3. Structural characteristics of food protein-derived renin inhibitory peptides

To reduce the time and cost-intensive steps in the peptide discovery with the conventional pathway, it is important to understand the relationship between peptide structure and subsequent bioactivity. By utilizing the knowledge of structure activity relationship putatively, active peptide sequences can be released in a targeted manner. To date, the research has focused in production and characterization of bioactive peptides, and data concerning the structure-activity relationship are still quite limited.

Renin is a 335-amino acid, glycosylated aspartic protease belonging to pepsin-like family [14, 69]. In contrast to other aspartic proteases such as pepsin, which cleaves a wide variety of substrates, renin specificity is very restricted. The high specificity of renin catalysis is explained by the restricted three-dimensional space of the active site. The C- and N-terminal domains of renin form a deep cleft constructing the active site in which the inhibitors bind [34, 60]. Angiotensinogen is the highly specific physiological substrate of renin, but new renin inhibitors—among which the best known is nonpeptidic Aliskiren—have been developed based on the structural data of the active site [34]. Aliskiren is an orally active renin inhibitor with a very high binding affinity for renin [77], but it is a complicated molecule and thus, drugs simpler in structure and with high bioavailability are desirable in the drug market.

The structure of the active site of renin and the binding of Aliskiren is illustrated in Figure 1 [58]. It is known that the binding to the catalytic aspartate residues is vital for all the protease inhibitors [9]. The active renin inhibitors seem to presuppose interactions with the aspartate residues of renin (Asp 32 or Asp 215) and the S3sp sub pocket unique for renin. Thus, it has been suggested that any new renin inhibitor should interact with these sites in the active site of renin [58].

Several renin inhibitor peptide sequences have been identified thus far (Table 1), however, quite little is known on detailed structure-activity relationship (SAR). Some general characteristics, such as hydrophobicity and molecular size of the peptide fractions, are suggested to correlate with the renin inhibitory activity, but the results are somewhat contradictory [2, 31, 36, 40, 44]. Taken together, the position of amino acid residues in the peptide sequence is more important for the renin inhibition capacity than the actual molecular size or total net charge.

The presence of N-terminal aliphatic (e.g., leucine, isoleucine, valine) and C-terminal bulky amino acid residues (e.g., phenylalanine, tryptophan) has been suggested to contribute to

Figure 1. Binding mode of aliskiren as produced from crystallographic data. The protein backbone is shown in ribbons. Residues of the binding site are displayed as gray sticks and aliskiren as ball and sticks. The right panel shows a zoom in the active site and the formed H-bonds with aliskiren [58].


Renin is a 335-amino acid, glycosylated aspartic protease belonging to pepsin-like family [14, 69]. In contrast to other aspartic proteases such as pepsin, which cleaves a wide variety of substrates, renin specificity is very restricted. The high specificity of renin catalysis is explained by the restricted three-dimensional space of the active site. The C- and N-terminal domains of renin form a deep cleft constructing the active site in which the inhibitors bind [34, 60]. Angiotensinogen is the highly specific physiological substrate of renin, but new renin inhibitors—among which the best known is nonpeptidic Aliskiren—have been developed based on the structural data of the active site [34]. Aliskiren is an orally active renin inhibitor with a very high binding affinity for renin [77], but it is a complicated molecule and thus, drugs simpler in structure and with high bioavailability are desirable in the drug market. The structure of the active site of renin and the binding of Aliskiren is illustrated in Figure 1 [58]. It is known that the binding to the catalytic aspartate residues is vital for all the protease inhibitors [9]. The active renin inhibitors seem to presuppose interactions with the aspartate residues of renin (Asp 32 or Asp 215) and the S3sp sub pocket unique for renin. Thus, it has been suggested that any new renin inhibitor should interact with these sites in the active site of renin [58].

Several renin inhibitor peptide sequences have been identified thus far (Table 1), however, quite little is known on detailed structure-activity relationship (SAR). Some general characteristics, such as hydrophobicity and molecular size of the peptide fractions, are suggested to correlate with the renin inhibitory activity, but the results are somewhat contradictory [2, 31, 36, 40, 44]. Taken together, the position of amino acid residues in the peptide sequence is more important for the renin inhibition capacity than the actual molecular size or

The presence of N-terminal aliphatic (e.g., leucine, isoleucine, valine) and C-terminal bulky amino acid residues (e.g., phenylalanine, tryptophan) has been suggested to contribute to

Figure 1. Binding mode of aliskiren as produced from crystallographic data. The protein backbone is shown in ribbons. Residues of the binding site are displayed as gray sticks and aliskiren as ball and sticks. The right panel shows a zoom in

the active site and the formed H-bonds with aliskiren [58].

total net charge.

244 Renin-Angiotensin System - Past, Present and Future



higher renin inhibitory activity of dipeptides [71]. For example, dipeptides Leu-Tyr, Ile-Trp, and Thr-Phe have been reported to inhibit renin activity with IC50 values of 1.8, 2.3, and 3.7 mM, respectively [30]. The structures of these peptides mostly agree with the characteristics proposed to contribute to renin inhibition. The importance of C-terminal bulky hydrophobic amino acid residue was also observed by changing the position of amino acids residues from Thr-Phe to Phe-Thr, which resulted to substantial decrease in renin inhibition [30, 71]. However, highly hydrophilic peptides, such as Gly-His-Ser, have also been reported to inhibit renin with IC50 value of 1.09 mM [31]. Also, a cationic tetrapeptide Arg-Ala-Leu-Pro and a 13-amino acid residue, Ile-Arg-Leu-Ile-Ile-Val-Leu-Met-Pro-Ile-Leu-Met-Ala, have also shown rather high renin inhibitory potency [22, 30]. The highest renin inhibitory activity among the reported food protein-derived peptides thus far is 0.054 mM for Trp-Tyr-Thr produced from hemp seed protein [27]). Taken together, more research is needed to gain more knowledge on detailed SAR for designing potential renin inhibitory peptide sequences as physiological antihypertensive agents.

Quantitative computational tools are increasingly applied in medicinal and pharmaceutical drug discovery. At present, the relationship of peptide structure and bioactivity, especially the enzyme inhibitors of ACE are known in some extent. The knowledge of the active peptide sequences enables utilization of quantitative structure-activity relationship modeling (QSAR) for evaluating the crucial physicochemical features of the peptide for the effective bioactivity. A small number of QSAR studies have been carried out on ACE-inhibitory peptides [59, 66] however, no studies have been carried out with seeking potential renin inhibitory peptides.

## 4. Bioavailability

Origin Hemp seed protein

Pea protein African yam bean seed

 Alcalase RP-HPLC fraction of the

hydrolysate

Alcalase, pepsin, trypsin,

RALP, LY, TF

15.80% at 1 mg/ml

ΔSBP

(Alcalase) after 4 h of oral

kg bw

ΔSBP

mmHg (RALP) after 6 h of oral

mg/kg bw

ΔSBP

30 mg/kg bw

17 mmHg after 6 h of oral

12 mmHg (TF),

26 mmHg (LY) and

16

administration,

 30 administration,

25 mmHg (pepsin) and

34 mmHg

[2, 30, 31]

administration,

 200 mg/

0.968 mM (RALP), 1.868

mM (LY), 3.061 mM (TF)

0.320 mM

GHS

pancreatin

Alcalase

Pepsin + pancreatin

> Table 1. Food

protein-derived

 renin inhibitory peptides and

antihypertensive

 effects in vivo.

Rapeseed and canola

protein

 Alcalase, pepsin, papain,

pepsin + pancreatin

Thermolysin,

fraction of the hydrolysate

<3kDa MWCO

17% at 1 mg/ml

Treatment

Identified

Renin inhibitory

Antihypertensive

 effects in vivo, SHRs

Reference

[44]

activity in vitro IC50

0.08–0.24 mg/ml

ΔSBP

25.33 mmHg after 4 h of oral

administration,

ΔSBP

200 mg/kg bw

ΔSBP

29 mmHg after 8 weeks of oral

administration

expression of renin mRNA levels was reduced

significantly.

ΔSBP

trial

35% at 1 mg/ml

55% at 1mg/ml

6 mmHg in a 3-week human

intervention

[1]

 to

Han:SPRD-cy,

 0.1% of diet. Renal

19 mmHg after 4 h of oral

administration,

[41]

246 Renin-Angiotensin System - Past, Present and Future

 200 mg/kg bw

sequences

To induce health effects in vivo, peptides need to reach the physiological target organs in intact and active conformation. Considering the renin inhibition, there are three main barriers and hydrolytic threats on the way to the in vivo outcome: the digestive proteinases in the gastrointestinal tract, enzymes in the site of absorption, and serum peptidases in the circulation. Thus far, the published data concerning the bioavailability of the peptides, which have shown in vitro renin inhibitory activity, are very limited. This makes it very difficult to predict the in vivo antihypertensive effect of the in vitro renin inhibitory peptides. However, some structural characteristics have been shown to correlate with the bioavailability of, e.g., ACEinhibitory peptides. These general peptidic characteristics can be considered with renin inhibitory peptides as well.

At first, after oral ingestion bioactive peptides need to resist the hydrolytic actions in stomach by pepsin and pancreatic peptidases, including trypsin, elastase, and chymotrypsin, and further, carboxypeptidases in the small intestine. Several different methods have been applied to model the gastrointestinal digestion in vitro. Most of the methods not only concern utilization of commercial porcine enzyme mixtures (e.g., Refs. [42, 46, 75]) but also human digestive liquids have been utilized [19, 49]. Due to the variation in the methods, the comparison of the results across the studies is difficult and thus, a harmonization of the various in vitro methods would be important. A consensus for a static process to model the digestion of plant secondary metabolites has been constructed based on in vivo data [3]. Indeed, the future research should focus more on the in vivo bioavailability of the peptides and based on the correlation with in vivo data, a harmonized in vitro method could be proposed.

The peptides are exposed to peptidolytic digestion also on the brush border membrane of the intestine. There are number of peptidases with varying specificities bound on the intestinal epithelial cells. It has been suggested that dipeptide and tripeptide tend to resist the gastric and duodenal digestion and also the hydrolytic action of peptidases at the brush border membrane. These small peptides can be absorbed by active transcellular transport or by passive process [63]. To study the absorption in vitro, the monolayer of intestinal cell lines, such as Caco-2 cells, simulating intestinal epithelium, is commonly utilized. Clinical data concerning the bioavailability of bioactive peptides are very restricted; however, ACE-inhibitory lactotripeptides, Ile-Pro-Pro and Val-Pro-Pro, have been detected in human and animal circulatory system after oral ingestion [25].
