Solid-Phase Synthesis

This is one of the most commonly used methods for peptide synthesis. It is based on the stepwise addition of amino acid units on a solid-phase support to build peptide chains. The solid phase support materials typically used are highly reactive resins such as Rink-Amide or Fmoc-AM resin. During the synthesis process, the amino acid unit is immobilized on the solid phase by a reactive protecting group, followed by coupling, deprotection and elution to finally obtain the target peptide.

Solution-Phase Synthesis

This method synthesizes peptides in solution. It is usually applied to shorter peptide chains. In solution-phase synthesis, the amino acid unit is coupled to other amino acids in a coupling reaction via an activator (e.g., an activated acid or an activated carboxylic acid). After the reaction, steps such as removal of protective groups and purification are required to obtain a pure peptide product.

Chemical Synthesis

Chemical synthesis is a technique for synthesizing peptide by methods of organic synthesis. It usually involves a reaction using chemical reagents such as protecting groups, activators and solvents. Chemical synthesis methods can be used to synthesize complex peptide structures, but are more challenging for long chain peptides due to the number of steps and the potential for side reactions.

Biosynthesis

Biosynthesis is the synthesis of peptides using cellular mechanisms and enzymes in the organism. This method is commonly used in the synthesis of natural peptides, such as in the production of hormones and peptide drugs. Through genetic engineering techniques, genes encoding peptide are introduced into appropriate expression systems to synthesize the target peptide within the cell.

These synthetic methods are widely used in the field of peptide drugs. Each method has its advantages and limitations, and the choice of synthesis depends on factors such as peptide structural complexity, target purity requirements, scale and economics. Researchers choose the most suitable synthesis strategy and technique based on the specific synthesis needs.

What  is Solid-Phase Synthesis?

Currently, solid-phase synthesis is the mainstay of peptide drug synthesis processes. The synthesis process of peptide drugs is mainly categorized into chemical synthesis method and biosynthesis method in consideration of technology application, production process, production cost, quality management and environmental protection requirements.

The chemical synthesis method is mainly used to synthesize peptide through amino acid condensation reaction. Such methods include solid-phase synthesis and solution-phase synthesis. The solid-phase synthesis method is mainly used for the synthesis of medium-length peptides, which is based on the gradual addition of amino acid units on a solid-phase support to build peptide chains. The solution-phase synthesis method is mainly used for the synthesis of short peptides, in which the amino acid units are coupled to other amino acids by activators in a coupling reaction.

Biosynthesis includes enzymatic hydrolysis, fermentation and genetic engineering methods. These methods utilize cellular mechanisms and enzymes in the organism to synthesize peptides. Enzymatic hydrolysis is the enzymatic cleavage of raw peptides into target peptides by the action of enzymes. Fermentation is used to synthesize peptides through the process of fermentation by microorganisms. The genetic engineering method utilizes genetic engineering techniques to introduce the gene encoding the target peptide into an appropriate expression system that will synthesize the target peptide within the cell.

At present, the solid-phase synthesis method is relatively mature, and most of the marketed peptide drugs use this process route. This is due to the advantages of solid-phase synthesis for peptide synthesis, which offers a high degree of control, high yields and high purity. However, with the continuous development and improvement of biosynthesis technology, biosynthesis also has a broad application prospect in the synthesis of peptide drugs.

Solid-phase synthesis is currently the most commonly used peptide synthesis process, which has become quite mature after years of development. In 1963, Merrifield proposed the classical solid-phase peptide synthesis (SPPS) method, a milestone that marked a new era in peptide synthesis. After decades of improvement and refinement, the solid-phase synthesis has formed a mature process with the Boc method and the Fmoc method as the main systems.

The core idea of the solid-phase synthesis is to combine the carboxyl group of the first amino acid of the target peptide with a solid-phase carrier (usually a resin) through covalent bonding. Then, starting with the amino group of that amino acid, the acylation reaction is carried out by removing the aminoprotecting group and the excess carboxyl group of the second amino acid. This step is repeated over and over again, resulting in the target peptide.

The main difference between the different solid-phase synthesis methods is the amine protecting group used. The Boc method uses Boc as the amino protecting group, while the side chain protecting group is usually a benzyl alcohol compound. The Fmoc method, on the other hand, uses Fmoc as the aminoprotecting group. Compared with the Boc method, the Fmoc method has the advantages of stable acidity, mild reaction conditions, fewer side reactions, and higher yields, so it has been widely used in the field of peptide solid-phase synthesis.

The advantage of the solid-phase synthesis is that it simplifies and automates the subsequent processing of each step. It avoids manual handling and losses in material transfer. However, the solid-phase synthesis has some drawbacks, such as shorter synthesis sequences, longer time-consumption, lower product purity, higher cost, and higher toxicity of reagents.

Difference Between Boc and Fmoc Methods

The Boc method and the Fmoc method are two amino-protected group methods commonly used in solid-phase synthesis, and they have some specific differences in peptide synthesis, as shown below:

1. Amino Protecting Group

Boc method (t-Butyloxycarbonyl): The Boc protecting group is obtained by reacting t-Butanol acid with the amino group of an amino acid, protecting the amino group of the amino acid. This protective group is stable under alkaline conditions and can be removed under strongly acidic conditions.

Fmoc method (Fluorenylmethyloxycarbonyl): The Fmoc protecting group is obtained by reacting the amino group of an amino acid with Fmoc-Cl, which also protects the amino group of the amino acid. Fmoc protecting groups are stable under acidic conditions and can be removed under alkaline conditions.

• De-protection Methods

Boc Methods: Boc protective groups can be removed under strong acidic conditions. Commonly used deprotection reagents are acids, such as trifluoroacetic acid (TFA). The process of removing the Boc protecting group requires the use of organic solvents such as dimethylformamide (DMF) or dichloromethane (DCM).

Fmoc method: Fmoc protective groups can be removed under alkaline conditions. Commonly used deprotection reagents are alkaline solutions, such as alkaline acetone (piperidine) solution. The process of removing Fmoc protecting groups usually requires the use of organic solvents such as N,N-dimethylformamide (DMF) or dichloromethane (DCM).

• Photosensitivity

Boc method: The Boc protecting group is not photosensitive, so photolithography operations can be performed using UV light under the Boc method.

Fmoc method: The Fmoc protecting group is photosensitive and photolytic reaction occurs when exposed to UV light, so protection and deprotection operations under photosensitizing conditions are required under the Fmoc method.

• Reaction Conditions

Boc method: The reaction conditions under the Boc method are relatively mild and suitable for general amino acid condensation reactions.

Fmoc method: The reaction conditions under the Fmoc method are also relatively mild, and it is suitable for general amino acid condensation reactions, but it is necessary to pay attention to the characteristics of the photosensitive protective group.

Overall, the Boc and Fmoc methods differ in their aminoprotecting groups and deprotection methods, and the choice of which method to use depends on the specific synthetic needs and conditions. In practical applications, the Fmoc method has become one of the more commonly used methods in solid-phase synthesis of peptides due to its advantages of acidic stability and few side reactions.

What is Solution-Phase Synthesis?

Solution-phase synthesis is a widely used method for peptide synthesis with obvious advantages and disadvantages.

The solution-phase synthesis can be subdivided into stepwise and segmented synthesis methods. Stepwise synthesis usually starts from the C-terminus of the peptide and adds amino acids one by one until the target peptide is synthesized. This method is generally applicable to the synthesis of short peptides or various bioactive peptide fragments. Segmental synthesis is a fast developing peptide synthesis method in recent years, and its chemical specificity has a prominent role in the field of long peptide synthesis. The core idea of the segmental synthesis method is to rationally divide the target peptide into a number of fragments, and after synthesizing these fragments, carry out linkage and condensation to finally obtain the target peptide. The major linkage modes for segmental synthesis include natural chemical linkage, chemical regioselective linkage, removable auxiliary group linkage, light-sensitive auxiliary group linkage, Staudinger linkage, and orthogonal chemical linkage.

Solution-phase synthesis offers many advantages such as high purity, ease of purification and lower cost. However, it is also somewhat complex and cumbersome, requiring time and effort. At the same time, the segmental synthesis technique has limitations in its application because it is based on natural chemical linkages, which must depend on cysteine (Cys) residues.

Solution-phase synthesis is a widely used method for peptide synthesis with the advantages of high purity, easy purification and lower cost. Segmental synthesis, as one of the strategies, is rapidly developing in the field of long peptide synthesis, which improves the efficiency and specificity of synthesis by rationally segmenting the target peptide and selecting the appropriate linkage. However, the solution-phase synthesis method has some challenges such as high complexity, cumbersome operation, time consumption, and the application of the segmental synthesis is limited by cysteine residues.

Common Methods for Solution-phase Synthesis

1. Natural Chemical Connection

Natural chemical connection are those that utilize chemical reactions between natural amino acids in two fragments. The most common natural chemical connection is the formation of a connection through an amide bond between an amine group (N-terminal) and a carboxyl group (C-terminal). This connection is made through a nucleophilic substitution reaction between the amine group and the carboxyl group, in which the nitrogen atom of the amine group attacks the carbon atom of the carboxyl group to produce an amide bond. Natural chemical connection methods are widely used in peptide synthesis because they are reliable, efficient, and have good compatibility.

• Chemical Region Selection Connection

Chemical region selection connection refers to the joining of unnatural chemical regions in two fragments of a peptide by selecting a specific chemical reaction between the two fragments. This linkage method does not rely on the way natural amino acids are linked to each other, but rather utilizes specific reactions between non-natural chemical groups in the fragments to occur the linkage. Common Chemical region selection connection methods include the use of specific functional groups, protecting groups, and ligands to achieve the linkage. This connection method can occur in specific chemical environments and is highly selective and specific, making it useful in the synthesis of complex peptides or peptides containing unnatural chemical groups.

• Removable Auxiliary Group Connection

The removable auxiliary group connection is a method of realizing a Connection by introducing a removable auxiliary group. These auxiliary groups can be easily removed after the connection is complete, leaving no trace. Commonly used methods of removable auxiliary group connection include the use of acrylic auxiliary groups and ester auxiliary groups. After the connection is completed, the target peptide can be obtained by cleavage of the auxiliary group through appropriate conditions, such as acidic or basic conditions.

• Light-sensitive Auxiliary Group Connection

Light-sensitive auxiliary group connection is a method that utilizes a light-sensitive compound as an auxiliary group, and the linkage occurs under the illumination of light at a specific wavelength. This connection method utilizes the photoactivity of the photosensitive compound to activate the auxiliary group by light to make the connection to another fragment. The light-sensitive auxiliary group connection method is highly spatially and temporally controllable, allows connection to be realized under specific conditions, and avoids the use of other chemical reagents.

• Staudinger Connection

A Staudinger connection is a method of making a connection through the use of a Staudinger reaction. The Staudinger reaction is a chemical reaction in which a new bond is formed between two functional groups. In peptide synthesis, the Staudinger linkage can be used to connect unnatural amino acid residues in two fragments. This linkage method has been widely used in the field of peptide synthesis.

• Orthogonal chemical Connection

Orthogonal chemical connection is a method of achieving linkage through the use of highly selective chemical reactions. These reactions enable interference-free linkage of fragments in peptide synthesis, i.e., connection occurs only with the target fragment and not with other functional groups under specific conditions. The advantage of this connection method is that the connection can be realized in a complex chemical environment, avoiding the occurrence of side reactions.

What is Biosynthesis?

Enzymatic Hydrolysis

This is a method for degrading large proteins into active peptides through the use of biological enzymes. This method can maintain the natural properties of peptides and retain the original nutritional value of proteins, and is commonly used in food, cosmetic, feed and other industries. However, the peptides obtained after enzymatic hydrolysis are unsuitable for the synthesis of specific peptide sequences because of the difficulty in separating them. Therefore, enzymatic hydrolysis is not a commonly used method in the field of peptide synthesis.

Fermentation Method

This is a method method of obtaining a natural peptide by a metabolic process of a microorganism. The production cost of this method is low, but its application is more limited. Currently, the main peptides that can be synthesized independently by fermentation method include ε-polylysine (ε-PL), γ-polyglutamic acid (γ-PGA), and cyanobacterial peptides.

Genetic Engineering Method

Peptides are synthesized by controlling the DNA template using DNA recombination technology. This method is suitable for the preparation of long peptides. The genetic engineering method has the advantages of strong expression targeting, high safety and low cost. However, it also has some drawbacks, such as a long development cycle, difficulty in isolation, low yield, and difficulty in large-scale production. Enzymatic hydrolysis is mainly used in food, cosmetic, feed and other industries; fermentation method is suitable for obtaining specific peptides, but the scope of application is narrower; genetic engineering method is suitable for the preparation of long peptides, which has certain advantages and disadvantages. In the field of peptide synthesis, the commonly used methods are ribosomal translation and chemical synthesis, which enable the synthesis and customization of specific peptide sequences.

Mechanism of DKP Formation

DKP can be formed through acid- or base-catalyzed reactions. The nucleophilic attack by the Nα group of the peptide on the carbonyl group between the second and third amino acid residues, either as an amide or ester carbonyl, leads to the cleavage of the amide or ester bond. The N-terminal dipeptide, as a derivative of the six-membered ring DKP, is separated from the peptide molecule. Consequently, the peptide molecule is truncated at its N-terminus by two amino acid residues, forming a byproduct characteristic of DKP. When the involved peptide is a depsipeptide (X=O in Figure 1), the formation of DKP is accelerated, as hydroxy derivatives act as better leaving groups than their amino counterparts.

FDA-approved Peptide Drugs

Considering the common occurrence of degradation in peptides with Nα groups, whether the N-terminus should be blocked to prevent DKP formation becomes a crucial consideration in peptide drug design. In fact, there are peptide drugs on the market that have a free Nα group. It is noteworthy that several peptide entities at their N-terminus are blocked through acylation, cyclization, or pyroglutamation. In peptide drugs where Nα is not blocked, they are often cyclic peptides with disulfide bonds or amide cyclization, or they may be prodrugs. Cyclization is known to effectively limit Nα activity, thereby reducing the formation of DKP. There are also three peptide drugs with free Nα, namely Golotimod, Thymodepressin, and NOV-002, which are actually derivatives modified from (L/D)-Glu side chain carboxylate salts. In these cases, the formation of DKP is also hindered.

Characteristics of Peptides Prone to DKP Formation

• N-terminal Secondary Amino Acid (Proline, Sarcosine)

When Pro or Sar is positioned as the second residue at the N-terminus of a peptide, the cis configuration of the peptide bond between the second and first amino acid residues is strengthened due to the properties of these secondary amino acids. This stimulation leads to DKP formation in peptides with sequences such as Phe-Pro at the N-terminus, as seen in recombinant human growth hormone (rhGH). Substituting Pro with other amino acids significantly inhibits DKP formation. N-alkyl amino acids appear at the first amino acid position in some marketed peptide drugs, such as oritavancin (N-Me-D-Val) and saralasin (Sar-Arg). It is noteworthy that despite the potential high risk of DKP with Xaa-Pro as the peptide N-terminus, approved peptide drugs like nesiritide (32-amino acid disulfide-bonded peptide) and bivalirudin (20-amino acid linear peptide) have N-terminal sequences containing H-Ser-Pro and H-D-Phe-Pro, respectively.

• Cα,α-Dialkylated Amino Acids

Cα,α-dialkylated amino acids synergistically contribute to peptide DKP formation. Although they do not promote cis amide bonds, they impose enhanced conformational restrictions on Cα-C’ and N-Cα bonds, favoring the helical secondary conformation. This conformational preference brings Nα closer spatially to the unstable amide carbonyl, promoting DKP formation. While introducing Cα,α-dialkylated amino acids may enhance susceptibility to DKP formation, some marketed peptide drugs like semaglutide (H-His-Aib, GLP-1 analog, 31 amino acids) and macimorelin (H-Aib-D-Trp) contain Aib (2-aminoisobutyric acid, Cα,α-dialkylated amino acid) residues near the N-terminus structural domain.

• N-terminal Glycine (Gly):

The correlation between DKP formation and N-terminal residues can be influenced by pKa, volume, amino acid configuration, and the conformational stability of DKP molecules. H-Gly-Pro-peptide is a highly sensitive N-terminal sequence that readily forms DKP due to the spatial promotion effect of Gly as the N-terminal residue and the cis configuration driven by Pro. H-Gly-Gly is another sequence highly sensitive to DKP formation. Marketed peptide drug peginesatide has a branched double N-terminal sequence Gly-Gly, but its N-terminal Gly is acetylated. Another marketed peptide drug terlipressin (cyclic peptide with disulfide bonds) has the N-terminal sequence H-Gly-Gly-Gly, and it is found to be susceptible to DKP formation during the formulation process. Peptide sequences with Gly as the N-terminal third residue also undergo DKP formation. Other marketed peptide drugs with free N-terminal Gly residues include desmopressin (H-Gly-Arg) and PepGen P-15 (H-Gly-Thr).

• N-terminal Histidine (His)

Peptides with the sequence H-His-Pro at the N-terminus are highly sensitive to DKP formation, attributed to the catalytic role of the imidazole group in the His side chain. In this structure, the His-Pro peptide bond adopts a cis conformation favorable for DKP, promoting nucleophilic attack by His-Nα on -Pro-Xaa3- main chain amide. The enhanced nucleophilicity of His-Nα is facilitated by the deprotonation of His’s basic imidazole group. This His-mediated catalytic process results in the cleavage of the Pro-Xaa3 peptide bond, ultimately forming the cyclic derivative His-Pro-DKP and complementary truncated peptide fragments.

In terms of DKP formation, the H-His-Gly sequence appears more stable than H-His-Pro. Many approved GLP-1/2 analog peptides, such as exenatide, dulaglutide, lixisenatide, and teduglutide, feature N-terminal H-His-Gly sequences. Additionally, Alloferon also has an H-His-Gly N-terminal sequence.

• Opposite Configuration of N-terminal Amino Acids

Peptide sequences composed of alternating L- and D-amino acids significantly affect DKP formation. In this case, the formed DKP molecule is stabilized thermodynamically due to the positioning of the two side chain groups, R1 and R2, on both sides of the DKP molecule plane, favoring DKP formation.

N-terminal dipeptide sequences with opposite configurations of amino acid residues are found in some marketed peptide drugs, such as icatibant (H-D-Arg-Arg) and bivalirudin (H-D-Phe-Pro).

• Disulfide-Bridged Peptides

Disulfide bonds can effectively suppress DKP formation by limiting the spatial position of the nucleophilic Nα group. This feature may explain the higher proportion of free Nα groups in marketed disulfide-bridged peptides, such as octreotide, oxytocin, and lanreotide. Compared to N-terminal unacetylated short linear peptides, they are less likely to form DKP.

• N-terminal H-Xaa-Asp Peptides

If Asp or Asn is located at the second position of the peptide N-terminus, the formation of aspartimide can promote peptide DKP formation. This aspartimide-induced DKP formation has been detected in many studies. Asn/Asp at the second N-terminal position initially forms aspartimide, and the formed aspartimide peptide intermediate can undergo differential isomerization into the corresponding non-enantiomeric isomer aspartimide or hydrolyze into Asp- or β-Asp peptides. Additionally, the aspartimide at the second N-terminal position can undergo nucleophilic attack from Nα and convert into the corresponding cis-DKP compound. Under neutral to alkaline conditions at room temperature or elevated temperatures, cis-DKP can rapidly isomerize into trans-DKP. The sensitivity of aspartimide at the second N-terminal position to DKP is attributed to the increased electrophilicity of the acyl imide part and the easy racemization of the aspartimide intermediate. In this mode, the six-membered ring of DKP is not excluded from the peptide molecules, resulting in the presence of DKP isomers in the peptides.

Plecanatide is a marketed peptide drug with both linear and Asp as the second N-terminal residue (H-Asn-Asp). Thymosin α1 has a Ser-Asp N-terminus, but its Nα group is acetylated.

Summary

• In FDA-approved short linear peptide drugs, the majority of Nα groups are acetylated and capped.
• Nα acetylation and capping can have a significant impact on the physicochemical properties of the parent peptide molecule, such as secondary structure, isoelectric point (PI), solubility, etc., particularly for short peptides.
• Long peptide drugs tend to have a relatively higher proportion of free Nα groups.
• The occurrence of unacetylated Nα is more common in disulfide-bridged peptides and amide-cyclized peptides.
• Special attention should be given to the possibility of DKP formation for short peptides containing sensitive N-terminal dipeptide sequences.

In the past 2022, the FDA has approved only three peptide drugs: Tirzepatide, Lutetium 177Lu Vipivotide Tetraxetan and Terlipressin, of which terlipressin was previously available outside the United States as Glypressin®. There is also literature that classifies Gadopiclenol approved in 2022 as a peptide drug, but this classification is not rigorous. Gadopiclenol contains only an amide bond, but it is not a condensation of two amino acids and should not be considered a peptide.

Trofinetide

Trofinetide is a novel synthetic analog of glypromate, also known as glycine-proline-glutamate (GPE). GPE is an N-terminal tripeptide of a naturally occurring protein and insulin-like growth factor 1 (IGF-1) in the brain. Compared with the proline Cα of GPE, trofinetide has one more methyl group.

Trofinetide (Daybue^TM), developed by Acadia, is a peptide drug approved by the FDA for the treatment of Rett syndrome on March 10, 2023, making it the first FDA-approved Rett syndrome drug in history. Syndrome drugs. Rett syndrome is a rare disease with a spectrum of cognitive, motor, and autonomic symptoms. It is a complex, rare neurodevelopmental disorder usually caused by genetic mutations in the MECP2 gene. It is characterized by a period of normal development before 6 to 18 months of age, followed by significant developmental regression, with loss of acquired communication skills and use of the hands. Symptoms of Rett syndrome may also include hand rigidity, such as wringing and clapping, and abnormal gait. In the United States, Rett syndrome affects 6,000 to 9,000 patients, of whom approximately 4,500 have been diagnosed. The approval of Trofinetide is undoubtedly a milestone event in the history of human struggle against Rett syndrome.

• Mechanism of Action of Trofinetide

Most cases of Rett syndrome are associated with loss-of-function mutations in the gene encoding methyl CpG-binding protein 2 (MECP2). MECP2 is a DNA-binding protein that plays a role in the epigenetic regulation of gene expression. These mutations lead to immature synapses in the cerebral cortex, abnormal metabolism of cholesterol in the brain leading to abnormal neuronal development, and abnormal neuronal signaling. Rett syndrome is more common in girls than boys.

The exact mechanism of action of Trofinetide in Rett syndrome has not been fully elucidated. In mouse studies, GPE peptides improved exercise and cardiorespiratory function, increased brain weight, and extended the lifespan of MECP2-deficient mice. As a synthetic analogue of GPE, trofinetide similarly attenuates apoptosis and reduces infarct size in a dose-dependent manner in a rat model of hypoxic injury. Trofinetide plays a multifaceted role in reducing inflammation, excitotoxic-induced tissue damage and apoptosis, thereby protecting neurons and their surrounding underlying tissues.

Rezafungin

New Echinocandin Peptide Drugs

On March 22, 2023, the FDA approved Cidara and Melinta’s Rezzayo^TM (rezafungin for injection) for the treatment of candidemia and invasive candidiasis in patients 18 years of age or older. Rezafungin is a new generation of broad-spectrum, long-acting echinocandin. Rezafungin has shown effective antifungal activity against Candida, Aspergillus and pneumospora sp. Rezafungin also became the first new echinocandin to receive FDA approval in more than a decade. Unlike other echinocinins such as caspofungin and micafungin, rezafungin has long-acting pharmacokinetics and high stability, allowing it to be administered at long intervals and maintain high plasma exposure. Rezafungin has a half-life of more than 130 hours and can be administered once a week instead of daily.

• Candidemia and Invasive Candidiasis

Candidemia and invasive candidiasis are rare, serious and life-threatening infections. According to the U.S. Centers for Disease Control and Prevention, the average rate of new infections in the United States is about 9 per 100,000 people, or about 25,000 cases per year. These infections are prone to occur in patients who already have other diseases and can lead to high morbidity and mortality. Patients at risk for invasive fungal infections include severely ill patients, immunosuppressed patients, patients after surgery, and patients using central venous catheters. In this already vulnerable patient population, invasive infections with candida often lead to serious illness and death. Patients with these invasive infections may develop a range of comorbidities on top of their underlying disease, including fever and septic shock. An additional hospital stay of 3 to 13 days is estimated after diagnosis of candidemia and invasive candidiasis. In addition, the mortality rate of these infected patients exceeds 40%. Echinocandins are used as first-line antifungals in practice guidelines for candidemia and invasive candidiasis because of their proven efficacy and safety as well as their powerful fungicidal activity.

• Another Cyclic Peptide

Rezafugin was discovered during the development of an orally administered Echinocin. Its outstanding structural feature is its chemical modification, mainly the addition of the choline moiety to the cyclic hexapeptide ring. This significantly improves molecular stability and biological properties. Compared to other echinocinins, the reduced metabolism and degradation of rezafungin enhances its stability and prolongates the half-life of the drug. This made it possible to administer the drug intravenously once a week.

• Mechanism of Action of Rezafungin

Rezafungin is an echinocandin antifungal drug. It inhibits the 1,3-β-D-glucan synthase complex in the fungal cell wall, which is responsible for the formation of 1,3-β-D-glucan. Since 1,3-β-D-glucan is an important component of fungal cell walls, rezafungin disrupts the cell walls of fungal species including Candida and acts as a concentration-dependent fungicide in vitro. Mammalian cells do not express β-1-3-glucan synthase, therefore, the action of rezafungin is specific to fungi.

Posluma (flotufolastat F 18)

Posluma (flotufolastat F 18) is an 18F-labeled ligand and an imaging agent for the detection and localization of prostate cancer in PET (positron emission tomography). Posluma contains an imaging agent (18F) whose active ingredient is flotufolastat F 18 gallium bound to non-radioactive gallium. Flotufolastat F 18 binds to prostate-specific membrane antigen (PSMA) (IC50= 4.4 nM) expressed on cells, including prostate cancer cells. In patients with recurrent prostate cancer who require local treatment, positron emission tomography (PET) with an 18F-labeled ligand provides accurate diagnostic imaging. The 18F group in Flotufolastat is a beta + emitting radionuclide that can be detected using PET. Since prostate cancer cells overexpress PSMA and flotufolastat F-18 is internalized by the cells, this radioactive agent can be used for diagnostic imaging in prostate cancer patients. Posluma received FDA regulatory approval on May 25, 2023 for PET of PSMA-positive lesions in men with suspected metastatic prostate cancer. It is the first FDA-approved PSMA-targeted imaging agent developed using a proprietary radiohybridization technology.

Paxlovid (nirmatrelvir, ritonavir)

The two active substances in Paxlovid, nirmatrelvir and ritonavir, are both peptidomimetics. Nirmatrelvir acts as a protease inhibitor to block the activity of proteases required for virus reproduction. It is an inhibitor of 3C-like protease (3C-like protease, 3CLPRO, also known as Mpro), which plays a key role in the replication of SARS-CoV-2 virus. The second peptidomimetic active substance ritonavir in Paxlovid is used as a pharmacokinetic enhancer of nirmatrelvir, which increases the half-life and bioavailability of nirmatrelvir by inhibiting the degradation of nirmatrelvir by CYP3A4.

Currently, available peptide nasal spray products include the following products:

Calcitonin Salmon (trade name Miacalcin®) 

The active ingredient in Miacalcin^® is the peptide calcitonin salmon (salmon calcitonin), a calcitonin hormone found in salmon. Like human calcitonin, calcitonin salmon is a peptide hormone that is secreted by the parafollicular cells of the thyroid gland in response to hypercalcemia and reduces blood calcium and phosphorus by promoting renal excretion. Miacalcin® nasal spray is used to treat osteoporosis in women who have been menopausal for at least 5 years. It should work in conjunction with adequate calcium and vitamin D intake. Miacalcin® nasal spray is aimed at patients who are not candidates for alternative therapies, such as those for whom other therapies are unsuitable or those who are intolerant or unwilling to use other therapies.

Desmopresin (trade name Stimate®)

Stimate^® Nasal Spray is indicated for patients with haemophilia with coagulation factor VIII activity levels greater than 5% (mild). Stimate^® Nasal Spray contains desmopressin acetate, a synthetic analog of the natural hormone Arginine vasopressin, as the active substance. A single (0.1 mL, 150 μg) spray of Stimate® nasal spray solution has an anti-diuretic activity of approximately 600 IU. The half-life of Stimate® Nasal Spray is between 3.3 and 3.5 hours and the intranasal dose ranges from 150 to 450 μg. The plasma concentration of Stimate® Nasal Spray reaches a maximum of approximately 40 to 45 minutes after administration. When administered by the intranasal route as a 1.5 mg/mL solution, the bioavailability of Stimate^® nasal spray ranged from 3.3% to 4.1%.

Desmopressin (trade name Octostim®)

Also desmopressin, its original developer Ferring Pharmaceuticals developed it as a different nasal spray. Octostim^® is an anti-diuretic, it can be used to treat central insipidus, a condition in which the kidneys are unable to retain water due to a deficiency of antidiuretic hormone (ADH). This condition causes frequent urination and thirst. It is also used to treat temporary thirst and increased urination that may occur after a head injury or certain types of brain surgery. For these uses, Desmopressin Nasal Solution works by helping to reduce the amount of urine produced by the kidneys.

Desmopressin (trade name Minirin®)

Primary nocturnal enuresis: Minirin^® nasal spray is indicated for the management of primary nocturnal enuresis. It can be used alone or as an adjunct to other non-pharmacological interventions for behavioral modification. It has been shown to be effective in certain cases that are difficult to treat with conventional therapy.

Central intracranial insipidus: Minirin^® nasal spray is indicated for the treatment of central intracranial diabetes insipidus and for anti-diuretic replacement therapy for temporary polyuria following head trauma or surgery in the pituitary region. It is not effective in treating nephrogenic diabetes insipidus.

The Minirin^® nasal spray compression pump delivers 0.1 mL (10 μg) of desmopressin acetate per spray. In addition to the nasal spray, Minirin^® is also available in the form of an oral tablet.

Desmopressin (trade name DDAVP)

DDAVP contains desmopressin acetate as the active substance with an anti-diuretic effect and 1 mL (0.1 mg) of intranasal DDAVP is active at approximately 400 IU. The biphasic half-lives of intranasal DDAVP were 7.8 minutes and 75.5 minutes in the fast and slow phases respectively, compared to 2.5 and 14.5 minutes for the other form of lysine vasopressin. Thus, intranasal DDAVP produces a rapid antidiuretic effect and a long duration of action after each dose.

Buserelin (trade name Suprefact®)

Buserelin is used in men to treat advanced prostate cancer but is not a cure. Most types of prostate cancer require the male hormone testosterone to grow and spread. Buserelin works by reducing the amount of testosterone produced by the body. This action helps to slow or stop the growth of cancer cells and helps to relieve symptoms such as painful/difficult urination.

Buserelin is also used to treat endometriosis in women. It works by reducing the body’s production of the female hormone estrogen. This effect helps to shrink abnormal tissue and reduce the symptoms of endometriosis (e.g. pelvic pain, dysmenorrhoea).

Buserelin is an artificial hormone, similar to the natural hormone “gonadotropin-releasing hormone” (GnRH) produced by the human body. Although the use of buserelin promotes the secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) (agonists), however, with prolonged administration of buserelin, the GnRH receptor becomes desensitized and completely stops responding to buserelin and endogenous GnRH. The profound desensitization of the GnRH receptor leads to a reduction in LH and FSH secretion from the anterior pituitary gland, followed by a cessation of gonadal sex hormone production, a significant reduction or loss of spermatogenesis in men, and an absence of ovulation in women, and the goal of “starving” cancer cells.

Suprefact contains 1.06 mg buserelin acetate (equivalent to 1 mg buserelin free base) per mL of the intranasal aqueous solution. For prostate cancer, the usual starting dose is 500 µg buserelin, by subcutaneous injection every 8 hours for 7 days. This is followed by maintenance therapy with a single daily injection of 200 µg buserelin or 400 µg of nasal spray delivered three times daily (2 sprays in each nostril). Women treated with buserelin for endometriosis are usually treated with buserelin nasal solution at a dose of 400 µg (2 sprays per nostril) 3 times a day. Treatment usually lasts for 6 months, but should not last longer than 9 months.

Nafarelin (trade name Synarel®)

Like buserelin, nafarelin is a GnRH (gonadotropin-releasing hormone) analog (agonist). It is used to treat endometriosis and precocious puberty. It is also used to treat uterine fibroids, to control ovarian stimulation in in vitro fertilization (IVF), and as part of cross-sex hormone therapy.

Synarel^® is indicated for the treatment of central precocious puberty (CPP) (gonadotropin-dependent precocious puberty) in both male and female children. It is used as a nasal spray 2 to 3 times a day. Synarel^® nasal solution contains nafarelin acetate (2 mg/mL). After filling the Synarel^® pump, each start of the unit delivers a spray of approximately 100 μL containing approximately 200 μg nafarelin free base. The contents of a spray bottle are provided to deliver at least 60 sprays.

After intranasal administration, nafarelin acetate is rapidly absorbed into the systemic circulation. The maximum serum concentrations were reached between 10 and 40 minutes. The average peak concentration was 0.6 ng/mL (range 0.2 to 1.4 ng/mL), after a single dose of 200 μg free base nafarelin and after a single dose of 400 μg free base nafarelin, the mean peak concentration observed was 1.8 ng/mL (range 0.5 to 5.3 ng/mL). The bioavailability of the 400 μg dose averaged 2.8% (range 1.2 to 5.6%). The mean serum half-life of nafarelin after intranasal administration is about 3 hours. Approximately 80% of nafarelin acetate is bound to plasma proteins at 4°C.^[3]

Oxytocin (trade name Syntocinon)

The uses of oxytocin nasal spray range from treating anxiety disorders to autism. In clinical trials, oxytocin has shown promise in the treatment of obesity and may have other super-indicated uses. It is a naturally occurring hormone that can also be used as a supplement.

Semax

Semax peptides are known for their neuroprotective, educational, and neurorestorative properties. Traditionally, Semax has been used to treat depression and anxiety. Semax was originally developed based on the molecular structure of ACTH (Adrenocorticotropic Hormone). Ongoing research suggests that Semax may be useful for individuals with cognitive disorders (including dementia, stroke, ADHD, etc.) and may better improve mental clarity and concentration, reduce anxiety and depressive symptoms, improve the clarity of thought and focus, and reduce the effects of stress, hence Semax is given the free translation of “brain peptide” in Chinese. “Semax is used for a wide range of indications, and in the medical setting, Semax has been prescribed for the treatment of anxiety disorders, ischaemic events, stroke, neuroregeneration, ADHD, opioid withdrawal, Parkinson’s disease, Alzheimer’s disease, thrombosis, and gastric disorders. It is worth noting that Cerebrolysin is mainly used in countries such as Russia and Ukraine and is not approved in most countries around the world.

Advantages of intranasal delivery of peptide drugs

Potential nose-to-brain route: The blood-brain barrier (BBB) is a major barrier to the delivery of therapeutic agents to the central nervous system (CNS), as most large molecular weight substances are severely restricted from crossing the BBB under normal circumstances. For biological agents, such as peptides, proteins, monoclonal antibodies, oligonucleotides, and gene and cell therapies via the nasal-brain pathway, nasal delivery offers a potential strategy to bypass the BBB and reach the brain, thereby providing new therapeutic approaches for indications such as Alzheimer’s disease, Parkinson’s disease, and antipsychotic drug-induced symptoms.

High efficacy and fast onset: Most biologicals are readily degraded enzymatically in the gastrointestinal tract, therefore the most common route of administration is by injection. However, when drugs are administered by the intranasal route, they enter through the respiratory zone around the inferior turbinate, where the nasal mucosa in the respiratory zone is richly vascularised and lined with columnar epithelial cells with a large surface area (> 150 cm² ), which is conducive to drug absorption and has strong permeability. ^[5] As the intranasal route avoids enzymatic degradation in the gastrointestinal tract and first-pass metabolism in the liver, as a result, it reduces the common barriers that limit drug absorption and facilitates the rapid onset of action.

High patient dependence: Intranasal delivery will undoubtedly result in higher patient compliance compared to injection. Good patient compliance is important for therapeutic outcomes, especially for intranasal drug products, which must be administered regularly and consistently to ensure sustained therapeutic outcomes. Patients’ satisfaction and comfort with the administration of medication is highly dependent on their compliance. The intranasal route is both non-invasive and highly tolerable, expanding the possibilities and compliance of patient self-administration

References

1. Fosgerau, K. et al. Peptide therapeutics: current status and future directions. Drug Discov. Today. 2015, 20, 122-128.
2. Arora, P. et al. Permeability issues in nasal drug delivery. Drug Discov. Today. 2002, 7, 967-975.
3. Darshana, S. et al. Nasal Delivery of Proteins and Peptides. Glob. J. Pharm. Sci. 2017, 1, 555569.
4. Grassin-Delyle, S. et al. Intranasal drug delivery: an efficient and non-invasive route for systemic administration: focus on opioids. Pharmacol. Ther. 2012, 134, 366-379.
5. Ward, D. Optimising Nasal Drug Products for Systemic Delivery.
6. Bakri, W. A., et al. Overview of intranasally delivered peptides: key considerations for pharmaceutical development. Expert Opinion on Drug Delivery. 2018, 15, 991-1005.
7. Basu, S. et al. Preparation and characterization of mucoadhesive nasal gel of venlafaxine hydrochloride for treatment of anxiety disorders. Indian J Pharm Sci. 2012, 74, 428-433.

Tab 1. FDA-approved covalent peptide drugs from 2011 to 2019

Name	Therapeutic Field	Active Gene	Chemical Structure
Telaprevir	Anti-hepatitis C virus	α-ketoamide
Boceprevir	Anti-hepatitis C virus	α-ketoamide
Narlaprevir	Anti-hepatitis C virus	α-ketoamide

Covalent peptide drugs usually have electrophilic groups (because the reactive groups on the corresponding protein receptor are generally nucleophilic groups), and the reaction between the electrophilic group and the nucleophilic group of the receptor leads to covalent binding between the drug molecule and the receptor, thus achieving the therapeutic effect. The invention of covalent peptide drugs has led to the re-emergence of many protein receptors previously considered “undruggable”.

One of the advantages of covalent peptide drug development is that it can combine molecules or groups based on the previous results of non-covalent drugs and introduce active groups into the lead of non-covalently bonded peptides that bind closely to the target protein. When non-covalent binding occurs, the active groups are introduced into the vicinity of the target amino acid residue of the target protein in the three-dimensional space. In this case, the amino acid residue reacts irreversibly with the active groups on the drug molecule to achieve the ultimate goal of inhibiting the target protein (Fig 1).

Fig. 1. The covalent binding of a peptide drug to a target protein based on non-covalent interaction

From a protein receptor point of view, cysteine is the most studied target for drug designers because its sulfhydryl group acts as an excellent nucleophile. In the process of drug design, acrylamide groups are often attached to the lysine residues in the original non-covalent peptide drugs, and covalent bonds are formed by the reaction between the sulfhydryl group of the receptor cysteine and the acrylamide of the drug molecules. A study showed that a modified peptide drug (a 13-peptide containing acrylamide lysine) was more effective at inhibiting Siah (E3 ubiquitin-protein ligase) and that the peptide drug was specifically bound to cysteines at key sites. However, when the acrylamide group is replaced by the more active chloroacetamide group, the resulting peptide drug loses its cysteine selectivity and can instead react with other cysteines on the Siah protein receptor. This result confirmed that the reactivity of the electrophilic groups of covalent drugs is very important for drug selectivity.

Since cysteine is scarce in native protein receptors, covalent peptide drugs targeting lysine and histidine have gained attention. In one study, a peptide drug with a benzoyl fluoride group formed a strong covalent binding to its receptor p53-MDM2/4 protein, effectively inhibiting the activity of this protein. The activity of the modified peptide was 5-fold enhanced compared with that of the peptide precursor without benzenesulfonyl fluoride. Notably, the SuFEx reaction between benzenesulfonyl fluoride and the amino group of the lysine side chain is considered a new type of click reaction.

Encouraged by the sulfur-fluorine exchange reactions, different active groups of benzenesulfonyl fluoride and fluorosulfonic acid aromatic esters were of interest. They can covalently bind to lysine, tyrosine, and histidine.

In the search for drugs to treat COVID-19, covalent peptide drugs have also emerged. A peptide molecule named VIR251, containing methyl acrylate structure, can covalently interact with papain-like protease (PLpro) of SARS-CoV-2 protein mediating COVID-19 through Michael’s addition. Thus, the physiological function of the protein was inhibited.

Conclusion

With the continuous development of small molecule covalent drugs and the R&D of non-covalent peptide drugs, the resources available for the combination of the two products, covalent peptide drugs, will continue to expand, and promote the R&D and marketing of new drugs in this field. However, while covalently bonded drugs have the advantage of long duration and low dose, practitioners are still concerned about the unanticipated side effects of these active substances indiscriminately binding to proteins in the body, as well as the possible immune response to these drugs. Therefore, safety has become a top priority in the design and development of covalently bonded drugs.

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References

[1] Dong J, Krasnova L, Finn MG, et al. Sulfur (VI) Fluoride Exchange (SuFEx): Another Good Reaction for Click Chemistry. Angewandte Chemie Int Ed. 2014, 53,9430-9448.

[2] Hoppmann C, Wang L. Proximity-enabled bioreactivity to generate covalent peptide inhibitors of p53-Mdm4. Chem. Commun. 2016, 52, 5140-5143.

[3] Pietro Delre, Fabiana Caporuscio, Michele Saviano, Giuseppe Felice Mangiatordi, Repurposing Known Drugs as Covalent and Non-covalent Inhibitors of the SARS-CoV-2 Papain-Like Protease. J. Med. Chem. 2019, 62, 9188-9200.

[4] Stebbins JL, Santelli E, Feng Y, et al. Structure-Based Design of Covalent Siah Inhibitors. Chem Biol. 2013, 20, 973-982.

[5] Wang N, Yang B, Fu C, et al. Genetically Encoding Fluorosulfate-l-tyrosine To React with Lysine, Histidine, and Tyrosine via SuFEx in Proteins in Vivo. J. Am. Chem. Soc. 2018, 140, 4995-4999.

1. Peptide drugs for cancer immunotherapy

The treatment of cancer by controlling the function of immune cells is a research hotspot of peptide drugs. PD-L1 is typically overexpressed on many cancer cells and when it binds to PD-1 expressing on activated T cells, the cancer cells can escape from the immune response. Therefore, inhibition of PD-1/PD-L1 interaction could be a method of tumor immunotherapy. Studies have shown that hydrophilic D-type peptide (D-PPA) itself has no cytotoxicity. When blocking the PD-1/PD-L1 interaction, it can inhibit tumor growth in CT26 tumor-bearing mice and prolong their lifespan.

Macrophages are immune cells that play an important role in innate and acquired immunity. The CSF-1/CSF-1R pathway is related to the survival and differentiation of macrophages. In a recent study, a peptide called M2pep[Cys-YeqDPwGVKWWY] was modified to improve specificity against M2 TAMs. The experimental results confirmed that co-encapsulation with CSF-1R siRNA and M2pep could target M2 TAM to remodel into the M1 phenotype and increase anti-tumor immune response.

2. Apoptosis-related peptide drugs

Certain peptides can induce apoptosis in cancer cells. For example, an anticancer peptide called RA-V (deoxybouvardin) can induce mitochondrial apoptosis in tumor cells, followed by loss of mitochondrial function. Another pentapeptide, Dolastatin 10, was able to induce apoptosis by up-regulating cytochrome C and Bax.

KLA (KLAKLAKLAK) is also one of the pro-apoptotic peptides, which induces programmed cell death by disrupting the mitochondrial membrane. Bioconjugates containing KLA peptides can be used in the development of various anticancer drugs. For example, KLA can bind to the tumor homing peptide IRGD(CRGDKGPDC) to improve the permeability of tumor tissues and cells. The potent anti-tumor effect of the recombinant KLA-IRGd conjugate as well as the high tumor selectivity and low systemic toxicity have been demonstrated in mouse models. These studies may lead to the development of novel targeted PDCS.

Another example of a KLA-based therapy is the penetratin (a cell-penetrating peptide)-KLA conjugate. To overcome the low cellular permeability of KLA peptides, penetratin was linked to KLA peptides via disulfide bonds, resulting in high cellular permeability and cytotoxicity, even at low concentrations. The penetratin-KLA conjugate has little effect on the mitochondria of normal cells, showing its therapeutic potential as a novel peptide conjugate.

3. Peptides for tumor enrichment

There have also been some new studies describing various methods of targeting tumors with peptides. For example, it was reported that the S-S-bridged cyclic peptide (ALOS-4) is specific for the integrin AVβ3, which is highly expressed in human metastatic melanoma. In this study, ALOS-4 was coupled to the topoisomerase I inhibitor camptothecin. The results showed that the conjugate had potent anti-tumor activity in human metastatic melanoma cells, but low toxicity to normal cells. In addition, a researcher team conjugated the four-branched peptide NT4 to paclitaxel (PTX). NT4 peptides can selectively bind to tumor membrane glycosaminoglycans sulfate. It is highly selective for cancer cells when combined with paclitaxel, and this combination allows it to exhibit more potent anti-tumor activity than paclitaxel alone.

In another study, the tumor targeting of albumin-binding peptides was investigated. In vitro studies, the bioconjugation of DICLPRWGCLW to form a stable albumin complex can play a role in tumor targeting, and also showed high tumor targeting and significantly increased half-life in the SCC7 tumor-bearing mouse model. Based on the positive correlation between albumin uptake and tumor growth, albumin binding is expected to become a novel therapeutic strategy for targeting tumor tissues.

4. Peptide drugs conjugated with small cytotoxic drugs

Under the concept of drug conjugates, the combination of peptides with cytotoxic drugs or other therapeutic agents can be used to develop peptide-based therapeutic agents.

Several studies have shown that cytotoxic drugs can bind to peptides with additional functions. For example, in anticancer therapy, platinum-based cisplatin chemotherapy is one of the most widely used cancer treatment strategies. However, because of cisplatin resistance, long-term use of cisplatin in patients reduces its efficacy. The use of peptides may be an important way to solve the problems related to drug resistance. When cisplatin is combined with peptides, the binding induces a high accumulation of cisplatin in tumors and metastasis-containing organs and significantly reduces the systemic toxicity of cisplatin.

Several peptide drug conjugations are currently under clinical investigation for the treatment of refractory tumors. On February 27, 2021, Melflufen, the first peptide-coupled drug, was approved by FDA for the treatment of multiple myeloma. Because melflufen is highly lipophilic, it is readily ingested by targeted cancer cells. Once internalized, melflufen molecules can be cleaved by aminopeptidase, which is highly expressed in multiple myeloma cells, and release alkylating agent toxic drugs that cause DNA damage and apoptosis in tumor cells. Now, many other forms of peptide-drug conjugates are also under active development as anticancer drugs.

Creative Peptides’ Perspectives

Due to the advantages of peptides, peptide drugs or peptide-drug conjugates have been extensively explored for new drug R&D. Previously, peptide drugs were seen as simply mimicking hormones or endogenous peptides in the body, but they are now being reborn to be designed into new therapies. However, as the limitations of peptide drugs have been solved, the powerful anti-tumor effect of peptide drugs has attracted the attention of researchers and the pipeline layout of more and more companies. Various therapeutic strategies that combine cytotoxic antitumor drugs with functional peptides come to a reality and are expected to be more effective than small-molecule drugs, antibodies, and ADCs.

References:

1. Blocking of the PD-1/PD-L1 Interaction by ad-Peptide Antagonist for Cancer Immunotherapy.
2. Tumor-penetrating peptide fused to a pro-apoptotic peptide facilitates effective gastric cancer therapy.

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What is Cyclic Peptide?

Cyclic peptides are peptide chains with a cyclic structure, mostly composed of 5-14 amino acids, with a molecular weight of about 500-2000 Da. Cyclic peptides have several advantages over linear peptides. Linear peptides as drugs may be inherently unstable and have a high probability of intracellular proteolysis. Considering that the free acid and free amine are at both ends, they are also usually polar. Cyclization promotes intramolecular hydrogen bonding within the ring structure and reduces the external hydrogen bonding capacity of the molecule, which reduces the polarity of the molecule compared to the acyclic precursor and increases the membrane permeability of the compound. The two β-turn local secondary structures caused by cyclization also reduce polar surface area, which generally enhances cell permeability.

Cyclic peptides are usually produced by end-to-end, cephalic, or side-chain-to-side-chain cyclization reactions. Introducing cyclization can make the conformation of the peptide chain more stable, thereby increasing the binding affinity to the target protein and reducing nonspecific binding due to fewer alternative conformations. Reduced conformational flexibility reduces the chance of molecules fitting to protease catalytic sites and improves proteomic resistance. Intervention in protein-protein interaction (PPI) by forming larger interaction surfaces to improve peptide chain efficacy.

Chem. Eur. J. 2021, 27, 1487-1513

Cell Permeability of Cyclic Peptides

There are three pathways by which cyclic peptides enter cells: passive diffusion pathway, carrier-mediated transport pathway, and endocytosis pathway. The passive diffusion pathway usually requires good amphiphilicity and low molecular weight (<1000Da) of cyclic peptide molecules to enter cells. Some peptide-based drugs are recognized by transporters and subsequently introduced into cells, and some cyclic peptides enter cells through PepT1 and PepT2 transporters. However, these receptors are expressed primarily in the gut (absorption) and kidney (excretion), so they are important for oral bioavailability and elimination of peptide drugs but are unlikely to be involved in the cellular penetration of target tissues. Moreover, the membrane of the endocytic sac is similar to the cell membrane, and non-passive permeable molecules may not be able to effectively escape the endosome and lysosome into the cytoplasm. In order to enhance the cell permeability of peptide drugs, it is necessary to improve the molecular weight and amphiphilicity of drugs. Cyclic peptide drugs, however, are flexible enough to meet these needs. First, controlling the sequence length of the cyclic peptide molecule can control the molecular weight requirements. Secondly, by modifying the side chains of amino acids (introducing unnatural amino acids, forming disulfide bonds, etc.) or main chains (n-methylation, etc.), introducing unnatural hydrophobic groups can enhance the hydrophobicity of cyclic peptide molecules, thus improving the cellular permeability of cyclic peptide drug molecules. Furthermore, the hydrophilic and hydrophobic properties of cyclic peptides can be further optimized according to the symmetry of the framework (d-type amino acids).

Chem. Eur. J. 2021, 27, 1487-1513

FDA-Approved Cyclic Peptide Drugs

Cyclic peptide drugs targeting intracellular proteins

1. Romidepsin

Romidepsin is a natural bicyclic peptide discovered in Chromobacterium violaceum (1994), containing a head-to-tail lactone cyclization and a pair of disulfide bonds, mainly produced by fermentation. Romidepsin, a histone deacetylase inhibitor (HDACi), was approved by the FDA in 2009 for cutaneous T-cell lymphoma (CTCL) that has received at least one systemic therapy, primarily as an antineoplastic drug. It is a prodrug in which disulfide is reduced to two mercaptols in the intracellular matrix that chelates zinc ions at the zinc-dependent active site of histone deacetylase (HDAC), thereby inhibiting histone deacetylase and inducing apoptosis.

2. Voclosporin

Voclosporin has 11 residues, including 1 D-type amino acid, which is head and tail cyclization. The N-terminus of some amino acids is methylated by S-adenosine methionine. Volcyclosporine was obtained by optimizing cyclosporin A. Voclosporin has better efficacy and metabolic stability than cyclosporin A. As a novel best-in-class calcineurin inhibitor (CNI), voclosporin has a synergistic and dual mechanism of action: it stabilizes renal podocyte by inhibiting CALcineurin (CN), blocking IL-2 expression and T cell-mediated immune response.

Cyclic peptide drugs targeting extramembrane proteins

1. Ziconotide

Ziconotide is a synthetic omega-MVIIA toxin isolated from the venom of Conus Magus. It contains 25 residues, of which six amino acids are cysteine, linked by three pairs of disulfide bonds. As a potent selective blocker of N-type calcium channels, Ziconotide was approved in 2004 for the treatment of severe chronic pain.

2. Linaclotide

Linaclotide is an oral peptide composed of 14 amino acids developed in the 1990s and belongs to the Cyclic Guanosine Monophosphate (cGMP) regulatory peptide family. In 2012, linaclotide was approved by the FDA for the treatment of irritable bowel syndrome with constipation (IBS-C).

3. Plecanatide

Pukanatide is an oral guanylate cyclase C(GC-C) agonist developed by Synergy that regulates acid and base ions in the gastrointestinal tract, inducing fluid transport into the gastrointestinal tract and increasing peristalsis. Pukanatide, a 16-amino acid analog of uranyin, is produced by replacing Human uranyin Asp3 with Glu3 and binds to guanylate cyclase C(GC-C) receptors at pH 5.0 (duodenum and proximal jejunum pH) bound by 2 pairs of disulfide bonds. On 2017-01-19, Plcanatide was first approved by the FDA for the treatment of gastrointestinal disorders such as constipated irritable bowel syndrome (IBS-C) and chronic idiopathic constipation (CIC).

4. Pasireotide

Pasireotide is a second-generation analog of somatostatin, a naturally occurring inhibitory hormone that blocks the release of several other hormones by interacting with the somatostatin receptor (coupled to G protein), including growth hormone, thyroid-stimulating hormone (TSH), insulin, and glucagon. Compared with other somatostatin analogs, paretide has a 40-fold increase in affinity for somatostatin receptor 5. Pasireotide is a head-to-tail cyclized hexapeptide. Pasireotide has a higher binding affinity to the somatostatin receptors SSTR1, SSTR3, and SSTR5 than octreotide and lanreotide.

5. Lanreotide

Lanretide is the first sustained-release, long-acting somatostatin octapeptide analog, which is produced by cyclization of a disulfide bond between cysteine and cysteine, and binds stronger to somatostatin receptors on adenohypophysial cells than octreotide. Studies have shown that lanreotide mainly acts through somatostatin receptor-2 and somatostatin receptor-5, with a strong affinity for peripheral somatostatin receptors and a weaker affinity for central receptors.

6. Vasopressin

Vasopressin, discovered in 1928 and its sequence elucidated in 1951, is an antidiuretic hormone with nine amino acids, cyclized by a disulfide bond between Cys4 and Cys9.

7. Terlipressin

Terisopressin is a synthetic, long-acting vasopressin analog that acts on the vasopressin receptors V1a, V1b, and V2. It is a prodrug that is degraded in vivo by endopeptidase to form lysine-vasopressin by removing the trisglycine residue at the N-terminal of the cyclic peptide, which is the main active metabolite and is further degraded in the liver, kidney, and other tissues. As a result, teripressin acts slowly but has a longer duration, with a half-life of 6hr, which is longer than that of vasopressin (10min), and is safer with fewer side effects.

8. Bremelanotide

Bremelanotide is a synthetic peptide molecule consisting of seven amino acids cycled between side chains Asp2 and Lys7 to form the lactam analog (α-MSH) of the alpha-melanocyte-stimulating hormone.

9. Setmelanotide

Setmelanotide is a disulfide-cyclized octapeptide that preferentially agonizes the melanocortin 4 receptor (MC4R) with an EC50 of 0.2 nM.

Antimicrobial Cyclic Peptides

1. Daptomycin

Daptomycin is a cyclic lipopeptide antibiotic isolated from Streptomyces rosiana. It contains 13 residues, 2 of which are non-natural amino acids, and 10 of which form lactone rings through the side chain and C-terminal of Thr4.

2. Telavacin

Telavancin is a semi-synthetic vancomycin derivative, which is based on the vancomycin structure. It is obtained by chemical modification to introduce an aliphatic chain on the amino group of the sugar group and to introduce methylamine methyl phosphate on the seventh aromatic amino acid. Through modification, the binding force with the cell membrane and affinity with the antibacterial target is enhanced, and teravanin is a fast bactericidal antibiotic for injection.

3. Dalbavancin

Dalbavancin is a second-generation semi-synthetic lipoglycopeptide antibiotic that is more potent than vancomycin and inhibits bacterial cell wall synthesis. The vancomycin family of natural antimicrobial glycopeptides is highly modifiable heptapeptides in which 5 amino acids remain unchanged, while amino acids 1 and 3 are highly differentiated. The heptapeptide core is used to bind the C-terminal D-Ala-D-Ala of the peptidoglycan chain and the side chain functional groups are derived to improve the properties of the peptide molecule.

4. Oritavancin

Oritavancin, a second-generation, semi-synthetic lipoglycopeptide, is derived from chloroeremomycin, an analog of vancomycin. Similar to vancomycin, it contains a core heptapeptide.

5. Caspofungin, Micafungin and Anidulafungin

Capofungin, micafengin, and anifengin all belong to the cyclic hexaliphatic peptide family. They share the same cyclic peptide core structure and are cyclized by amidation between the n-end of the side chain of the first lysine residue and the c-end of the peptide chain, and all target the 1,3-β -glucan synthase in the cell wall for the synthesis of glucan. All three antifungal agents have clinical efficacy against invasive candidiasis and other forms of systemic mycosis.

References

Cyclic peptide drugs approved in the last two decades (2001-2021)

Peptide is a type of compound formed by connecting multiple amino acids through peptide bonds, usually composed of 10-100 amino acid molecules, and its connection method is the same as that of protein. The relative molecular mass is less than 10,000. In recent years, with the development and maturity of peptide synthesis technology, peptide drugs have become one of the hot spots of drug research and development. Because of their wide indications, high safety, and remarkable efficacy, they have been widely used in the prevention, diagnosis and treatment of tumors, cardiovascular and cerebrovascular, hepatitis, diabetes, AIDS and other diseases.

Characteristics of peptide drugs

Peptide drugs have high safety and high target affinity. They are mainly eliminated by proteolytic degradation and renal filtration. The products of hydrolysis are amino acids. Therefore, it is generally not considered whether the metabolites of peptide drugs are toxic.

The developed peptide drugs often use endogenous peptides as templates, which usually have a higher target affinity and a smaller risk of off-target.

The probability of peptide drugs being approved for marketing through clinical trials is twice higher than that of small-molecule drugs, and the average R&D cycle is 0.7 years shorter. The development of peptide drugs has also extended to many disease fields, including anti-infection, anti-tumor, physiological regulation, pain, heart failure, osteoporosis, diabetes, vaccines, etc.

Compared with protein, peptide chemical synthesis technology is mature, easy to separate from impurities or by-products, high purity, and easy to introduce unnatural amino acids. However, the quality, purity and yield of recombinant proteins are difficult to guarantee. And the recombinant proteins cannot be introduced with unnatural amino acids and cannot be amidated at the ends. At the same time, the production cycle is long and the cost is high. Peptides are generally lower in cost than protein and antibody drugs, and higher in synthesis cost than most small molecule drugs.

Peptides are easily degraded, have a short half-life and a high plasma clearance rate. The biggest problem with peptides is that they cannot be taken orally, mainly because they are easily degraded and difficult to cross the intestinal mucosa. In addition, peptides are easily hydrolyzed by proteolytic enzymes in the blood after entering the blood, and have a short plasma half-life and plasma clearance rate, which affects the efficacy of the drug and the choice of the route of administration, and therefore does not produce drug accumulation.

Peptide drugs may develop immunogenicity as the number of amino acids increases.

Table 1 Comparison of peptide with small-molecule and protein

Marketed Peptide Drugs

In the future, the number of peptide drugs in the world will continue to increase. There is no shortage of innovations in peptide drugs and they have great potential. In the future, emerging peptide drugs such as antimicrobial peptides, peptide vaccines, peptide carriers, PDC drugs, etc., are still in the clinical research stage and have very good market prospects. Antimicrobial peptides, as new and powerful broad-spectrum antibiotics, are different from previous antibiotics, and have the advantages of improving immunity and not easy to develop drug resistance. Peptide vaccines use certain peptides to have immunogenicity. They are currently on the market in the field of hepatitis treatment, and a new generation of peptide vaccines in the field of Ebola and AIDS are under development.

• Snake toxin peptide drug.

Snake venom is a mixture of enzyme and non-enzyme protein or peptide. Many drugs derived from snake toxin peptide have been used in clinical treatment.

A pentapeptide compound, which is an enhancer of bradykinin, was extracted from the venom of Agkistrodon halys Bothrops jaracaca. The structure of the pentapeptide was cut and the minimum pharmacophore Ala-Pro dipeptide was modified to synthesize an oral drug “captopril”. Captopril reduces blood pressure by increasing bradykinin activity and inhibiting thrombin (ACE). Captopril is a routine drug for the treatment of hypertension. In order to modify the structure of captopril, enalapril, lisinopril, Ramipril, and fosinopril were developed.

• Scorpion toxin peptide drug

It was found that chlorotoxin peptides in scorpion Leiurus quinquestriatus venom could delay the activity of human glioma cells in vitro. The venom of Venezuelan scorpion Tityus dispans, which has antibacterial and antiparasitic activity, can inhibit the growth of Leishmania in vitro. The toxin protein Bengalin of Indian scorpion can induce apoptosis of human leukemia cells in vitro and improve the biochemical indexes of osteoporosis in female rats. The α-neurotoxin found in scorpion venom can delay the inactivation of sodium channel at the neuromuscular junction, and the inactivation of the sodium channel may enhance neuromuscular reflex and airway contraction, which can treat sleep apnea theoretically.

• Sea anemone toxin peptide drug

Sea anemone is one of the oldest poisonous animals in existence. Like other members of the phylum, sea anemones have many special thorn cells and are widely distributed throughout the body. ShK toxin isolated from anemone is an effective Kv1.3 channel blocker. Kv1.3 channel plays an important role in the activation (proliferation and cytokine production) of human effector memory T cells. ShK can treat T-cell-mediated autoimmune diseases. Such as multiple sclerosis and rheumatoid arthritis. Kv1.3 receptor blockers are also considered to be targets for the treatment of obesity. ShK toxin also has potential applications in the treatment of obesity and insulin resistance. ShK-186, an analog of ShK toxin, now known as Dalazatide, has successfully completed clinical trials and entered the drug market for the treatment of autoimmune diseases.

• Leech toxin peptide drug

Leech is an important traditional medicine, which has many functions in the treatment of cerebral hemorrhage and other thrombus-related diseases. Hirudin is an effective anticoagulant in the salivary gland of leeches. Hirudin can inhibit the formation of thrombus and prevent the clotting of host blood. Pharmacological studies have shown that hirudin has the functions of anticoagulation, antithrombus, anti-atherosclerosis, anti-platelet aggregation, anti-tumor, and anti-inflammation, improving hemorheology and protecting the cerebral ischemia-reperfusion injury.

Conclusion

Although a large number of animal toxins have not yet been developed, animal toxin peptide drugs have made great achievements in clinical application, which makes more people realize the great potential and prospect of animal toxin polypeptide drug research and development. However, the effectiveness and safety of many animal toxins in clinical treatment need to be further verified, so the research and development of drugs based on animal toxins is also a great challenge.

References

1. Wieland, T. (2012). Peptides of poisonous Amanita mushrooms. Springer Science & Business Media.

2. Gopalakrishnakone, P., Cruz, L. J., & Luo, S. (Eds.). (2017). Toxins and Drug Discovery. Springer.