Oral Simnotrelvir for Adult Patients with Mild-to-Moderate Covid-19.

BACKGROUND: Simnotrelvir is an oral 3-chymotrypsin-like protease inhibitor that has been found to have in vitro activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and potential efficacy in a phase 1B trial. METHODS: In this phase 2-3, double-blind, randomized, placebo-controlled trial, we assigned patients who had mild-to-moderate coronavirus disease 2019 (Covid-19) and onset of symptoms within the past 3 days in a 1:1 ratio to receive 750 mg of simnotrelvir plus 100 mg of ritonavir or placebo twice daily for 5 days. The primary efficacy end point was the time to sustained resolution of symptoms, defined as the absence of 11 Covid-19-related symptoms for 2 consecutive days. Safety and changes in viral load were also assessed. RESULTS: A total of 1208 patients were enrolled at 35 sites in China; 603 were assigned to receive simnotrelvir and 605 to receive placebo. Among patients in the modified intention-to-treat population who received the first dose of trial drug or placebo within 72 hours after symptom onset, the time to sustained resolution of Covid-19 symptoms was significantly shorter in the simnotrelvir group than in the placebo group (180.1 hours [95% confidence interval {CI}, 162.1 to 201.6] vs. 216.0 hours [95% CI, 203.4 to 228.1]; median difference, -35.8 hours [95% CI, -60.1 to -12.4]; P = 0.006 by Peto-Prentice test). On day 5, the decrease in viral load from baseline was greater in the simnotrelvir group than in the placebo group (mean difference [±SE], -1.51±0.14 log10 copies per milliliter; 95% CI, -1.79 to -1.24). The incidence of adverse events during treatment was higher in the simnotrelvir group than in the placebo group (29.0% vs. 21.6%). Most adverse events were mild or moderate. CONCLUSIONS: Early administration of simnotrelvir plus ritonavir shortened the time to the resolution of symptoms among adult patients with Covid-19, without evident safety concerns. (Funded by Jiangsu Simcere Pharmaceutical; ClinicalTrials.gov number, NCT05506176.).

Structure of Mpro from SARS-CoV-2 and discovery of its inhibitors.

A new coronavirus, known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is the aetiological agent responsible for the 2019-2020 viral pneumonia outbreak of coronavirus disease 2019 (COVID-19)1-4. Currently, there are no targeted therapeutic agents for the treatment of this disease, and effective treatment options remain very limited. Here we describe the results of a programme that aimed to rapidly discover lead compounds for clinical use, by combining structure-assisted drug design, virtual drug screening and high-throughput screening. This programme focused on identifying drug leads that target main protease (Mpro) of SARS-CoV-2: Mpro is a key enzyme of coronaviruses and has a pivotal role in mediating viral replication and transcription, making it an attractive drug target for SARS-CoV-25,6. We identified a mechanism-based inhibitor (N3) by computer-aided drug design, and then determined the crystal structure of Mpro of SARS-CoV-2 in complex with this compound. Through a combination of structure-based virtual and high-throughput screening, we assayed more than 10,000 compounds-including approved drugs, drug candidates in clinical trials and other pharmacologically active compounds-as inhibitors of Mpro. Six of these compounds inhibited Mpro, showing half-maximal inhibitory concentration values that ranged from 0.67 to 21.4 µM. One of these compounds (ebselen) also exhibited promising antiviral activity in cell-based assays. Our results demonstrate the efficacy of our screening strategy, which can lead to the rapid discovery of drug leads with clinical potential in response to new infectious diseases for which no specific drugs or vaccines are available.

Bioactive compounds from Huashi Baidu decoction possess both antiviral and anti-inflammatory effects against COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic is an ongoing global health concern, and effective antiviral reagents are urgently needed. Traditional Chinese medicine theory-driven natural drug research and development (TCMT-NDRD) is a feasible method to address this issue as the traditional Chinese medicine formulae have been shown effective in the treatment of COVID-19. Huashi Baidu decoction (Q-14) is a clinically approved formula for COVID-19 therapy with antiviral and anti-inflammatory effects. Here, an integrative pharmacological strategy was applied to identify the antiviral and anti-inflammatory bioactive compounds from Q-14. Overall, a total of 343 chemical compounds were initially characterized, and 60 prototype compounds in Q-14 were subsequently traced in plasma using ultrahigh-performance liquid chromatography with quadrupole time-of-flight mass spectrometry. Among the 60 compounds, six compounds (magnolol, glycyrrhisoflavone, licoisoflavone A, emodin, echinatin, and quercetin) were identified showing a dose-dependent inhibition effect on the SARS-CoV-2 infection, including two inhibitors (echinatin and quercetin) of the main protease (Mpro), as well as two inhibitors (glycyrrhisoflavone and licoisoflavone A) of the RNA-dependent RNA polymerase (RdRp). Meanwhile, three anti-inflammatory components, including licochalcone B, echinatin, and glycyrrhisoflavone, were identified in a SARS-CoV-2-infected inflammatory cell model. In addition, glycyrrhisoflavone and licoisoflavone A also displayed strong inhibitory activities against cAMP-specific 3',5'-cyclic phosphodiesterase 4 (PDE4). Crystal structures of PDE4 in complex with glycyrrhisoflavone or licoisoflavone A were determined at resolutions of 1.54 Å and 1.65 Å, respectively, and both compounds bind in the active site of PDE4 with similar interactions. These findings will greatly stimulate the study of TCMT-NDRD against COVID-19.

Assessing Genetic Diversity in Endangered Plant Orchidantha chinensis: Chloroplast Genome Assembly and Simple Sequence Repeat Marker-Based Evaluation.

Orchidantha chinensis T. L. Wu, an endemic species in China, is listed as a key protected wild plant in Guangdong Province. However, the lack of reports on the chloroplast genome and simple sequence repeat (SSR) markers has hindered the assessment of its genetic diversity and conservation strategies. The limited number of molecular markers to assess the genetic diversity of this species, and thus develop proper conservation strategies, highlighted the urgent need to develop new ones. This study developed new SSR markers and investigated genetic variation using 96 samples of O. chinensis from seven populations. Through high-throughput sequencing, a complete chloroplast genome of 134,407 bp was assembled. A maximum-likelihood phylogenetic tree, based on the chloroplast genome, showed that O. chinensis is closely related to Ravenala madagascariensis. The study identified 52 chloroplast SSRs (cpSSRs) and 5094 expressed sequence tag SSRs (EST-SSRs) loci from the chloroplast genome and leaf transcriptome, respectively. Twenty-one polymorphic SSRs (seven cpSSRs and fourteen EST-SSRs) were selected to evaluate the genetic variation in 96 accessions across seven populations. Among these markers, one cpSSR and 11 EST-SSRs had high polymorphism information content (>0.5). Cluster, principal coordinate, and genetic structure analyses indicated that groups G1 and G6 were distinct from the other five groups. However, an analysis of molecular variance showed greater variation within groups than among groups. The genetic distance among the populations was significantly positively correlated with geographical distance. These findings provide new markers for studying the genetic variability of O. chinensis and offer a theoretical foundation for its conservation strategies.

Pigment Diversity in Leaves of Caladium × hortulanum Birdsey and Transcriptomic and Metabolic Comparisons between Red and White Leaves.

Leaf color is a key ornamental characteristic of cultivated caladium (Caladium × hortulanum Birdsey), a plant with diverse leaf colors. However, the genetic improvement of leaf color in cultivated caladium is hindered by the limited understanding of leaf color diversity and regulation. In this study, the chlorophyll and anthocyanin content of 137 germplasm resources were measured to explore the diversity and mechanism of leaf color formation in cultivated caladium. Association analysis of EST-SSR markers and pigment traits was performed, as well as metabolomics and transcriptomics analysis of a red leaf variety and its white leaf mutant. We found significant differences in chlorophyll and anthocyanin content among different color groups of cultivated caladium, and identified three, eight, three, and seven EST-SSR loci significantly associated with chlorophyll-a, chlorophyll-b, total chlorophyll and total anthocyanins content, respectively. The results further revealed that the white leaf mutation was caused by the down-regulation of various anthocyanins (such as cyanidin-3-O-rutinoside, quercetin-3-O-glucoside, and others). This change in concentration is likely due to the down-regulation of key genes (four PAL, four CHS, six CHI, eight F3H, one F3'H, one FLS, one LAR, four DFR, one ANS and two UFGT) involved in anthocyanin biosynthesis. Concurrently, the up-regulation of certain genes (one FLS and one LAR) that divert the anthocyanin precursors to other pathways was noted. Additionally, a significant change in the expression of numerous transcription factors (12 NAC, 12 bZIP, 23 ERF, 23 bHLH, 19 MYB_related, etc.) was observed. These results revealed the genetic and metabolic basis of leaf color diversity and change in cultivated caladium, and provided valuable information for molecular marker-assisted selection and breeding of leaf color in this ornamental plant.

Diversity, classification, and EST-SSR-based association analysis of caladium ornamental traits.

Caladium (Caladium × Hortulanum Birdsey) is a popular ornamental plant with a wide range of vibrant leaf color among Araceae. Even after years of breeding, creating new caladium leaf color variations is extremely difficult. Molecular marker-assisted selection is an effective approach for accelerating breeding, but few studies on the molecular markers associated with caladium traits have been performed. In the current study, 144 caladium accessions were used to examine 12 phenotypic characteristics. The coefficient of variation for four numerical characters ranged from 23.94% to 43.22%, and the Shannon-Wiener indexes for eight descriptive characters ranged from 0.13 to 1.52. Based on L*, a*, b*, C, h° values determined by a colorimeter and hierarchical cluster analysis, the leaf color can be divided into four groups: pale green, green, light pink, and red. Furthermore, 7708 new SSR loci were identified by transcriptome sequencing, and 26 SSR markers with high polymorphism and reproducibility were screened. Genetic structure, NJ clustering, and PCoA analysis revealed that 144 accessions could be divided into three clusters, with genetic structure being closely related to germplasm origin. An association analysis revealed that the SSR markers 2, 1, 1, 1, 1, and 1 were mainly associated with petiole color, main vein color, blade upperside glossiness, and C, b*, and L* of leaf color (p < 0.01). These findings will serve as a valuable reference for evaluating germplasm resources and caladium molecular marker-assisted breeding.

Design, synthesis and biological evaluation of covalent peptidomimetic 3CL protease inhibitors containing nitrile moiety.

In this paper, a series of peptidomimetic SARS-CoV-2 3CL protease inhibitors with new P2 and P4 positions were synthesized and evaluated. Among these compounds, 1a and 2b exhibited obvious 3CLpro inhibitory activities with IC50 of 18.06 nM and 22.42 nM, respectively. 1a and 2b also showed excellent antiviral activities against SARS-CoV-2 in vitro with EC50 of 313.0 nM and 170.2 nM, respectively, the antiviral activities of 1a and 2b were 2- and 4-fold better than that of nirmatrelvir, respectively. In vitro studies revealed that these two compounds had no significant cytotoxicity. Further metabolic stability tests and pharmacokinetic studies showed that the metabolic stability of 1a and 2b in liver microsomes was significantly improved, and 2b had similar pharmacokinetic parameters to that of nirmatrelvir in mice.

Discovery of novel cGAS inhibitors based on natural flavonoids.

Cyclic GMP-AMP synthase (cGAS) plays an important role in the inflammatory response. It has been reported that aberrant activation of cGAS is associated with a variety of immune-mediated inflammatory disorders. The development of small molecule inhibitors of cGAS has been considered as a promising therapeutic strategy for the diseases. Flavonoids, a typical class of natural products, are known for their anti-inflammatory activities. Although cGAS is closely associated with inflammation, the potential effects of natural flavonoid compounds on cGAS have been rarely studied. Therefore, we screened an in-house natural flavonoid library by pyrophosphatase (PPiase) coupling assay and identified novel cGAS inhibitors baicalein and baicalin. Subsequently, crystal structures of the two natural flavonoids in complex with human cGAS were determined, which provide mechanistic insight into the anti-inflammatory activities of baicalein and baicalin at the molecular level. After that, a virtual screening based on the crystal structures of baicalein and baicalin in complex with human cGAS was performed. As a result, compound C20 was identified to inhibit both human and mouse cGAS with IC50 values of 2.28 and 1.44 µM, respectively, and its detailed interactions with human cGAS were further revealed by the X-ray crystal structure determination. These results demonstrate the potential of natural products used as hits in drug discovery and provide valuable hints for further development of cGAS inhibitors.

Structure-Based Design of Potent Peptidomimetic Inhibitors Covalently Targeting SARS-CoV-2 Papain-like Protease.

The papain-like protease (PLpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a critical role in the proteolytic processing of viral polyproteins and the dysregulation of the host immune response, providing a promising therapeutic target. Here, we report the structure-guide design of novel peptidomimetic inhibitors covalently targeting SARS-CoV-2 PLpro. The resulting inhibitors demonstrate submicromolar potency in the enzymatic assay (IC50 = 0.23 µM) and significant inhibition of SARS-CoV-2 PLpro in the HEK293T cells using a cell-based protease assay (EC50 = 3.61 µM). Moreover, an X-ray crystal structure of SARS-CoV-2 PLpro in complex with compound 2 confirms the covalent binding of the inhibitor to the catalytic residue cysteine 111 (C111) and emphasizes the importance of interactions with tyrosine 268 (Y268). Together, our findings reveal a new scaffold of SARS-CoV-2 PLpro inhibitors and provide an attractive starting point for further optimization.

Structure-based design of a novel inhibitor of the ZIKA virus NS2B/NS3 protease.

Zika virus (ZIKV) has been a serious public health problem, and there is no vaccine or drug approved for the prevention or treatment of ZIKV yet. The ZIKV NS2B/NS3 protease plays an important role in processing the virus precursor polyprotein and is thus a promising target for antiviral drugs development. In order to discover novel inhibitors of this protease, we carried out a fragment-based hit screening and characterized protein-inhibitor interactions using the X-ray crystallography together with isothermal titration calorimetry. We reported two high-resolution crystal structures of the protease (bZiProC143S) in complex with an active fragment as well as a tetrapeptide, revealing that there is domain swapping in the protein structures and two ligands only occupy the substrate-binding pocket of one copy in a symmetric unit. Based on the detailed binding modes of two ligands revealed by crystal structures, we designed a novel inhibitor which inhibits the NS2B/NS3 protease with a higher potency than the fragment and possesses a higher ligand-binding efficiency and a comparable IC50 compared to the tetrapeptide. These results thus provide a structural basis and valuable hint for development of more potent inhibitors of the ZIKV NS2B/NS3 protease.

A multi-targeting drug design strategy for identifying potent anti-SARS-CoV-2 inhibitors.

The COVID-19, caused by SARS-CoV-2, is threatening public health, and there is no effective treatment. In this study, we have implemented a multi-targeted anti-viral drug design strategy to discover highly potent SARS-CoV-2 inhibitors, which simultaneously act on the host ribosome, viral RNA as well as RNA-dependent RNA polymerases, and nucleocapsid protein of the virus, to impair viral translation, frameshifting, replication, and assembly. Driven by this strategy, three alkaloids, including lycorine, emetine, and cephaeline, were discovered to inhibit SARS-CoV-2 with EC50 values of low nanomolar levels potently. The findings in this work demonstrate the feasibility of this multi-targeting drug design strategy and provide a rationale for designing more potent anti-virus drugs.

Identification of Cysteine 270 as a Novel Site for Allosteric Modulators of SARS-CoV-2 Papain-Like Protease.

The coronavirus papain-like protease (PLpro ) plays an important role in the proteolytic processing of viral polyproteins and the dysregulation of the host immune response, providing a promising therapeutic target. However, the development of inhibitors against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) PLpro is challenging owing to the restricted S1/S2 sites in the substrate binding pocket. Here we report the discovery of two activators of SARS-CoV-2 PLpro and the identification of the unique residue, cysteine 270 (C270), as an allosteric and covalent regulatory site for the activators. This site is also specifically modified by glutathione, resulting in protease activation. Furthermore, a compound was found to allosterically inhibit the protease activity by covalent binding to C270. Together, these results elucidate an unrevealed molecular mechanism for allosteric modulation of SARS-CoV-2 PLpro and provid a novel site for allosteric inhibitors design.

Potent and Selective RIPK1 Inhibitors Targeting Dual-Pockets for the Treatment of Systemic Inflammatory Response Syndrome and Sepsis.

Sepsis, characterized with high risk of life-threatening organ dysfunction, represents a major cause of health loss and the World Health Organization (WHO) labelled sepsis as the most urgent unmet medical need in 2017. The emerging biological understanding of the role of RIPK1 in sepsis has opened up an exciting opportunity to explore potent and selective RIPK1 inhibitors as an effective therapeutic strategy for SIRS and sepsis therapy. Herein, we have synthesized a class of highly potent dual-mode RIPK1 inhibitors occupying both the allosteric and the ATP binding pockets, exemplified by compound 21 (ZB-R-55) which is about 10-fold more potent than GSK2982772, and exhibits excellent kinase selectivity, good oral pharmacokinetics and good therapeutic effects in the LPS-induced sepsis model, suggesting that compound ZB-R-55 is a highly promising preclinical candidate.

What coronavirus 3C-like protease tells us: From structure, substrate selectivity, to inhibitor design.

The emergence of a variety of coronaviruses (CoVs) in the last decades has posed huge threats to human health. Especially, the ongoing pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to more than 70 million infections and over 1.6 million of deaths worldwide in the past few months. None of the efficacious antiviral agents against human CoVs have been approved yet. 3C-like protease (3CLpro ) is an attractive target for antiviral intervention due to its essential role in processing polyproteins translated from viral RNA, and its conserved structural feature and substrate specificity among CoVs in spite of the sequence variation. This review focuses on all available crystal structures of 12 CoV 3CLpro s and their inhibitors, and intends to provide a comprehensive understanding of this protease from multiple aspects including its structural features, substrate specificity, inhibitor binding modes, and more importantly, to recapitulate the similarity and diversity among different CoV 3CLpro s and the structure-activity relationship of various types of inhibitors. Such an attempt could gain a deep insight into the inhibition mechanisms and drive future structure-based drug discovery targeting 3CLpro s.

Molecular evolution of chloroplast genomes in subfamily Zingiberoideae (Zingiberaceae).

BACKGROUND: Zingiberoideae is a large and diverse subfamily of the family Zingiberaceae. Four genera in subfamily Zingiberoideae each possess 50 or more species, including Globba (100), Hedychium (> 80), Kaempferia (50) and Zingiber (150). Despite the agricultural, medicinal and horticultural importance of these species, genomic resources and suitable molecular markers for them are currently sparse. RESULTS: Here, we have sequenced, assembled and analyzed ten complete chloroplast genomes from nine species of subfamily Zingiberoideae: Globba lancangensis, Globba marantina, Globba multiflora, Globba schomburgkii, Globba schomburgkii var. angustata, Hedychium coccineum, Hedychium neocarneum, Kaempferia rotunda 'Red Leaf', Kaempferia rotunda 'Silver Diamonds' and Zingiber recurvatum. These ten chloroplast genomes (size range 162,630-163,968 bp) possess typical quadripartite structures that consist of a large single copy (LSC, 87,172-88,632 bp), a small single copy (SSC, 15,393-15,917 bp) and a pair of inverted repeats (IRs, 29,673-29,833 bp). The genomes contain 111-113 different genes, including 79 protein coding genes, 28-30 tRNAs and 4 rRNA genes. The dynamics of the genome structures, gene contents, amino acid frequencies, codon usage patterns, RNA editing sites, simple sequence repeats and long repeats exhibit similarities, with slight differences observed among the ten genomes. Further comparative analysis of seventeen related Zingiberoideae species, 12 divergent hotspots are identified. Positive selection is observed in 14 protein coding genes, including accD, ccsA, ndhA, ndhB, psbJ, rbcL, rpl20, rpoC1, rpoC2, rps12, rps18, ycf1, ycf2 and ycf4. Phylogenetic analyses, based on the complete chloroplast-derived single-nucleotide polymorphism data, strongly support that Globba, Hedychium, and Curcuma I + "the Kaempferia clade" consisting of Curcuma II, Kaempferia and Zingiber, form a nested evolutionary relationship in subfamily Zingiberoideae. CONCLUSIONS: Our study provides detailed information on ten complete Zingiberoideae chloroplast genomes, representing a valuable resource for future studies that seek to understand the molecular evolutionary dynamics in family Zingiberaceae. The identified divergent hotspots can be used for development of molecular markers for phylogenetic inference and species identification among closely related species within four genera of Globba, Hedychium, Kaempferia and Zingiber in subfamily Zingiberoideae.

Structural basis for ligand recognition of the human thromboxane A2 receptor.

Stimulated by thromboxane A2, an endogenous arachidonic acid metabolite, the thromboxane A2 receptor (TP) plays a pivotal role in cardiovascular homeostasis, and thus is considered as an important drug target for cardiovascular disease. Here, we report crystal structures of the human TP bound to two nonprostanoid antagonists, ramatroban and daltroban, at 2.5 Å and 3.0 Å resolution, respectively. The TP structures reveal a ligand-binding pocket capped by two layers of extracellular loops that are stabilized by two disulfide bonds, limiting ligand access from the extracellular milieu. These structures provide details of interactions between the receptor and antagonists, which help to integrate previous mutagenesis and SAR data. Molecular docking of prostanoid-like ligands, combined with mutagenesis, ligand-binding and functional assays, suggests a prostanoid binding mode that may also be adopted by other prostanoid receptors. These insights into TP deepen our understanding about ligand recognition and selectivity mechanisms of this physiologically important receptor.

Discovery of novel inhibitors against main protease (Mpro) of SARS-CoV-2 via virtual screening and biochemical evaluation.

SARS-CoV-2 is the pathogen that caused the global COVID-19 outbreak in 2020. Promising progress has been made in developing vaccines and antiviral drugs. Antivirals medicines are necessary complements of vaccines for post-infection treatment. The main protease (Mpro) is an extremely important protease in the reproduction process of coronaviruses which cleaves pp1ab over more than 11 cleavage sites. In this work, two active main protease inhibitors were found via docking-based virtual screening and bioassay. The IC50 of compound VS10 was 0.20 µM, and the IC50 of compound VS12 was 1.89 µM. The finding in this work can be helpful to understand the interactions of main protease and inhibitors. The active candidates could be potential lead compounds for future drug design.

Species differences in the CYP3A-catalyzed metabolism of TPN729, a novel PDE5 inhibitor.

TPN729 is a novel phosphodiesterase 5 (PDE5) inhibitor used to treat erectile dysfunction in men. Our previous study shows that the plasma exposure of metabolite M3 (N-dealkylation of TPN729) in humans is much higher than that of TPN729. In this study, we compared its metabolism and pharmacokinetics in different species and explored the contribution of its main metabolite M3 to pharmacological effect. We conducted a combinatory approach of ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry-based metabolite identification, and examined pharmacokinetic profiles in monkeys, dogs, and rats following TPN729 administration. A remarkable species difference was observed in the relative abundance of major metabolite M3: i.e., the plasma exposure of M3 was 7.6-fold higher than that of TPN729 in humans, and 3.5-, 1.2-, 1.1-fold in monkeys, dogs, and rats, respectively. We incubated liver S9 and liver microsomes with TPN729 and CYP3A inhibitors, and demonstrated that CYP3A was responsible for TPN729 metabolism and M3 formation in humans. The inhibitory activity of M3 on PDE5 was 0.78-fold that of TPN729 (The IC50 values of TPN729 and M3 for PDE5A were 6.17 ± 0.48 and 7.94 ± 0.07 nM, respectively.). The plasma protein binding rates of TPN729 and M3 in humans were 92.7% and 98.7%, respectively. It was astonishing that the catalyzing capability of CYP3A4 in M3 formation exhibited seven-fold disparity between different species. M3 was an active metabolite, and its pharmacological contribution was equal to that of TPN729 in humans. These findings provide new insights into the limitation and selection of animal model for predicting the clinical pharmacokinetics of drug candidates metabolized by CYP3A4.

In Silico Screening-Based Discovery of Novel Inhibitors of Human Cyclic GMP-AMP Synthase: A Cross-Validation Study of Molecular Docking and Experimental Testing.

Cyclic GMP-AMP synthase (cGAS) has been recently uncovered to be a promising therapeutic target for immune-associated diseases. Until now, only a few inhibitors have been identified through high-throughput screening campaigns. Here, we reported the discovery of novel inhibitors for the catalytic domain of human cGAS (h-cGASCD) by virtual screening for the first time. To generate a reliable docking mode, we first obtained a high-resolution crystal structure of h-cGASCD in complex with PF-06928215, a known inhibitor of h-cGAS, followed by molecular dynamics simulations on this complex structure. Four fragment hits were identified by the virtual screening together with a thermal shift assay. The crystal structures of these four compounds in complex with h-cGASCD were subsequently determined, and the binding modes of the compounds were similar to those predicted by molecular docking, supporting the reliability of the docking model. In addition, an enzyme activity assay identified compound 18 (IC50 = 29.88 ± 3.20 µM) from the compounds predicted by the virtual screening. A similarity search of compound 18 followed by a second virtual screening led to the discovery of compounds S2 (IC50 = 13.1 ± 0.09 µM) and S3 (IC50 = 4.9 ± 0.26 µM) as h-cGAS inhibitors with improved potency. Therefore, the present study not only provides the validated hit compounds for further development of h-cGAS inhibitors but also demonstrates a cross-validation study of virtual screening, in vitro experimental assays, and crystal structure determination.

Diterpenoids from the Root Bark of Pinus massoniana and Evaluation of Their Phosphodiesterase Type 4D Inhibitory Activity.

Thirty-two diterpenoids were obtained from the root bark of Pinus massoniana, and, among them, five compounds (pinmassins A-E) were identified as undescribed analogues. Spectroscopic methods, X-ray single-crystal diffraction analysis, and ECD calculations were applied to establish the structure of the new isolates. Pinmassin D (4) and abieta-8,11,13,15-tetraen-18-oic acid (23) showed moderate phosphodiesterase type 4D (PDE4D) inhibitory effects with IC50 values of 2.8 ± 0.18 and 3.3 ± 0.50 µM, respectively, and their binding modes were investigated by a molecular docking study.