Protease Inhibitors: Ways to Choose Them and Use Them

Abstract

Though proteases—enzymes that cleave proteins—are instrumental in the proper functioning of all living cells, there are times when scientists would prefer to halt their activity. When purifying proteins, for example, the last thing you want are proteases chewing up your target protein (especially if your target is low in abundance to begin with). A range of protease inhibitors is available today to help stop the action of proteases while you purify your target. Researchers also use protease inhibitors in experiments examining cellular functions and even in therapeutic applications. For example, the family of angiotensin-converting enzyme (ACE) inhibitor drugs acts to relax blood vessels and lower blood pressure, and several inhibitors of the HIV-1 protease are used to treat HIV patients.

To address the diverse array of proteases present in cells and tissue samples, the options for inhibitors continue to increase—so it can be hard to know where to start. Basically, there are four types of proteases (named for the amino acids within their active sites): serine proteases, cysteine proteases, aspartic proteases and metalloproteases. Many individual proteases fall under each type, and protease inhibitors can be specific to one protease or broad enough to affect many proteases. Most inhibitors are classified according to the type of protease they act upon (e.g., serine protease inhibitor) and may be reversible or irreversible. To shed more light on finding inhibitors to guard your tissue lysate from proteolysis, here is some advice from protease inhibitor vendors on how best to choose them and use them.

Start with a cocktail

Choice of protease inhibitors depends on each user’s individual experiments, but for applications such as lysing cells or homogenizing tissues—basically, any protocol that involves compromising cell membranes, thereby releasing proteases from lysosomes and other cellular compartments—a cocktail or mixture of inhibitors is often a solid defense to block the range of different proteases present. Abbexa’s most frequently sold inhibitors include two cocktail products, the Protease Inhibitor Cocktail Multipurpose, and the Protease, Phosphatase and PMSF (phenylmethane sulfonyl fluoride) Combined; both are suitable for applications using lysates. “They are more universally applicable and can work for many samples, no matter whether the tissues are fresh or FFPE, or from different species,” says Abbexa’s manager, Sabrina Calabressi.

Chandra Mohan, senior manager of technical writing and documentation development at EMD Millipore, also recommends an inhibitor cocktail for situations in which a researcher wants to preserve a protein of interest while also maintaining cell health. She suggests choosing a cocktail that includes inhibitors for all four protease types, such as EMD Millipore’s Protease Inhibitor Cocktail III. And sometimes it’s appropriate to use a less specific inhibitor rather than a cocktail of many specific ones. “When lysing cells or making tissue homogenates, it is best to use inhibitors with broad specificity,” says Mohan. “They should react rapidly and irreversibly with proteases and [be] easily removed following protein purification.”

To find a protease inhibitor or cocktail that’s right for you, Abbexa maintains that trying many different types is the vendor’s, not the researcher’s, responsibility. The company recommends talking with vendors and taking advantage of their knowledge about the strengths of different inhibitors for different conditions. Abbexa, for example, recommends the best inhibitors for a researcher to use only after learning details of the researcher’s particular assays, sample types and applications.

Consider the mechanism of action

With some types of experiments, it may help to approach inhibitor choice in a more focused manner. Mohan recommends determining what kind of protease you need to block and then choosing an appropriate inhibitor—because different types of inhibitors have different mechanisms of inhibition. “Protease inhibitors may behave as tight-binding reversible or pseudo-irreversible inhibitors, and prevent substrate access to the active site through steric hindrance,” says Mohan. Other inhibitors may exert their effects through modification of an amino acid in the protease’s active site. “For example, the serine proteases are inactivated by PMSF, which reacts with the active serine, whereas the chloromethylketone derivatives react with the histidine of the catalytic triad [within the protease’s active site],” says Mohan.

Start broad, then focus

Inhibitors are used for a surprising number of applications today, which is one reason to choose carefully. “Downstream processing and analysis will determine how protease inhibitors are selected and used,” says Robert Gates, market segment manager at Sigma-Aldrich. For example, he adds, “liquid-phase proteomics analysis [of serum from blood samples] may require immediate inhibition of proteases that are hydrolyzing low-abundance target proteins.”

Indeed, there are so many types of inhibitors available that it can be overwhelming to sort them out. For choosing a protease inhibitor, Gates recommends a strategy of starting broad and then narrowing the focus. “The first step would be to try a pre-formulated cocktail, such as Sigma-Aldrich’s P8340, specifically formulated for mammalian tissues,” he says. “If proteolysis is still problematic, then a targeted approach for the specific tissue and protein of interest can be taken.”

Vendors continue to develop inhibitors to address the ever-changing needs of scientists. Vendors such as Sigma-Aldrich, Roche Life Sciences, AbCam, Thermo Fisher Scientific, Promega, EMD-Millipore and others all provide online guides to educate and guide researchers to make better selections. “Our MS-Safe inhibitor cocktail eliminates the covalent protein modifications seen with inhibitors such as PMSF and AEBSF and also addresses phosphatase inhibition,” says Gates. “Ideally, in the future, we could add a cocktail to a sample and stop all proteolysis as well as freeze post-translational modifications by inhibiting phosphatase, kinase, glycosytransferase and glycosidase activities.” It remains to be seen whether we can inhibit more proteases by using fewer inhibitors with broader specificity, or by using specific inhibitors of many individual proteases. In the meantime, there’s little doubt that you’ll be able to find an effective protease inhibitor among the many candidates and options available.