Development of a protease production platform for structure-based drug design

Area of Science:

• Biochemistry
• Structural Biology
• Drug Discovery

Background:

• Structure-based drug design (SBDD) relies on detailed knowledge of target proteins, particularly proteases.
• Developing potent and selective protease inhibitors requires a consistent supply of high-quality protein for assays and structural studies.
• Traditional protein production methods often present bottlenecks, hindering rapid drug discovery efforts.

Purpose of the Study:

• To redesign and optimize the protein production pipeline for proteases.
• To establish a rapid and reliable method for generating diverse human and viral proteases.
• To support structure-based drug design programs with a continuous supply of purified proteins.

Main Methods:

• Designed a multi-system expression vector for both eukaryotic and prokaryotic hosts.
• Implemented high-throughput cloning, expression, and purification strategies.
• Utilized salvage screens for detergent extraction and refolding, coupled with classical biophysical techniques (light-scattering, analytical ultracentrifugation).

Main Results:

• Successfully expressed and purified all mechanistic classes of active human and viral proteases.
• Established a rapid protein production route, overcoming traditional bottlenecks.
• Ensured high protein quality for enzymatic assays and crystallization studies.

Conclusions:

• The developed process-orientated approach significantly accelerates protease production for SBDD.
• This strategy enables a consistent supply of diverse proteases, crucial for developing selective inhibitors.
• The method integrates high-throughput techniques with classical biophysics to ensure protein quality and facilitate drug discovery.