An Integrated Strategy for the Detection, Dereplication, and Identification of DNA-Binding Biomolecules from Complex Natural Product Mixtures

Abstract

A fundamental factor in natural product drug discovery programs is the necessity to identify the active component(s) from complex chemical mixtures. Whereas this has traditionally been accomplished using bioassay-guided fractionation, we questioned whether alternative techniques could supplement and, in some cases, even supplant this approach. We speculated that a combination of ligand-fishing methods and modern analytical tools (e.g., LC-MS and online natural product databases) offered a route to enhance natural product drug discovery. Herein, a candidate solution referred to as the lickety-split ligand-affinity-based molecular angling system (LLAMAS) is described. This approach utilizes an ultrafiltration-based LC-PDA-MS/MS-guided DNAbinding assay in combination with the (i) Global Natural Products Social Molecular Networking, (ii) Dictionary of Natural Products, and (iii) SciFinder platforms to identify DNA binders in complex chemical mixtures. LLAMAS was initially vetted in tests using known small-molecule DNA binders and then optimized to a 96-well plate-based format. A set of 332 plant samples used in traditional Chinese medicine was screened for DNA-binding activity with LLAMAS, resulting in the identification of seven DNAbinding molecules, including berberine (12), palmatine (13), coptisine (14), fangchinoline (15), tetrandrine (16), daurisoline (17), and dauricine (18). These results demonstrate that LLAMAS is an effective natural product discovery platform for the efficient identification and dereplication of DNA-binding molecules from complex mixtures.N atural products are an important source of therapeutic drug leads due to the incredible diversity of their structures and bioactivities. 1−3 Many of the success stories to emerge from natural product drug development are legendary: the discovery of the antibiotic penicillin in 1928, 4 the identification and subsequent clinical approval of the anticancer drug Taxol (paclitaxel) in 1992, 5 the development of the antimalarial drug artemisinin (a discovery that was acknowledged by the 2015 Nobel Prize in Physiology or Medicine), 6 and more. 7 These milestones in drug discovery established natural products as an unparalleled resource for identifying therapeutically useful compounds to combat a wide range of diseases.Strikingly, a majority of these and other iconic natural products that are used as medicines 1,8,9 were discovered using the well-established, yet powerful technique known as bioassay-guided purification. 10,11 This approach relies on subjecting mixtures of compounds (e.g., extracts and fractions) to iterative steps of fractionation and biological testing with the

Altertoxin II, a Highly Effective and Specific Compound against Ewing Sarcoma

“…Based on our observations that ATXII induces DNA damage and activates DNA damage response pathways, we sought to determine if these effects were the result of the direct binding of ATXII to DNA. We recently developed a technique for identifying DNA-binding molecules in complex mixtures called lickety-split ligand-affinity-based molecular angling system (LLAMAS) [ 48 ]. Using this assay, no binding of ATXII to purified DNA was observed, suggesting that ATXII-induced DNA damage is not due to direct DNA binding ( Figure 6 A,B).…”

Ligand fishing as a tool to screen natural products with anticancer potential

“…The authors argued that this method has easy operation and fast response with few parameters to adjust [18]. Ma et al [12] applied UF to identify DNA-binding substances from several herbal extracts. After evaluating different membranes and conditions, they chose a modified poly(ether sulfone) membrane that was able to retain the target and operated under a variety of eluting solvents.…”

“…Based on our survey, eight works used DNA strands (double, triple, and quadruple) as smart baits to fish out ligands from natural products with anticancer potential. Different approaches were used to apply DNA strands into ligand fishing settings, including dialysis [186], UF [12], MPs [187], and microfluid chips [188,189] as well as other less trivial supports such as streptavidin-coated 96-well plates [49,50] and agarose beads [47]. The DNA-bound ligands were tested in preclinical settings in two specific reports.…”

“…To date, a large number of ligand fishing techniques have been developed with various protocols and efficiently optimized to identify ligands from plant, animal, or bacterial extracts. Combined with highly sensitive high-resolution analytical methods (e.g., LC-HRMS and NMR), ligand fishing allows for the rapid identification and purification of fished ligands, accelerating the pharmacological evaluation and characterization of bioactive natural products [11][12][13].…”

Exploring Multifunctional Bioactive Components from Podophyllum sinense Using Multi-Target Ultrafiltration

“…Affinity ultrafiltration can facilitate the rapid separation of small molecule ligands bound with large molecular receptors (drug targets) from unbound molecules, and LC–MS can enable the quick identification of potential bioactive ligands after they are released from the targets ( Qin et al, 2015 ). In this regard, the combination of the two techniques is not only vital to reveal the effective phytochemicals of natural products, such as medicinal plants, but also conducive to drug discovery ( Qin et al, 2015 ; Ma et al, 2020 ). To date, UF–LC/MS technology has been successfully applied to screen bioactive ingredients in natural products by employing various disease-related drug targets ( Mulabagal and Calderón, 2010 ; Lavecchia et al, 2013 ; Li et al, 2015 ; Chen et al, 2020 ; Zhang et al, 2021 ).…”