TRMOME-974; No. of Pages 8

Cyclic dipeptides: from bugs to brainIlaria Bellezza, Matthew J. Peirce, and Alba Minelli

Experimental Medicine Department, Polo Unico S. Andrea delle Fratte, University of Perugia, 06124 Perugia, Italy

Opinion

Cyclic dipeptides (CDPs) are a group of hormone-likemolecules that are evolutionarily conserved from bacte-ria to humans. In bacteria, CDPs are used in quorumsensing (QS) to communicate information about popu-lation size and to regulate a behavioural switch fromsymbiosis with their host to virulence. In mammals,CDPs have been shown to act on glial cells (macro-phage-like cells) to control a conceptually homologousbehavioural switch between homeostatic and inflamma-tory modes, with implications for the control of neuro-degenerative disease. Here we argue that, because oftheir capacity to regulate inflammation via glial cells andinduce a protective response in neuronal cells, CDPshave potential therapeutic utility in an array of inflam-matory diseases.

QSQS (see Glossary) is a mechanism of cell–cell communica-tion via secreted, hormone-like signalling molecules that istraditionally associated with bacteria. QS is often used toconvey information about population density and has beenimplicated in the switch between symbiosis and virulenceby regulating the expression of a particular set of genes[1]. Initially QS was described as a means of communica-tion between individual members of the same bacterialpopulation but it has become apparent that some elementsof QS can be shared between different bacterial species [2],providing a mechanism by which one bacterial populationmay modify the function of a second, possibly distinctbacterial species. Moreover, there is a growing body ofdata indicating that molecules functioning as QS signalshave the capacity to modulate both plant [3] and mamma-lian [4,5] signal transduction. With growing awareness ofthe importance of the diversity of bacterial populations(microbiome) to the physiology of their mammalian hosts,understanding QS and the molecules it employs acquiresadded, clinical significance.

QS was first described in Vibrio fischeri [6], a luminoussymbiotic species that provides its marine eukaryotic hostswith light (Figure 1). After the discovery of the genes (luxIand luxR) responsible for the phenomenon in V. fischeri,homologous systems with diverse biological roles werefound in other proteobacterial species [7–14].

In Vibrio harveyi, a free-living marine bacterium lack-ing the luxI/R system, light production is controlled by two

1471-4914/

� 2014 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.molmed.2014.08.003

Corresponding author: Minelli, A. (alba.minelli@unipg.it).Keywords: diketopiperazines; quorum sensing; neuroinflammation; neurodegenera-tion; neuroprotection.

nonhomologous parallel pathways. The autoinducer of oneof these pathways is a furanosyl borate diester [14], synthe-sised from the gene luxS, whose receptor is a membrane-associated sensor kinase, LuxQ. Several Gram-negativeand Gram-positive bacteria use this inducer and all bacte-rial species with luxS show a similar biosynthetic pathway.Hence, it might be assumed that this system is used ininterspecies communication [15,16]. The gene luxS con-trols toxin production in Clostridium perfringens and thevirulence cascade in Vibrio cholerae, Escherichia coli, andSalmonella typhimurium [17,18].

The genes responsible for QS are distributed in a dis-continuous manner among bacteria [19]. Phylogeneticanalyses of the genes underlying the two QS systems(i.e., LuxI/R and LuxS) show that these systems are an-cient and were established early in evolution with a com-plex history of lateral transfer, ancestral duplication, andgene loss within the genus [19–22].

While most of the intracellular architecture of QS hasalready been clarified, the use of multiple signals by manybacterial species has complicated the task of defining theirfunctional roles [23]. Recently, however, a new functionalmodel of combinatorial QS has been proposed showing thatbacteria can sense their social and physical environmentusing combinatorial (nonadditive) responses to multiplesignals with distinct half-lives [24]. When multiple mole-cules are secreted, by combinatorial processing of theinformation a bacterium can greatly increase the rangeof its responses to social and physical environment cues.

The QS mechanism has been lately extended to signal-ling between phyla or inter-kingdom signalling [4,25] andforms part of the extensive communication between micro-organisms and their hosts. Bacterial products can modu-late mammalian cell-signal transduction, while hosthormones can cross-signal with QS signals to modulatebacterial gene expression [5], suggesting a complex con-nection between host stress signalling, bacterial QS, andpathogenesis. It has been proposed that decoding thecommunication between bacteria and their hosts will leadto the design and implementation of novel strategies fortherapeutic control of the human microbiome to optimiseits performance as well as to improve the diagnosis andtreatment of pathologies resulting from dysregulation [12].

CDPs: structural considerationsCDPs, or 2,5-diketopiperazines (DKPs), a recently discov-ered family of biologically active small molecules, aresynthesised by proteobacterial species as well as byhumans [26–28]. CDPs are generated by tRNA-dependentcyclodipeptide synthases and contain a family-definingCDP ‘core’ or ‘scaffold’ structure (Figure 2). Diversity

Trends in Molecular Medicine xx (2014) 1–8 1

Glossary

Alzheimer’s disease (AD): the most common form of dementia. In the early

stages, the most prominent symptom is short-term memory loss. As the

disease advances, symptoms can include confusion, irritability, aggression,

mood swings, trouble with language, and long-term memory loss. Gradually

bodily functions are lost, leading to death. AD is classified as a neurodegen-

erative disorder, associated with plaques and tangles in the brain.

Amyotrophic lateral sclerosis (ALS): a neurodegenerative disease charac-

terised by muscle spasticity, rapidly progressive weakness due to muscle

atrophy and difficulty in speaking (dysarthria), swallowing (dysphagia), and

breathing (dyspnea) due to degeneration of the upper and lower motor

neurons. Individuals affected by the disorder may ultimately lose the ability to

control all voluntary movement, although bladder and bowel function and the

muscles responsible for eye movement are usually spared until the final stages

of the disease. Cognitive function is generally spared for most patients.

Biofilms: a structured community of bacterial cells enclosed in a self-produced

protective polymeric matrix and adherent to an inert or living surface.

Blood–brain barrier (BBB): a highly selective permeability barrier that separates

the circulating blood from the brain extracellular fluid (BECF) in the CNS.

Formed by endothelial cells that are connected by tight junctions, it allows the

passage of molecules crucial to neural function and prevents the entry of

potential neurotoxins.

Cyclic dipeptides (CDPs), or 2,5-diketopiperazines: relatively simple com-

pounds resulting from nonenzymatic cyclisation of dipeptides and their

amides. They are the most common peptide derivatives found in nature and

are synthesised by proteobacterial species as well as by humans. CDPs are

characterised by stability to proteolysis and promotion of interactions with

biological targets.

Cyclic scaffold: a six-membered ring that, due to its stable structural

characteristics, represents a significant pharmacophore in medicinal chemis-

try.

Endoplasmic reticulum (ER) stress response: the ER, the organelle essential for

calcium storage, lipid synthesis, and protein folding and secretion, can

accommodate increases in the demand for protein folding. Situations where

extracellular stimuli and changes in intracellular homeostasis cause protein

misfolding characterise the ER stress response.

Human microbiome: the human body comprises around ten trillion cells but

harbours 100 trillion bacteria; for example, on the skin and in the gut. This is

the human ‘microbiome’ and has a huge impact on human health. Never-

theless, humans, in turn, can affect their microbiome by influencing the species

of bacteria that take up residence in and on their bodies.

Inflammation: a response of the innate immune system to harmful stimuli such

as pathogens, damaged cells, or irritants. It is a protective attempt by the

organism to remove the injurious stimuli and to initiate the healing process.

Classical signs are pain, heat, redness, swelling, and loss of function.

Microglia: a type of non-neural cell that constitutes the resident macrophages

of the brain and spinal cord and acts as the first and main form of active

immune defence in the CNS.

Parkinson’s disease (PD): a progressive neurodegenerative disease that

belongs to the group of conditions called motor system disorders. PD often

occurs after the age of 50 years and is one of the most common nervous

system disorders of the elderly. PD is caused by slow deterioration of the

dopamine-forming nerve cells in the brain. Dopamine is a natural substance

found in the brain that helps control muscle movement throughout the body.

Pharmacophore: a part of a molecular structure that is responsible for a

particular biological or pharmacological interaction.

Quorum sensing (QS): a mechanism of cell–cell communication via secreted

signalling molecules. Secreted autoinducers regulate the expression of a

particular set of genes once the cell population density is sufficient to produce

a threshold accumulation of the secreted autoinducer.

Reactive oxygen species (ROS): a number of reactive molecules and free

radicals derived from molecular oxygen, such as singlet oxygen, superoxides,

peroxides, the hydroxyl radical, and hypochlorous acid.

Thyrotropin-releasing hormone (TRH): a tripeptide hormone produced by the

hypothalamus that stimulates the release of thyroid-stimulating hormone and

prolactin from the anterior pituitary.

Unfolded-protein response (UPR): an evolutionarily conserved response

related to the ER stress response. The initial intent of the UPR is to adapt to

the changing environment and re-establish normal ER function. When

adaptation fails, ER-initiated pathways signal alarm by inducing the expression

of genes encoding mediators of host defence. Excessive and prolonged ER

stress triggers cell suicide, usually in the form of apoptosis, representing a last

resort of multicellular organisms to dispense with dysfunctional cells.

Virulence factors: molecules expressed and secreted by pathogens (bacteria,

viruses, fungi, and protozoa) that enable them to replicate and disseminate

within a host in part by subverting or eluding host defences.

Opinion Trends in Molecular Medicine xxx xxxx, Vol. xxx, No. x

TRMOME-974; No. of Pages 8

2

within this molecular family is generated by the combinedaction of a cyclodipeptide oxidase and two distinct S-ade-nosyl-methionine (SAM)-dependent O/N-methyltrans-ferases [29], which yields various chemically diverse sidechains. The potential to use CDPs as pharmacologicalagents is aided by the fact that they can be obtained byextraction from natural sources or prepared by relativelystandard chemical syntheses [30]. In addition, the coreCDP scaffold makes these molecules resistant to proteoly-sis and enables them to cross the intestinal barrier andblood–brain barrier (BBB) [31,32]. Finally, the chemicalflexibility afforded by the addition of various side chainsenables ‘tailoring’ of these compounds to interact with anarray of biological target molecules. This combination ofstructural stability and flexibility means that CDPs areideal pharmacophores.

CDPs as QS signalsCDPs represent a new class of QS signal molecule and,potentially, interspecies or even inter-kingdom signals[33–35]. Cyclo(Phe–Pro) produced by Vibrio vulnificus in-duced the expression of V. fischeri lux genes. Cyclo(Phe–Pro) also enhanced the expression of the ctx genes in V.cholera, which are known to be ToxR regulated [36]. Thisindicated that cyclo(Phe–Pro) is a signal molecule that cancontrol the expression of genes important for Vibrio path-ogenicity, acting as a QS signal [36]. Confirmatory resultson the capacity of QS signalling by CDPs were laterobtained by further studies on V. cholerae. The ability ofV. cholerae to cause disease depends on the production oftwo critical virulence determinants, cholera toxin (CT) andtoxin-coregulated pilus (TCP), whose expression is con-trolled by the ToxR regulon. Cyclo(Phe–Pro) produced byV. cholera inhibited the production of the virulence factorsby activating the expression of leuO, a LysR-family regu-lator. Increased leuO expression represses aphA transcrip-tion, which results in downregulation of the ToxR regulonand attenuates CT and TCP production [36]. Also, a syn-thetic CDP, cyclo(Val–Val) [37], was capable of inhibitingthe virulence factor production by a ToxR-dependent pro-cess. These results suggest that CDPs might be used astherapeutics for cholera treatment and that hydrophobicamino acid side chains on both arms of the cyclic scaffoldare a structural requirement for its inhibitory activity.

The human vaginal commensal bacterium Lactobacillusreuteri RC-14 produces two CDPs, cyclo(Phe–Pro) andcyclo(Tyr–Pro), both capable of interfering with the staph-ylococcal agr QS system, a key regulator of virulence genes.Moreover, the same CDPs repress the expression of toxicshock syndrome toxin-1 in Staphylococcus aureus MN8, aprototype of menstrual toxic shock syndrome [15]. Theseresults suggest that there may be some redundancy amongCDPs and, besides providing a better understanding of thecommunication between Lactobacillus and Staphylococ-cus, also attest to a unique mechanism by which endoge-nous or probiotic strains may attenuate the production ofvirulence factors by pathogenic bacteria.

Biofilms are architecturally complex bacterial commu-nities held together by an extracellular matrix andare found in more than 80% of human Staphylococcusepidermidis infections. The dipeptide cis-cyclo(Leu–Tyr)

Low cell density

LuxRLuxR

LuxR

LuxR Luxl LuxC LuxD LuxA LuxB LuxE LuxR Luxl LuxC

Gene expression

Luciferase

Light

LuxD LuxA LuxB LuxE

Luxl Luxl

High cell densityVibrio fischeri

TRENDS in Molecular Medicine

Figure 1. Lux pathway controlling bioluminescence in Vibrio fischeri. At low cell density, the level of the autoinducer (yellow circles) synthesised by LuxI is below the

threshold to activate LuxR. At high cell density, the autoinducer reaches the threshold level and binds to LuxR protein, leading to transcription of the lux operon and

bioluminescence.

Opinion Trends in Molecular Medicine xxx xxxx, Vol. xxx, No. x

TRMOME-974; No. of Pages 8

can inhibit biofilm formation [38] and may potentially beused as a therapeutic for these infections. S. aureus, anotherimportant human pathogen responsible for nosocomial andcommunity-acquired infections associated with high mor-bidity and mortality, also forms biofilms and is generallyassociated with chronic skin ulcers [39]. CDPs represent themolecules by which S. aureus modulates its pathogenicity;therefore CDPs, may be exploited as potential therapeuticagents for chronic S. aureus biofilm-based infections[15,40]. In general, it is clear that these compounds couldbe used to potentially block or mimic native autoinducersignals and attenuate QS and thus regulate the expressionof virulence genes and reduce the impact of bacterial infec-tion on human health [41].

Cyclic scaffold

O

O

HNHN

NN

His�dine side-chain

Proline side-chain

TRENDS in Molecular Medicine

Figure 2. Chemical structure of cyclo(His–Pro). The cyclic scaffold (red), histidine

side chain with unsaturated system (blue), and proline side chain (green) are

marked.

CDPs: an ancient behavioural switch?The studies highlighted above indicate a role for CDPs inQS, an ancient and evolutionarily conserved signallingpathway controlling a switch in behaviour between symbi-osis and virulence. We present evidence below that CDPsare also generated endogenously in higher mammals andthat CDPs can change the state of responding cells. CDPscan promote the restoration of a homeostatic behaviouralprogram in glial cells from an inflammatory program, aswell as inducing a protective state in neuronal cells. Whilethe molecular details of this pathway in higher animalsremain to be elucidated, there are conceptual parallelsbetween the switch in bacteria between peaceful coexis-tence with the host (symbiosis) and aggressive invasion(virulence) and that in mammalian macrophage-like cellsbetween inflammatory and homeostatic modes (Table 1).

Interestingly, bidirectional communication between thegut microbiome and the central nervous system (CNS) hasrecently been reported [42–45], raising the possibility thatCDPs might also act on the CNS indirectly, by interveningin the human gut microbiome–brain axis. However, thistopic is beyond the scope of this Opinion.

CDPs as CNS agentsNeurodegenerative diseases such as Alzheimer’s disease(AD), Parkinson’s disease (PD), and amyotrophic lateralsclerosis (ALS) are age-dependent multifactorial disorderscharacterised by neuronal death and degeneration leadingto progressive functional decline. A growing body of datasuggests that these diseases result, at least in part, from

3

Table 1. Do bacterial CDPs parallel human CDPs?

CDPs in bacteria CDPs in mammals

Endogenous compounds [15] Endogenous

compounds [26,27]

Cell–cell communication [1,6,34–40] ?

Synthesis ex novo [35,38] Synthesis ex novo/

proteolytic cleavage [26]

Binding protein (LuxR) [6–14] ?

Gene expression regulation [6–14] Gene expression

regulation [54,63,67,72]

Environment sensing [24] ?

Switch between virulence/symbiosis

[37,38]

Switch between

homeostasis/

inflammation [67]

Cyclo(Phe–Pro) [15,36]

Cyclo(Tyr–Pro) [15]

Cyclo(Leu–Tyr) [38]

Tyrvalin and phevalin [40]

Cyclo(His–Pro) [54,63,67,72]

Cyclo(Pro–Gly) [47]

Opinion Trends in Molecular Medicine xxx xxxx, Vol. xxx, No. x

TRMOME-974; No. of Pages 8

inappropriate neuroinflammation [46]. The current avail-able treatments are symptomatic and do not address theunderlying inflammatory cause of the disease. Moreover,some of the more effective therapies targeting inflamma-tory diseases of the periphery such as rheumatoid arthritis[e.g., antitumor necrosis factor (TNF)] may be ineffectivebecause they fail to cross the BBB. There currently exists aclear unmet clinical need for novel modalities specificallyto target neuroinflammatory disease.

Naturally occurring hormones such as corticosteroids,progesterone, and thyrotropin-releasing hormone (TRH)have been among the first compounds to be studied fortheir multipotential neuroprotective effects. Evidence hasbeen found for a significant neuroprotective role of TRH(reviewed in [27]) and TRH or TRH analogues can signifi-cantly improve neurological recovery after traumatic brainor spinal cord injuries. TRH is a tripeptide present in theCNS, where it acts as a hypothalamic neuroendocrinesignal eliciting several behavioural responses. The mainmechanism responsible for the extracellular catabolism ofTRH within the CNS is the hydrolytic removal of theamino-terminal pyroglutamic acid residue by pyrogluta-myl aminopeptidases (PPs) (reviewed in [27]). After thecleavage, there is a subsequent cyclisation of the dipeptideHis–Pro-NH2 that produces the CDP histidyl–proline[cyclo(His–Pro)]. The process of cyclisation confers highstability against the activity of peptidases and is a struc-tural prerequisite for its active transport in the intestine[31] and its passage through the BBB [32], a key charac-teristic for the delivery and specific targeting of cyclo(His–Pro) therapy in the CNS.

Cyclo(His–Pro) has been demonstrated to be minimallyderived from TRH metabolism and is indeed synthesisedde novo endogenously in vivo [26]. Cyclo(His–Pro) is ubiq-uitous in the CNS and has been found in the gastrointes-tinal tract and prostate, as well as in several body fluidssuch as blood, semen, cerebrospinal fluid, and urine[26]. The only other endogenous CDP reported in theCNS is cyclo(Pro–Gly). Found in rat brain, it is a memo-ry-facilitating substance with anxiolytic activity [47]. Noeffects related to inflammation or neuroprotection havebeen reported.

4

Cyclo(His–Pro) and the mammalian brainOrganic cation transporter (OCT) 2 and cyclo(His–Pro)

transport

To exert a functional effect, cyclo(His–Pro) must first beinternalised by cells. Until recently the role of transportersduring reabsorption and excretion of drugs received onlyminor attention. This changed after the discovery of mam-malian drug efflux transporters of the ATP-binding cas-sette (ABC) family, such as polyspecific OCTs[48,49]. Members of the OCT family share functional prop-erties such as: (i) the ability to translocate organic cationswith differing molecular structures; and (ii) bidirectionaltranslocation across the plasma membrane independent ofthe transmembrane sodium gradient [50]. Cyclo(His–Pro),with its positive charge from one of two imidazolic nitro-gens, is a selective endogenous substrate of the organiccation transporter OCT2 in the brain [49]. OCT2 is pref-erentially expressed in the dopaminergic brain regions,with the highest central expression in the substantia nigrapars compacta (SNc), the area with the highest density ofdopamine cell bodies in the CNS [49]. Analysis of thedistribution of cyclo(His–Pro) in the rat brain indicatesthat its prevalence is greatest in dopaminergic areas,supporting the highly specific function for OCT2 in thenigral dopaminergic system [49]. In vitro data generatedusing OCT2-transfected HEK-293 cells and SH-SY5Y andHTZ-146 cells also support the role of OCT2 in internalis-ing cyclo(His–Pro) [49].

Cyclo(His–Pro) and glial cell-mediated inflammation

Chronic neuroinflammation underlies many neurodegen-erative conditions (Box 1). Lipopolysaccharide (LPS) is acommon inflammogen that, by interacting with the mem-brane receptor Toll-like receptor 4 (TLR4), triggers a di-verse array of microglial responses leading to theproduction of proinflammatory mediators and nuclear fac-tor kappa B (NF-kB) activation [51]. Moreover, LPS spe-cifically activates part of the endoplasmic reticulum (ER)stress response pathway, contributing to its proinflamma-tory effect [52–54]. LPS leads to neuronal damage only inthe presence of microglia, whose activation is toxic to theneighbouring neurons. This toxicity can in turn causefurther microglial activation and a self-propelling progres-sive cycle of inflammation and neuron damage [55]. It hasbeen shown that systemic administration of cyclo(His–Pro)exerts anti-inflammatory effects in vivo in the CNS bycounteracting the LPS-induced reactive gliosis [54].

Nuclear factor-like 2 (Nrf2) is a transcription factor thatregulates the constitutive and inducible expression of an-tioxidant and phase 2 detoxification enzymes via a cis-acting DNA element called EpRE (ARE). The Nrf2–AREpathway represents a physiological adaptation to oxidativestress and its activation is the major mechanism in termi-nating the NF-kB-driven immune response. NF-kB is apleiotropic transcription factor that regulates the expres-sion of genes involved in the innate and the acquiredimmune response and the associated inflammation re-sponse. Several phosphorylation cascades activate NF-kB in response to stimuli such as stress, cytokines, freeradicals, UV irradiation, oxidised low-density lipoprotein(LDL), and bacterial or viral antigens. Activated NF-kB

Box 1. Neuroinflammation

Neuroinflammation, usually triggered by peripheral inflammation,

describes a broad range of immune responses of the CNS by

microglia, astrocytes, and the BBB, with each element linked by

dynamic crosstalk. The BBB is permeable to proinflammatory

mediators derived from peripheral inflammation and is able to

release and transmit these mediators and allow leucocyte migra-

tion. Thus peripheral inflammation may cause prolonged and

damaging neuroinflammation. The neuroinflammatory response

results in synaptic impairment, neuronal death, and eventually

neurodegeneration. A crucial role in the process of neuroinflamma-

tion is played by microglial cells, the resident macrophages of the

CNS. In response to cytokines and other signalling molecules from

acute inflammation, microglia convert to an activated phagocytic

state and release proinflammatory mediators. Microglial activation

is toxic to neighbouring neurons, which causes further microglial

hyperactivation and a self-propelling progressive cycle of inflam-

mation and neuron damage [55]. Astrocytes, the other class of glial

cell, are essential in synaptic transmission and information proces-

sing by neural circuit functions. Astrocytes respond to all forms of

CNS insult through reactive astrogliosis, a finely graded range of

progressive changes in gene expression and other cellular changes

[74,75]. Excessive and prolonged neuroinflammation damages brain

function and is relevant to CNS disease progression, from acute

delirium and postoperative cognitive dysfunction to AD, PD, multi-

ple sclerosis (MS), ALS, and AIDS dementia. In general, neuroin-

flammation not only causes and accelerates long-term

neurodegenerative diseases but plays a central role in the early

development of chronic conditions such as dementia.

ROS

ROS

CDPsAn�oxidant

defenceNrf2

NF-κB

Nrf2

NF-κB

Pro-inflammatorycytokines

Pro-inflammatorycytokines

CDP-induced neuroprotec�on

Neuronal cell death

TRENDS in Molecular Medicine

Figure 3. Cyclic dipeptides (CDPs) as neuroprotective agents. Inflammatory

mediators acting on microglial cells, via nuclear factor kappa B (NF-kB)

activation, increase production of reactive oxygen species (ROS) (red circles),

nitric oxide (NO), and cytokines (blue circles), which cause neuronal cell death.

CDPs (yellow circles), by intervening in the crosstalk between nuclear factor-like 2

(Nrf2) and NF-kB signalling, enhance antioxidant protection while depressing the

proinflammatory response, thus resulting in neuronal protection.

Opinion Trends in Molecular Medicine xxx xxxx, Vol. xxx, No. x

TRMOME-974; No. of Pages 8

triggers the self-propelling expression of proinflammatorycytokines and the synthesis of enzymes responsible forcrosstalking to antagonising systems such as the anti-inflammatory Nrf2 system. The NF-kB system thus gen-erates the signals required for Nrf2 activation, which inturn dampens proinflammatory signalling by upregulatingperoxidases and other anti-inflammatory proteins [56–61]. While persistently elevated reactive oxygen species(ROS) levels activate NF-kB and lead to inflammation [62],a moderate level of ROS is able to activate Nrf2, leading tothe upregulation of stress-inducible genes including hemeoxygenase-1 (HO-1) [63]. Increased HO activity confersboth anti-inflammatory and adaptive survival responseson in vitro or in vivo oxidative insults [63–65]. According tothe dual-key mechanism of inflammatory neurodegenera-tion [66], inflammatory mediators, via NF-kB activation,cause inducible nitric oxide synthase (iNOS) expression,which is responsible for high levels of NO, and acutelystimulate NADPH oxidase (NOX) enzymes, responsible forhigh levels of ROS, resulting in neuronal damage [66].

Cyclo(His–Pro) intervenes in the modulation of the twosystems by activating Nrf2, and thereby HO-1 expressionand activity, and suppressing NF-kB activity [58,63,67]. Bysimultaneously regulating Nrf2 and NF-kB, cyclo(His–Pro)dictates the cellular protective response, confirming thecrucial role of Nrf2 activation and HO-1 activity in contrib-uting to the anti-inflammatory activity of cells [68]. Cyclo(-His–Pro) downregulates iNOS, gp91 phox, and 47phoxgene expression, confirming that the induction of Nrf2-mediated antioxidant enzymes and the reduction ofNF-kB-mediated inflammatory mediators exert beneficialeffects in microglial BV-2 cells [54]. Beneficial cyclo(His–Pro) effects were also mediated by its ability to reducethe effects of ER stress, an integral component of

neuroinflammation [69,70], by launching the unfolded-protein response (UPR) and increasing protein-foldingcapacity through upregulation of ER chaperones such asBip/GRP78 [71]. These results have been the first clearevidence that the cytoprotective effects of cyclo(His–Pro)can be ascribed to crosstalk between the suppression of NF-kB signalling and the activation of the Nrf2 pathway, theformer depressing the proinflammatory response and thelatter enhancing the antioxidant defensive response.

Cyclo(His–Pro) and neuroprotection

In addition to regulating inflammation at the level of glialcells, cyclo(His–Pro) is able to exert protective effectsfollowing internalisation by neurons. Pretreatment ofOCT2-transfected cells with cyclo(His–Pro) before neuro-nal damage (induced using salsolinol) markedly dimin-ished cell degeneration [49]. This effect was achieved byinhibiting excitotoxic calcium influx, thus preventing mi-tochondrial impairment and apoptosis. Therefore, expres-sion of OCT2, as well as of cyclo(His–Pro), appears to becrucial for maintenance of dopaminergic cell integrity. Adecline in intracellular cyclo(His–Pro) levels caused calci-um-triggered apoptotic cell death, which is analogous tothe selective chronic nigral degeneration observed in PD

5

Box 3. Outstanding questions

� Can CDPs be released in response to environmental stimuli in

mammals?

� Do CDPs exert their effects through a binding protein homologous

to LuxR in mammals?

� Can CDPs secreted by the human microbiome interfere with

inflammatory reactions in humans?

� Are CDPs molecules that connect the human microbiome to CNS

health?

Opinion Trends in Molecular Medicine xxx xxxx, Vol. xxx, No. x

TRMOME-974; No. of Pages 8

[49]. Additional reports have confirmed the role of cyclo(-His–Pro) in dopaminergic cell integrity [72]. Cyclo(His–Pro) was shown to protect PC12 cells from the neurotoxiceffects of glutamate, rotenone, paraquat (PQ), and b-amy-loid [72]. Hence, cyclo(His–Pro) might be regarded as one ofthe most promising candidates for neuroprotective inter-vention (Figure 3).

The protective effect mediated by cyclo(His–Pro) mightinclude multiple pathways and/or mechanisms. Proteomicscreening of cyclo(His–Pro)-induced phosphorylationshowed that, in serum-starved PC12 cells (a condition thatcauses oxidative stress), cyclo(His–Pro) activated p38 mi-togen-activated protein kinase (MAPK) while reducing theextent of phosphorylation of extracellular signal-regulatedkinase (ERK) 1/2 and g and d protein kinase C isoforms,thus restoring basal cell viability [73]. By decreasing ROSgeneration and NO levels, cyclo(His–Pro) fully counter-acted the cytotoxic effects of PQ in PC12 cells [63]. Thecytoprotective effect of cyclo(His–Pro) against a wide rangeof toxic pro-oxidant insults to dopaminergic PC12 cells wasshown to occur through a mechanism involving activationof Nrf2, which results in decreased oxidative stress [63,72].

The results described unveil a potential dual-prongedtherapeutic use of cyclo(His–Pro) against neuroinflamma-tion-related diseases through interfering with the NRF2–NF-kB axis. By downregulating the inflammation path-ways of glial cells, along with inducing a neuroprotectiveresponse in neurons, cyclo(His–Pro) may be a therapeuticadvance in counteracting neuroinflammation-based degen-erative pathologies.

Concluding remarks and future perspectivesThe linear forms of dipeptides are often less stable thantheir cyclic counterparts in vivo and therefore CDPs are farmore promising in terms of therapeutic application byboth parenteral and oral administration routes (Box 2).

Box 2. Administering CDPs as therapeutics

Oral administration

CDPs occur as chemical degradation products in roasted coffee,

stewed beef, and beer and cause a bitter taste in these foods

[76]. Cyclo(His–Pro) was first found in the low-phenylalanine dietary

preparation Lofenelac (Mead Johnson, New York, NY, USA) and

subsequently in several common nutritional supplements. One

study on the pharmacokinetics and possible toxicity of acute oral

consumption of cyclo(His–Pro) (24 mg/dose) showed that the CDP is

absorbed from the gastrointestinal tract, with plasma cyclo(His–Pro)

levels peaking at 4 h and returning to normal by 24 h. No side effects

were experienced in any of the subjects based on physical and

blood chemistry examinations [77].

Topic treatment

An in vivo skin oedema test showed that mice that underwent

cyclo(His–Pro) pretreatment had a significant decrease in the

oedematogenic response, indicating that topical application of

CDP can cross the epidermal barrier and exert anti-inflammatory

effects [67].

Carriers for medical substances

Phenylalanine-containing CDPs have been discovered in a new type

of hydrogelator. The gelators comprise one or more gelling agents

in aqueous solution and behave as viscoelastic materials due to the

immobilisation of solvent molecules in a 3D network. These

hydrogels are suited as carriers for medical substances and, when

mechanically damaged, have excellent self-healing capacity and are

therefore suitable for injection-based drug delivery [78].

6

Moreover, conventional anti-inflammatory therapeuticsare unsuitable for treating neuroinflammation since theycannot cross the BBB. CDPs can be regarded as BBB-permeable drugs with remarkable bioactivity in reducinginflammation at glial cells and inducing a protective statein neurons. However, their utility might be extended be-yond CNS applications, as their capacity to control macro-phage-like cells could also be useful in peripheralinflammatory diseases (Box 2).

The capacity of CDPs to act as QS signals and tomodulate the microbiome argues for their considerationas therapeutic agents in infectious diseases (e.g., toxicshock syndrome or S. aureus infection as discussed above).Moreover, given the growing appreciation that changes tothe microbiome can impact importantly on the functionand development of both the immune system and the CNS,it is tempting to speculate that these microbiome-modu-lating effects of CDPs might contribute to their therapeuticutility as agents in neurological and peripheral inflamma-tory disease (Box 3).

References1 Fuqua, C. et al. (2001) Regulation of gene expression by cell-to-cell

communication: acyl-homoserine lactone quorum sensing. Annu. Rev.Genet. 35, 439–468

2 Sun, J. et al. (2004) Is autoinducer-2 a universal signal for interspeciescommunication: a comparative genomic and phylogenetic analysis ofthe synthesis and signal transduction pathways. BMC Evol. Biol. 4, 36

3 Ortiz-Castro, R. et al. (2011) Transkingdom signaling based onbacterial cyclodipeptides with auxin activity in plants. Proc. NatlAcad. Sci. U.S.A. 108, 7253–7258

4 Hughes, D.T. and Sperandio, V. (2008) Inter-kingdom signalling:communication between bacteria and their hosts. Nat. Rev.Microbiol. 6, 111–120

5 Sperandio, V. et al. (2003) Bacteria–host communication: the languageof hormones. Proc. Natl Acad. Sci. U.S.A. 100, 8951–8956

6 Nealson, K.H. and Hastings, J.W. (1979) Bacterial bioluminescence: itscontrol and ecological significance. Microbiol. Rev. 43, 496–518

7 Moophayak, K. et al. (2013) Evaluation of an I-box wind tunnel modelfor assessment of behavioral responses of blow flies. Parasitol. Res. 112,3789–3798

8 O’Loughlin, C.T. et al. (2013) A quorum-sensing inhibitor blocksPseudomonas aeruginosa virulence and biofilm formation. Proc.Natl. Acad. Sci. U.S.A. 110, 17981–17986

9 Lesic, B. et al. (2009) Quorum sensing differentially regulatesPseudomonas aeruginosa type VI secretion locus I and homologousloci II and III, which are required for pathogenesis. Microbiology 155,2845–2855

10 Chatterjee, A. et al. (2010) Regulation of motility in Erwinia carotovorasubsp. carotovora: quorum-sensing signal controls FlhDC, the globalregulator of flagellar and exoprotein genes, by modulating theproduction of RsmA, an RNA-binding protein. Mol. Plant MicrobeInteract. 23, 1316–1323

11 Lang, J. and Faure, D. (2014) Functions and regulation of quorum-sensing in Agrobacterium tumefaciens. Front. Plant Sci. 5, 14

12 Human Microbiome Project Consortium (2012) Structure, function anddiversity of the healthy human microbiome. Nature 486, 207–214

Opinion Trends in Molecular Medicine xxx xxxx, Vol. xxx, No. x

TRMOME-974; No. of Pages 8

13 Chen, R. et al. (2012) Dissection of quorum-sensing genes inBurkholderia glumae reveals non-canonical regulation and the newregulatory gene tofM for toxoflavin production. PLoS ONE 7, e52150

14 Chen, X. et al. (2002) Structural identification of a bacterial quorum-sensing signal containing boron. Nature 415, 545–549

15 Li, J. et al. (2011) Lactobacillus reuteri-produced cyclic dipeptidesquench agr-mediated expression of toxic shock syndrome toxin-1 instaphylococci. Proc. Natl. Acad. Sci. U.S.A. 108, 3360–3365

16 Trejo-Hernandez, A. et al. (2014) Interspecies competition triggersvirulence and mutability in Candida albicans–Pseudomonasaeruginosa mixed biofilms. ISME J. http://dx.doi.org/10.1038/ismej.2014.53

17 Jacobi, C.A. (2012) Quorum sensing in the probiotic bacteriumEscherichia coli Nissle 1917 (Mutaflor) – evidence that furanosylborate diester (AI-2) is influencing the cytokine expression in theDSS colitis mouse model. Gut Pathog. 4, 8

18 Higgins, D.A. et al. (2007) The major Vibrio cholerae autoinducer andits role in virulence factor production. Nature 450, 883–886

19 Lerat, E. and Moran, N.A. (2004) The evolutionary history of quorum-sensing systems in bacteria. Mol. Biol. Evol. 21, 903–913

20 Schuster, M. et al. (2013) Acyl-homoserine lactone quorum sensing:from evolution to application. Annu. Rev. Microbiol. 67, 43–63

21 Tannieres, M. et al. (2013) A metagenomic study highlightsphylogenetic proximity of quorum-quenching and xenobiotic-degrading amidases of the AS-family. PLoS ONE 8, e65473

22 Pollitt, E.J. et al. (2014) Cooperation, quorum sensing, and evolution ofvirulence in Staphylococcus aureus. Infect. Immun. 82, 1045–1051

23 West, S.A. et al. (2012) Quorum sensing and the confusion aboutdiffusion. Trends Microbiol. 20, 586–594

24 Cornforth, D.M. et al. (2014) Combinatorial quorum sensing allowsbacteria to resolve their social and physical environment. Proc. Natl.Acad. Sci. U.S.A. 111, 4280–4284

25 Gonzalez, J.F. and Venturi, V. (2013) A novel widespread interkingdomsignaling circuit. Trends Plant Sci. 18, 167–174

26 Prasad, C. (1995) Bioactive cyclic dipeptides. Peptides 16, 151–16427 Minelli, A. et al. (2008) Focus on cyclo(His–Pro): history and

perspectives as antioxidant peptide. Amino Acids 35, 283–28928 Cornacchia, C. et al. (2012) 2,5-Diketopiperazines as neuroprotective

agents. Mini Rev. Med. Chem. 12, 2–1229 Lahoud, G. and Hou, Y.M. (2010) Biosynthesis: a new (old) way of

hijacking tRNA. Nat. Chem. Biol. 6, 795–79630 Kukla, M.J. et al. (1985) Synthesis, characterization, and anorectic

testing of the four stereoisomers of cyclo(histidylproline). J. Med.Chem. 28, 1745–1747

31 Beck, J.G. et al. (2012) Intestinal permeability of cyclic peptides:common key backbone motifs identified. J. Am. Chem. Soc. 134,12125–12133

32 Teixido, M. et al. (2007) Diketopiperazines as a tool for the study oftransport across the blood–brain barrier (BBB) and their potential useas BBB-shuttles. J. Am. Chem. Soc. 129, 11802–11813

33 de Carvalho, M.P. and Abraham, W.R. (2012) Antimicrobial and biofilminhibiting diketopiperazines. Curr. Med. Chem. 19, 3564–3577

34 Borthwick, A.D. (2012) 2,5-Diketopiperazines: synthesis, reactions,medicinal chemistry, and bioactive natural products. Chem. Rev.112, 3641–3716

35 Gu, Q. et al. (2013) Identification and characterization of extracellularcyclic dipeptides as quorum-sensing signal molecules from Shewanellabaltica, the specific spoilage organism of Pseudosciaena crocea during4 8C storage. J. Agric. Food Chem. 61, 11645–11652

36 Park, D.K. et al. (2006) Cyclo(Phe–Pro) modulates the expression ofompU in Vibrio spp. J. Bacteriol. 188, 2214–2221

37 Vikram, A. et al. (2014) Cyclo(valine–valine) inhibits Vibrio choleraevirulence gene expression. Microbiology 160, 1054–1062

38 Scopel, M. et al. (2013) Dipeptide cis-cyclo(leucyl–tyrosyl) produced bysponge associated Penicillium sp. F37 inhibits biofilm formation of thepathogenic Staphylococcus epidermidis. Bioorg. Med. Chem. Lett. 23,624–626

39 James, G.A. et al. (2008) Biofilms in chronic wounds. Wound RepairRegen. 16, 37–44

40 Secor, P.R. et al. (2012) Phevalin (aureusimine B) production byStaphylococcus aureus biofilm and impacts on human keratinocytegene expression. PLoS ONE 7, e40973

41 Gonzalez, J.E. and Keshavan, N.D. (2006) Messing with bacterialquorum sensing. Microbiol. Mol. Biol. Rev. 70, 859–875

42 Borre, Y.E. et al. (2014) Microbiota and neurodevelopmental windows:implications for brain disorders. Trends Mol. Med. 20, 509–518

43 De Palma, G. et al. (2014) The microbiota–gut–brain axis ingastrointestinal disorders: stressed bugs, stressed brain or both? J.Physiol. 592, 2989–2997

44 Holzer, P. and Farzi, A. (2014) Neuropeptides and the microbiota–gut–brain axis. Adv. Exp. Med. Biol. 817, 195–219

45 Mann, E.R. and Li, X. (2014) Intestinal antigen-presenting cells inmucosal immune homeostasis: crosstalk between dendritic cells,macrophages and B-cells. World J. Gastroenterol. 20, 9653–9664

46 Gonzalez, H. et al. (2014) Neuroimmune regulation of microglialactivity involved in neuroinflammation and neurodegenerativediseases. J. Neuroimmunol. 274, 1–13

47 Boiko, S.S. et al. (2010) Regional and subcellular localization ofcycloprolylglycine in rat brain. Bull. Exp. Biol. Med. 149, 709–711

48 Pelis, R.M. and Wright, S.H. (2014) SLC22, SLC44, and SLC47transporters – organic anion and cation transporters: molecular andcellular properties. Curr. Top. Membr. 73, 233–261

49 Taubert, D. et al. (2007) Identification of the endogenous key substratesof the human organic cation transporter OCT2 and their implication infunction of dopaminergic neurons. PLoS ONE 2, e385

50 Koepsell, H. (2013) The SLC22 family with transporters of organiccations, anions and zwitterions. Mol. Aspects Med. 34, 413–435

51 Chung, D.W. et al. (2010) Systemic administration oflipopolysaccharide induces cyclooxygenase-2 immunoreactivity inendothelium and increases microglia in the mouse hippocampus.Cell. Mol. Neurobiol. 30, 531–541

52 Nakayama, Y. et al. (2010) Molecular mechanisms of the LPS-inducednon-apoptotic ER stress–CHOP pathway. J. Biochem. 147, 471–483

53 Hotamisligil, G.S. (2010) Endoplasmic reticulum stress and theinflammatory basis of metabolic disease. Cell 140, 900–917

54 Bellezza, I. et al. (2014) Neuroinflammation and endoplasmicreticulum stress are coregulated by cyclo(His–Pro) to prevent LPSneurotoxicity. Int. J. Biochem. Cell Biol. 51, 159–169

55 Cunningham, C. (2013) Microglia and neurodegeneration: the role ofsystemic inflammation. Glia 61, 71–90

56 Brigelius-Flohe, R. and Flohe, L. (2011) Basic principles and emergingconcepts in the redox control of transcription factors. Antioxid. RedoxSignal. 15, 2335–2381

57 Bellezza, I. (2010) Nrf2 and NF-kB and their concerted modulation incancer pathogenesis and progression. Cancers (Basel) 2, 483–497

58 Bellezza, I. et al. (2012) Inhibition of NF-kB nuclear translocation viaHO-1 activation underlies a-tocopheryl succinate toxicity. J. Nutr. 23,1583–1591

59 Bellezza, I. et al. (2013) Furanodien-6-one from Commiphora erythraeainhibits the NF-kB signalling and attenuates LPS-inducedneuroinflammation. Mol. Immunol. 54, 347–354

60 Ganesh Yerra, V. et al. (2013) Potential therapeutic effects of thesimultaneous targeting of the Nrf2 and NF-kB pathways in diabeticneuropathy. Redox Biol. 1, 394–397

61 Cuadrado, A. et al. (2014) Transcription factors NRF2 and NF-kB arecoordinated effectors of the Rho family, GTP-binding protein RAC1during inflammation. J. Biol. Chem. 289, 15244–15258

62 Leavy, O. (2014) Inflammation: regulating ROS. Nat. Rev. Immunol.14, 357

63 Minelli, A. et al. (2009) Cyclo(His–Pro) up-regulates heme oxygenase1 via activation of Nrf2–ARE signalling. J. Neurochem. 111, 956–966

64 Motterlini, R. and Foresti, R. (2014) Heme oxygenase-1 as a target fordrug discovery. Antioxid. Redox Signal. 20, 1810–1826

65 Koskenkorva-Frank, T.S. et al. (2013) The complex interplay of ironmetabolism, reactive oxygen species, and reactive nitrogen species:insights into the potential of various iron therapies to induce oxidativeand nitrosative stress. Free Radic. Biol. Med. 65, 1174–1194

66 Mander, P. and Brown, G.C. (2005) Activation of microglial NADPHoxidase is synergistic with glial iNOS expression in inducing neuronaldeath: a dual-key mechanism of inflammatory neurodegeneration. J.Neuroinflammation 2, 20

67 Minelli, A. et al. (2012) Cyclo(His–Pro) exerts anti-inflammatory effectsby modulating NF-kB and Nrf2 signalling. Int. J. Biochem. Cell Biol.44, 525–535

7

Opinion Trends in Molecular Medicine xxx xxxx, Vol. xxx, No. x

TRMOME-974; No. of Pages 8

68 Jadhav, A. et al. (2008) Interaction among heme oxygenase, nuclearfactor-kappaB, and transcription activating factors in cardiachypertrophy in hypertension. Hypertension 52, 910–917

69 Hosoi, T. et al. (2014) Inhibition of inducible nitric oxide synthase andinterleukin-1(expression by tunicamycin in cultured glial cells exposedto lipopolysaccharide. Brain Res. 1558, 11–17

70 Afrazi, A. et al. (2014) Toll-like receptor 4-mediated endoplasmicreticulum stress in intestinal crypts induces necrotizingenterocolitis. J. Biol. Chem. 289, 9584–9599

71 Tam, A.B. et al. (2012) ER stress activates NF-kB by integratingfunctions of basal IKK activity, IRE1 and PERK. PLoS ONE 7,e45078

72 Minelli, A. et al. (2009) Cyclo(His–Pro) promotes cytoprotection byactivating Nrf2-mediated up-regulation of antioxidant defence. J.Cell. Mol. Med. 13, 1149–1161

8

73 Minelli, A. et al. (2006) Phosphoproteomic analysis of the effect of cyclo-[His–Pro] dipeptide on PC12 cells. Peptides 27, 105–113

74 Sofroniew, M.V. and Vinters, H.V. (2010) Astrocytes: biology andpathology. Acta Neuropathol. 119, 7–35

75 Lyman, M. et al. (2014) Neuroinflammation: the role and consequences.Neurosci. Res. 79, 1–12

76 Perzborn, M. et al. (2013) Enzymatical and microbial degradation ofcyclic dipeptides (diketopiperazines). AMB Express 3, 51

77 Uyemura, K. et al. (2010) Metabolism and toxicity of high doses ofcyclo(His–Pro) plus zinc in healthy human subjects. J. Drug Metab.Toxicol. 1, 105

78 Kleinsmann, A.J. and Nachtsheim, B.J. (2013) Phenylalanine-containing cyclic dipeptides – the lowest molecular weighthydrogelators based on unmodified proteinogenic amino acids.Chem. Commun. (Camb.) 49, 7818–7820