High-throughput fragment screening by affinity LC-MS.

Area of Science:

• Biochemistry
• Analytical Chemistry
• Drug Discovery

Background:

• Fragment screening is crucial for identifying novel drug leads.
• Established techniques like NMR and SPR have limitations in throughput and cost.
• Vemurafenib, a cancer drug, highlights the success of fragment-based drug discovery.

Purpose of the Study:

• To introduce and validate weak affinity LC/MS as a high-throughput fragment screening method.
• To assess the efficiency and effectiveness of LC/MS for screening fragment libraries against a specific target.

Main Methods:

• Utilized affinity-based capillary columns with immobilized thrombin for screening.
• Screened a library of 590 compounds divided into 11 mixtures using MS detection.
• Performed primary screening in under 4 hours, achieving a throughput of over 3500 fragments per day.
• Conducted secondary screening with an active site-blocked thrombin column to confirm specificity.

Main Results:

• Identified 30 initial 'hits' from the primary screen.
• One compound demonstrated selective binding to thrombin with a dissociation constant (Kd) in the 0.1 mM range.
• Achieved high throughput screening (>3500 fragments/day) with minimal target and fragment consumption.

Conclusions:

• Weak affinity LC/MS is a high-throughput, user-friendly, and resource-efficient method for fragment screening.
• This technique shows significant promise as a valuable tool in drug discovery pipelines.
• The study validates LC/MS as a viable alternative to traditional fragment screening methods.