Structural Basis for Coronaviral Main Proteases Inhibition by the 3CLpro Inhibitor GC376.

The main protease (Mpro) of coronaviruses participates in viral replication, serving as a hot target for drug design. GC376 is able to effectively inhibit the activity of Mpro, which is due to nucleophilic addition of GC376 by binding covalently with Cys145 in Mpro active site. Here, we used fluorescence resonance energy transfer (FRET) assay to analyze the IC50 values of GC376 against Mpros from six different coronaviruses (SARS-CoV-2, HCoV-229E, HCoV-HUK1, MERS-CoV, SARS-CoV, HCoV-NL63) and five Mpro mutants (G15S, M49I, K90R, P132H, S46F) from SARS-CoV-2 variants. The results showed that GC376 displays effective inhibition to various coronaviral Mpros and SARS-CoV-2 Mpro mutants. In addition, the crystal structures of SARS-CoV-2 Mpro (wide type)-GC376, SARS-CoV Mpro-GC376, MERS-CoV Mpro-GC376, and SARS-CoV-2 Mpro mutants (G15S, M49I, S46F, K90R, and P132H)-GC376 complexes were solved. We found that GC376 is able to fit into the active site of Mpros from different coronaviruses and different SARS-CoV-2 variants properly. Detailed structural analysis revealed key molecular determinants necessary for inhibition and illustrated the binding patterns of GC376 to these different Mpros. In conclusion, we not only proved the inhibitory activity of GC376 against different Mpros including SARS-CoV-2 Mpro mutants, but also revealed the molecular mechanism of inhibition by GC376, which will provide scientific guidance for the development of broad-spectrum drugs against SARS-CoV-2 as well as other coronaviruses.

Crystal structure of SARS-CoV-2 main protease (Mpro) mutants in complex with the non-covalent inhibitor CCF0058981.

SARS-CoV-2 constantly circulates and evolves worldwide, generating many variants and posing a menace to global health. It is urgently needed to discover effective medicines to treat the disease caused by SARS-CoV-2 and its variants. An established target for anti-SARS-CoV-2 drug discovery is the main protease (Mpro), since it exerts an irreplaceable action in viral life cycle. CCF0058981, derived from ML300, is a non-covalent inhibitor that exhibits low nanomolar potency against SARS-CoV-2 Mpro and submicromolar anti-SARS-CoV-2 activity, thereby providing a valuable starting point for drug design. However, structural basis underlying inhibition of SARS-CoV-2 Mpro by CCF0058981 remains undetermined. In this study, the crystal structures of CCF0058981 in complex with two SARS-CoV-2 Mpro mutants (M49I and V186F), which have been identified in the recently emerged Omicron subvariants, were solved. Structural analysis defined the pivotal molecular factors responsible for the interactions between CCF0058981 and these two Mpro mutants, and revealed the binding modes of CCF0058981 to Mpro M49I and V186F mutants. These data not only provide structural insights for SARS-CoV-2 Mpro inhibition by CCF0058981, but also add to develop effective broad-spectrum drugs against SARS-CoV-2 as well as its variants.

Crystal structures of main protease (Mpro) mutants of SARS-CoV-2 variants bound to PF-07304814.

There is an urgent need to develop effective antiviral drugs to prevent the viral infection caused by constantly circulating SARS-CoV-2 as well as its variants. The main protease (Mpro) of SARS-CoV-2 is a salient enzyme that plays a vital role in viral replication and serves as a fascinating therapeutic target. PF-07304814 is a covalent inhibitor targeting SARS-CoV-2 Mpro with favorable inhibition potency and drug-like properties, thus making it a promising drug candidate for the treatment of COVID-19. We previously solved the structure of PF-07304814 in complex with SARS-CoV-2 Mpro. However, the binding modes of PF-07304814 with Mpros from evolving SARS-CoV-2 variants is under-determined. In the current study, we expressed six Mpro mutants (G15S, K90R, M49I, S46F, V186F, and Y54C) that have been identified in Omicron variants including the recently emerged XBB.1.16 subvariant and solved the crystal structures of PF-07304814 bound to Mpro mutants. Structural analysis provided insight into the key molecular determinants responsible for the interaction between PF-07304814 and these mutant Mpros. Patterns for PF-07304814 to bind with these investigated Mpro mutants and the wild-type Mpro are generally similar but with some differences as revealed by detailed structural comparison. Structural insights presented in this study will inform the development of novel drugs against SARS-CoV-2 and the possible conformation changes of Mpro mutants when bound to an inhibitor.

Structural basis for the inhibition of coronaviral main proteases by ensitrelvir.

Main protease (Mpro) is a highly conserved cysteine protease that plays a vital role in the replication of coronaviruses, making it an attractive pan-coronaviral therapeutic target. Ensitrelvir (S-217622), developed by Shionogi, is the first orally active non-covalent, non-peptidic SARS-CoV-2 Mpro inhibitor, which also displays antiviral efficacy against other human coronaviruses as well as SARS-CoV-2 variants of concern (VOCs) and variants of interest (VOIs). Here, we report the crystal structures of the main proteases from SARS-CoV-2, SARS-CoV-2 VOC/VOIs, SARS-CoV, MERS-CoV, and HCoV-NL63 bound to the inhibitor S-217622. A detailed analysis of these structures illuminates key structural determinants essential for inhibition and elucidates the binding modes of the main proteases from different coronaviruses. Given the importance of the main protease for the treatment of coronaviral infection, structural insights obtained from this study could accelerate the design of novel antivirals with broad-spectrum efficacy against different human coronaviruses.

Crystal structures of main proteases of SARS-CoV-2 variants bound to a benzothiazole-based inhibitor.

Main protease (M pro) serves as an indispensable factor in the life cycle of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as well as its constantly emerging variants and is therefore considered an attractive target for antiviral drug development. Benzothiazole-based inhibitors targeting M pro have recently been investigated by several groups and proven to be promising leads for coronaviral drug development. In the present study, we determine the crystal structures of a benzothiazole-based inhibitor, YH-53, bound to M pro mutants from SARS-CoV-2 variants of concern (VOCs) or variants of interest (VOIs), including K90R (Beta, B.1.351), G15S (Lambda, C.37), Y54C (Delta, AY.4), M49I (Omicron, BA.5) and P132H (Omicron, B.1.1.529). The structures show that the benzothiazole group in YH-53 forms a C-S covalent bond with the sulfur atom of catalytic residue Cys145 in SARS-CoV-2 M pro mutants. Structural analysis reveals the key molecular determinants necessary for interaction and illustrates the binding mode of YH-53 to these mutant M pros. In conclusion, structural insights from this study offer more information to develop benzothiazole-based drugs that are broader spectrum, more effective and safer.

RNA-binding protein DHX9 promotes glioma growth and tumor-associated macrophages infiltration via TCF12.

BACKGROUND: Glioma is the most common malignant tumor of the central nervous system, with high heterogeneity, strong invasiveness, high therapeutic resistance, and poor prognosis, comprehending a serious challenge in neuro-oncology. Until now, the mechanisms underlying glioma progression have not been fully elucidated. METHODS: The expression of DExH-box helicase 9 (DHX9) in tissues and cells was detected by qRT-PCR and western blot. EdU and transwell assays were conducted to assess the effect of DHX9 on proliferation, migration and invasion of glioma cells. Cocultured model was used to evaluate the role of DHX9 on macrophages recruitment and polarization. Animal study was performed to explore the role of DHX9 on macrophages recruitment and polarization in vivo. Bioinformatics analysis, dual-luciferase reporter assay and chromatin immunoprecipitation (ChIP)-qPCR assay was used to explore the relation between DHX9 and TCF12/CSF1. RESULTS: DHX9 was elevated in gliomas, especially in glioblastoma multiforme (GBM). Besides promoting the proliferation, migration, and invasion of glioma cells, DHX9 facilitated the infiltration of macrophages into glioma tissues and polarization to M2-like macrophages, known as tumor-associated macrophages (TAMs). DHX9 silencing decreased the expression of colony-stimulating factor 1 (CSF1), which partially restored the inhibitory effect on malignant progress of glioma and infiltration of TAMs caused by DHX9 knockdown by targeting the transcription factor 12 (TCF12). Moreover, TCF12 could directly bind to the promoter region of CSF1. CONCLUSION: DHX9/TCF12/CSF1 axis regulated the increases in the infiltration of TAMs to promote glioma progression and might be a novel potential target for future immune therapies against gliomas.

Structural Basis for the Inhibition of SARS-CoV-2 Mpro D48N Mutant by Shikonin and PF-07321332.

Preventing the spread of SARS-CoV-2 and its variants is crucial in the fight against COVID-19. Inhibition of the main protease (Mpro) of SARS-CoV-2 is the key to disrupting viral replication, making Mpro a promising target for therapy. PF-07321332 and shikonin have been identified as effective broad-spectrum inhibitors of SARS-CoV-2 Mpro. The crystal structures of SARS-CoV-2 Mpro bound to PF-07321332 and shikonin have been resolved in previous studies. However, the exact mechanism regarding how SARS-CoV-2 Mpro mutants impact their binding modes largely remains to be investigated. In this study, we expressed a SARS-CoV-2 Mpro mutant, carrying the D48N substitution, representing a class of mutations located near the active sites of Mpro. The crystal structures of Mpro D48N in complex with PF-07321332 and shikonin were solved. A detailed analysis of the interactions between Mpro D48N and two inhibitors provides key insights into the binding pattern and its structural determinants. Further, the binding patterns of the two inhibitors to Mpro D48N mutant and wild-type Mpro were compared in detail. This study illustrates the possible conformational changes when the Mpro D48N mutant is bound to inhibitors. Structural insights derived from this study will inform the development of new drugs against novel coronaviruses.

Genome-wide detection of RNA editing events during the hair follicles cycle of Tianzhu white yak.

BACKGROUND: The hair coat is available for the yak to live in the harsh environment of the plateau. Besides, improving the hair production of yak is necessary for its textile industry development. Hair grows from hair follicles (HFs). The HFs undergo periodic growth after birth and are regulated by the complex gene regulatory network. However, the molecular mechanism of HFs regeneration in the Tianzhu white yak remains unclear. RNA editing is a post-transcriptional mechanism that regulates gene expression and produces new transcripts. Hence, we investigated the influence of the A-to-I RNA editing events on the HFs cycle of the Tianzhu white yak. RESULTS: We finally identified 54,707 adenosine-to-inosine (A-to-I) RNA editing sites (RESs) from RNA sequencing data of the HFs cycle in the Tianzhu white yak. Annotation results showed RESs caused missense amino acid changes in 7 known genes. And 202 A-to-I editing sites altered 23 target genes of 140 microRNAs. A total of 1,722 differential RESs were identified during the HFs cycle of Tianzhu white yak. GO and KEGG enrichment analysis revealed several signaling pathways and GO terms involved skin development, hair growth, and HFs cycle. Such as genes with differential RNA editing levels were significantly enriched in the peroxisome, metabolic pathways, Notch signaling pathway, and PPAR signaling pathway. Besides, the editing sites in HFs development-related genes FAS, APCDD1, WWOX, MPZL3, RUNX1, KANK2, DCN, DSC2, LEPR, HEPHL1, and PTK2B were suggested as the potential RESs involving HFs development. CONCLUSION: This study investigated the global A-to-I RNA editing events during the HFs cycle of yak skin tissue and expanded the knowledge of A-to-I RNA editing on the HFs cycle. Furthermore, this study revealed that RNA editing-influenced genes may regulate the HFs cycle by participating in the HFs development-related pathways. The findings might provide new insight into the regulation of RNA editing in hair growth.

Understanding Mammalian Hair Follicle Ecosystems by Single-Cell RNA Sequencing.

Single-cell sequencing technology can fully reflect the heterogeneity of cell populations at the single cell level, making it possible for us to re-recognize various tissues and organs. At present, the sequencing study of hair follicles is transiting from the traditional ordinary transcriptome level to the single cell level, which will provide diverse insights into the function of hair follicle cells. This review focuses on research advances in the hair follicle microenvironment obtained from scRNA-seq studies of major cell types in hair follicle development, with a special emphasis on the discovery of new subpopulations of hair follicles by single-cell techniques. We also discuss the problems and current solutions in scRNA-seq observation and look forward to its prospects.

Structural Basis for the Inhibition of Coronaviral Main Proteases by a Benzothiazole-Based Inhibitor.

The ongoing spread of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused hundreds of millions of cases and millions of victims worldwide with serious consequences to global health and economies. Although many vaccines protecting against SARS-CoV-2 are currently available, constantly emerging new variants necessitate the development of alternative strategies for prevention and treatment of COVID-19. Inhibitors that target the main protease (Mpro) of SARS-CoV-2, an essential enzyme that promotes viral maturation, represent a key class of antivirals. Here, we showed that a peptidomimetic compound with benzothiazolyl ketone as warhead, YH-53, is an effective inhibitor of SARS-CoV-2, SARS-CoV, and MERS-CoV Mpros. Crystal structures of Mpros from SARS-CoV-2, SARS-CoV, and MERS-CoV bound to the inhibitor YH-53 revealed a unique ligand-binding site, which provides new insights into the mechanism of inhibition of viral replication. A detailed analysis of these crystal structures defined the key molecular determinants required for inhibition and illustrate the binding mode of Mpros from other coronaviruses. In consideration of the important role of Mpro in developing antivirals against coronaviruses, insights derived from this study should add to the design of pan-coronaviral Mpro inhibitors that are safer and more effective.

Whole transcriptome analyses and comparison reveal the metabolic differences between oxidative and glycolytic skeletal muscles of yak.

Mammalian skeletal muscle is composed of various muscle fibers that exhibit different physiological and metabolic features. Muscle fiber type composition has significant influences on the meat quality of livestock. In this study, we comprehensively analyzed the whole transcriptome profiles of the oxidative muscle biceps femoris (BF) and the glycolytic muscle obliquus externus abdominis (OEA) of yak. A total of 1436 mRNAs, 1172 lncRNAs, and 218 circRNAs were differentially expressed in the oxidative muscles compared with the glycolytic muscles. KEGG annotation showed that differentially expressed mRNAs regulated by lncRNA and circRNA were mainly involved in PPAR signaling pathway, citrate cycle (TCA cycle), and PI3K-Akt signaling pathway, which reflect the different metabolic properties between oxidative and glycolytic muscles. In addition, regulatory networks associated with muscle fiber type conversion and mitochondria energy metabolism in muscles were constructed. Our study provides new evidence for a better understanding of the molecular mechanisms underlying skeletal muscle fiber determination and meat quality traits of yak.

Pupil Diameter Changes after Anesthesia with Different Doses of Sufentanil under Ultrasound Monitoring.

Objective: This study aims to observe the changes in pupil diameter (PD) after anesthesia with different doses of sufentanil with the ultrasound method and observe whether pupil contraction is correlated with hemodynamic changes and bispectral index (BIS) values. Methods: A total of 124 patients between the ages of 18-65 with ASA I-II undergoing general anesthesia for surgery were enrolled in the study. According to the sufentanil dose initially injected, they were randomly divided into groups P, S1, S2, and S3, with 31 cases in each group. Group P was injected with normal saline. Group S1 was injected with 0.2 µg/kg of sufentanil. Group S2 was injected with 0.4 µg/kg of sufentanil. Group S3 was injected with 0.6 µg/kg of sufentanil. Following propofol administration and eye closure, the pupil diameter (PD) of the patients in the four groups was observed and measured by ultrasound after the loss of consciousness (T1) and within 3 min after the sufentanil injection at an interval of 30 s (30 s (T2), 1 min (T3), 1 min 30 s (T4), 2 min (T5), 2 min 30 s (T6), and 3 min (T7)). PD, systolic blood pressure (SBP), diastolic blood pressure (DBP), heart rate (HR), and BIS values at T1-T7 were recorded. Results: The ultrasonic method was used to observe that different doses of sufentanil could make the patients' pupils contract. During anesthesia induction, the changes in PD have a positive correlation with SBP, DBP, HR, and BIS values. Conclusion: Ultrasound can become a new noninvasive method to monitor pupil changes during general anesthesia, and ultrasonic observation of pupil changes has great potential for individualized analgesia management in the perioperative period.

Crystal structures of the catalytic domain of human PARP15 in complex with small molecule inhibitors.

PARP15, or ARTD7, is an enzyme carrying out mono-ADP-ribosylation and regulating activities of a range of cellular proteins. This enzyme belongs to the family of the poly(ADP-ribose) polymerases (PARPs), which comprises of proteins with various potential disease indications. Due to their involvement in a number of cellular processes and important role in DNA repair and regulation, PARPs have been considered attractive therapeutic targets over the past few years. The pursuit of small molecule PARP inhibitors has resulted in several FDA approved drugs for multiple cancers so far. As the use of PARP inhibitors as drug scaffolds is actively explored recently, there is increasing interest in the design of selective inhibitors based on the structural features of the PARP proteins. Here, we solved high-resolution crystal structures of the human PARP15 catalytic domain in complex with three marketed drugs of PARP inhibitors, which includes compounds 3-AB, iniparib and niraparib. The structures reported here contribute to our understanding of the ligand binding modes and structural features in the PARP15 catalytic domain, which can be employed to guide the rational design of selective inhibitors of PARPs.

Structural Basis of Main Proteases of Coronavirus Bound to Drug Candidate PF-07304814.

New variants of the severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) emerged and spread rapidly all over the world, which strongly supports the need for pharmacological options to complement vaccine strategies. Main protease (Mpro or 3CLpro) is a critical enzyme in the life cycle of SARS-CoV-2 and appears to be highly conserved among different genera of coronaviruses, making it an ideal target for the development of drugs with broad-spectrum property. PF-07304814 developed by Pfizer is an intravenously administered inhibitor targeting SARS-CoV-2 Mpro. Here we showed that PF-07304814 displays broad-spectrum inhibitory activity against Mpros from multiple coronaviruses. Crystal structures of Mpros of SARS-CoV-2, SARS-CoV, MERS-CoV, and HCoV-NL63 bound to the inhibitor PF-07304814 revealed a conserved ligand-binding site, providing new insights into the mechanism of inhibition of viral replication. A detailed analysis of these crystal structures complemented by comprehensive comparison defined the key structural determinants essential for inhibition and illustrated the binding mode of action of Mpros from different coronaviruses. In view of the importance of Mpro for the medications of SARS-CoV-2 infection, insights derived from the present study should accelerate the design of pan-coronaviral main protease inhibitors that are safer and more effective.

Structural Basis of the Main Proteases of Coronavirus Bound to Drug Candidate PF-07321332.

The high mutation rate of COVID-19 and the prevalence of multiple variants strongly support the need for pharmacological options to complement vaccine strategies. One region that appears highly conserved among different genera of coronaviruses is the substrate-binding site of the main protease (Mpro or 3CLpro), making it an attractive target for the development of broad-spectrum drugs for multiple coronaviruses. PF-07321332, developed by Pfizer, is the first orally administered inhibitor targeting the main protease of SARS-CoV-2, which also has shown potency against other coronaviruses. Here, we report three crystal structures of the main protease of SARS-CoV-2, SARS-CoV, and Middle East respiratory syndrome (MERS)-CoV bound to the inhibitor PF-07321332. The structures reveal a ligand-binding site that is conserved among SARS-CoV-2, SARS-CoV, and MERS-CoV, providing insights into the mechanism of inhibition of viral replication. The long and narrow cavity in the cleft between domains I and II of the main protease harbors multiple inhibitor-binding sites, where PF-07321332 occupies subsites S1, S2, and S4 and appears more restricted than other inhibitors. A detailed analysis of these structures illuminated key structural determinants essential for inhibition and elucidated the binding mode of action of the main proteases from different coronaviruses. Given the importance of the main protease for the treatment of SARS-CoV-2 infection, insights derived from this study should accelerate the design of safer and more effective antivirals. IMPORTANCE The current pandemic of multiple variants has created an urgent need for effective inhibitors of SARS-CoV-2 to complement vaccine strategies. PF-07321332, developed by Pfizer, is the first orally administered coronavirus-specific main protease inhibitor approved by the FDA. We solved the crystal structures of the main protease of SARS-CoV-2, SARS-CoV, and MERS-CoV that bound to the PF-07321332, suggesting PF-07321332 is a broad-spectrum inhibitor for coronaviruses. Structures of the main protease inhibitor complexes present an opportunity to discover safer and more effective inhibitors for COVID-19.

A self-developed contained bag for laparoscopic myomectomy morcellation.

OBJECTIVES: Open power morcellation during a laparoscopic myomectomy (LM) can result in the dissemination of benign or occult malignant tumor cells in the abdominopelvic cavity. The development of a new contained collection bag for power morcellation is now favored by gynecologic surgeons worldwide. MATERIAL AND METHODS: This study was a single-arm trial comprising 20 women who consecutively underwent an LM involving the use of a newly designed contained collection bag for power morcellation between November 3rd 2017 and April 31st 2018. There was also a historical control group consisting of 30 women who underwent open power morcellation during an LM between May 1st 2017 and October 31st 2017. All the essential information concerning the patients and surgically related data, including the myoma size, the operation duration, and the cell count of the intraperitoneal irrigating fluid, were collected and analyzed. RESULTS: The uterus size and the maximum diameters of the uterus and the myoma of the two groups were not significantly different (p = 0.65, p = 0.71, and p = 0.31, respectively). Pseudopneumoperitoneum was established and clear visualization was guaranteed in all 20 cases in the experimental group. The remaining fragment tissue amount (mean ± SD) and weight (mean ± SD) in the collection bag after morcellation in the experimental group were 5.00 ± 1.48 and 3.87 ± 1.31 (g). All the collection bags were routinely examined after the LM using normal saline, and no leaks or lesions were found. The cell counts of the intraperitoneal irrigating fluid both before and after morcellation were less than 105-106/L. The pathology of all the tissues confirmed that there were no malignant tumors. The operation of the experimental group was 18 mins longer than that of the historical control group (p = 0.00). CONCLUSIONS: This newly designed collection bag system for LM morcellation is effective, feasible, and safe.

Gliosarcoma with osteosarcomatous component: A case report and short review illustration.

BACKGROUND: Gliosarcoma (GS) represents a rare variant of glioblastoma in the central nervous system, characterized by biphasic histopathological features of gliomatous and sarcomatous components. Here, we present an unusual case of GS, which also demonstrated osteosarcomatous differentiation. CASE PRESENTATION: A 65-year-old female patient underwent gross total resection (GTR) of the right temporal lobe lesion. Subsequently received 60 Gy external beam radiation therapy and chemotherapy. Postoperative histopathological analysis indicated that the sarcomatous portion of the typical fibrosarcoma pattern mingled with areas of osteoid structure. The molecular pathological analysis demonstrated IDH1/2 wild-type and MGMT promoter island methylated phenotype. Target Enrichment Sequencing (TES) was performed on the gliomatous and sarcomatous components of the tumor tissues. TERT promoter, RB1, NF1, TP53 mutations and copy number variations (CNVs) on chromosome 7, 10q, 11q, 12, 13, 17 and 22 were observed in gliomatous and fibro-sarcomatous mixed tumor tissue; While we found TERT promoter, RB1, TP53 mutations and CNVs on chromosome 2q, 3q, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19 and 22 in osteosarcomatous component. Noteworthy, EGFR amplification was not observed in both gliomatous/fibro-sarcomatous and osteosarcomatous components. CONCLUSIONS: Integrated with histopathology, molecular pathology, and genomic alteration analysis, we report a case of GS with an extremely rare histopathologic phenotype of osteosarcomatous differentiation, who also suffered lung multi-metastases. Additionally, synthesizing the literature review, our study of this unusual differentiation of GS into osteosarcoma may provide novel insight into the natural history of GS.

Structure-Based Discovery and Structural Basis of a Novel Broad-Spectrum Natural Product against the Main Protease of Coronavirus.

Over the past 20 years, the severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome CoV (MERS-CoV), and SARS-CoV-2 emerged, causing severe human respiratory diseases throughout the globe. Developing broad-spectrum drugs would be invaluable in responding to new, emerging coronaviruses and to address unmet urgent clinical needs. Main protease (Mpro; also known as 3CLpro) has a major role in the coronavirus life cycle and is one of the most important targets for anti-coronavirus agents. We show that a natural product, noncovalent inhibitor, shikonin, is a pan-main protease inhibitor of SARS-CoV-2, SARS-CoV, MERS-CoV, human coronavirus (HCoV)-HKU1, HCoV-NL63, and HCoV-229E with micromolar half maximal inhibitory concentration (IC50) values. Structures of the main protease of different coronavirus genus, SARS-CoV from the betacoronavirus genus and HCoV-NL63 from the alphacoronavirus genus, were determined by X-ray crystallography and revealed that the inhibitor interacts with key active site residues in a unique mode. The structure of the main protease inhibitor complex presents an opportunity to discover a novel series of broad-spectrum inhibitors. These data provide substantial evidence that shikonin and its derivatives may be effective against most coronaviruses as well as emerging coronaviruses of the future. Given the importance of the main protease for coronavirus therapeutic indication, insights from these studies should accelerate the development and design of safer and more effective antiviral agents. IMPORTANCE The current pandemic has created an urgent need for broad-spectrum inhibitors of SARS-CoV-2. The main protease is relatively conservative compared to the spike protein and, thus, is one of the most promising targets in developing anti-coronavirus agents. We solved the crystal structures of the main protease of SARS-CoV and HCoV-NL63 that bound to shikonin. The structures provide important insights, have broad implications for understanding the structural basis underlying enzyme activity, and can facilitate rational design of broad-spectrum anti-coronavirus ligands as new therapeutic agents.

Crystal structures of human coronavirus NL63 main protease at different pH values.

Human coronavirus NL63 (HCoV-NL63), which belongs to the genus Alphacoronavirus, mainly infects children and the immunocompromized and is responsible for a series of clinical manifestations, including cough, fever, rhinorrhoea, bronchiolitis and croup. HCoV-NL63, which was first isolated from a seven-month-old child in 2004, has led to infections worldwide and accounts for 10% of all respiratory illnesses caused by etiological agents. However, effective antivirals against HCoV-NL63 infection are currently unavailable. The HCoV-NL63 main protease (Mpro), also called 3C-like protease (3CLpro), plays a vital role in mediating viral replication and transcription by catalyzing the cleavage of replicase polyproteins (pp1a and pp1ab) into functional subunits. Moreover, Mpro is highly conserved among all coronaviruses, thus making it a prominent drug target for antiviral therapy. Here, four crystal structures of HCoV-NL63 Mpro in the apo form at different pH values are reported at resolutions of up to 1.78 Å. Comparison with Mpro from other human betacoronaviruses such as SARS-CoV-2 and SARS-CoV reveals common and distinct structural features in different genera and extends knowledge of the diversity, function and evolution of coronaviruses.

Effects of Stellate Ganglion Block Through Different Approaches Under Guidance of Ultrasound.

OBJECTIVE: This study aimed to investigate the effects of stellate ganglion block (SGB) through different approaches under guidance of ultrasound. METHODS: A total of 130 patients undergoing SGB in our hospital between February 2019 and February 2020 were enrolled as the research subjects. According to the random number table method, these subjects were divided into two groups: a modified 6th cervical vertebra (C6) group (n = 65) and a 7th cervical vertebra (C7) group (n = 65). Under the guidance of ultrasound, the subjects in the modified C6 group were punctured at the level of the C6 transverse process, and the subjects in the C7 group were punctured at the level of the C7 transverse process. The operation duration, number of puncture angle adjustments, block effects, and adverse reactions for SGB were compared between the two groups. RESULTS: The modified C6 group showed shorter SGB operation duration and a lower number of puncture angle adjustments than the C7 group, and the differences were statistically significant (P < 0.05). Horner Syndrome occurred in both groups after SGB. The incidence of adverse reactions in the modified C6 group was 4.62%, comprising 1 case of hoarseness and 2 cases of slowed pulse, while that in the C7 group was 6.15%, with 1 case of hoarseness and 3 cases of slowed pulse; the difference between the two groups was not statistically significant (P > 0.05). CONCLUSION: The operation duration for modified SGB guided by ultrasound puncturing at the C6 transverse process is shorter and requires fewer puncture angle adjustments than puncturing at the C7 transverse process; however, there is no significant difference between the incidence of adverse reactions or the blocking effects of the two methods.