Dicyclic Peptide Drugs and Synthesis Routes
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What are dicyclic peptide drugs?
Due to their biocompatibility with proteins, structural diversity, and rigidity, dicyclic peptides are an attractive class of potential therapeutic molecules and are promising as effective alternatives to small molecule and antibody drugs. At present, dicyclic peptides have been widely used in protein interaction inhibitors, enzyme inhibitors, receptor agonists, and antagonists.
Since the discovery of the first bioactive cyclic peptide (bacitracin S) in 1947, more and more Marine organisms or microbial-derived cyclic peptides have attracted the attention of researchers all over the world for their potential value in the field of medicinal chemistry. For example, cyclopeptides are cyclic peptides found in sponges, cyanobacteria, and fungi that have been shown to exhibit a variety of biological activities (such as anti-inflammatory, anti-HIV, and anti-tumor). These biogenic cyclic peptides have inspired peptide researchers to chemically modify defective natural linear peptides to construct cyclic peptides.
In the past, small molecule and antibody drugs held great promise, while peptides were considered "undruggable" because they targeted the surface of proteins. The first two decades of the 21st century were a golden age for small-molecule medicine. In terms of current FDA-approved drugs, subclasses of drugs with a relative molecular weight of less than 500 are overwhelmingly dominant. Small-molecule drugs that are optimally designed to easily penetrate the cell membrane can therefore target proteins within the membrane; However, the nature of subclasses of drugs means that they usually have a deep "pocket" near the active site of the target protein for the small molecule to bind to the protein. It is difficult to find pockets suitable for small molecules to bind to 90% of the proteins in a cell, so only about 10% of the proteins in a cell are druggable for small-molecule drugs.
In recent years, due to the rapid development of peptide chemistry and the urgent need for protein-targeting drugs, some peptide fragments with fewer amino acids have been found to have similar activities as proteins, or even more prominent functions. In addition, they possess excellent binding properties due to their relatively large size and can interact with extended surfaces that cannot be targeted by conventional small molecules.
In recent years, monoclonal antibodies have developed rapidly. In addition to directly inhibiting its function by binding to target proteins, monoclonal antibodies can also use its Fc domain to bind to immune cells such as NK cells and macrophages to kill tumor cells through ADCC and ADCP. But like small-molecule inhibitors, monoclonal antibodies have an Achilles heel. Because of their high molecular weight, monoclonal antibodies can't get inside cells at all, so they can only target proteins on the cell membrane. Thus, for monoclonal antibodies, more than 80% of the protein is still not available.
The advantages of cyclic peptides over liner peptides
Compared with traditional drugs, peptide drugs have the following advantages:
- High efficiency: at a low dose or concentration can show significant activity;
- The molecule size is moderate: it is between small molecule and antibody, which may have the characteristics of small molecule permeable membrane and antibody mimicking protein function;
- Low immunogenicity: Because peptides are composed of amino acids, peptide sequences are often designed with homologous sequences with humans, so there are few side effects;
- High synthesis efficiency: at present, peptide fragments with less than 50 amino acids can be quantitatively synthesized by the solid-phase synthesis method.
Although peptide drugs have a promising prospect, they still have some shortcomings such as
- short half-life in vivo, easy to be degraded by various proteases, but lead to low oral utilization;
- unstable binding to the target;
- not easy to penetrate the cell membrane into the cell to play a role.
To overcome the limitation above, the focus of current research and development of peptide drugs is mainly to maintain all the activities of the original organisms and achieve the purpose of stabilizing the structure and enhancing the binding force of the target through external modification. Cyclization has become one of the best strategies to lock conformation and stabilize the structure.
More and more studies have found that cyclic peptides can significantly improve the activity of peptides. Compared with linear analogs, cyclic peptides can enhance target affinity, increase stability against proteolytic enzymes, and, in some cases, easier entry into cells. At present, there are many discussions in the academic circle about the reason why cyclization can effectively improve the physicochemical properties of linear peptides. The classical explanation is that the conformation of linear peptides is relatively free and disordered, and cyclization endows linear peptides with a certain degree of conformational locking and functional group preorganization, thus reducing the entropy loss caused by binding with the target.
Cyclic peptide drugs have the characteristics of good stability, high specificity, high affinity, and low toxicity, which make them popular in the field of new drug development. At present, more than 40 cyclic peptide drugs have been widely used in the clinic, among which there are many natural cyclic peptide antibiotics such as vancomycin, daptomycin, and polymyxin B, which can be used for inflammation caused by bacterial infection. There are also cyclic peptide drugs derived from endogenous somatostatin derivatives such as Octreotide and its analogs Pasireotide, which have a longer half-life in vivo than somatostatin and can be used to treat acromegaly; There are also dual-target cyclopeptide inhibitors of MDM2 and MDMX, such as ALRN-6924, which are optimized by the cyclopeptide drug ATSP-7041 and can block p53 binding to MDM2 and MDMX and restore p53-mediated apoptosis of tumor cells.
There are two main ways of cyclization of peptides:
- Use functional side chains of natural amino acids for chemical modification and modification, such as lactam cyclic peptide, and cysteine side chain sulfhydryl for the synthesis of disulfide cyclic peptide and thioether cyclic peptide.
- Introduce orthogonal non-natural amino acids or groups to assist the synthesis of cyclic peptides, such as the cycloaddition reaction between alkynes and azides and the Michael addition reaction between mercaptans and alkenes in click chemistry.
When coupling cyclic peptides with natural amino acids, it is necessary not only to consider appropriate orthogonal protection groups to protect the special functional groups but also to avoid the influence of other same amino acids in the peptide sequence. These problems have limited the development of natural amino acid cyclization to construct cyclic peptides. Therefore, peptide researchers envision introducing a series of unnatural amino acids with special functional groups to develop methods for building various cyclic peptides.
In addition to diolefin, the olefin complex decomposition reaction with a closed loop and the 'Click' reaction with alkyne and azide to form triazole are the most widely used in the construction of novel cyclic peptides.
1. Synthesis and application of the lactam process
Due to the easy availability of cyclization precursor carboxylic acids and primary amines in the peptide chain, the lactam method is one of the earliest cyclization methods studied and can be used for the synthesis of head-tail macrocyclic peptides. The synthesis of lysine with aspartic acid or glutamic acid lactam into a cyclic peptide is one of the most common methods in the synthesis of the cyclic peptide. In the process of synthesis, a variety of selective amino and carboxyl protective groups and deprotection mechanisms are selected to realize the lactam cyclization at specific sites and even the synthesis of multiple lactam rings. Lactam cyclization can effectively improve the binding affinity between peptide drugs and receptors and also show higher resistance to protease. In this process, the reaction sites of lactam cyclization are mainly i,i+3; i,i+4 or i,i+7.
In the peptide helix, every 3.6 amino acids rise around the circle. Although amino acids i,i+3 and i,i+4 are both likely to be on the same plane in helical conformation,i,i+3 lactam cyclization is less effective in stabilizing helical conformation than i,i+4 lactam cyclization. The lactam cyclization of Aspi-Lys i+3 was initially applied to amphiphilic apo E peptides, and the results of the circular dichroic spectrum and nuclear magnetic resonance spectroscopy showed that i,i+3 lactam cyclization has a stable effect on helical conformation comparable to Lysi-Asp i+4 lactam cyclization.
2. Synthesis and application of disulfide bonding
The disulfide bond is an important part of the secondary or tertiary structure of natural proteins, and it is an essential link to play the function of proteins. Many natural proteins (such as insulin and neurotoxin, etc.) contain multiple disulfide bonds. Currently, two disulfide cyclic peptides, Ciconotide and linaclotide, are approved by the FDA for the treatment of severe chronic pain and constipation and intestinal stress syndrome caused by chronic idiopathic constipation. In addition, disulfide-rich peptides have been widely used in chronic diseases, cancer, bacterial and viral infections, acute heart failure, and coagulation disorders, many of which have entered clinical studies.
Since the formation of disulfide bonds is reversible and usually occurs in multiple pairs, the main challenge in the synthesis of such peptides is the ability to form disulfide Bridges at specified locations during folding. At present, the most popular synthesis method is to introduce orthogonal cysteine side chain protection groups, which are carried out by step removal and step oxidation. However, in the synthesis of multi-pair disulfide bonds, there are some shortcomings such as isomerization, long step, and low yield.
3. Synthesis and application of thioether method
Although disulfide cyclic peptides are effective in stabilizing the structure and enhancing the affinity of the target, they are reducing environment or the presence of isomerase, resulting in different degrees of weakening of biological activity. In the past 20 years, many research groups have been seeking more stable and more active disulfide substitutes based on maintaining the original activity. In addition to the diamino disulfide bridge mentioned above, the sulfide bond is often designed as a disulfide substitute because of its similar structural parameters to the disulfide bond.
In recent years, the main synthesis methods of thioether cyclic peptides include the Michael addition reaction between mercaptan and olefin, disulfide desulfurization, diamino diacid process, and mercaptan dialkylation. Among them, mercaptan dialkylation reaction forms sulfur ether bond through SN2 reaction between two cysteine side chain sulfhydryl groups and two halogen atoms. This reaction is favored by many research groups because of its unique chemical stability and easy operation. More importantly, a wide variety of small molecules containing dihalides are readily available, most of which are commercially available, and a large number of mono-dicyclic thioether peptides with different ligands can be obtained in a short time.
4. Synthesis and application of the Alkyne-azide method
In addition to the cyclization methods described above, the cycloaddition of alkynes to azides catalyzed by copper is also used in the synthesis of cyclic peptides. One of the advantages of this reaction is that it is orthogonal to all unprotected natural amino acid side chains.
5. Synthetic application of Michael addition method
In addition to the cyclization method described above, the Michael addition reaction between electron-deficient center olefin and mercaptan molecules is also often used in the synthesis of cyclic peptides. For example, bismaleimide derivatives can form dis-succinimide complexes with mercaptan through Michael addition.
In addition to the introduction of olefin by exogenous small molecules, the Michael addition can also modify the peptide chain by converting the side chain of cysteine to dehydroalanine (Dha). However, the drawback is that two different stereoisomers are still obtained in the addition process with mercaptan.
