2.4.8: X-Ray Diffraction Analysis - Biology


  • Summarize the methods used for x-ray diffraction analysis and the contributions they have made to science

X-ray diffraction (XRD) is a tool for characterizing the arrangement of atoms in crystals and the distances between crystal faces. The technique reveals detailed information about the chemical composition, crystallography, and microstructure of all types of natural and manufactured materials, which is key in understanding the properties of the material being studied.

Since many materials can form crystals—such as salts, metals, minerals, semiconductors, as well as various inorganic, organic, and biological molecules —X-ray crystallography has been fundamental in the development of many scientific fields. The method determined the size of atoms, the lengths and types of chemical bonds, and the atomic-scale differences among various materials, especially minerals and alloys. The method also revealed the structure and function of many biological molecules, including vitamins, drugs, proteins, and nucleic acids such as DNA.

Samples are commonly analyzed in a crystal form. X-ray diffraction is caused by constructive interference of x-ray waves that reflect off internal crystal planes. A thin film or layer of powder is fixed in the path of monochromatic x-rays. A detector measures x-rays from the sample over a range of angles. The powder consists of tiny crystals randomly oriented. At certain angles of the sensor, populations of crystals have the correct angle so that Bragg’s equation is satisfied for one of the crystal planes, resulting in a spike in X-rays.

The output graph displays x-ray intensity over 2 theta, the angle of the detector. The data generated with this technique requires extensive mathematical analysis that is now made easier by available computer algorithms. The analysis consists of indexing, merging, and phasing variations in electron density. It begins with the identification of molecules using the international center for diffraction database (ICDD). This is an organization dedicated to collecting, editing, publishing, and distributing powder diffraction data for the identification of crystalline materials. Further analysis involves structure refinement and quantitative phase using the general structure analysis system (GSAS), which ultimately leads to the identification of the amorphous or crystalline phase of a matter and helps construct its three dimensional atomic model.

Key Points

  • X-ray diffraction utilizes x-ray beams targeted to hit crystallized matter and generates a diffraction pattern.
  • Data collected using this method undergo a systematic analytical process that employes mathematical models and computer algorithms to obtain the final 3D atom model of a matter.
  • X-ray diffraction analysis identifies composition and chemical bonds between atoms of crystal, liquid, powder, or amorphous samples.

Correlative microscopy approach for biology using X-ray holography, X-ray scanning diffraction and STED microscopy

We present a correlative microscopy approach for biology based on holographic X-ray imaging, X-ray scanning diffraction, and stimulated emission depletion (STED) microscopy. All modalities are combined into the same synchrotron endstation. In this way, labeled and unlabeled structures in cells are visualized in a complementary manner. We map out the fluorescently labeled actin cytoskeleton in heart tissue cells and superimpose the data with phase maps from X-ray holography. Furthermore, an array of local far-field diffraction patterns is recorded in the regime of small-angle X-ray scattering (scanning SAXS), which can be interpreted in terms of biomolecular shape and spatial correlations of all contributing scattering constituents. We find that principal directions of anisotropic diffraction patterns coincide to a certain degree with the actin fiber directions and that actin stands out in the phase maps from holographic recordings. In situ STED recordings are proposed to formulate models for diffraction data based on co-localization constraints.

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2. Experimental methods and results

2.1. Expression-vector construction and protein expression

The DNA encoding the human PILRα V-set domain (residues 13�, designated PILRα) was amplified by the polymerase chain reaction (PCR) using the full-length cDNA of human PILRα as a template (AF16080): 5′-GGA ATT CCA TAT GCT TTA TGG GGT CAC TCA ACC AAA AC-3′ as the forward primer and 5′-CCA AGC TTA CTA GGT GAT GGA GAG TTT GGT CCC-3′ as the reverse primer. The resultant PCR fragment including the V-set domain with additional N-terminal methionine was digested with the NdeI and HindIII restriction enzymes and was cloned into the pGMT7 vector (Reid et al., 1996 ▶), which contains the T7 promoter for overexpression in Escherichia coli.

The plasmid was transformed into E. coli Rosetta (DE3) competent cells (Novagen) and a single colony was inoculated into 5 ml 2YT medium at 310 K and cultured overnight. The overnight culture was then transferred into a flask containing 1 l 2YT medium with 100 mg l 𢄡 ampicillin (Nacalai Tesque, Japan). When the OD600 reached 0.6, the culture was supplemented with isopropyl β- d -1-thiogalactopyranoside (IPTG Nacalai Tesque, Japan) to a final concentration of 1 mM for induction. The cells were further cultured for 6 h at 310 K and were harvested by centrifugation. The cell pellet was suspended in resuspension buffer (50 mM Tris–HCl pH 8.0, 100 mM NaCl), sonicated and centrifuged. The pellet, including inclusion bodies, was washed with Triton buffer (0.5% Triton X-100, 50 mM Tris–HCl pH 8.0, 100 mM NaCl) and with resuspension buffer. The purified inclusion bodies of PILRα were then dissolved in guanidine buffer (6 M guanidine–HCl, 50 mM MES–NaOH pH 6.5, 100 mM NaCl, 10 mM EDTA).

2.2. Protein refolding and purification

For refolding, 20 mg solubilized inclusion bodies of the human PILRα V-set domain were rapidly diluted into 1 l refolding buffer (1 M l -arginine–HCl, 0.1 M Tris–HCl pH 8.0, 2 mM EDTA) at 277 K. The solution was stirred at 277 K for 2𠁝 and concentrated to 5� ml. The refolded protein was filtered (0.22 µm cutoff) and purified by gel-filtration chromatography (HiLoad 26/60 Superdex 75pg, GE Healthcare) with elution buffer comprising 20 mM Tris–HCl pH 8.0, 100 mM NaCl. The PILRα protein eluted as a monomer, even though it has one free cysteine (Cys106 Fig. 1 ▶ a). Further purification was performed by ion-exchange chromatography (Resource S, GE Healthcare) with elution buffer comprising 20 mM succinate pH 6.0, 0� mM NaCl. The purified PILRα protein was analyzed by SDS–PAGE with Coomassie Brilliant Blue (Qiagen) staining (Fig. 1 ▶ b), which confirmed its high purity. Because PILRα is unstable at high pH, dialysis was conducted against 20 mM succinate pH 5.0, 100 mM NaCl buffer and the solution was concentrated to approximately 6 mg ml 𢄡 . The selenomethionyl derivative of PILRα (referred to hereafter as SeMet PILRα) was prepared in the same manner, except that M9 medium containing selenomethionine (25 mg l 𢄡 ) was used instead of 2YT medium.

Gel-filtration chromatography and SDS–PAGE analysis of PILRα. (a) Gel-filtration chromatogram of PILRα. The arrow indicates the peak for the PILRα protein. (b) SDS–PAGE of the purified PILRα protein. Under reducing conditions, PILRα migrated as a 14 kDa band (the black arrow). Lane 1, molecular-weight markers (kDa) lane 2, PILRα protein after ion-exchange chromatography, ready for crystallization.

2.3. Crystallization

Initial crystallization trials were performed with Crystal Screens 1 and 2 (Hampton Research) and The Classics Suite (Qiagen) on Intelliplates (Art Robbins) using the automatic Hydra-Plus-One crystallization-setup robot (Art Robbins). The drop was a mixture of 0.2 µl protein solution (6 mg ml 𢄡 PILRα, 20 mM succinate pH 5.0, 100 mM NaCl) and 0.2 µl reservoir solution and the crystallization plates were incubated with 90 µl reservoir buffer at 293 K. Crystals of native PILRα were obtained using The Classics Suite solution No. 7 (0.1 M trisodium citrate pH 5.6, 20% 2-propanol, 20% PEG 4000 Fig.ਂ ▶ a). However, the SeMet PILRα protein did not crystallize under the same conditions and extensive crystallization trials were therefore performed using the same commercially available screening kits as for the native PILRα. Crystals of the SeMet PILRα protein were successfully obtained using The PEGs Suite (Qiagen) solution No. 91 (0.2 M ammonium phosphate, 20% PEG 3350 Fig. 2 ▶ b).

PILRα crystals grown by sitting-drop vapour diffusion. (a) Native crystals of PILRα. (b) SeMet PILRα crystals. The scale bars represent 0.1 mm.

2.4. X-ray diffraction analysis

Prior to data collection, the crystals of native PILRα were soaked in a cryoprotectant solution (0.1 M trisodium citrate pH 5.6, 20% 2-­propanol, 30% PEG 4000) and flash-cooled. A 1.3 Å diffraction data set was collected at 100 K from the single largest crystal at beamline BL41 of SPring-8. Harima, Japan (λ = 0.9 Å). Data were processed and scaled with the HKL-2000 program package (Otwinowski & Minor, 1997 ▶). The PILRα structure could not be solved by molecular replacement using the crystal structure of sialoadhesin, which has 23% sequence similarity to PILRα, as a search model. Therefore, we prepared crystals of SeMet PILRα and MAD data sets were collected. However, this was also unsuccessful because of the low phasing power of the Se atoms (0.739 and 0.400 at the Se edge and peak, respectively). Finally, we prepared an iodide-anion derivative by soaking the native crystals in cryoprotectant solution including 1 M KI for 30 s prior to flash-cooling. A 1.8 Å diffraction SAD data set was collected at 100 K at beamline BL17A of the Photon Factory, Tsukuba, Japan (λ = 1.8 Å). The SAD data were processed and scaled with the HKL-2000 program package. The crystals belonged to space group P212121, with unit-cell parameters a =ꁀ.4, b = 45.0, c =ꁖ.9 Å, and contained one molecule per asymmetric unit. Five iodine sites were identified, refined and used for phase calculation with the autoSHARP package (Vonrhein et al., 2006 ▶). The phases were further improved using the density-modification program DM (Cowtan, 1994 ▶) with a mean figure of merit of 0.574 for acentric reflections and of 0.213 for centric reflections. The statistics of data collection and SAD phasing are summarized in Tableਁ ▶ . The resulting electron-density map allowed the tracing of the main chain of the polypeptide. Model building and structure refinement are now in progress.

Table 1

Values in parentheses are for the outer shell.

Space groupP212121P212121
Unit-cell parameters
a (Å)40.3340.77
b (Å)44.9445.51
c (Å)56.8755.94
X-ray sourceBL41XU, SPring-8BL17A, PF
Wavelength (Å)0.90001.8000
Resolution (Å)20𠄱.3 (1.33𠄱.30)20𠄱.8 (1.86𠄱.80)
Observed reflections183584 (17370)67494 (6382)
Unique reflections26166 (2555)10063 (967)
Redundancy7.0 (6.8)6.7 (6.6)
Completeness (%)99.9 (99.8)99.4 (98.0)
I/σ(I)〉13.1 (4.1)10.2 (7.56)
Rmerge (%) † 5.8 (37.5)8.6 (15.9)
ASU content1 subunit1 subunit
SAD phasing statistics
 Number of I sites in ASU 5
 Phasing power (centric/acentric)  ‡ /2.486
 𣊟OM〉 (centric/acentric) 0.21311/0.57388
R merge = , where I i(hkl) is the observed intensity and is the average intensity obtained from multiple observations of symmetry-related reflections.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome is initially expressed as two large polyproteins. Its main protease, M pro , is essential to yield functional viral proteins, making it a key drug target. Günther et al. used x-ray crystallography to screen more than 5000 compounds that are either approved drugs or drugs in clinical trials. The screen identified 37 compounds that bind to M pro . High-resolution structures showed that most compounds bind at the active site but also revealed two allosteric sites where binding of a drug causes conformational changes that affect the active site. In cell-based assays, seven compounds had antiviral activity without toxicity. The most potent, calpeptin, binds covalently in the active site, whereas the second most potent, pelitinib, binds at an allosteric site.

The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous human suffering. To date, no effective drug is available to directly treat the disease. In a search for a drug against COVID-19, we have performed a high-throughput x-ray crystallographic screen of two repurposing drug libraries against the SARS-CoV-2 main protease (M pro ), which is essential for viral replication. In contrast to commonly applied x-ray fragment screening experiments with molecules of low complexity, our screen tested already-approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds that bind to M pro . In subsequent cell-based viral reduction assays, one peptidomimetic and six nonpeptidic compounds showed antiviral activity at nontoxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2.

Infection of host cells by SARS-CoV-2 is governed by the complex interplay of molecular factors from both the host and the virus (1, 2). Coronaviruses are RNA viruses with a genome of approximately 30,000 nucleotides. The viral open reading frames are expressed as two overlapping large polyproteins which must be separated into functional subunits for replication and transcription activity (1). This proteolytic cleavage is primarily accomplished by the main protease (M pro ), also known as 3C-like protease 3CL pro or nsp5. M pro cleaves the viral polyprotein pp1ab at 11 distinct sites. The core cleavage motif is Leu-Gln↓(Ser/Ala/Gly) (1). M pro possesses a chymotrypsin-like fold appended with a C-terminal helical domain and harbors a catalytic dyad comprised of Cys 145 and His 41 in its active site, which is formed by four major pockets that are labeled according to their position relative to the scissile bond of the substrate (Fig. 1) (1). The active site is located in a cleft between the two N-terminal domains of the three-domain structure of the monomer, whereas the C-terminal helical domain is involved in regulation and dimerization of the enzyme (Fig. 1A). Because of its central involvement in virus replication, M pro is recognized as a prime target for antiviral drug discovery and compound screening activities aiming to identify and optimize drugs which can tackle coronavirus infections (3). Indeed, a number of recent publications confirm the potential of targeting M pro for inhibition of virus replication (1, 2, 4).

(A) Schematic drawing of M pro dimer structure. Protomer A is shown in white, and protomer B is in red. For clarity, the 29 binding compounds (yellow sticks) are only depicted on one of the two protomers. Catalytic residues His 41 (H41) and Cys 145 (C145), the active site, and two allosteric drug binding sites are highlighted. (B) Close-up view of the active site with peptide substrate bound (blue sticks), modeled after SARS-CoV M pro (PDB 2Q6G). The scissile bond is indicated in yellow and with the green arrowhead. Substrate binding pockets S1ʹ, S1, S2, and S4 are indicated by colored regions.

In order to find drug candidates against SARS-CoV-2, we performed a large-scale x-ray crystallographic screen of M pro against two repurposing libraries containing 5953 compounds from the Fraunhofer IME Repurposing Collection and the Safe-in-man library from Dompé Farmaceutici S.p.A. (5).

In contrast to crystallographic fragment screening experiments, compounds in repurposing libraries are chemically more complex (fig. S1A) (6, 7). Thus, these compounds likely bind more specifically and with higher affinity (8). Because of the higher molecular weights, we performed cocrystallization experiments at a physiological pH of 7.5 instead of compound soaking into native crystals (9).

From the 5953 compounds in our screen, we obtained x-ray diffraction datasets for 2381 compounds, which we subjected to automated structure refinement followed by cluster analysis (10) and pan dataset density analysis (PanDDA) (11) (table S1). We observed additional electron density, indicating binding to M pro , for 43 compounds, which were classified as hits, representing 37 distinct compounds (tables S1, S2, and S3). From these, the binding mode could be unambiguously determined for 29 molecules (Fig. 1A and table S4). The majority of hits were found in the active site of the enzyme. Of the 16 active site binders, six covalently bind as thioethers to Cys 145 , one compound binds covalently as a thiohemiacetal to Cys 145 , one is zinc-coordinated, and eight bind noncovalently. The remaining 13 compounds bind outside the active site at various locations (Fig. 1A).

Of the 43 hits from our x-ray screen, 37 compounds were available in quantities required for testing their antiviral activity against SARS-CoV-2 in cell assays (table S2). Nine compounds that reduced viral RNA (vRNA) replication by at least two orders of magnitude in Vero E6 cells (fig. S2) were further evaluated to determine the effective concentrations that reduced not only vRNA but also SARS-CoV-2 infectious particles by 50% (EC50) (Fig. 2). Additionally, AT7519 and ifenprodil, which showed slightly lower vRNA level reduction, were included because of their distinct binding sites outside of the active site. From these 11, seven compounds (AT7519, calpeptin, ifenprodil, MUT056399, pelitinib, tolperisone, and triglycidyl isocyanurate) exhibited a ≥100-fold reduction in infectious particles in combination with either a selectivity index [SI calculated as the 50% cytotoxic concentration (CC50) divided by the EC50] of >5 or no cytotoxicity in the tested concentration range and are considered antivirally active (table S5).

The vRNA yield (solid circles), viral titers (half-solid circles), and cell viability (empty circles) were determined by reverse transcription–quantitative polymerase chain reaction, immunofocus assays, and the CCK-8 method, respectively. EC50 for the viral titer reduction is shown. Individual data points represent means ± SD from three independent replicates in one experiment.

Here, we focus on a more detailed description of the 11 compounds analyzed in the secondary screen, which are grouped according to their different binding sites. The remaining hits are described in the supplementary text and figs. S3 to S5.

Tolperisone, 2-[β-(4-hydroxyphenyl)-ethylaminomethyl]-tetralone (HEAT), and isofloxythepin bind covalently to the active site. Tolperisone is antivirally active (EC50 = 19.17 μM) and shows no cytotoxicity (CC50 > 100 μM) (Fig. 2), whereas HEAT (EC50 = 24.05 μM, CC50 = 55.42 μM) and isofloxythepin (EC50 = 4.8 μM, CC50 = 17 μM) show unfavorable cytotoxicity. For all three compounds, only breakdown products are observed in the active site. Tolperisone and HEAT are β-aminoketones, but we only observe the part of the drug containing the ketone (2,4′-dimethylpropiophenone and 2-methyl-1-tetralone), whereas the remaining part with the amine group is missing. The breakdown product binds as a Michael acceptor to the thiol of Cys 145 , independently confirmed for HEAT by mass spectrometry (fig. S6 and table S6). The decomposition of tolperisone and HEAT was detected in both the crystallization and cell culture conditions (fig. S7) and is reported to be pH dependent (12). The parent compounds can be regarded as prodrugs (13, 14). In the x-ray structures the aromatic ring systems of tolperisone (Fig. 3A) and HEAT (Fig. 3B) protrude into the S1 pocket and form van der Waals contacts with the backbone of Phe 140 and Leu 141 and the side chain of Glu 166 . In addition, the keto group accepts a hydrogen bond from the imidazole side chain of His 163 . Tolperisone is used as a skeletal muscle relaxant (15). The x-ray structure suggests that isofloxythepin binds similarly as a fragment to Cys 145 (Fig. 3C).

Bound compounds are depicted as colored sticks, and the surface of M pro is shown in gray with selected interacting residues shown as sticks. Substrate binding pockets are colored as in Fig. 1. Hydrogen bonds are depicted by dashed lines. (A) Tolperisone. (B) HEAT. (C) Isofloxythepin. (D) Triglycidyl isocyanurate. (E) Calpeptin. (F) MUT056399.

Triglycidyl isocyanurate has antiviral activity (EC50 = 30.02 μM, CC50 > 100 μM) and adopts covalent and noncovalent binding modes to the active site. In both modes, the compound’s central ring sits on top of the catalytic dyad (His 41 , Cys 145 ), and its three epoxypropyl substituents reach into subsites S1′, S1, and S2. The noncovalent binding mode is stabilized by hydrogen bonds to the main chain of Gly 143 and Gln 166 and to the side chain of His 163 . In the covalently bound form, one oxirane ring is opened by nucleophilic attack of Cys 145 , forming a thioether (Fig. 3D). Triglycidyl isocyanurate has been tested as an antitumor agent (16).

Calpeptin shows the highest antiviral activity in the screen (EC50 = 72 nM, CC50 > 100 μM). It binds covalently via its aldehyde group to Cys 145 , forming a thiohemiacetal. This peptidomimetic inhibitor occupies substrate pockets S1 to S3, similar to the peptidomimetic inhibitors GC-376 (17, 18), calpain inhibitors (19), N3 (2), and the α-ketoamide 13b (1). The peptidomimetic backbone forms hydrogen bonds to the main chain of His 164 and Glu 166 , whereas the norleucine side chain maintains van der Waals contacts with the backbone of Phe 140 , Leu 141 , and Asn 142 (Fig. 3E). Calpeptin has known activity against SARS-CoV-2 M pro in enzymatic assays (17). The structure is highly similar to the common protease inhibitor leupeptin (fig. S3A), which served as a positive control in our x-ray screen but was not tested further in antiviral assays. In silico docking experiments also suggested calpeptin as a possible M pro binding molecule (table S7). Calpeptin also inhibits cathepsin L (20), and dual targeting of cathepsin L and M pro is suggested as an attractive path for SARS-CoV-2 inhibition (19).

MUT056399 binds noncovalently to the active site (EC50 = 38.24 μM, CC50 > 100 μM). The diphenyl ether core of MUT056399 blocks access to the catalytic site, which consists of Cys 145 and His 41 . The terminal carboxamide group occupies pocket S1 and forms hydrogen bonds to the side chain of His 163 and the backbone of Phe 140 (Fig. 3F). The ethyl phenyl group of the molecule reaches deep into pocket S2, which is enlarged by a shift of the side chain of Met 49 out of the substrate binding pocket. MUT056399 was developed as an antibacterial agent against multidrug-resistant Staphylococcus aureus strains (21).

Quipazine maleate showed moderate antiviral activity (EC50 = 31.64 μM, CC50 > 100 μM). In the x-ray structure, only the maleate counterion is observed covalently bound as a thioether (supplementary text and fig. S3B). Maleate is observed in structures of six other compounds showing no antiviral activity. The observed antiviral activity is thus likely caused by an off-target effect of quipazine.

In general, the enzymatic activity of M pro relies on the architecture of the active site, which critically depends on the dimerization of the enzyme and the correct relative orientation of the subdomains. This could allow ligands that bind outside of the active site to affect activity. In fact, we identified two such allosteric binding sites of M pro .

Five compounds of our x-ray screen bind in a hydrophobic pocket in the C-terminal dimerization domain (Fig. 4, A and B), located close to the oxyanion hole in pocket S1 of the substrate binding site. One of these showed strong antiviral activity (Fig. 2). Another compound binds between the catalytic and dimerization domains of M pro .

(A) Close-up view of the binding site in the dimerization domain (protomer A, gray cartoon representation), close to the active site of the second protomer (protomer B, surface representation) in the native dimer. Residues forming the hydrophobic pocket are indicated. Pelitinib (dark green) binds to the C-terminal α-helix at Ser 301 and pushes against Asn 142 and the β-turn of the pocket S1 of protomer B (residues marked with an asterisk). The inset shows the conformational change of Gln 256 (gray sticks) compared with the M pro apo structure (white sticks). (B) RS-102895 (purple), ifenprodil (cyan), PD-168568 (orange), and tofogliflozin (blue) occupy the same binding pocket as pelitinib. (C) AT7519 occupies a deep cleft between the catalytic and dimerization domain of M pro . (D) Conformational changes in the AT7519-bound M pro structure (gray) compared with those in the apo structure (white).

Central to the first allosteric binding site is a hydrophobic pocket formed by Ile 213 , Leu 253 , Gln 256 , Val 297 , and Cys 300 within the C-terminal dimerization domain (Fig. 4A). Pelitinib, ifenprodil, RS-102895, PD-168568, and tofogliflozin all exploit this site by inserting an aromatic moiety into this pocket.

Pelitinib shows the second highest antiviral activity in our screen (EC50 = 1.25 μM, CC50 = 13.96 μM). Its halogenated benzene ring binds to the hydrophobic groove in the helical domain, which becomes accessible by movement of the Gln 256 side chain (Fig. 4A). The central 3-cyanoquinoline moiety interacts with the end of the C-terminal helix (Ser 301 ). The ethyl ether substituent pushes against Tyr 118 and Asn 142 (from loop 141–144 of the S1 pocket) of the opposing protomer within the native dimer. The integrity of this pocket is crucial for enzyme activity (22). Pelitinib is an amine-catalyzed Michael acceptor (23) and was developed as an anticancer agent to bind to a cysteine in the active site of the tyrosine kinase epidermal growth factor receptor inhibitor (24). However, from its observed binding position, it is impossible for it to reach into the active site, and no evidence for covalent binding to Cys 145 is found in the electron density maps.

Ifenprodil and RS-102895 bind to the same hydrophobic pocket in the dimerization domain as pelitinib (Fig. 4B fig. S4, A and B and supplementary text). Only ifenprodil (EC50 = 46.86 μM, CC50 > 100 μM) shows moderate activity. RS-102895 (EC50 = 19.8 μM, CC50 = 54.98 μM) interacts, similar to pelitinib, with the second protomer by forming two hydrogen bonds to the side and main chains of Asn 142 , whereas the other compounds exhibit weaker or no interaction with the second protomer. PD-168568 and tofogliflozin bind the same site but are inactive (Fig. 4B and fig. S4, C and D).

The second allosteric site is formed by the deep groove between the catalytic domains and the dimerization domain. AT7519 is the only compound in our screen that we identified bound to this site (Fig. 4C). Though it has only moderate activity, we discuss it here because this site may be a target. The chlorinated benzene ring is engaged in various van der Waals interactions to loop 107-110, Val 202 , and Thr 292 . The central pyrazole has van der Waals contacts to Ile 249 and Phe 294 , and its adjacent carbonyl group forms a hydrogen bond to the side chain of Gln 110 . The terminal piperidine sits on top of Asn 151 and forms hydrogen bonds to the carboxylate of Asp 153 . This results in a displacement of loop 153-155, slightly narrowing the binding groove. The Cα atom of Tyr 154 moves 2.8 Å, accompanied by a conformational change of Asp 153 (Fig. 4D). This allows hydrogen bonding to the compound and the formation of a salt bridge to Arg 298 . Arg 298 is crucial for dimerization (25). The mutation Arg 298 Ala causes a reorientation of the dimerization domain relative to the catalytic domain, leading to changes in the oxyanion hole and destabilization of the S1 pocket by the N terminus. AT7519 was evaluated for treatment of human cancers (26). The potential of allosteric inhibition of M pro through modulation of Arg 298 has been independently demonstrated by mass spectrometry (27).

Our x-ray screen revealed 43 compounds binding to M pro , with seven compounds showing antiviral activity against SARS-CoV-2. We present structural evidence for interaction of these compounds at active and allosteric sites of M pro , although we cannot exclude that off-target effects played a role in the antiviral effect in cell culture, in particular for compounds with a low selectivity index. Conversely, an absence of antiviral activity of compounds binding clearly to M pro in the crystal might be due to rapid metabolization in the cellular environment. Calpeptin and pelitinib showed strong antiviral activity with low cytotoxicity and are suitable for preclinical evaluation. In any case, all hit compounds are valuable lead structures with potential for further drug development, especially because drug-repurposing libraries offer the advantage of proven bioactivity and cell permeability (28).

The most active compound, calpeptin, binds in the active site similar to other members of the large class of peptide-based inhibitors that bind as thiohemi-acetals or -ketals to M pro (29). In addition to this peptidomimetic inhibitor, we discovered several nonpeptidic inhibitors. Those compounds binding to the active site of M pro contained new Michael acceptors based on β-aminoketones (tolperisone and HEAT). These compounds lead to the formation of thioethers and have not been described as prodrugs for viral proteases. We also identified a noncovalent binder, MUT056399, that blocked the active site. In addition to this common active site inhibition, we identified compounds that inhibit the enzyme through binding at two allosteric sites of M pro .

The first allosteric site (dimerization domain) is in the direct vicinity of the S1 pocket of the adjacent monomer within the native dimer. The potential for antiviral inhibition through this site is demonstrated by pelitinib. The hydrophobic nature of the residues forming the main pocket is conserved in all human coronavirus M pro (fig. S8). Consequently, potential drugs targeting this binding site may be effective against other coronaviruses. The potential of the second allosteric site as a druggable target is demonstrated by the observed moderate antiviral activity of AT7519.

User's Guide - Sample Collection and Preparation

Determination of an unknown requires: the material, an instrument for grinding, and a sample holder.

  • Obtain a few tenths of a gram (or more) of the material, as pure as possible
  • Grind the sample to a fine powder, typically in a fluid to minimize inducing extra strain (surface energy) that can offset peak positions, and to randomize orientation. Powder less than

  • smear uniformly onto a glass slide, assuring a flat upper surface
  • pack into a sample container
  • sprinkle on double sticky tape

Silica Hosts for Acid and Basic Organosilanes: Preparation, Characterization, and Application in Catalysis

Katarzyna Stawicka , Maria Ziolek , in Chemistry of Silica and Zeolite-Based Materials , 2019 Characterization of Surface Properties

The presence and properties of anchored sulfonic species on mesoporous silica may be investigated by numerous techniques like N2 adsorption/desorption isotherm, XRD (X-ray diffraction), FTIR (Fourier Transform Infrared Spectroscopy), Raman spectroscopy, UV–Vis (Ultraviolet and Visible spectroscopy), XPS (X-ray Photoelectron Spectroscopy), DTA/TG ( Differential Thermal Analysis / Themogravimetry), 29 Si MAS NMR (Magic Angle Spinning Nuclear Magnetic Resonance), 13 C MAS NMR, elemental analysis, FTIR combined with pyridine adsorption or acid–base titration.

The organosilanes introduced to silica cause changes in textural/structural properties of supports which may be detected by N2 adsorption/desorption and XRD techniques. It has been proved that addition of organosilanes with sulfonic species by grafting into mesoporous silica led to a decrease in surface area, pore volume, and diameter due to filling of pores by organic molecules. 8 Thus it is important to select the appropriate silica support for modification to avoid the blocking of pores by the modifier. On the other hand, the addition of organosilane, that is, MPTMS during the synthesis of silica may cause an increase in pore size, which was observed for SBA-15. 13 As mentioned above, the differences in structure between the parent silica and the sample modified with organosilanes containing sulfonic species may be also detected by XRD. The decrease in intensity of peaks characteristic of ordered structure are frequently visible after silica modification which proves the organosilane anchoring. Such behavior was observed for SBA-15 and HMS after functionalization with arenesulfonic species or MPTMS. 3,8

The direct evidence of the presence of incorporated sulfonic species can be provided by FTIR, UV–Vis, DTA/TG, 29 Si MAS NMR, and 13 C MAS NMR. In FTIR spectra the band typical of –C–S stretching vibration appears at 603 cm −1 , 16 while the bands characteristic of S=O and S–OH vibrations are generally observed at 1380 and 840 cm −1 , respectively. 12,16 In the spectra of the sample with MPTMS anchored, the bands in the region 2850–2950 cm −1 assigned to C−H stretching vibration of the propyl chain 3 and at 2500 cm −1 corresponding to nonoxidized –SH groups were frequently observed, 15 while for the samples with CSPTMS the bands at 1567, 1460, and 682 cm −1 typical of phenyl ring were visible. 8 Besides, for both modifiers the characteristic IR band at 1455 cm −1 assigned to methoxy species vibration may be detected. 12 The Raman spectra confirmed the presence of anchored thiol and sulfone species by the presence of bands at 1254 cm −1 assigned to CH2–S, at 1043 and 1159 cm −1 typical of sulfonic groups and 2585 cm −1 characteristic of thiol species vibration. 3

Very useful information about the materials containing thiol and sulfonic species can be obtained from UV–Vis analysis. In the UV–Vis spectrum of the sample functionalized with MPTMS followed by its oxidation, both, –SH and –SO3H species can be detected. The band characteristic of electron transfer in thiol species is observed at 220 nm, while the band assigned to charge transfer in sulfonic groups is detected at 255 nm. 13 Thus this technique allows the estimation of the efficiency of thiol oxidation to sulfonic species. The oxidation of thiol species from MPTMS to sulfonic groups may be also monitored by XPS. In the S2p core-level spectra a component corresponding to –SH groups is visible at 164 eV, while the component at 169 eV is typical of sulfonic species. 3 Similar information can be obtained by DTA/TG analysis. In the DTA curve the exothermic effect of thiol species decomposition appeared at ca 326 o C, while the exothermic effect at ca 512 o C was due to the decomposition of sulfonic species. At the same time the TG curve revealed the mass loss that corresponded to exothermic effects detected by DTA. 12 More evidence of anchoring of organosilanes containing sulfonic groups is provided by NMR spectroscopy. As a result of functional groups introduction, the 29 Si MAS NMR spectra displayed additional signals, in comparison to those in the spectrum of parent silica support, between −40 and −80 ppm corresponding to T3 and T2 species in organosilanes anchored to silica by three or two siloxane bonds, respectively. 3,6 In the 13 C MAS NMR spectra, different signals may be detected depending on the modifier used for silica modification. For MPTMS functionalized silica, the NMR signals assigned to C3 in propyl chain were shifted by 13 ppm, while those assigned to C2 and C1 carbons were detected at 19 and 55 ppm. For the samples modified with CSPTMS the signals typical of C1, C2, C3, C4, and C5 appeared at 138, 128, 150, 15, and 29 ppm, respectively. The signals detected for CSPTMS correspond to ethane arenesulfonic acid species. 17

The analytical techniques mentioned above allow determination of changes in silica structure after its modification with organosilane and are able to prove the presence of active species at the support. However, it is also important to estimate the number of introduced sulfonic species in mesoporous silica which have an impact on catalytic activity. This information can be achieved from elemental analysis, acid/base titration and FTIR spectroscopy with pyridine adsorption used as a probe of acid sites.

The elemental analysis is widely used for estimation of the number of included organosilanes with sulfonic groups by detection of sulfur and carbon content in a given solid. However, in the samples modified with MPTMS, some part of detected sulfur may come from thiol species not oxidized to sulfonic groups. 4,5 Thus a better option for determination of total number of sulfonic species anchored to silica is acid–base titration of acid sites. 3 The concentration of sulfonic groups may be also established by pyridine adsorption followed by FTIR spectroscopy. It is well known that –SO3H groups play the role of Brønsted acid sites (BAS) and thus they can be easily detected by pyridine chemisorption. An example of such studies is presented in Ref. [4] . The band at 1547 cm −1 in the FTIR spectra of modified mesoporous silica assigned to symmetric vibrations in pyridine cations indicated that pyridinium ions were formed by the abstraction of proton from BAS. This band was accompanied by antisymmetric vibration band at 1639 cm −1 . The number of BAS can be calculated as the number of pyridine molecules chemisorbed on modified mesoporous silica, knowing the extinction coefficient of the band at 1547 cm −1 . 7 Moreover, the FTIR measurement combined with pyridine adsorption provides information on the acidity strength of sulfonic species. A comparison of the ratio of absorbance for the band corresponding to pyridinium cations recorded after desorption of pyridine at different temperatures can be used to measure relative strength of BAS. On the other hand, the position of the band at ca 1639 cm −1 corresponds to acid strength of BAS. The shift of the band position to higher wavenumbers indicates a higher strength of BAS. 7 Conclusions about the acidic strength of sulfonic species may be also drawn from the position of exothermic effect on the DTA curve assigned to –SO3H decomposition. If this effect appears at a higher temperature, the sulfonic species are more stabilized by silica support and thus exhibit higher acidity strength. 4

DIALS development at Diamond Light Source is performed by employees of Diamond Light Source, and STFC via CCP4.

We are supported by funding from Wellcome for grant 218270/Z/19/Z: DIALS: making serial crystallography data analysis accessible for biomedical researchers.

DIALS development at Lawrence Berkeley National Laboratory is supported by National Institutes of Health / National Institute of General Medical Sciences grant R01-GM117126. Work at LBNL is performed under Department of Energy contract DE-AC02-05CH11231.

2.4.8: X-Ray Diffraction Analysis - Biology

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