Informatics and Computational Approaches for the Discovery and Optimization of Natural Product-Inspired Inhibitors of the SARS-CoV-2 2'-O-Methyltransferase.

The urgent need for new classes of orally available, safe, and effective antivirals─covering a breadth of emerging viruses─is evidenced by the loss of life and economic challenges created by the HIV-1 and SARS-CoV-2 pandemics. As frontline interventions, small-molecule antivirals can be deployed prophylactically or postinfection to control the initial spread of outbreaks by reducing transmissibility and symptom severity. Natural products have an impressive track record of success as prototypic antivirals and continue to provide new drugs through synthesis, medicinal chemistry, and optimization decades after discovery. Here, we demonstrate an approach using computational analysis typically used for rational drug design to identify and develop natural product-inspired antivirals. This was done with the goal of identifying natural product prototypes to aid the effort of progressing toward safe, effective, and affordable broad-spectrum inhibitors of Betacoronavirus replication by targeting the highly conserved RNA 2'-O-methyltransferase (2'-O-MTase). Machaeriols RS-1 (7) and RS-2 (8) were identified using a previously outlined informatics approach to first screen for natural product prototypes, followed by in silico-guided synthesis. Both molecules are based on a rare natural product group. The machaeriols (3-6), isolated from the genus Machaerium, endemic to Amazonia, inhibited the SARS-CoV-2 2'-O-MTase more potently than the positive control, Sinefungin (2), and in silico modeling suggests distinct molecular interactions. This report highlights the potential of computationally driven screening to leverage natural product libraries and improve the efficiency of isolation or synthetic analog development.

Platanosides from Platanus × acerifolia: New molecules, SAR, and target validation of a strong lead for drug-resistant bacterial infections and the associated sepsis.

Three undescribed (1-3) and nine known (4-12) platanosides were isolated and characterized from a bioactive extract of the May leaves of Platanus × acerifolia that initially showed inhibition against Staphylococcus aureus. Targeted compound mining was guided by an LC-MS/MS-based molecular ion networking (MoIN) strategy combined with conventional isolation procedures from a unique geographic location. The novel structures were mainly determined by 2D NMR and computational (NMR/ECD calculations) methods. Compound 1 is a rare acylated kaempferol rhamnoside possessing a truxinate unit. 6 (Z,E-platanoside) and 7 (E,E-platanoside) were confirmed to have remarkable inhibitory effects against both methicillin-resistant S. aureus (MIC: ≤ 16 µg/mL) and glycopeptide-resistant Enterococcus faecium (MIC: ≤ 1 µg/mL). These platanosides were subjected to docking analyses against FabI (enoyl-ACP reductase) and PBP1/2 (penicillin binding protein), both of which are pivotal enzymes governing bacterial growth but not found in the human host. The results showed that 6 and 7 displayed superior binding affinities towards FabI and PBP2. Moreover, surface plasmon resonance studies on the interaction of 1/7 and FabI revealed that 7 has a higher affinity (KD = 1.72 µM), which further supports the above in vitro data and is thus expected to be a novel anti-antibacterial drug lead.

Boesenbergia stenophylla-Derived Stenophyllol B Exerts Antiproliferative and Oxidative Stress Responses in Triple-Negative Breast Cancer Cells with Few Side Effects in Normal Cells.

Triple-negative breast cancer (TNBC) is insensitive to target therapy for non-TNBC and needs novel drug discovery. Extracts of the traditional herb Boesenbergia plant in Southern Asia exhibit anticancer effects and contain novel bioactive compounds but merely show cytotoxicity. We recently isolated a new compound from B. stenophylla, stenophyllol B (StenB), but the impact and mechanism of its proliferation-modulating function on TNBC cells remain uninvestigated. This study aimed to assess the antiproliferative responses of StenB in TNBC cells and examine the drug safety in normal cells. StenB effectively suppressed the proliferation of TNBC cells rather than normal cells in terms of an ATP assay. This preferential antiproliferative function was alleviated by pretreating inhibitors for oxidative stress (N-acetylcysteine (NAC)) and apoptosis (Z-VAD-FMK). Accordingly, the oxidative-stress-related mechanisms were further assessed. StenB caused subG1 and G2/M accumulation but reduced the G1 phase in TNBC cells, while normal cells remained unchanged between the control and StenB treatments. The apoptosis behavior of TNBC cells was suppressed by StenB, whereas that of normal cells was not suppressed according to an annexin V assay. StenB-modulated apoptosis signaling, such as for caspases 3, 8, and 9, was more significantly activated in TNBC than in normal cells. StenB also caused oxidative stress in TNBC cells but not in normal cells according to a flow cytometry assay monitoring reactive oxygen species, mitochondrial superoxide, and their membrane potential. StenB induced greater DNA damage responses (γH2AX and 8-hydroxy-2-deoxyguanosine) in TNBC than in normal cells. All these StenB responses were alleviated by NAC pretreatment. Collectively, StenB modulated oxidative stress responses, leading to the antiproliferation of TNBC cells with little cytotoxicity in normal cells.

Platanosides, a Potential Botanical Drug Combination, Decrease Liver Injury Caused by Acetaminophen Overdose in Mice.

Oxidative stress plays an important role in acetaminophen (APAP)-induced hepatotoxicity. Platanosides (PTSs) isolated from the American sycamore tree (Platanus occidentalis) represent a potential new four-molecule botanical drug class of antibiotics active against drug-resistant infectious disease. Preliminary studies have suggested that PTSs are safe and well tolerated and have antioxidant properties. The potential utility of PTSs in decreasing APAP hepatotoxicity in mice in addition to an assessment of their potential with APAP for the control of infectious diseases along with pain and pyrexia associated with a bacterial infection was investigated. On PTS treatment in mice, serum alanine aminotransferase (ALT) release, hepatic centrilobular necrosis, and 4-hydroxynonenal (4-HNE) were markedly decreased. In addition, inducible nitric oxide synthase (iNOS) expression and c-Jun-N-terminal kinase (JNK) activation decreased when mice overdosed with APAP were treated with PTSs. Computational studies suggested that PTSs may act as JNK-1/2 and Keap1-Nrf2 inhibitors and that the isomeric mixture could provide greater efficacy than the individual molecules. Overall, PTSs represent promising botanical drugs for hepatoprotection and drug-resistant bacterial infections and are effective in protecting against APAP-related hepatotoxicity, which decreases liver necrosis and inflammation, iNOS expression, and oxidative and nitrative stresses, possibly by preventing persistent JNK activation.

Structure and Antimicrobial Activity of Rare Lactone Lipids from the Sooty Mold (Scorias spongiosa).

Two new lactone lipids, scoriosin (1) and its methyl ester (2), with a rare furylidene ring joined to a tetrahydrofurandione ring, were isolated from Scorias spongiosa, commonly referred to as sooty mold. The planar structure of these compounds was assigned by 1D and 2D NMR. The conformational analysis of these molecules was undertaken to evaluate the relative and absolute configuration through GIAO NMR chemical shift analysis and ECD calculation. In addition to the potent antimicrobial activities, compound 2 strongly potentiated the activity of amphotericin B against Cryptococcus neoformans, suggesting the potential utility of this compound in combination therapies for treating cryptococcal infections.

Manzamine-A Alters In Vitro Calvarial Osteoblast Function.

Manzamine-A is a marine-derived alkaloid which has anti-viral and anti-proliferative properties and is currently being investigated for its efficacy in the treatment of certain viruses (malaria, herpes, HIV-1) and cancers (breast, cervical, colorectal). Manzamine-A has been found to exert effects via modulation of SIX1 gene expression, a gene critical to craniofacial development via the WNT, NOTCH, and PI3K/AKT pathways. To date little work has focused on Manzamine-A and how its use may affect bone. We hypothesize that Manzamine-A, through SIX1, alters bone cell activity. Here, we assessed the effects of Manzamine-A on cells that are responsible for the generation of bone, pre-osteoblasts and osteoblasts. PCR, qrtPCR, MTS cell viability, Caspase 3/7, and functional assays were used to test the effects of Manzamine-A on these cells. Our data suggests Six1 is highly expressed in osteoblasts and their progenitors. Further, osteoblast progenitors and osteoblasts exhibit great sensitivity to Manzamine-A treatment exhibited by a significant decrease in cell viability, increase in cellular apoptosis, and decrease in alkaline phosphatase activity. In silico binding experiment showed that manzamine A potential as an inhibitor of cell proliferation and survival proteins, i.e., Iκb, JAK2, AKT, PKC, FAK, and Bcl-2. Overall, our data suggests Manzamine-A may have great effects on bone health overall and may disrupt skeletal development, homeostasis, and repair.

Stenophyllols A-C, new compounds from Boesenbergia stenophylla.

Three new compounds, i.e. stenophyllols A-C (1-3), were isolated from the rhizome of Boesenbergia stenophylla. The structures were determined by spectroscopic analysis (UV, IR, NMR and HRESIMS). In-vitro neuroblastoma cell viability assay showed stenophyllol A (1) was able to reduce the N2A cell viability to 20% within 24 h.

Contemporary Approaches to the Discovery and Development of Broad-Spectrum Natural Product Prototypes for the Control of Coronaviruses.

The pressing need for SARS-CoV-2 controls has led to a reassessment of strategies to identify and develop natural product inhibitors of zoonotic, highly virulent, and rapidly emerging viruses. This review article addresses how contemporary approaches involving computational chemistry, natural product (NP) and protein databases, and mass spectrometry (MS) derived target-ligand interaction analysis can be utilized to expedite the interrogation of NP structures while minimizing the time and expense of extraction, purification, and screening in BioSafety Laboratories (BSL)3 laboratories. The unparalleled structural diversity and complexity of NPs is an extraordinary resource for the discovery and development of broad-spectrum inhibitors of viral genera, including Betacoronavirus, which contains MERS, SARS, SARS-CoV-2, and the common cold. There are two key technological advances that have created unique opportunities for the identification of NP prototypes with greater efficiency: (1) the application of structural databases for NPs and target proteins and (2) the application of modern MS techniques to assess protein-ligand interactions directly from NP extracts. These approaches, developed over years, now allow for the identification and isolation of unique antiviral ligands without the immediate need for BSL3 facilities. Overall, the goal is to improve the success rate of NP-based screening by focusing resources on source materials with a higher likelihood of success, while simultaneously providing opportunities for the discovery of novel ligands to selectively target proteins involved in viral infection.

RSK1 vs. RSK2 Inhibitory Activity of the Marine ß-Carboline Alkaloid Manzamine A: A Biochemical, Cervical Cancer Protein Expression, and Computational Study.

Manzamines are complex polycyclic marine-derived ß-carboline alkaloids with reported anticancer, immunostimulatory, anti-inflammatory, antibacterial, antiviral, antimalarial, neuritogenic, hyperlipidemia, and atherosclerosis suppression bioactivities, putatively associated with inhibition of glycogen synthase kinase-3, cyclin-dependent kinase 5, SIX1, and vacuolar ATPases. We hypothesized that additional, yet undiscovered molecular targets might be associated with Manzamine A's (MZA) reported pharmacological properties. We report here, for the first time, that MZA selectively inhibited a 90 kDa ribosomal protein kinase S6 (RSK1) when screened against a panel of 30 protein kinases, while in vitro RSK kinase assays demonstrated a 10-fold selectivity in the potency of MZA against RSK1 versus RSK2. The effect of MZA on inhibiting cellular RSK1 and RSK2 protein expression was validated in SiHa and CaSki human cervical carcinoma cell lines. MZA's differential binding and selectivity toward the two isoforms was also supported by computational docking experiments. Specifically, the RSK1-MZA (N- and C-termini) complexes appear to have stronger interactions and preferable energetics contrary to the RSK2-MZA ones. In addition, our computational strategy suggests that MZA binds to the N-terminal kinase domain of RSK1 rather than the C-terminal domain. RSK is a vertebrate family of cytosolic serine-threonine kinases that act downstream of the ras-ERK1/2 (extracellular-signal-regulated kinase 1/2) pathway, which phosphorylates substrates shown to regulate several cellular processes, including growth, survival, and proliferation. Consequently, our findings have led us to hypothesize that MZA and the currently known manzamine-type alkaloids isolated from several sponge genera may have novel pharmacological properties with unique molecular targets, and MZA provides a new tool for chemical-biology studies involving RSK1.

Forrestiacids†A and†B, Pentaterpene Inhibitors of ACL and Lipogenesis: Extending the Limits of Computational NMR Methods in the Structure Assignment of Complex Natural Products.

Forrestiacids A (1) and B (2) are a novel class of [4+2] type pentaterpenoids derived from a rearranged lanostane moiety (dienophile) and an abietane unit (diene). These unprecedented molecules were isolated using guidance by molecular ion networking (MoIN) from Pseudotsuga forrestii, an endangered member of the Asian Douglas Fir Family. The intermolecular hetero-Diels-Alder adducts feature an unusual bicyclo[2.2.2]octene ring system. Their structures were elucidated by spectroscopic analysis, GIAO NMR calculations and DP4+ probability analyses, electronic circular dichroism calculations, and X-ray diffraction analysis. This unique addition to the pentaterpene family represents the largest and the most complex molecule successfully assigned using computational approaches to predict accurately chemical shift values. Compounds 1 and 2 exhibited potent inhibitory activities (IC50 s <5 µM) of ATP-citrate lyase (ACL), a new drug target for the treatment of glycolipid metabolic disorders including hyperlipidemia. Validating this activity 1 effectively attenuated the de novo lipogenesis in HepG2 cells. These findings provide a new chemical class for developing potential therapeutic agents for ACL-related diseases with strong links to traditional medicines.

The Marine Natural Product Manzamine A Inhibits Cervical Cancer by Targeting the SIX1 Protein.

Natural products remain an important source of drug leads covering unique chemical space and providing significant therapeutic value for the control of cancer and infectious diseases resistant to current drugs. Here, we determined the antiproliferative activity of a natural product manzamine A (1) from an Indo-Pacific sponge following various in vitro cellular assays targeting cervical cancer (C33A, HeLa, SiHa, and CaSki). Our data demonstrated the antiproliferative effects of 1 at relatively low and non-cytotoxic concentrations (up to 4 µM). Mechanistic investigations confirmed that 1 blocked cell cycle progression in SiHa and CaSki cells at G1/S phase and regulated cell cycle-related genes, including restoration of p21 and p53 expression. In apoptotic assays, HeLa cells showed the highest sensitivity to 1 as compared to other cell types (C33A, SiHa, and CaSki). Interestingly, 1 decreased the levels of the oncoprotein SIX1, which is associated with oncogenesis in cervical cancer. To further investigate the structure-activity relationship among manzamine A (1) class with potential antiproliferative activity, molecular networking facilitated the efficient identification, dereplication, and assignment of structures from the manzamine class and revealed the significant potential in the design of optimized molecules for the treatment of cervical cancer. These data suggest that this sponge-derived natural product class warrants further attention regarding the design and development of novel manzamine analogues, which may be efficacious for preventive and therapeutic treatment of cancer. Additionally, this study reveals the significance of protecting fragile marine ecosystems from climate change-induced loss of species diversity.

A coumarin-based fluorescent probe as a central nervous system disease biomarker.

Homocysteine and methylmalonic acid are important biomarkers for diseases associated with an impaired central nervous system (CNS). A new chemoassay utilizing coumarin-based fluorescent probe 1 to detect the levels of homocysteine is successfully implemented using Parkinson's disease (PD) patients' blood serum. In addition, a rapid identification of homocysteine and methylmalonic acid levels in blood serum of PD patients was also performed using the liquid chromatography-mass spectrometry (LC-MS). The results obtained from both analyses were in agreement. The new chemoassay utilizing coumarin-based fluorescent probe 1 offers a cost- and time-effective method to identify the biomarkers in CNS patients.

Antimalarial Drug Discovery from Natural and Synthetic Sources.

Malaria remains a significant global health threat despite extensive efforts aimed at its eradication. Numerous challenges persist in eliminating the disease, chief among them being the parasite's ability to mutate, resulting in drug resistance. The discovery of antimalarial drugs has relied on both phenotypic and target-based approaches. While phenotypic screening has identified promising candidates, target-based methods offer a more precise approach by leveraging chemically validated targets and computational tools. Analysis of Plasmodium spp. protein structures reveal druggable targets, offering opportunities for in silico screening. Combining compounds from natural and synthetic sources in a target-based approach accelerates the discovery of new antimalarial agents. This review explores previous breakthroughs in antimalarial drug discovery from natural products and synthetic origins, emphasizing their specific target proteins within Plasmodium species.

Manzamine A reduces androgen receptor transcription and synthesis by blocking E2F8-DNA interactions and effectively inhibits prostate tumor growth in mice.

The androgen receptor (AR) is the main driver in the development of castration-resistant prostate cancer, where the emergence of AR splice variants leads to treatment-resistant disease. Through detailed molecular studies of the marine alkaloid manzamine A (MA), we identified transcription factor E2F8 as a previously unknown regulator of AR transcription that prevents AR synthesis in prostate cancer cells. MA significantly inhibited the growth of various prostate cancer cell lines and was highly effective in inhibiting xenograft tumor growth in mice without any pathophysiological perturbations in major organs. MA suppressed the full-length AR (AR-FL), its spliced variant AR-V7, and the AR-regulated prostate-specific antigen (PSA; also known as KLK3) and human kallikrein 2 (hK2; also known as KLK2) genes. RNA sequencing (RNA-seq) analysis and protein modeling studies revealed E2F8 interactions with DNA as a potential novel target of MA, suppressing AR transcription and its synthesis. This novel mechanism of blocking AR biogenesis via E2F8 may provide an opportunity to control therapy-resistant prostate cancer over the currently used AR antagonists designed to target different parts of the AR gene.

N-acyl homoserine lactone-producing Pseudomonas putida strain T2-2 from human tongue surface.

Bacterial cell-to-cell communication (quorum sensing) refers to the regulation of bacterial gene expression in response to changes in microbial population density. Quorum sensing bacteria produce, release and respond to chemical signal molecules called autoinducers. Bacteria use two types of autoinducers, namely autoinducer-1 (AI-1) and autoinducer-2 (AI-2) where the former are N-acylhomoserine lactones and the latter is a product of the luxS gene. Most of the reported literatures show that the majority of oral bacteria use AI-2 for quorum sensing but rarely the AI-1 system. Here we report the isolation of Pseudomonas putida strain T2-2 from the oral cavity. Using high resolution mass spectrometry, it is shown that this isolate produced N-octanoylhomoserine lactone (C8-HSL) and N-dodecanoylhomoserine lactone (C12-HSL) molecules. This is the first report of the finding of quorum sensing of P. putida strain T2-2 isolated from the human tongue surface and their quorum sensing molecules were identified.

In-Silico Approaches for the Screening and Discovery of Broad-Spectrum Marine Natural Product Antiviral Agents Against Coronaviruses.

The urgent need for SARS-CoV-2 controls has led to a reassessment of approaches to identify and develop natural product inhibitors of zoonotic, highly virulent, and rapidly emerging viruses. There are yet no clinically approved broad-spectrum antivirals available for beta-coronaviruses. Discovery pipelines for pan-virus medications against a broad range of betacoronaviruses are therefore a priority. A variety of marine natural product (MNP) small molecules have shown inhibitory activity against viral species. Access to large data caches of small molecule structural information is vital to finding new pharmaceuticals. Increasingly, molecular docking simulations are being used to narrow the space of possibilities and generate drug leads. Combining in-silico methods, augmented by metaheuristic optimization and machine learning (ML) allows the generation of hits from within a virtual MNP library to narrow screens for novel targets against coronaviruses. In this review article, we explore current insights and techniques that can be leveraged to generate broad-spectrum antivirals against betacoronaviruses using in-silico optimization and ML. ML approaches are capable of simultaneously evaluating different features for predicting inhibitory activity. Many also provide a semi-quantitative measure of feature relevance and can guide in selecting a subset of features relevant for inhibition of SARS-CoV-2.

Characterization of quorum sensing and quorum quenching soil bacteria isolated from Malaysian tropical montane forest.

We report the production and degradation of quorum sensing N-acyl-homoserine lactones by bacteria isolated from Malaysian montane forest soil. Phylogenetic analysis indicated that these isolates clustered closely to the genera of Arthrobacter, Bacillus and Pseudomonas. Quorum quenching activity was detected in six isolates of these three genera by using a series of bioassays and rapid resolution liquid chromatography analysis. Biosensor screening and high resolution liquid chromatography-mass spectrometry analysis revealed the production of N-dodecanoyl-L-homoserine lactone (C12-HSL) by Pseudomonas frederiksbergensis (isolate BT9). In addition to degradation of a wide range of N-acyl-homoserine lactones, Arthrobacter and Pseudomonas spp. also degraded p-coumaroyl-homoserine lactone. To the best of our knowledge, this is the first documentation of Arthrobacter and Pseudomonas spp. capable of degrading p-coumaroyl-homoserine lactone and the production of C12-HSL by P. frederiksbergensis.

Coexistence of quorum-quenching and quorum-sensing in tropical marine Pseudomonas aeruginosa strain MW3A.

A chemically defined medium called KGm medium was used to isolate from a sample of sea water a bacterial strain, MW3A, capable of using N-3-oxohexanoyl-L: -homoserine lactone as the sole carbon source. MW3A was clustered closely to Pseudomonas aeruginosa by 16S ribosomal DNA sequence analysis. It degraded both N-acylhomoserine lactones (AHLs) with a 3-oxo group substitution and, less preferably, AHLs with unsubstituted groups at C3 position in the acyl side chain, as determined by Rapid Resolution Liquid Chromatography. Its quiP and pvdQ homologue gene sequences showed high similarities to those of known acylases. Spent supernatant of MW3A harvested at 8-h post inoculation was shown to contain long-chain AHLs when assayed with the biosensor Escherichia coli [pSB1075], and specifically N-dodecanoyl-L: -homoserine lactone and N-3-oxotetradecanoyl-L: -homoserine lactone by high resolution mass spectrometry. Hence, we report here a novel marine P. aeruginosa strain MW3A possessing both quorum-quenching and quorum-sensing properties.

Glabraquinone A and B, new bisanthraquinones from Prismatomeris glabra (Korth.) Valeton.

Two new bisanthraquinones, glabraquinone A and B (1-2) were isolated from the root of Prismatomeris glabra (Korth.) Valeton. In addition to the new glabraquinones, six known anthraquinones, that is, 1-hydroxy-2-methoxy-6-methylanthraquinone (3), 1,2-dimethoxy-7-methylanthraquinone (4), lucidin (5), nordamnacanthal (6), damnacanthal (7) and 2-carboxaldehyde-3-hydroxyanthraquinone (8)) and an aromatic compound, that is, catechol diethyl ether (9) were isolated and characterized in this study. Compounds 1, 4 and 9 showed mild activity, reducing N2A cell viability to 77%, 82% and 77%, respectively, in anti-neuroblastoma assay.

Facile Solid-Phase Synthesis of Well-Defined Defect Lysine Dendrimers.

An efficient solid-phase method has been reported to prepare well-defined lysine defect dendrimers. Using orthogonally protected lysine residues, pure G2 to G4 lysine defect dendrimers were prepared with 48-95% yields within 13 h. Remarkably, high-purity products were collected via precipitation without further purification steps. This method was applied to prepare a pair of 4-carboxyphenylboronic acid-decorated defect dendrimers (16 and 17), which possessed the same number of boronic acids. The binding affinity of 16, in which the ε-amines of G1 lysine are fractured, for glucose and sorbitol was 4 times that of 17. This investigation indicated the role of allocation and distribution of peripheries for the dendrimer's properties and activity.