Crystallographic Methods Research Articles

A New Drug Discovery Platform: Application to DNA Polymerase Eta and Apurinic/Apyrimidinic Endonuclease 1

The ability to quickly discover reliable hits from screening and rapidly convert them into lead compounds, which can be verified in functional assays, is central to drug discovery. The expedited validation of novel targets and the identification of modulators to advance to preclinical studies can significantly increase drug development success. Our SaXPyTM (“SAR by X-ray Poses Quickly”) platform, which is applicable to any X-ray crystallography-enabled drug target, couples the established methods of protein X-ray crystallography and fragment-based drug discovery (FBDD) with advanced computational and medicinal chemistry to deliver small molecule modulators or targeted protein degradation ligands in a short timeframe. Our approach, especially for elusive or “undruggable” targets, allows for (i) hit generation; (ii) the mapping of protein–ligand interactions; (iii) the assessment of target ligandability; (iv) the discovery of novel and potential allosteric binding sites; and (v) hit-to-lead execution. These advances inform chemical tractability and downstream biology and generate novel intellectual property. We describe here the application of SaXPy in the discovery and development of DNA damage response inhibitors against DNA polymerase eta (Pol η or POLH) and apurinic/apyrimidinic endonuclease 1 (APE1 or APEX1). Notably, our SaXPy platform allowed us to solve the first crystal structures of these proteins bound to small molecules and to discover novel binding sites for each target.

Structural and functional investigations of syn-copalyl diphosphate synthase from Oryza sativa

The large superfamily of labdane-related diterpenoids is defined by the cyclization of linear geranylgeranyl pyrophosphate (GGPP), catalyzed by copalyl diphosphate synthases (CPSs) to form the basic decalin core, the copalyl diphosphates (CPPs). Three stereochemically distinct CPPs have been found in plants, namely (+)-CPP, ent-CPP and syn-CPP. Here, we used X-ray crystallography and cryo-EM methods to describe different oligomeric structures of a syn-copalyl diphosphate synthase from Oryza sativa (OsCyc1), and provided a cryo-EM structure of OsCyc1D367A mutant in complex with the substrate GGPP. Further analysis showed that tetramers are the dominant form of OsCyc1 in solution and are not necessary for enzyme activity in vitro. Through rational design, we identified an OsCyc1 mutant that can generate ent-CPP in addition to syn-CPP. Our work provides a structural and mechanistic basis for comparing different CPSs and paves the way for further enzyme design to obtain diterpene derivatives with specific chirality.

Bis(acylthiourea) compounds as enzyme inhibitors: Synthesis, characterization, crystal structures and in silico molecular docking studies

A series of bis(acylthiourea) compounds was synthesized and characterized by various spectroscopic and analytical methods. The structures of the three compounds were determined by X-ray crystallographic method and refined to good R factors. Surface properties, and intermolecular interactions were studied by Hirshfeld surface analysis. The binding data revealed urease as one of the enzyme targets for the bis(acylthiourea) derivatives. As thiourea derivatives possess various pharmacological properties like anticancer, anti-inflammatory, antibacterial and antiviral, we have attempted to understand the binding potential of the bis(acylthiourea) derivatives with VEGFR2, EGFR (anticancer targets) and SARS-CoV-2 main protease (antiviral target) in addition to urease (antibacterial target) through in silico molecular docking tool. The results revealed good binding potential of the compounds with these enzyme targets.

Ab Initio Shape of Supramolecular Complexes of Cucurbit[8]uril in Solution Found from Small-Angle X-ray Scattering Data

Previous studies of the spatial structure of the guest–host complexes of macrocyclic cavitands cucurbiturils with a number of nitroxyl radicals by ESR, NMR, and crystallographic methods showed that, in aqueous solutions containing a number of nitroxyl radicals as guest molecules, ordered aggregates in the form of an equilateral triangle, with three guest–host monocomplexes located in its vertices, may arise. We performed experiments on small-angle X-ray scattering of aqueous solutions of guest–host cucurbit[8]uril complexes with a stable nitroxyl radical (protonated tempoamine) and, based on the experimental results, carried out ab initio modeling of the shape of aggregates of complexes in the natural state in solution. The search for models of the shape of aggregates was performed either using no additional information about their structure or assuming the presence of a threefold axis. ESR is applied as an independent method for studying the aggregation of complexes in solution. It is shown that the shape of the particles constituting complexes at high cavitand and guest concentrations in an aqueous solution is close in its parameters to an equilateral triangle, which is in agreement with the known crystallographic and ESR data.

Determination of Digoxin in Biological Samples and Medicinal Plants by Electrochemical and Chromatographic Methods

AbstractIn this study, the nanocomposite with FeS2 nanoparticles and sulfur‐doped reduced graphene oxide (SRGO) were synthesized and characterized with appropriate spectroscopic, crystallographic and morphological methods. The FeS2‐SRGO nanocomposite was coated on a glass‐carbon (GC) electrode as working electrode for the electrochemical determination of digoxin. The method was linear in the range of 3–25 μgL−1 with the detection limit of the method is 0.54 μgL−1. The plant‘s active ingredient was extracted from Leonurus cardiac and Valeriana officinalis. Three important types of real samples including plants extracted and urine samples were selected to measure the amounts of digoxin. The electrochemical sensing results were evaluated by studying the amount of spiked recovery and statistical test with comparison to the high‐performance liquid chromatography (HPLC) method results and the accuracy and precision of the method for digoxin determination in real samples were confirmed. The results show the plant extracted contained a significant amount of digoxin. The presence of digoxin in valerian was investigated for the first time in this study.

The dimerized pentraxin-like domain of the adhesion G protein–coupled receptor 112 (ADGRG4) suggests function in sensing mechanical forces

Adhesion G protein-coupled receptors (aGPCRs) feature large extracellular regions with modular domains that often resemble protein classes of various function. The pentraxin (PTX) domain, which is predicted by sequence homology within the extracellular region of four different aGPCR members, is well known to form pentamers and other oligomers. Oligomerization of GPCRs is frequently reported and mainly driven by interactions of the seven-transmembrane region and N or C termini. While the functional importance of dimers is well-established for some class C GPCRs, relatively little is known about aGPCR multimerization. Here, we showcase the example of ADGRG4, an orphan aGPCR that possesses a PTX-like domain at its very N-terminal tip, followed by an extremely long stalk containing serine-threonine repeats. Using X-ray crystallography and biophysical methods, we determined the structure of this unusual PTX-like domain and provide experimental evidence for a homodimer equilibrium of this domain which is Ca2+-independent and driven by intermolecular contacts that differ vastly from the known soluble PTXs. The formation of this dimer seems to be conserved in mammalian ADGRG4 indicating functional relevance. Our data alongside of theoretical considerations lead to the hypothesis that ADGRG4 acts as an invivo sensor for shear forces in enterochromaffin and Paneth cells of the small intestine.

Aluminium and Gallium Silylimides as Nitride Sources.

Terminal aluminium and gallium imides of the type K[(NON)M(NR)], bearing heteroatom substituents at R, have been synthesised via reactions of anionic aluminium(I) and gallium(I) reagents with silyl and boryl azides (NON=4,5-bis(2,6-diisopropyl-anilido)-2,7-di-tert-butyl-9,9-dimethyl-xanthene). These systems vary significantly in their lability in solution: the N(Sii Pr3 ) and N(Boryl) complexes are very labile, on account of the high basicity at nitrogen. Phenylsilylimido derivatives provide greater stabilization through the π-acceptor capabilities of the SiR3 group. K[(NON)AlN(Sit BuPh2 )] offers a workable compromise between stability and solubility, and has been completely characterized by spectroscopic, analytical and crystallographic methods. The silylimide species examined feature minimal π-bonding between the imide ligand and aluminium/gallium, with the HOMO and HOMO-1 orbitals effectively comprising orthogonal lone pairs centred at N. Reactivity-wise, both aluminium and gallium silylimides can act as viable sources of nitride, [N]3- , with systems derived from either metal reacting with CO to afford cyanide complexes. By contrast, only the gallium system K[(NON)Ga{N(SiPh3 )}] is capable of effecting a similar transformation with N2 O to yield azide, N3 - , via formal oxide/nitride metathesis. The aluminium systems instead generate RN3 via transfer of the imide fragment [RN]2- .

Novel spirooxindole alkaloid derivatives from the medicinal insect Blaps japanensis and their biological evaluation

Blapspirooxindoles A–C (1–3), three novel spirooxindole alkaloids with a unique spiro[chromane-4,3′-indoline]-2,2′-dione motif, blapcumaranons A and B (4 and 5), two new 2-cumaranon derivatives, blapoxindoles A–J (6–15), ten new oxindole alkaloid derivatives, along with one known compound (16), were isolated from the whole bodies of Blaps japanensis. Their structures including absolute configurations were determined by using spectroscopic, X-ray crystallographic, and computational methods. Compounds 1–11 and 13 exist as racemic mixtures in nature, and their (–)- and (+)-antipodes were separated by chiral HPLC. Biological evaluations of these compounds were determined with multiple assays including anti-tumor, anti-inflammatory, and renal protection activities in vitro. Several compounds displayed effective activity in one or more assays.

(Invited) Crystallographic Characterization of U@C2 n (2n = 82−86): Insights about Metal-Cage Interactions for Mono-Metallofullerenes

Endohedral mono-metallofullerenes are the prototypes to understand the fundamental nature and the unique interactions between the encapsulated metals and the fullerene cages. Recently, we succeeded in the synthesis, isolation, and characterization of four new U-based mono-metallofullerenes, including U@Cs (6)-C82, U@C 2(8)-C84, U@Cs (15)-C84, and U@C 1(12)-C86. Furthermore, a new finding concerning the metal positions inside the cages reveals that the encapsulated metal ions are always located on symmetry planes of the fullerene cages, as long as the fullerene cages possess mirror planes. Symmetrical patterns, such as indacene, sumanene, and phenalene, and charge transfer are found to determine the metal positions inside the fullerene cages. DFT calculations support that the metal-fullerene motif interaction determines the stability of the metal position. In fullerenes containing symmetry planes, the metal prefers to occupy a symmetrical arrangement with respect to the interacting motifs, which share one of their symmetry planes with the fullerene. This finding provides new insights into the nature of metal-cage interactions and gives new guidelines for structural determinations using crystallographic and theoretical methods.

Remote laboratory training for high school students: grocery store based hands-on project in protein crystallography.

The COVID-19 pandemic measures forced students to stay home and confined them to remote learning. This had a large impact on laboratory experiments, which are often impossible to complete from home. This article is a resource for instructors/educators to introduce the topic of structural biology and crystallographic methods. The main focus is to describe a hands-on crystallization laboratory exercise that can be carried out remotely at home with safe household products. X-ray crystallography is a vital technique for determining protein structure and function. This information can be used to understand fundamental biological processes and to help in the design of life-saving medications. Here, a method was developed to teach crystallography using reagents and equipment that can be found in grocery stores. The steps involved in a crystallography experiment are detailed with links and references to additional resources.

Heterobimetallic Base Pair Programming in Designer 3D DNA Crystals.

Metal-mediated DNA (mmDNA) presents a pathway toward engineering bioinorganic and electronic behavior into DNA devices. Many chemical and biophysical forces drive the programmable chelation of metals between pyrimidine base pairs. Here, we developed a crystallographic method using the three-dimensional (3D) DNA tensegrity triangle motif to capture single- and multi-metal binding modes across granular changes to environmental pH using anomalous scattering. Leveraging this programmable crystal, we determined 28 biomolecular structures to capture mmDNA reactions. We found that silver(I) binds with increasing occupancy in T-T and U-U pairs at elevated pH levels, and we exploited this to capture silver(I) and mercury(II) within the same base pair and to isolate the titration points for homo- and heterometal base pair modes. We additionally determined the structure of a C-C pair with both silver(I) and mercury(II). Finally, we extend our paradigm to capture cadmium(II) in T-T pairs together with mercury(II) at high pH. The precision self-assembly of heterobimetallic DNA chemistry at the sub-nanometer scale will enable atomistic design frameworks for more elaborate mmDNA-based nanodevices and nanotechnologies.

Distance-based global analysis of consistent cis-bonds in protein backbones

Biological polypeptides are known to contain cis-linkage in their main chain as a minor but important feature. Such anomalous connection of amino acids has different structural and functional effects on proteins. Experimental evidence of cis-bonds in proteins is mainly obtained using X-ray crystallography and other methods in the field of structural biology. To date, extensive analyses have been carried out on the experimentally found cis-bonds using the Protein Data Bank (PDB) entry-wise or residue-wise; however, their consistency in each protein has not been examined on a global scale. Data accumulation and advances in computational methodology enable the use of new approaches from a proteomic point of view. Here, we sought to carry out protein-wise analysis and describe a simple procedure for the detection and confirmation of cis-bonds from a set of experimental PDB chains for a protein to discriminate this type of bond from isomerizable and/or misassigned bonds. The resulting set of consistent cis bonds (found at identical positions in multiple chains) provides unprecedented insights into the trend of “high cis content” proteins and the upper limit of consistent cis bonds per polypeptide length. Recognizing such limit would not only be important for a practical check of upcoming structures, but also for the design of novel protein folds beyond the evolutionally-acquired repertoire.

Boron citrate esters as boron daily food supplementing agents. Synthesis and structural characterizations

Boric acid, H3BO3, is a weak acid and is in the form of an uncharged small molecule at physiological pH. Acting as a Lewis acid, it forms complexes (esters) with amino and hydroxy acids, carbohydrates, nucleotides and vitamins through electron donor-acceptor interactions. These interactions are believed to be beneficial for human health. Therefore, synthetic bichelate complexes of boric acid with organic biomolecules are considered for nutritional and/or pharmaceutical applications. Research is needed to better elucidate the mechanisms of action of boron and to identify requirements in different areas. In this study, the complexation of citric acid, which is widely used in the food industry, with boric acid was investigated considering that it would be more beneficial in bio applications. Mono-chelate (1:1) and bichelate (1:2) anionic complexes of citric acid and boric acid, which are thought to be used as food supplements, were isolated from aqueous solutions in salt form with K+, Mg+2, Na+ and Ca+2 ions. The complexes were characterized by elemental analysis, FT-IR, X-ray crystallography and thermal methods.

The Best of Both Worlds: Combining the Power of MICs and WCAs To Generate Stable and Crystalline CrI -Tetracarbonyl Complexes with π-Accepting Ligands.

Here we present stable and crystalline chromium(I) tetracarbonyl complexes with pyridyl-MIC (MIC=mesoionic carbene) ligands and weakly coordinating anions (WCA=[Al(ORF )4 ]- , RF =C(CF3 )3 and BArF =[B(ArF )4 ]- , ArF =3,5-(CF3 )2 C6 H3 ). The complexes were fully characterized via crystallographic, spectroscopic and theoretical methods. The influence of counter anions on the IR and EPR spectroscopic properties of the CrI complexes was investigated, and the electronic innocence versus non-innocence of WCAs was probed. These are the first examples of stable and crystalline [Cr(CO)4 ]+ complexes with a chelating accepting ligand, and the data presented here are of relevance for both the photochemical and the electrochemical properties of these classes of compounds.

Determination of hydrogen atom position in Stryker's reagent through the application of quantum crystallographic methods on MicroED data

Determination of hydrogen atom position in Stryker's reagent through the application of quantum crystallographic methods on MicroED data

3-acetyl-coumarin-substituted thiosemicarbazones and their ruthenium, rhodium and iridium metal complexes: An investigation of the antibacterial, antioxidant and cytotoxicity activities

A series of ruthenium, iridium and rhodium half-sandwich complexes containing 3-acetyl chromen-2-one-4(N)-substituted thiosemicarbazone derivatives have been prepared for use as antibacterial and anticancer agents. These complexes have been isolated as cationic bidentate (N, S) and neutral bidentate (N, S) complexes. The complexes have been characterized by spectral techniques (FT-IR, UV-Vis, NMR and HRMS) and single-crystal X-ray crystallography methods. The structure of complex 6 has been determined by a single-crystal X-ray diffraction study, which shows that coordination of the 3-acetyl chromen-2-one-4(N)-substituted thiosemicarbazone ligand to the metal occurs via azomethine nitrogen and thiolate sulfur atoms. Vitro antibacterial activity against gram-positive Staphylococcus aureus and gram-negative Escherichia coli, Klebsiella pneumonia strains and antioxidant activity have been evaluated. Also, the said complexes were tested for anticancer efficacy (in vitro for 48h) in Dalton's lymphoma (DL) cancer cell lines using cell cytotoxicity (MTT) and apoptosis tests. The results were compared with cisplatin (standard drug) under identical experimental conditions. The cell cytotoxicity results showed that complex 5 induced higher cytotoxicity in DL cells, followed by complex 4, 1, and 2.

Reactions of lithium amidinates or guanidinates toward carbodiimides: Synthesis and characterization of multi-site triazapentadienyllithium compounds

Lithium amidinates or guanidinates form novel complexes 1–4 with N,N′-dicyclohexylcarbodiimide (CyN=C=NCy, DCC) or N,N′-diisopropylcarbodiimide (iPrN=C=NiPr, DIC). Their structures were characterized by spectroscopic and X-ray crystallographic method. The X-ray diffraction analysis revealed that heteroleptic complexes 1–3 were mononuclear with a four-co-ordinate metal center and homoleptic 4 was dimeric. Both the W-shaped 1,3,5-triazapentadienato lithium 1–3 and U-shaped 4 could be regarded as good intermediates en route to their metal complexes with varied substrates.

Quantum crystallographic method improves the accuracy of screening of inhibitors: A set of Alzheimer’s disease responsible protein‐ligand complexes

AbstractBackgroundIn drug design and development, identification of lead structure is a crucial step to predict the ligand binding affinity and functional potency with any of target enzyme/protein. The recently developed quantum crystallographic method (fragment‐based approach) has been used as a substitutional way to calculate the interaction energies. This quantum mechanical level information gives a deeper understanding of structural facts with the position of hydrogen atoms and valence electron distributions. It is very much essential to understanding the electronic properties of drug‐receptor interactions. Therefore, the quantum crystallographic based interaction energy calculation is a better alternative method to docking score‐based modelling.MethodA set of AD responsible protein‐ligand complexes were selected for the quantitative analysis. Molecular docking of selected compounds with four different enzymes (AChE/BChE/BACE1/GSK3β) was carried out and then, all the selected complexes have been subjected to MD simulation. Later, the 3D coordinates of ligand with active site residues (around 4Å from the ligand) were used for quantum crystallographic studies.ResultThe screened molecules are forming strong hydrogen bonding and hydrophobic interactions with active site residues of all four enzymes; particularly, π‐π stacking, π‐cationic, π‐alkyl and π‐σ types of interactions, which are highly stable during dynamic behavior. After the quantum crystallographic studies, the interaction energies of selected compounds with different active site residues of all four enzymes were analyzed; it is ranged from ‐20.83 to ‐3.92 kcal/mol and the total interaction energies of each complex are higher than binding energy gained from MD simulation. And, the RMSD of each molecule was compared with the corresponding geometry of solid‐state and dynamic state; it is found to be significantly low (>0.5Å) and similar. Interestingly, the correlation between experimental IC50 and averaged interaction energies of selected compounds exhibits good agreement.ConclusionThe in‐silico results revealed conformational flexibility, high stability and compactness, binding affinity and orientation of the target molecules in the active site residues of all enzymes in the physiological nature (water‐medium). Quantum crystallographic method is a novel approach that opens new window to delineate the different type of interactions at electronic level. And, it may allow us to design new drug against target disease.

The effects of nature-inspired amino acid substitutions on structural and biochemical properties of the E. coli L-asparaginase EcAIII.

The Escherichia coli enzyme EcAIII catalyzes the hydrolysis of L-Asn to L-Asp and ammonia. Using a nature-inspired mutagenesis approach, we designed and produced five new EcAIII variants (M200I, M200L, M200K, M200T, M200W). The modified proteins were characterized by spectroscopic and crystallographic methods. All new variants were enzymatically active, confirming that the applied mutagenesis procedure has been successful. The determined crystal structures revealed new conformational states of the EcAIII molecule carrying the M200W mutation and allowed a high-resolution observation of an acyl-enzyme intermediate with the M200L mutant. In addition, we performed structure prediction, substrate docking, and molecular dynamics simulations for 25 selected bacterial orthologs of EcAIII, to gain insights into how mutations at the M200 residue affect the active site and substrate binding mode. This comprehensive strategy, including both experimental and computational methods, can be used to guide further enzyme engineering and can be applied to the study of other proteins of medicinal or biotechnological importance.

Loss Determination Techniques for Piezoelectrics: A Review

Nowadays, heat dissipation in electronic devices is one of the serious issues to be resolved in energy and environmental terms. Piezoelectric materials are being utilized in many electronic devices, yet the roadblock toward further miniaturization of piezoelectric devices was identified as heat dissipation. Three types of losses (dielectric, elastic, and piezoelectric) are known to be related to the heat dissipation mechanism of piezoelectric materials, therefore obtaining accurate values of the loss factors is essential for minimizing the heat dissipation of piezoelectric devices. The purpose of this review is to introduce several loss determination techniques for piezoelectric materials. The review starts with brief discussions of the loss factors and of the importance of piezoelectric loss that is related to the antiresonance frequency. Then, the review covers the methods developed by our research group, including High Power Piezoelectric Characterization Systems (HiPoCSTM), the crystallographic orientation method and the partial electrode method, as well as other methods such as the pulse-echo method and computer-based approaches. The review continues with a discussion of piezoelectric device modeling (analytical solution and equivalent circuits) that considers loss factors. Finally, the review provides concluding remarks for addressing current issues and suggesting possible solutions.