Cyclic immonium ion of lactyllysine reveals widespread lactylation in the human proteome.

Lactylation was initially discovered on human histones. Given its nascence, its occurrence on nonhistone proteins and downstream functional consequences remain elusive. Here we report a cyclic immonium ion of lactyllysine formed during tandem mass spectrometry that enables confident protein lactylation assignment. We validated the sensitivity and specificity of this ion for lactylation through affinity-enriched lactylproteome analysis and large-scale informatic assessment of nonlactylated spectral libraries. With this diagnostic ion-based strategy, we confidently determined new lactylation, unveiling a wide landscape beyond histones from not only the enriched lactylproteome but also existing unenriched human proteome resources. Specifically, by mining the public human Meltome Atlas, we found that lactylation is common on glycolytic enzymes and conserved on ALDOA. We also discovered prevalent lactylation on DHRS7 in the draft of the human tissue proteome. We partially demonstrated the functional importance of lactylation: site-specific engineering of lactylation into ALDOA caused enzyme inhibition, suggesting a lactylation-dependent feedback loop in glycolysis.

Chemoproteomic mapping of the glycolytic targetome in cancer cells.

Hyperactivated glycolysis is a metabolic hallmark of most cancer cells. Although sporadic information has revealed that glycolytic metabolites possess nonmetabolic functions as signaling molecules, how these metabolites interact with and functionally regulate their binding targets remains largely elusive. Here, we introduce a target-responsive accessibility profiling (TRAP) approach that measures changes in ligand binding-induced accessibility for target identification by globally labeling reactive proteinaceous lysines. With TRAP, we mapped 913 responsive target candidates and 2,487 interactions for 10 major glycolytic metabolites in a model cancer cell line. The wide targetome depicted by TRAP unveils diverse regulatory modalities of glycolytic metabolites, and these modalities involve direct perturbation of enzymes in carbohydrate metabolism, intervention of an orphan transcriptional protein's activity and modulation of targetome-level acetylation. These results further our knowledge of how glycolysis orchestrates signaling pathways in cancer cells to support their survival, and inspire exploitation of the glycolytic targetome for cancer therapy.

A plant-specific SWR1 chromatin-remodeling complex couples histone H2A.Z deposition with nucleosome sliding.

Deposition of H2A.Z in chromatin is known to be mediated by a conserved SWR1 chromatin-remodeling complex in eukaryotes. However, little is known about whether and how the SWR1 complex cooperates with other chromatin regulators. Using immunoprecipitation followed by mass spectrometry, we found all known components of the Arabidopsis thaliana SWR1 complex and additionally identified the following three classes of previously uncharacterized plant-specific SWR1 components: MBD9, a methyl-CpG-binding domain-containing protein; CHR11 and CHR17 (CHR11/17), ISWI chromatin remodelers responsible for nucleosome sliding; and TRA1a and TRA1b, accessory subunits of the conserved NuA4 histone acetyltransferase complex. MBD9 directly interacts with CHR11/17 and the SWR1 catalytic subunit PIE1, and is responsible for the association of CHR11/17 with the SWR1 complex. MBD9, TRA1a, and TRA1b function as canonical components of the SWR1 complex to mediate H2A.Z deposition. CHR11/17 are not only responsible for nucleosome sliding but also involved in H2A.Z deposition. These results indicate that the association of the SWR1 complex with CHR11/17 may facilitate the coupling of H2A.Z deposition with nucleosome sliding, thereby co-regulating gene expression, development, and flowering time.

Application of Aptamer-SERS Nanotags for Unveiling the PD-L1 Immunomarker Progression Correlated to the Cell Metabolic Bioprocess.

In recent years, the expression and progression of programmed cell death ligand 1 (PD-L1) as an immunomarker in the context of a cell metabolic environment has gained significant attention in cancer research. However, intercellular bioprocesses that control the dynamics of PD-L1 have been largely unexplored. This study aimed to explore the cell metabolic states and conditions that govern dynamic variations of PD-L1 within the cell metabolic environment using an aptamer-based surface-enhanced Raman scattering (SERS) approach. The aptamer-SERS technique offers a sensitive, rapid, and powerful analytical tool for targeted and nondestructive detection of an immunomarker with high sensitivity and specificity. By combining aptamer-SERS with cell state profiling, we investigated the modulation in PD-L1 expression under different metabolic states, including glucose deprivation, metabolic coenzyme activity, and altered time/concentration-based cytokine availability. The most intriguing features in our findings include the cell-specific responses, cell differentiation by revealing distinct patterns, and dynamics of PD-L1 in different cell lines. Additionally, the time-dependent variations in PD-L1 expression, coupled with the dose-dependent relationship between glucose concentration and PD-L1 levels, underscore the complex interplay between immune checkpoint regulation and cellular metabolism. Therefore, this work demonstrates the advantages of using highly-sensitive and specific aptamer-SERS nanotags for investigating the immune checkpoint dynamics and related metabolic bioprocess.

Carbene Footprinting Directs Design of Genetically Encoded Proximity-Reactive Protein Binders.

Genetically encoding proximal-reactive unnatural amino acids (PrUaas), such as fluorosulfate-l-tyrosine (FSY), into natural proteins of interest (POI) confer the POI with the ability to covalently bind to its interacting proteins (IPs). The PrUaa-incorporated POIs hold promise for blocking undesirable POI-IP interactions. Selecting appropriate PrUaa anchor sites is crucial, but it remains challenging with the current methodology, which heavily relies on crystallography to identify the proximal residues between the POIs and the IPs for the PrUaa anchorage. To address the challenge, here, we propose a footprinting-directed genetically encoded covalent binder (footprinting-GECB) approach. This approach employs carbene footprinting, a structural mass spectrometry (MS) technique that quantifies the extent of labeling of the POI following the addition of its IP, and thus identifies the responsive residues. By genetically encoding PrUaa into these responsive sites, POI variants with covalent bonding ability to its IP can be produced without the need for crystallography. Using the POI-IP model, KRAS/RAF1, we showed that engineering FSY at the footprint-assigned KRAS residue resulted in a KRAS variant that can bind irreversibly to RAF1. Additionally, we inserted FSY at the responsive residue in RAF1 upon footprinting the oncogenic KRASG12D/RAF1, which lacks crystal structure, and generated a covalent binder to KRASG12D. Together, we demonstrated that by adopting carbene footprinting to direct PrUaa anchorage, we can greatly expand the opportunities for designing covalent protein binders for PPIs without relying on crystallography. This holds promise for creating effective PPI inhibitors and supports both fundamental research and biotherapeutics development.

Targeting delivery of miR-146a via IMTP modified milk exosomes exerted cardioprotective effects by inhibiting NF-κB signaling pathway after myocardial ischemia-reperfusion injury.

Reperfusion therapy is critical for saving heart muscle after myocardial infarction, but the process of restoring blood flow can itself exacerbate injury to the myocardium. This phenomenon is known as myocardial ischemia-reperfusion injury (MIRI), which includes oxidative stress, inflammation, and further cell death. microRNA-146a (miR-146a) is known to play a significant role in regulating the immune response and inflammation, and has been studied for its potential impact on the improvement of heart function after myocardial injury. However, the delivery of miR-146a to the heart in a specific and efficient manner remains a challenge as extracellular RNAs are unstable and rapidly degraded. Milk exosomes (MEs) have been proposed as ideal delivery platform for miRNA-based therapy as they can protect miRNAs from RNase degradation. In this study, the effects of miR-146a containing MEs (MEs-miR-146a) on improvement of cardiac function were examined in a rat model of MIRI. To enhance the targeting delivery of MEs-miR-146a to the site of myocardial injury, the ischemic myocardium-targeted peptide IMTP was modified onto the surfaces, and whether the modified MEs-miR-146a could exert a better therapeutic role was examined by echocardiography, myocardial injury indicators and the levels of inflammatory factors. Furthermore, the expressions of miR-146a mediated NF-κB signaling pathway-related proteins were detected by western blotting and qRT-PCR to further elucidate its mechanisms. MiR-146 mimics were successfully loaded into the MEs by electroporation at a square wave 1000 V voltage and 0.1 ms pulse duration. MEs-miR-146a can be up-taken by cardiomyocytes and protected the cells from oxygen glucose deprivation/reperfusion induced damage in vitro. Oral administration of MEs-miR-146a decreased myocardial tissue apoptosis and the expression of inflammatory factors and improved cardiac function after MIRI. The miR-146a level in myocardium tissues was significantly increased after the administration IMTP modified MEs-miR-146a, which was higher than that of the MEs-miR-146a group. In addition, intravenous injection of IMTP modified MEs-miR-146a enhanced the targeting to heart, improved cardiac function, reduced myocardial tissue apoptosis and suppressed inflammation after MIRI, which was more effective than the MEs-miR-146a treatment. Moreover, IMTP modified MEs-miR-146a reduced the protein levels of IRAK1, TRAF6 and p-p65. Therefore, IMTP modified MEs-miR-146a exerted their anti-inflammatory effect by inhibiting the IRAK1/TRAF6/NF-κB signaling pathway. Taken together, our findings suggested miR-146a containing MEs may be a promising strategy for the treatment of MIRI with better outcome after modification with ischemic myocardium-targeted peptide, which was expected to be applied in clinical practice in future.

Study on the Anti-Mycobacterium marinum Activity of a Series of Marine-Derived 14-Membered Resorcylic Acid Lactone Derivatives.

With the emergence of drug-resistant strains, the treatment of tuberculosis (TB) is becoming more difficult and there is an urgent need to find new anti-TB drugs. Mycobacterium marinum, as a model organism of Mycobacterium tuberculosis, can be used for the rapid and efficient screening of bioactive compounds. The 14-membered resorcylic acid lactones (RALs) have a wide range of bioactivities such as antibacterial, antifouling and antimalarial activity. In order to further study their bioactivities, we initially constructed a 14-membered RALs library, which contains 16 new derivatives. The anti-M. marinum activity was evaluated in vitro. Derivatives 12, 19, 20 and 22 exhibited promising activity with MIC90 values of 80, 90, 80 and 80 µM, respectively. The preliminary structure-activity relationships showed that the presence of a chlorine atom at C-5 was a key factor to improve activity. Further studies showed that 12 markedly inhibited the survival of M. marinum and significantly reduced the dosage of positive drugs isoniazid and rifampicin when combined with them. These results suggest that 12 is a bioactive compound capable of enhancing the potency of existing positive drugs, and its effective properties make it a very useful leads for future drug development in combating TB resistance.

A suggested mortality benefit with integrated health care versus conventional home health care in Taiwan.

In Taiwan, the Integrated Home Care (IHC) project was introduced for medically compromised patients living at home receiving Home Health Care (HHC) in 2016. The focus of the project was on organizing care teams and managing care for patients. The aim of this study was to investigate the benefits and impacts of IHC in Taiwan. The primary outcome measure was the mortality rate of patients who received IHC versus those who did not receive IHC (non-IHC). The secondary outcomes were medical utilization and expenditure. The results showed that IHC was associated with a statistically significant reduction in mortality compared to non-IHC for home-dwelling patients over 90-, 180-, and 365-days periods. Additionally, IHC users were less likely to be hospitalized and had shorter hospitalization times compared to non-IHC users. Furthermore, IHC was found to reduce medical expenditure compared to non-IHC.

Inhibition of influenza A virus replication by a marine derived quinolone alkaloid targeting virus nucleoprotein.

Owing to the emergence of drug resistance and high morbidity and mortality, the need for novel anti-influenza A virus (IAV) drugs with divergent targets is highly sought after. Herein, a novel quinolone alkaloid (QLA) derived from marine fungus was discovered with broad-spectrum anti-IAV activities with low toxicity. Distinct from current anti-IAV drugs, QLA may block virus replication and viral RNA (vRNA) export from the nucleus by targeting virus nucleoprotein (NP). QLA can block the binding of chromosome region maintenance 1 to nuclear export signal 3 of NP to inhibit the nuclear export of NP and vRNP. QLA may also affect vRNP assembly by interfering with the binding of NP to RNA rather than NP oligomerization. Arg305 and Phe488-Gly490 may be required for the interaction between QLA and NP, and the binding pocket around these amino acids may be a promising target for anti-IAV drugs. Importantly, oral administration of QLA can protect the mice against IAV-induced death and weight loss, superior to the effects of the clinical drug oseltamivir. In summary, the marine derived compound QLA has the potential to be developed into a novel anti-IAV agent targeting virus NP protein in the future.

Dual Recognition of H3K4me3 and DNA by the ISWI Component ARID5 Regulates the Floral Transition in Arabidopsis.

Chromatin remodeling and histone modifications are important for development and floral transition in plants. However, it is largely unknown whether and how these two epigenetic regulators coordinately regulate the important biological processes. Here, we identified three types of Imitation Switch (ISWI) chromatin-remodeling complexes in Arabidopsis (Arabidopsis thaliana). We found that AT-RICH INTERACTING DOMAIN5 (ARID5), a subunit of a plant-specific ISWI complex, can regulate development and floral transition. The ARID-PHD dual domain cassette of ARID5 recognizes both the H3K4me3 histone mark and AT-rich DNA. We determined the ternary complex structure of the ARID5 ARID-PHD cassette with an H3K4me3 peptide and an AT-containing DNA. The H3K4me3 peptide is combinatorially recognized by the PHD and ARID domains, while the DNA is specifically recognized by the ARID domain. Both PHD and ARID domains are necessary for the association of ARID5 with chromatin. The results suggest that the dual recognition of AT-rich DNA and H3K4me3 by the ARID5 ARID-PHD cassette may facilitate the association of the ISWI complex with specific chromatin regions to regulate development and floral transition.

Trends of antimalarial marine natural products: progresses, challenges and opportunities.

Covering 1972 to 2021Malaria remains a significant public health problem in some regions of the world. The great efforts to control malaria have been severely compromised due to the widespread resistance of Plasmodium falciparum to nearly all frontline drugs. Pursuit of novel molecules from the sea will potentially result in new interventions against malaria, which are urgently needed to combat the increase of resistance. Focusing on the strategy of the "Blue Drug Bank", the molecules highlighted here can serve as an inspiration for future medicinal chemistry campaigns. This review covers the developments in the field of antimalarial marine lead compounds reported between 1972 and July 2021, and offers a comprehensive overview on their progresses and potentials. We selected 60 representative potential candidate molecules from 361 marine natural products, and highlighted their structure-activity relationships, molecular mechanisms of targets, and drug-like properties in order to assess their full potential to be developed. We summarized 107 clinically proven or potential antimalarial targets and their subcellular locations in the relevant target proteins, which linked the molecules to the target proteins at the subcellular level. Hence, it could be expected that natural products targeting different mechanisms may prove to be an effective strategy in antimalarial drug research and development in the future.

The PEAT protein complexes are required for histone deacetylation and heterochromatin silencing.

In eukaryotes, heterochromatin regions are typically subjected to transcriptional silencing. DNA methylation has an important role in such silencing and has been studied extensively. However, little is known about how methylated heterochromatin regions are subjected to silencing. We conducted a genetic screen and identified an epcr (enhancer of polycomb-related) mutant that releases heterochromatin silencing in Arabidopsis thaliana We demonstrated that EPCR1 functions redundantly with its paralog EPCR2 and interacts with PWWP domain-containing proteins (PWWPs), AT-rich interaction domain-containing proteins (ARIDs), and telomere repeat binding proteins (TRBs), thus forming multiple functionally redundant protein complexes named PEAT (PWWPs-EPCRs-ARIDs-TRBs). The PEAT complexes mediate histone deacetylation and heterochromatin condensation and thereby facilitate heterochromatin silencing. In heterochromatin regions, the production of small interfering RNAs (siRNAs) and DNA methylation is repressed by the PEAT complexes. The study reveals how histone deacetylation, heterochromatin condensation, siRNA production, and DNA methylation interplay with each other and thereby maintain heterochromatin silencing.

Living Cell-Target Responsive Accessibility Profiling Reveals Silibinin Targeting ACSL4 for Combating Ferroptosis.

We report a living cell-target responsive accessibility profiling (LC-TRAP) approach to identify the targetome of silibinin (SIL), a well-established hepatoprotective natural product (NP), in HepG2 cells. Proteins showing accessibility changes, probed by covalent lysine labeling reagents and leveraged by multiplexed quantitative proteomics, following the administration of SIL to the living cells were assigned as potential targets. Among the assigned targetome, ACSL4, an enzyme essential for ferroptosis induction, might be involved in the hepatoprotective effects of SIL and hence was intensively validated. We first demonstrated that SIL protected HepG2 cells from ferroptosis dependent on ACSL4. Then, we used biophysical assays and a SIL-derivatized chemical probe to corroborate that SIL can bind to ACSL4. The ensuing enzymatic assays showed that SIL inhibited ACSL4 enzymatic activity, thereby mitigating the ACSL4-mediated ferroptosis. As such, we revealed that ACSL4 inhibition, using SIL as a model compound, represents a promising hepatoprotective strategy. Further, since TRAP probes the accessibility changes of reactive proteinaceous lysines, it can pinpoint the proximal regions where the ligand engagement may occur. Thus, the LC-TRAP analysis of SIL, the newly discovered ligand of ACSL4, and arachidonic acid (AA), the substrate, intriguingly showed that SIL and AA both affected the conformation of the K536-proximal region of ACSL4, albeit through distinct binding patterns. Collectively, we describe a straightforward LC-TRAP workflow that does not involve ligand-derived probe synthesis and is widely applicable to target discovery of NPs.

Analysis on short-term change of macular function and the correlates after intravitreal conbercept for CRVO-ME.

AIM: To study the short-term change of macular function and the correlates after intravitreal conbercept for CRVO-ME. STUDY DESIGN: Prospective, clinical study. METHODS: Twenty Three patients(23 eyes) were recruited, who were non-ischemia central retinal vein occlusion diagnosed by FFA (fundus fluorescein angiography) and treated with intravitreal conbercept for macular edema, best - corrected visual acuity ( BCVA), central macular thickness(CMT), amplitude density of P1 wave and implicit time of P1,N1 wave from ring 1 and ring 2 of mf-ERG were measured before and 1 week、2 month after treatment. RESULTS: Compared to the baseline, BCVA、CMT、amplitude density of P1 wave and implicit time of P1,N1 wave from ring 1 and ring 2 were greatly improved at 1 W、2 M after treatment; better results were gained at 2 M compared to 1 W; Pearson correlation analysis shows no significantly correlation between the improvement of mf-ERG with the change of BCVA、CMT. CONCLUSION: The BCVA、the structure and the function of macular were greatly improved after intravitreal conbercept for central retinal vein occlusion induced macular edema; however no significantly correlation between the improvement of the function of macular with the strcture of macular and BCVA.

Integrating Activity-Guided Strategy and Fingerprint Analysis to Target Potent Cytotoxic Brefeldin A from a Fungal Library of the Medicinal Mangrove Acanthus ilicifolius.

Mangrove-associated fungi are rich sources of novel and bioactive compounds. A total of 102 fungal strains were isolated from the medicinal mangrove Acanthus ilicifolius collected from the South China Sea. Eighty-four independent culturable isolates were identified using a combination of morphological characteristics and internal transcribed spacer (ITS) sequence analyses, of which thirty-seven strains were selected for phylogenetic analysis. The identified fungi belonged to 22 genera within seven taxonomic orders of one phyla, of which four genera Verticillium, Neocosmospora, Valsa, and Pyrenochaeta were first isolated from mangroves. The cytotoxic activity of organic extracts from 55 identified fungi was evaluated against human lung cancer cell lines (A-549), human cervical carcinoma cell lines (HeLa), human hepatoma cells (HepG2), and human acute lymphoblastic leukemia cell lines (Jurkat). The crude extracts of 31 fungi (56.4%) displayed strong cytotoxicity at the concentration of 50 µg/mL. Furthermore, the fungus Penicillium sp. (HS-N-27) still showed strong cytotoxic activity at the concentration of 25 µg/mL. Integrating cytotoxic activity-guided strategy and fingerprint analysis, a well-known natural Golgi-disruptor and Arf-GEFs inhibitor, brefeldin A, was isolated from the target active strain HS-N-27. It displayed potential activity against A549, HeLa and HepG2 cell lines with the IC50 values of 101.2, 171.9 and 239.1 nM, respectively. Therefore, combining activity-guided strategy with fingerprint analysis as a discovery tool will be implemented as a systematic strategy for quick discovery of active compounds.

Design, Semisynthesis, Insecticidal and Antibacterial Activities of a Series of Marine-Derived Geodin Derivatives and Their Preliminary Structure-Activity Relationships.

To enhance the biological activity of the natural product geodin (1), isolated from the marine-derived fungus Aspergillus sp., a series of new ether derivatives (2-37) was designed and semisynthesized using a high-yielding one-step reaction. In addition, the insecticidal and antibacterial activities of all geodin congeners were evaluated systematically. Most of these derivatives showed better insecticidal activities against Helicoverpa armigera Hübner than 1. In particular, 15 showed potent insecticidal activity with an IC50 value of 89 µM, comparable to the positive control azadirachtin (IC50 = 70 µM). Additionally, 5, 12, 13, 16, 30 and 33 showed strong antibacterial activity against Staphylococcus aureus and Aeromonas salmonicida with MIC values in the range of 1.15-4.93 µM. The preliminary structure-activity relationships indicated that the introduction of halogenated benzyl especially fluorobenzyl, into 1 and substitution of 4-OH could be key factors in increasing the insecticidal and antibacterial activities of geodin.

Nigrosporins B, a Potential Anti-Cervical Cancer Agent, Induces Apoptosis and Protective Autophagy in Human Cervical Cancer Ca Ski Cells Mediated by PI3K/AKT/mTOR Signaling Pathway.

Nigrosporins B, an anthraquinone derivative obtained from the secondary metabolites of marine fungus Nigrospora oryzae. In this study, we characterized the distinctive anti-cancer potential of Nigrosporins B in vitro and underlying molecular mechanisms in human cervical cancer Ca Ski cells for the first time. The results of MTT assay showed that Nigrosporins B significantly inhibited the proliferation of multiple tumor cells in a dose-dependent manner, especially for the Ca Ski cells with an IC50 of 1.24 µM. Nigrosporins B exerted an apoptosis induction effect on Ca Ski cells as confirmed by flow cytometry, AO/EB dual fluorescence staining, mitochondrial membrane potential analysis and western blot assay. In addition, Nigrosporins B induced obvious autophagy accompanied with the increase of autophagic vacuoles and the acceleration of autophagic flux as indicated by Cyto-ID staining, mRFP-GFP-LC3 adenovirus transfection and western blot analysis. Interestingly, the combination of Nigrosporins B with the three autophagy inhibitors all significantly enhanced the cytotoxicity of Nigrosporins B on Ca Ski cells, indicating that the autophagy induced by Nigrosporins B might protect Ca Ski cells from death. Furthermore, we found that Nigrosporins B inhibited the phosphorylation of PI3K, AKT, mTOR molecules and increased the protein expression levels of PTEN and p-AMPKα in a dose-dependent manner, suggesting that Nigrosporins B induced apoptosis and protective autophagy through the suppression of the PI3K/AKT/mTOR signaling pathway. Together, these findings revealed the anti-cervical cancer effect of Nigrosporins B and the underlying mechanism of action in Ca Ski cells, it might be as a promising alternative therapeutic agent for human cervical cancer.

Brefeldin A delivery nanomicelles in hepatocellular carcinoma therapy: Characterization, cytotoxic evaluation in vitro, and antitumor efficiency in vivo.

Hepatocellular carcinoma (HCC) is one of the major cancers with high mortality rate. Traditional drugs used in clinic are usually limited by the drug resistance and side effect and novel agents are still needed. Macrolide brefeldin A (BFA) is a well-known lead compound in cancer chemotherapy, however, with poor solubility and instability. In this study, to overcome these disadvantages, BFA was encapsulated in mixed nanomicelles based on TPGS and F127 copolymers (M-BFA). M-BFA was conferred high solubility, colloidal stability, and capability of sustained release of intact BFA. In vitro, M-BFA markedly inhibited the proliferation, induced G0/G1 phase arrest, and caspase-dependent apoptosis in human liver carcinoma HepG2 cells. Moreover, M-BFA also induced autophagic cell death via Akt/mTOR and ERK pathways. In HepG2 tumor-bearing xenograft mice, indocyanine green (ICG) as a fluorescent probe loaded in M-BFA distributed to the tumor tissue rapidly, prolonged the blood circulation, and improved the tumor accumulation capacity. More importantly, M-BFA (10 mg/kg) dramatically delayed the tumor progression and induced extensive necrosis of the tumor tissues. Taken together, the present work suggests that M-BFA has promising potential in HCC therapy.

Targeted Isolation of Asperheptatides from a Coral-Derived Fungus Using LC-MS/MS-Based Molecular Networking and Antitubercular Activities of Modified Cinnamate Derivatives.

Under the guidance of MS/MS-based molecular networking, four new cycloheptapeptides, namely, asperheptatides A-D (1-4), were isolated together with three known analogues, asperversiamide A-C (5-7), from the coral-derived fungus Aspergillus versicolor. The planar structures of the two major compounds, asperheptatides A and B (1 and 2), were determined by comprehensive spectroscopic data analysis. The absolute configurations of the amino acid residues were determined by advanced Marfey's method. The two structurally related trace metabolites, asperheptatides C and D (3 and 4), were characterized by ESI-MS/MS fragmentation methods. A series of new derivatives (8-26) of asperversiamide A (5) were semisynthesized. The antitubercular activities of 1, 2, and 5-26 against Mycobacterium tuberculosis H37Ra were also evaluated. Compounds 9, 13, 23, and 24 showed moderate activities with MIC values of 12.5 µM, representing a potential new class of antitubercular agents.

17-Hydroxybrevianamide N and Its N1-Methyl Derivative, Quinazolinones from a Soft-Coral-Derived Aspergillus sp. Fungus: 13S Enantiomers as the True Natural Products.

Under the guidance of MS/MS-based molecular networking and HPLC-UV, two new alkaloid racemates, (±)-17-hydroxybrevianamide N (1) and (±)-N1-methyl-17-hydroxybrevianamide N (2), featuring a rare o-hydroxyphenylalanine residue and an imide subunit, were isolated from a soft-coral-derived Aspergillus sp. fungus. The true natural products (+)-1 and (+)-2 were further monitored and obtained from the freshly prepared EtOAc extracts, while (-)-1 and (-)-2 are artifacts generated during extraction and purification processes. Simultaneously, the structures including absolute configurations of (+)-13S-1, (-)-13R-1, (+)-13S-2, and (-)-13R-2 were elucidated on the basis of comprehensive spectroscopic analysis, ECD calculations, and X-ray diffraction data. Interestingly, basic solution promotes the racemization of (+)-1 and (-)-1, whereas acidic solution suppresses the transformation. The current research was concerned with the true natural products and their artifacts, providing critical insight into the isolation and identification of natural products.