Mechanistic characterization of cyclic peptide drugs is one of the key goals of modern structure-based drug design, as the knowledge of the details of binding, conformation, and modulation of downstream signaling can be utilized to directly inform the optimization of potency, selectivity, and developability of new drug candidates. Cyclic peptides can act via a broad spectrum of mechanisms, and while many small molecules typically bind in a deep and specific pocket of their target protein, cyclic peptides have been observed to allosterically modulate enzymes, antagonize/agonize receptor signaling, and perturb protein–protein interactions, which had previously been considered "undruggable" for small molecules. This is likely a consequence of macrocyclization that imposes significant topological constraints, which naturally preorganizes the peptide into folded and stable secondary structural motifs such as α-helices, β-hairpins, and loop structures that are similar to the common binding elements at physiological binding surfaces. This leads to high affinity binding, as less entropy is lost upon complexation, as well as proteolytic stability and cell permeability, which are often poor in their linear counterparts. This mechanism of action of cyclic peptides is relevant to a number of therapeutic areas, including oncology, immunology, and metabolic disease, as cyclic peptides have been shown to target targets previously considered to be undruggable, expanding the druggable genome and providing new opportunities to modulate disease signaling.
Understanding the mode of action of a cyclic peptide drug is essential for switching from a discovery phase, which is based on high-throughput screening approaches, to a development phase, in which rational design, based on validated hypotheses, is more likely to succeed. Indeed, if the pharmacophoric features of a compound are related to known binding sites (so-called "hot spots"), alterations in the peptide ring size, residue sequence, and backbone chirality can then be guided by structure-activity relationships (SAR) in order to improve affinity, selectivity (in particular, towards anti-targets), and physico-chemical attributes (water solubility, membrane permeability, etc.). Mode of action may also affect dose, and in clinical drug development, it is critical for identification of biomarkers and stratification of patient populations. There are several mechanisms of action by which cyclic peptides are known to work: inhibition of protein–protein interactions (binding to the target), enzyme inhibition (binding to the active site), receptor binding (extracellular or transmembrane domains), and membrane binding (antimicrobial peptides, pore formation). Elucidating mode of action is required in drug development and various types of assays may be useful in validating a given mechanism.
Fig. 1 Effects of cyclization on (a) binding affinity, (b) stability, and (c) membrane permeability.1,5
Protein–protein interactions (PPIs) play a crucial role in various biological processes and have become important drug targets. However, PPIs are often regarded as "undruggable" with small molecules due to their large, flat and featureless binding surfaces. This led to the utilization of other modalities, such as cyclic peptides, to effectively target PPIs. Cyclic peptides have emerged as a potential therapeutic strategy to interfere with PPIs. Their conformationally constrained and diverse structures enable them to mimic key secondary structural elements, such as α-helices or β-sheets, to disrupt PPIs. Additionally, cyclic peptides have the advantage of being able to interact with the extended surface areas of PPI interfaces. Successful examples of cyclic peptide PPI inhibitors include MDM2, integrin and PD-1/PD-L1. MDM2 inhibitors have shown promise in cancer therapy, while integrin inhibitors and PD-1/PD-L1 inhibitors have demonstrated potential in immunotherapy and treatment of inflammatory diseases, respectively.
Protein–protein interactions (PPIs) present a set of attributes that are in many cases antithetical to the historical concept of a drug target, and for decades PPIs were viewed as "undruggable". Inhibitors and modulators of PPIs typically must span large interaction surfaces, and many PPI surfaces are flat and featureless, lacking the concavity and shape complementarity of more typical small molecule binding sites. The surfaces are typically highly distributed with little overall change in shape, making it difficult for small molecules to gain entropic compensation when binding to PPIs. Furthermore, given the relatively large area (often >3000 Å2) of PPI interfaces and relatively low surface-to-volume ratio, small molecules typically bind to a relatively small fraction of the surface with correspondingly weak affinity, and often exhibit poor selectivity among different proteins of the same family. The relative flatness of PPI surfaces, and the prevalence of large hydrophobic residues (valine, leucine, isoleucine, phenylalanine, tryptophan, and methionine) within these surfaces, typically interspersed with more polar or charged patches, present a different topology from that typically found in enzymatic active sites. This has presented a special challenge for small molecules, which cannot span the entire interface, and until recently have required a single scaffold to interact simultaneously with at least two discrete subsites within the binding interface. Alternative approaches, such as peptides, peptidomimetics, aptamers and antibodies have an extended or multiple recognition surface and present a more promiscuous set of chemical functionalities that can be exploited to overcome these issues.
Cyclic peptides can become powerful inhibitors of protein–protein interactions because they can be designed to mimic the secondary recognition elements that naturally participate in protein–protein interactions, allowing for sufficient surface complementarity to be achieved. A peptide segment of an interacting protein surface that is involved in protein–protein interactions can be isolated and macrocyclized to form a constrained α-helix, β-hairpin, or loop structure. The cyclic structure of such peptides will preorganize the side-chains into a specific three-dimensional orientation, as in the native protein–protein interaction, and will also often exhibit higher protease resistance and cell permeability. For example, the MDM2–p53 protein–protein interaction can be inhibited by cyclic peptides that are designed to mimic p53's transactivation domain. Such cyclic peptides fold into helical structures that position the key hydrophobic residues, phenylalanine, tryptophan, and leucine, into MDM2's binding cleft. Cyclic RGD-macrocycles mimic the constrained loop structure found in several extracellular matrix proteins such as fibronectin or vitronectin, to bind to the integrin headpiece with high affinity. In another case, cyclic peptides are used to block the immune checkpoint by directly binding to PD-1, thereby relieving the inhibition on T-cells mediated by PD-L1. The cyclic peptides used as PD-1 antagonists were designed to mimic the β-strand and the connecting loop of PD-1, thereby sterically occluding the PD-1–PD-L1 interaction. The macrocyclic scaffold itself also contributes additional contacts and rigidity that lead to increased affinity for the target protein compared to the linear counterpart. Moreover, the inclusion of non-natural amino acids and D-stereochemistry further increases their metabolic stability and cell permeability. Taken together, cyclic peptides have found utility in both mimicking protein–protein interactions and offering a minimal scaffold that can be used to dissect protein interaction networks for the development of small molecules and biologics.
Characterized by an inherent ability to interact with both enzymes and receptors in a highly specific and robust manner, cyclic peptides represent a unique class of therapeutics that function at the interstice of small molecules and biologics. The design of macrocycles focuses on binding to the active sites of enzymes or receptors' binding pockets which enables them to regulate enzyme activity and signal pathways through inhibition and activation mechanisms. In addition, cyclic peptides allow for interesting chemical space. This is because the cyclic nature of the peptide affords certain levels of conformational preorganization, enabling the functional group preorientation needed to be optimally complementary to that of the target site. This renders the compound more susceptible to the necessary structural requirements for optimal target interaction. As a result, cyclic peptides display several advantages over their linear counterparts, such as greater resistance to proteolytic degradation in some cases as well as an ability to cross cell membranes in other instances. Moreover, their potential to target both intracellular and cell-surface proteins has made them a potentially useful therapeutic for a number of different disease states such as cancer, infectious diseases, and metabolic disorders where improper enzyme activity or deregulated signaling pathways play a pivotal role in disease progression. The following sections will explore the mechanisms by which these peptides and macrocycles interact with and inhibit enzymes, as well as regulate receptor activity.
Cyclic peptides have been employed as protease, kinase, and histone deacetylase inhibitors. The preorganization of the macrocyclic backbone can be leveraged to present the peptidic component into an enzyme active site with high affinity and specificity. Proteases can be inhibited by cyclic peptides that bind either the catalytic triad or the metal center required for peptide bond hydrolysis. By designing cyclic peptide macrocycles with backbones that replicate transition-state geometry or substrate recognition motifs researchers can achieve steric inhibition of catalysis. Cyclic backbones are especially well-suited to proteasome inhibition. Because the peptidic backbone needs to traverse a narrow catalytic chamber, it is constrained to adopt conformations that place electrophilic warheads such as epoxyketone moieties in positions where they are susceptible to irreversible active-site nucleophilic attack. Cyclic peptides have been used to overcome the selectivity issues posed by targeting kinases, which bind ATP in shallow, highly conserved pockets. By binding outside the ATP cleft and making extended contacts with distal hydrophobic patches and allosteric pockets, cyclic inhibitors can gain selectivity by binding to non-conserved epitopes flanking the kinase active site. The binding of the macrocycle enforces an inactive conformation that is unable to phosphorylate substrates and trigger a downstream signaling event. Histone deacetylases represent a challenging target for cyclic peptide drug discovery. These enzymes have deep, hydrophobic tunnels that bind the acetylated lysine side chain and require zinc for catalysis. Cyclic peptides have been used as prodrugs that are activated upon reduction of a disulfide bond, which releases a free thiol group that tightly chelates the catalytic zinc.
Cyclic peptides can be G-protein-coupled receptor (GPCR) agonists or antagonists as well as ion channel modulators. These are two different reasons they are used in the field of precision therapeutics. In terms of GPCR agonists or antagonists, it was recently found that a class of cyclic tetra-peptides modulate opioid receptors, the mechanism of which is said to be "binding, activating, blocking, or modulating opioid receptor-mediated downstream signalling pathways" in this article. It was found that they can be both antagonists, agonists, or partial agonists. The other case is being ion channel modulators. Cyclic peptides can bind to different regions of the ion channel, for example, the transmembrane domains, this interaction causes a conformational change in the channel that alters the opening or closing of the channel, which in turn can modulate neuronal excitability and neurotransmission, making cyclic peptides a potential tool for the treatment of pain and neurological disorders.
Fig. 2 Overview of the screening platform developed to discover agonist or antagonist peptides of human GPCRs.2,5
For cyclic peptide therapeutics, translating in vitro potency to in vivo efficacy requires the drug to pass through the cellular membrane and remain intact in the intracellular environment. These factors, among other pharmacokinetic obstacles, have often been limiting for the development of macrocycle drugs. Cyclic peptides do not diffuse through cell membranes as easily as many small molecules due to size, backbone polarity, and protease susceptibility, making membrane penetration one of the key design trade-offs when developing these drugs. Cyclic peptides enter cells either through passive diffusion (down a concentration gradient) or through a transport mediated uptake, both of which have associated challenges for delivery. For example, passive diffusion relies on a careful balance of a drug's polarity, H-bond donors/acceptors, and lipophilicity to permit transient insertion into the membrane as well as aqueous solubility, while transporter mediated uptake can lead to endosomal trapping of the drug and inefficient escape to the cytosol. Cyclic peptides can also be prone to a number of degradation pathways once inside the cell, and intracellular targets are often modified by proteases, reductases, or oxidative moieties. When creating cyclic peptides scientists need to consider multiple factors because modifications for higher cell permeability often reduce metabolic stability and target binding potential while targeting improvements can affect permeability. Recently, strategies have been developed to make peptide backbones chameleonic such that they can tune their conformational and polarity dynamics in response to their environment. In particular, cationic residues, polar groups, and hydrogen bond donors/acceptors can be hidden from the cellular membrane by making them part of an intramolecular hydrogen bonding network. These peptide backbones can then be designed to become more open, exposing these functional groups, once the drug has reached its cytosolic or nuclear target.
Passive diffusion and transport-mediated uptake are two principal mechanisms by which cyclic peptides can enter cells. Passive diffusion is the primary mechanism for the absorption of small, nonpolar, and lipophilic substances. This process allows such molecules to cross cell membranes directly without the need for energy or transport proteins. The rate and extent of passive diffusion are influenced by the concentration gradient of the molecule across the membrane, with the movement being from an area of higher concentration to one of lower concentration. Larger or charged molecules, which are common among cyclic peptides, may not readily diffuse passively due to the hydrophobic nature of the cell membrane. In such cases, transport-mediated uptake is crucial. This process involves specific proteins in the cell membrane, such as channels or carriers, which facilitate the movement of substances across the membrane. These proteins can aid in passive transport by providing a hydrophilic path for polar molecules, or they can actively transport molecules against their concentration gradient, a process that requires cellular energy. The physicochemical properties of a cyclic peptide, such as its polarity, hydrogen bond capacity, and lipophilic balance, influence whether passive diffusion or transport-mediated uptake is the predominant uptake mechanism. For example, cyclic peptides with high polarity and hydrogen bond donors are more likely to rely on transport-mediated uptake, whereas those with higher lipophilicity may more readily undergo passive diffusion.
After cellular uptake, a cyclic peptide still needs to escape the endosome and remain intracellularly stable. Cellular uptake often results in the encapsulation of the cyclic peptide into membrane-bound endosomes, which can mature and subsequently fuse with lysosomes, the cell's main degradation organelles. This series of processes and barriers that the cyclic peptide must pass after cellular uptake has been called the "endosomal escape problem". There are several proposed endosomal escape mechanisms of a natural basis. One such proposal is that the biological membranes that make up the endosomal membrane experience spontaneous fluctuations, which create temporary openings in the membrane through which some amount of the encapsulated cyclic peptides can escape. Another proposed mechanism is that membrane fusion events that occur during the maturation process may create opportunities for the intraluminal contents of the endosome, including the cyclic peptide, to escape. The precise location of endosomal escape and the mechanisms by which this occurs is a subject of ongoing research. Cyclic peptide intracellular stability is also highly dependent on its resistance to proteolytic degradation and maintaining structural integrity within the intracellular environment. The sequence, conformation, and post-translational modifications of a cyclic peptide can all influence its intracellular stability.
Multi-mechanistic peptide drugs are those drugs that can act at more than one target. A common strategy is the merging of a receptor modulation (agonist or antagonist) and an enzyme inhibition moiety into a single molecular platform. Cyclic peptides, with their unique structural properties and high specificity, are particularly well-suited for this purpose. They can simultaneously interact with multiple targets, such as receptors and enzymes, to modulate complex biological processes. For instance, cyclic peptides can be designed to act as protease inhibitors while also modulating the activity of kinases or histone deacetylases (HDACs). This dual action can be especially valuable in diseases where multiple pathways contribute to the pathology, such as cancer, inflammatory disorders, and neurodegenerative diseases. The design of such peptides often involves careful consideration of their structural features to ensure they can effectively engage with both types of targets. This not only increases their therapeutic potential but also reduces the likelihood of resistance development, as multiple targets are addressed simultaneously. Lanreotide and Cyclosporin A are examples of multi-mechanistic peptide drugs with significant therapeutic value. Lanreotide, a somatostatin analog, is used to target endocrine tissues and modulate hormone secretion. Lanreotide acts by binding to somatostatin receptors, particularly in the pituitary gland and gastrointestinal tract, inhibiting the release of growth hormone and other hormones. This makes it effective in treating conditions like acromegaly and neuroendocrine tumors. Cyclosporin A, an immunosuppressive agent, inhibits the phosphatase activity of calcineurin. By doing so, it prevents the dephosphorylation of the nuclear factor of activated T-cells (NFAT), suppressing the transcription of interleukins and other pro-inflammatory cytokines. This dual mechanism of action—targeting both enzymatic activity and receptor-mediated signaling—makes Cyclosporin A highly effective in preventing organ transplant rejection and treating autoimmune diseases. These examples illustrate how multi-mechanistic peptide drugs can harness their unique structural and functional properties to address complex disease mechanisms, providing significant therapeutic benefits.
Cyclic peptide topology is directly related to the mechanism of pharmacological action and selectivity, which are essential for modulating cellular functions. The closed-loop structure of cyclic peptides confers conformational rigidity, stability, and bioavailability. The presence of a fixed cyclic backbone reduces the entropy loss upon binding and allows for the spatial orientation of functional groups to interact with specific targets. The lack of N and C termini in cyclic peptides minimizes proteolytic degradation, resulting in a longer half-life in vivo. The conformational pre-organization in cyclic peptides can improve the binding affinity and selectivity towards target receptors or enzymes. In the case of PPIs, the extended and flat interaction surfaces often require structurally defined compounds for high-affinity binding. The structural constraints and modifications in cyclic peptides can be tailored through chemical synthesis or bioengineering, offering further pharmacological potential. The optimization of the ring size, the incorporation of non-natural amino acids, or the inclusion of chemical linkers can enhance the desired therapeutic properties of cyclic peptides. By combining these structural advantages with a multi-mechanistic approach, cyclic peptides remain a valuable tool for the discovery of novel therapeutic agents.
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