Research Progress in Structure-Activity Relationship of Bioactive Peptides

Abstract

Bioactive peptides are specific protein fragments that have positive impact on health. They are important sources of new biomedicine, energy and high-performance materials. The beneficial effects of bioactive peptides are due to their antioxidant, antihypertensive, anticarcinogenic, antimicrobial, and immunomodulatory activities. The structure-activity relationship of bioactive peptides plays a significant role in the development of innovative and unconventional synthetic polymeric counterparts. It provides the basis of the stereospecific synthesis, transformation, and development of bioactive peptide products. This review covers the progress of studies in the structure-activity relationship of some bioactive peptides including antioxidant peptides, angiotensin-I-converting enzyme-inhibitory peptides, and anticarcinogenic peptides in the past decade.

Introduction

Bioactive peptides are short sequences of approximately 2–20 amino acids in length that exert physiological benefits when consumed in vivo. They are inactive within the sequence of the parent proteins and may be released by proteolytic hydrolysis using commercially available enzymes or proteolytic microorganisms and fermentation methods.¹ Bioactive peptides can be absorbed in the intestine and enter the blood stream directly, which ensures their bioavailability in vivo and a physiological effect at the target site.²

There are wide varieties of bioactive peptides in the nature and nearly thousands of bioactive peptides have been discovered. According to their forming process, bioactive peptides can be divided into endogenous and exogenous bioactive peptides. The endogenous peptides are the peptides hydrolyzed from protein by endopetidase, and the exogenous peptides are the peptides hydrolyzed from protein by exopeptidase. According to the materials from which bioactive peptides are produced, they can be divided into marine and terrestrial bioactive peptides. According to their origin, they can be divided into natural and artificial bioactive peptides. For instance, antioxidant peptides isolated from rapeseed proteins³ and angiotensin-I-converting enzyme (ACE) inhibitory peptides Gly-Pro-Leu and Gly-Pro-Met extracted from the skin of Theragra chalcogramma ⁴ are natural peptides, while antioxidant peptide Trp-Tyr-Pro-Ala-Ala-Pro synthesized on the basis of solid phase peptide synthesis using ASP48S is an artificial peptide.⁵ Antihypertensive drugs such as Captopril, Enalapril, and Fosenpril used now are all synthetic peptides. According to the physiological functions, bioactive peptides can be divided into antihypertensive peptides, antioxidant peptides, antimicrobial peptides, anticancer peptides, immunomodulatory peptides, opioid peptides, mineral binding peptides, antithrombotic peptides, thymosin, hypnosis peptide, neuropeptides, and so on.^6,7

In recent years, consumer interest in the relationship between diet and health has increased substantially. It has been recognized that functional foods may make a positive contribution to health and well-being when combined with a healthy lifestyle. Food-derived bioactive peptides exerting physiological effect in humans have attracted more attention by either consumers or scientists. The food-derived bioactive peptides are found in milk, egg, meat, and fish of various kinds and in many plants.⁸ It has been found that the addition of appropriate active peptides in the diet is very beneficial to human health because they can regulate physiological functions.⁹ Depending on their amino acid sequences, food-derived peptides can exhibit diverse activities, including opiate-like, mineral binding, immunomodulatory, antimicrobial, antioxidant, antithrombotic, hypocholesterolemic, anticancer, and antihypertensive actions.¹⁰ Moreover, many milk or milk protein-derived peptides exhibit multifunctional properties.¹¹ Some bioactive peptides as functional foods have been in commercial process, especially in Japan.¹² Examples are low antigen food, mobile food, baby products, food products for sports nutrition, blood pressure regulation, calcium-absorption promotion, and other products.

Bioactive peptide with specific physiological activity is composed of certain amino acid residues linked in specific sequences. The activities or functions of the bioactive peptides depend on their structures, such as the amino acid composition, the type of amino acid in C- and N-terminal, the length and weight of the peptide chain, the hydrophobic property, charge character of amino acid, spatial structure, and so on.¹³ The relationship between activity and structure of peptide is still in the exploratory stage. Further exploring the structure-activity relationship and revealing the mechanism of bioactive peptide will provide important theoretical basis for synthesis, transformation, development of bioactive foods, or complementary medicines. In this review, the available information regarding the relationship between structure and activity of bioactive peptide was summarized. The antihypertensive, antioxidative, and anticarcinogenic mechanisms of different bioactive peptides were also covered in this review.

Structure-Activity Relationship of Ace Inhibitory Peptides

Hypertension refers to arterial systolic and/or diastolic blood pressure increased (≥140/90 mmHg) in the resting status. It is a systemic disease characterized by organ remodeling, often accompanied by fat and glucose metabolism disorder, and heart, brain, kidneys, retina, and other organs functional changes.¹⁴ At present, hypertensive diseases endanger human health seriously. The average prevalence rate of hypertension is about 15% worldwide.¹⁵

Human body's blood pressure is regulated by many factors. The most important one is the balance of boost system (Renin-Angiotensin System, RAS) and depressurization system (Kallikrein-Kinin System, KKS). RAS and KKS is a pair of mutually antagonistic systems in blood pressure regulation, in which, the ACE (EC 3.4.15.1) balance the boost and depressurization system as a rate-limiting enzyme. In RAS system, renin stimulates angiotensinogen to release a nonactive peptide-angiotensin I. ACE catalyzes angiotensin I (10 peptides) into angiotensin II (8 peptides). The latter has a strong vasoconstrictor and boost effect, leading blood volume and blood pressure increased.¹⁶ In the KKS system, ACE acts on the vasodilator bradykinin Phe-Arg or Ser-Pro at C-terminal and is inactivated, causing high blood pressure.¹⁷ If ACE activity is inhibited, biosynthesis of angiotensin II is reduced and the bradykinin is inactivated. Subsequently, blood pressure goes down. Therefore, ACE becomes an ideal target for the treatment of hypertension disease.

By now, more than 16 kinds of ACE inhibitors such as captopril, enalapril, benazepril, and cilazapril are officially used for clinical treatment of hypertension and 80 kinds have been studied.¹⁸ Most of these drugs are synthetic drugs, often accompanied by many side effects such as cough, pruritus, taste disorder, or low blood pressure. Many studies confirm that a reasonable diet can prevent and relieve hypertension.^19,20 Therefore, more attentions are on finding and developing safe, nontoxic, and efficient alternative antihypertensive drugs from food sources. It has become a research hotspot.

There is a class of peptides with good ACE inhibitory activity from food sources. Normally, these peptides are in relative small molecular weight.^21
–23 The amino acid sequence and chain length of these peptides have different characteristics. Only when it combines with ACE active site closely, ACE inhibitory peptides can show inhibitory activity. The catalytic site of ACE is composed of three subunits, which are corresponding to three hydrophobic amino acids (Pro, His, and Phe) of angiotensin I in C-terminal district. It catalyzes angiotensin I into bradykinin and leads to blood pressure rise. Therefore, the three amino acids in C-terminal district of bioactive peptides greatly influence ACE activity.

Amino acids composition and ACE inhibitory activity

The primary structure and amino acid composition of ACE inhibitory peptides are closely related to its inhibitory activity. Research showed that the interactions between peptides and ACE were greatly influenced by the sequences of three amino acids in C-terminal district of peptides.^24,25 Aromatic or alkaline amino acids in N-terminal of ACE inhibitory peptides can improve its ACE inhibitory activity. It was also reported that the peptides containing leucine, isoleucine, and valine in N-terminal exhibited good antihypertensive effect, but the ACE inhibitory activity was lowered in the presence of proline in N-terminal of ACE inhibitory peptide.

Two hundred seventy ACE inhibitory peptides were collected and their amino acid compositions were analyzed.²⁶ The amino acids tyrosine (Tvr), proline (Pro), tryptophan (Trp), phenylalanine (Phe), and leucine (Leu) are more likely in the C-terminal of these ACE inhibitory peptides while arginine (Arg), Tvr (Gly), glycine, valine (Va1), alanine (Ala), and isoleucine (Ile) are more likely in the N-terminal. The characteristics of C-terminal amino acids contribute more to the antihypertensive effects. Therefore, the activity of ACE inhibitory peptide is closely related to the terminal amino acid composition.

Hydrophobicity/hydrophilicity of peptide and ACE inhibitory activity

The hydrophobic/hydrophilic property of polypeptide is another important factor influencing its activity. There is a positive correlation between hydrophobicity of C-terminal amino acids and ACE inhibitory activity.^27,28 The high hydrophilicity makes the peptide hardly close to the ACE active sites and results in the weaker activity. The relationship between mass percentage of hydrophobic amino acids in C-terminal tripeptides and ACE inhibiting activity is shown in Table 1. It can be seen that there is a large amount of hydrophobic amino acids in the primary structure of ACE inhibitory peptides, especially in C-terminal tripeptides. The relationship between content of hydrophobic amino acids and ACE inhibitory activity showed that most of the antihypertensive effect of ACE inhibitory peptides was in accordance with the law above. In addition, studies also show that N-terminal hydrophobic amino acid or aliphatic amino acids in side chain can promote the peptide binding with ACE.²⁹ We can see from Table 1, peptides with hydrophobic amino acids in C-terminal had higher ACE inhibitory activity, but the activity did not increase with increasing percentage of hydrophobic amino acids. The same percentage of different hydrophobic amino acids had different activity. IC₅₀ of AHLL with 67% hydrophobic amino acids of two leucine was 40.2 μM; IC₅₀ of NGTWFEPP with 67% hydrophobic amino acids of two proline was 0.63 μM. This means the percentage of the hydrophobic amino acids in C-terminal tripeptide is not the only factor influencing the ACE inhibitory activity, the type of amino acid in C-terminal and the length of peptide also play important roles.

Table 1.

Relationship Between Percentage of the Hydrophobic Amino Acids in C-Terminal Tripeptide and Angiotensin-I-Converting Enzyme Inhibitory Activity

Sequence	Hydrophobic amino acids in C-terminal tripeptide (%)	IC₅₀ (μM)	Reference
AHLL	67	40.2	18
RYLGY	67	0.71	30
LIWKL	67	0.47	31
YPHK	33	2.31	32
VKAGF	100	20.3	33
DERF	33	37.08	34
RYPSYG	33	65.71	34
NGTWFEPP	67	0.63	35
LVYPFPGPIPNSLPQNIPP	100	5	36
VAP	100	5.34	37
VYPFPGPIPNSLPQNIPP	100	4	28
GPL	100	2.6	4
GPM	100	17.13	4
ALPMHIR	33	42.6	38

IC₅₀ value: the concentration of peptide required to inhibit 50% of the activity.

Positive charged amino acids in C-terminal and ACE inhibitory activity

Research showed that lysine(ɛ-amino positive charge) and arginine (guanidine positive charge) in C-terminal of peptide can promote the ACE inhibitory activity.³⁸ Table 1 shows that the activity of Ala-Leu-Pro-Met-His-Ile-Arg with arginine in C-terminal is stronger compared with other peptides. Also, Ala-Ile-Tyr-Lys with lysine in C-terminal has higher activity.³⁹ Additionally, Pro in C-terminal with strong affinity on active site can promote the binding with ACE.⁴⁰

In summary, peptides with higher ACE inhibitory activity usually have aromatic or alkaline amino acids in N-terminal, higher quantity of hydrophobic and positively charged amino acids in C-terminal.

Structure-Activity Relationship of Antioxidant Peptides

Many health problems stem from the effect of oxidation process in human body. Oxidation is a vital process in all living organisms, but the free radicals produced by oxidation could be very destructive. The attacks of free radicals lead cells to continuous damage, known as oxidative stress or oxidation.⁴¹ Under normal circumstances, oxidation is a dynamic balance between continuous emergence and elimination of free radicals in vivo. The excessive free radicals would cause oxidation damage of cell tissues, which leads to human aging and many other diseases, such as cancer, high blood pressure, artery hardening, inflammation, and infertility. Heart, lungs, and brain are the main targets of free radicals because they are heavy users of oxygen.

Oxidation in foods is one of the major causes of food deterioration.⁴² It affects lipids, proteins, and carbohydrates. However, lipid oxidation is the main cause of deterioration of food quality, leading to rancidity and shortening of shelf life.⁴³ Oxidation of proteins in foods is influenced by lipid oxidation, where lipid oxidation products react with proteins causing their oxidation.⁴⁴ Carbohydrates are also susceptible to oxidation, but they are less sensitive than lipids and proteins.⁴⁵

Antioxidant is a substance that significantly decreases the adverse effects of reactive species, such as reactive oxygen and nitrogen.⁴⁶ In terms of foods, antioxidants are compounds that are able to delay, retard, or prevent autooxidation processes.⁴⁷ At present, the antioxidants are mainly from chemical synthesis, such as butylated hydroxyanisole, butylated hydroxutoluene, propyl gallate, and tertiary butylhydroquinone. They are widely used as preservatives in food and cosmetics. Due to the potential adverse effects of these synthetic antioxidants on the human liver, spleen, lungs, and other viscera organs, food industry has been actively seeking natural antioxidants. Natural antioxidants widely used in foods include vitamin C, vitamin E, and polyphenols. In the case of vitamin C and vitamin E, the antioxidant molecule is either recharged by accepting an electron from another type of antioxidant or it is recycled into building tissue repair.

Since Prokorny first reported that some peptides and proteins hydrolysate could reduce autooxidation rate and the peroxide content of fat in 1991, more and more research has focused on searching for antioxidant peptides with high antioxidant activity.⁴⁸ Whey, soy, and egg yolk hydrolysates have shown to inhibit lipid oxidation in various muscle foods, such as beef, pork, and tuna.^49,50 Some histidine (His) containing peptides such as carnosine and anserine that are naturally present in skeleton muscle have antioxidant activity. Most experts believe that getting antioxidant peptide from food is the most healthful way. Antioxidant peptide with small molecular weight is easy to be absorbed and has excellent antioxidant effect. The antioxidative pathways mainly include the active oxygen inactivation, scavenging free radicals, chelating metal ions, reducing the formation of hydrogen peroxide, and changing physical properties of food system.⁵¹ The antioxidant activity of antioxidant peptide is related to the amino acid composition, the amino acid sequence of the peptides, the molecular weight of the peptide chain, the N- and C-terminal amino acid residues, and hydrophilic properties.

Amino acid composition and antioxidant activity of bioactive peptides

Hydrophobic amino acids and aromatic amino acids have close correlation to the antioxidant activity. Generally, the more hydrophobic amino acids is, the stronger antioxidant is. The reason is that fatty acid free radicals are hydrophobic and they tend to combine with hydrophobic antioxidant peptides first.⁵²

First discovered peptide with antioxidant activity was an acidic peptide.⁵³ There is an interactive passivation of oxidation between some metal ion (Fe²⁺ and Cu⁺) and carboxyl residue of side chain in acidic amino acid, which decreases the free radical chain reaction and exerts the antioxidant effect. The proton donor in amino acid residues such as Tyr and Trp is a necessary component of antioxidant peptide, in which, hydrogen atoms were supplied to free radical with its hydrogen donating capacity. Finally, the indole free radicals and benzene-oxygen free radical is stable by means of resonance. The free radical chain reaction is thus slowed or stopped.⁴¹ Peptide Leu-Asp-Tyr-Glu was an antioxidative peptide prepared from corn.⁵⁴ Tyrosine in the peptide provided proton quenching free radicals and the carboxylate base of its adjacent aspartic acid (Asp) and glutamate (Glu) possessed the electron-withdrawing effect, which enhanced the proton donation of Tyr. Furthermore, Leu of the N-terminal was a hydrophobic amino acid, which enhanced the interaction of antioxidant peptides with fatty acids and improved the capture of lipid free radical.

Some amino acids have the antioxidative activity. Cysteine (Cys) and methionine (Met) can directly react with the corresponding free radicals. Met can easily be oxidized to Met sulfoxide, the latter can be deoxidated to Met by Met sulfoxide reductase, which resume the antioxidant activity of Met. The antioxidant activity of single amino acid is far lower than that of peptide containing the amino acid.⁵⁵ It is believed that the His of the peptide can be used as a metal ion chelating agent, active oxygen quencher, and hydrogen free radical scavenger, which enhance the antioxidant activity of the peptide.⁵⁶ Oxidation of amino acid residues is one of the causes that antioxidant peptide could scavenge free radicals. Although His is important to the antioxidant activity of the peptide, the free amino acid doesn't exhibit good antioxidant activity because the primary structure of peptides is a necessary factor of antioxidant activity. Obviously, the protection of lipid from oxidation by antioxidant peptides is at the expense of specific amino acids such as His and Met in the peptides.

Hydrophobic/hydrophilic amino acids and antioxidant activity

Nonpolar aliphatic side chain can strengthen the interaction between antioxidant peptides and hydrophobic polyunsaturated fatty acids. Peptides with hydrophobic amino acids such as His, Pro, Cys, Tyr, Trp, Phe, and Met delay the lipid peroxidation chain reaction by combining with oxygen or inhibition of hydrogen release in lipid, thus producing the antioxidant effect.⁵⁷

Hydrophobic amino acids in the side chain or C-terminal and N-terminal of peptides display strong antioxidative activity. It is believed that the antioxidant tripeptide will present high antioxidative activity if its N-terminal is hydrophobic amino acids with low isoelectric point, such as Ala, Gly, Val and Leu.⁵⁸ N-terminals of six antioxidant peptides isolated from soybean protein enzymatic hydrolysates are all hydrophobic amino acids.⁵⁹ The more hydrophobic amino acids and aromatic amino acids the peptide contained, the more significant antioxidant activity it showed.⁵² The antioxidant peptides identified in recent years and their hydrophobic amino acids contents are summarized in Table 2.

Table 2.

Antioxidant Peptides and Percentage of the Hydrophobic Amino Acids

Sequence	Hydrophobic amino acids content (%)	Reference
HGPLGPL	86	60
HPHPHLSF	50	61
GSTVPERTHPACPDFN	43	62
PSYV	50	63
IVGGFPHYL	78	64
RPDFDLEPPY	50	65
CAAP	75	66
VCSV	50	66
VKAGFAWTANQQLS	57	67
WYSLAMA	71	68
INYW	50	69
LDQW	50	69
ISELGW	50	70
LPHSGY	50	71
LLGPGLTNHA	70	72
DLGLGLPGAH	80	72
LPHSGY	50	71

Other factors

The correlation between the structural conformation of amino acid of peptides and their antioxidant activity was reported. When second L-histidine in Pro-His-His was replaced by D-histidine, the antioxidant activity of the peptide decreased obviously.⁷³ There was a close relationship between antioxidant activity of peptides and their amino acid structures. Suetsuna synthetized peptides lack of Tyr, Tyr-Phe, Tyr-Phe-Tyr, and Tyr-Phe-Tyr-Pro based on the antioxidant peptide Tyr-Phe-Tyr-Pro-Glu-Leu from casein. The activities of preferred sequences were Glu-Leu (EL)>Tyr-Phe-Tyr-Pro-Glu-Leu (YFYPEL)>Phe-Tyr-Pro-Glu-Leu (FYPEL)>Tyr-Pro-Glu-Leu (YPEL)>Pro-Glu-Leu (PEL), suggesting that the EL sequence is important for the activity.⁷⁴ Also, there is close relationship between molecular weight and the antioxidant activity. Usually, the smaller the molecular weight is, the easier that peptide goes through the biological membrane to the effective site.⁵⁵

Although significant progress has been made in the structure-activity relationship of antioxidant peptides, there is undoubtedly still an enormous amount to be learned. At present, the active sites of antioxidant peptides cannot be analyzed and confirmed accurately. There is no final conclusion about the relation between the molecular conformation and activity of antioxidant peptides. How much the interaction of amino acids affects the antioxidant activity needs further illustration. With the development of bioengineering and new types of separate technology, the relationship of the structure-activity of antioxidant peptides will be more and more visualized.

Structure-Activity Relationship of Anticancer Peptides

Nearly half a century, through the tireless efforts of scientists, gradually realized that cancer is caused by complicated reasons including environmental, nutritional, dietary, genetic, viral infections, and lifestyle. The occurrence of cancer is a multifactor, multistep complex biological process, which can be divided into three different and successive stages including initiation, promotion, and progression, and involving a variety of complex mechanism, such as oncogene activation, inactivation of cancer suppressor genes, and their interactions.⁷⁵ Cancer remains a major source of morbidity and mortality throughout the world and it is predicted to displace heart diseases as the leading cause of death worldwide.⁷⁶ In the United States, one person is diagnosed with cancer every 30 sec and one person dies of cancer every 35 sec.⁷⁷

Although much progress has been achieved in respect of cancer treatments and therapies in recent decades, chemotherapy remains the choice of usual treatment for advanced or metastatic disease. However, the use of conventional chemotherapeutic agents that typically target rapidly dividing cancer cells is often associated with deleterious side effects due to drug-induced damage to normal cells and tissues.⁷⁸ Moreover, cancer cells develop resistance to these drugs that is mediated by the over expression of multidrug-resistance proteins that pump the drugs out of cells and thus render the drugs ineffective.⁷⁹ Therefore, the research and development of more effective and less toxic anticancer agents has become necessary. Anticancer peptides have recently received attention as alternative chemotherapeutic agents that overcome the limits of current drugs. These peptides have several advantages over currently used anticancer therapeutics, such as selective cytotoxicity for cancer cells, bypass of the multidrug-resistance mechanism, and additive effects in combination of therapy.⁸⁰

Hundreds of natural peptides have been found to show antimicrobial properties that can kill a wide spectrum of Gram-positive and Gram-negative bacteria, protozoa, and fungi. Some antimicrobial peptides exhibit anticancer activity, thus called anticancer peptides.⁸¹ Because many antimicrobial peptides may be toxic to human being, anticancer peptides derived from foods with less toxicity have attracted more and more attention of researchers and pharmaceutical industry. Kannan observed the high antiproliferative activity in human colon and liver cancer cells in the low molecular weight fraction (<5 kDa) of an Alcalase hydrolysate from rice bran.⁸² A 440.9 Da anchovy hydrophobic peptide was found to be able to induce apoptosis in human U937 lymphoma cells by increasing caspase-3 and caspase-8 activity.⁸³ Epinecidin-1, a peptide from fish (Epinephelus coioides) showed an antitumor effect similar to lytic peptides in human fibrosarcoma cells.⁸⁴ These anticancer peptides usually have cationic and small molecule. In this regard, electrostatic interactions between cationic anticancer peptides and anionic cell membrane components are believed to be a major factor in the selective killing of cancer cells. Although some researchers have suggested that these peptides exert activity against cancer cells through ion-permeable channel formation in the cell membrane,⁸⁵ the structure-activity relationship and the precise mechanism of the cancer cell-killing action of the peptides remains to be elucidated.

Spatial structure and anticancer activity

α-helical anticancer peptides

α-helical structure is the main structural characteristics of anticancer peptides. The high amphipathic and stable helical regions tend to be the heart of anticancer activity. Hydrophilic and hydrophobic amino acid side chains are arranged in two side of α-helical structure and form clear hydrophilic and hydrophobic surfaces. Or they are concentrated in the N-terminal and C-terminal to format distinct hydrophilic and hydrophobic sides. The amphiphilic structure is conducive for the membrane binding of anticancer peptides.⁸⁶ The anticancer activity of alpha-anticancer peptides (α-ACPs) normally occurs at micromolar levels but is not accompanied by significant levels of hemolysis or toxicity to other mammalian cells. Structure/function studies have established that architectural features of α-ACPs such as amphiphilicity levels and hydrophobic arc size are of major importance to the ability of these peptides to invade cancer cell membranes.⁸⁷

Various structure-activity studies have been conducted on the α-helical peptide. The 26-residue amphipathic α-helical peptide A12L/A20L (Ac- KWKSFLKTFKSLKKTVLHTLLKAISS - amide) with strong anticancer activity and specificity was used as the framework to study the effects of helicity of α-helical anticancer peptides on biological activities. Strong hemolytic activity of peptides generally correlated with high hydrophobicity, high amphipathicity, and high helicity. Lower helicity caused the decrease of anti-HeLa activity of peptides.⁸⁸ In most cases, the D-amino acid substituted peptides possessed an enhanced activity compared with L-diastereomers.⁸⁹ Magainin 2, N-Terminal truncation of the first 3 residues GIG does not much influence the anticancer activity, but the residue 4 (K) from the N-terminal is critical to the anticancer activity of magainin. The deletion of residue 4 (K) greatly reduces activity and further deletion of residues 5 and 6 (F and L) eliminates activity altogether.⁹⁰ That is because the truncated peptide is unable to span the lipid bilayer corresponding to loss of anticancer activity. LL-37 could interact with the alert signal and result in more NK cells (cells of immune system) that are capable of destroying the gastric cancer cells (Table 3). Cecropins (N-terminal α-helix of antimicrobial protein) kill neoplastic cells at concentrations lower than those required to lyse normal cells such as peripheral blood lymphocytes.⁹¹ BMAP-28 could kill various human leukemia cell lines at 1.5–6 μM concentrations. The hydrophobic tail (residues 19 to 27 or 28) at the COOH-terminus is important for BMAP peptides to mediate their cytotoxic effect.⁷⁸ Melittin, a 26 amino acid channel-forming cationic amphiphilic peptide, specifically counter selects for cells in culture that express high levels of the oncogene (Table 3). The structure facilitates the formation of ion-permeable pores in membranes, consequently inducing depolarization and cytolysis.⁸⁶

Table 3.

Summary of Selected Cationic Amphipathic Peptides with Anticancer Activity

Peptide	Sequence	Reference
α-helical
LL-37	LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES	96
BMAP-28	GGLRSLGRKILRAWKKYGPIIVPIIRI	78
Temporin-1CEa	FVDLKKIANIINSIFGK	97
Aureins	GLFDIIKKIAESF	98
Magainin	GIGKFLKKAKKFGKAFVKILKK	99
Melittin	GIGAVLKVLTTGLPALISWIKRKRQQ	100
Cecropin A	KWKLFKKIEKVGQNIRDGIIKAGPAVAVVGQATQIAK	101
Cecropin B	KWKIFKKIEKVGRNIRNGIIKAGPAVAVLGEAKAL	101
Citropins	GLFDVIKKVASVIGGL	102
β-sheet
β-defensin	NPVSCVRNKGICVPIRCPGSMKQIGTCVGRAVKCCRKK	96
Anginex	LHKRWKKFILIKVMKQLVNSDLKINA	103
Lactoferricin B	FKCRRWQWRMKKLGAPSITCVRRAF	104
Gomesin	ECRRLCYKQRCVTYCRGR	105
Tachyplesin	KWCFRVCYRGICYRRCR	93
Buforin II	RAGLQFPVGRLLRRLLRRLLR	85
Protegrin-1	RGGRLCYCRRRFCVCVGR	102

β-sheet anticancer peptides

β-sheet anticancer peptides are generally stabilized by the disulfide bonds, characterized by the presence of antiparallel β-sheets. In this family, defensins, lactoferricin, and tachyplesin are constrained by disulfide bonds. Tachyplesins represent a convenient scaffold for structure-activity studies due to their small size. Tachyplesin I could be against human gastric adenocarcinoma⁹² and human hepatocarcinoma cells⁹³ with an antiparallel β-sheet (residues 3–8 and 11–16) connected by a type I β-turn (residues 8–11) stabilized by two disulfide bonds.⁹⁴ Buforin IIb could be against 62 cancer cell lines by specifically targeting cancer cells through interaction with cell surface gangliosides. It deduced mitochondria-dependent apoptosis in the cells.⁹⁵ Protegrin-1 displayed cytotoxicity in human histiocytic lymphoma cell lines (Table 3). Bovine lactoferricin (LfcinB) consists of 25 amino acid residues including two Cys residues that create a disulfide bond linking the highly positively charged NH₂-terminal region and the COOH-terminal region of the peptide.⁵⁶ It could kill leukemia cells, fibrosarcoma cells, various carcinomas, and neuroblastoma cells through suppressing both basic fibroblast growth factor bFGF- and VEGF- driven proliferation and migration of human endothelial cells, including α-defensins and β-defensin (Table 3). The disulfide connections in α-defensins are Cys1–Cys6, Cys2–Cys4, and Cys3–Cys5 (the number indicates the location of the Cys residue in the amino acid sequence from the N-terminus), while in β-defensin are Cys1–Cys5, Cys2–Cys4, and Cys3–Cys6.⁹⁵ Three pairs of disulfide bonds are important to stabilize the spatial configuration of defensin. The structures of the two main defensin subfamilies, α and β-defensins, were analyzed by two-dimensional NMR and X-ray crystallography were used for analysis and both were found consisting of a triple-stranded β-sheet. Various types of α-helical and β-sheet anticancer peptides are summarized below.

Anticancer peptides and cancer cell membrane

The bilayer phospholipids of cell membrane function as a permeability barrier and regulate the flux of metabolites between the external environment and the intracellular content. Therefore, membrane is the first and main factor for the anticancer activity exertion. Cancer cell membranes typically carry a net negative charge due to a higher than normal expression of anionic molecules. The initial association of peptides with the cancer cell membrane occurs through electrostatic interactions between the cationic peptide and anionic lipopolysaccharide in the outer membrane leading to membrane perturbation.⁸⁶ The peptides associate with the outer monolayer of the cytoplasmic membrane. If this reorientation leads to perturbation of the integrity of the cytoplasmic membrane, cancer cell membrane is disruptive.¹⁰⁶ The net charge and the number of positive charge also influence the activity of peptides. There is a sharp transition of hemolytic activity on the polar face of V13K, the change from +8 to +9 resulted in greater than 32-fold increase in hemolytic activity.¹⁰⁷ In contrast to neoplastic cells, electrostatic interactions between anticancer peptides and untransformed cells are not favored because of the overall neutral charge conferred on healthy cells by the zwitterionic nature of their major membrane components. Besides electrostatic interactions, molecular properties of lipids such as their molecular shape and structural folding play a significant role for the aggregation state of lipids to affect interactions between membrane and active peptides.¹⁰⁸ Apart from the surface characteristics, membrane fluidity also appears to be altered in tumorigenic cells. The increased fluidity of the cancer cell membrane may enhance the lytic activity of ACPs by facilitating membrane destabilization for lymphomas, lung carcinomas, and neural tumors.¹⁰⁹

Anticancer peptides induce cell death with different mechanisms, including apoptosis, affecting the tubulin-microtubule equilibrium, or inhibiting angiogenesis. Anticancer peptide executes its function depends on a number of physicochemical properties: the amino acid sequence, net charge, amphipathicity, hydrophobicity, structural folding (includes secondary structure, dynamics, and orientation) in membranes, oligomerization, peptide concentration, and membrane composition.¹¹⁰ They are normally characterized by low molecular weight (in the majority of cases less than 30 amino acids) and exhibit a predominantly cationic amphipathic structure making them prone to interact with anionic cell membrane surfaces.^111,112 Some cationic amphipathic peptides with anticancer activity are summarized in Table 3. Much progress has been achieved in the structure-activity relationship of anticancer peptides in recent decades, which contributes both to an increased understanding of the immune system and to their potential as clinical antibiotics. Nonetheless, most studies on anticancer peptide are still in vitro. More in vivo experiments of anticancer peptides are needed to bridge across the clinical application.

Conclusions

The studies cited in this review indicated that biological activities of peptides are closely associated with molecular structure and molecular size. Peptides with higher ACE inhibitory activity usually have aromatic or alkaline amino acids in N-terminal, higher quantity of hydrophobic and positively charged amino acids in C-terminal. Nonpolar aliphatic peptides showed higher antioxidant activity. Both primary and secondary structures are important to the anticancer peptides. Due to the difficulty to isolate and purify specific bioactive peptides from the mixture of natural protein hydrolysates, the progress in structure-activity research of bioactive peptides will play an important role in synthesis of more specific, high bioactive, and low toxic peptides for prevention and treatment of diseases such as hypertension and different types of cancers. Currently, products of bioactive peptide only take up a small market place. With the urgent demand of human natural medicine, research on active peptides will continue to advance.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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