BACKGROUND:Conversion of cholesterol into bile acids is a central pathway for cholesterol disposal, which was mainly controlled by cholesterol 7alpha-hydroxylase (Cyp7a1). In present study, we aimed to investigate the effect and the potential underlying mechanism of microRNA-96 (miR-96) on atherosclerosis development. RESEARCH DESIGN AND METHODS:The anti-atherosclerosis effects of a miR-96 inhibitor (miR-96i) were evaluated using ApoE KO mice fed a high-fat diet, which was treated with miR-96i for 8 weeks. The regulatory mechanism was revealed and validated by RNA-seq transcriptomics, quantitative PCR and western blotting analyses in hepatic cells. RESULTS:The authors identified that miR-96i significantly decreased serum cholesterol and bile acid levels and attenuated arterial plaque in mice. We further revealed that miR-96 regulated Cyp7a1 via a FOXO1-involved indirect pathway, in which miR-96 directly modulated FOXO1 in a posttranscriptional manner. A coordinated regulatory effect of miR-96 and miR-185 on FOXO1 was also observed. The full spectrum of mechanisms underlying the antiatherosclerotic activity beside miR-96-FOXO1-CYP7A1 axis remains to be elucidated. CONCLUSIONS:This study provides convincing evidence for the pivotal role of miR-96 in FOXO1 modulation and CYP7A1-involved cholesterol-bile acid metabolism, suggesting that miR-96 is a novel therapeutic target for the discovery and development of drugs against ACVD.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a well-established target for lowering cholesterol and is abundantly present in the extracellular space. Inhibitors of PCSK9 have achieved marked success in the clinic, but an alternative strategy for therapeutic modulation is emerging through the degradation of PCSK9. This novel strategy has been enabled by the identification of cell surface receptors such as the asialoglycoprotein receptor (ASGPR), which mediates the lysosomal degradation of extracellular ligands. Given the importance of this therapeutic mechanism, we investigated the synthesis of bifunctional molecules comprising Tri-GalNAc (an ASGPR binder) with a peptide inhibitor we previously reported. In addition to chemical synthesis, we report a novel method for the production of Tri-GalNAc-conjugated peptides, involving the use of enzymatically mediated ligation postsynthesis. We demonstrate that both the synthetic constructs and chemoenzymatic constructs have the intended structures and in vitro activities. While these molecules did not show cellular activities, the chemical and biochemical methods reported here could be broadly applied to the construction of LYTACs in general. One significant challenge that this work overcomes is the C-terminal attachment of Tri-GalNAc, which remains hitherto a difficult experimental task for not only peptides but also larger biologics in particular.

In the omics era, confident high-throughput analytical tools are crucial for the efficient identification of metabolites. Here, we present DeepHalo, a deep learning-integrated and hierarchically optimized workflow designed for high-throughput exploration of halogenated metabolites from high-resolution mass spectrometry-based metabolomics. DeepHalo leverages deep learning models combined with a comprehensive scoring to enhance the reliability of halogen predictions. It integrates PyOpenMS for fast isotope pattern detection and incorporates a halogen-based dereplication algorithm with GNPS molecular networking to efficiently exploit and annotate halogenates from complex biological matrices. To validate its performance, DeepHalo was applied to explore halogenated metabolites from 1296 microbial culture crudes, leading to the discovery of six families of structurally diverse halogenated molecules. This included a new class of cyclic depsipeptides, aglomycins A‒E, featuring rare 3-chloroanthranilic acid and/or epoxyvaline blocks. Additionally, a plausible biosynthetic pathway of aglomycins was proposed through bioinformatics analyses and targeted gene knockout experiments. Bioassays revealed that aglomycin A exhibits synergistic antibacterial activity with linezolid against vancomycin-resistant Enterococcus faecium (VRE) both in vitro and in vivo. We envision that DeepHalo, a user-friendly standalone executable freely available at https://github.com/xieyying/deephalo/releases/tag/DeepHalo_V1.0.0, will become a powerful tool for accelerating the discovery of halogenated “dark matter”.

Microbial sulfur-containing secondary metabolite thienodolin (1) features a unique tricyclic thieno[2,3-b]indole scaffold, yet its biosynthesis has remained enigmatic. Here, we uncover an unexpected enzymatic logic in which a consortium of distinct oxidoreductases cooperatively orchestrates indolethiophene skeleton formation and subsequent bioactivation, ultimately generating the authentic antibacterial metabolite thienoxidolin (10). Following thiotryptophan formation by SDR enzyme TndE, the heme-dependent DUF6875 enzyme TndD initiates C-S bond formation via N-hydroxylation to yield a dearomatized tricyclic species, which is efficiently driven forward and stabilized by aromatization catalyzed by the FAD-dependent oxidoreductase TndG. Notably, TndD functions as a bidirectional redox enzyme, reverting the N-hydroxyl group to the stable N-H form to complete the indolethiophene scaffold construction. After amide formation, late-stage N-hydroxylation by the cytochrome P450 enzyme TndC, previously misassigned as the C-S bond-forming enzyme, produces the bioactive product 10. Intriguingly, TndD may also mediate the deactivation of 10 back to 1, representing an intrinsic self-protection mechanism. Together, these results expand the catalytic repertoire of heme-dependent enzymes and highlight reversible N-hydroxylation as a pivotal strategy for heterocycle formation and bioactivity regulation in microbial secondary metabolism.

Omicsynins are a group of pseudo-tetrapeptides produced by Streptomyces sp. 1647, which exhibited potent anti-influenza A virus and anti-coronavirus activities. However, its biosynthesis mechanism of C-terminus reduction remains unknown. In this work, we explored two short-chain dehydrogenase/reductase (SDR) superfamily encoding genes in the omicsynin biosynthetic gene cluster (BGC) and confirmed the necessity of omnF, rather than omnG, in the biosynthesis of omicsynins through gene deletion in vivo. Subsequently, Feature-Based Molecular Networking (FBMN) analysis revealed three pseudo-tetrapeptides with C-terminal carboxyl group and four unexpected analogues encoded by the omicsynin BGC in the omnF reductase (R) domain knockout mutant strain. This led to the isolation and structural characterization of a group of novel pseudo-tripeptide compounds. Compared to the known omicsynins, these pseudo-tripeptides lack the second amino acid unit and the C-terminal aldehyde group, and consequently lose their anti-coronavirus activity. In conclusion, our work highlights the effectiveness of FBMN in unveiling cryptic analogues and clearly underscores the essential role of the R domain of OmnF in the biosynthesis of the C-terminal aldehyde warhead.

Chuangxinmycin (CM) is an old antibiotic from Actinoplanes tsinanensis CPCC, 200056, characterized by a dihydrothiopyrano[4,3,2-cd]indole scaffold and potent activity against Mycobacterium tuberculosis. Its congener norchuangxinmycin (NCM), which lacks antibacterial activity against various bacteria, unexpectedly retains antitubercular activity, indicating new mechanisms of action against M. tuberculosis in addition to tryptophan-tRNA synthetase inhibition. However, the variable low productivity and the limited number of active structural analogues represent a significant challenge for the future discovery and development of new anti-tuberculosis drugs involving CM and its derivatives. Based on the elucidation of CM biosynthetic pathway, we employed a stepwise strategy by combining heterologous expression, activator overexpression, promoter optimization and fermentation media screening to achieve directed and high-level production of CM and its congener NCM. The highest yields achieved were 301 mg/L (a 20.1-fold increase) for CM and 117.6 mg/L (a 13.7-fold increase) for NCM. Furthermore, eleven halogenated CM derivatives were obtained through precursor-directed biosynthesis, with six of them being purified and structurally confirmed by HR-MS, HR-MS/MS and NMR. Bioactivity testing against M. tuberculosis H37Rv and clinical isolates of isoniazid/rifampin-resistant M. tuberculosis showed potent activity for 5-F-CM and 7-F-NCM. Synthetic biology techniques are well-suited for the targeted and high-level biosynthesis of CM and its derivatives. This study reports the highest laboratory-level yields of CM and NCM to date. This is the first instance of obtaining CM derivatives by biosynthesis rather than chemical synthesis, and it also marks the first report of halogenated NCM derivatives. High-level production of CM and its diverse analogues will provide a solid material foundation for advancing CM and its derivatives as potential anti-tuberculosis drug candidates.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), represents a global health challenge, necessitating new treatments with distinct mechanisms of action (MOA) to combat drug resistance. Chuangxinmycin (CM), characterized by its indole-dihydrothiopyran heterocyclic skeleton, exhibits potent antibacterial activity both in vitro and in vivo, with a minimum inhibitory concentration (MIC) of 0.25 μg/mL against Mtb. However, the MOA of CM against Mtb has remained obscure. Through comprehensive genetic, chemical rescue, and protein-drug interaction studies, coupled with biochemical analyses, we reveal that CM selectively binds and inhibits tryptophanyl-tRNA synthetase (TrpRS) encoded by trpS, rather than anthranilate synthase (TrpE). Overexpression of trpS in Mtb results in a 128-fold increase in the MIC of CM, indicating a fundamental cause of resistance, whereas overexpression of trpE leads to modest resistance, suggesting a secondary effect. Conversely, knockdown of trpS or trpE enhances the susceptibility of Mtb to CM. Meanwhile, promoters of trpS in CM-resistant Mtb mutants exhibit increased activity compared to the wild type. Furthermore, drug-protein interaction and biochemical assays have confirmed that while CM effectively inhibits TrpRS, mutants of TrpE show decreased affinity for tryptophan. These results establish that CM exerts its anti-Mtb effects by interfering with the tryptophan-tRNA linkage essential for protein synthesis.

Cobalamin (Cbl)-dependent radical S-adenosyl-L-methionine (SAM) proteins constitute the largest collection of the radical SAM superfamily that has hundreds of thousands of individual members. Many of these proteins are involved in the biosynthesis of pharmaceutically important natural products to catalyze chemically demanding reactions. In the biosynthetic pathway of chuangxinmycin (CXM), a unique indole alkaloid antibiotic with potent anti-infective activity, functionalization of the characteristic thiopyrano[4,3,2-cd]indole scaffold by regio- and stereoselective C3-methylation is believed to rely on a Cbl-dependent radical process, which, however, remained to be reconstituted biochemically. We here report the dissection of this enzymatic process, which requires the incorporation of Cxm8, a Cbl-dependent radical SAM protein, with Cxm9, a DUF5825 family protein that shares no homology to any proteins of known functions. Cxm8 and Cxm9 function together by forming an unexpected heterodimeric complex that selectively catalyzes C3-methylation of the tricyclic indole-S-hetero ring system in a successive manner, achieving CXM and a recently identified, C3-dimethylated congener. Detailed biochemical characterization, isotope labeling, structural simulation and bioinformatics analysis rationalized the catalysis of the Cxm8/Cxm9 complex and particularly the necessity of the DUF5825 protein for C3-methylase activity. This is the first example that a Cbl-dependent protein acts with a partner to exhibit radical SAM activity.

The pandemic of SARS-CoV-2 worldwide with successive emerging variants urgently calls for small-molecule oral drugs with broad-spectrum antiviral activity. Here, we show that carrimycin, a new macrolide antibiotic in the clinic and an antiviral candidate for SARS-CoV-2 in phase III trials, decreases the efficiency of programmed –1 ribosomal frameshifting of coronaviruses and thus impedes viral replication in a broad-spectrum fashion. Carrimycin binds directly to the coronaviral frameshift-stimulatory element (FSE) RNA pseudoknot, interrupting the viral protein translation switch from ORF1a to ORF1b and thereby reducing the level of the core components of the viral replication and transcription complexes. Combined carrimycin with known viral replicase inhibitors yielded a synergistic inhibitory effect on coronaviruses. Because the FSE mechanism is essential in all coronaviruses, carrimycin could be a new broad-spectrum antiviral drug for human coronaviruses by directly targeting the conserved coronaviral FSE RNA. This finding may open a new direction in antiviral drug discovery for coronavirus variants.

Illuminated by insights into the hijacking of host cellular metabolism by coronaviruses, we identified an initial hit compound 7030B-C5, characterized by a xanthine scaffold, via a cellular-level phenotypic screening from a domestic repertoire of lipid-modulating agents. A series of derivatives were synthesized and optimized through comprehensive structure-activity relationship (SAR) studies focusing on the N-1, C-8, and N-7 positions of xanthine and preliminary exploration on the N-3 position and parent nucleus. Compounds 10e, 10f and 10o, featuring modifications at the N-7 position, showed inhibitory activity with half maximal effective concentration (EC50) values in the three-digit nanomolar range against human coronavirus-229E (HCoV-229E). In particular, compound 10o exerted superior potency across various coronavirus strains, including HCoV-229E, HCoV-OC43, and the Omicron variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Further investigations revealed that 10o acted on the post-entry stages of virus replication and exhibited a distinctive antiviral mechanism from that of clinically approved nirmatrelvir and molnupiravir. Moreover, drug combination study indicates that 10o operates additively with nirmatrelvir, molnupiravir or omicsynin B4, a dual inhibitor of host proteases for S protein priming. Additionally, in vivo assessments show that 10o has favorable pharmacokinetic and safety profiles compared to its parent compound 7030B-C5. These findings underscore the potential of 10o as a promising antiviral candidate for the treatment of current and potential future coronavirus infections.

Objective: Type 2 diabetes mellitus (T2DM) is a common chronic metabolic disease. Peroxisome proliferator-activated receptors (PPARs) play crucial roles in regulating glucolipid metabolism. Previous studies showed that E17241 could ameliorate atherosclerosis and lower fasting blood glucose levels in ApoE−/− mice. In this work, we investigated the role of E17241 in glycolipid metabolism in diabetic KKAy mice. Approach and results: We confirmed that E17241 is a powerful pan-PPAR agonist with a potent agonistic activity on PPARγ, a high activity on PPARα, and a moderate activity on PPARδ. E17241 also significantly increased the protein expression of ATP-binding cassette transporter 1 (ABCA1), a crucial downstream target gene for PPARs. E17241 clearly lowered plasma glucose levels, improved OGTT and ITT, decreased islet cholesterol content, improved β-cell function, and promoted insulin secretion in KKAy mice. Moreover, E17241 could significantly lower plasma total cholesterol and triglyceride levels, reduce liver lipid deposition, and improve the adipocyte hypertrophy and the inflammatory response in epididymal white adipose tissue. Further mechanistic studies indicated that E17241 boosts cholesterol efflux and insulin secretion in an ABCA1 dependent manner. RNA-seq and qRT-PCR analysis demonstrated that E17241 induced different expression of PPAR target genes in liver and adipose tissue differently from the PPARγ agonist rosiglitazone. In addition, E17241 treatment was also demonstrated to have an exhilarating cardiorenal benefits. Conclusions: Our results demonstrate that E17241 regulates glucolipid metabolism in KKAy diabetic mice while having cardiorenal benefits without inducing weight gain. It is a promising drug candidate for the treatment of T2DM.

Background Isatropolone A and C, produced by Streptomyces sp. CPCC 204095, belong to an unusual class of non-benzenoid aromatic compounds and contain a rare seven-membered ring structure. Isatropolone A exhibits potent activity against Leishmania donovani, comparable to the only oral drug miltefosine. However, its variably low productivity represents a limitation for this lead compound in the future development of new anti-leishmaniasis drugs to meet unmet clinical needs. Results Here we first elucidated the regulatory cascade of biosynthesis of isatropolones, which consists of two SARP family regulators, IsaF and IsaJ. Through a series of in vivo and in vitro experiments, IsaF was identified as a pathway-specific activator that orchestrates the transcription of the gene cluster essential for isatropolone biosynthesis. Interestingly, IsaJ was found to only upregulate the expression of the cytochrome P450 monooxygenase IsaS, which is crucial for the yield and proportion of isatropolone A and C. Through targeted gene deletions of isaJ or isaS, we effectively impeded the conversion of isatropolone A to C. Concurrently, the facilitation of isaF overexpression governed by selected promoters, prompted the comprehensive activation of the production of isatropolone A. Furthermore, meticulous optimization of the fermentation parameters was conducted. These strategies culminated in the attainment of an unprecedented maximum yield-980.8 mg/L of isatropolone A-achieved in small-scale solid-state fermentation utilizing the genetically modified strains, thereby establishing the highest reported titer to date. Conclusion In Streptomyces sp. CPCC 204095, the production of isatropolone A and C is modulated by the SARP regulators IsaF and IsaJ. IsaF serves as a master pathway-specific regulator for the production of isatropolones. IsaJ, on the other hand, only dictates the transcription of IsaS, the enzyme responsible for the conversion of isatropolone A and C. By engineering the expression of these pivotal genes, we have devised a strategy for genetic modification aimed at the selective and high-yield biosynthesis of isatropolone A. This study not only unveils the unique regulatory mechanisms governing isatropolone biosynthesis for the first time, but also establishes an essential engineering framework for the targeted high-level production of isatropolone A.

Optimization of peptide stability is essential for the development of peptides as bona fide alternatives to approved monoclonal antibodies. This is clearly the case for the many peptides reported to antagonize proprotein convertase subtilisin-like/kexin type 9 (PCSK9), a clinically validated target for lowering cholesterol. However, the effects of optimization of stability on in vivo activity and particularly the effects of binding to albumin, an emerging drug design paradigm, have not been studied for such peptide leads. In this study, we optimized a PCSK9 inhibitory peptide by mutagenesis and then by conjugation to a short lipidated tag to design P9-alb fusion peptides that have strong affinity to human serum albumin. Although attachment of the tag reduced activity against PCSK9, which was more evident in surface plasmon resonance binding and enzyme-linked immunosorbent competition assays than in cellular assays of activity, activity remained in the nanomolar range (∼40 nM). P9-alb peptides were exceptionally stable in human serum and had half-lives exceeding 48 h, correlating with longer half-lives in mice (40.8 min) compared to the unconjugated peptide. Furthermore, the decrease in in vitro binding was not deleterious to in vivo function, showing that engendering albumin binding improved low-density lipoprotein receptor recovery and cholesterol-lowering activity. Indeed, the peptide P9-albN2 achieved similar functional endpoints as the approved anti-PCSK9 antibody evolocumab, albeit at higher doses. Our study illustrates that optimization of stability instead of binding affinity is an effective way to improve in vivo function.

Sansanmycins represent a family of uridyl peptide antibiotics with antimicrobial activity specifically against Mycobacterium tuberculosis (including drug-resistant M. tuberculosis) and Pseudomonas aeruginosa. They target translocase I (MraY) to inhibit bacterial cell wall assembly. Given the unique mechanism of action, sansanmycin has emerged as a potential lead compound for developing new anti-tuberculosis drugs, while the 5′-aminouridine moiety plays a crucial role in the pharmacophore of sansanmycin. For expanding the structural diversity of the 5′-aminouridine moiety of sansanmycin through biosynthetic methods, we firstly demonstrated that SsaM and SsaK are responsible for the biosynthesis of the 5′-aminouridine moiety of sansanmycin in vivo. Using the ssaK deletion mutant (SS/KKO), we efficiently obtained a series of new analogues with modified 5′-aminouridine moieties through mutational biosynthesis. Based on molecular networking analysis of MS/MS, twenty-two new analogues (SS-KK-1 to -13 and SS-KK-A to -I) were identified. Among them, four new analogues (SS-KK-1 to -3 and SS-KK-C) were purified and bioassayed. SS-KK-2 showed better antibacterial activity against E. coli ΔtolC than the parent compound sansanmycin A. SS-KK-3 showed the same anti-TB activity as sansanmycin A against M. tuberculosis H37Rv as well as clinically isolated, drug-sensitive and multidrug-resistant M. tuberculosis strains. Furthermore, SS-KK-3 exhibited significantly improved structural stability compared to sansanmycin A. The results suggested that mutasynthesis is an effective and practical strategy for expanding the structural diversity of 5′-aminouridine moiety in sansanmycin.

The emergence of SARS-CoV-2 variants represents a major threat to public health and requires identification of novel therapeutic agents to address the unmet medical needs. Small molecules impeding viral entry through inhibition of spike protein priming proteases could have potent antiviral effects against SARS-CoV-2 infection. Omicsynin B4, a pseudo-tetrapeptides identified from Streptomyces sp. 1647, has potent antiviral activity against influenza A viruses in our previous study. Here, we found omicsynin B4 exhibited broad-spectrum anti-coro-navirus activity against HCoV-229E, HCoV-OC43 and SARS-CoV-2 prototype and its variants in multiple cell lines. Further investigations revealed omicsynin B4 blocked the viral entry and might be related to the inhibition of host proteases. SARS-CoV-2 spike protein mediated pseudovirus assay supported the inhibitory activity on viral entry of omicsynin B4 with a more potent inhibition of Omicron variant, especially when overexpression of human TMPRSS2. Moreover, omicsynin B4 exhibited superior inhibitory activity in the sub-nanomolar range against CTSL, and a sub-micromolar inhibition against TMPRSS2 in biochemical assays. The molecular docking analysis confirmed that omicsynin B4 fits well in the substrate binding sites and forms a covalent bond to Cys25 and Ser441 in CTSL and TMPRSS2, respectively. In conclusion, we found that omicsynin B4 may serve as a natural protease inhibitor for CTSL and TMPRSS2, blocking various coronavirus S protein-driven entry into cells. These results further highlight the potential of omicsynin B4 as an attractive candidate for broad-spectrum antiviral therapy that could rapidly respond to emerging variants of SARS-CoV-2.

Chuangxinmycin is an antibiotic isolated from Actinoplanes tsinanensis in the 1960s with a novel indole-dihydrothiopyran heterocyclic skeleton. It showed good antibacterial activity and unique antibacterial mechanism. However, its biosynthetic gene cluster has remained obscure for over 50 years. The historical evolution of chuangxinmycin research was introduced, and the latest advances in the understanding of its biosynthetic mechanism were reviewed in detail from the perspectives of biosynthetic gene cluster, biosynthetic pathway, the novel mechanism for sulfur incorporation catalyzed by a deubiquitinase-like sulfurtransferase and the rare C-S bond formation mechanism catalyzed by cytochrome P450 enzyme. The elucidation of the biosynthetic mechanism of chuangxinmycin provides the basis for applying combinatorial biosynthesis and synthetic biology technology in generating diverse structures for new drug discovery and development.

Two novel cinnamoyl-containing nonribosomal peptides (CCNPs) grisgenomycin A and B were identified in Streptomyces griseus NBRC 13350 (CGMCC 4.5718) and ATCC 12475, through genome mining using conserved adjacent LuxR family regulators as probes and activators. Notably, grisgenomycins represent a new group of bicyclic decapeptides featuring an unprecedented C-C bond between the tryptophan carbocycle and the cinnamoyl group. A plausible biosynthetic pathway for grisgenomycins was deduced by a bioinformatics analysis. Grisgenomycins exhibited activity against human coronaviruses at the micromolar level.

A new congener of chuangxinmycin (CM) was identified from Actinoplanes tsinanensis CPCC 200056. Its structure was determined as 3-methylchuangxinmycin (MCM) by 1D and 2D NMR. MCM could be generated in vivo from CM by heterologous expression of the vitamin B12-dependent radical SAM enzyme CxnA/A1 responsible for methylation of 3-demethylchuangxinmycin (DCM) in CM biosynthesis, indicating that CxnA/A1 could perform iterative methylation for MCM production. In vitro assays revealed significant activities of CM, DCM, and MCM against Mycobacterium tuberculosis H37Rv and clinically isolated isoniazid/rifampin-resistant M. tuberculosis, suggesting that CM and its derivatives may have potential for antituberculosis drug development.

Developing non-statin small molecules for the treatment of hypercholesterolemia remains challenging. The proprotein convertase subtilisin/kexin type 9 (PCSK9)-targeted therapies have attracted considerable attentions. Forty-five 7030B-C5 derivatives were synthesized and evaluated for the PCSK9 repression activity, taking the PCSK9 transcriptional inhibitor 7030B-C5 as the lead. Structure-activity relationship (SAR) analysis at C8 and N7-position was carried out, and compound 3s and 5r exhibited comparable PCSK9 transcriptional inhibitory activity but much lower cytotoxicity with the therapeutic index (TI) values doubled of that of 7030B-C5. In the in vitro assay, both compounds significantly reduced the level of PCSK9 protein and increased LDL receptor (LDLR) protein level. What's more, both compounds promoted LDL cholesterol (LDL-C) clearance more efficiently than 7030B-C5 in HepG2 cells. Most importantly, compound 3s reduced the atherosclerotic plaque areas with promising lipid-lowing effects in ApoE KO mice with a higher in vivo activity and lower toxicity. The regulatory mechanism of 3s was explored that it might target the transcription factor HNF1α and/or HINFP upstream of PCSK9 transcription, similar to that of 7030B-C5. Thus, 3s was considered as a potential anti-atherosclerosis drug candidate as a novel PCSK9 down-regulatory agent, worthy of further investigations.

Three isocoumarins, including two new compounds, (±) pestalactone D ( 1 ) and pestapyrone F ( 2 ), as well as one known compound, pestapyrone D ( 3 ), were isolated from the culture of the endolichenic Pestalotiopsis rhododendri LF-19-12. The planar structures of all compounds were elucidated by NMR and MS spectra. And the absolute configurations of 1 were confirmed by single crystal X-ray diffraction analysis, indicative of it as a racemate of 4 S /12 S and 4 R /12 R enantiomers. Compound 1 exhibited weak anti-coronaviral activity against human coronavirus HCoV-229E with an EC 50 of 77.61 μM. Based on the bioinformatics analysis, the biosynthetic pathway of 1 has been proposed.