How to choose appropriate protease inhibitors in clinical trials?

Even though proteases are important for cellular function, there are times when you don’t want to see them. For example, when purifying proteins, you don’t want proteases to devour your target protein, especially low abundance proteins. Nowadays, there are various protease inhibitors available on the market to help you stop protease activity when necessary.
In order to deal with various proteases present in cell and tissue samples, the selection of inhibitors is also constantly increasing. At present, there are four main types of proteases, named after the amino acids in their active sites: serine protease, cysteine protease, aspartate protease, and metalloproteinase. Protease inhibitors can target a specific protease or be broad-spectrum.
People usually classify protease inhibitors based on the type of protease they act on and whether they are reversible or irreversible. How to choose and use it? This seems to be a difficult problem, just looking at the name can already make people dizzy. Now let’s listen to the advice of professionals.
Starting with cocktails
The selection of protease inhibitors depends on the personalized experiments of each user, but for the application of cell lysis or homogenate tissue, inhibitor mixtures (or cocktails) are usually a safer choice to block different proteases. The best-selling inhibitors of Abexa are two mixtures: Protein Inhibitor Cocktail Multipurpose and Protein, Phosphatase & PMSF； Both of these apply to the cracking solution. “They are universally applicable and suitable for many samples, whether fresh or FFPE tissues, or different species,” said Sabrina Calabresi, Abbexa’s manager.
If researchers want to preserve the target protein while maintaining cell health, Chandra Mohan, senior manager of Merck Millipore, also suggests using inhibitor mixtures. She suggests choosing inhibitors targeting four types of proteases, such as Merck’s Protein Inhibitor Cocktail III. It can inhibit aspartic acid, cysteine, serine proteases, and aminopeptidases in mammalian cells and tissues.
Sometimes, less specific inhibitors seem to be a better choice. “When lysing cells or preparing tissue homogenates, it is best to use inhibitors with broad-spectrum specificity,” Mohan said. “They should react quickly, bind irreversibly with proteases, and be easily removed after protein purification.”
Nevertheless, the difficulty of choice still revolves around you. Abbexa believes that trying different types is the responsibility of the supplier, not the researcher. This company suggests that you communicate with suppliers so that they can use their professional knowledge to help you choose. Usually, Abbexa recommends the best inhibitor only after understanding the specific analysis, sample type, and application of the researchers.
Consider the mechanism of action
For certain types of experiments, you may need to search for inhibitors on a smaller scale. According to Mohan’s suggestion, you need to first determine which type of protease is being blocked, and then choose the appropriate inhibitor. Because different inhibitors have different mechanisms of action.
“Protease inhibitors can be reversible or irreversible, as they prevent substrates from approaching active sites through steric hindrance,” Mohan said. Other inhibitors may exert their effects by modifying amino acids at the active site of the protease. “For example, PMSF can inactivate serine proteases, which react with active serine, where chloride+ketone derivatives react with catalytic triplets at the active site,” Mohan said.
First broad, then specific
Nowadays, inhibitors are used in various applications, which is also a reason why people need to choose carefully. “The downstream processing and analysis will determine the selection and use of protease inhibitors,” said Robert Gates, Marketing Manager at Sigma Aldrich. For example, he added, “Liquid phase proteomic analysis (such as serum) may require immediate inhibition of proteases as they hydrolyze low abundance target proteins.”
In fact, there are currently so many types of inhibitors that it is really difficult to categorize them. When choosing protease inhibitors, Gates suggests starting from a broad range and then continuously narrowing down the range. “The first step may be to try pre configured cocktails, such as Sigma Aldrich’s P8340, designed specifically for mammalian tissues,” he said. “If protein hydrolysis still exists, methods targeting specific tissues and proteins can be adopted.”
Whether we can use fewer inhibitors to inhibit more proteases remains to be seen. However, among so many choices, there is no doubt that you can find an effective protease inhibitor.