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

In contrast to the major families of small molecules and antibodies, cyclic peptides, as a family of synthesizable macromolecules, have distinct biochemical and therapeutic properties for pharmaceutical applications. Cyclic peptide-based drugs have increasingly been developed in the past two decades, confirming the common perception that cyclic peptides have high binding affinities and low metabolic toxicity as antibodies, good stability and ease of manufacture as small molecules. Natural peptides were the major source of cyclic peptide drugs in the last century, and cyclic peptides derived from novel screening and cyclization strategies are the new source. In this review, we will discuss and summarize 18 cyclic peptides approved for clinical use in the past two decades to provide a better understanding of cyclic peptide development and to inspire new perspectives. The purpose of the present review is to promote efforts to resolve the challenges in the development of cyclic peptide drugs that are more effective.

Developing cell permeable cyclic peptides that can reach intracellular targets is a challenging task. 10 Unlike small molecules that enter cells primarily through passive diffusion, peptide-based molecules greater than 1000 Da have distinct physical properties, mechanisms, and cell permeability ability, and currently available theories for improving the ADMET (absorption, distribution, metabolism, excretion, and toxicity) properties of small molecules do not apply to the development of cyclic peptides. 24 Available theories for designing cell permeable peptides, such as passive diffusion, endocytosis and carrier-mediated transport with (CPPs), 25,26 are limited in many aspects. It is well known that CPPs bind to several extracellular receptors and can be actively imported. 27,28 It is necessary to incorporate specific elements in the sequence of the peptide chain, and these chemical alterations can impact the conformation of peptide drugs and further reduce the target binding affinity. Improving the hydrophilic and lipophilic characteristics of peptides with modification strategies such as N -methylation, side chain lipidation, 29,30 and introduction of d -amino acid substituents 31 can all increase the general ability of peptides to pass through lipid bilayer-based cell membranes. However, the fundamental difficulty associated with developing cell-permeable peptides is the lack of knowledge on the mechanisms associated with cell penetration. 10 Five properties, including hydrogen-bonding potential, conformation, charge, size, and hydrophobicity, were observed to influence cell permeability. 24 The extra stability of cyclization introduced into cyclic peptides makes them ideal candidates for entering cells through endocytosis, which requires a resistance to the variety of enzymes present in lysosomes. 32 Romidepsin (1, Table 1 and Fig. 1a ) and voclosporin (2, Table 1 and Fig. 1a ) are the only two cyclic peptides approved in the past two decades that target intracellular protein targets. 33,34 Romidepsin is a natural bicyclic peptide obtained from the bacterium Chromobacterium violaceum in 1994 and approved by the FDA in 2009 for cutaneous T-cell lymphoma (CTCL). 35,36 Romidepsin is a rather small depsipeptide that contains a head–tail lactone cyclization and a pair of disulfide bridges. 37 Although romidepsin can be chemically synthesized, the primary source of romidepsin was manufactured by fermentation. Romidepsin is a prodrug in which the disulfide is reduced to two thiols in the intracellular matrix. 38 Once reduced, thiols can chelate zinc in the zinc-dependent active site of histone deacetylase (HDAC) enzymes, thereby inhibiting histone deacetylases and inducing cell apoptosis. 39 The half-life of intravenously administered romidepsin is approximately 3.5 hours. 40 In rats, the primary route of elimination of romidepsin and its metabolites is the bile with subsequent excretion in feces, and approximately 20% hepatic clearance is metabolized. 41 Because it does not rely on enzymatic degradation or hepatic or renal excretion, the drug is safe to use in patients with any degree of hepatic or renal impairment. 42 Recently, Istodax was withdrawn as a treatment for peripheral T-cell lymphoma, while it remains on the market for treatment of patients with cutaneous T-cell lymphoma (CTCL) who have received at least one prior systemic therapy. 43 The cyclic peptide drugs approved in the last two decades that target intracellular proteins No. Trade name Generic name Target Indication Approval PDB Cycle # Ring size (AA) Cyclization type Hydrophobic a Polar Basic Acidic N-Me Route of admin. Company Sales 2020 1 Istodax Romidepsin Histone deacetylases Anticancer 2009 3RQD 2 3–2 Lactone and disulfide 4 0 0 0 0 IV fusion Celgene Inc. N. A. 2 Lupkynis Voclosporin Calcineurin Lupus nephritis 2021 3ODI 1 11 Head–tail 11 0 0 0 7 Oral Aurinia Inc. N. A. a Based on the physical properties of the sidechains of the canonical amino acids, Ala, Gly, Ile, Leu, Met, Phe, Val, Pro and cystine are categorized as hydrophobic; Asn, Gln, Ser, Thr, Trp and Tyr are categorized as polar; Arg, His and Lys are categorized as basic; and Asp and Glu are categorized as acidic. The noncanonical amino acids are categorized similarly as the canonical analogs. Fig. 1 The chemical structures of cyclic peptides from Tables 1 and 2 that target (a) intracellular proteins and (b) extracellular proteins. Voclosporin was recently approved by the FDA for the treatment of lupus nephritis (LN) on January 22, 2021. 44 Voclosporin was developed from ciclosporin with enhanced potency and metabolic stability. 45 The rationale of the optimization is to replace the butenyl group of the first residue 4-[( E )-2-butenyl]-4, N -dimethyl-l-threonine (Bmt1) in cyclosporin A (CsA) with 4-[(2 E ,4 EZ )-2,4-pentadienyl]-4, N -dimethyl-l-threonine (E-MePmt1) to optimize its fit against the hydrophobic Cn surface to increase the binding affinity. 46,47 Voclosporin is 1

The high potency and low toxicity of cyclic peptides make them ideal for developing antimicrobials for targeting microbial proteins that have no or low similarity to human proteins. 104 In the past two decades, four antibacterial and three antifungal cyclic peptides were approved for clinical use ( Table 3 and Fig. 2 ). All of these cyclic peptides were developed from natural metabolic products that were evolved and produced by organisms for eliminating competitors. 105 The application of cyclic peptides in developing antimicrobials demonstrates the strength of their stable structure in binding to relatively small targets, which can only be achieved by large proteins in traditional beliefs. 106 The action of cyclic peptides depends on a combination of secondary structure, charge, and hydrophobicity and amphipathicity properties, distinct from small molecules’ binding mode. 107 In this section, the structures and action mechanisms of selected antimicrobials will be described and discussed for unveiling the importance of cyclic peptides. Antibiotics and antifungals derived from cyclic peptides approved in the last two decades No. Trade name Generic name Target Indication Approval PDB Route of admin. Cmax a (nM) HT (h) Company Sales 2020 12 Cubicin Daptomycin Membrane pore formation Antibiotic 2005 1T5N IV 11 3400 7.7 Merck Sharp & Dohme Corp. $ 152 M 13 Vibativ Telavancin Cell wall synthesis Antibiotic 2009 IV fusion 57 400 8 Cumberland Inc. N. A. 14 Dalvance Dalbavancin Cell wall synthesis Antibiotic 2014 3RUL IV fusion 15 8000 346 Allergan Ltd. N. A. 15 Orbactiv Oritavancin Cell wall synthesis Antibiotic 2014 IV fusion 77 000 245 Melinta Inc. N. A. 16 Cancidas Caspofungin 1,3-Beta-glucan synthase Antifungal 2001 2N2Q IV 8450 27 MERCK & CO., Inc. $ 430 M 17 Mycamine Micafungin 1,3-Beta-glucan synthase Antifungal 2005 IV 7815 13.4 Astellas Inc. $ 349 M 18 Eraxis Anidulafungin 1,3-Beta-glucan synthase Antifungal 2006 IV fusion 9500 50.3 Pfizer N. A. a Pharmacokinetic data was cited from FDA approval summaries. Fig. 2 The chemical structures of cyclic peptide antimicrobial agents from Table 3 . Daptomycin (12, Table 3 and Fig. 2 ) is a cyclic lipopeptide antibiotic isolated from Streptomyces roseosporus . 108,109 Daptomycin is 13 residues in length with two noncanonical amino acids, 10 of which are arranged in a cyclic format by forming a lactone structure between the sidechain of Thr4 and the C-terminus. Daptomycin acts by inserting its decanoic acid into the cell wall of Gram-positive bacteria in a phosphatidylglycerol-dependent fashion. 110 Aggregated daptomycin creates holes in the membrane of targeted bacteria and disrupts the curvature of the membrane, resulting in ion leakage and a loss of membrane potential. Consequently, the cell activities of protein, DNA, and RNA synthesis are inhibited and apoptotic cell death is induced. 111 The half-life of daptomycin in adult patients is around 8 hours and the majority (78%) remains unchanged and eliminated by excretion into the urine. 112 Telavancin (13, Table 3 and Fig. 2 ) is a semisynthetic derivative of vancomycin obtained by alkylating the vancosamine nitrogen with a decylaminoethyl side chain and a hydrophilic phosphonomethylaminomethyl group on the cyclic peptide residue for achieving improved potency and faster action. 113–117 Telavancin inhibits Gram-positive bacterial cell wall synthesis by binding to the d -Ala- d -Ala terminus of peptidoglycan in the growing cell wall and interfering with the polymerization and cross-linking of peptidoglycan. The MIC 90 of telavancin against S. aureus and coagulase-negative staphylococci strains is ≤1 μg ml −1 , regardless of methicillin resistance. 118 Telavancin was also reported to bind to the cell wall precursor lipid II and disrupts the membrane barrier function. 119 Telavancin can also inhibit liver enzymes cytochrome P450 3A4 and 3A5, and should not be prescribed for patients with pre-existing moderate to severe renal impairment. 120 Telavancin has poor oral bioavailability and is administered by intravenous infusion with a half-life of approximately 6.5 h. 121 Dalbavancin (14, Table 3 and Fig. 2 ) is a novel second-generation lipoglycopeptide antibiotic medication that is more potent than vancomycin and also targets cell wall biosynthesis. 122 The vancomycin family of natural antimicrobial glycopeptides are highly modified heptapeptides that have five invariant amino acids, while amino acids 1 and 3 are highly differentiated. 123 The constancy of the peptide is responsible for binding to the d -alanyl- d -alanine motif, and the sidechain functional groups are derivatized to improve the properties of the peptide. 122 Dalbavancin possesses a potent and rapid anti-bactericidal activity in vitro against a broad spectrum of both resistant and susceptible Gram-positive bacteria, including Staphylococcus aureus , MRSA, enterococci , and streptococci . 124,125 Precursor glycopeptides of dalbavancin is manufac

Cyclic peptides have received increasing attention as a unique category of substances with many advantageous characteristics as drugs in comparison to small molecules and macromolecular antibodies. Natural peptides have been the major source of approved cyclic peptides in the last century due to the large number of cyclic peptides present in nature. The generation of specific biological functions by natural evolution also contributed to the drug development of cyclic peptides. Cyclic peptide hormones, such as oxytocin and vasopressin, are potent and selective agonizers and were used in clinics for a long time. Antagonizing cyclic peptides, such as the large number of toxins used by diverse species against competitors and prays, are widely present in nature and are a rich source for drug development. In the past two decades, the primary trend in peptide drug discovery has been the shift of naturally isolated cyclic peptides toward cyclic peptide analogs optimized for improving potency, stability and pharmacokinetic properties. By incorporating aromatic naphthalene and d -tryptophan into lanreotide, its nanotubes with supramolecular packing were stabilized, which contributed to its extended half-life and ability to suppress hormone levels and activities. From a technical perspective, cyclic peptide drug development benefited from rapid lead discovery with adjustable and expandable optimization by chemical synthesis, which was the milestone in peptide drug development. The mutation of the lipophilic side chain of oritavancin prolonged its half-life and enhanced coverage against several vancomycin-resistant bacteria. Fermentation of cyclic peptides also benefited from modern molecular biology technologies, and the primary sequences of enzymes could be rapidly mutated and optimized for achieving a higher production yield. Increased expression of echinocandin B deacylase in the production host significantly increased the transcription and bioconversion efficiency of anidulafungin. The convenience to incorporate nonnatural elements helped to improve not only the potency, but also the stability and pharmacokinetic/pharmacodynamic properties. Noncanonical amino acids were introduced to improve proteomic resistance. Amino acids with hydrophobic long side chains were incorporated for extending plasma retention time and introducing extra biological activities of cyclic peptides. The population of Fmoc based solid-phase peptide synthesis technologies also greatly reduced the cost of production and promoted cyclic peptide research in individual groups. The combination of modern recombinant technologies and peptide chemistry has solved the major problems of cyclic peptide production, mutation and optimization of the primary sequences. However, the space of optimizing natural cyclic peptides for achieving cyclic peptide drugs is limited due to the expanded desire to develop cyclic peptide ligands targeting diverse proteins. Recent development of cyclic peptide discovery was characterized by the generation of combinatory libraries that can cover a much larger chemical space and the development of novel screening methodologies for identifying binders and inhibitors. 4,159,160 Split-and-mix cyclic peptide libraries could be synthesized with millions of diversities and screened against various targets. 161–167 However, the difficulties associated with sequence identification of cyclic peptides by partial Edman degradation or mass spectrometry limited the applications of one bead-one peptide library. Directed evolutions with genetically displayed cyclic peptide recombinant libraries are increasingly playing more important roles in next-generation cyclic peptide drug discovery. Display systems such as mRNA display, 168–171 DNA display 172 and phage display 22,173,174 offer the advantages of large library diversity and rapid screening, and have resulted in the discovery of a good portion of cyclic peptides in clinic development. Compared with the typical diversity of only a few thousands for conventional natural and synthetic cyclic peptide libraries, the diversity of cyclic peptide libraries built by genetic recombinant technologies is several orders higher. The introduction of various cyclization strategies, noncanonical amino acids and even functional moieties have further increased the functionality of recombinant libraries, such as chemical stability, metabolic stability and uniform conformation. The development of selection strategies also enabled the discovery of cyclic peptides that have special properties such as cell membrane permeation ability and oral bioavailability. A second trend in cyclic peptide drug discovery is the development of cyclic peptide conjugates to achieve a selective delivery of different effector molecules to target tissue. Besides the drug itself, which induces the desired biological functions, and the linker, which controls drug release, cyclic peptides have been investigated intensively to facili