Bowman-Birk inhibitors (BBIs) are found primarily in seeds of legumes and in cereal grains. These canonical inhibitors share a highly conserved nine-amino acids binding loop motif CTP1SXPPXC (where P1 is the inhibitory active site, while X stands for various amino acids). They are natural controllers of plants’ endogenous proteases, but they are also inhibitors of exogenous proteases present in microbials and insects. They are considered as plants’ protective agents, as their elevated levels are observed during injury, presence of pathogens, or abiotic stress, i.a. Similar properties are observed for peptides isolated from amphibians’ skin containing 11-amino acids disulfide-bridged loop CWTP1SXPPXPC. They are classified as Bowman-Birk like trypsin inhibitors (BBLTIs). These inhibitors are resistant to proteolysis and not toxic, and they are reported to be beneficial in the treatment of various pathological states.
The complete set of proteases in an organism, known as a human degradome, is encoded by over 550 genes and represents more than 2% of the whole human genome . Although proteases are exclusively specialized in the hydrolysis of peptide bonds, they are classified into several groups, applying different modes of action: metalloproteases (the most abundant), serine proteases, cysteine proteases, aspartyl proteases, threonine proteases, glutamic proteases, and asparagine lyases . Proteases are implicated in numerous key biological processes, such as cell development and apoptosis, tissue modeling, angiogenesis, blood coagulation, wound healing, protein turnover, zymogen activation, and regulation of signaling cascades. Their dysregulated activity can bring destructive effects, as reported for various disorders, including cancers, inflammatory, and cardiovascular diseases  . Notably, there are more than 130 hereditary diseases related to mutations in proteases’ genes . In order to control proteases action, sophisticated mechanisms are utilized, including posttranslational modifications, production of inactive zymogens, and their rational conversion into active forms, as well as binding of enzymes with endogenous inhibitors.
The protease inhibitors offer high pharmaceutical potential in the treatment of diseases in which upregulated proteolytic activity is observed. Drugbank online database, which gathers drug compounds approved by the U.S. Food and Drug Administration (FDA), provides data concerning 108 natural and synthetic protease inhibitors . According to the comprehensive MEROPS database , protease inhibitors are classified into 38 clans and subdivided into 78 families. Among them, various families of inhibitors, including serpins, phytocystatins, Kunitz-type inhibitors (KTIs), Bowman-Birk inhibitors (BBIs), bifunctional α-amylase-trypsin inhibitors, mustard-type inhibitors, potato type-I and potato type-II inhibitors, potato metallocarboxypeptidase inhibitors, and squash and cyclotide inhibitors have gained much attention recently . This is mostly due to their potential application in the treatment of neurodegenerative disease, cancer, and autoimmune disorders . Such inhibitors are found mostly in seeds, leaves, and tubers of plants. They are supposed to regulate the activity of both endogenous proteases and exogenous digestive enzymes produced by phytopathogens. Thus, plant-derived inhibitors are considered as plant defense system components. Moreover, they are also regarded as storage of sulfur-containing amino acids.
Two major clusters of plant inhibitors are KTIs and BBIs families. The main difference between their members is the number of disulfide linkages—BBIs contain usually seven, while most KTIs two disulfide bonds. KTIs contain a single reactive site, while, in some BBIs, there are two reactive sites. Interestingly, both families share a similar mechanism of inhibition . They are found in legumes; some plants contain members of both families, while, in others, only one of them occurs, e.g., BBIs are present exclusively in common bean and lentil. BBIs are found primarily in the seeds of legumes and in cereal grains.
According to the MEROPS database, BBIs are coded as I12 (holotype: Bowman-Birk trypsin/chymotrypsin inhibitor unit 1) and I99 (holotype: Bowman-Birk-like trypsin inhibitor; Odorrana versabilis) . There are 611 BBIs out of 6720 identified inhibitors in plants, which account for 9.1% . The phrase “Bowman-Birk serine protease inhibitor family” (used without additional restrictions) results in 49 reviewed and 779 unreviewed records in the web database www.uniprot.org, which collects comprehensive protein sequence and functional information.
The first representative of the BBI family was isolated from soybean (Glycine max) by Donald E. Bowman in 1946  and further characterized in 1963 by Yehudith Birk et al. . Currently, it is often referred to as ‘classical BBI’ (here, it is abbreviated as BBI). BBIs are usually isolated from plants using multi-step chromatographic procedures . Interestingly, Fields et al.  proposed a novel purification approach based on high gradient magnetic separation and synthetic dodecapeptides, identified by phage display technology, targeting specifically BBI. Upon immobilization on superparamagnetic microbeads, the selected peptides were able to bind and isolate BBI from crude soy whey extracts. On the other hand, Palavalli et al.  demonstrated that active BBI and other proteins might be released into the surrounding media from seeds upon 4-8 h incubation in the water at 50 °C.
BBIs are one of the best recognized and characterized natural protease inhibitors family, as evidenced by the presence of several comprehensive review articles . However, their role in plants is not unequivocally defined. Their elevated expression is observed in various situations considered dangerous for plants, such as injury, the presence of fungus and pathogens, or abiotic stress .
Plant derived BBIs from dicotyledonous usually have a low molecular weight between 6–9 kDa and two homologous and independent binding loops located at the opposite sites of the molecules. Such ‘double-headed’ inhibitors are capable of inhibiting two, the same or different, enzyme molecules either simultaneously or independently. In contrast, BBIs from monocotyledonous are more diverse and are divided into two subclasses containing either ‘mono-headed’ inhibitors with molecular weight of about 8 kDa or ‘double-headed’ with molecular weight ~16 kDa. In ‘double-headed’ inhibitors the first binding loop is usually involved in inhibition of trypsin and the second is mostly associated with chymotrypsin . The family of BBI contains also a strong trypsin inhibitor named sunflower trypsin inhibitor SFTI-1 composed of just 14 amino acids (~1.5 kDa). Even though this backbone-cyclized peptide containing a single disulfide bond is not genetically related to other BBIs , they share an almost identical binding loop . It is worth noting that SFTI-1 is one of the most popular starting structures to produce potent inhibitors of a wide range of biologically relevant proteases .
As mentioned before, the BBIs family was established as a bunch of plant-derived inhibitors; however, a novel group of peptides originating from animals, which imitates the BBI’s trypsin inhibitory loop (TIL), has been recently identified . These peptides were isolated from frogs’ skin, and similarly to plant BBIs, they present strong trypsin inhibitory activity. Their disulfide-bridged loop contains 11 residues, with the general formula CWTP1SX1PPX2PC (where P1 is the inhibitory active site usually occupied by Lys, while X1 and X2 are variable). This loop is longer than that found in plant BBIs composed of 9-amino acids (CTP1SX1PPX2C). Since the spatial structures of both binding loops are highly similar, although not identical, these trypsin inhibitors are termed as BBI-like trypsin inhibitors (here, abbreviated as BBLTIs).
Different isoforms of BBIs are frequently present in the same plant. It was proposed that isoinhibitors are produced due to co-evolution of the plants and insects . Such a strategy is apparently applied to increase efficiency in combating pathogens. This minimizes a risk of hydrolysis of all inhibitors by the pest enzymes as well as helps to deal with inhibitor-insensitive or inhibitor-degrading proteinases .
BBIs’ defensive function is reflected in an insecticidal activity, as various members of this family display antifeedant activity against insects . Thus, the transfer of the BBI gene into plants with economic importance is a promising strategy to produce transgenic plants resistant to insects . Some BBIs are also blocking proteases produced by pathogens; thus, they have the potential to be used as antimicrobials . BBI was proved to display antiviral activity toward bovine herpes virus-1 , herpes simplex virus type 2 (HSV-2)  and HIV . It also possess antifungal activity  and activity against human pathogenic Gram-positive bacteria Staphylococcus aureus . Also some frog skin-derieved BBLTIs exhibit moderate antibacterial activity, e.g. Ranacyclin T (lethal concentration for Escherichia coli 30 μM; Yersinia pseudotuberculosis 5 μM; Bacillus megaterium 3 μM) , inhibitor from skin of Odorrana grahami frog (S. aureus MIC=5.83 mg/mL, E. coli MIC=3.20 mg/mL) .
Their intrinsic ability to inhibit serine proteases is thought to be resposible for antiproliferative activities of BBIs. It was hypothesized that strong inhibitory activity, induced particularly by chymotrypsin‐binding site, is necessary to evoke effective anti-carcinogenic actions . Indeed, various BBIs and BBLTIs were proved to exhibit anticancerogenic potency e.g. BBI from soybean Glycine max , kidney bean Phaseolus vulgari , black-eyed pea Vigna unguiculata , chickpea Cicer arietinum , and the skin secretion of frog Pelophylax esculentus .
BBI structure, making it resistant to proteolysis in digestive system makes it a good candidate to treat inflammatory diseases, as serine proteases (neutrophil serine proteases, coagulation factors, granzymes, etc) are known to be involved in tissue damage during inflammation . BBI’s anti-inflammatory properties were confirmed for inflammatory disorders of gastrointestinal tract (GI) such as inflammatory bowel disease usually standing for ulcerative colitis or Crohn disease . It was suggested that soybean BBI could be effective in inhibition of the Alzheimer's disease , animal model of multiple sclerosis  and animal model of Guillain-Barre syndrome . Also some of amphibian skin derived BBLTIs are reported to have anti-inflammatory properties. pLR was described as the first and the most potent, noncytolytic histamine-liberating peptide of natural origin, it inhibits granulopoiesis, but unlikely other inhibitors, its activity is directed only against myeloid progenitor cells and no effect was observed in case of mature neutrophils .
It is worth noting that due to their high stability in GI tract, BBIs alongside with tannins and phytic acid, are considered as antinutritional factors. They may reduce activity of pivotal enzymes within the gastrointestinal tract of animals, leading to lower digestion and adsorption of dietary proteins. This may result in inhibition of organism growth and pancreatic disorders, such as hypertrophy and hyperplasia .
BBIs’ intrinsically high inhibitory activity combined with extreme thermal, proteolytic, and pH stability build the fundaments of their potential for diverse applications. Even though the classical soybean BBI does not meet high initial expectations to become an effective, natural anticancer agent, it is shown that it might be considered as a complement for other molecules endowed with more evident anti-cancer properties, such as α-tocopheryl succinate or bioactive peptide lunasin.Noteworthy, BBIs’ biomedical application in the treatment of various diseases related to dysregulated proteolytic activity, not only cancers but also metabolic and inflammatory disorders, is still under examination. Moreover, various BBIs may be utilized as efficient tools for learning the exact role of proteolytic enzymes involved in diseases’ progress and development. They are also attractive starting structures for designing novel, potent, synthetic inhibitors and other compounds, displaying a combination of various capabilities. The later merit has been shown for the smallest BBI's member SFTI-1, in which simultaneous rational modifications of both loops have resulted in novel bifunctional bioactive peptides. In the case of some BBIs and BBLTIs, this unique combination of strong inhibitory activity towards proteolytic enzymes with bactericidal potency and low toxicity may result in novel antimicrobial agents. In the light of growing antibiotic resistance and the high propensity of known antimicrobial peptides to hydrolytic breakdown, such compounds seem to be of particular interest. Despite the physiological role of BBIs in plants and animals is still vague, it is their multifaceted biological activity that draws a lot of researchers’ attention.