Miaomiao Liu

Mudjala bark, prepared from Barringtonia acutangula, is an Australian Aboriginal medicine traditionally used for pain relief, and previous studies have associated its activity with triterpenoid-rich fractions. However, the low-molecular-weight triterpenoids, which were major chromatographic constituents in these fractions, remained chemically unresolved. In this study, we define the aglycone-rich triterpenoid profile of Australian Mudjala bark and resolve its dominant inseparable aglycone mixture. The two major chromatographic isomeric acidic triterpenes could not be separated directly by reversed-phase HPLC, but isolation and hydrolysis of their corresponding C-28 glucosides enabled their assignment as bartogenic acid (1) and trachelosperogenin A (2). Overall, thirteen triterpenoids were assigned, including one new acylated barringtogenol C-type aglycone (5) and twelve known compounds (1–4, 6–13). The profile comprises three main chemical domains: 24,28-dioic acid aglycones and C-28 glucosides, acylated barringtogenol C-type oleananes, and common pentacyclic triterpene acids. These findings establish a compound-level chemical framework for the previously bioactive low-molecular-weight Mudjala fraction and support future quantitative and biological investigation.

Identification of ligands targeting essential enzymes in Mycobacterium species remains an important strategy for anti-tuberculosis drug discovery. Here, a native mass spectrometry approach was employed using pooled 100-compound mixtures, enabling the direct detection of intact HPPK–ligand complexes in solution. Dual-mode MS acquisitions (low collision energy for complex detection and high collision energy for ligand confirmation), combined with an automated data analysis workflow, ensured robust identification of binding events from these complex samples. This strategy led to the identification of several HPPK-binding small molecules, all belonging to the dammarane triterpene glycoside (ginsenoside) class. Subsequent analysis of the hits revealed clear structure–affinity relationships, highlighting how specific aglycone modifications and glycosylation patterns influence binding to HPPK. Our findings expand the known chemical space of HPPK ligands and demonstrate the utility of native MS-based screening coupled with automated data analysis to uncover new ligand scaffolds for challenging enzyme targets.

Native mass spectrometry (MS) is a powerful technique for studying protein–ligand interactions in their native states by employing soft electrospray ionization (ESI). It enables the direct observation of all species in equilibrium within a solution. Building on these advantages, Collision-Induced Affinity Selection MS (CIAS-MS) was introduced as an alternative approach, incorporating quadrupole mass selection, controlled complex dissociation, and ligand detection to enhance the study of protein–ligand interactions. This method retains the native ionization properties of proteins while enabling high-sensitivity detection of released ligands. This study presents an optimized CIAS-MS platform for improved ligand binding detection and quantitative affinity ranking. The integration of both positive and negative ion modes significantly broadens the detection of structurally diverse ligands, overcoming biases from single-mode analysis. More importantly, the collision-induced dissociation (CID) slope, derived from dissociation curves, is introduced as a robust parameter for affinity ranking. Unlike absolute intensities or dissociation thresholds, the CID slope reflects solution-phase affinity order across ligands and remains accurate in highly complex backgrounds, including 100-compound mixtures and bacterial lysates. These advances highlight the robustness of CIAS-MS for detecting bound ligands and ranking their affinities even under challenging conditions where traditional native MS fails. These findings establish CIAS-MS as a scalable and efficient platform for high-throughput ligand discovery and proteome-wide target engagement studies, offering a powerful addition to the biophysical toolkit for modern drug discovery.

While immunophilins are well-recognized therapeutic targets, several members of this family of peptidyl-proline isomerases (PPIases) have yet to be subjected to ligand discovery efforts. In this study, we demonstrate a cost-effective means to identify ligands to the insufficiently investigated two-domain PPIase human Cyclophilin40 (Cyp40). Central to this effort was the use of beads, wherein a confocal nanoscanning (CONA) approach was used to rapidly probe candidates. Here, we describe how one can adapt the physical nature of microsized beads as a means to strategically reduce cost and ultimately make the discovery of small molecule hit and lead compounds more accessible to everyone irrespective of financial status (democratization).

Native mass spectrometry (MS) is a powerful analytical technique that enables the direct observation of protein-ligand interactions under near-native conditions. In this study, we utilized native MS to investigate the catalytic cycle of Mycobacterium smegmatis leucyl-tRNA synthetase (LeuRS), a key enzyme involved in protein biosynthesis. The leucine-AMP intermediate in complex with LeuRS was successfully detected and shown to occupy the amino acid binding site, preventing further substrate binding and halting the reaction. MS was used to study the intermediate stability and optimize purification protocols, including incubation times and buffer exchange. A binding competition experiment with leucine confirmed the intermediate's role and provided mechanistic insights into the enzyme's catalytic cycle. Additionally, we demonstrated that the intermediate could be eliminated by supplementing the lysate with tRNA, facilitating the completion of the reaction. These findings highlight the potential of native MS for studying enzyme mechanisms in real time, offering valuable insights into enzyme-substrate interactions.

There is an urgent need for novel therapeutics to combat Plasmodium falciparum, especially in light of increasing drug resistance. Here, we present a multiplexed native mass spectrometry (MS) platform capable of simultaneously screening multiple protein targets against chemically diverse crude extracts with minimal sample preparation. A mixture of seven malarial proteins was analyzed under optimized native MS conditions, enabling the detection of specific ligand binding events. Using this platform, lithospermate B from Salvia miltiorrhiza (Danshen) was identified as a novel ligand for a malarial ubiquitin-conjugating enzyme with moderate affinity (Kd = 30.5 ± 2.5 μM). This is the first report linking lithospermate B to a malarial protein target, highlighting the potential of native MS to uncover new bioactivities of known natural products. This approach significantly enhances the throughput of protein–ligand screening and offers a powerful tool for early-stage natural product-based drug discovery.

Live-cell transcription factor (TF) activity reporting is crucial for synthetic biology, drug discovery and functional genomics. Here we present dFLASH (dual FLuorescent transcription factor Activity Sensor for Histone-integrated live-cell reporting), a modular, genome-integrated TF sensor. dFLASH homogeneously and specifically detects endogenous Hypoxia Inducible Factor (HIF) and Progesterone Receptor (PGR) activities, as well as coactivator recruitment to synthetic TFs. The dFLASH system produces dual-color nuclear fluorescence, enabling normalized, dynamic, live-cell TF activity sensing with strong signal-to-noise ratios and robust screening performance (Z’ = 0.61–0.74). We validate dFLASH for functional genomics and drug screening, demonstrating HIF regulation via CRISPRoff and application to whole-genome CRISPR KO screening. Additionally, we apply dFLASH for drug discovery, identifying HIF pathway modulators from a 1600-compound natural product library using high-content imaging. Together, this versatile platform provides a powerful tool for studying TF activity across diverse applications.

Background: The increasing prevalence of drug-resistant tuberculosis (TB) underscores the urgent need for novel antimicrobial agents. Methods: This study integrates cultivation optimization, nuclear magnetic resonance (NMR) fingerprinting, and principal component analysis (PCA) to explore microbial secondary metabolites as potential anti-TB agents. Results: Using the combined approach, 11 bioactive compounds were isolated and identified, all exhibiting anti-Mycobacterium bovis BCG activity. Notable findings include borrelidin, a potent threonyl-tRNA synthetase inhibitor with broad biological activities, and L-O-Lac-L-Val-D-O-Hiv-D-Val, a peptide isolated for the first time from a plant endophyte, demonstrating broad-spectrum antimicrobial activity. Additionally, elaiophylin and polycyclic tetramate macrolactams (PTMs) displayed significant bactericidal effects, with elaiophylin achieving complete BCG inhibition at 72 h and PTMs marking their first reported anti-TB activity. The study also identified bafilomycins as potent scaffolds for anti-TB drug development, showcasing rapid bactericidal activity at low MIC values. Conclusions: These findings emphasize the value of microbial metabolites as a reservoir of bioactive compounds and provide new avenues for developing next-generation anti-TB therapies.

Ferroptosis is an iron-dependent programmed cell death that is characterized by the dysregulation of lipid reactive oxygen species (ROS) production, causing abnormal changes in hypochlorous acid (HClO) levels in lysosomes. Super-resolution imaging can observe the fine structure of the lysosome at the nanometer level; therefore, it can be used to detect lysosome HClO levels during ferroptosis at the suborganelle level. Herein, we utilize a ratiometric fluorescent probe, SRF-HClO, for super-resolution imaging of lysosome HClO. Structured-illumination microscopy (SIM) improves the accuracy of lysosome targeting and enables the probe SRF-HClO to be successfully applied to rapidly monitor the up-regulated lysosome HClO at the nanoscale during inflammation and ferroptosis. Importantly, the probe SRF-HClO can also detect HClO changes in inflammatory and ferroptosis mice and evaluate the inhibitory effect of ferroptosis on mice tumors.