How to Improve the Activity of Peptide?

Peptides are organic compounds formed by dehydration between various amino acid molecules, linked by peptide bonds. Their molecular weight ranges from 1 to 10 kDa, falling between small molecules and biological macromolecules. Many signaling molecules in the body are peptides or proteins, and the onset and progression of diseases are closely related to these peptides or proteins. Among the known bioactive peptides, most are secreted or metabolically converted by the body. Based on the source of bioactive peptides, they can be classified into two categories: the first category includes proteins and bioactive peptides derived from the organism itself, known as endogenous bioactive peptides. Endogenous bioactive peptides have a low concentration in the body, but are widely distributed and highly effective. The second category includes bioactive peptides and antibiotics derived from plants and animals, known as exogenous bioactive peptides. Exogenous bioactive peptides are potent and widely distributed. The peptide library consisting of both endogenous and exogenous bioactive peptides provides novel structural frameworks for drug development, and many marketed drugs originate from the discovery of peptide compounds.

However, the classical peptide structure is not stable against in vivo proteases and is quickly degraded after entering the body. In addition, most bioactive peptides have poor bioavailability, are not orally bioavailable, and require modifications to the formulation to identify suitable administration routes. Due to these factors, structural modification and chemical alteration of bioactive peptides are necessary. The objectives of bioactive peptide modifications are diverse, mainly including improving the affinity and selectivity of the peptide for its receptor; enhancing the pharmacokinetic stability of the peptide; reducing degradation or elimination of the peptide in the body; improving the membrane permeability of the peptide; and improving the water solubility of hydrophobic peptides. This article summarizes and categorizes strategies for modifying peptide molecular structures based on different modification goals. These strategies are divided into two categories: one focuses on modifying the peptide backbone, including non-natural amino acid modifications, pseudo-peptoid strategies, inverse peptide strategies, cyclization strategies, and terminal structure modifications; the other category involves introducing other groups for structural optimization and functional modification without changing the peptide backbone, including modifications with long-chain fatty acids, polyethylene glycol, protein fusion strategies, and cholesterol modifications. By applying these structural modification strategies to lead compounds, the drug-like properties of peptide compounds can be significantly improved, providing theoretical guidance and practical experience for the development of innovative peptide-based drugs.

The relationship between the chemical structure of a drug and its pharmacological activity has always been an important area of research in medicinal chemistry. Activity is a prerequisite for a compound to be developed into a drug, and the same applies to peptides. Some natural peptides or artificially synthesized peptide molecules have poor bioactivity, requiring chemical modification to improve their affinity for receptors and enhance their activity. Main methods for improving the activity of peptide molecules include modifications of the terminal structure, concatenation strategies, cyclization strategies, non-natural amino acid modifications, pseudo-peptide strategies, and cholesterol modifications.

Peptide Backbone Modification

Modifying and remodeling the peptide backbone to enhance the activity of peptide molecules involves several main strategies, including terminal structure modifications, concatenation strategies, cyclization strategies, non-natural amino acid modifications, and pseudo-peptide strategies.

N-Cap Structural Modification

Peptides with exposed N- and C-termini are prone to recognition by exopeptidases, leading to cleavage and degradation, which results in the loss of activity. However, by modifying the N-terminal and C-terminal structures, one can improve the metabolic stability of peptide molecules while maintaining or even enhancing their activity. For example, Stoermer et al. reported a tripeptide (KKR sequence) aldehyde compound as a West Nile virus (WNV) protease inhibitor. Similarly, Yin Zheng's research group reported a tripeptide (KRR sequence) aldehyde compound as a Dengue virus (DENV) protease inhibitor, and its activity was significantly enhanced compared to other tetrapeptide aldehyde compounds. Based on these studies, Andreas et al. found that different structural modifications at the N-terminal of tripeptide aldehyde compounds had varying effects on their activity. They explored modifications to the N-terminal cap region and synthesized various acylated tripeptide aldehyde compounds. The study showed that different acylation modifications had a significant impact on the antiviral activity of these compounds. For example, the N-terminal phenylacetyl-modified tripeptide aldehyde (compound 2) exhibited good inhibitory activity against both Dengue and West Nile viruses, while the N-terminal 4-phenylphenylacetyl-modified tripeptide aldehyde (compound 11) showed nearly a 7-fold increase in inhibitory activity against West Nile virus compared to compound 2. This suggests that structural modifications at the N-terminal have a significant impact on the activity of peptide compounds and can be used as a strategy for peptide modification.

Inhibitory concentrations of X-KRR-H and X-KKR-H aldehyde inhibitors where X is a varying N-terminal cap group (n = norleucine). (PENG Jing-jing, et al. 2020)

C-Terminal Structural Modification

In contrast to N-terminal structural modifications, C-terminal structural modifications also have broad applications in the modification of peptide molecules. C-terminal modification strategies have been successfully used in the structural remodeling of various viral protease inhibitors. Hepatitis C virus (HCV) NS3/4A protease is a serine protease, and most serine protease inhibitors contain electrophilic groups that form covalent bonds with the hydroxyl group of the catalytic triad serine. Researchers started with a decapeptide substrate, simplified the peptide chain, and modified the C-terminal structure to obtain the active peptide aldehyde 19, which has a binding constant of 12 μmol·L^-1 against HCV NS3/4A protease. However, the aldehyde group showed poor chemical and metabolic stability. Therefore, researchers modified the aldehyde group by replacing it with α-halogenated ketones, heteroatom-substituted ketones, α-diketones, and α-ketoamides. The resulting ketoamide compound 20 exhibited a 12-fold increase in the binding constant against the serine protease NS3/4A.

Upon analyzing the interaction between the ketoamide 20 and serine protease, researchers found that the ketoamide structure could covalently bind to the serine at position 139 and form hydrogen bonds with the nearby glutamic acid at position 137 and serine at position 138. This enhanced the compound's binding to the NS3/4A protease, thereby improving its antiviral activity.

C-terminal structure modification to improve activity against serine protease (PENG Jing-jing, et al. 2020)

EV71 3C protease is a cysteine protease. Yin Zheng et al. discovered a peptide aldehyde molecule 21 with good inhibitory activity against the EV71 virus (EC50 = 0.11 μmol·L^-1). However, due to the poor stability of the aldehyde group, the drug-like properties of the compound were suboptimal. In their subsequent structural modifications, they focused on optimizing the aldehyde group and synthesized a hydroxycyanide compound 22, which showed an improved activity against EV71 with an EC50 of 0.056 μmol·L^-1.

Through molecular docking, the interaction between compound 22 and EV71 3C protease was analyzed. The molecular docking results indicated that, compared to the aldehyde group, the cyano group in the hydroxycyanide structure formed hydrogen bonds with glutamine at position 146 and glutamic acid at position 24 through water molecules, thereby enhancing the binding between the compound and EV71 3C protease. This interaction led to the improved antiviral activity of the compound.

C-terminal structure modification to improve activity against EV71 3C protease( Zhai Y, et al. )

Concatenation Strategy

In the modification of peptide compounds, it is often necessary to optimize and modify different sites simultaneously. The concatenation strategy is an efficient method for structural optimization. First, active compounds are obtained by separately modifying and optimizing the N- and C-terminal structures. Then, the advantageous segments are concatenated to quickly generate compounds with higher activity.

For example, the pseudo-peptide compound 23 is an inhibitor of Dengue virus protease, with an inhibitory activity IC50 of 13.3 μmol·L^-1. During the modification of this compound, researchers adopted a strategy of optimizing the N- and C-terminal structures separately. In the N-terminal structural modification, researchers found that the peptide molecule 24, which was modified with an N-terminal cap structure, exhibited enhanced activity, with an IC50 of 2.5 μmol·L^-1. In the C-terminal side-chain modification, they discovered that replacing the n-butyl side chain with a phenyl group in compound 25 also improved the inhibition of Dengue virus, with a nearly 4-fold increase in activity.

Considering that both modification strategies could improve the activity of the compound, the researchers concatenated the two advantageous segments to form compound 26. This compound exhibited an inhibitory activity against Dengue virus with an IC50 of 0.6 μmol·L^-1, representing a nearly 20-fold increase in activity.

Splicing strategy to improve DENV inhibitory activity (PENG Jing-jing, et al. 2020)

Cyclization Strategy

In many cases, the molecular flexibility of linear peptides causes conformational changes, which reduce their binding strength and selectivity with the receptor. Furthermore, exopeptidases such as aminopeptidases and carboxypeptidases in the body are also prone to gradually cleave the peptide chain from both ends, leading to degradation. Therefore, cyclization of the peptide chain, which restricts its conformation, is an important structural modification strategy to improve the biological stability and bioactivity of peptide molecules. Studies have shown that after converting linear peptides into cyclic peptides, the bioactivity of many compounds increases by tens to hundreds of thousands of times. Many natural product peptides with antimicrobial, antiviral, antitumor, and immunomodulatory activities often contain different types of backbone cyclization structures. Thus, cyclization is a crucial strategy in peptide structural modification.

For example, a linear octapeptide compound 27 showed weak binding activity to Dengue virus NS2B-NS3 protease (Ki = 42 μmol·L^-1). Xu et al. hypothesized that the poor binding activity of the linear peptide might be due to its larger spatial occupation, which prevents effective binding to the protease. In contrast, cyclizing the peptide could reduce the spatial occupation and improve the compound's binding activity to NS2B-NS3 protease. They designed and synthesized a series of cyclic peptide structures and tested their binding activities. They found that cyclic peptide 28, with its specific conformation, could bind more effectively to Dengue virus NS2B-NS3 protease. Compared to the linear peptide, its activity improved nearly 20-fold, with a Ki value of 2.2 μmol·L^-1.

Cyclization strategy to improve activity against NS2B-NS3 protease (PENG Jing-jing, et al. 2020)

In addition to the head-to-tail cyclic strategy, local cyclization often stabilizes the conformation of peptide compounds by limiting the cyclization region, enhancing the interaction between the peptide and the receptor, and improving the peptide's activity. The signal transducer and activator of transcription 3 (STAT3) is a transcription factor that directly transmits extracellular receptor signals to the nucleus. Persistent activation of STAT3 promotes cell proliferation, leading to tumor formation, and inhibits apoptosis in tumor cells.

In early research, Wang Shaomeng and colleagues at the University of Michigan found that the gp130 pYLPQTV peptide 29 exhibited strong affinity for the STAT3 receptor. Studies showed that the threonine and valine residues in the gp130 phosphopeptide could be replaced by benzylamine without affecting the peptide's binding activity to STAT3. As a result, they truncated the phosphopeptide by removing threonine and valine, using benzylamine to cap the C-terminus, which resulted in truncated phosphopeptide 30. The binding affinity of peptide 30 to STAT3 was measured with a Ki of 350 nmol·L^-1. Through molecular docking, they found that the isobutyl side chain of leucine and the five-membered ring of proline could cyclize to form a bicyclic lactam structure without disrupting the β-turn conformation of peptide 30.

Thus, applying the local cyclization strategy, they designed and synthesized the constrained bicyclic peptide 31, which exhibited a significantly improved binding affinity to STAT3 with a Ki of 17 nmol·L^-1, nearly 20 times stronger than peptide 30. Molecular docking results showed that the bicyclic lactam structure maintained the β-turn conformation of peptide 30 effectively. To verify whether the Cbz protecting group was related to the peptide's binding with STAT3, they replaced the benzyl carbamate group with an acetyl group, producing peptide 32, which also had a similar binding affinity for STAT3 (Ki = 15 nmol·L^-1), indicating that the Cbz group was not essential for activity.

Subsequently, they evaluated the inhibitory activity of peptide 32 on two human breast cancer cell lines, MDA-MB-231 and MDA-MB-468, which overexpress high levels of phosphorylated STAT3. However, at 100 μmol·L^-1, peptide 32 showed no inhibitory activity against these two cancer cell lines, likely due to the high polarity of the phosphopeptide, which prevents its passage through the cell membrane. To enhance the inhibitory activity of peptide 32, they introduced a fatty acid at the N-terminus of the phosphopeptide, resulting in fatty acid-modified phosphopeptide 33. This peptide showed a binding affinity of Ki = 10 nmol·L^-1 for STAT3, and its inhibitory activity against the two cell lines, with IC50 values of 25 and 35 μmol·L^-1, demonstrated some suppression at the cellular level. This also suggested that fatty acid modification could alter the properties of the peptide and improve its membrane permeability.

Partial cyclization strategy to improve peptide binding affinity with STAT3 receptor (PENG Jing-jing, et al. 2020)

Non-Natural Amino Acid Modification

Captopril was the first reported angiotensin-converting enzyme (ACE) inhibitor, approved by the U.S. Food and Drug Administration (FDA) in 1981 for the treatment of hypertension. However, clinical studies indicated that the thiol group in captopril might cause adverse reactions such as rashes and loss of appetite in patients. To address this issue, researchers sought to develop new ACE inhibitors as antihypertensive drugs.

In the early development of captopril, active compound 34 exhibited some ACE inhibitory activity with an IC₅₀ of 4.9 μmol·L^-1. To improve this activity, researchers replaced the methylene group with a nitrogen atom through bioisosteric replacement, resulting in the dipeptide lead compound 35, which showed a doubling of activity. The introduction of the nitrogen atom increased the hydrophilicity of the compound. To balance the enhanced hydrophilicity caused by the nitrogen, the researchers introduced an alkyl side chain at the α-position of the amino acid, further improving the activity to 0.09 μmol·L^-1.

Subsequent studies on the structure-activity relationship (SAR) of the α-alkyl side chain found that the compound with a phenylethyl group was optimal for activity. The researchers then esterified the carboxyl group to develop the prodrug enalapril (compound 37), which was approved by the FDA in 1985 for the treatment of hypertension and heart failure. Enalapril exhibited a 74-fold increase in ACE inhibitory activity compared to compound 36, demonstrating that the introduction of non-natural amino acids can significantly enhance the pharmacological activity of peptide-based molecules.

Introduction of unnatural amino acid to improve ACE inhibitory activity (PENG Jing-jing, et al. 2020)

Pseudopeptide Strategy

Pseudopeptides mimic the transition state of peptide hydrolysis and utilize bioelectronic repulsion principles to replace the easily hydrolyzed amide bond. This prevents peptides from being cleaved by proteases, thereby preserving or even enhancing the pharmacological activity of peptide compounds. Figure lists some representative structures of pseudopeptides.

Representative structures of pseudopeptides (PENG Jing-jing, et al. 2020)

Fragment 38 (hydroxymethylene) is a common structural fragment shared by many HIV protease inhibitors, renin inhibitors, and β-secretase inhibitors. The basic design principle is to use the pseudopeptide strategy, simulating the transition state during amide bond hydrolysis and replacing the easily hydrolyzed amide bond. Szelke et al. developed a pseudopeptide inhibitor 43 by replacing the amide bond with hydroxymethylene in the leucine-valine (Leu-Val) segment of renin substrate 42. This modification significantly improved the HIV-1 protease inhibitory activity, with an IC50 value of 0.0007 μmol·L^-1.

Pseudopeptide Strategy to improve anti-HIV-1 protease inhibitory (PENG Jing-jing, et al. 2020)

The siloxane fragment 41 also has widespread applications. Since carbon and silicon atoms belong to the same main group, their properties are quite similar. Silicon atoms, compared to carbon atoms, tend to favor sp³ hybridization. As a result, fragment 41 is less likely to undergo elimination reactions to form siloxane, and its stability is higher than that of ketones. Therefore, when designing and modifying active peptides, introducing fragment 41 can enhance interactions with the enzyme's active site while also providing chemical stability. This makes it widely used in drug design. Fragment 41 has shown good activity in many peptide analogs. For example, an ACE inhibitor 44 containing fragment 41 exhibits an inhibition activity of 3.8 nmol·L^-1 against ACE; similarly, an HIV protease inhibitor 45 containing fragment 41 also demonstrates an inhibition activity of 2.7 nmol·L^-1 against the protease. These results indicate that this type of structure plays an important role in the modification of active peptide structures.

Representative silicon containing pseudopeptide inhibitor (PENG Jing-jing, et al. 2020)

Conjugation Group Modification

The primary method of enhancing the activity of peptide molecules through conjugation group modification is cholesterol modification.

Cholesterol modification is also an important structural modification strategy for peptide molecules. The introduction of cholesterol often improves the pharmacokinetics of peptides, particularly by increasing their in vivo half-life, while also enhancing their pharmacological activity. Wang et al. evaluated the antiviral activity of peptides through cell-cell fusion experiments and found that the peptide m4HR exhibited some anti-HIV-1 activity (IC50 = 36,910 nmol·L^-1). When a cholesterol molecule was conjugated to the C-terminus of m4HR to form compound 46, its antiviral activity increased by 200-fold (IC50 = 57.2 nmol·L^-1). Further modification at the N-terminus led to an even greater enhancement in HIV-1 inhibition. Among these, the most active peptide molecule was 47, which showed an IC50 of 8.3 nmol·L^-1. These examples of structural optimization further demonstrate the important application of cholesterol modification in the enhancement of peptide drug activity.

Cholesterol modification to improve anti-viral activity against HIV-1

Replacing Phe/Tyr with pyridine motif to improve water-solubility of glucagon (Aib, aminoisobutyric acid)

Peptide modifications services at Creative Peptides

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