Molecules interacting on a microchip with data streams, symbolizing high throughput screening and the elimination of false signals in drug discovery.

Drug Discovery Breakthrough: New High-Throughput Screening Method Eliminates False Results

Mira Elwood in Science & Nature December 2025 • 5 min read.

"Revolutionary LC-MS based technique identifies a novel carbonic anhydrase inhibitor, paving the way for faster and more reliable drug development."

In the high-stakes world of drug discovery, speed and accuracy are paramount. High-throughput screening (HTS) is the cornerstone of early-stage drug development, allowing researchers to sift through vast libraries of compounds to identify potential drug candidates. However, traditional HTS methods are plagued by inaccuracies, generating both false positives (identifying inactive compounds as active) and false negatives (missing potentially effective compounds). These errors lead to wasted resources, delayed progress, and missed opportunities for treating diseases.

Current HTS assays often rely on methods like fluorescence, chemiluminescence, or surface plasmon resonance, which require modifications to either the target protein or the compounds being screened. These modifications can interfere with the natural binding process, leading to unreliable results. While label-free techniques like nuclear magnetic resonance (NMR) exist, they often require large amounts of protein and are limited by low throughput, making them impractical for screening large compound libraries.

Now, a groundbreaking study published in Scientific Reports presents a novel liquid chromatography-mass spectrometry (LC-MS) based HTS method that addresses these challenges head-on. This innovative approach not only minimizes false positives and negatives but also boasts high throughput and requires only small amounts of protein, offering a significant leap forward in drug discovery. The research team, led by Kasun P. Imaduwage and Heather Desaire, has validated this method and even identified a new inhibitor for carbonic anhydrase, an enzyme with therapeutic potential for a range of diseases.

How This New Screening Method Works: Accuracy and Efficiency Combined

Molecules interacting on a microchip with data streams, symbolizing high throughput screening and the elimination of false signals in drug discovery.

The key to this method lies in its unique approach to detecting binding events. Instead of directly detecting the binding of a test compound to the target protein, the researchers introduce a known "reporter molecule" – a weak binder that is easily detectable by mass spectrometry. The target protein is first incubated with this reporter molecule.

Next, the protein-reporter complex is exposed to a batch of library compounds. If a compound in the library binds more strongly to the target protein than the reporter molecule, it displaces the reporter. This displacement increases the concentration of the reporter molecule in the surrounding solution, which is then easily detected by LC-MS. This indirect detection method offers several key advantages:

Eliminating False Positives: Only compounds that truly bind to the target protein and displace the reporter molecule are identified as hits. Non-specific binding is avoided because these compounds don't effectively compete with the reporter.
Mitigating False Negatives: Because the reporter molecule is easily detectable, the method doesn't rely on the test compounds themselves being readily ionized. This is crucial because many potentially active compounds are missed by traditional MS-based assays due to their poor ionization efficiency.
High Throughput and Low Protein Consumption: The method is fast, with each batch of compounds screened in just 10 minutes. Moreover, it requires only nanograms of protein per compound, making it ideal for targets that are difficult to produce in large quantities.

To validate their method, the researchers tested it on three well-characterized proteins: pepsin, maltose-binding protein (MBP), and carbonic anhydrase (CA). In each case, they were able to successfully identify known binders in the presence of hundreds of non-binding compounds, demonstrating the method's accuracy and reliability.

A New Inhibitor Discovered: Pifithrin-µ Targets Carbonic Anhydrase

In a compelling demonstration of the method's power, the researchers screened a library of 1200 compounds against carbonic anhydrase and identified a novel inhibitor, pifithrin-µ. This compound had previously gone undetected by other MS-based assays due to its poor ionization efficiency.

Further testing confirmed that pifithrin-µ effectively inhibits carbonic anhydrase activity, with an IC50 of approximately 25 nM. This discovery highlights the ability of the new method to identify previously overlooked drug candidates, expanding the possibilities for treating a range of diseases, including cancer and glaucoma, where CA inhibitors have shown promise.

This new LC-MS based HTS method represents a significant advancement in drug discovery. By minimizing false positives and negatives, increasing throughput, and reducing protein consumption, it offers a more efficient and reliable way to identify potential drug candidates, ultimately accelerating the development of new therapies for a wide range of diseases. The discovery of pifithrin-µ as a carbonic anhydrase inhibitor further underscores the method's potential to unlock new avenues for drug development.

About this Article -

This article was crafted using a human-AI hybrid and collaborative approach. AI assisted our team with initial drafting, research insights, identifying key questions, and image generation. Our human editors guided topic selection, defined the angle, structured the content, ensured factual accuracy and relevance, refined the tone, and conducted thorough editing to deliver helpful, high-quality information.See our About page for more information.

This article is based on research published under:

DOI-LINK: 10.1038/s41598-017-08602-w, Alternate LINK

Title: Rapid Lc-Ms Based High-Throughput Screening Method, Affording No False Positives Or False Negatives, Identifies A New Inhibitor For Carbonic Anhydrase

Subject: Multidisciplinary

Journal: Scientific Reports

Publisher: Springer Science and Business Media LLC

Authors: Kasun P. Imaduwage, Jude Lakbub, Eden P. Go, Heather Desaire

Published: 2017-09-04

Everything You Need To Know

What is high-throughput screening (HTS), and why are traditional methods often inaccurate?

High-throughput screening (HTS) is a method used in early-stage drug development to quickly test a large number of compounds for their potential to interact with a specific target protein. Traditional HTS methods often rely on techniques like fluorescence or chemiluminescence, but these can produce inaccurate results because they may interfere with the natural binding process. This can lead to false positives (identifying inactive compounds as active) and false negatives (missing potentially effective compounds), wasting time and resources.

How does the new LC-MS based HTS method work to improve drug discovery?

The new liquid chromatography-mass spectrometry (LC-MS) based HTS method uses a 'reporter molecule'—a weak binder to the target protein that's easily detectable by mass spectrometry. The target protein is incubated with this reporter molecule, and then exposed to library compounds. If a compound binds more strongly than the reporter, it displaces the reporter, increasing the reporter's concentration which is then detected by LC-MS. This indicates that the compound is a potential drug candidate. The advantage of this approach is that this new approach minimizes false positives and negatives, provides high throughput, and requires only small amounts of protein.

How does this new LC-MS method minimize false positives in drug screening?

This new LC-MS method minimizes false positives by identifying only those compounds that truly bind to the target protein and displace the reporter molecule. Compounds that bind non-specifically don't compete effectively with the reporter and are therefore not identified as hits. This reduces the chance of pursuing inactive compounds.

How does the LC-MS method mitigate false negatives, ensuring potentially effective compounds aren't missed?

This LC-MS method mitigates false negatives because it relies on detecting the reporter molecule, which is easily detectable, rather than directly detecting the test compounds themselves. This is important because many potentially active compounds might be missed by traditional mass spectrometry (MS)-based assays due to their poor ionization efficiency, meaning they don't readily form ions for detection. By focusing on the reporter, the method can identify compounds that might otherwise be overlooked.

What was the result of screening compounds against carbonic anhydrase?

The researchers screened a library of 1200 compounds against carbonic anhydrase (CA) and identified pifithrin-µ as a novel inhibitor. Pifithrin-µ had previously gone undetected by other MS-based assays because of its poor ionization efficiency. Carbonic anhydrase is an enzyme with therapeutic potential for treating a range of diseases, including cancer and glaucoma, so the discovery of a new inhibitor is promising for future drug development efforts targeting these conditions. The discovery of pifithrin-µ as a carbonic anhydrase inhibitor highlights the method's ability to find previously missed compounds.