Cyclic Dipeptides: The Biological and Structural Landscape with Special Focus on the Anti-Cancer Proline-Based Scaffold

Cyclic dipeptides, also know as diketopiperazines (DKP), the simplest cyclic forms of peptides widespread in nature, are unsurpassed in their structural and bio-functional diversity. DKPs, especially those containing proline, due to their unique features such as, inter alia, extra-rigid conformation, high resistance to enzyme degradation, increased cell permeability, and expandable ability to bind a diverse of targets with better affinity, have emerged in the last years as biologically pre-validated platforms for the drug discovery. Recent advances have revealed their enormous potential in the development of next-generation theranostics, smart delivery systems, and biomaterials. Here, we present an updated review on the biological and structural profile of these appealing biomolecules, with a particular emphasis on those with anticancer properties, since cancers are the main cause of death all over the world. Additionally, we provide a consideration on supramolecular structuring and synthons, based on the proline-based DKP privileged scaffold, for inspiration in the design of compound libraries in search of ideal ligands, innovative self-assembled nanomaterials, and bio-functional architectures.


Introduction
Cyclic dipeptides, also known as cyclo-dipeptides, diketopiperazines (DKPs), piperazinediones, dioxopiperazines, or dipeptide anhydrides, are the simplest, naturally occurring cyclic forms of peptides, commonly biosynthesized by a large variety of living organisms [1][2][3][4] and conserved in bacteria to humans [5,6]. They were first discovered in 1880 and later studied by E. Fischer [7]. Once believed to be only protein artifacts or degradation products, and therefore neglected, DKPs are now considered essential metabolic intermediates, and an interesting platform for therapeutic exploration [8]. DKPs possess all advantages of cyclic peptides. DKPs are an unsurpassed class of bio-molecules in their structural and bio-functional diversity. Moreover, the 'biosynthetic hooks' are a useful strategy for the identification of the genes modifying the DKP ring to expand the chemical space of cyclic dipeptides [9,10]. Moreover, modified DKPs have recently emerged as an important pharmacophore in a number of theranostic settings. Notably, proline motif introduces additional conformational and bio-functional value into the DKP-derived structures. The attractive features, such as extra rigidity, structural stability, and consequently, greater resistance to degradation by enzymes, higher bioactivity, specificity, selectivity, and efficacy, increased cell permeability, or binding affinity to specific targets, inherent biocompatibility, structural importance to biological systems [11,12] make proline-based cyclic dipeptides a promising alternative to currently used small molecule and macromolecule pharmaceuticals.
The mechanism of proline-based DKPs formation is described elsewhere [13]. DKPs are 'diamonds in the rough', offering endless possibilities in future innovative therapies [14]. Therefore, since the earliest report on DKP in 1924 [15], an increasing degree of attention to DKPs has been observed, and numerous scientific findings revealing their broad-spectrum biological activities have been reported in recent years. In particular, proline-based DKPs have diverse properties depending on structure and application, such as anticancer, antioxidant, neuroprotective, antiviral, antibacterial, anti-inflammatory, antihyperglycemic, antiarrhythmic, immunomodulatory, antiparasitic, anthelmintic, insecticidal, antifouling, vasorelaxant, and metabolic regulatory activity [16][17][18][19][20][21][22]. They have the potential to be antibiotics of the future. Moreover, they act as antagonists of human oxytocin receptors [23], inhibitors of platelet aggregation [24], calpain inhibitors against stroke [25]. DKPs have relevance in the prevention of cell division [26], cell-cell signaling, or quorum sensing [27]. They are useful in smart delivery systems of drugs that have low permeability to cross the blood-brain barrier [19]. As a curiosity, DKPs are a hot topic in ecological chemistry [28]. Furthermore, DKP-containing compounds are used as catalysts or chiral auxiliaries in synthetic organic chemistry, in the formation of alkaloids [29][30][31].
They are an excellent model in theoretical studies on the constrained structural scaffold with a relevant pharmacophore [17,32,33]. In the future, cyclic-dipeptide-based compounds will find a wider use in various fields as perfect tools for probing specific proteins or metabolites in vivo, or as building blocks for macromolecules.
Here, we present a comprehensive overview of the recent progress on bio-landscape and structural diversity of compounds containing proline-based DKP motif, which are exploited as privileged peptidomimetic scaffolds for future innovative drug discovery, smart delivery systems, and modern bio-control agents [34][35][36]. We pay special attention to anticancer proline-based DKPs since cancers are main cause of death all over the world, with nearly 10 million deaths in 2020 according to the WHO [37]. Therefore, novel, effective, and safe therapeutics are extremely needed. In this context, the supramolecular structuring and the role of versatile synthons for inspiration in the design of compound libraries in search of ideal ligands with unique proline-DKP motif are also discussed.

Occurence and Biosynthesis of DKPs
DKP skeleton is observed in micro-species, bacteria, such as Bacillus subtilis, Streptomyces, Pseudomonas aeruginosa, or Lactobacillus plantarum [38][39][40], marine, and terrestrial fungi [41] as Aspergillus flavus or Alternaria alternata, and Penicillium, respectively [42,43], marine sponges such as Dysidea herbacea, and fragilis [44], or proteobacteria Alcaligenes faecalis, algae, lichens, gorgonians, tunicates, plants, or animals venoms. Remarkably, DKPs were found in human central nervous system, gastrointestinal tract, or blood [45]. They occur in food and beverages, such as pu-erh tea, cocoa, dried bonito, roasted coffee, sake, beer, cheese, casein, chicken extract, or stewed beef, giving a special metallic bitter taste [13,18,31,32,[46][47][48][49], but also in culture broths fermented with lactic acid bacteria. Products containing both L-and D-proline-based DKPs are common in nature, and their structural and biological complexity is highly impressive [50]. As a curiosity, 90% of DKPs in foods contain proline [28]. DKPs provide an eco-friendly approach to food and feed preservation [51]. On the other hand, DKP framework is present in drugs, e.g., in aminopenicillin, amoxicillin, ACE inhibitors [52,53] as by-products of spontaneous intramolecular cyclization of the dipeptidyl moiety in active peptide-based substances. Degradation via DKPs formation is observed during long-term storage. Moreover, DKPs can appear as a result of chemical peptide synthesis, or hydrolysis of functional peptides and proteins [54,55]. The cyclization is facilitated when a proline is present at the second position from the N-terminus (i.e., penultimate proline) [55].
From the biosynthetic point of view, naturally produced DKPs are known to be effective and biodegradable, however their production yield is low [56,57]. Laboratory trials for DKPs-microbial induction have faced some constraints. Although producing DKPs from microorganisms via an expression system is feasible; the optimization is long and not a straightforward process [58]. The biosynthesis of DKPs relies mainly on two enzymes, non-ribosomal peptide synthetases (NRPs) and tRNA-dependent cyclodipeptide synthases (CDPs) [59]. Both enzymes are part of a biosynthetic gene cluster that targets DKPs scaffold modifications necessary for the stability of the produced DKPs [60]. Metagenomics and next generation sequencing enhanced the biosynthetic gene clusters encoding DKP tailoring enzymes [61]. As reported, the microbial genes responsible for a specific secondary metabolite were found to be close to other genes in the dedicated biosynthetic gene clusters [62]. Since the genes responsible for DKPs biosynthesis are clustered on the microbial chromosome, therefore, the encoding of the biosynthetic genes depends on the discovery of a single gene in the pathway. There are approximately 700 known CDPsencoding genes clustered with the predicted tailoring genes [9]. Recently, heterologous expression, zinc finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN) have been used to edit target genes for secondary metabolite induction in microorganisms [63]. However, these approaches found some limitations in their efficiency and productivity level [64][65][66]. Thus, clustered regularly interspersed short palindromic repeats (CRISPR)/associated protein (Cas) system has been recently used as a new approach for the biosynthesis of secondary metabolites and for activation of silent biosynthetic gene clusters [67]. CRISPR/Cas system has outmatched other techniques due to its possible multi-gene editing and high efficiency. Precisely, type II CRISPR/Cas system has been successfully applied for the biosynthesis of secondary metabolites [67]. Previous studies reported the use of CRISPR/Cas9 system in the filamentous fungus Trichoderma reesei, achieving the homologous recombination of > 93% efficiencies [68]. Similarly, Nodvig et al. [69] obtained a genome-edited phenotype by targeting the yA gene in the model fungus Aspergillus nidulans. Thus, CRISPR/Cas system could be a potential mechanism for the efficient biosynthesis of DKPs.

DKP Scaffold
The concept of scaffold is useful in medicinal chemistry and drug design to generate, characterize, and compare cores of bio-active substances and their analogs [70]. The scaffold is the main fragment of compound (e.g., ring system) after removal of substituents (R-groups) [71,72]. The privileged molecular scaffold is defined as a core structure, which forms structurally diverse bio-molecules via introducing different functional groups [73,74]. DKPs can play the role of a privileged, multi-functionalized scaffold for the design and development of advanced therapeutic agents, drug delivery systems, biomaterials, or bio-imaging to mitigate numerous disease conditions, also as for the synthesis of complex natural products [74] because of their specific conformational and physico-chemical attributes. DKPs are heterocyclic compounds consisting of two amino acid residues linked to a central six-membered lactam ring core with (or without) various substituents, providing the control of the substituent's stereochemistry at up to four positions, chiral nature, three-dimensionality, and consequently leading to the promotion of the intermolecular Hbonding interactions with bio-target sites via the corresponding sites of donors and acceptors [17]. Thus, the rigid DKP core allows either a constrained or flexible behavior of amino acids, mimicking preferential peptide conformation [59]. It makes DKP molecules perfect to predicting properties of larger peptides with multiple H-bond acceptors, and donor functionality, and multiple sites for the structural elaboration of diverse functional groups. These characteristics do not only enable them to bind with high affinity to a large variety of receptors and enzymes [32], showing multifarious biological activities, but they also allow a more predictable receptor interaction and the development of the drug-like physicochemical properties that are required for the multi-objective optimization process of transforming lead to a drug product. The general structure for DKP cores can be seen in Figure 1. It should be highlighted that even though 2,5-DKPs are the most popular [32,75], other two regioisomers, such as 2,3-DKPs, and 2,6-DKPs, are also possible as important pharmacophores [76]. All isomers can be found in natural sources in the course of biochemical synthesis. Interestingly, the first crystal structure of DKP, 2,5-DKP, was reported in 1938 [19,77]. More specifically, 2,5-DKPs are common, naturally occurring peptide derivatives (and are frequently generated as unwanted by-products in the synthesis of oligopeptides). The 2,5-DKP core is present in the structure of known drugs, such as Tadalafil, phosphodiesterase-5 inhibitor for the treatment of pulmonary arterial hypertension and erectile dysfunction [78][79][80], Retosiban, an oxytocin antagonist for preterm labor [81], Epelsiban, an oxytocin antagonist in premature ejaculation in men [32], Aplaviroc against HIV [82], in the vascular disruption, and tubulin-depolymerizing Plinabulin, on the basis of marine fungal Halimide, a potential therapeutical drug in lung cancer [83,84], and other anticancer natural agents as Ambewelamide, Phenylahistin, Dehydrophenylahistin [85], Verticillin A [86], antiviral and immunosuppressive Sirodesmin, a type of phytotoxin, anti-inflammatory agents, e.g., FR106969 [59], antibacterial Bicyclomycin, Brevianamide S, Avrainvillamide [87] or Albonoursin, antifungal Maremycin, mycotoxins such as Roquefortine C [22] or Gliotoxin, which is also a potent inducer of apoptotic, and necrotic cell death [88,89]. Thaxtomin A [90], phytotoxin and insecticidal okaramine, and so on. Furthermore, 2,5-DKPs are present in food, e.g., in fermented olives and beverages. Thus, they have the potential to be used in the development of new functional foods [91]. Diverse 2,5-DKPs have been discovered from marine habitats (sponges, and microorganisms) in recent years [14]. These DKPs have cytotoxic (~36%), antimicrobial (~19%), antiviral (~13%), antioxidant (10%), enzyme inhibition (~5%), and other activities (18%) [14].
Proline-based DKPs have a special characteristic [12]. Proline is a unique amino acid due to its specific structure. The DKP nucleus is fused to the pyrrolidine ring, resulting in eminent bio-properties, mentioned earlier. Both L-and D-proline, and to a lesser extent hydroxyproline-based DKPs, exhibit bio-activity. Moreover, proline cis-trans isomerization play a role inter alia in controlling auto-inhibition of signaling proteins [12]. In nature, proline-based DKPs (e.g., cyclo(L-Pro-L-Pro), cyclo(L-His-L-Pro), cyclo(L-Phe-L-Pro), cyclo(L-Tyr-L-Pro), cyclo(L-Leu-L-Pro), and cyclo(L-Val-L-Pro)) are common, which is translated into the structural complexity and impressive bio-activities of this important class of DKPs [51,91,[100][101][102][103], which are thoroughly described in the next subsection. Interestingly, cyclo(Pro-Pro) is as an archaic precursor in the early evolution of life process [5,38]. Notably, many biologically important cyclic peptide sequences, and natural products contain multiple proline residues. The 'proline-rich cyclic structures' have started gaining the attention of the pharmaceutical industry, but their true potential is still very much unknown. Analogs of proline in DKPs should not be overlooked. As an example, silaproline exhibits similar conformational properties, but confers higher lipophilicity and improved resistance to biodegradation [104].

Other Bio-Activities
In the following sub-sections, key issues are only discussed briefly since a growing potential in terms of broad spectrum bio-activities of proline-based DKPs have been observed only recently.

Neuroprotection
Neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS) are age-dependent multifactorial pathologies [19] with various etiopathogenetic sources, but sharing common pathogenic mechanisms: neuroinflammation, oxidative and ER stress; all characterized by neuronal death and degeneration leading to a progressive functional decline. There is no currently available treatment to cure any of those diseases with only symptomatic or slowing down the progress of mental regression is offered.
Therefore, there is a demand for the new class of pharmaceutics providing the neuroprotection and preventing neurons damage. Neurodegenerative diseases could be treated DKP, and their analogs, as they can cross the BBB, because of their unique cyclic structure, and inhibit neuronal degeneration due to their good pharmacokinetic profile and long-lasting neuroprotection. There are mainly two classes of DKP: 1., the TRH-related and 2., unsaturated compounds, proposed recently as potential drug candidates possessing remarkable neuroprotective profile.

TRH-Related DKP
Naturally occurring hormone, thyrotropin-releasing hormone (TRH), L-pyroglutamyl-L-histidyl-L-prolineamide, was the first characterised as playing neuromodulatory role within the central nervous system (CNS) with noticing its rapid enzymatic degradation, what is a big disadvantage for clinical use [176,177]. Therefore, during the last two decades researchers have focused on the cyclic derivatization of the linear TRH peptides, naturally occurring and synthetic [19] with the most investigated cyclo(His-Pro), (CHP), produced by the metabolism of TRH as regulatory agent mediating brain activities [176,177]. CHP is ubiquitous in the cerebrospinal fluid (CSF), blood, and several other body fluids [178] playing important roles in pain awareness, body core temperature, food intake, and modulating prolactin secretion, acting as an endocrine effector. There is a correlation between the decrease CHP level and increased age of patients reaching a value in adulthood of 300 pg/mL in adult age, while for the preterm babies it is 1909 pg/mL. CHP and CHP-like compounds, when increased the presence in CSF, they can improve cognitive function and enhance neurological recovery after trauma due to their molecular mechanisms of neuroprotection to be related to apoptosis and necrosis; DNA repair; oxidative stress; ER stress and unfolded protein response; mitochondrial energy metabolism; together with immunity pathways. Additionally, CHP was found to protect neurons from cytotoxicity induced by salsolinol, a metabolite of L-DOPA linked to Parkinson. CHP was proven to inhibit the pro-inflammator NF-kappaB pathway and its isomers were tested against neuropathological conditions showing an ameliorating potential via elevating METAP2 (responsible for promoting cell proliferation in SH-SY5Y neuroblastoma cells and for exhibiting immune-modulatory activity) expression decreased by Abeta 1-42 [179]. Moreover, the CHP isomer exhibited noncytotoxic and non-mutagenic features in cultured human blood cells and DKP derivatives are promising therapeutics against neurodegeneration-related pathologies [19,179]. They are multipotent anti-Alzheimer drug candidates [179,180]. They also are promising in the treatment of other neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS, mentioned previously) [179]. The TRH-related DKPs are reported to prevent or reduce both necrotic and apoptotic cell death, they were tested in diverse in vitro models to present significantly improved cognitive and motor outcomes and to reduce lesion volumes following traumatic brain injury. From all the bio-evaluations reported in the literature, it could be concluded that the proline residue is a structural element contributing significantly to the DKPs biopotency [19].

Unsaturated DKPs
DKPs consisting of unsaturated units possess the ability to enhance somatosensorymotor function and long-term histological outcome to exhibit a significant radical scavenging activity. The naturally occurring and synthetic DHP derivatives are comprehensively described by Pinnen and colleagues in their mini-review [19]. They concluded that in those DKP scaffolds, the unsaturated motifs play a pivotal role in the protective and reparative properties of these DKPs, suggesting that the increase of bioactivity is strictly related to the presence of unsaturated groups in the DKP and such design can contribute significantly to the development of the potential therapeutic intervention in neurodegenerative diseases.

Other DKPs
The cyclic dipeptides not structurally related to TRH or with unsaturated fragment have also been shown to exhibit neuroprotective and nootropic properties. The first example is cyclo(Leu-Gly), the cyclic analog of H-Pro-Leu-Gly-NH2, the C-terminal neurohypophyseal tripeptide of oxytocin, reducing the blockade of memory induced by puromycin, a protein synthesis inhibiting drug which can impair memory processes. The second example is cyclo(Pro-Gly) that was found to reduce motor neuron death induced by glutamate, probably due to the presence of neuroprotective Gly in the structure and potentially slowing down the progression of ALS [181].
Cyclo(Pro-Gly) is the only, apart from cyclo(His-Pro), cyclic dipeptide in the central nervous system. It can be derived from glycine-proline-glutamate. It has anxiolytic activity [181]. It is an endogenous prototype of Piracetam (derivative of gamma-aminobutyric acid), a nootropic drug. Cyclo(Pro-Gly) and its analog cyclo-L-glycyl-L-2-allylproline are neuroprotective after ischaemic brain injury [182]. Moreover, it has the antinociceptive effect that is mediated through the interaction with opioid system, with fewer side effects [183]. Cyclic glycyl proline compounds are promising in the treatment of Pitt Hopkins Syndrome, a rare, genetic neurological disorder [184], for which there is no cure so far. Moreover, cyclo(Pro-Gly) is a nature nutrient of the blackcurrant anthocyanins. It can improve the function of insulin-like growth factor-1, in Parkinson disease [185,186]. In the latter case, cyclo-L-glycyl-L-2-allylproline has relevance as well [187]. Interestingly, cyclo(Gly-Pro) is a major bioactive component of raksi, an ethnic beverage, considered as a remedy for every possible disorder related to high-altitude sicknesses. This cyclo-dipeptide, reported earlier as antibiotic, is also a potential antioxidant, reported as promising anti-inflammatory, pain reliever, neuroprotective, and antinociceptive [27,188,189].
The derivatives of gliotoxin, without a disulfide bridge, are a new promising class of opioid antagonists with neuroprotective properties. Other DKP-based compounds are inhibitors of opioid receptors as well. DKPs show clinical implication in neurological recovery after a traumatic brain or spinal cord injuries [191][192][193].
2,6-DKPs display a broad anticonvulsant activity in epilepsy, a major neurological disorder, exhibiting a broad spectrum of seizure-suppressing effect. Notably, traditional antiseizure drugs lead to severe side effects and drug-drug interactions caused by enzyme induction. Keeping in mind the drug-resistance as growing problem, DKPs are a good promise for the need of either more effective, safer, or 'disease modifying drugs' that inhibit or reverse the progression of the sickness [96,194].

Mode of Actions
Due to their heterocyclic structure, DKPs possess high biostability against the proteolysis and bioavailability for the active intestinal absorption and crossing the blood-brain barrier (BBB), both challenging targets for natural linear peptides. DKPs can reduce oxidative and ER stress as well as inflammation, the main culprits of several neurological disorders. Some of DKPs can act in the bacterial quorum sensing (QS), therefore they can influence the gut microbiome regulation, quite important in any clinical dysregulations, including neuropathology. This section is divided into four components, each presenting the individual aspect of the DKP properties.

The Blood-Brain Barrier Transporters
The drugs delivery to the specific target sites is a challenge. Biological barriers such as blood brain barrier (BBB) prevent the passage of nearly all drugs to the brain [195]. Most of the DKPs have an ability to cross the BBB [74,196]. More specifically, the ring structure allows permeability, as well as protects against proteolytic degradation. Thus, DKPs are perfect candidates for new therapeutics to treat brain diseases by oral administration [19,197]. The DKP-derived template has been also investigated as a brain shuttle for the delivery of medicinal agents with limited ability to cross the BBB, bypassing the limited bioavailability of several drugs [19,196].

Antioxidant Activity
DKPs are a source of natural antioxidants [198][199][200] due to their significant radical scavenging ability. The spiro-anthronopyranoid DKPs, namely variecolortides, isolated from the mycelia of the halotolerant fungal strain Aspergillus variecolor, exhibit antioxidant activity [4]. The DKPs with the DOPA catechol entity [201], such as cyclo(His-Pro), CHP [176] were isolated from Bacillus sp. N strain [202], as an example. CHP reduces oxidative and endoplasmic reticulum stress by increasing antioxidant capacity in the potential treatment of neurodegenerative diseases [179]. The treatment with CHP isomers provided significant positive oxidative alterations via reducing oxidant status and supporting antioxidant capacity in the cellular AD model by protecting rat insulinoma cells (RINm5F) from streptozotocin (STZ)-induced in vitro cytotoxicity by minimizing of nitric oxide (NO) production and lipid peroxidation [179]. The proline structural element in CHP is responsible for its neuroprotective, nootropic, and cognitive-enhancing properties due to its specific conformation [203,204]. The DKP compounds enhance memory function and the incorporation of the proline in the 2,3-or 2,5-DKP skeleton presents a crucial aspect of neuroprotective action of theses cyclic peptides [19]. They are multipotent anti-Alzheimer drug candidates [179,180]. They also are promising in the treatment of other neurodegenerative diseases, such as ALS [179].

DKP and Gut Microbiota
DKP and their analogs have been isolated from Gram-positive and Gram-negative marine bacteria and identified as a promising source of bioactive compounds with potential pharmacologic activity. These cyclic dipeptides are involved in the bacterial quorum sensing (QS), a cell-to-cell communication system based on the production, secretion, and detection of signals, QS effectors, that contain a family-defining cyclic dipeptide core/scaffold and are produced by proteobacterial species as well as by humans [205]. Therefore, they could play a crucial role in gut dysbiosis, a primary factor behind various gastrointestinal disorders causing increased intestinal and BBB permeability via microbiota-gutbrain axis. Such uncontrolled accumulation of misfolded proteins can facilitate the axonal damage and neuronal demyelination in the pathogenesis of neurodegenerative disorders such as Parkinson's disease, Alzheimer's disease, multiple sclerosis, and amyotrophic lateral sclerosis [206].

Protein-protein Interaction Mechanism
The role of DKP derivatives can be especially important in relation to 'difficult targets' such as protein-protein interactions. More specifically, DKP scaffolds can play a lead role as antiprion agents [195]. Prion diseases, allo known as transmissible spongiform encephalopathies, are fatal infectious and neurodegenerative disorders, while effective drugs are lacking. Both prion and Alzheimer's diseases are protein misfolding disorders. The interplay of prion and Alzheimer's diseases, which cause dementia in over 40 milion people all over the world, revealed an urgent need for antiprion treatment. The progression of the disease is caused by the conformational change of the prion protein, from alpha-helical to beta-sheeted form, forming amyloid aggregates. A planar conformation of DKP core is the main determinant for this kind of bio-activity [207]. DKPs can modulate protein-protein interactions. They can optimize the desired anti-aggregating properties [195].

Metalloproteinase Inhibitors
DKPs, such as cyclo(His-Pro), are important agents in terms of matrix metalloproteinases (and collagenase inhibitors), zinc-based enzymes, engaged in the degradation and remodeling of the extracellular matrix, mainly with regard to cancer or arthritis [32,208], but also multiple sclerosis.

Antibacterial Activity
Novel effective antibacterial agents are extremely needed to fight the increasing serious problem of drug-resistant bacterial infections, claiming the lives of millions of people worldwide [209]. It is the alarming global problem and serious challenge. Short peptides, due to their unique features, are considered as antibiotics of the future [11]. DKPs have great potential in this area due to their specific features (Table 7), mentioned earlier. In particular, proline-based DKP, a known example of DKP classic antibiotic, is bicyclomycin, which is produced by Streptomyces sapporonensis and S. aizunensis against diarrhea [210][211][212][213]. It is also a rho (a member of the RecA-type ATPase) inhibitor [213]. Cyclo(L-Leu-L-Pro) was found to inhibit the growth of Vancomycin-resistant Enterococcus faecalis strains with MIC value of 12.5 μg/mL, an important finding as these types of strains are sometimes untreatable by any current antibiotic or antibiotic combinations [214]. The combination of cyclo(L-Leu-L-Pro) and cyclo(L-Phe-L-Pro) was synergistic with MIC values of 0.25 to 1 μg/mL and was found to be active also against Micrococcus luteus, Escherichia coli, Candida albicans, Staphylococcus aureus, and Cryptococcus neoformans with MIC values of 0.25 to 0.5 μg/mL [32,215]. Other antibiotics, such as avrainvillamide, speramide A, fumitremorgin C, tryprostatin B, spirotryprostatin, are active against multidrug-resistant bacteria [32,41]. Cyclo(L-Trp-L-Pro), known as brevianamide F, isolated from marine fungi Aspergillus fumigatus and Aspergillus sp. and from the bacterium Streptomyces sp., has activity against Gram-positive bacteria [32,216]. Brevianamide S, dimer cyclo(Pro-Trp)-cyclo(Pro-Trp), with antimycobacterial effect, should not be overlooked. Verpacamides, e.g., cyclo(L-Arg-L-Pro), have both antimicrobial and anticancer activity [217]. In another report, seven DKPs from the fermentation broth of Aspergillus fumigatus isolate, apart from cyclo(L-Leu-L-Pro) and cyclo(L-Phe-L-Pro), cyclo(L-Gly-L-Pro), cyclo(L-Pro-L-Pro), cyclo(L-Pro-L-Val), cyclo(L-Leu-L-trans-4-OH-Pro), and cyclo(L-Phe-L-trans-4-OH-Pro), were found to exhibit weak antibacterial activity inhibiting the growth of Staphylococcus aureus and Micrococcus luteus [218]. Additionally, cyclo(L-Pro-L-Pro) isolated from Lucilia sericata demonstrated antibacterial activities against Pseudomonas aeruginosa and Micrococcus luteus [219].  10 mm zone of inhibition [222] 3.

Quorum-Sensing Agonists and Antagonists
In regard to quorum sensing, proline-and hydroxyproline-based DKPs were found to influence cell-cell signaling, offering alternative ways of biofilm control by interfering with microbial communication [225][226][227][228][229]. Cyclo(L-Leu-L-Pro) and cyclo(L-Pro-L-Tyr) found in Pseudomonas putida, were able to activate the biosensor strain Agrobacterium tumefaciens NT1 [221,230]. Holden et al. reported the activation of quorum sensing in Chromobacterium violaceum by cyclo(L-Pro-L-Val) [39]. In another study, cyclo(L-Phe-L-Pro) produced by Vibrio vulnificus was found to activate the quorum sensing on an E. coli biosensor strain by modulating the expression of the gene encoding OmpU protein [231]. Finally, cyclo(L-Phe-L-Pro) and cyclo(L-Pro-L-Tyr) produced by Lactobacillus reuteri, were reported to interfere with quorum sensing in the Gram-positive S. Aureus [41,221,227,[232][233][234].
Moreover, DKPs, especially those containing proline, as chitinase inhibitors, can act as insecticides, fungicides, and antimalarials. DKPs are chitinase inhibitors. Chitin, polymer of N-acetylglucosamine, is a key element of diverse pathogens (fungal, nematodal, and insect). Chitinases, hydrolyzing chitin, play an essential function in the life cycle of pathogens and pathogenesis [32,[238][239][240]. Cyclo(L-Arg-D-Pro) and cyclo(L-His-L-Pro), produced by the marine bacteria Pseudomonas, inhibit the growth of Saccharomyces cerevisiae through mimicking an intermediate structure in the pathological cycle of chitin [241]. It is an appealing lead compound drug [74]. In this context, cyclo(L-Phe-L-Pro) and cyclo(L-Phe-trans-4-OH-L-Pro) are antifungal compounds as well [40].

Antiviral Activity
DKPs containing proline motif exhibit diverse antiviral activities. In this context, there are intriguing indole DPK alkaloids, which are widely distributed secondary metabolites of microorganisms in fungi Aspergillus, Penicillium, Pestalotiopsis, and Chromocleista, and have diverse bio-activities [57]. Marine-derived proline-based DKP alkaloids, such as haenamindole, produced by Penicillium sp. [14,242], raistrickindole from P. raistrickii [113], showing activity against the hepatitis C virus, are good examples. Furthermore, rubrumlines from E. rubrum [14], the indolyl DKP derivatives, and aspamide DKPs, isolated from the Aspergillus versicolor, endophyte from the sea crab, Chiromantes haematocheir, revealing anti-influenza A activity [243] are worthwile too. The latter may be helpful in the treatment of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) [243]. Moreover, eutypellazines, thiodiketopiperazine-type alkaloids from Eutypella sp. present an inhibitory effect in relation to the human immunodeficiency virus (HIV) with low cytotoxicity [244]. Epicoccins isolated from the Epicoccum nigrum are another example of prolinecontaining DKP derivatives causing HIV inhibitory effect [245]. Notably, DKPs are good anti-HIV drugs with high selectivity and few side effects. They inhibit HIV infection and replication [8]. DKPs, as chemokine receptor antagonists, act against multidrug-resistant strains of HIV [41]. Other examples of antiviral DKPs are cis-cyclo(L-Leu-L-Pro), cis-cyclo(L-Phe-L-Pro), and cis-cyclo(L-Val-L-Pro) acting mainly as inhibitors to the proliferation of influenza A virus (H3N2), but also against plant and human pathogenic fungi [246]. Cyclo(L-Tyr-L-Pro) isolated from the Red Sea Spongia officinalis, reveals a strong effect as a hepatitis C virus protease inhibitor [247]. It should be mentioned that haenamindole has weak inhibitory activity against the hepatitis C virus with the IC50 value of 76.3 μM [14]. Cyclo(L-Phe-trans-4-OH-L-Pro), the same as cyclo(L-Pro-L-Leu), cyclo(4-transhydroxy-L-Pro-L-Leu), and cyclo(4-trans-hydroxy-L-Pro-L-Phe) isolated from Streptomyces gougerotii GT and Microbulbifer variabilis exhibited a significant reduction effect on the dengue virus type 2 replication [248].
Furthermore, we can highlight that gliotoxin, mycotoxin containing proline-based DKP core, is an either antiviral or anticancer, antimicrobial, antifungal, and immunomodulatory agent.
Clomycin, albonoursin, ambewelamides, and phenylahistin were reported as agents with antibacterial, antiviral, and immunosuppressive properties [249]. DKP derivatives containing proline moiety play a role of inhibitors of viral haemorrhagic septicemia virus (VHSV), a deadly salmonid rhabdovirus of economic importance in the aquaculture industry [250].

Nucleoside Natural Products
Nucleosides, as the parts of DNA/RNA, are important elements for cells and their metabolic pathways, e.g., energy donors, second messengers, enzyme cofactors. Nucleoside natural products are secondary metabolites of microbes. They offer wide range activity, not only antibacterial, antifungal, antiviral, but also antitumor. In this context, prolinecontaining DKPs are promising for drug discovery, especially antibiotic development [251,252].
Fumitremorgin B and verruculogen are potential growth inhibitors of the parasites Leishmania donovani as well as Trypanosoma brucei brucei [57].

Antitoxin Activity
Cyclo(L-Leu-L-Pro) and cyclo(L-Ala-L-Pro) are inhibitors of production of aflatoxin, carcinogenic toxin, produced by Aspergillus sp. [265,266]. Notably, proline moiety is critical to inhibitory activity. The cis-trans proline isomerism of the N-alkylamide bond in the DKPs is involved in the receptor-mediated bio-activity [267,268]. The relationship between this activity and molecular configuration can be crucial in defining the inhibition mechanism of DKPs [265].

Antagonists of G protein-Coupled Receptors
G protein-coupled receptors (GPCRs) are the largest family in the genome, implicated in different diseases, such as cancer, Alzheimer's disease, depression, type 2 diabetes mellitus, obesity etc., and represent therapeutic targets.
Interestingly, the chemokine receptor 5 (CCR5) antagonists, on the basis of 2,5-DKP scaffold, which was developed for the anti-HIV drug Aplaviroc, belong to a GPCR class [32,82]. The potential of DKPs in relation to classical GPCRs is described below.

Inhibitors of GABAergic Receptors
GABA (gamma-aminobutyric acid) antagonists are drugs inhibiting the action of GABA inhibitory neurotransmitters in the central nervous system. Cyclo(His-Pro) is a cyclic dipeptide endogenous to the brain due to its structural similarity to tyreotropin-releasing hormone [269]. Its aza-analogs are a promising scaffold in the discovery of nextgeneration GABA inhibitors [270]. Piperazinediones reveal anxiolytic activity through the modulation of GABAergic transmission [271].

Antagonists of Oxytocin Receptor
Oxytocin, parathyroid hormone, and alpha-melanotropin have relevance in the occurrence of preterm birth. DKP core facilitates the design and optimization of novel agents to either complement or antagonize the action of the parent hormone [17,23,[274][275][276][277][278]. DKP-based retosiban, and epelsiban, more potent than early non-DKP-based atosiban, was previously developed as an oral drug for the prevention of premature labor [278][279][280]. However, proline-based DKPs as potent and selective antagonists provide effective approaches [278,280] in the treatment of sexual dysfunction, e.g., premature ejaculation, either the treatment or prevention of benign prostate hyperplasia is also worthwhile [32,281,282].

PDE-5 Inhibitors
DKPs can act as efficient inhibitors of phosphodiesterase-5 (PDE-5) useful in the treatment of sexual/erectile dysfunction [78,283,284] as a revolutionary approach. A wellknown example is tadalafil (and its analogs), which is a highly potent and highly selective drug [79,80,[285][286][287] with limited adverse effects [288] and a prolonged action [289,290]. Notably, tadalafil has the longest half-life of the PDE5 inhibitors.
In the case of asthma, we should mention theophylline, a natural substance also used in the treatment of other respiratory disorders, such as chronic obstructive pulmonary disease [303].

Cardio-Metabolic Disorders
There are several reports in the literature describing the relevance of proline-based DKPs in cardio-metabolic dysfunctions. Firstly, the calcium-channel-blocking effect of DKPs was proposed [304] with cyclo(Trp-Pro) blocking channels, and confirmed later [192]. In addition, verrucologens from fungus Penicillium verruculosum are neurotoxins that can block Ca 2+ activated K + channels [305]. The ability of blocking sodium channels by DKPs should not be neglected [306]. Next, the potential of DKPs in the treatment of cardiological dysfunctions with an antiarrhythmic effect of cyclo(Trp-Pro) was presented [307]. They have relevance in the treatment of hypertension, and congestive heart failure [306]. Cyclic glycine-proline, a natural nutrient, normalizes blood pressure in obese rats [186]. Cyclo(L-His-L-Pro), mentioned earlier, also plays a cardiovascular role. It exhibits endocrine and electrophysiological effects [176]. Notably, ~430 million people worldwide suffer from diabetes, and it will reach the seventh cause of death by 2030 [308,309]. Current drugs are not sufficient because of the side effects, such as weight gain, liver damage, and allergic reactions [309]. Antihyperglycaemic activity of DKPs is well-documented in the literature [310,311]. Proline-based DKP alkaloids, from marine fungi, can provide a new platform for the discovery of a drug [312]. Cyclo(His-Pro) decreases the level of blood glucose [176].
Pancreatic lipase is an important enzyme in relation to the digestion of dietary triglycerides. Thus, its inhibition is a promising target in the context of obesity treatment. Cyclo(D-8-acetoxyl-Pro-L-Leu), isolated from the Streptomyces sp. revealed preferable antilipase activity, and could be well bound with the catalytic pocket of the pancreatic lipase [14,313].
Interestingly, cyclo(L-His-L-Pro) inhibits mainly appetite but also the desire for alcohol [21,314].

FSH Receptor Antagonists
Among the glycoprotein hormones used in infertility treatment, follicle-stimulating hormone (FSH) is the major value driver for therapeutic intervention. Without the FSH treatment, there is no ovarian hyperstimulation. DKPs can modulate FSH receptor and were considered as lead structures [8,316,317].

Immunosupressants
Some DKPs possess immunosuppressive properties. In particular, chetomin and gliotoxin are of relevance to the inhibition of macrophage phagocytosis, mitogen-activated T cell proliferation [283,318]. Other known examples are tardioxopiperazines, from the Ascomycete, and cristatin A, from the plant Lepidagathis cristata [32].

Heat Shock Response
Asparaprolines, asparagus-derived proline-containing 3-alkyldiketopiperazines, mainly cyclo(L-Phe-L-Pro), cyclo(L-Tyr-L-Pro), and cyclo(L-Leu-L-Pro), exhibit heat shock protein 70 mRNA induction activity. In more details, the heat shock response, a highly conserved mechanism in organisms, play important role in resetting of the circadian clock in relation to extreme proteotoxic insults (heat, oxidative stress, ultraviolet radiation, heavy metals, toxins, and bacterial infection). The response is mediated at the transcriptional level via heat shock elements, in the upstream area of genes encoding heat shock proteins. Chaperons, members of heat shock protein 70, cause cytoprotective activity through increased cell viability and promotion of protein damage repair. Thus, thanks to antiapoptotic and anti-inflammatory properties, heat shock proteins are therapeutic targets in relation to inter alia regulation of circadian rhythm and sleep, antiulcer drugs, prevention/treatment of diseases caused by protein misfolding such as neurodegenerative disorders or cystic fibrosis [319].

Photoprotective Agents
DKP derivatives have skin photoprotective potential as safe and more eco-friendly agents [320].

Activators of Dioxygen in Oxidative Processes
Proline-based DKPs, including aromaric derivatives, act as catalytic mediators for chemoselective aerobic oxidation processes, such as sulfides oxidation, alkenes epoxidation, or the oxidative coupling of phenols [321]. Cyclo(Pro-Pro) is an archaic precursor in the evolution of life, rendering its potential role in the activation of dioxygen of tremendous importance. Proline 'could condense to afford DKPs under potentially prebiotic alkaline aqueous conditions with high yield' [5,38].

Biological Herbicides and Plant Growth Regulation
Synthetic herbicides can lead to increased human health risks, environmental pollution, and the evolution of resistant weeds. Thus, bio-herbicides from natural sources are required. DKPs are promising nature-inspired herbicides due to their environmental friendliness, safety, high selectivity, and probable new target sites [322][323][324]. Proline-derived DKPs have specific herbicide activities [32]. Notably, proline is linked to diverse plant stresses as defense against toxicity [325]. It is used by a plethora of organisms in protection against the cellular imbalance resulting from environmental stress [326]. The proline improves the formation of reactive oxygen species, signaling, cellular apoptosis [327][328][329]. The maculosin-like peptides cyclo(L-Tyr-L-Pro), but also cyclo(L-Phe-L-Pro) produced by Alternaria alternata were investigated as potent safe, and environmentally friendly agents [32,91]. Thus, proline-based maculosin and its analogs are a specific herbicide [21].
DKPs containing acylhydrazone exhibit insecticidal activity to Plutella xylostella and Culex pipiens pallens [330]. DKP alkaloids are important secondary metabolites of microbes. Indole DKP alkaloids are the condensation products of tryptophan with a second amino acid-like L-proline [105,312,331].

Biotechnological Applications Bone Tissue Engineering
It is a rapidly growing field with the objective of generating a bio-functional tissue, which is able to treat diseased or damaged tissues. The number of orthopedic surgery procedures is projected to ~28 million by 2022 [333]. Therefore, alternative scaffold fabrication techniques are extremely needed in organizing the final, better structure-mechanical properties, and biological response of the implanted biomaterials [334]. DKPs are potential scaffolds for bone regeneration. They can be used in various bone-related applications, inter alia bone defect, and fracture healing, osteoporosis, osteopenia, and many more [333]. DKPs induce osteogenic differentiation, and support osteogenesis with no cytotoxicity. DKP-like compounds (e.g., protuboxepins) are promising inhibitors of induced osteoblastic differentiation. They can offer benefits for fibrodysplasia ossificans progressiva. It is a rare disease causing progressive and widespread heterotopic ossification in soft tissues (muscle, tendons, and ligaments), but surgery is prohibited due to severe heterotopic ossification induced by injury to soft tissues. On the other hand, no drugs have been approved so far. Therefore, DKP-derived agents give a glimmer of hope [335].

Vaccinology
Vaccines are among most successful medical interventions. Vaccines recruit the body's immune system to protect the host from infections or cancer. Modern vaccine formulations are typically comprised of antigen (immunogen) and adjuvant (immune stimulating component) [336]. The antigens are fragments of the targeted pathogen against which an immune system builds the response. Peptide epitopes are minimal antigens and can be divided into two groups: T-cell epitopes, which trigger either cellular or assisting/helper immunity, and B-cell epitopes, which induce humoral immunity [337,338]. Protein antigens typically include several of these epitopes [337,339]. Both peptides and proteins are often inadequately immunogenic, thus adjuvant are required to boost the immunogenicity of vaccine antigen(s) [340]. A wide variety of molecules have been investigated as potential adjuvants for vaccine delivery, including peptides [341][342][343][344][345][346][347][348][349].
DKPs have not been used as vaccine antigens to date. However, they are structurally rigid and easy of modification, thus, could act as peptidomimetic antigens in the future [55,350]. B-Cell epitopes are predominantly discontinuous, and the quality of the antibody response depends on the antigen structural conformation. Therefore, the conformational properties and customization ability of DKP might be employed for antigen design [11,351]. DKP can facilitates orienting amino acid's side chains at 'stereo-correct' distances and angles to superimpose those of native epitopes. Such antigens could additionally be tailored to resist enzymatic degradation [352], thus adapted for oral delivery, and can bear additional targeting moieties for improved delivery to lymph nodes. Further, the rigid structures of DKP-based peptidomimetic antigens could improve immune recognition of epitope's parent protein compared to the highly flexible standard peptide antigens.
In the vaccine design DKP can be employed not only as peptidomimetic antigens. For example, DKPs have been recently used as transfection agents in mRNA-based vaccine formulations [353]. The formulation was based on a phospholipid-based lipid nanoparticles (LNPs) loaded with mRNA that encoded a pathogenic antigen, intended to transfect host cells. Upon immunization with such vaccine, host cell should express the antigen, which is then recognized by immune cells initiating the immune response. To achieve it, the vaccine formulation must escape endosomes, where mRNA could degrade if exposed to enzymes. Therefore, charge altering DKPs were used as endosome escape agents in this mRNA-LNP formulation to generate proton sponge effect enabling translation of mRNA cargo [353]. The DKP-bearing vaccine was highly immunogenic and triggered a long-lasting Th1-responses, with increased IFN-γ + CD4 + and CD8 + T-cell counts and generated high IgG titers.
Natively derived DKPs were recently employed as immunomodulatory agents; to alleviate adverse events or disorders arising from inflammatory immune responses. Aspartyl-alanyl DKP fragment in human serum albumin was found to trigger immunomodulatory effect, via RIP-1-dependent suppression of IFN-γ expression from T-cells, rendering them anergic [354]. A self-administrable, anti-inflammatory intranasal product based on this DKP fragment was launched (Ampion ® ) [355], and currently being investigated for alleviating of severe symptoms in COVID-19 patients (Phase-1, NCT04839965).

DNA Delivery
DKP-based cell penetrating peptidomimetics enable efficient cellular uptake and DNA delivery, via specific noncovalent interactions [74,356]. Incorporation of cyclic peptides improves tolerability against proteases [45]. These mimetics contain lysine and aspartic acid [356]. However, proline-containing cell-penetrating peptides present the greatest penetrating activity, among amino acids, in relation to effective transport of DNA [357,358].

Fluorescent DKPs
They are excellent future scaffolds to form optical agents useful in inter alia smart molecular bio-imaging or activatable fluorescent pro-drugs, while other applications will appear in the coming years. More specifically, drug discovery programs for cell reprogramming or mechanistic studies to characterize cells in tissue microenvironments and clinical applications are the main areas where highly specific probes are needed. They can be helpful in building dual probes with a multimodal character, compatible with diverse imaging modalities, as well as probes of theranostics to deliver imaging reporters, or therapeutic loads. Fluorescent DKPs will play an important role in the development of new smart therapies for personalized medicine. Moreover, proline-containing DKPs, providing extra bio-and conformational features, have potential. We can mention nano-chromophores on based on the self-assembled proline-based cyclo-dipeptides or fluorophorebearing oligomers of DKPs for the intracellular DNA delivery [359]. Notably, the self-assembled, well-ordered structure of DKPs leads to much better fluorescent properties [360].

Inhalable Powder Formulations
DKPs are utilized in the development of innovative forms of delivery systems, such as technosphere dry powder formulation for drugs administration into the pulmonary system. The formulation of insulin-loaded fumaryl DKP microparticles suitable for inhaled delivery (Afrezza) is a good example [74,361]. It is an interesting, new option for subcutaneous injection, which is an inconvenience for patients, and lead to many side effects such as fat atrophy or fat hyperplasia at the injection site, and unsatisfactory blood glucose control [362]. In comparison with other non-injection routes (oral preparation, transdermal patch, pulmonary inhalation, or nasal mucosal administration) it is better due to its physiological advantage. More specifically, alveoli have a large surface area, high permeability, low toxicity, and large circulation perfusion, good lung deposition inhalation, which could rapidly decrease the blood glucose level without immune stimulation, and the drug can be quickly absorbed into the blood circulation, preventing both drug decomposition by digestive intestine enzyme and 'first pass effect' of the liver [74,361,362].

Pheromonal Scaffold
Diatoms, an important ecological group of phytoplankton, an extremely diverse group of microalgae, offer possibilities for biotechnological applications, e.g., sustainable pest control in agriculture [363,364]. The proline derived DKP, cyclo(L-Pro-L-Pro), has been recently reported as the first molecular structure of sex-inducing pheromone from marine diatom, Seminavis robusta sp., with moderate to good bioactivity, and low to no phytotoxicity [365]. Interestingly, diatom-based DKPs have relevance in biofuel production [366].

Supramolecular Structuring of DKPs
Cyclo-dipeptides, as important biomolecules, provide promising minimalistic scaffolds for self-assemblies in terms of molecular recognition, aqueous processability, thermal stability, biocompatibility, structural, and functional versatility towards developing innovative therapeutic modalities, with increased efficacy and reduced side effects. A selfassembly is a process of linking molecules via noncovalent (supramolecular) interactions into (bio)functional systems (assemblies, called supermolecules). It can be observed in living organisms, from bacteria to humans. It is useful in arranging bio-machinery, including the DNA double helix, ribosomes, the quaternary structure of enzymes as well as bioentities, such as cell membranes, the helical structure of collagen, or cytoskeleton [201,367]. Thus, from the supramolecular point of view, non-covalent interactions provide special systems which help to understand bio-processes and create innovative bio-materials that are able to promote 3D cell growth with increased proliferation and differentiation [368]. In this field, DKPs have an inherent tendency to take part in intermolecular interactions driven by specific hydrogen bonding patterns leading to highly organized artificial nanostructures. In particular, DKPs contain four main H-bonding sites, well-oriented H-bond donors, and acceptors, enabling the formation of gelators. In addition, weak forces, such as π … π stacking, hydrophobic effect, electrostatic interactions, act as catalysts by templating unique arrangements toward easier gellation [369]. Notably, peptide-based hydrogels are a new great promise for diverse bio-nano-applications, inter alia in tissue engineering or therapeutics delivery [369]. DKP-based supramolecular hydrogelators are promising in anti-infectious vaccination, also in cancer immunotherapy, mainly because they enhance either the vaccines potency or delivery. They can be excellent novel, safer, and more potent adjuvants. In this context, they have high stability, while the costs of production are low. Moreover, they can be produced and modified in large quantities in an easy way [370]. DKP-based low molecular weight gelators also find applications in encapsulation of drugs [74,370]. Self-assembled functional proline containing DKP-based bio-nano-materials have superior structural rigidity and in vivo stability provided by the DKP ring. On the other hand, they display dynamic features such as morphological flexibility [369]. They can cross the blood-brain barrier, and reach difficult pathological targets, offering versatility of desirable biophysical properties. They have broad-spectrum prospectives in bio-medicine, such as high strength biomaterials, isothermal, and injectable gelation in (stimuli-responsive) the delivery of drugs and other bioactive molecules (as well as gene), or 3D bio-printing, smart bio-nano-architectonics, microfluidic devices, bio-sensing, and point-of-care diagnostics, hierarchical biomimetics [367,370]. DKP nanostructures can encapsulate biomolecules, fluorescent dyes and deliver them inside cells. These theranostics have enhanced efficacy [368]. In addition, we should mention proline-rich cyclic peptides which due to size and complexity, can provide useful scaffolds for modulating more challenging biological targets, such as protein-protein interactions and allosteric binding sites [371]. The proline-based DKP scaffold is highly promising and should inspire researchers to develop other appealing smart self-assembled bio-applications toward an effective clinical translation [372]. In particular, targeting domains to promote receptor binding and to achieve cytoplasmic/nuclear engaging drug delivery applications, can be included [368]. Therefore, better understanding of the nature of self-assembly behavior is required [368]. The design of DKP-based self-assembled materials still is a challenge.
From this point of view, a deep insight into the structural landscape of these supramolecular materials can help to understand the modulation mechanism of DKP self-assemblies. The nature of self-assembly depends on the information encoded in the molecular building motifs called synthons [367]. The term 'molecular synthon' was introduced by Corey as 'structural unit within molecules which can be assembled by known or conceivable synthetic operations' [373], and developed to 'supramolecular synthon' by Desiraju as 'structural units formed with intermolecular interactions' [374]. Both the self-assembly process and applications of the final supramolecular systems (materials) can be modulated by a suitable recognition and design of synthons [367]. Synthons should be robust enough to exchange from one network to another. The proline-based DKP synthons are a bottom-up approach of great importance, providing inspiring self-assembly building blocks to develop bio-functional architectures with appealing, innovative applications of advanced, next generation theranostics, biomimetics, biomaterials, as well as to tune their features via a proper choice of amino acid and stereochemistry of DKPsynthons [32]. Supramolecular interactions forming building motifs (synthons) are increasingly regarded as useful in the design of ideal ligands, inside the protein pocket [375]. However, they have potential in the design of bio-functional materials, with desired and controllable properties, too. Supramolecular interactions, despite their importance in bio-systems, have been mostly behind the scenes because of the difficulty of correct detection. Nevertheless, recent advances in structural biology, and better resolution of 3D structures, open a new avenue to deeper insight into supramolecular bio-complexes leading to better knowledge on the bio-supramolecular interactions engaged in the synthons formation.
In this context, supramoleculas studies on peptides, simple biomolecules, have primary importance. In the course of our ongoing project, focused on the supramolecular perspective of peptide-based systems [6,11,12,52,53,, a thorough screening of the structural databases revealed an appealing sub-family of proline-based DKPs and its analogs. Here, we provide a brief overview of structural databases and library of family of proline-based DKP structures as well as key non-covalent interaction motifs in found compounds, included in the Supplementary Materials (Table S1-S3). Notably, the same motifs are observed in DKP-based bio-complexes ( Figure 2). These findings can be helpful in the design of both more effective drugs and smart supramolecular bio-materials.

Conclusions
To sum up, cyclic dipeptides offer appealing structural and biological diversity. Most recently, more and more interesting DKPs and their derivatives have been isolated from natural sources and investigated in relation to novel, impressive bio-functionalities. The proline-based DKPs are valuable molecular and supramolecular scaffolds, 'programmed' by nature, in synthetic biology and protein engineering, toward tuning either the desirable features of modern theranostics, biomimetics, biomaterials, or interactions via a proper choice of additional amino acid and stereochemistry of DKP-synthons, which may be further chemically modified to increase their bio-activity spectrum. Here, we summarize an overview on both the bio-landscape and supramolecular structuring of proline-based DKPs and their derivatives on the basis of the latest scientific and patent literature as well as structural databases. Proline-based cyclo-dipeptides are extremely compact and stable bio-molecules, making noncovalent self-assemblies extra rigid, biomimetic and smart materials to address complex biological problems, towards innovative biomedical and biotechnological applications for personalized medicine. Taken all together, we hope that this guide will deepen the knowledge on proline-containing DKPs and catalyse further intriguing studies in the field of drug discovery utilizing proline-DKP as a versatile scaffold for the molecular and supramolecular design of innovative smart therapeutics and biofunctional self-assembled (nano)materials that have been challenging so far.
Supplementary Materials: The following are available online at www.mdpi.com/2218-273X/11/10/1515/s1. Table S1: Crystal structures containing DKP moiety, retrieved from the Cambridge Structure Databases. Table S2. Graphical schemes of selected proline-DKP-based supramolecular synthons Table S3. Library of supramolecular synthons, and interactions engaged in the synthons formation, found in proline-based DKP compounds and their derivatives, according to the Cambridge Structure Databases. Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.