigenicity data based on antibodies with shorter CDR3s (e.g., murine) may be misleading. This dataset reveals neutralization mechanisms for recently circulating EV-A71 genotypes, which will inform immunotherapies. We demonstrate synergy in vitro between canyon binding and both two- and threefold binding antibodies to increase neutralization potency. Less
Ribosome stalling caused by polyproline PPs motifs is common Their translation is enhanced by accessory proteins such as YebC in bacteria whose homolog TRANSLATIONAL ACTIVATOR OF CYTOCHROME C OXIDASE TACO aids the translation of mitochondria-encoded proteins The prevalence of PP motifs across plastid-encoded genes and their impact on the translation of photosynthesis-relevant proteins remains unexplored Equally a translation-enhancer of PP motifs equivalent to TACO for plastid ribosomes has not been reported Here we show that plastid genomes encode proteins with a minimum of one PP motif on average half of which are conserved in their cyanobacterial homologs and that the ... More
Ribosome stalling caused by polyproline (PPs) motifs is common. Their translation is enhanced by accessory proteins such as YebC in bacteria, whose homolog, TRANSLATIONAL ACTIVATOR OF CYTOCHROME C OXIDASE 1 (TACO1), aids the translation of mitochondria-encoded proteins. The prevalence of PP motifs across plastid-encoded genes and their impact on the translation of photosynthesis-relevant proteins remains unexplored. Equally, a translation-enhancer of PP motifs equivalent to TACO1 for plastid ribosomes has not been reported. Here, we show that plastid genomes encode 24 proteins with a minimum of one PP motif on average, half of which are conserved in their cyanobacterial homologs, and that the vast majority of eukaryotes, including plants, encode a single TACO1 that we demonstrate to be dually targeted to mitochondria and plastids of Marchantia polymorpha. We resolved the MpTACO1 structure at 2.34 Å by X-ray crystallography and the flexibility by small-angle X-ray scattering. Through modelling, we demonstrate that MpTACO1 can fit into the peptidyl transfer centre of plant chlororibosomes in a similar manner as human TACO1 in the mitoribosome. The identification and structure determination of the first plastid-targeted YebC/TACO1 allows us to sketch a unified model for the function and evolution of this ancient family of ribosomal accessory proteins, underscoring their indispensable role in the translation of bioenergetic membrane proteins reaching back almost 4 billion years. Less
Structural biology has fundamentally influenced pharmaceutical research and development at Merck Sharp Dohme LLC Rahway New Jersey USA progressing from pioneering macromolecular crystallography in the s to a fully integrated platform that today encompasses X-ray crystallography cryo-electron microscopy micro-electron diffraction and cryo-electron tomography In this review we present a comprehensive overview of how these complementary methods have advanced drug discovery across diverse therapeutic areas We illustrate how atomic to cellular structural insights inform drug discovery and development from target identification and validation hit finding lead identification through lead optimization and clinical progression Furthermore we describe how structural biology techniques aid ... More
Structural biology has fundamentally influenced pharmaceutical research and development at Merck Sharp & Dohme LLC, Rahway, New Jersey, USA, progressing from pioneering macromolecular crystallography in the 1980s to a fully integrated platform that today encompasses X-ray crystallography, cryo-electron microscopy, micro-electron diffraction and cryo-electron tomography. In this review, we present a comprehensive overview of how these complementary methods have advanced drug discovery across diverse therapeutic areas. We illustrate how atomic to cellular structural insights inform drug discovery and development, from target identification and validation, hit finding, lead identification through lead optimization and clinical progression. Furthermore, we describe how structural biology techniques aid in formulation strategies of antibodies and vaccines. Finally, we highlight the growing integration of ex situ and in situ approaches as a paradigm shift towards elucidating drug mechanisms in native cellular contexts, a transition poised to accelerate the discovery and development of next-generation therapeutics. Less
Linking of fragments in neighboring binding sites is one of the optimization strategies in fragment-based drug discovery where additive or even more substantial bioactivity improvements can be realized However such efforts present a considerable challenge when one fragment binds covalently to the target protein as small modifications can influence the correct positioning of the covalent warhead toward the targeted nucleophilic residue Here we present a case study of fragment linking that yielded single-digit micromolar covalent inhibitors of the SARS-CoV- main protease starting from fragments that were inactive in the biochemical assay Using structural information from a recent high-throughput crystallographic fragment ... More
Linking of fragments in neighboring binding sites is one of the optimization strategies in fragment-based drug discovery, where additive or even more substantial bioactivity improvements can be realized. However, such efforts present a considerable challenge when one fragment binds covalently to the target protein, as small modifications can influence the correct positioning of the covalent warhead toward the targeted nucleophilic residue. Here, we present a case study of fragment linking that yielded single-digit micromolar, covalent inhibitors of the SARS-CoV-2 main protease, starting from fragments that were inactive in the biochemical assay. Using structural information from a recent, high-throughput crystallographic fragment screen, we show that the success of fragment linking in the design of targeted covalent inhibitors is heavily impacted by several factors, including the warhead type, the labeling chemistry, and even subtle changes in the designed linker. Notably, we observe that induced fit effects might override the original fragment orientations in the linked molecule, highlighting the need for reliable structure verification, especially in consecutive rounds of fragment elaboration. Less
To establish infection phytopathogens deploy effectors to compromise host defences and facilitate invasive growth As part of this the battle for control of symplastic connectivity via plasmodesmata is a key determinant of infection outcomes yet little is known about how fungal effectors directly exploit these channels and in turn how hosts defend them Here we have identified ChEC as a plasmodesmal-targeting cell-to-cell mobile effector from the anthracnose fungus Colletotrichum higginsianum ChEC binds the plasmodesmal protein HEAVY METAL-ASSOCIATED HMA ISOPRENYLATED PLANT PROTEIN HIPP from Arabidopsis via a tetrahedral metal ion coordination site with either of its HMA domains Constitutive in planta ... More
To establish infection, phytopathogens deploy effectors to compromise host defences and facilitate invasive growth. As part of this, the battle for control of symplastic connectivity via plasmodesmata is a key determinant of infection outcomes, yet little is known about how fungal effectors directly exploit these channels, and in turn, how hosts defend them. Here, we have identified ChEC108 as a plasmodesmal-targeting, cell-to-cell mobile effector from the anthracnose fungus, Colletotrichum higginsianum. ChEC108 binds the plasmodesmal protein HEAVY METAL-ASSOCIATED (HMA) ISOPRENYLATED PLANT PROTEIN 6 (HIPP6) from Arabidopsis via a tetrahedral metal ion coordination site with either of its HMA domains. Constitutive in planta expression of ChEC108 induces plasmodesmal closure and the upregulation of defence-associated genes in a manner dependent on its capacity to bind HIPP6. Further, HIPP6 binding impairs cell-to-cell mobility of ChEC108. Alongside the finding that loss of ChEC108 favoured C. higginsianum infection, this suggests ChEC108-HIPP6 interaction at plasmodesmata positively regulates defence. Less
Visualizing and manipulating proteins in live cells is crucial for studying complex biological processes Self-labelling protein SLP tags such as HaloTag and SNAP-tag are widely used for protein labelling and new systems are needed to expand multiplexing capabilities and broaden the scope of applications Here we present BromoCatch a small kDa bromodomain BD -based SLP platform engineered with a nucleophilic cysteine for covalent ligand engagement A structure-based designed library of electrophilic ligands was screened against two cysteine-containing mutants using differential scanning fluorimetry and intact protein mass spectrometry to assess covalent complex formation We identified a para-acrylamide bumped derivative MR and ... More
Visualizing and manipulating proteins in live cells is crucial for studying complex biological processes. Self-labelling protein (SLP) tags such as HaloTag and SNAP-tag are widely used for protein labelling, and new systems are needed to expand multiplexing capabilities and broaden the scope of applications. Here we present BromoCatch, a small ~13 kDa bromodomain (BD)-based SLP platform, engineered with a nucleophilic cysteine for covalent ligand engagement. A structure-based designed library of electrophilic ligands was screened against two cysteine-containing mutants using differential scanning fluorimetry and intact protein mass spectrometry to assess covalent complex formation. We identified a para-acrylamide bumped derivative MR116 and the Brd4-BD2 double mutant L387A,E438C as the optimal protein-ligand pair, and reveal the binding mode through an X-ray co-crystal structure solved to 1.3 Å resolution. BromoCatch demonstrated potent and irreversible cellular target engagement in NanoBRET and residence-time assays. Its versatility was demonstrated through the design of a biotinylated conjugate, PROTAC-based degraders, and fluorescent full-on and “switch-on” probes for ex-cellulo and live-cell imaging, including side-by-side comparison and orthogonality with HaloTag. Together, these results establish BromoCatch as a robust, modular, and orthogonal SLP tool with broad potential for multiplexed labelling and targeted protein manipulation. Less
Background and objectives The PWWP domain of lens epithelium-derived growth factor p LEDGF p mediates chromatin engagement through recognition of histone H lysine di- and trimethylation H K me and nucleosomal DNA LEDGF p plays a role in multiple human diseases In particular its interaction with HIV- integrase enables viral genome integration However the LEDGF PWWP domain remains difficult to target with small molecules as it lacks optimally shaped binding pockets Here we report the generation of high-affinity nanobodies Nbs to investigate the structure and function of this domain Methods Camelids were immunized with recombinant LEDGF PWWP domain and immune ... More
Background and objectives: The PWWP domain of lens epithelium-derived growth factor p75 (LEDGF/p75) mediates chromatin engagement through recognition of histone H3 lysine 36 di- and trimethylation (H3K36me2/3) and nucleosomal DNA. LEDGF/p75 plays a role in multiple human diseases. In particular, its interaction with HIV-1 integrase enables viral genome integration. However, the LEDGF PWWP domain remains difficult to target with small molecules as it lacks optimally shaped binding pockets. Here we report the generation of high-affinity nanobodies (Nbs) to investigate the structure and function of this domain. Methods: Camelids were immunized with recombinant LEDGF PWWP domain, and immune phage display libraries were screened for affinity. Selected Nbs were recombinantly expressed in E. coli and purified. Their interaction with the PWWP domain of LEDGF and its close homolog HRP-2 was characterized using size-exclusion chromatography and surface plasmon resonance. Structural characterization of the Nbs was performed by X-ray crystallography. Functional effects on chromatin engagement were evaluated using the AlphaScreen assay. Results: Nine sequence-distinct Nbs were identified, seven of which were confirmed to bind the LEDGF PWWP domain with nanomolar affinities. Five Nbs also bound the HRP-2 domain, consistent with conserved functional surfaces, while two showed reduced affinity. Crystal structures of two Nbs (NbC03 and NbH10) confirmed canonical immunoglobulin folds, while the latter additionally revealed a domain-swapped dimer. Moreover, NbH10 dose-dependently inhibited the interaction between full-length LEDGF/p75 and H3K36me3-modified nucleosomes in vitro. Conclusions: This work establishes a validated panel of Nbs targeting the LEDGF PWWP domain and demonstrates their ability to functionally disrupt the LEDGF-chromatin interaction. These Nbs serve as valuable tools towards functional studies and structure-based drug design. Less
TL A is a proinflammatory cytokine in the tumor necrosis factor TNF superfamily that signals via DR on T helper cells innate lymphoid cells and fibroblasts Dysregulated TL A signaling has been hypothesized to affect multiple immune-mediated diseases with clinical proof of concept demonstrated in ulcerative colitis and Crohn s disease We characterized the binding affinity specificity structure pharmacodynamics pharmacokinetics and toxicity profiles of SPY and SPY two novel extended half-life monoclonal antibodies that inhibit TL A SPY and SPY demonstrated selective high-affinity binding to human TL A KD pM and potent functional inhibition of DR signaling Based on Fc ... More
TL1A is a proinflammatory cytokine in the tumor necrosis factor (TNF) superfamily that signals via DR3 on T helper cells, innate lymphoid cells, and fibroblasts. Dysregulated TL1A signaling has been hypothesized to affect multiple immune-mediated diseases, with clinical proof of concept demonstrated in ulcerative colitis and Crohn’s disease. We characterized the binding affinity, specificity, structure, pharmacodynamics, pharmacokinetics, and toxicity profiles of SPY002 and SPY072, two novel extended half-life monoclonal antibodies that inhibit TL1A. SPY002 and SPY072 demonstrated selective, high-affinity binding to human TL1A (KD ≈ 31–35 pM) and potent functional inhibition of DR3 signaling. Based on Fc modifications, SPY002 and SPY072 showed attenuated Fc effector function and increased FcRn binding at acidic pH (5.8). Both antibodies exhibited enhanced PK profiles in nonhuman primates, resulting in predicted human half-lives that support quarterly or biannual dosing. In toxicity studies, no drug-related adverse effects were observed with either antibody at exposures >10 times those anticipated in clinical trials. In a rat collagen-induced arthritis model, anti-TL1A antibody treatment effectively reduced arthritis severity, with similar efficacy to the TNF antagonist etanercept. In humanized mouse Imiquimod-induced psoriasis and 2,4,6‑trinitrobenzene sulfonic acid colitis models, anti-TL1A demonstrated similar efficacy to anti–IL-23 and anti-TNF antibodies. These findings characterize two novel extended half-life TL1A antibodies and support the ongoing Phase 2 clinical development of SPY002 and SPY072 for immune-mediated diseases such as inflammatory bowel disease and rheumatic diseases. Less
Melanin binding of small molecule drugs can lead to targeted disposition to the pigmented tissues and prolonged pharmacological responses in the eye Melanin binding of drugs in vitro correlates with in vivo binding but current workflows for binding affinity require multiple slow steps and analytical method development and or they may result in high data variability We developed tangential flow filtration-based methodology to produce size-specific fractions of water-soluble melanin nanoparticles MNPs reducing production time from - days to just a few hours and yielding MNPs with enhanced fluorescence signal Improved MNPs enabled modifications to a previously published microscale thermophoresis-based melanin ... More
Melanin binding of small molecule drugs can lead to targeted disposition to the pigmented tissues and prolonged pharmacological responses in the eye. Melanin binding of drugs in vitro correlates with in vivo binding, but current workflows for binding affinity require multiple slow steps, and analytical method development, and/or they may result in high data variability. We developed tangential flow filtration-based methodology to produce size-specific fractions of water-soluble melanin nanoparticles (MNPs), reducing production time from 2-3 days to just a few hours and yielding MNPs with enhanced fluorescence signal. Improved MNPs enabled modifications to a previously published microscale thermophoresis-based melanin binding protocol, shifting analytical focus toward thermophoretic behavior, reducing data variability, and improving reproducibility. The process was tested with nine compounds with varying melanin binding affinities, and the results were consistent with literature, confirming the ability of the method to differentiate compounds based on melanin binding. Fast and reliable workflow will be useful in screening binding affinity for therapeutics and new drug candidates to melanin thereby facilitating ocular drug discovery and construction of predictive pharmacokinetic simulation models. Less
Background Calcium-dependent protein kinase CDPK has emerged as a protozoan specific target for the treatment of cryptosporidiosis A previous study identified pyridopyrimidinones as new Cryptosporidium parvum Cp CDPK inhibitors with potent growth inhibition against C parvum and C hominis Docking analyses suggested the unique positioning of the kinase s Chelix could present refinement opportunities Methods Compounds designed to optimize the pyridopyrimidinones focused on the back-pocket region predicted to be proximal to the C-helix the solvent exposed region and the ATP ribose-binding site Designed derivatives were synthesized and assessed for CpCDPK and Src kinase inhibition and for Cryptosporidium spp growth inhibition ... More
Background: Calcium-dependent protein kinase 1 (CDPK1) has emerged as a protozoan specific
target for the treatment of cryptosporidiosis. A previous study identified pyridopyrimidinones as
new Cryptosporidium parvum (Cp) CDPK1 inhibitors with potent growth inhibition against C.
parvum and C. hominis. Docking analyses suggested the unique positioning of the kinase’s αChelix could present refinement opportunities.
Methods: Compounds designed to optimize the pyridopyrimidinones focused on the back-pocket region predicted to be proximal to the αC-helix, the solvent exposed region and the ATP ribose-binding site. Designed derivatives were synthesized and assessed for CpCDPK1 and Src kinase inhibition and for Cryptosporidium spp., growth inhibition in mammalian cells. AMP/Mg+2 and three inhibitors were co-crystalized with CpCDPK1, and two inhibitors were profiled for kinase selectivity.
Results: WIN 4-88 was identified with CpCDPK1 (IC50 = 0.056 μM), and growth inhibition of zoonotic C. parvum (NLuc EC50 = 0.042 μM), anthroponotic C. parvum (Tu114 EC50 = 0.030 μM), and C. hominis Tu502 (EC50 = 0.062 μM), as well as enhanced kinome selectivity. The crystal structures confirmed the predicted binding mode, indicating key interactions with hinge residue Y155, similar orientations of the solvent expose moieties, occupancy of the back-pocket near the αC-helix and for one inhibitor containing a solubilizing hydroxyethyl attached to the central heterocycle extension into the ATP ribose-binding site.
Conclusions: The expanded structure-activity relationship and structural insights will potentially be applicable to other chemotypes with similar binding modes and will enhance development of CpCDPK1 inhibitors for the treatment of cryptosporidiosis. Less
target for the treatment of cryptosporidiosis. A previous study identified pyridopyrimidinones as
new Cryptosporidium parvum (Cp) CDPK1 inhibitors with potent growth inhibition against C.
parvum and C. hominis. Docking analyses suggested the unique positioning of the kinase’s αChelix could present refinement opportunities.
Methods: Compounds designed to optimize the pyridopyrimidinones focused on the back-pocket region predicted to be proximal to the αC-helix, the solvent exposed region and the ATP ribose-binding site. Designed derivatives were synthesized and assessed for CpCDPK1 and Src kinase inhibition and for Cryptosporidium spp., growth inhibition in mammalian cells. AMP/Mg+2 and three inhibitors were co-crystalized with CpCDPK1, and two inhibitors were profiled for kinase selectivity.
Results: WIN 4-88 was identified with CpCDPK1 (IC50 = 0.056 μM), and growth inhibition of zoonotic C. parvum (NLuc EC50 = 0.042 μM), anthroponotic C. parvum (Tu114 EC50 = 0.030 μM), and C. hominis Tu502 (EC50 = 0.062 μM), as well as enhanced kinome selectivity. The crystal structures confirmed the predicted binding mode, indicating key interactions with hinge residue Y155, similar orientations of the solvent expose moieties, occupancy of the back-pocket near the αC-helix and for one inhibitor containing a solubilizing hydroxyethyl attached to the central heterocycle extension into the ATP ribose-binding site.
Conclusions: The expanded structure-activity relationship and structural insights will potentially be applicable to other chemotypes with similar binding modes and will enhance development of CpCDPK1 inhibitors for the treatment of cryptosporidiosis. Less
Insomnia is a widespread sleep disorder that significantly impairs quality of life and imposes a societal burden Although benzodiazepines and Z-drugs are commonly used for treating insomnia these drugs often have side effects such as excessive muscle relaxation and dependency The orexin signaling pathway has emerged as a promising therapeutic target for insomnia with dual orexin receptor antagonists DORAs offering an alternative approach to treatment To reduce the potential of these drugs for next-day residual effects we developed vornorexant a novel DORA with a high receptor affinity and a short elimination half-life In this study we investigated the molecular interactions ... More
Insomnia is a widespread sleep disorder that significantly impairs quality of life and imposes a societal burden. Although benzodiazepines and Z-drugs are commonly used for treating insomnia, these drugs often have side effects, such as excessive muscle relaxation and dependency. The orexin signaling pathway has emerged as a promising therapeutic target for insomnia, with dual orexin receptor antagonists (DORAs) offering an alternative approach to treatment. To reduce the potential of these drugs for next-day residual effects, we developed vornorexant, a novel DORA with a high receptor affinity and a short elimination half-life. In this study, we investigated the molecular interactions of this drug with human orexin receptors through crystal structure analysis of its binding to orexin type 2 receptor (OX2R) and a docking simulation of the drug with orexin type 1 receptor (OX1R). The crystal structure of the OX2R–vornorexant complex revealed a conserved U-shaped conformation stabilized by hydrophobic and hydrogen-bonding interactions, including key contacts with Asn324, His350 and Pro131. OX1R–vornorexant docking simulations indicated a similar binding mode to OX1R, with no steric hindrance observed, supporting a balanced dual antagonism. These results provide a structural basis for the high-affinity dual antagonism of vornorexant and offer insights for the design of orexin receptor antagonists. Less
SummaryBACH is a transcriptional regulator that modulates various cytoprotective pathways Among these pathways BACH regulates the cellular oxidative stress responses by suppressing the expression of cytoprotective genes Dysregulated BACH activity has been implicated in a range of pathologies including chronic inflammatory diseases fibrosis and cancer making it a promising therapeutic target However BACH remains an underexploited drug target with limited pharmacological inhibitors available We have developed a novel luciferase-based reporter cell line enabling quantitative high-throughput assessment of BACH inhibition Using this platform we rigorously screened two small-molecule libraries with compounds and identified four structurally distinct compounds that robustly inhibit BACH ... More
SummaryBACH1 is a transcriptional regulator that modulates various cytoprotective pathways. Among these pathways BACH1 regulates the cellular oxidative stress responses by suppressing the expression of cytoprotective genes. Dysregulated BACH1 activity has been implicated in a range of pathologies, including chronic inflammatory diseases, fibrosis, and cancer, making it a promising therapeutic target. However, BACH1 remains an underexploited drug target, with limited pharmacological inhibitors available. We have developed a novel luciferase-based reporter cell line enabling quantitative, high-throughput assessment of BACH1 inhibition. Using this platform, we rigorously screened two small-molecule libraries with 2,046 compounds and identified four structurally distinct compounds that robustly inhibit BACH1 function. Notably, these compounds simultaneously activate transcription factor NRF2, suggesting the potential for a broader modulation of oxidative stress pathways.Importantly, we demonstrate that commonly used 2D migration assays may fail to detect phenotypes consistent with BACH1 inhibition, resulting in false negatives. In contrast, we establish that 3D invasion assays more robustly capture anti-invasive effects of BACH1 functional inhibition. Using this 3D system, we validate the identified compounds as potent suppressors of lung cancer cell invasion in vitro.This study delivers a novel screening platform for BACH1-targeted drug discovery, and challenges current in vitro standards by establishing 3D invasion assays as a more accurate functional readout for BACH1-targeting compounds. Additionally, it identifies new dual functional BACH1 inhibitors/NRF2 activators, offering novel chemical scaffolds for the development of anti-metastatic therapies and potentially treatments for diseases driven by oxidative stress and inflammation. Less
Conversion of prothrombin to thrombin occurs in the final step of the blood coagulation cascade and depends on association of the serine protease factor f Xa and the cofactor fVa on activated cell surfaces to form the prothrombinase complex Prothrombinase cleaves prothrombin at two sites in a processive manner -times faster than fXa on its own How fVa confers rapid and processive cleavage of prothrombin is an enzymatical mystery with profound consequence We created a variant of fXa that binds to fVa with high affinity in the absence of phospholipids that preserves the activity of wild-type prothrombinase and recently reported ... More
Conversion of prothrombin to thrombin occurs in the final step of the blood coagulation cascade and depends on association of the serine protease, factor (f) Xa, and the cofactor fVa on activated cell surfaces to form the prothrombinase complex. Prothrombinase cleaves prothrombin at two sites in a processive manner ∼500,000-times faster than fXa on its own. How fVa confers rapid and processive cleavage of prothrombin is an enzymatical mystery with profound consequence. We created a variant of fXa that binds to fVa with high affinity in the absence of phospholipids that preserves the activity of wild-type prothrombinase, and recently reported on the cryo-EM structure of the complex. It revealed an extensive interface between the two proteins, including a critical interaction between the first acidic region C-terminal to the A2 domain of fVa (the N-terminal portion of the a2-loop) with the heparin binding site of fXa. Here we present the cryo-EM structures of prothrombinase bound to prothrombin and the intermediate meizothrombin, both to 3.1 Å resolution. The prothrombin complex revealed a surprising interaction between the second acidic region of the a2-loop with exosite I of prothrombin, accounting for 70% of the total buried surface area. Cleavage at Arg320 triggers the zymogen-to-protease conformational change in meizothrombin which alters all domain-domain and fVa interactions, and results in the presentation of the second cleavage site (Arg271) for processing. Together, these structures reveal a remarkable enzymatic mechanism that depends on the active participation of the substrate itself, and introduce the new paradigm of substrate allostery. Less
The precise and selective transport of protons across cellular membranes relies on the dynamic formation and dissipation of hydrogen-bonding networks involving water molecules protein sidechains and backbone carbonyls As in aqueous solution protons are conducted over long distances along chains of hydrogen-bonded water molecules within narrow protein pores To engineer proton-conductive pathways therefore we must explicitly account for the dynamic behavior of these networks In previous work we showed that incorporation of polar Gln residues into hydrophobic pores drives formation of transient single-file water wires that enable proton-selective transport Here we sought to enhance conduction by introducing targeted Ile-to-Ser substitutions ... More
The precise and selective transport of protons across cellular membranes relies on the dynamic formation and dissipation of hydrogen-bonding networks involving water molecules, protein sidechains, and backbone carbonyls. As in aqueous solution, protons are conducted over long distances along chains of hydrogen-bonded water molecules within narrow protein pores. To engineer proton-conductive pathways, therefore, we must explicitly account for the dynamic behavior of these networks. In previous work, we showed that incorporation of polar Gln residues into hydrophobic pores drives formation of transient, single-file water wires that enable proton-selective transport. Here, we sought to enhance conduction by introducing targeted Ile-to-Ser substitutions to extend connectivity across the pore. We find that the position of Ser relative to Gln modulates sidechain dynamics and, in turn, channel hydration. Although increased polarity reduces hydrophobic length and enhances hydration, these effects alone do not explain the observed conduction rates. Instead, asymmetry in the arrangement and dynamics of polar sidechains emerges as a key determinant of proton conductivity. Together, these results demonstrate that proton conduction is governed not only by pore polarity and hydration, but also by the dynamic and asymmetric organization of hydrogen-bonding networks. This work establishes design principles for engineering proton-selective channels and reveals how asymmetry enables efficient proton transport across biological membranes. Less
The evolution of transcription factor TF DNA-binding specificity is a major driver of gene regulatory innovation Unlike most TFs which diversify through gene duplication and neofunctionalization the plant-specific LEAFY LFY TF evolved novel binding specificities without extensive duplication Here we combine experimental structural determination and biochemical assays to reveal how LFY s dimerization and DNA-binding preferences shifted during the water-to-land transition We present crystal structures of the LFY DNA-binding domain DBD from the hornwort Nothoceros aenigmaticus and the alga Interfilum paradoxum bound to DNA demonstrating two distinct dimerization mechanisms one mediated by direct protein-protein interactions and another driven by DNA-mediated ... More
The evolution of transcription factor (TF) DNA-binding specificity is a major driver of gene regulatory innovation. Unlike most TFs, which diversify through gene duplication and neofunctionalization, the plant-specific LEAFY (LFY) TF evolved novel binding specificities without extensive duplication. Here, we combine experimental structural determination and biochemical assays to reveal how LFY’s dimerization and DNA-binding preferences shifted during the water-to-land transition. We present crystal structures of the LFY DNA-binding domain (DBD) from the hornwort Nothoceros aenigmaticus and the alga Interfilum paradoxum bound to DNA, demonstrating two distinct dimerization mechanisms: one mediated by direct protein-protein interactions and another driven by DNA-mediated cooperativity. In the ancestral state, LFY likely bound DNA as a dimer through DNA-mediated cooperativity, with protein-protein dimerization emerging later, enforcing new DNA-binding preferences. Our findings support a revised evolutionary scenario for LFY, highlighting the dynamic interplay between protein-DNA and protein-protein interactions as key drivers of TF binding specificity. This work deepens our understanding of how structural adaptations in TFs underpin evolutionary transitions in gene regulation. Less
The richness of our somatosensory experience is reflected in the functional diversity of somatic sensory neurons Single-cell RNA sequencing of sensory neurons has revealed a molecular basis for such diversity However sensory neuron diversity has yet to be captured at the level of the proteome Here we combined electrophysiology with deep visual proteomics to quantify over proteins from phenotypically-defined sensory neurons in mice and identified proteomic markers of sensory neuron subtypes Comparative analysis revealed both concordance and meaningful divergence between transcriptomes and proteomes We further show that up to proteins can be quantified from one-fourth of a single neuron demonstrating ... More
The richness of our somatosensory experience is reflected in the functional diversity of somatic sensory neurons. Single-cell RNA sequencing of sensory neurons has revealed a molecular basis for such diversity1,2,3. However, sensory neuron diversity has yet to be captured at the level of the proteome. Here, we combined electrophysiology with deep visual proteomics 4 to quantify over 6000 proteins from phenotypically-defined sensory neurons in mice and identified proteomic markers of sensory neuron subtypes. Comparative analysis revealed both concordance and meaningful divergence between transcriptomes and proteomes. We further show that up to 3000 proteins can be quantified from one-fourth of a single neuron, demonstrating subset-specific protein signatures. In culture, nociceptive neurons can be acutely sensitized to mechanical stimuli by nerve growth factor (NGF) which normally drives inflammatory pain in vivo5. Indeed, overnight exposure of peptidergic nociceptors to NGF and a protein kinase C (PKC) activator produced functional sensitization associated with proteome changes. Functional knockdown experiments identified the up-regulated B3GNT2 enzyme as a potential effector of nociceptor sensitization. In summary, we present a high-resolution proteomic resource linking molecular identity to function, enabling the discovery of mechanisms underlying somatic sensation and pain sensitization. Less
Constant changes in SARS-CoV- in human populations as well as potential future spillovers from animal coronaviruses have provided the impetus for the development of additional direct-acting antivirals We describe herein the discovery of a new class of broad-spectrum inhibitors of coronavirus C-like protease CLpro a cysteine protease essential for viral replication and a validated drug target that incorporate in their structure a -oxazaphospholidin- -one scaffold Inhibitors and were found to have EC values of and nM against SARS-CoV- CLpro respectively and CC values M These compounds also potently inhibited MERS-CoV CLpro IC nM and nM respectively Importantly several of the ... More
Constant changes in SARS-CoV-2 in human populations as well as potential future spillovers from animal coronaviruses have provided the impetus for the development of additional direct-acting antivirals. We describe herein the discovery of a new class of broad-spectrum inhibitors of coronavirus 3C-like protease (3CLpro), a cysteine protease essential for viral replication and a validated drug target, that incorporate in their structure a 1,3,2-oxazaphospholidin-3-one scaffold. Inhibitors 1 and 2 were found to have EC50 values of 60 and 50 nM against SARS-CoV-2 3CLpro, respectively, and CC50 values >100 µM. These compounds also potently inhibited MERS-CoV 3CLpro (IC50 120 nM and 90 nM, respectively). Importantly, several of the synthesized compounds inhibited recombinant human cathepsin L with IC50 values in the low nM to sub-nM range. Thus, the compounds can potentially exhibit high antiviral potency by abrogating viral entry via the inhibition of cathepsin L and viral replication by inhibition of 3CLpro. High resolution cocrystal structures were determined to elucidate the mechanism of action, identify the molecular determinants associated with binding, and to inform the optimization process. Less
Virtual reality VR devices are increasingly being utilized within operating theaters and intensive care units where appropriate sanitation is vital to ensure that patients do not unnecessarily acquire hospital-associated infections The morphology of VR devices in conjunction with the variety of materials and internal components provides challenges to their repurposing This study aimed to evaluate the microorganisms remaining on VR headsets following sanitation by laboratory staff in a medical education anatomy teaching facility The external components and internal facial interface were swabbed and separately cultured on four AGAR plates Horse Blood Nutrient bile Esculin and Mannitol Salt Colonies were counted ... More
Virtual reality (VR) devices are increasingly being utilized within operating theaters and intensive care units where appropriate sanitation is vital to ensure that patients do not unnecessarily acquire hospital-associated infections. The morphology of VR devices in conjunction with the variety of materials and internal components provides challenges to their repurposing. This study aimed to evaluate the microorganisms remaining on VR headsets following sanitation by laboratory staff in a medical education anatomy teaching facility. The external components and internal facial interface were swabbed and separately cultured on four AGAR plates (Horse Blood, Nutrient, bile Esculin, and Mannitol Salt). Colonies were counted, sampled, pooled and subsequently processed for shotgun metagenomic sequencing. A higher number of colonies were present on surfaces closest to the eyes and facial interface compared to the external components. Metagenomic analysis identified 27 pathogenic bacteria including 4 “ESKAPE” pathogens (Enterobacter sp., Staphylococcus aureus, Klebsiella spp. and, Escherichia coli) and numerous organisms associated with ocular infections. A broad range of antimicrobial resistance genes were identified conveying resistance to Methicillin, Aminoglycosides, Macrolides, Tetracyclines, and Polymixins. Further research is required to ensure that current sanitization practices of VR head mounted displays are appropriate within high-risk hospital settings. Less
The PD- PD-L immune checkpoint is a pivotal target for cancer immunotherapy Monoclonal antibodies mAbs targeting the PD- PD-L interaction have achieved clinical success but face limitations including high production costs suboptimal tumor penetration and potential immunogenicity To address these challenges we present the DNA-linked Inhibitor Antibody Assay DIANA a robust high-throughput screening platform optimized for identifying and characterizing low-molecular-weight inhibitors of human PD-L DIANA integrates competitive binding with qPCR detection enabling single-well determination of dissociation constants Kd and rapid screening of thousands of compounds The assay was validated using three FDA-approved mAbs atezolizumab avelumab and durvalumab the PD-L -binding ... More
The PD-1/PD-L1 immune checkpoint is a pivotal target for cancer immunotherapy. Monoclonal antibodies (mAbs) targeting the PD-1/PD-L1 interaction have achieved clinical success but face limitations, including high production costs, suboptimal tumor penetration, and potential immunogenicity. To address these challenges, we present the DNA-linked Inhibitor Antibody Assay (DIANA)─a robust, high-throughput screening platform optimized for identifying and characterizing low-molecular-weight inhibitors of human PD-L1. DIANA integrates competitive binding with qPCR detection, enabling single-well determination of dissociation constants (Kd) and rapid screening of thousands of compounds. The assay was validated using three FDA-approved mAbs (atezolizumab, avelumab, and durvalumab), the PD-L1-binding macrocyclic peptide WL12, and the native PD-1 receptor, yielding Kd values consistent with the literature. DIANA demonstrated a broad dynamic range spanning more than 4 orders of magnitude, excellent robustness (Z′-factor = 0.94), and high tolerance to DMSO (up to 10%). We applied DIANA to screen two libraries: a 5,280-compound in-house library (pooled format) and a 1,298-compound commercial peptidomimetic library (individual format). While very weak initial hits were detected, none were confirmed in follow-up manual (non-HTS) experiments or in an orthogonal cell-based assay. Nonetheless, DIANA’s sensitivity, scalability, and minimal sample requirements establish it as a powerful tool for accelerating the discovery of next-generation PD-1/PD-L1 inhibitors and overcoming key limitations of conventional screening methods. Less
Structural analyses of Trichomonas vaginalis pyrophosphate-dependent phosphofructokinase (TvPPi-PFK)
Trichomonas vaginalis causes trichomoniasis the most common non-viral sexually transmitted disease in humans T vaginalis pyrophosphate-dependent phosphofructokinase TvPPi-PFK is a putative target for rational structure-based drug discovery given its absence in mammals and its importance for parasite survival TvPPi-PFK is a cytosolic enzyme that catalyzes the phosphorylation of fructose- -phosphate using pyrophosphate PPi as the phosphoryl donor This reversible reaction catalyzed by TvPPi-PFK is the first committed step in glycolysis Its reverse reaction is vital for gluconeogenesis in T vaginalis The purification crystallization structure determination and preliminary structure-functional analyses of three crystal structures of TvPPi-PFK are presented All three structures ... More
Trichomonas vaginalis causes trichomoniasis, the most common non-viral sexually transmitted disease in humans. T. vaginalis pyrophosphate-dependent phosphofructokinase (TvPPi-PFK) is a putative target for rational, structure-based drug discovery, given its absence in mammals and its importance for parasite survival. TvPPi-PFK is a cytosolic enzyme that catalyzes the phosphorylation of fructose-6-phosphate using pyrophosphate (PPi) as the phosphoryl donor. This reversible reaction, catalyzed by TvPPi-PFK, is the first committed step in glycolysis. Its reverse reaction is vital for gluconeogenesis in T. vaginalis. The purification, crystallization, structure determination, and preliminary structure-functional analyses of three crystal structures of TvPPi-PFK are presented. All three structures organize as tetramers with the conserved motifs essential for pyrophosphate binding and PPi-PFK catalytic activity. Comparative analysis with structural neighbors from other organisms demonstrated that despite sharing <29% sequence identity, TvPPi-PFK’s protomer shares overall topology with both PPi- and ATP-dependent PFKs. Mass photometry confirmed that TvPPi-PFK formed tetramers under near-physiological conditions. Unexpectedly, TvPPi-PFK crystals dephosphorylate ATP to AMP during soaking. In all three structures, either ATP or AMP is bound at the enzyme’s dimer interface, typical of ATP-PFKs, but a novel finding for PPi-PFKs. Furthermore, a sugar phosphate binding site was observed in proximity to the ATP-binding site. Thus, the three reported TvPPi-PFK structures validate its established PPi-dependent activity while revealing previously unreported ATP and sugar phosphate binding. This study also lays a foundation for future research into putative ATP-dependent activity of TvPPi-PFK and for evaluating known phosphofructokinase inhibitors as potential therapeutics for trichomoniasis. These findings expand our understanding of PFK superfamily diversity and support the continued exploration of TvPPi-PFK as a drug target for trichomoniasis Less
The ToxRS system belongs to a family of co-component transmembrane transcription regulators that act as sensors of environmental cues and regulate virulence gene expression in several bacterial pathogens These systems are thought to operate by sensing environmental stimuli and transmitting signals through periplasmic domains to activate DNA-binding transcription factors In the enteric pathogens Vibrio parahaemolyticus and Vibrio cholerae the ToxRS system regulates virulence factors responsible for severe gastrointestinal symptoms in humans ToxR is a DNA-binding regulator associated in the periplasm with ToxS a protein of poorly understood function ToxS modulates the activity of its binding partner ToxR in the presence ... More
The ToxRS system belongs to a family of co-component transmembrane transcription regulators that act as sensors of environmental cues and regulate virulence gene expression in several bacterial pathogens. These systems are thought to operate by sensing environmental stimuli and transmitting signals through periplasmic domains to activate DNA-binding transcription factors. In the enteric pathogens Vibrio parahaemolyticus and Vibrio cholerae, the ToxRS system regulates virulence factors responsible for severe gastrointestinal symptoms in humans. ToxR is a DNA-binding regulator associated in the periplasm with ToxS, a protein of poorly understood function. ToxS modulates the activity of its binding partner ToxR in the presence of bile salts, antimicrobial cholesterol metabolites secreted into the gut. To date, the molecular mechanism underlying this regulation remains unclear. We present crystal structures of the V. parahaemolyticus ToxS periplasmic domain (ToxSp) with and without the bile salt glycocholate. ToxSp forms an 8-stranded broken β-barrel with a central α-helix and is structurally homologous to a group of chaperone proteins. ToxSp has a highly conserved hydrophobic core that stabilizes the β-barrel fold, while the binding pocket tolerates substantial variation, consistent with binding hydrophobic ligands. Strikingly, we discovered that Vp-ToxSp binds three molecules of glycocholate and the presence of this bile salt leads to the formation a strand-swapped ToxS homodimer. Finally, modeling two ToxR periplasmic domains in complex with the glycocholate-bound ToxSp homodimer provides a structure-based model for bile salt-mediated heterotetramerization of the ToxRS system. Overall, our study addresses a major longstanding question in the field of Vibrio virulence regulation providing a scenario that could apply to other pathogens that utilize these membrane-bound family transcriptional regulators. Less
Nucleic acid nanoparticles NANPs fabricated by using DNA origami are an emerging delivery vector for nucleic acid therapeutics Despite their advantages over other nanomaterials that include controlled spatial presentation of targeting ligands such as lipids and sugars understanding their cell targeting and uptake mechanisms remains limited Here we investigated NANP cellular targeting uptake and delivery of small interfering RNAs siRNAs to liver and neuronal cell models in vitro Using a rational design approach we targeted NANPs to two clinically validated receptors the asialoglycoprotein receptor ASGPR and the low-density lipoprotein receptor LDLR respectively using GalNAc and lipidation We systematically evaluated how ... More
Nucleic acid nanoparticles (NANPs) fabricated by using DNA origami are an emerging delivery vector for nucleic acid therapeutics. Despite their advantages over other nanomaterials that include controlled spatial presentation of targeting ligands such as lipids and sugars, understanding their cell targeting and uptake mechanisms remains limited. Here, we investigated NANP cellular targeting, uptake, and delivery of small interfering RNAs (siRNAs) to liver and neuronal cell models in vitro. Using a rational design approach, we targeted NANPs to two clinically validated receptors, the asialoglycoprotein receptor (ASGPR) and the low-density lipoprotein receptor (LDLR), respectively, using GalNAc and lipidation. We systematically evaluated how the ligand valency, interligand spacing, linker length, and ligand chemistry affected NANP association with on- and off-target liver cell types, revealing the relative roles of the biomolecular corona, receptor engagement, and endocytosis in these targeting strategies. We found that lipidation enhanced NANP uptake into HepG2 cells, a model cell line for hepatocytes, by promoting apolipoprotein recruitment, LDLR engagement, and clathrin-mediated endocytosis and also increased association with nonparenchymal cells. HepG2 uptake was further improved by conjugating NANPs to lipids with higher valency provided that lipids were adequately displayed away from the surface of NANP edges with more lipophilic lipids yielding greater cell association. We then benchmarked the potential for NANPs to deliver siRNAs to HepG2 cells in comparison with lipid nanoparticle and conjugate technologies and explored lipid functionalization as a strategy for nonhepatic NANP targeting to model neuronal cells. Overall, this study advances the foundational understanding of how clinically relevant targeting ligands mediate NANP interactions with both on- and off-target liver cell types in vitro, offering insights into potential design criteria for nucleic acid therapeutic delivery. Less
Recoverin is a key calcium sensor that controls the desensitization of the visual rhodopsin by GRK Previous studies have traditionally been conducted on bovine protein bRec while data on human ortholog hRec remain scarce Here we combine X-ray crystallography Xray absorption spectroscopy XANES quantum mechanical calculations molecular dynamics and functional assays to provide an integrated characterization of hRec The Ca -bound hRec structure was solved at showing that unlike bRec hRec interacts with ROS membranes at physiologically relevant submicromolar Ca levels due to a speciesspecific charge distribution that might influence membrane interactions Both recoverins form a set of Ca Zn ... More
Recoverin is a key calcium sensor that controls the desensitization of the visual rhodopsin
by GRK1. Previous studies have traditionally been conducted on bovine protein (bRec), while
data on human ortholog (hRec) remain scarce. Here, we combine X-ray crystallography, Xray absorption spectroscopy (XANES), quantum mechanical calculations, molecular
dynamics, and functional assays to provide an integrated characterization of hRec. The
2Ca2+-bound hRec structure was solved at 1.60 Å, showing that, unlike bRec, hRec interacts
with ROS membranes at physiologically relevant submicromolar Ca2+ levels, due to a speciesspecific charge distribution that might influence membrane interactions. Both recoverins
form a set of Ca2+/Zn2+-bound conformers with improved functional performance. X-ray
crystallography (1.85 Å) and XANES revealed a specific tetrahedral Zn2+ site in 1Ca2+-bound
hRec, the first such site reported in the NCS family. In 1Ca2+-bound hRec, zinc promotes the
formation of active state, whereas in 2Ca2+-state of bRec, it significantly enhances GRK1
binding, as the latter can complement the Zn2+ coordination. These data refine our
understanding of recoverin function in humans and highlight its role as a key link between
calcium and zinc signaling in mammalian photoreceptors under normal and pathological
conditions. Less
by GRK1. Previous studies have traditionally been conducted on bovine protein (bRec), while
data on human ortholog (hRec) remain scarce. Here, we combine X-ray crystallography, Xray absorption spectroscopy (XANES), quantum mechanical calculations, molecular
dynamics, and functional assays to provide an integrated characterization of hRec. The
2Ca2+-bound hRec structure was solved at 1.60 Å, showing that, unlike bRec, hRec interacts
with ROS membranes at physiologically relevant submicromolar Ca2+ levels, due to a speciesspecific charge distribution that might influence membrane interactions. Both recoverins
form a set of Ca2+/Zn2+-bound conformers with improved functional performance. X-ray
crystallography (1.85 Å) and XANES revealed a specific tetrahedral Zn2+ site in 1Ca2+-bound
hRec, the first such site reported in the NCS family. In 1Ca2+-bound hRec, zinc promotes the
formation of active state, whereas in 2Ca2+-state of bRec, it significantly enhances GRK1
binding, as the latter can complement the Zn2+ coordination. These data refine our
understanding of recoverin function in humans and highlight its role as a key link between
calcium and zinc signaling in mammalian photoreceptors under normal and pathological
conditions. Less
AXIS: a Lab-in-the-Loop machine learning approach for automated detection of macromolecular crystals
Macromolecular crystallography provides mechanistic understanding of biological processes and can be applied in drug design Nowadays the use of robotic systems for crystal growth and diffraction analysis is widespread and high-throughput protein-to-structure pipelines for ligand and fragment screening are revolutionizing the field However the identification of crystals is still largely carried out through manual inspection sometimes involving tens of thousands of images which represents a bottleneck in an otherwise highly automated process Here we describe AXIS an AI-based Crystal Identification System combining the DINOv computer vision model state-of-the-art transfer learning and MARCO the largest crystallization dataset available to date for ... More
Macromolecular crystallography provides mechanistic understanding of biological processes and can be applied in drug design. Nowadays, the use of robotic systems for crystal growth and diffraction analysis is widespread and high-throughput protein-to-structure pipelines for ligand and fragment screening are revolutionizing the field. However, the identification of crystals is still largely carried out through manual inspection, sometimes involving tens of thousands of images, which represents a bottleneck in an otherwise highly automated process. Here we describe AXIS, an AI-based Crystal Identification System combining the DINOv2 computer vision model, state-of-the-art transfer learning and MARCO, the largest crystallization dataset available to date, for automated crystal detection. AXIS can operate with both visible and UV light images and integrates a Lab-in-the-Loop approach combining ML and expert inputs for iterative learning and specialization. AXIS enables automated annotation of large crystallization image datasets with performance and accuracy comparable to that of human experts, and the Lab-in-the-Loop approach introduced here enables efficient adaptation to local conditions, facilitating widespread application, which has been a major limitation to date. AXIS can help to correct human errors in image annotation and removes critical bottlenecks, particularly in the context of extensive crystallization screens or high-throughput applications like fragment and ligand screening, unlocking the potential for higher levels of automation that are key in both fundamental and translational research. Less
Antisense oligonucleotides ASOs are promising therapeutics but safety concerns such as liver toxicity and off-target OffT effects necessitate thorough evaluation during the compound selection process This study leverages time course global proteomics and transcriptomics to assess ASO-induced changes in vitro comparing liver toxic versus non-liver toxic ASOs The research confirms that ASOs perturb different cellular pathways at both RNA and protein levels effectively discriminating between liver toxic and non-liver toxic ASOs Contrary to expectations protein level reduction isn t delayed relative to ASO-induced RNA reduction highlighting the importance of understanding RNA and protein level relationships in specific model systems Furthermore ... More
Antisense oligonucleotides (ASOs) are promising therapeutics, but safety concerns such as liver toxicity and off-target (OffT) effects necessitate thorough evaluation during the compound selection process. This study leverages time course global proteomics and transcriptomics to assess ASO-induced changes in vitro, comparing liver toxic versus non-liver toxic ASOs. The research confirms that ASOs perturb different cellular pathways at both RNA and protein levels, effectively discriminating between liver toxic and non-liver toxic ASOs. Contrary to expectations, protein level reduction isn’t delayed relative to ASO-induced RNA reduction, highlighting the importance of understanding RNA and protein level relationships in specific model systems. Furthermore, many OffT effects observed at the RNA level do not directly translate to corresponding protein level changes. These findings suggest that current RNA-focused OffT assessment strategies capture predicted OffTs but could benefit from protein level studies that could potentially de-risk oligonucleotide drug (OND) candidates with seemingly problematic OffT profiles at the RNA level. The study underscores the value of global proteomics as a complement to RNAseq in ASO drug development, refining safety assessment and improving candidate selection. Less
UreE is a nickel chaperone that is required for the safe and efficient delivery of nickel to the active site of the metalloenzyme urease which is a key virulence factor of the urinary-tract pathogen Proteus mirabilis We investigated the structural features of P mirabilis UreE PmUreE using protein X-ray crystallography and its nickel-binding capacity by inductively coupled plasma mass spectrometry Here we report a resolution crystal structure of homodimeric PmUreE and show that it has the capacity to bind five Ni II ions per dimer Truncation of the histidine-rich C-terminus reduced the nickel-binding capacity by two Ni II ions per ... More
UreE is a nickel chaperone that is required for the safe and efficient delivery of nickel to the active site of the metalloenzyme urease, which is a key virulence factor of the urinary-tract pathogen Proteus mirabilis. We investigated the structural features of P. mirabilis UreE (PmUreE) using protein X-ray crystallography and its nickel-binding capacity by inductively coupled plasma mass spectrometry. Here, we report a 2.0 Å resolution crystal structure of homodimeric PmUreE and show that it has the capacity to bind five Ni(II) ions per dimer. Truncation of the histidine-rich C-terminus reduced the nickel-binding capacity by two Ni(II) ions per dimer, and comparison with homologous UreE structures allowed the assignment of putative nickel-binding sites within the PmUreE structure. These findings increase our understanding of how PmUreE binds nickel and ultimately prevents this toxic metal from causing significant cellular damage in P. mirabilis. Less
The Smart-seq family of methods represents the gold standard for high-sensitivity full-length single-cell RNA sequencing Despite iterative improvements fundamental challenges remain the generation of non-specific PCR products that limit sensitivity the inability to capture precise Transcription End Sites TES and the insidious generation of phantom UMIs artificial molecular barcodes created during PCR that systematically inflate molecular counts Here we present ESPeR-seq a novel architecture that resolves these barriers To enable precise stranded TES capture we developed an Omega-dT primer that bypasses synthetic poly-T tracts restoring high-quality sequencing directly at transcript termini To eliminate both PCR background and phantom UMIs we ... More
The Smart-seq family of methods represents the gold standard for high-sensitivity, full-length single-cell RNA sequencing. Despite iterative improvements, fundamental challenges remain: the generation of non-specific PCR products that limit sensitivity, the inability to capture precise Transcription End Sites (TES), and the insidious generation of “phantom UMIs”—artificial molecular barcodes created during PCR that systematically inflate molecular counts. Here, we present ESPeR-seq, a novel architecture that resolves these barriers. To enable precise, stranded TES capture, we developed an “Omega-dT” primer that bypasses synthetic poly-T tracts, restoring high-quality sequencing directly at transcript termini. To eliminate both PCR background and phantom UMIs, we implemented a biochemical “multi-lock” mechanism utilizing uracil-containing TSOs and a uracil-intolerant DNA polymerase. We validate this approach using the logQ-slope, a novel metric that sensitively diagnoses UMI fidelity. Benchmarking reveals that while state-of-the-art methods still exhibit signs of UMI inflation, ESPeR-seq strictly prevents it. Furthermore, the strandedness and precise end-delineation provided by TSO and dT reads support robust de novo gene model reconstruction, enabling the discovery of novel multi-exon genes, unannotated 3’ UTR extensions, and candidate eRNAs across aggregated single-cell populations. Thus, ESPeR-seq establishes a robust framework for absolute quantitative accuracy and full-length isoform resolution. Less
Mass spectrometry-based proteomics increasingly demands platforms that combine quantitative rigor with the discovery capabilities of accurate mass systems Here we present the ZenoTOF system a compact mass spectrometry system that integrates enhanced ion capture and transmission optics with an optical detection system Zeno trap-enhanced MS MS electron-activated dissociation and scanning quadrupole data-independent acquisition ZT Scan DIA We show that ZT Scan DIA outperforms conventional variable-window DIA Zeno SWATH DIA in both identifications and quantitative reproducibility and demonstrate the platform s versatility across proteomics applications thousands of protein groups from bulk samples at up to samples per day single-cell proteomics yielding ... More
Mass spectrometry-based proteomics increasingly demands platforms that combine quantitative rigor with the discovery capabilities of accurate mass systems. Here we present the ZenoTOF 8600 system, a compact mass spectrometry system that integrates enhanced ion capture and transmission optics with an optical detection system, Zeno trap-enhanced MS/MS, electron-activated dissociation, and scanning quadrupole data-independent acquisition (ZT Scan DIA). We show that ZT Scan DIA outperforms conventional variable-window DIA (Zeno SWATH DIA) in both identifications and quantitative reproducibility, and demonstrate the platform’s versatility across proteomics applications: thousands of protein groups from bulk samples at up to 500 samples per day, single-cell proteomics yielding up to 4,700 proteins, accurate ratio recovery in mixed-species quantitative benchmarks, low-attomole targeted quantitation, and detection of disease-relevant phosphorylation in a Parkinson’s disease cellular model using complementary CID and EAD fragmentation. The instrument’s compact footprint makes it attractive for settings where both analytical breadth and operational robustness are required. Less
The chikungunya virus CHIKV outbreak imposes a significant burden on healthcare systems and raises an urgent need for effective antiviral therapies So far there are no specific drugs against CHIKV A CHIKV macrodomain is critical for virulence and counteracts the host immune response representing a promising antiviral drug target Here we describe small molecule inhibitors targeting the CHIKV macrodomain Compound MDOLL- was identified through a high-throughput screening using a fluorescence resonance energy transfer FRET -based assay and its inhibitory activity was validated through multiple orthogonal assays Compound has a dual thiobarbiturate-indole scaffold and exhibits an IC of M X-ray crystallography ... More
The chikungunya virus (CHIKV) outbreak imposes a significant burden on healthcare systems and raises an urgent need for effective antiviral therapies. So far there are no specific drugs against CHIKV. A CHIKV macrodomain is critical for virulence and counteracts the host immune response, representing a promising antiviral drug target. Here, we describe small molecule inhibitors targeting the CHIKV macrodomain. Compound 1 (MDOLL-0273) was identified through a high-throughput screening using a fluorescence resonance energy transfer (FRET)-based assay, and its inhibitory activity was validated through multiple orthogonal assays. Compound 1 has a dual thiobarbiturate-indole scaffold and exhibits an IC50 of 8.9 µM. X-ray crystallography revealed that the inhibitor occupies an adenine binding site of the macrodomain and extends into a novel cryptic pocket. Notably, the inhibitor shows high selectivity for the CHIKV macrodomain over a panel of human and viral ADP-ribosyl binding and hydrolyzing proteins. Structure-activity relationship studies and medicinal chemistry efforts provide a promising starting point for further hit optimization. Less
The advancement of single-crystal structural analysis has emerged as a pivotal technology surpassing spectroscopic methods in revealing the intricate structural details of organic small molecules including crystal packing and stereochemical configurations It plays a critical role across scientific domains such as chemistry biology agronomy and medicine Traditional single-crystal X-ray diffraction SCXRD has always been restricted by its stringent requirements on the physical state size and quality of crystals This review discusses the arsenal of equipment and theoretical techniques for obtaining single-crystal structures including SCXRD PXRD CSP and more recently Micro-ED It further explores the significant crystal growth techniques based on ... More
The advancement of single-crystal structural analysis has emerged as a pivotal technology surpassing spectroscopic methods in revealing the intricate structural details of organic small molecules, including crystal packing and stereochemical configurations. It plays a critical role across scientific domains such as chemistry, biology, agronomy, and medicine. Traditional single-crystal X-ray diffraction (SCXRD) has always been restricted by its stringent requirements on the physical state, size, and quality of crystals. This review discusses the arsenal of equipment and theoretical techniques for obtaining single-crystal structures, including SCXRD, PXRD & CSP, and more recently, Micro-ED. It further explores the significant crystal growth techniques based on three foundational methods: solution-based crystallization, melt crystallization, and sublimation crystallization. Detailed discussion is provided on the crystallizability of molecules and the refinement of crystal growth methods. Specifically, for crystallizable analytes, a combination of crystal growth enhancement techniques and high-throughput technologies (under-oil) can compensate for poor crystallinity, small size, and defects under normal conditions. For molecules inherently resistant to crystallization, a “crystallization chaperone”, such as a MOFs as a crystalline sponge or tetraaryladamantane as a cocrystallization chaperone, can determine absolute configurations. Looking ahead, this review emphasizes the potential of artificial intelligence and machine learning approaches for crystal growth and structural prediction. The development of integrated analysis strategies combining SCXRD, PXRD, and Micro-ED is identified as a future trend for providing comprehensive structural insights. This review highlights the significance of advancements in single-crystal structural analysis techniques, paving the way for groundbreaking innovations in molecular design and materials science, and predicts a bright future for the field with new technologies. Less
-Hemolysin Ahly is a major Staphylococcus aureus virulence determinant implicated in tissue injury and immune dysregulation antibody inhibitors have reached clinical trials but alternatives with improved ease of manufacture and tissue penetration are desirable Here we demonstrate that phage-derived bicyclic peptides can serve as compact chemically tractable Ahly neutralisers Using TATB-scaffolded M phage libraries we identified WNP-motif containing bicyclic binders with a lead hit of Peptide KD nM and progressed the lead by iterative affinity maturation to Peptide KD nM and by incorporation of strategically chosen non-canonical amino acids to yield Peptide KD nM A co-crystal structure with AhlyH A ... More
α-Hemolysin (Ahly) is a major Staphylococcus aureus virulence determinant implicated in tissue injury and immune dysregulation; antibody inhibitors have reached clinical trials but alternatives with improved ease of manufacture and tissue penetration are desirable. Here we demonstrate that phage-derived bicyclic peptides can serve as compact, chemically tractable Ahly neutralisers. Using TATB-scaffolded M13 phage libraries we identified WNP-motif containing bicyclic binders, with a lead hit of Peptide 14 (KD = 1792nM) and progressed the lead by iterative affinity maturation to Peptide 20 (KD = 609 nM) and by incorporation of strategically chosen non-canonical amino acids to yield Peptide 88 (KD = 96 nM). A 2.2 Å co-crystal structure with AhlyH35A locates the binding footprint on the rim domain and explains the critical role of the WNP motif in target engagement. Functional assays show that the Peptide 88 blocks Ahly mediated hemolysis, inhibits Ahly driven ADAM10 activation, and elucidate its inhibitory mechanism of preventing Ahly binding to human A549 epithelial cells. Peptide 88 protects A549 cells from recombinant toxin and attenuates cytotoxicity in S. aureus co-culture experiments, whilst showing no toxicity to A549 cells. Bicyclic peptides thus represent a new and promising anti-virulence modality: small, synthetically accessible molecules that mimic antibody recognition, with therapeutic potential against S. aureus infections. Less
Safe and effective vaccines against co-circulating mosquito-borne orthoflaviviruses such as Zika virus ZikV and the four serotypes of Dengue virus DenV - must elicit broadly neutralizing antibodies bnAbs to prevent the risk of enhancement of infection by non-neutralizing antibodies We recently discovered new orthoflavivirus-directed bnAbs including F S which neutralizes DenV - and ZikV with comparable or superior potency to the previously characterized E dimer epitope EDE bnAbs Here we used cryoEM and X-ray crystallography to understand the basis of cross-neutralization of F S at the molecular level We obtained a cryoEM structure of F S Fab bound to a ... More
Safe and effective vaccines against co-circulating mosquito-borne orthoflaviviruses such as Zika virus (ZikV) and the four serotypes of Dengue virus (DenV1-4) must elicit broadly neutralizing antibodies (bnAbs) to prevent the risk of enhancement of infection by non-neutralizing antibodies. We recently discovered new orthoflavivirus-directed bnAbs, including F25.S02, which neutralizes DenV1-4 and ZikV with comparable or superior potency to the previously characterized E dimer epitope (EDE) bnAbs. Here, we used cryoEM and X-ray crystallography to understand the basis of cross-neutralization of F25.S02 at the molecular level. We obtained a ~ 4.2 Å cryoEM structure of F25.S02 Fab bound to a stabilized DenV3 soluble E protein dimer and a 2.3 Å crystal structure of F25.S02 Fab bound to ZikV soluble E protein dimer. Like previously described EDE1 bnAbs, the structural epitope of F25.S02 is at the E dimer interface, encompassing predominantly conserved regions in domain II, including the fusion loop. However, unlike EDE1 bnAbs, F25.S02 binding is almost entirely dependent on the heavy chain and is shifted slightly away from the dimer symmetry axis. Our findings emphasize the importance of this cross-neutralizing site of vulnerability for DenV and ZikV that can facilitate rational design of vaccines and therapeutics. Less
Imidazolium LipidBrick cationic lipid nanoparticles LNPs provide a pH-independent alternative to conventional ionizable systems for nucleic acid delivery Through a high-throughput screen of formulations spanning eight imidazolium cores three helper lipids and varying PEG densities we found that more than half of the library outperformed the clinical ionizable benchmark ALC- in multiple representative mammalian cell types Top-performing candidates showed robust cellular uptake efficient endosomal escape and strong transgene expression both in vitro and following intramuscular administration A lead formulation C LNP incorporating an imidazolium lipid core bearing a hydroxyethyl substituent with mol DOPE achieved comparable intramuscular luciferase expression and antibody ... More
Imidazolium LipidBrick® cationic lipid nanoparticles (LNPs) provide a pH-independent alternative to conventional ionizable systems for nucleic acid delivery. Through a high-throughput screen of 1,944 formulations spanning eight imidazolium cores, three helper lipids, and varying PEG densities, we found that more than half of the library outperformed the clinical ionizable benchmark ALC-0315 in multiple representative mammalian cell types. Top-performing candidates showed robust cellular uptake, efficient endosomal escape, and strong transgene expression both in vitro and following intramuscular administration. A lead formulation (C3 LNP), incorporating an imidazolium lipid core bearing a hydroxyethyl substituent, with 30 mol% DOPE, achieved comparable intramuscular luciferase expression and antibody titers to ALC-0315, while eliciting ∼ 3-fold stronger ovalbumin-specific IFN-γ+ T-cell responses and maintaining low cytotoxicity. Machine-learning analysis of the dataset further distilled transferable design rules to inform future formulation strategies. Collectively, these findings establish cationic LipidBrick® LNPs as a versatile platform for mRNA delivery, offering a generalizable framework for the high-throughput discovery of ionization-independent systems that effectively prime adaptive immune responses. Less
Tankyrases are poly-ADP-ribosyltransferases that orchestrate numerous biological processes involved in disease Their established regulatory roles particularly within the WNT -catenin pathway have driven notable drug discovery efforts aimed at inhibiting their catalytic activity Targeting tankyrases interaction with proteins through their ARC domains represents an alternative strategy to be explored as a therapeutic approach against specific protein-protein interactions In this article we employed a pre-established FRET-based assay to screen the EU-OPENSCREEN libraries for identification of ARC inhibitors We discovered a series of pyrrolone-based compounds and we synthesized compound S ARCher- which binds selectively to ARC with a potency of M NMR ... More
Tankyrases are poly-ADP-ribosyltransferases that orchestrate numerous biological processes involved in disease. Their established regulatory roles, particularly within the WNT/β-catenin pathway, have driven notable drug discovery efforts aimed at inhibiting their catalytic activity. Targeting tankyrases’ interaction with proteins through their ARC domains represents an alternative strategy to be explored as a therapeutic approach against specific protein-protein interactions. In this article, we employed a pre-established FRET-based assay to screen the EU-OPENSCREEN libraries for identification of ARC4 inhibitors. We discovered a series of pyrrolone-based compounds, and we synthesized compound S8 (ARCher-142), which binds selectively to ARC4 with a potency of 8 μM. NMR analysis and X-ray crystallography allowed us to identify the binding site and to rationalize the observed selectivity. Despite binding exclusively to ARC4, the inhibitor can attenuate the WNT/β-catenin signaling pathway in cells. Our work demonstrates that targeting single ARC domains is possible, offering an inhibition approach tailored to tankyrase ARC4. Less
Improving health and quality of life in our society is a key focus of drug development Methods for drug discovery are being optimized in multiple ways to reduce costs and timelines Crystallographic fragment screening CFS is increasingly being employed as an early screening method in drug discovery projects Here we demonstrate that selecting the optimal protein crystal form can significantly impact hit rates Two CFS campaigns are carried out against the two crystal forms of the SARS-CoV- main protease using the same fragment library and an almost identical experimental setup Although both crystal forms exhibit similar diffraction properties the observed ... More
Improving health and quality of life in our society is a key focus of drug development. Methods for drug discovery are being optimized in multiple ways to reduce costs and timelines. Crystallographic fragment screening (CFS) is increasingly being employed as an early screening method in drug discovery projects. Here, we demonstrate that selecting the optimal protein crystal form can significantly impact hit rates. Two CFS campaigns are carried out against the two crystal forms of the SARS-CoV-2 main protease, using the same fragment library and an almost identical experimental setup. Although both crystal forms exhibit similar diffraction properties, the observed hit rates in the two campaigns differ significantly. A hit rate of 3% is determined for the monoclinic crystals, while a hit rate of 16% is observed for the orthorhombic crystals. These findings are consistent with the more open molecular packing in the orthorhombic crystals, where the solvent channels leading to the active sites are approximately twice the size of those in the monoclinic crystal form. Our results highlight the critical importance of the crystal form in a crystallographic screening, identifying it as one of the most important parameters to optimize when preparing a CFS campaign. Less
Complement and pathogenic antibodies act independently and together to mediate the pathology of many autoimmune diseases To address these drivers of disease we generated a monoclonal antibody mAb CSL that binds and inhibits both complement and the neonatal Fc fragment crystallizable receptor FcRn The fragment antigen binding Fab portion of CSL was engineered to bind both human C huC zymogen and the active fragment huC b to inhibit the classical and lectin complement pathways in vitro and C b deposition on primary lung endothelial cells using a -dimensional microvascular model system Engineering of a triple amino acid mutation YPY motif ... More
Complement and pathogenic antibodies act independently and together to mediate the pathology of many autoimmune diseases. To address these drivers of disease, we generated a monoclonal antibody (mAb), CSL305, that binds and inhibits both complement and the neonatal Fc (fragment crystallizable) receptor FcRn. The fragment antigen binding (Fab) portion of CSL305 was engineered to bind both human C2 (huC2) zymogen and the active fragment huC2b to inhibit the classical and lectin complement pathways in vitro, and C3b deposition on primary lung endothelial cells using a 3-dimensional microvascular model system. Engineering of a triple amino acid mutation (“YPY” motif) into the Fc region of CSL305 increased its affinity to FcRn at both acidic and neutral pH, allowing it to also act as a potent FcRn antagonist. Intracellular trafficking experiments demonstrated that CSL305, but not the wild-type (WT) mAb lacking the YPY motif, was able to block immunoglobulin G (IgG) recycling in vitro. The generation of a high resolution 2.6Å crystal structure of CSL305 Fab region bound to huC2b showed that the epitope lies directly over the huC2b catalytic triad, providing evidence of its complement mechanism of action as a neutralising mAb. Early pharmacokinetic (PK)/pharmacodynamic (PD) studies using CSL305 in cynomolgus monkeys demonstrated both complement inhibition and FcRn antagonism in vivo, with reductions in complement classical pathway activity and endogenous IgG observed following single intravenous (IV) administration. CSL305 thus represents a dual-functional mAb as a potential therapeutic candidate. Less
Light Oxygen Voltage LOV domains are important widespread receptors of blue light that also found applications in optogenetics and imaging While LOV domains from mesophiles are relatively well characterized their counterparts from thermophilic microorganisms remain understudied Here we express two constructs of a LOV domain belonging to a histidine kinase from Meiothermus ruber MrLOV and MrLOVe and show that they are photoactive with recovery time values of and min respectively and thermostable Crystal structures reveal that MrLOV which lacks helices A and J forms a parallel dimer whereas MrLOVe is a tetramer organized as an antiparallel dimer of two parallel ... More
Light Oxygen Voltage (LOV) domains are important widespread receptors of blue light that also found applications in optogenetics and imaging. While LOV domains from mesophiles are relatively well characterized, their counterparts from thermophilic microorganisms remain understudied. Here, we express two constructs of a LOV domain belonging to a histidine kinase from Meiothermus ruber, MrLOV and MrLOVe, and show that they are photoactive, with recovery time values of 21 and 27 min, respectively, and thermostable. Crystal structures reveal that MrLOV, which lacks helices A’α and Jα, forms a parallel dimer, whereas MrLOVe is a tetramer organized as an antiparallel dimer of two parallel dimers interacting via helices Jα. One MrLOVe dimer is symmetric, and the other is asymmetric, with conformational differences mirroring activation-related changes in other LOV domains. Our data provide the structural basis for understanding and engineering of thermophilic LOVs and pave the way for development of thermostable and photostable LOV-derived optogenetic tools and flavin-based fluorescent proteins. Less
Gene expression is governed by dynamic switches between repressive and activating transcriptional states Among the molecules mediating these transitions chromatin readers and transcription factors play pivotal roles However how they assemble with regulatory machineries to enable crosstalk between gene repression and activation remains unknown Here we use an integrative structural dynamics approach combining cryo-EM crosslinking mass spectrometry fragment-resolved protein interactome mapping and crystallography to show how the dual-role chromatin reader Cti and transcription factors Ash and Ume engage the Sin deacetylase complex a major regulatory hub in eukaryotes We find that Cti competes with Ash to drive its dynamic recruitment ... More
Gene expression is governed by dynamic switches between repressive and activating transcriptional states1,2. Among the molecules mediating these transitions, chromatin readers and transcription factors play pivotal roles3,4. However, how they assemble with regulatory machineries to enable crosstalk between gene repression and activation remains unknown. Here, we use an integrative structural dynamics approach – combining cryo-EM, crosslinking mass spectrometry, fragment-resolved protein interactome mapping and crystallography – to show how the dual-role chromatin reader Cti6 and transcription factors Ash1 and Ume6 engage the Sin3 deacetylase complex, a major regulatory hub in eukaryotes5. We find that Cti6 competes with Ash1 to drive its dynamic recruitment to a shared peripheral module, while Ume6 engages the Sin3 scaffold through a defined, minimal interface. Using high-throughput mutational scanning, we reveal deleterious and gain-of-function mutations in Sin3, identifying evolutionarily conserved residues essential for anchoring transcription factors. Together, these results provide structural and functional insights into how dual-role regulators engage the central Sin3 complex, revealing subtle assembly principles that may facilitate crosstalk between gene repression and activation. They also establish an integrative multidisciplinary framework to dissect the dynamics of macromolecular assemblies across biological systems. Less
Filament-forming proteins such as TasA Bacillus subtilis and camelysins CalY CalY Bacillus cereus pose a particular challenge for structural analysis due to their strong tendency to self-association and their polydispersity which severely limits their ability to crystallize or to be a target for NMR-spectroscopy To address this it is necessary to modify the amino acid sequence to prevent filamentation Engineering a series of N- and C-terminal truncated variants by removing flexible parts is often key to success N-terminal extensions are also a powerful tool for obtaining crystals of fiber-forming proteins
Epstein-Barr virus EBV causes infectious mononucleosis and contributes to neurodegenerative disorders and malignancies particularly in immune-compromised hosts Transplant patients face high risk of post-transplant lymphoproliferative disease a life-threatening EBV-driven lymphoma There are no EBV-specific vaccines or treatments however neutralizing antibodies against EBV glycoproteins may offer utility as therapeutic agents EBV entry into B cells involves gp which binds complement receptors and gp which engages HLA class II to trigger fusion Most existing monoclonal antibodies mAbs against these antigens are non-human limiting clinical use Using a transgenic mouse model we generate two gp and eight gp genetically human neutralizing mAbs that ... More
Epstein-Barr virus (EBV) causes infectious mononucleosis and contributes to neurodegenerative disorders and malignancies, particularly in immune-compromised hosts. Transplant patients face high risk of post-transplant lymphoproliferative disease, a life-threatening EBV-driven lymphoma. There are no EBV-specific vaccines or treatments; however, neutralizing antibodies against EBV glycoproteins may offer utility as therapeutic agents. EBV entry into B cells involves gp350, which binds complement receptors, and gp42, which engages HLA class II to trigger fusion. Most existing monoclonal antibodies (mAbs) against these antigens are non-human, limiting clinical use. Using a transgenic mouse model, we generate two gp350 and eight gp42 genetically human neutralizing mAbs that block receptor binding. Structural analyses reveal extended sites of vulnerability relevant to vaccine development. Delivery of a gp42 mAb protects humanized mice from EBV challenge, while a gp350 mAb provides partial protection. These mAbs highlight the utility of transgenic mice to produce therapeutic mAbs for preventing EBV-driven disease. Less
HECT E ligases regulate many cellular processes yet how they recognise their substrates and synthesise specific types of poly-ubiquitin chains is still incompletely understood HECTD a member of the other HECT family is implicated in the regulation of inflammation apoptosis and infection and highly expressed in several cancers These functions are largely attributed to its ligase activity and modification of diverse substrates with different types of ubiquitin chains We present a detailed analysis of the ligase activity of HECTD including its ubiquitin linkage preferences oligomeric state and substrate ubiquitination Using cryo-EM we provide the full-length structures of HECTD in both ... More
HECT E3 ligases regulate many cellular processes, yet how they recognise their substrates and synthesise specific types of poly-ubiquitin chains is still incompletely understood. HECTD3, a member of the “other HECT” family, is implicated in the regulation of inflammation, apoptosis, and infection and highly expressed in several cancers. These functions are largely attributed to its ligase activity and modification of diverse substrates with different types of ubiquitin chains. We present a detailed analysis of the ligase activity of HECTD3, including its ubiquitin linkage preferences, oligomeric state and substrate ubiquitination. Using cryo-EM, we provide the full-length structures of HECTD3 in both apo and ubiquitin-loaded forms, revealing key insights into its domain organisation, including discovery of a distinct fold of the N-terminal region, and mechanistic features. Some of these are shared with other HECT ligases, while others are unique to HECTD3 and contribute to differences in its catalytic mechanisms and functional diversity. Less
Autosomal recessive congenital ichthyosis ARCI refers to a group of rare highly debilitating skin disorders that significantly impair patients quality of life and lack any effective treatment options Here we report clinically relevant in situ correction of the most common ARCI-causing mutation TGM c - A G a splice-site aberration in human disease models Targeted skin barrier modulation followed by topical application of the cytosine base editor eTd packaged into lipid nanoparticles yielded functional restoration of of wild-type transglutaminase activity in skin tissue Toxicity studies and comprehensive off-target analysis demonstrated an excellent safety profile even after repeated application without systemic ... More
Autosomal recessive congenital ichthyosis (ARCI) refers to a group of rare, highly debilitating skin disorders that significantly impair patients’ quality of life and lack any effective treatment options. Here, we report clinically relevant in situ correction of the most common ARCI-causing mutation, TGM1 c.877-2A>G, a splice-site aberration, in human disease models. Targeted skin barrier modulation followed by topical application of the cytosine base editor eTd packaged into lipid nanoparticles yielded functional restoration of ∼30% of wild-type transglutaminase 1 activity in skin tissue. Toxicity studies and comprehensive off-target analysis demonstrated an excellent safety profile even after repeated application, without systemic distribution of the lipid nanoparticles or the genetic cargo as determined via highly sensitive methods, including desorption electrospray ionization (DESI) metabolic imaging. This study presents comprehensive preclinical data on the feasibility of in situ gene correction of genodermatoses-causing mutations, showcasing its therapeutic potential and paving the way for curative next-generation treatments for severe genetic skin diseases. Less
Transformation of agro-industrial products into value-added products such as prebiotic oligosaccharides is a key element of the emerging bioeconomy Here we characterized a new GH glucuronoxylanase from Bacillus pumilus BpXyn A for its potential in producing xylooligosaccharides XOS BpXyn A showed tolerance to ethanol and NaCl and released both linear and branched XOS containing MeGlcA at the penultimate nonreducing end residue Its X-ray structure determined at resolution revealed high similarity to other glucuronoxylanases Furthermore BpXyn A achieved higher xylan conversion yields from corn cob and Eucalyptus sawdust than Ruminococcus champanellensisRcXyn A Finally fermentation assays showed that Bifidobacterium adolescentis metabolized neutral ... More
Transformation of agro-industrial products into value-added products, such as prebiotic oligosaccharides, is a key element of the emerging bioeconomy. Here, we characterized a new GH30_8 glucuronoxylanase from Bacillus pumilus (BpXyn30_8A) for its potential in producing xylooligosaccharides (XOS). BpXyn30_8A showed tolerance to ethanol and NaCl and released both linear and branched XOS containing MeGlcA at the penultimate nonreducing end residue. Its X-ray structure, determined at 2.16 Å resolution, revealed high similarity to other glucuronoxylanases. Furthermore, BpXyn30_8A achieved higher xylan conversion yields from corn cob and Eucalyptus sawdust than Ruminococcus champanellensisRcXyn30A. Finally, fermentation assays showed that Bifidobacterium adolescentis metabolized neutral XOS to acetate and lactate, whereas acidic XOS were poorly utilized. These results highlight the potential of BpXyn30_8A as a valuable enzyme for the green transformation of plant biomass into prebiotic oligosaccharides with promising applications in human and animal nutrition, health, and biotechnology. Less
Stable carbon and nitrogen isotope ratios are widely used in the life sciences to investigate diet trophic interactions and metabolic fluxes but conventional isotope ratio mass spectrometry requires milligram-scale samples limiting its applicability to small or rare biological specimens Fourier Transform Isotopic Ratio Mass Spectrometry FT IsoR MS enables amino acid resolved isotope analysis in a proteomics-compatible workflow and has previously been demonstrated at the microgram scale Here we assess the lower sample limit of FT IsoR MS by integrating it with single-cell proteomics style sample preparation Using human HeLa cells cultured in C-glucose enriched and control media we show ... More
Stable carbon and nitrogen isotope ratios are widely used in the life sciences to investigate diet, trophic interactions, and metabolic fluxes, but conventional isotope ratio mass spectrometry requires milligram-scale samples, limiting its applicability to small or rare biological specimens. Fourier Transform Isotopic Ratio Mass Spectrometry (FT IsoR MS) enables amino acid–resolved isotope analysis in a proteomics-compatible workflow and has previously been demonstrated at the microgram scale. Here, we assess the lower sample limit of FT IsoR MS by integrating it with single-cell proteomics–style sample preparation. Using human HeLa cells cultured in 13C-glucose–enriched and control media, we show that reliable relative δ13C measurements can be obtained from as few as 50 cells, corresponding to <10 ng of total protein, with a precision of approximately ±9‰. The observed amino acid–specific labeling patterns are metabolically coherent and consistent with bulk measurements, while smaller cell numbers (≤10 cells) do not yield statistically robust results. These findings establish the practical sensitivity threshold of FT IsoR MS at the low-nanogram level and demonstrate its suitability for isotope-resolved analyses of small cell populations, micro-organoids, and other low-input biological samples, thereby extending stable isotope analysis toward single-cell–scale applications. Less