Discovery of Anti-SARS-CoV-2 Nsp9 Binders from Natural Products by a Native Mass Spectrometry Approach.

The search for effective antiviral agents against SARS-CoV-2 remains a critical global endeavor. In this study, we focused on the viral nucleocapsid protein Nsp9, which is a key player in viral RNA replication and an attractive drug target. Employing a two-pronged approach, an in-house natural product library was screened using native mass spectrometry to identify compounds capable of binding to Nsp9. From the initial screening, apart from the previously reported hit oridonin (protein binding ratio of 0.56 in the initial screening, Kd = 7.2 ± 1.0 µM), we have identified a second Nsp9-interacting compound, the diterpenoid ryanodine, with a protein binding ratio of 0.3 and a Kd of 48.05 ± 5.03 µM. To gain deeper insights into the binding interactions and to explore potential structural requirements, the collision-induced affinity selection mass spectrometry (CIAS-MS) approach allowed us to identify six known oridonin analogues produced by the plant Rabdosia rubescens, each with varying affinities to Nsp9. Native MS validation of their individual binding activities to Nsp9 revealed that all analogues exhibited reduced affinity compared to oridonin. Structural-activity relationship analysis highlighted key functional groups, including 1-OH, 6-OH, 7-OH, and the enone moiety, which are crucial for Nsp9 binding. Combined data from our native mass spectrometry and CIAS-MS approaches provide valuable insights into the molecular interactions between Nsp9 and these compounds.

Collision-Induced Affinity Selection Mass Spectrometry for Identification of Ligands.

Hyphenated mass spectrometry has been used to identify ligands binding to proteins. It involves mixing protein and compounds, separation of protein-ligand complexes from unbound compounds, dissociation of the protein-ligand complex, separation to remove protein, and injection of the supernatant into a mass spectrometer to observe the ligand. Here we report collision-induced affinity selection mass spectrometry (CIAS-MS), which allows separation and dissociation inside the instrument. The quadrupole was used to select the ligand-protein complex and allow unbound molecules to be exhausted to vacuum. Collision-induced dissociation (CID) dissociated the protein-ligand complex, and the ion guide and resonance frequency were used to selectively detect the ligand. A known SARS-CoV-2 Nsp9 ligand, oridonin, was successfully detected when it was mixed with Nsp9. We provide proof-of-concept data that the CIAS-MS method can be used to identify binding ligands for any purified protein.

Discovery of a Natural Product That Binds to the Mycobacterium tuberculosis Protein Rv1466 Using Native Mass Spectrometry.

Elucidation of the mechanism of action of compounds with cellular bioactivity is important for progressing compounds into future drug development. In recent years, phenotype-based drug discovery has become the dominant approach to drug discovery over target-based drug discovery, which relies on the knowledge of a specific drug target of a disease. Still, when targeting an infectious disease via a high throughput phenotypic assay it is highly advantageous to identifying the compound's cellular activity. A fraction derived from the plant Polyalthia sp. showed activity against Mycobacterium tuberculosis at 62.5 µge/µL. A known compound, altholactone, was identified from this fraction that showed activity towards M. tuberculosis at an minimum inhibitory concentration (MIC) of 64 µM. Retrospective analysis of a target-based screen against a TB proteome panel using native mass spectrometry established that the active fraction was bound to the mycobacterial protein Rv1466 with an estimated pseudo-Kd of 42.0 ± 6.1 µM. Our findings established Rv1466 as the potential molecular target of altholactone, which is responsible for the observed in vivo toxicity towards M. tuberculosis.

A Phenotarget Approach for Identifying an Alkaloid Interacting with the Tuberculosis Protein Rv1466.

In recent years, there has been a revival of interest in phenotypic-based drug discovery (PDD) due to target-based drug discovery (TDD) falling below expectations. Both PDD and TDD have their unique advantages and should be used as complementary methods in drug discovery. The PhenoTarget approach combines the strengths of the PDD and TDD approaches. Phenotypic screening is conducted initially to detect cellular active components and the hits are then screened against a panel of putative targets. This PhenoTarget protocol can be equally applied to pure compound libraries as well as natural product fractions. Here we described the use of the PhenoTarget approach to identify an anti-tuberculosis lead compound. Fractions from Polycarpa aurata were identified with activity against Mycobacterium tuberculosis H37Rv. Native magnetic resonance mass spectrometry (MRMS) against a panel of 37 proteins from Mycobacterium proteomes showed that a fraction from a 95% ethanol re-extraction specifically formed a protein-ligand complex with Rv1466, a putative uncharacterized Mycobacterium tuberculosis protein. The natural product responsible was isolated and characterized to be polycarpine. The molecular weight of the ligand bound to Rv1466, 233 Da, was half the molecular weight of polycarpine less one proton, indicating that polycarpine formed a covalent bond with Rv1466.

Development of an HPLC-based guanosine monophosphate kinase assay and application to Plasmodium vivax guanylate kinase.

The development of a high-performance liquid chromatography (HPLC)-based method, for guanosine monophosphate kinase activity assays, is presented. The method uses the intrinsic UV absorption (at 260 nm) of substrates and products of the enzymatic reaction (GMP, ATP, ADP and GDP) to unambiguously determine percent conversion of substrate into product. It uses a commercially available C18 column which can separate reaction samples by elution under isocratic conditions in 12 min per run. The kinetics of the forward reaction catalyzed by Plasmodium vivax guanylate kinase (PvGK), a potential drug target against malaria, was determined. The relative concentrations of the two substrates (GMP and ATP) have a distinct effect on reaction velocity. Kinetic analyses showed the PvGK-catalyzed reaction to be associated with atypical kinetics, where substrate inhibition kinetics and non-Michaelis-Menten (sigmoidal) kinetics were found with respect to GMP and ATP, respectively. Additionally, the method was used in inhibition assays to screen twenty fragment-like compounds. The assays were robust and reproducible, with a signal window of 3.8 and a Z' factor of 0.6. For the best inhibitor, an IC50 curve was generated.

Fragment-Based Screening of a Natural Product Library against 62 Potential Malaria Drug Targets Employing Native Mass Spectrometry.

Natural products are well known for their biological relevance, high degree of three-dimensionality, and access to areas of largely unexplored chemical space. To shape our understanding of the interaction between natural products and protein targets in the postgenomic era, we have used native mass spectrometry to investigate 62 potential protein targets for malaria using a natural-product-based fragment library. We reveal here 96 low-molecular-weight natural products identified as binding partners of 32 of the putative malarial targets. Seventy-nine (79) fragments have direct growth inhibition on Plasmodium falciparum at concentrations that are promising for the development of fragment hits against these protein targets. This adds a fragment library to the published HTS active libraries in the public domain.