1209 Citations
Aconitate decarboxylase an enzyme member of the MmgE-PrpD family of proteins has gained significant attention in the last decade as a therapeutic target for cancer and inflammatory diseases Its product itaconate is a multifunctional metabolite shown to drive several disease states Though extensively studied in cellulo and in vivo this protein is biochemically and mechanistically under characterized and although a family of inhibitors has been described no ligand-bound structures have yet been determined In this work we present a thorough structural investigation that yielded the first ligand-bound structure of this protein family which required the generation of artifact-free apo crystals ... More
Aconitate decarboxylase 1, an enzyme member of the MmgE-PrpD family of proteins, has gained significant attention in the last decade as a therapeutic target for cancer and inflammatory diseases. Its product, itaconate, is a multifunctional metabolite shown to drive several disease states. Though extensively studied in cellulo and in vivo, this protein is biochemically and mechanistically under characterized and although a family of inhibitors has been described, no ligand-bound structures have yet been determined. In this work we present a thorough structural investigation that yielded the first ligand-bound structure of this protein family, which required the generation of artifact-free apo crystals. We also developed a novel, low-consumption, robust kinetic assay and investigated active site and allosteric mutants to further elucidate structural and dynamic activity relationships of this protein. Less
Fc RIIa encoded by FCGR A is a widely expressed Fc receptor implicated in autoimmunity and infectious disease susceptibility To fine-map the rheumatoid arthritis RA association at the complex FCGR locus we combined gene-specific resequencing genetic association studies in UK and Spanish European cohorts functional genomics structural biology biophysical analyses and cellular assays We identified a common European FCGR A haplotype A defined by Q W H H and the RA-associated SNP rs which showed the strongest association with RA Multi-omics analyses demonstrated that A is associated with reduced expression of the soluble FCGR A splice variant and lower circulating ... More
FcγRIIa, encoded by FCGR2A, is a widely expressed Fc receptor implicated in autoimmunity and infectious disease susceptibility. To fine-map the rheumatoid arthritis (RA) association at the complex FCGR locus, we combined gene-specific resequencing, genetic association studies in UK and Spanish European cohorts, functional genomics, structural biology, biophysical analyses, and cellular assays. We identified a common European FCGR2A haplotype (2A.3), defined by Q27W, H131H, and the RA-associated SNP rs12746613, which showed the strongest association with RA. Multi-omics analyses demonstrated that 2A.3 is associated with reduced expression of the soluble FCGR2A splice variant and lower circulating soluble FcγRIIa levels. Functional studies revealed altered IgG interactions and delayed FcγRIIa signal transduction associated with Q27W, while structural analyses found no evidence for stable ectodomain dimerisation. Together, these findings identify 2A.3 as an important functional contributor to RA susceptibility and provide mechanistic insight into how FCGR2A variation may influence immune regulation and disease risk in Europeans. Less
The ribosome is a universally conserved and essential protein complex but its biogenesis in mammals is more complex than in single-celled eukaryotes To explore this added complexity we conducted a protein protein interaction screen in human cells This led to the identification of the eumetazoan-specific SPATA SPATA L CINP C ORF LCC complex as a key regulator of ribosome biogenesis Structural analyses using cryo-EM and X-ray crystallography defined the architecture of LCC Functional studies following acute depletion revealed that each component is essential for pre- S maturation Swapping endogenous LCC components with mutant versions pinpointed critical functional interactions and showed ... More
The ribosome is a universally conserved and essential protein complex, but its biogenesis in mammals is more complex than in single-celled eukaryotes. To explore this added complexity, we conducted a protein–protein interaction screen in human cells. This led to the identification of the eumetazoan-specific SPATA5–SPATA5L1–CINP–C1ORF109 (55LCC) complex as a key regulator of ribosome biogenesis. Structural analyses using cryo-EM and X-ray crystallography defined the architecture of 55LCC. Functional studies following acute depletion revealed that each component is essential for pre-60S maturation. Swapping endogenous 55LCC components with mutant versions pinpointed critical functional interactions and showed that SPATA5’s ATPase activity is more important than SPATA5L1’s. Our findings support that SPATA5 evolved from the solitary yeast ATPase Drg1 into the multiprotein 55LCC complex in metazoans. This work provides insights into the complexity of ribosome biogenesis and lays the foundation for deeper exploration of 55LCC’s role in pre-60S maturation. Less
Babesia bovis is a species of apicomplexan hemoparasitic protozoa that can be transmitted by ticks causing a global cattle disease As it depends mainly on the glycolytic pathway for energy production and life cycle maintenance glycolytic enzymes are possible targets for drug development against Babesia Glyceraldehyde- -Phosphate Dehydrogenase GAPDH has been one of such targets against several parasitic organisms It performs the reversible oxidative phosphorylation of glyceraldehyde- -phosphate to -bisphospho-D-glycerate in the presence of nicotinamide adenine dinucleotide The protocol for Babesia bovis Glyceraldehyde- -Phosphate Dehydrogenase BbGAPDH expression and purification has been developed to yields of mg of pure protein per ... More
Babesia bovis is a species of apicomplexan hemoparasitic protozoa that can be transmitted by ticks, causing a global cattle disease. As it depends mainly on the glycolytic pathway for energy production and life cycle maintenance, glycolytic enzymes are possible targets for drug development against Babesia. Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) has been one of such targets, against several parasitic organisms. It performs the reversible oxidative phosphorylation of glyceraldehyde-3-phosphate to 1,3-bisphospho-D-glycerate in the presence of nicotinamide adenine dinucleotide. The protocol for Babesia bovis Glyceraldehyde-3-Phosphate Dehydrogenase (BbGAPDH) expression and purification has been developed to yields of 28 mg of pure protein per liter of culture medium, with a specific activity of 55.5 ± 6.99 U mg−1 after His-tag removal, indicating preservation of enzymatic activity. For crystallization, the His-tag removal proved essential. Crystals diffracted to 3.12 Å resolution in the P3121 space group; NAD+ cofactor molecules are observed in their respective sites. Comparisons to the theoretical model indicate a number of side chain conformation differences. These results provide support for future enzyme inhibition assays, in addition to crystallization assays with potential inhibitors. Less
NADP-dependent malic enzyme NADP-ME has been repeatedly co-opted into distinct metabolic roles across plants most prominently as the decarboxylase of NADP-ME-type C photosynthesis In maize the plastidic C - and nonC -NADP-ME isoforms are closely related in sequence yet display strikingly different catalytic properties suggesting that small changes in ligand recognition can re-tune reaction chemistry However mechanistic interpretation has been hampered by the scarcity of plant NADP-ME structures captured in catalytically informative ligand-bound states Here we integrate X-ray crystallography with structure-guided docking and atomistic molecular dynamics MD to resolve ligand-site interaction networks across reaction states We determined a structure of ... More
NADP-dependent malic enzyme (NADP-ME) has been repeatedly co-opted into distinct metabolic roles across plants, most prominently as the decarboxylase of NADP-ME-type C4 photosynthesis. In maize, the plastidic C4- and nonC4-NADP-ME isoforms are closely related in sequence yet display strikingly different catalytic properties, suggesting that small changes in ligand recognition can re-tune reaction chemistry. However, mechanistic interpretation has been hampered by the scarcity of plant NADP-ME structures captured in catalytically informative, ligand-bound states. Here, we integrate X-ray crystallography with structure-guided docking and atomistic molecular dynamics (MD) to resolve ligand-site interaction networks across reaction states. We determined a 2.55 Å structure of maize plastidic nonC4-NADP-ME bound to NADP+, pyruvate, and Mg2+, revealing a conserved NADP-ME fold with localized active-site flexibility. Comparison with maize C4-NADP-ME uncovers isoform-specific rewiring of NADP+ and pyruvate contacts, with the nonC4 enzyme forming a denser product–cofactor interaction network. To access substrate-bound states, we reconstructed malate-NADP+-Mg2+ complexes by docking followed by MD, identifying distinct malate-Mg2+ coordination geometries and alternative NADP+ positioning between isoforms. Together, these structures and simulations provide a network-level framework for plastidic NADP-ME functional diversification and generate testable hypotheses for how ligand coordination drives isoform-specific catalysis. Less
Peptide-activated G protein-coupled receptors GPCRs play crucial roles in numerous diseases but remain difficult therapeutic targets due to the challenges in developing small-molecule drugs Here we explore structure-based strategies to identify small-molecule agonists of neurotensin NTS receptors which hold promise for developing non-opioid analgesics Chemical libraries of drug-like molecules are first designed based on a receptor-peptide complex and then million compounds are computationally docked to the orthosteric binding site of the NTS receptor A set of top-ranked compounds is synthesized and seven of these are experimentally confirmed to activate the NTS receptor Structure-guided optimization yields NTS ligands with signaling signatures ... More
Peptide-activated G protein-coupled receptors (GPCRs) play crucial roles in numerous diseases, but remain difficult therapeutic targets due to the challenges in developing small-molecule drugs. Here, we explore structure-based strategies to identify small-molecule agonists of neurotensin (NTS) receptors, which hold promise for developing non-opioid analgesics. Chemical libraries of drug-like molecules are first designed based on a receptor-peptide complex, and then 14.5 million compounds are computationally docked to the orthosteric binding site of the NTS1 receptor. A set of 39 top-ranked compounds is synthesized, and seven of these are experimentally confirmed to activate the NTS1 receptor. Structure-guided optimization yields NTS1 ligands with signaling signatures distinct from the endogenous peptide, and these compounds also exhibit high affinity for the NTS2 receptor. High-resolution crystal structures of two agonists bound to the NTS1 receptor confirm predicted binding modes and reveal key determinants of activation. In vivo, the compounds produce robust antinociception in rodents without inducing hypotension, consistent with a contribution of NTS2 receptor activity. To facilitate broader application of our virtual screening approach to peptide-binding GPCRs, we provide access to tailored chemical libraries containing billions of readily synthesizable compounds. Less
The evolution of C photosynthesis required extensive modification of ancestral enzymes enabling the development of an efficient carbon concentrating mechanism A key example is NADP-malic enzyme NADP-ME which in maize and sorghum members of the same C lineage underwent gene duplication and neofunctionalization resulting in plastidic isoforms with distinct oligomeric states a tetrameric C -specific isoform and a dimeric housekeeping nonC isoform In this study we resolve the structural basis of this oligomeric divergence using X-ray crystallography cryo-electron microscopy and molecular modeling combined with targeted biochemical analysis Our findings demonstrate that the N-terminal region of nonC -NADP-ME is involved in ... More
The evolution of C4 photosynthesis required extensive modification of ancestral enzymes enabling the development of an efficient carbon concentrating mechanism. A key example is NADP-malic enzyme (NADP-ME), which, in maize and sorghum—members of the same C4 lineage—underwent gene duplication and neofunctionalization, resulting in 2 plastidic isoforms with distinct oligomeric states: a tetrameric C4-specific isoform and a dimeric housekeeping (nonC4) isoform. In this study, we resolve the structural basis of this oligomeric divergence using X-ray crystallography, cryo-electron microscopy, and molecular modeling combined with targeted biochemical analysis. Our findings demonstrate that the N-terminal region of nonC4-NADP-ME is involved in its oligomeric organization, whereas a suite of adaptive substitutions at the dimer interface drives the transition to the stable tetramer characteristic of the C4 isoform. Moreover, the C-terminal region stabilizes the oligomeric states of C4- and nonC4-NADP-ME through specific interactions with adaptive residues. We propose that tetramerization mitigates aggregation at the high expression levels demanded by the C4 cycle and likely creates a scaffold for the emergence of regulatory properties. Collectively, the data show that remodeling of terminal domains and inter-subunit interfaces rewires the quaternary architecture of the enzymes, illustrating how subtle structural changes can drive the evolution of complex innovations such as C4 photosynthesis. Less
Four I domain-containing integrins and have evolved to recognize various members of the collagen family A defining structural feature of these receptors is the presence of the C helix within their I domains Glu Arg in the I domain a structure solely found in collagen-binding integrins whose functional mechanism has remained unclear To elucidate the functional role of the C helix and to assess the contribution of the mechanistically important Arg -Glu ion pair in I domain activation we created two variants IR A and IE A Functional solid-phase binding assays and surface plasmon resonance revealed that these variants exhibit ... More
Four αI domain-containing integrins (α1β1, α2β1, α10β1, and α11β1) have evolved to recognize various members of the collagen family. A defining structural feature of these receptors is the presence of the αC helix within their αI domains (Glu284–Arg288 in the α2I domain), a structure solely found in collagen-binding integrins, whose functional mechanism has remained unclear. To elucidate the functional role of the αC helix, and to assess the contribution of the mechanistically important Arg288-Glu318 ion pair in α2I domain activation, we created two variants α2IR288A and α2IE318A. Functional solid-phase binding assays and surface plasmon resonance revealed that these variants exhibit strikingly increased avidity for collagens I and IV, whereas affinities for triple-helical GFOGER peptides, representing a single binding motif, were only slightly increased. The variants displayed distinct ligand-binding profiles, including differences in association and dissociation constants. To understand these differences at a structural level, we determined four novel X-ray crystal structures of the variants, including both ligand-free and ligand-bound (succinic acid or malic acid) states. These structures revealed differences in the folding of the αC helix region, suggesting that its conformation regulates α2I domain activation and ligand specificity, consistent with our binding assays. Additionally, the structures captured the movements of the catalytic metal ion in the metal ion dependent adhesion site, providing a detailed view of the sophisticated activation mechanism of the α2I domain. Less
Fragment-based drug discovery FBDD is an effective approach for exploring chemical space using small low-affinity fragments as starting points to facilitate development of lead compounds Strategies to improve fragment potency include fragment merging and linking to generate higher-affinity inhibitors Recently artificial intelligence AI and machine learning ML have accelerated this process through structure-based optimization and generative compound design Here we present an AI-assisted FBDD workflow applied to the SARS-CoV- macrodomain Mac a conserved viral protein involved in immune evasion and ADP-ribose metabolism Using available structural data and previously identified fragments we combined deep learning with molecular docking to design novel ... More
Fragment-based drug discovery (FBDD) is an effective approach for exploring chemical space using small, low-affinity fragments as starting points to facilitate development of lead compounds. Strategies to improve fragment potency include fragment merging and linking to generate higher-affinity inhibitors. Recently, artificial intelligence (AI) and machine learning (ML) have accelerated this process through structure-based optimization and generative compound design. Here, we present an AI-assisted FBDD workflow applied to the SARS-CoV-2 macrodomain (Mac1), a conserved viral protein involved in immune evasion and ADP-ribose metabolism. Using available structural data and previously identified fragments, we combined deep learning with molecular docking to design novel Mac1 binders. Selected compounds were synthesized and validated by NMR spectroscopy and X-ray crystallography, demonstrating improved binding relative to the original fragment hits with KD values in the range of 299-990 µM. This study demonstrates the advantages of integrating AI with FBDD to streamline molecular design, providing a data-driven framework for discovering new Mac1 inhibitors and guiding future antiviral drug development. Less
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
Background Transforming Growth Factor Beta TGF plays a dual role in cancer acting as a tumor suppressor early in disease but promoting progression and immune evasion when dysregulated In pancreatic ductal adenocarcinoma PDAC TGF -driven desmoplasia fosters chemoresistance and immunosuppression limiting therapeutic efficacy NIS a fully human monoclonal antibody targeting TGF demonstrated anti-fibrotic and immunomodulatory activity in preclinical models and early-phase trials Methods We conducted a randomized open-label phase II study in treatment-na ve metastatic PDAC patients to evaluate NIS spartalizumab anti-PD- combined with nab-paclitaxel gemcitabine ABRA GEM versus ABRA GEM alone Primary endpoint was progression-free survival PFS secondary endpoints ... More
Background: Transforming Growth Factor Beta (TGFβ) plays a dual role in cancer, acting as a tumor suppressor early in disease but promoting progression and immune evasion when dysregulated. In pancreatic ductal adenocarcinoma (PDAC), TGFβ-driven desmoplasia fosters chemoresistance and immunosuppression, limiting therapeutic efficacy. NIS793, a fully human monoclonal antibody targeting TGFβ, demonstrated anti-fibrotic and immunomodulatory activity in preclinical models and early-phase trials. Methods: We conducted a randomized, open-label, phase II study in treatment-naïve metastatic PDAC patients to evaluate NIS793 ± spartalizumab (anti-PD-1) combined with nab-paclitaxel/gemcitabine (ABRA/GEM) versus ABRA/GEM alone. Primary endpoint was progression-free survival (PFS); secondary endpoints included overall survival (OS), safety, pharmacokinetics, and biomarker analyses. Exploratory assessments included paired tumor RNA sequencing, cfDNA profiling, and plasma proteomics. Results: NIS793 demonstrated target engagement and suppression of TGFβ signaling, confirmed by transcriptomic and proteomic analyses. Stromal remodeling was evident, with significant downregulation of CAF markers (ACTA2, FAP) and collagen-related signatures. Despite proof-of-mechanism, clinical efficacy was not observed: median PFS and OS were comparable or numerically worse in NIS793 arm versus control (HR for OS in NIS793+ABRA/GEM vs ABRA/GEM: 1.32; 95% CI: 0.84–2.07). Safety profile was manageable, with no unexpected toxicities. Biomarker data revealed increased expression of neutrophil-related genes post-treatment, suggesting potential induction of tumor-promoting inflammation. Conclusions: NIS793 effectively inhibited TGFβ signaling and led to stroma remodeling but failed to improve outcomes in metastatic PDAC. These findings highlight the complexity of TGFβ biology and caution against its blockade in combination with chemotherapy for PDAC. Future strategies should consider context-dependent effects of TGFβ inhibition(NCT04390763). 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
Extracellular vesicles EVs are versatile biological nanoparticles with applications in therapeutics diagnostics and biotechnology Current production methods relying on transient transfection or chemical conjugation suffer from high variability limited scalability and heterogeneous EV populations Here we present a synthetic-biology-based biomaterial manufacturing platform that uses CRISPR-Cas genome editing to generate stable HEK T cell lines for continuous production of surface-functionalized EVs A fusion construct encoding mCherry-C C was site-specifically integrated into the AAVS safe-harbor locus enabling consistent and heritable expression of EV membrane proteins without repeated transfection Engineered cells produced EVs with uniform size nm preserved canonical markers CD and ALIX ... More
Extracellular vesicles (EVs) are versatile biological nanoparticles with applications in therapeutics, diagnostics, and biotechnology. Current production methods relying on transient transfection or chemical conjugation suffer from high variability, limited scalability, and heterogeneous EV populations. Here, we present a synthetic-biology-based biomaterial manufacturing platform that uses CRISPR-Cas9 genome editing to generate stable HEK293T cell lines for continuous production of surface-functionalized EVs. A fusion construct encoding mCherry-C1C2 was site-specifically integrated into the AAVS1 safe-harbor locus, enabling consistent and heritable expression of EV membrane proteins without repeated transfection. Engineered cells produced EVs with uniform size (120−130 nm), preserved canonical markers (CD63 and ALIX), and enhanced surface-display efficiency compared with transiently transfected controls. These vesicles exhibited robust cellular uptake and maintained structural and functional stability for over 25 passages (∼3 months), confirming durable genome-encoded production. Overall, this platform eliminates batch-to-batch variability inherent to transient systems and provides a genetically defined route to biofunctional nanomaterial fabrication. This approach links genetic design to nanoscale surface functionality, establishing a versatile foundation for reproducible biomanufacturing of engineered EVs for biomaterial, therapeutic, and diagnostic applications. 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
The Zika virus protease composed of the cofactor region from NS B and the N-terminal region of NS plays a critical role in viral polyprotein maturation and represents an attractive therapeutic target However developing small-molecule inhibitors for its highly hydrophilic active site remains challenging highlighting the importance of pursuing allosteric inhibition strategies In this study we engineered an NS B-NS protease containing an -residue NS B sequence linked to the N-terminal region of NS via a glycine-rich linker We determined its crystal structure and obtained the solution NMR spectrum with backbone resonance assigned This new construct was used in fragment ... More
The Zika virus protease, composed of the cofactor region from NS2B and the N-terminal region of NS3, plays a critical role in viral polyprotein maturation and represents an attractive therapeutic target. However, developing small-molecule inhibitors for its highly hydrophilic active site remains challenging, highlighting the importance of pursuing allosteric inhibition strategies. In this study, we engineered an NS2B-NS3 protease containing an 18-residue NS2B sequence linked to the N-terminal region of NS3 via a glycine-rich linker. We determined its crystal structure and obtained the solution NMR spectrum with backbone resonance assigned. This new construct was used in fragment screening and two new fragments were identified. This design excludes the C-terminal part of NS2B cofactor region, whose conformation is influenced by substrate or inhibitor binding, making the construct particularly valuable for screening and characterizing allosteric inhibitors. 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
This protocol was used to grow coxsackievirus A CVA A protease crystals that were used as a surrogate for enterovirus A EV-A A protease in high-throughput crystallographic fragment screening and in the crystallographic screening of follow-up compounds against the target PDB ID of apo-structure solved using sulfur phasing pdb jc Picornaviridae primarily CVA and EV-A are the causative agents of paediatric hand-foot-and-mouth disease These viruses are a target for pandemic preparedness due to the risk of higher-order complications in a large-scale outbreak The A protease of the viruses is responsible for self-cleavage from the polyprotein allowing for correct folding and ... More
This protocol was used to grow coxsackievirus A16 (CVA16) 2A protease crystals that were used as a surrogate for enterovirus A71 (EV-A71) 2A protease in high-throughput crystallographic fragment screening and in the crystallographic screening of follow-up compounds against the target. (PDB ID of apo-structure solved using sulfur phasing: pdb_000029jc)
Picornaviridae, primarily CVA16 and EV-A71, are the causative agents of paediatric hand-foot-and-mouth disease. These viruses are a target for pandemic preparedness due to the risk of higher-order complications in a large-scale outbreak. The 2A protease of the viruses is responsible for self-cleavage from the polyprotein, allowing for correct folding and assembly of capsid proteins in the final stages of viral replication. Inhibition deranges capsid folding and assembly, preventing formation of mature virions in host cells and making the protease a valuable target for antiviral activity. Less
Picornaviridae, primarily CVA16 and EV-A71, are the causative agents of paediatric hand-foot-and-mouth disease. These viruses are a target for pandemic preparedness due to the risk of higher-order complications in a large-scale outbreak. The 2A protease of the viruses is responsible for self-cleavage from the polyprotein, allowing for correct folding and assembly of capsid proteins in the final stages of viral replication. Inhibition deranges capsid folding and assembly, preventing formation of mature virions in host cells and making the protease a valuable target for antiviral activity. 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
This protocol describes the testing of how protein crystals survive exposure to organic chemistry reagents and solvents with the purpose of reducing crystal attrition prior to high-throughput crystallographic screening of ligands from crude reaction mixtures containing the reagents A step-by-step description is included describing how to complete this process using the XChem laboratory and Protein Crystallisation Facility at Diamond Light Source The data analysis cutoffs rationale and the spreadsheet used during analysis are also included within the attachments
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