The COVID-19 pandemic has highlighted the need to develop broad-spectrum antiviral therapeutics targeting rapidly evolving coronaviruses. This research focuses on SARS-CoV-2 PLpro, a conserved viral protease that plays dual roles in viral polyprotein processing and host immune suppression. Using an integrated fragment-based drug discovery (FBDD) approach that combines high-throughput X-ray crystallography and biochemical assays, we systematically screened a diverse library of 800 fragment compounds. Structural characterization identified 129 validated binders occupying 12 distinct binding sites on PLpro. Remarkably, two fragments demonstrated potent micromolar inhibitory activity. Fr12895 inhibited SARS-CoV-2 PLpro protease activity (IC50 = 8.013 μM), as measured using the fluorogenic substrate RLRGG-AMC, while Fr12338 showed inhibition against PLpro deISGylase activity (IC50 = 4.5 μM), as determined with the substrate Ac-ISG15prox-Rh110MP. This provides a case for directly obtaining micromolar-active compounds through crystallographic fragment screening of a small number of random compounds. Detailed structural analysis revealed these fragments engage key functional regions including the blocking loop 2 (BL2) and ubiquitin/ISG15 binding interface through extensive hydrogen-bond networks and hydrophobic interactions. Our study reveals novel micromolar active inhibitors and druggable sites of SARS-CoV-2 PLpro by crystallographic fragment screening, provides crucial scaffold and a structural roadmap for developing broad-spectrum antivirals against coronaviruses.

The essential role of RIPK3 in necroptosis makes its inhibition a promising therapeutic strategy. However, the development of RIPK3 inhibitors has been hampered by on-target apoptosis and limited kinase selectivity. Inspired by the R69H mutation, which prevents on-target apoptosis by disrupting RIPK3 dimerization, we design LK-series inhibitors that effectively inhibit RIPK3 in biochemical assays and block TNF-α-induced necroptosis in both mouse L929 and human HT29 cells without inducing apoptosis. The representative compound, LK01003, shows high selectivity across a panel of 379 kinases. Our structural studies reveal that LK compounds act as Type I1/2 inhibitors, engaging a unique hydrophobic site and stabilizing an inactive conformation of RIPK3. Moreover, several type II inhibitors are also revealed to maintain RIPK3 in the inactive conformation and do not induce on-target apoptosis. These findings suggest a promising strategy for rational design of safe and selective inhibitors by locking the inactive conformation of RIPK3.

Maltooligosaccharides (MOs) have gained significant attention in the food and pharmaceutical industries owing to their valuable functional properties, including controlled sweetness, digestibility, and enhanced bioavailability. However, conventional MOs is production involves complex processing steps and significant production costs. A potential high-efficiency synthesis of specific MOs can be achieved through the ring-opening reaction of cyclodextrins (CDs) catalyzed by amylolytic enzymes. In this study, we analyze the catalytic conversion of α-, β-, and γ-CDs by a GH57 family amylopullulanase from Aquifex aeolicus (AaApu) using thin-layer chromatography (TLC). Our findings demonstrate that AaApu has a substrate specificity for γ-CD, while all three CDs exert competitive inhibition on pullulan hydrolysis. To elucidate the molecular mechanism of CDs as inhibitor and substrate of amylopullulanase, we determined high-resolution crystal structures of AaApu (wild-type and D352N) in complex with α-, β-, and γ-CD through co-crystallization. These findings establish a structure-function framework for understanding the bifunctional nature of CDs as both substrates and inhibitors in GH57 amylopullulanases.

Recurrence of coronavirus outbreaks and zoonotic origins of human coronaviruses underscore the importance of developing pan‐coronavirus antivirals. The highly conserved 3C‐like protease (3CL pro ) in coronaviruses, together with the well‐established druggability, makes it an ideal target for broad‐spectrum antiviral therapeutics. Here, the inhibitory activity of approved 3CL pro inhibitors, including nirmatrelvir, ensitrelvir, and simnotrelvir, against fifteen 3CL pro s is first reported by enzymatic assays. Despite their potent inhibition toward 3CL pro s of β‐CoVs, these inhibitors show reduced potency against 3CL pro s from the other three genera, particularly against two newly identified human coronaviruses (α‐CCoV‐HuPn‐2018 and δ‐PDCoV). In this context, continued efforts in structure‐based optimization of nirmatrelvir lead to the identification of compound 8 that potently inhibits a panel of 32 3CL pro s across all subgenera (IC 50 s: 19–146 n m ), with an IC 50 value of 61 and 81 n m against α‐CCoV‐HuPn‐2018 and δ‐PDCoV 3CL pro s, respectively. Moreover, it effectively inhibits nirmatrelvir‐resistant 3CL pro mutants and demonstrates broad‐spectrum antiviral efficacy in cells. These findings suggest an important rule that a small, non‐cyclic P2 segment and a P4 segment with a suitable size are preferred by the design of ultra‐broad‐spectrum 3CL pro inhibitors, and provide a proof‐of‐concept guide for developing broad‐spectrum antivirals as potential pan‐CoV therapeutics.

Glycoside Hydrolase Family 57 (GH57) amylopullulanase is a thermophilic endoamylase capable of hydrolyzing both α-1,4 and α-1,6-glycosidic bonds, demonstrating significant potential for one-step starch saccharification in industrial applications. However, the mechanisms underlying the dual catalytic activities of GH57 family amylopullulanase remain poorly understood. In this study, we report the first crystal structures of a GH57 amylopullulanase from Aquifex aeolicus (AaApu) in complex with oligosaccharides containing both α-1,4 and α-1,6 glycosidic bonds. Our structural analysis reveals that GH57 amylopullulanase features dual binding pockets arranged in a "Y"-shaped configuration, which accommodates branched-chain starches. The dual binding pockets share a common catalytic dyad composed of Glu256 and Asp352. Notably, unlike the typical retaining mechanism observed in many glycoside hydrolases, the distance between the catalytic residues in GH57 amylopullulanase is significantly larger (approximately 7 Å). This study provides critical insights into the structural basis of GH57 amylopullulanase activity and offers a foundation for the rational engineering of these enzymes for industrial applications.

Heat-shock protein 90 (HSP90) is a highly active molecular chaperone that plays a crucial role in cellular function. It facilitates the folding, assembly and stability of various oncogenic proteins, particularly kinases and transcription factors involved in regulating tumor growth and maintenance signaling pathways. Consequently, HSP90 inhibitors are being explored as drugs for cancer therapy. Crystallographic fragment screening is a novel screening method that has been developed in recent years for fragment-based drug discovery and is known for its high hit rate and its ability to provide direct insights into the complex structures of proteins and compounds. In this paper, high-diffraction-resolution crystals of the N-terminal domain of human HSP90α were employed in crystallographic fragment screening to discover binding fragments and binding sites. A diverse library of 800 structurally distinct fragments was screened, yielding 91 starting points for the fragment-based drug design of new HSP90α N-terminal inhibitors. Nearly a thousand crystals were measured, with 738 being processed and phased using a highly automated data-processing pipeline including data reduction and phasing, refinement and hit identification via PanDDA multi-data-set analysis. The 91 identified compounds bind to eight distinct regions of the HSP90α N-terminus, with 63 fragments located in the ATP-binding pocket and its surroundings, thus demonstrating the potential for the development of HSP90α- and ATP-binding inhibitors. This study emphasizes crystallographic fragment screening as a powerful method that can effectively identify fragment molecules and inhibitors that bind to HSP90α, contributing to ongoing efforts in cancer drug discovery.

Fragment-based lead discovery (FBLD) is an efficient and effective method for identifying novel chemical scaffolds that have advantages in drug development. X-ray crystallography has an inherent advantage in recognizing low-affinity fragments and integrates fragment identification with complex structure determination, making it an increasingly important tool for screening fragment compounds. Here, we introduce a crystallographic fragment-screening platform developed by the biological macromolecular crystallography group at Shanghai Synchrotron Radiation Facility, named the XFBLD-Platform. This platform promotes fragment-based lead discovery through a comprehensive workflow that includes high-throughput complex crystal preparation, crystal harvesting, diffraction data collection and analysis, and fragment-affinity estimation. It integrates advanced hardware, high-performance beamline facilities and specialized experimental design and data-management software. The platform provides a rapid and practical approach for structure-based drug development.

(Hyper)thermophilic enzymes derived from (hyper)thermophilic microorganisms have significant implications for both basic research and industrial applications. Investigating the thermal stability and catalytic mechanisms of thermophilic enzymes from a structural perspective is essential for the rational modification of these enzymes. Currently, X-ray crystallography remains one of the key techniques for determining the structures of biomolecules; however, protein crystallization is a multiparameter-regulated and often unpredictable process. Here, we present a method for the high-temperature crystallization of the GH57 family hyperthermophilic amylopullulanase from Aquifex aeolicus (AaApu), conducted at 338 K. In comparison to traditional crystallization methods performed at 277 or approximately 291 K, the crystals grown at high temperatures are larger, with dimensions increasing from 400 x 10 mu m2 to 200 x 150 mu m2. Additionally, the time required for crystal growth is significantly reduced from 5 days to 2 h, and there is a notable improvement in resolution, enhancing from 2.5 to 1.7 & Aring;. Furthermore, high-temperature crystallization facilitates the binding of larger molecular weight substrates to the protein molecules and reveals the catalytic mechanisms of enzymes. Overall, high-temperature crystallization preserves the original structure of thermophilic proteins, suggesting that it could be a promising method for thermophilic proteins.

An integrated computer software system for macromolecular crystallography (MX) data collection at the BL02U1 and BL10U2 beamlines of the Shanghai Synchrotron Radiation Facility is described. The system, Finback , implements a set of features designed for the automated MX beamlines, and is marked with a user-friendly web-based graphical user interface (GUI) for interactive data collection. The Finback client GUI can run on modern browsers and has been developed using several modern web technologies including WebSocket, WebGL, WebWorker and WebAssembly. Finback supports multiple concurrent sessions, so on-site and remote users can access the beamline simultaneously. Finback also cooperates with the deployed experimental data and information management system, the relevant experimental parameters and results are automatically deposited to a database.

To resolve photons hungry for weak diffraction samples by the crystallographic method, a double-multilayer monochromator (DMM) was employed on an undulator beamline (BL17UM) at the Shanghai Synchrotron Radiation Facility (SSRF) to provide a focused sub-micron beam with high brightness for macromolecular crystallography experiments. High-quality crystallographic datasets from model protein crystal samples were collected and processed by an existing crystallographic program for structure solution and refinement. The data quality was compared with datasets from a normal silicon crystal monochromator to evaluate the bandwidth of the DMM effect on these crystallographic data. This experiment demonstrates that multilayer optics on an undulator beamline may play a valuable role in satisfying the demands of structure-related research, which requires novel methods.

The persistent pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants accentuates the great demand for developing effective therapeutic agents. Here, we report the development of an orally bioavailable SARS-CoV-2 3C-like protease (3CL pro ) inhibitor, namely simnotrelvir, and its preclinical evaluation, which lay the foundation for clinical trials studies as well as the conditional approval of simnotrelvir in combination with ritonavir for the treatment of COVID-19. The structure-based optimization of boceprevir, an approved HCV protease inhibitor, leads to identification of simnotrelvir that covalently inhibits SARS-CoV-2 3CL pro with an enthalpy-driven thermodynamic binding signature. Multiple enzymatic assays reveal that simnotrelvir is a potent pan-CoV 3CL pro inhibitor but has high selectivity. It effectively blocks replications of SARS-CoV-2 variants in cell-based assays and exhibits good pharmacokinetic and safety profiles in male and female rats and monkeys, leading to robust oral efficacy in a male mouse model of SARS-CoV-2 Delta infection in which it not only significantly reduces lung viral loads but also eliminates the virus from brains. The discovery of simnotrelvir thereby highlights the utility of structure-based development of marked protease inhibitors for providing a small molecule therapeutic effectively combatting human coronaviruses.

BL10U2 is an undulator-based macromolecular crystallography (MX) beamline located at the 3.5-GeV Shanghai Synchrotron Radiation Facility. BL10U2 is specifically designed for conducting routine and bio safety level-2 (BSL-2) MX experiments utilizing high-flux tunable X-rays with energies from 7 to 18 keV, providing a beam spot size of 20 µm (horizontal) × 10 µm (vertical) at the sample point. Certification by the Shanghai Pudong Municipal Health Commission confirmed the capability to perform BSL-2 MX experiments. The beamline is currently equipped with an Eiger X 16M detector and two newly developed in-house high-precision diffractometers that can be switched to perform conventional or in situ crystal diffraction experiments. An automatic sample changer developed in-house allows fast sample exchange in less than 30s, supporting high-throughput MX experimentation and rapid crystal screening. Data collection from both the diffractometer and detector was controlled by an in-house developed data collection software (Finback) with a user-friendly interface for convenient operation. This study presents a comprehensive overview of the facilities, experimental methods, and performance characteristics of the BL10U2 beamline.

Macromolecular crystallography beamline BL17U1 at the Shanghai Synchrotron Radiation Facility has been relocated, upgraded, and given a new ID (BL02U1). It now delivers X-rays in the energy range of 6–16 keV, with a focused beam of 11.6 µm × 4.8 µm and photon flux greater than 10 12 phs/s. The high credibility and stability of the beam and good timing synchronization of the equipment significantly improve the experimental efficiency. Since June 2021, when it officially opened to users, over 4200 h of beamtime have been provided to over 200 research groups to collect data at the beamline. Its good performance and stable operation have led to the resolution of several structures based on data collected at the beamline.

MMACHC is an essential protein for the body to metabolise vitamin B12, and its deficiency will cause cblC-type methylmalonic aciduria and homocystinuria. MMACHC can interact with cyanocobalamin (a type of vitamin B12) cofactor and plays an important role in targeting cyanocobalamin to the enzyme of interest. In this paper, the GST-tag fusion-tagged MMACHC protein was successfully expressed by Escherichia coli (E. coli) low-temperature induction, and the high-purity MMACHC protein was successfully purified by affinity chromatography and gel filtration. Further, the crystal structure of MMACHC and cyanocobalamin complex was obtained with a resolution of 1.93 Å using X-ray diffraction. By analysing the complex structure of MMACHC and cyanocobalamin, we revealed the reasons for the diversity of MMACHC substrates and explained the reasons for the differences in disease conditions caused by different MMACHC site mutations. The acquisition of the complex structure of MMACHC and cyanocobalamin will play a significant role in promoting research on the metabolic pathway of vitamin B12.

DarR, a novel member of the LTTR family derived from Vibrio fischeri, activates transcription in response to D-Asp and regulates the overexpression of the racD genes encoding a putative aspartate racemase, RacD. Here, the crystal structure of full-length DarR and its mutant DarR-M202I were obtained by X-ray crystallography. According to the electron density map analysis of full-length DarR, the effector binding site of DarR is occupied by 2-Morpholinoethanesulfonic acid monohydrate (MES), which could interact with amino acids in the effector binding site and stabilize the effector binding site. Furthermore, we elaborated the structure of DarR-M202I, where methionine is replaced by isoleucine resulting in overexpression of the downstream operon. By comparing DarR-MES and DarR-M202I, we found similar behavior of DarR-MES in terms of the stability of the RD active pocket and the deflection angle of the DBD. The Isothermal titration calorimetry and Gel-filtration chromatography experiments showed that only when the target DNA sequence of a particular quasipalindromic sequence exceeds 19 bp, DarR can effectively bind to racD promoter. This study will help enhance our understanding of the mechanism in the transcriptional regulation of LTTR family transcription factors.

Macromolecular crystallography is commonly used to determine the structure of biological macromolecules. Currently the beamlines at synchrotron radiation facilities play an important role in macromolecular crystallography, and have produced an enormous number of molecular structures to help solve scientific questions and support applications. Structure information makes significant contributions to the virus-related research as well. However, it is mandatory to be protected the operators under a compatible biosafety infrastructure when a pathological agent is set up in a beamline. Here a level-2 biosafety protection for a macromolecular crystallography beamline at Shanghai Synchrotron Radiation Facility (SSRF) is introduced. To fulfill the biosafety in a radioactive environment, a dedicated design is implemented. Since the beamline will be opened to the external users from nationwide research units, the management process and experimental method are also drawn up.

The ongoing pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) urgently needs an effective cure. 3CL protease (3CL pro ) is a highly conserved cysteine proteinase that is indispensable for coronavirus replication, providing an attractive target for developing broad-spectrum antiviral drugs. Here we describe the discovery of myricetin, a flavonoid found in many food sources, as a non-peptidomimetic and covalent inhibitor of the SARS-CoV-2 3CL pro . Crystal structures of the protease bound with myricetin and its derivatives unexpectedly revealed that the pyrogallol group worked as an electrophile to covalently modify the catalytic cysteine. Kinetic and selectivity characterization together with theoretical calculations comprehensively illustrated the covalent binding mechanism of myricetin with the protease and demonstrated that the pyrogallol can serve as an electrophile warhead. Structure-based optimization of myricetin led to the discovery of derivatives with good antiviral activity and the potential of oral administration. These results provide detailed mechanistic insights into the covalent mode of action by pyrogallol-containing natural products and a template for design of non-peptidomimetic covalent inhibitors against 3CL pro s, highlighting the potential of pyrogallol as an alternative warhead in design of targeted covalent ligands.

A novel DNA polymerase found in the deep-sea vent phage NrS-1, was confirmed to have both DNA polymerase and primase activities. In this polymerase, the N-terminal residues 1-300 (referred to as N300) are the core region required for polymerizing DNA and catalyzing de novo DNA synthesis. Here, the crystal structure of N300 was solved at a resolution of 1.80 angstrom. The overall structure consists of a prim/pol domain and a helix bundle domain, which are separated by a 14-residue-long flexible tether (residues 177-190). Both the prim/pol domain of N300 and other primase-polymerases (prim-pol) encompass an analogous fold with conserved catalytic residues. Mutagenesis and enzymatic activity assays show that the acidic active-site residue E139 is required for both polymerase and primase activities. Functional assays confirm the essentiality of the helix bundle domain for primase activity. Furthermore, we identified a mutant (N300-Y261A) of the helix bundle domain, which probably plays an indispensable role in the primer initiation and recognition of template DNA. (C) 2019 Elsevier Inc. All rights reserved.

[Background] X-ray crystallography is still the most important means of obtaining atomic resolution structure of biological macromolecules. Obtaining protein crystals of sufficient size is the bottleneck of crystallography. Small crystals are easier to obtain than large crystals, and lattice packing is better. But small crystals are susceptible to radiation damage, and it is difficult to obtain complete structural information from a crystal even when data collection is performed at low temperatures. [Purpose] This paper aims to propose a method for acquiring small crystal data based on chip techniques at a microfocus beam line station of synchrotron radiation light source. [Methods] First of all, the chip was fabricated by laser lithography technology, and crystal was able to be deposited in the groove of the chip. Then, high-throughput data collection for diffraction of multi-crystals on the chip was achieved on the chip, and collected multiple chips data sets were emerged to get structure information of biological macromolecules. Finally, the feasibility of chip-based sample delivery method was verified by using the lysozyme crystal at the microfocus beamline (BL18U1) of SSRF. [Results] The quartz chip has low background scattering and high sample delivery efficiency. The structure of lysozyme with high resolution of 0.139 nm was obtained by chip loading and data acquisition. [Conclusions] The chip-based polycrystalline data acquisition method can obtain complete biomacromolecule structural information without obvious radiation damage by merging multiple data sets.

With the popularity of hybrid pixel array detectors, hundreds of diffraction data sets are collected at a biological macromolecular crystallography (MX) beamline every day. Therefore, the manual processing and recording procedure will be a very time-consuming and error-prone task. Aquarium is an automatic data processing and experiment information management system designed for synchrotron radiation source MX beamlines. It is composed of a data processing module, a daemon module and a web site module. Before experiments, the sample information can be registered into a database. The daemon module will submit data processing jobs to a high-performance cluster as soon as the data set collection is completed. The data processing module will automatically process data sets from data reduction to model building if the anomalous signal is available. The web site module can be used to monitor and inspect the data processing results.