Structure-based development of potent and selective type-II kinase inhibitors of RIPK1

Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) functions as a key regulator in inflammation and cell death and is involved in mediating a variety of inflammatory or degenerative diseases. A number of allosteric RIPK1 inhibitors (RIPK1i) have been developed, and some of them have already advanced into clinical evaluation. Recently, selective RIPK1i that interact with both the allosteric pocket and the ATP-binding site of RIPK1 have started to emerge. Here, we report the rational development of a new series of type-II RIPK1i based on the rediscovery of a reported but mechanistically atypical RIPK3i. We also describe the structure-guided lead optimization of a potent, selective, and orally bioavailable RIPK1i, 62, which exhibits extraordinary efficacies in mouse models of acute or chronic inflammatory diseases. Collectively, 62 provides a useful tool for evaluating RIPK1 in animal disease models and a promising lead for further drug development.

Flavonoids from the roots and rhizomes of Sophoratonkinensis and their in vitro anti-SARS-CoV-2 activity / 中国天然药物

Acute respiratory infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) had caused a global pandemic since 2019, and posed a serious threat to global health security. Traditional Chinese medicine (TCM) has played an indispensable role in the battle against the epidemic. Many components originated from TCMs were found to inhibit the production of SARS-CoV-2 3C-like protease (3CLpro) and papain-like protease (PLpro), which are two promising therapeutic targets to inhibit SARS-CoV-2. This study describes a systematic investigation of the roots and rhizomes of Sophora tonkinensis, which results in the characterization of 12 new flavonoids, including seven prenylated flavanones (1-7), one prenylated flavonol (8), two prenylated chalcones (9-10), one isoflavanone (11), and one isoflavan dimer (12), together with 43 known compounds (13-55). Their structures including the absolute configurations were elucidated by comprehensive analysis of MS, 1D and 2D NMR data, and time-dependent density functional theory electronic circular dichroism (TDDFT ECD) calculations. Compounds 12 and 51 exhibited inhibitory effects against SARS-CoV-2 3CLpro with IC50 values of 34.89 and 19.88 μmol·L-1, repectively while compounds 9, 43 and 47 exhibited inhibitory effects against PLpro with IC50 values of 32.67, 79.38, and 16.74 μmol·L-1, respectively.

High-throughput screening of SARS-CoV-2 main and papain-like protease inhibitors

The global COVID-19 coronavirus pandemic has infected over 109 million people, leading to over 2 million deaths up to date and still lacking of effective drugs for patient treatment. Here, we screened about 1.8 million small molecules against the main protease (Mpro) and papain like protease (PLpro), two major proteases in severe acute respiratory syndrome-coronavirus 2 genome, and identified 1851Mpro inhibitors and 205 PLpro inhibitors with low nmol/l activity of the best hits. Among these inhibitors, eight small molecules showed dual inhibition effects on both Mpro and PLpro, exhibiting potential as better candidates for COVID-19 treatment. The best inhibitors of each protease were tested in antiviral assay, with over 40% of Mpro inhibitors and over 20% of PLpro inhibitors showing high potency in viral inhibition with low cytotoxicity. The X-ray crystal structure of SARS-CoV-2 Mpro in complex with its potent inhibitor 4a was determined at 1.8 Å resolution. Together with docking assays, our results provide a comprehensive resource for future research on anti-SARS-CoV-2 drug development.

Identification of C270 as a novel site for allosteric modulators of SARS-CoV-2 papain-like protease

The papain-like protease (PLpro) in coronavirus is one of key cysteine proteases responsible for the proteolytic processing of viral polyproteins, and plays an important role in dysregulation of host immune response. PLpro is a promising therapeutic target with a major challenge in inhibitor design due to the restricted S1/S2 sites for two consecutive glycine of substrates. Here we reported the discovery of two activators of the SARS-CoV-2 PLpro from a biochemical screening, and the identification of the unique residue, C270, as an allosteric and covalent regulation site for the activators. This site was also specifically modified by glutathione oxidized, resulting in the S-glutathionylation and activation of the protease. Furthermore, one compound was found to allosterically inhibit the protease by covalent binding to this crucial site. Together, these results elucidated an unrevealed molecular mechanism for allosteric modulation of the proteases activity, and provided a new strategy for discovery of allosteric inhibitors of the SARS-CoV-2 PLpro.

Bispecific prodrug nanoparticles circumventing multiple immune resistance mechanisms for promoting cancer immunotherapy

Cancer immunotherapy is impaired by the intrinsic and adaptive immune resistance. Herein, a bispecific prodrug nanoparticle was engineered for circumventing immune evasion of the tumor cells by targeting multiple immune resistance mechanisms. A disulfide bond-linked bispecific prodrug of NLG919 and JQ1 (namely NJ) was synthesized and self-assembled into a prodrug nanoparticle, which was subsequently coated with a photosensitizer-modified and tumor acidity-activatable diblock copolymer PHP for tumor-specific delivery of NJ. Upon tumor accumulation via passive tumor targeting, the polymeric shell was detached for facilitating intracellular uptake of the bispecific prodrug. NJ was then activated inside the tumor cells for releasing JQ1 and NLG919 via glutathione-mediated cleavage of the disulfide bond. JQ1 is a bromodomain-containing protein 4 inhibitor for abolishing interferon gamma-triggered expression of programmed death ligand 1. In contrast, NLG919 suppresses indoleamine-2,3-dioxygenase 1-mediated tryptophan consumption in the tumor microenvironment, which thus restores robust antitumor immune responses. Photodynamic therapy (PDT) was performed to elicit antitumor immunogenicity by triggering immunogenic cell death of the tumor cells. The combination of PDT and the bispecific prodrug nanoparticle might represent a novel strategy for blockading multiple immune evasion pathways and improving cancer immunotherapy.

Structural characterization of cocktail-like targeting polysaccharides from Ecklonia kurome Okam and their anti-SARS-CoV-2 activities in vitro

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent responsible for the worldwide coronavirus disease 2019 (COVID-19) outbreak. Investigation has confirmed that polysaccharide heparan sulfate can bind to the spike protein and block SARS-CoV-2 infection. Theoretically, similar structure of nature polysaccharides may also have the impact on the virus. Indeed, some marine polysaccharide has been reported to inhibit SARS-Cov-2 infection in vitro, however the convinced targets and mechanism are still vague. By high throughput screening to target 3CLpro enzyme, a key enzyme that plays a pivotal role in the viral replication and transcription using nature polysaccharides library, we discover the mixture polysaccharide 375 from seaweed Ecklonia kurome Okam completely block 3Clpro enzymatic activity (IC50, 0.48 {micro}M). Further, the homogeneous polysaccharide 37502 from the 375 may bind to 3CLpro molecule well (kD value : 4.23 x 10-6). Very interestingly, 37502 also can potently disturb spike protein binding to ACE2 receptor (EC50, 2.01 {micro}M). Importantly, polysaccharide 375 shows good anti-SARS-CoV-2 infection activity in cell culture with EC50 values of 27 nM (99.9% inhibiting rate at the concentration of 20 {micro}g/mL), low toxicity (LD50: 136 mg/Kg on mice). By DEAE ion-exchange chromatography, 37501, 37502 and 37503 polysaccharides are purified from native 375. Bioactivity test show that 37501 and 37503 may impede SARS-Cov-2 infection and virus replication, however their individual impact on the virus is significantly less that of 375. Surprisingly, polysaccharide 37502 has no inhibition effect on SARS-Cov-2. The structure study based on monosaccharide composition, methylation, NMR spectrum analysis suggest that 375 contains guluronic acid, mannuronic acid, mannose, rhamnose, glucouronic acid, galacturonic acid, glucose, galactose, xylose and fucose with ratio of 1.86 : 9.56 : 6.81 : 1.69 : 1.00 : 1.75 : 1.19 : 11.06 : 4.31 : 23.06. However, polysaccharide 37502 is an aginate which composed of mannuronic acid (89.3 %) and guluronic acid (10.7 %), with the molecular weight (Mw) of 27.9 kDa. These results imply that mixture polysaccharides 375 works better than the individual polysaccharide on SARS-Cov-2 may be the cocktail-like polysaccharide synergistic function through targeting multiple key molecules implicated in the virus infection and replication. The results also suggest that 375 may be a potential drug candidate against SARS-CoV-2.

Discovery of baicalin and baicalein as novel, natural product inhibitors of SARS-CoV-2 3CL protease in vitro

Human infections with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cause coronavirus disease 19 (COVID-19) and there is currently no cure. The 3C-like protease (3CLpro), a highly conserved protease indispensable for replication of coronaviruses, is a promising target for development of broad-spectrum antiviral drugs. To advance the speed of drug discovery and development, we investigated the inhibition of SARS-CoV-2 3CLpro by natural products derived from Chinese traditional medicines. Baicalin and baicalein were identified as the first non-covalent, non-peptidomimetic inhibitors of SARS-CoV-2 3CLpro and exhibited potent antiviral activities in a cell-based system. Remarkably, the binding mode of baicalein with SARS-CoV-2 3CLpro determined by X-ray protein crystallography is distinctly different from those of known inhibitors. Baicalein is perfectly ensconced in the core of the substrate-binding pocket by interacting with two catalytic residues, the crucial S1/S2 subsites and the oxyanion loop, acting as a "shield" in front of the catalytic dyad to prevent the peptide substrate approaching the active site. The simple chemical structure, unique mode of action, and potent antiviral activities in vitro, coupled with the favorable safety data from clinical trials, emphasize that baicalein provides a great opportunity for the development of critically needed anti-coronaviral drugs.

Structural Basis for the Inhibition of the RNA-Dependent RNA Polymerase from SARS-CoV-2 by Remdesivir

The pandemic of Corona Virus Disease 2019 (COVID-19) caused by SARS-CoV-2 has become a global crisis. The replication of SARS-CoV-2 requires the viral RNA-dependent RNA polymerase (RdRp), a direct target of the antiviral drug, Remdesivir. Here we report the structure of the SARS-CoV-2 RdRp either in the apo form or in complex with a 50-base template-primer RNA and Remdesivir at a resolution range of 2.5-2.8 [A]. The complex structure reveals that the partial double-stranded RNA template is inserted into the central channel of the RdRp where Remdesivir is incorporated into the first replicated base pair and terminates the chain elongation. Our structures provide critical insights into the working mechanism of viral RNA replication and a rational template for drug design to combat the viral infection.

Structure-Based Design, Synthesis and Biological Evaluation of Peptidomimetic Aldehydes as a Novel Series of Antiviral Drug Candidates Targeting the SARS-CoV-2 Main Protease

SARS-CoV-2 is the etiological agent responsible for the COVID-19 outbreak in Wuhan. Specific antiviral drug are urgently needed to treat COVID-19 infections. The main protease (Mpro) of SARS-CoV-2 is a key CoV enzyme that plays a pivotal role in mediating viral replication and transcription, which makes it an attractive drug target. In an effort to rapidly discover lead compounds targeting Mpro, two compounds (11a and 11b) were designed and synthesized, both of which exhibited excellent inhibitory activity with an IC50 value of 0.05 M and 0.04 M respectively. Significantly, both compounds exhibited potent anti-SARS-CoV-2 infection activity in a cell-based assay with an EC50 value of 0.42 M and 0.33 M, respectively. The X-ray crystal structures of SARS-CoV-2 Mpro in complex with 11a and 11b were determined at 1.5 [A] resolution, respectively. The crystal structures showed that 11a and 11b are covalent inhibitors, the aldehyde groups of which are bound covalently to Cys145 of Mpro. Both compounds showed good PK properties in vivo, and 11a also exhibited low toxicity which is promising drug leads with clinical potential that merits further studies.

Structure-based drug design, virtual screening and high-throughput screening rapidly identify antiviral leads targeting COVID-19

A new coronavirus (CoV) identified as COVID-19 virus is the etiological agent responsible for the 2019-2020 viral pneumonia outbreak that commenced in Wuhan1-4. Currently there is no targeted therapeutics and effective treatment options remain very limited. In order to rapidly discover lead compounds for clinical use, we initiated a program of combined structure-assisted drug design, virtual drug screening and high-throughput screening to identify new drug leads that target the COVID-19 virus main protease (Mpro). Mpro is a key CoV enzyme, which plays a pivotal role in mediating viral replication and transcription, making it an attractive drug target for this virus5,6. Here, we identified a mechanism-based inhibitor, N3, by computer-aided drug design and subsequently determined the crystal structure of COVID-19 virus Mpro in complex with this compound. Next, through a combination of structure-based virtual and high-throughput screening, we assayed over 10,000 compounds including approved drugs, drug candidates in clinical trials, and other pharmacologically active compounds as inhibitors of Mpro. Six of these inhibit Mpro with IC50 values ranging from 0.67 to 21.4 M. Ebselen also exhibited promising antiviral activity in cell-based assays. Our results demonstrate the efficacy of this screening strategy, which can lead to the rapid discovery of drug leads with clinical potential in response to new infectious diseases where no specific drugs or vaccines are available.

Progress in the fragment-based drug discovery / 药学学报

As an extension of the structure-based drug discovery, fragment-based drug discovery is matured increasingly, and plays an important role in drug development. Fragments in a small library, with lower molecular mass and high "ligand efficiency", are detected by SPR, MS, NMR, X-ray crystallography technologies and other biophysical methods. Then they are considered as starting points for chemical optimization with the guidance of structural biology methods to get good "drug-like" lead and candidate compounds. In this article, we reviewed the current progress of fragment-based drug discovery and detailed a number of examples to illustrate the novel strategies.