X-ray Crystallography Research Articles

Optimized Second-Generation IL-4Rα Inhibition: Structural and Molecular Dynamics Properties of Rademikibart Fab-IL-4Rα Complex

Introduction: Global Burden of Disease studies showed atopic dermatitis (AD) and asthma are the top two immune-mediated inflammatory diseases at younger age [1]. Dupilumab was the initial first-generation interleukin-4 receptor alpha (IL-4Rα) inhibitor for treating both type I and type II IL-4Rα-dependent inflammatory disorders. Following recent clinical trials, rademikibart (previously, CBP-201) emerges as an optimized next-generation human monoclonal antibody with higher binding affinity to IL-4Rα compared to dupilumab [2]. It demonstrated better effect in inhibiting STAT6 intracellular signaling in vitro and provided similar potency inhibiting both IL-4 induced TARC release and IL-4 induced B cell activation [2]. Materials & Methods: X-ray crystallography was used to determine the atomic resolution 3D structure of rademikibart fragment antigen binding (Fab) bound to IL-4Rα. This structure was analyzed and compared computationally with the 2.82 Å resolution crystal structure of dupilumab Fab bound to IL-4Rα (Protein Data Bank Code 6WGL). Molecular dynamics studies on rademikibart and dupilumab bound to IL-4Rα examined the stability of the complexes and effects of amino acid mutations on complex formation. Results: The x-ray crystal structure of rademikibart Fab bound to IL-4Rα was determined at 2.71Å and compared to the complex of dupilumab Fab and IL-4Rα. The rotation angle between dupilumab and rademikibart bound to IL- 4Rα is 59.17°. This rotation enables the epitope of rademikibart, but not dupilumab, on IL-4Rα to overlap more closely with the conserved binding interface utilized by IL-4 and IL-13 cytokines. Molecular dynamics simulations of rademikibart Fab and dupilumab Fab complexed with IL-4Rα showed the third interface loop (residues 148 to 152 in domain 2) of IL-4Rα interacts directly with rademikibart, which is absent in dupilumab/IL- 4Rα complex. This finding is confirmed by analysis of the hydrogen bond interactions at the interface between the antibodies and IL-4Rα, demonstrating superior binding energy for rademikibart. Through single amino acid mutation analysis on rademikibart, we identified residue Y50 on rademikibart as the key residue interacting with IL- 4Rα’s third interface loop. Conclusion: Our data provide a molecular and structural rationale for the enhanced IL-4Rα inhibition by rademikibart over dupilumab, confirming rademikibart as an optimized second-generation IL-4Rα inhibitor.

Rational design of uncleaved prefusion-closed trimer vaccines for human respiratory syncytial virus and metapneumovirus.

Respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) cause human respiratory diseases and are major targets for vaccine development. In this study, we design uncleaved prefusion-closed (UFC) trimers for the fusion protein (F) of both viruses by examining mutations critical to F metastability. For RSV, we assess four previous prefusion F designs, including the first and second generations of DS-Cav1, SC-TM, and 847A. We then identify key mutations that can maintain prefusion F in a native-like, closed trimeric form (up to 76%) without introducing any interprotomer disulfide bond. For hMPV, we develop a stable UFC trimer with a truncated F2-F1 linkage and an interprotomer disulfide bond. Dozens of UFC constructs are characterized by negative-stain electron microscopy (nsEM), x-ray crystallography (11 RSV-F structures and one hMPV-F structure), and antigenic profiling. Using an optimized RSV-F UFC trimer as bait, we identify three potent RSV neutralizing antibodies (NAbs) from a phage-displayed human antibody library, with a public NAb lineage targeting sites Ø and V and two cross-pneumovirus NAbs recognizing site III. In mouse immunization, rationally designed RSV-F and hMPV-F UFC trimers induce robust antibody responses with high neutralizing titers. Our study provides a foundation for future prefusion F-based RSV and hMPV vaccine development.

PnictogenIII Dications Supported by BZIMPY Ligands.

Two homologous series of pnictogen(III) dications, stabilized by 2,6-bis(benzimidazole-2-yl)pyridine ligands have been prepared. Both series contain PnIII-X moieties (Pn = P, As, Sb, Bi; X = Cl or Ph) and have been fully characterized using spectroscopic methods including X-ray crystallography. The Lewis acidity of these compounds has also been probed by computational methods; the results suggest that the dictations are strong Lewis acids, with the PnCl2+ compounds being more acidic than the PnPh2+ compounds, and with Lewis acidity increasing from P to Bi, in both series. The PhP2+-containing compound was also found to be a versatile PIII transfer reagent, leading to new synthetic routes for various PhP-containing compounds. The redox chemistry of all compounds has also been probed using cyclic voltammetry and chemical reductions. In some cases the resulting PnI moieties could be trapped using diazabutadienes.

Unexpected XPS Binding Energy Observations Further Highlighted by DFT Calculations of Ruthenocene-Containing [IrIII(ppy)2(RCOCHCORc)] Complexes: Cytotoxicity and Crystal Structure of [Ir(ppy)2(FcCOCHCORc)

The series of iridium(III) complexes, [Ir(ppy)2(RCOCHCOR′)], with R = CH3 and R′ = CH3 (1), Rc (2), and Fc (3), as well as R = Rc and R′ = Rc (4) or Fc (5), and R = R′ = Fc (6), ppy = 2-phenylpyridinyl, Fc = FeII(η5–C5H4)(η5–C5H5), and Rc = RuII(η5–C5H4)(η5–C5H5), has been investigated by single-crystal X-ray crystallography and X-ray photoelectron spectroscopy (XPS) supplemented by DFT calculations. Here, in the range of 3.74 ≤ ΣχR ≤ 4.68, for Ir 4f, Ru 3d and 3p and N 1s orbitals, binding energies unexpectedly decreased with increasing ΣχR (ΣχR = the sum of Gordy group electronegativities of the R groups on β-diketonato ligands = a measure of electron density on atoms), while in Fe 2p orbitals, XPS binding energy, as expected, increased with increasing ΣχR. Which trend direction prevails is a function of main quantum level, n = 1, 2, 3…, sub-quantum level (s, p, d, and f), initial state energies, and final state relaxation energies, and it may differ from compound series to compound series. Relations between DFT-calculated orbital energies and ΣχR followed opposite trend directions than binding energy/ΣχR trends. X-ray-induced decomposition of compounds was observed. The results confirmed good communication between molecular fragments. Lower binding energies of both the Ir 4f7/2 and N 1s photoelectron lines are associated with shorter Ir-N bond lengths. Cytotoxic tests showed that 1 (IC50 = 25.1 μM) and 3 (IC50 = 37.8 μM) are less cytotoxic against HeLa cells than cisplatin (IC50 = 1.1 μM), but more cytotoxic than the free β-diketone FcCOCH2COCH3 (IC50 = 66.6 μM).

Covalent Folding of Fluorinated Polyphenylene by Sulfur Fluorine Annulative Substitution.

We report that fluorinated polyphenylene P50 undergoes folding-like sulfur fluorine annulative substitution (SFAS) to form a well-defined tubular helix H50. This is achieved with atomic precision in an exceptionally efficient manner, involving the simultaneous transformation of 98 C-F bonds with the yield per reaction site approaching 99.9%. The desired product H50 features a fully fused dibenzothiophene skeleton of 5.8 helical turns in total. It is truly monodisperse (Đ = 1.0) in nature, allowing for thorough spectroscopic characterizations. Unambiguous single crystal X-ray structure, distinct chiroptical properties, and intriguing through-cavity threading complexation are described. These results, in addition to those of H50's lower congeners, H34 and H18, illustrate the great potential of folding-like SFAS in shaping random-coiled polymer chains into higher-order functional structures.

Diarenofuran[b]-fused BODIPYs: One-Pot SNAr-Suzuki Synthesis and Properties.

We present a streamlined methodology that integrates regioselective tetrahalogen-BODIPY and o-hydroxyphenylboronic acid in a one-pot process, leveraging SNAr nucleophilic substitution in conjunction with Suzuki coupling to afford diarenofuran [b]-fused BODIPYs (DBFB1-9) with commendable yields (85-95%) and short reaction times (0.5-1.0 h). X-ray structure analyses of DBFB5,7-9 elucidate that these diarenofuran[b]-fused BODIPYs adopt a "butterfly" conformation, maintaining a highly rigid planarity. A comprehensive examination of the spectroscopic and electrochemical properties of these diarenofuran[b]-fused BODIPY derivatives, incorporating various substituents, reveals intriguing characteristics, including pronounced absorption and emission in the near-infrared region (NIR), elevated fluorescence quantum yields (ΦF = 75-89% in dichloromethane), and tunable HOMO-LUMO levels. Remarkably, compared to DBFB1-8, DBFB9 possesses a large extended π-conjugated system, resulting in significant red shifts in its absorption and emission maxima, reaching 623 and 635 nm, respectively, and a reduced HOMO-LUMO gap. Despite this substantial structural expansion, DBFB9 maintains a surprisingly high fluorescence quantum yield (ΦF = 80%), underscoring its exceptional photophysical performance and strong potential for applications requiring efficient NIR emission and robust fluorescence retention.

Discovery and Optimization of First-in-Class Molecular Glue Degraders of the WIZ Transcription Factor for Fetal Hemoglobin Induction to Treat Sickle Cell Disease.

Sickle cell disease (SCD) is a prevalent, life-threatening condition with few treatment options, attributed to a heritable mutation in β-hemoglobin. Therapeutic induction of fetal hemoglobin (HbF) with small molecules has been pursued as a treatment to ameliorate many disease complications but with limited success. Herein, we report the discovery of 10, a novel, potent, and selective molecular glue degrader of the transcription factor WIZ that robustly induces HbF expression as a potential treatment for SCD. 10 was optimized from a phenotypic screening hit utilizing insights from X-ray crystallography and computational modeling to improve potency, selectivity and in vivo exposure. In an hNBSGW mouse xenograft model, 10 demonstrated robust WIZ degradation and HbF induction. These results highlight the potential of WIZ degraders as a promising therapy for sickle cell disease.

Structural insights into thyroid hormone receptors.

Thyroid hormone receptors (TRs) are essential components of the endocrine system, mediating the cellular effects of thyroid hormones. The two TR genes, THRA and THRB, encode four isoforms, with TRα1 and TRβ1 being the most prevalent. TRs are ligand-dependent transcription factors and members of the nuclear receptor superfamily, indispensable for human growth, development and metabolism. Dysfunctional TR signaling can lead to conditions like resistance to thyroid hormone (RTH) syndrome, thyroid cancer, and metabolic disorders. Structurally, TRs comprise several domains: a variable N-terminal domain, a conserved DNA-binding domain, and a ligand-binding domain that mediates interaction with hormones and transcriptional coregulators. TRs predominantly function as heterodimers with the retinoid X receptor (RXR), binding to thyroid hormone response elements (TREs) in target genes to regulate their transcription. This review examines the structural studies on TRs, primarily performed through X-ray crystallography, that have provided detailed insights into TR functions, including DNA recognition, ligand binding, and coregulator interactions. We also discuss how these findings have deepened our understanding of TR mechanisms and contributed to the interpretation of pathogenic mutations.

Interplay of Stereochemistry and Charge Governs Guest Binding in Flexible ZnII4L4 Cages.

Here, we report the synthesis of a family of chiral ZnII4L4 tetrahedral cages by subcomponent self-assembly. These cages contain a flexible trialdehyde subcomponent that allows them to adopt stereochemically distinct configurations. The incorporation of enantiopure 1-phenylethylamine produced Δ4 and Λ4 enantiopure cages, in contrast to the racemates that resulted from the incorporation of achiral 4-methoxyaniline. The stereochemistry of these ZnII4L4 tetrahedra was characterized by X-ray crystallography and chiroptical spectroscopy. Upon binding the enantiopure natural product podocarpic acid, the ZnII stereocenters of the enantiopure Δ4-ZnII4L4 cage retained their Δ handedness. In contrast, the metal stereocenters of the enantiomeric Λ4-ZnII4L4 cage underwent inversion to a Δ configuration upon encapsulation of the same guest. Insights gained about the stereochemical communication between host and guest enabled the design of a process for acid/base-responsive guest uptake and release, which could be followed by chiroptical spectroscopy.

Utilizing MEDT analysis of [3 + 2] cycloaddition reaction: x-ray crystallography of spirooxindole linked with thiophene/furan heterocycles and triazole framework.

Hybridization of spirooxindole with different pharmacophores such as triazole and heterocycle such as thiophene and furan moiety was achieved by the [3 + 2] cycloaddition (32CA) reaction approach. Structural investigations of the compounds 4a and 4b were performed using X-ray single crystal structure determinations and Hirshfeld analysis. Both compounds crystallized in monoclinic crystal system. The space group is P21/c for 4a and P21/n for 4b. The crystal parameters are a = 10.2619(3) Å, b = 13.6776(3) Å, c = 10.9318(3), β = 116.640(4)° for the former while a = 13.0012(1) Å, b = 14.9692(1) Å, c = 14.1178(1) Å, β = 97.101(1)° for the latter. In both compounds, the aryl group and the triazole moieties are twisted from one another. The twist angle is 84.75˚for 4a while 86.64˚ for 4b. Based on Hirshfeld calculations, the Cl…H, O…H, N…H and C…H non-covalent interactions in 4a while the O…H interactions in 4b are the most important. The molecular mechanism of the key 32CA reaction between the in situ generated azomethine ylides and the corresponding chalcones has been studied within the Molecular Electron Density Theory (MEDT). The MEDT study reveals that the low activation energies and high experimental selectivity are the result of the supernucleophilic character of the ylides and the strong electrophilicity of the chalcones, which favour the process through a high polar character. This high polar character accounts for the total endo selectivity experimentally found.

In Silico Efficacy of Oleic Acid, n-Hexanoic acid, and Epiglobulol Obtained from Conocarpus Lancifolius against Class D β-lactamases

Introduction: Antimicrobial resistance poses an escalating global challenge, compounded by the sluggish pace of new antibiotic discovery. Objectives: This study undertook an in silico analysis of phytochemicals derived from Conocarpus lancifolius, a local plant in the Jazan region of Saudi Arabia, to investigate potential antibacterial agents. Methods: The 3D crystallographic structure of OXA-48 was sourced from the RCSB protein databank (PDBID: 3HBR) and pre-processed. The Lib Dock algorithm facilitated the analysis of optimal binding orientations and potential interactions between compounds and the 3HBR. Results: The docking analysis outcome revealed Oleic acid as the most active compound and the binding mode exhibited that the -OH group of the terminal carboxylic acid moiety formed hydrogen bonds with Thr197 and Ala207, and the oxygen atom showed bonding with Lys116. Moreover, N-hexanoic acid also demonstrated promising binding interactions with the 3HBR, displaying hydrogen bond interactions with Lys208, Tyr211, and Ser70, along with hydrophobic alkyl interactions with Lys208 and Met115 at the protein's active sites. Unfavourable donor-donor and acceptor-acceptor bindings were also observed with Ser70 and Arg250 residues. Conclusions: The OXA-48 and NDM-1 predominate in countries of the Arabian Peninsula. Our findings suggest oleic acid as a promising candidate for combating OXA-48 β-lactamase, pending further assessment of its atomic structure via X-ray crystallography.

Using a NewPt(II) Source to Make Pt(II) Lantern-Shaped Cages, Including Low-Symmetry, Heteroleptic, and Multicavity Examples.

Current synthetic methods towards Pt(II) lantern-shaped cages involve the use of dry solvent, inert atmosphere, lengthy reaction times, and highly variable yields if isolated. Starting materials such as [Pt(CH3CN)4](BF4)2 suffer from a poor shelf-life, reducing the synthetic accessibility of various Pt(II) architectures. A new Pt(II) source (with varied counterions), [Pt(3-ClPy)4](X)2 (3-ClPy = 3-chloropyridine, X = BF4-, OTf-, NO3-), is developed and characterised, showing greatly enhanced shelf-life characteristics under ambient atmospheric conditions. Using this starting material, the assembly of Pt(II) lantern-shaped cages was completed in as little as 1.5 hours with wet solvent and ambient atmosphere. The first examples of low-symmetry, heteroleptic and multicavity Pt(II) lantern-shaped cages are reported using this approach. Attempts towards an M6L8 octahedron using a tritopic ligand instead generate an interesting M2L4 cage with unbound pyridyl arms. Ligands and cages are characterised by NMR spectroscopy, IR spectroscopy, mass spectrometry, and X-ray crystallography in some cases.

Discovery of two new tirucallane-type triterpenes anomers from Toona ciliata

Toona ciliata, a member of the Meliaceae family, is valued for its high-quality timber and medicinal potential in treating various ailments. In this study, two triterpene isomers were isolated and characterised as an inseparable mixture from its chloroform extract using chromatography and spectroscopic techniques. Despite rigorous/extensive attempts to separate these isomers, they co-crystallised in a consistent 67:33 ratio, as verified by X-ray crystallography. The structure was further confirmed by ESI-MS, HRMS,1H NMR,13C NMR, DEPT 90, DEPT 135, COSY, NOESY, HSQC, and HMBC. Additionally, four more compounds were isolated successfully, out of that lutein and dyscusin C found for the first time from this plant.

Determinants of chemoselectivity in ubiquitination by the J2 family of ubiquitin-conjugating enzymes.

Ubiquitin-conjugating enzymes (E2) play a crucial role in the attachment of ubiquitin to proteins. Together with ubiquitin ligases (E3), they catalyze the transfer of ubiquitin (Ub) onto lysines with high chemoselectivity. A subfamily of E2s, including yeast Ubc6 and human Ube2J2, also mediates noncanonical modification of serines, but the structural determinants for this chemical versatility remain unknown. Using a combination of X-ray crystallography, molecular dynamics (MD) simulations, and reconstitution approaches, we have uncovered a two-layered mechanism that underlies this unique reactivity. A rearrangement of the Ubc6/Ube2J2 active site enhances the reactivity of the E2-Ub thioester, facilitating attack by weaker nucleophiles. Moreover, a conserved histidine in Ubc6/Ube2J2 activates a substrate serine by general base catalysis. Binding of RING-type E3 ligases further increases the serine selectivity inherent to Ubc6/Ube2J2, via an allosteric mechanism that requires specific positioning of the ubiquitin tail at the E2 active site. Our results elucidate how subtle structural modifications to the highly conserved E2 fold yield distinct enzymatic activity.

Synthesis of Stable Hypervalent Bromine(III) Complexes by in Situ Oxidation with Lewis Acids Containing sp-Hybridized Nitrogen.

Stable hypervalent bromine(III) compounds were synthesized via aryl bromine oxidation with sp-hybridized nitrogen cations generated by oxime N-O bond cleavage in trifluoroacetic acid. The resulting intramolecular N-Br hypervalent bond is effectively stabilized by the planar xanthone structure. The structures and physicochemical properties of these λ3-bromanes were characterized by X-ray crystallography, cyclic voltammetry, UV-vis spectroscopy, and computational analysis.

Correction: X-ray structures, thermal stabilities and kinetics of guest desolvation of complexes of three fluorenone-derived host compounds with the polar aprotic guest solvent, tetramethylurea

Correction: X-ray structures, thermal stabilities and kinetics of guest desolvation of complexes of three fluorenone-derived host compounds with the polar aprotic guest solvent, tetramethylurea

Nucleophilic Fluoride Anion Delivery from Triazacyclononane-Supported Molecular Ca-F Complexes.

The major source of fluoride, namely calcium difluoride (CaF2), is derived from the mineral fluorspar. Recent advances in the activation of CaF2 via mechanochemical methods have inspired investigation of the fundamental properties of Ca-F bonds in molecular complexes. However, the paucity of well-defined molecular Ca-F-containing complexes undermines systematic understanding of the factors governing nucleophilic fluoride delivery. Here we report the use of a multidentate bis(phenoxide) ligand based on a 1,4,7-triazacyclononane (TACN) scaffold for the formation of well-defined Ca-F complexes and demonstrate their capabilities in fluoride delivery. Key to this synthesis is a desymmetrization step carried out on the TACN ligand within the Ca coordination sphere. A series of stable anionic dinuclear calcium fluoride complexes has then been accessed and characterized by NMR spectroscopy (critically by 19F NMR) and X-ray crystallography. Nucleophilic fluoride delivery can be used to generate C-F, Si-F and S-F bonds, with a notable advance over amide-derived ligand scaffolds being the reduced extent of side reactions derived from competing attack on the electrophile by the less nucleophilic O-donor anionic ligand set.

Structure of human phospholipase D3, a single-strand exonuclease associated with Alzheimer's disease.

Phospholipase D3 (PLD3) has emerged as an important 5'-exonuclease in charge of removing single-stranded DNA in lysosomes. Rare genetic variants of the gene encoding PLD3 have been implicated in late-onset Alzheimer's disease (AD). Ishii etal. have produced the soluble domain of human PLD3 with the aim of determining its three-dimensional structure using X-ray crystallography. The high-resolution structure (2.3 Å) provides new insights into the biochemical properties of the enzyme and paves the way to a deeper understanding of amino acid replacements affecting the stability and activity of the enzyme.

Evaluating the antidiabetes and antioxidant activities of halogenated Schiff bases derived from 4-(diethylamino)salicylaldehyde: in vitro antidiabetes, antioxidant and computational investigation

Six Schiff bases with general name 5-(diethylamino)-2-(((halophenyl)imino)methyl)phenol (where halo = 4-fluoro (H1), 2-fluoro (H2), 2-bromo (H3), 4-bromo (H4), 4-chloro (H5) and 3-chloro-4-fluoro (H6)) were prepared by the condensation reaction between 4-(diethylamino)salicylaldehyde and suitable halogenated aromatic amines. The six halogenated Schiff bases were elucidated using different spectroscopic techniques and the structure of H3 and H6 were confirmed using single-crystal X-ray crystallography. The bond lengths of C7–N1, C7–C8 and C8–C9 obtained from structural analysis for both compounds depicted their enol-tautomeric characteristic form. The Hirshfeld analysis revealed that H‧‧‧H intermolecular contacts contributed most towards the Hirshfeld surfaces of both H3 (47.6%) and H6 (39.9%). Quantum chemical calculation studies showed that H1 and H2 have the highest and lowest energy band gap (∆E = 3.80 eV for H1 and ∆E = 3.73 eV for H2), depicting H2 and H1 as the most and least chemically reactive respectively among all the compounds. α-Amylase and α-glucosidase assay were used to evaluate the antidiabetes prowess of the synthesized compounds. All the compounds exhibited lower IC50 values than acarbose (reference drug) in α-amylase assay experiments and H5 with lowest IC50 value of 63.54 μM could be suggested to have the highest α-amylase inhibitory potential among the test samples. For α-glucosidase assay, H1–H6 displayed good antidiabetic potential. However, none of the compounds outshined acarbose with H6 having highest α-glucosidase inhibitory potential when compared to others i.e., IC50 of H6 = 60.89 μM and IC50 of acarbose = 51.42 μM. We measured the antioxidant potential of H1–H6 exploring 2,2-diphenyl-1-picrylhydrazyl (DPPH), nitric oxide (NO) and ferric reducing ability power (FRAP) assays. The DPPH as well as NO radical scavenging assay showed that all the compounds exhibited excellent antioxidant results with some of the compounds surpassing catechin (reference drug). Compound H5 with IC50 values of 30.32 mM and 31.73 mM outshined catechin with IC50 values of 31.17 mM and 140.62 mM for DPPH and NO assays respectively. All the compounds fell within the threshold of Lipinski’s Ro5 projecting them as orally bioavailable and less toxic future therapeutics.

Beyond expectations: the development and biological activity of cytokinin oxidase/dehydrogenase inhibitors.

Cytokinins are one of the main groups of plant hormones that regulate growth and development of plants. Cytokinin oxidase/dehydrogenase (CKX) is an enzyme that rapidly and irreversibly degrades cytokinins and thus directly affects their concentration and physiological effect. Genetically modified plants with reduced CKX activity in the shoot, i.e. with a higher concentration of cytokinins, showed e.g. increased tolerance to drought stress, formed larger inflorescences and had higher grain yield. For these reasons, chemical compounds capable of inhibiting the CKX activity (CKX inhibitors) were sought. First, they were identified among strong synthetic cytokinins, but their inhibitory activity was low. The trend has been to develop potent CKX inhibitors with minimal intrinsic cytokinin activity in the hope of avoiding the negative effect of cytokinins on root growth. Cloning CKX, production of key recombinant enzymes from Arabidopsis (AtCKX2) and maize (ZmCKX1 and ZmCKX4a), development of screening bioassays and progress in X-ray crystallography and synthetic organic chemistry led to extensive progress in the development of these compounds. Currently, the most suitable CKX inhibitors are seeking their application in research and the commercial sphere in two main areas - plant tissue cultures and agriculture. The key milestones that preceded it are summarized in this review.