Case story: CETSA for Lead Generation

Advancing Drug Discovery Against a Challenging Membrane-Associated Target

Targeting membrane-associated proteins with small molecules remains a significant hurdle in early-stage drug discovery. These proteins often require their native cellular context to maintain proper folding, localization, and functional interactions. Standard biochemical and biophysical assays cannot typically replicate these conditions, making it difficult to detect meaningful target engagement.

A client developing positive allosteric modulators of a membrane-associated enzyme linked to Alzheimer’s disease, encountered this challenge. Despite using multiple assay platforms, no evidence of in-cell engagement was observed. The client partnered with Pelago Bioscience to evaluate whether CETSA® could offer the missing mechanistic confirmation.

Two key translational questions needed to be addressed: Could CETSA demonstrate target engagement in intact cells, where other assays had failed? Could CETSA identify novel chemical matter suitable for further lead optimization? Given that target activity is dependent on native membrane localization and protein interactions, an assay system preserving the complete cellular architecture was essential.

The Solution

A CETSA assay was developed and applied directly in live, intact cells to meet these challenges. The process involved the following stages:

1. Intact-Cell Library Screening

CETSA was used to screen the client’s compound library in living cells, preserving the physiological environment necessary.

2. Hit Confirmation and Potency Determination

Initial hits were reconfirmed through independent CETSA runs and tested in full concentration–response curves to determine binding strength and specificity.

3. Comparative Assay Analysis

Hits were compared across three assay formats: CETSA in intact cells, a cell-based functional assay, and a biochemical assay using recombinant protein.

Translational Outcome

The CETSA-based approach produced several high-confidence leads. The results showed: A strong correlation between CETSA signals and outcomes from the cell-based functional assay. No correlation between CETSA data and the biochemical assay using recombinant protein. These findings demonstrated that capturing engagement required the integrity of the full cellular context. CETSA provided direct and actionable evidence where traditional methods were inadequate.

STRATEGIC INSIGHTS: This study highlights the unique value of CETSA in enabling drug discovery against complex, membrane-bound targets. By operating in a native cellular environment, CETSA identified novel lead compounds and validated their on-target activity. The client could proceed confidently, expand screening efforts to larger libraries, and reorient their lead generation strategy based on reliable, context-aware data.

"Pelago Bioscience offers exceptional technical expertise in all aspects of target deconvolution using CETSA – including study design, sample preparation, LC-MS data acquisition, and statistical processing. Our collaboration always proceeded effectively due to Pelago Bioscience's responsiveness, scientific commitment, and dedication to clients resulting in delivery of impactful scientific results."

Benjamin Fontaine, Senior Scientist

LifeMine Therapeutics

“As we approached late preclinical development, our lead compound demonstrated strong efficacy and a favorable toxicity profile.Partnering with Pelago Bioscience, we leveraged their expertise in CETSA technology to successfully identify and validate the protein target. With the comprehensive data package they provided, we met investor requirements and gained the support needed to move forward. The Pelago team´s swift and precise response was invaluable throughout the process, and their scientific excellence made a real difference. We’re highly encouraged by the results and grateful for their outstanding collaboration.”

Martin Bonde, CEO

Inthera Bioscience AG

"At AstraZeneca, we are applying CETSA in hit identification and lead optimization to identify and characterize compounds for a number of novel targets."

Thomas Lundbäck, Senior Director

AstraZeneca

"Our small molecule drug discovery project was seeking a robust assay allowing to screen against the target of interest expressed in its natural cellular environment. Pelago Bioscience's expertise in drug discovery projects, specifically in the CETSA platform and high throughput screens was the key to achieving our goals. We found Pelago's team to be professional, knowledgeable, flexible and dedicated. We highly recommend Pelago Bioscience for drug discovery projects."

Po-Shun Lee, Chief Medical Officer

Generian

"CETSA data is a keystone in our SAR analysis, and by assessing cellular target engagement of our compounds across multiple cell lines we gain confidence in our project prioritizations."

Peter Joyce, CEO and Co-Founder

Grey Wolf Therapeutics

"While ligand binding assays are commonly used for PK/PD studies in clinical trials of biologics and endogenous protein quantification, Lipum AB sought to establish an orthogonal quantitative LC-MS/MS method for our target protein, bile salt-stimulated lipase (BSSL). We chose Pelago Bioscience as our partner due to their rapid project initiation, collaborative approach, and extensive expertise in processing complex biological samples using high-resolution mass spectrometry. Working with one of the best proteomics labs in the Nordics, Pelago's team delivered high-quality results that aligned perfectly with our clinical research goals."

Ola Sandborgh, CEO

Lipum AB

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