Methods to Enhance the Metabolic Stability of Peptide-Based PET Radiopharmaceuticals

molecules

Review Methods to Enhance the Metabolic Stability of Peptide-Based PET Radiopharmaceuticals

Brendan J. Evans 1 , Andrew T. King 1 , Andrew Katsiﬁs 2, Lidia Matesic 3 and Joanne F. Jamie 1,*

1 Department of Molecular Sciences, Macquarie University, Sydney, NSW 2109, Australia; [email protected] (B.J.E.); [email protected] (A.T.K.) 2 Department of Molecular Imaging, Royal Prince Alfred Hospital, Camperdown, NSW 2050, Australia; [email protected] 3 Australian Nuclear Science and Technology Organisation (ANSTO), Lucas Heights, NSW 2234, Australia; [email protected] * Correspondence: [email protected]; Tel.: +61-2-9850-8283

Academic Editor: Anne Roivainen Received: 1 April 2020; Accepted: 13 May 2020; Published: 14 May 2020

Abstract: The high affinity and specificity of peptides towards biological targets, in addition to their favorable pharmacological properties, has encouraged the development of many peptide-based pharmaceuticals, including peptide-based positron emission tomography (PET) radiopharmaceuticals. However, the poor in vivo stability of unmodified peptides against proteolysis is a major challenge that must be overcome, as it can result in an impractically short in vivo biological half-life and a subsequently poor bioavailability when used in imaging and therapeutic applications. Consequently, many biologically and pharmacologically interesting peptide-based drugs may never see application. A potential way to overcome this is using peptide analogues designed to mimic the pharmacophore of a native peptide while also containing unnatural modifications that act to maintain or improve the pharmacological properties. This review explores strategies that have been developed to increase the metabolic stability of peptide-based pharmaceuticals. It includes modifications of the C- and/or N-termini, introduction of D- or other unnatural amino acids, backbone modification, PEGylation and alkyl chain incorporation, cyclization and peptide bond substitution, and where those strategies have been, or could be, applied to PET peptide-based radiopharmaceuticals.

Keywords: radiopharmaceuticals; peptides; positron emission tomography; proteolysis; metabolic stability

1. Introduction Positron emission tomography (PET) is a nuclear medicine imaging technique for the non-invasive quantitative measurement of specific biochemical, physiological, and pharmacological processes in vivo [1]. PET is useful in the diagnosis and staging of neurological, cardiovascular, and various oncology-based diseases [2]. PET imaging is achieved by administering a patient with a PET radiopharmaceutical which will localize into organs and/or tissues that express the desired biological target. Once localized, the distribution of the PET radiopharmaceutical throughout the body can be imaged with a PET scanner and a diagnosis can be made. PET radiopharmaceuticals are biologically active molecules that are labeled with positron-emitting radionuclides such as fluorine-18, gallium-68, or copper-64. A key component of a PET radiopharmaceutical is the targeting entity, which is designed to possess a pharmacophore that has high affinity and specificity towards a desired biological target present in an organ and/or tissue that is associated with a specific disease or malignancy [3]. The targeting entities of radiopharmaceuticals were initially developed as biologically active small

Molecules 2020, 25, 2314; doi:10.3390/molecules25102314 www.mdpi.com/journal/molecules MoleculesMolecules 20202020,, 2525,, x 2314 22 of of 24 24 biologically active small molecules, as is the case for the most widely used PET radiopharmaceutical 18 [molecules,18F]fluorodeoxyglucose as is the case for [4]. the However, most widely in recent used PETyears radiopharmaceutical there has been a rapid [ F]ﬂuorodeoxyglucose development in the use [4]. ofHowever, targeting in entities recent years developed there has from been biologics, a rapid developmentsuch as peptides, in the proteins, use of targeting antibodies, entities and developed antibody fragmentsfrom biologics, for the such use as PET peptides, radiopharmaceuticals proteins, antibodies, [5–7]. and antibody fragments for the use as PET radiopharmaceuticals [5–7]. 1.1. Peptides as Radiopharmaceuticals Peptides as Radiopharmaceuticals The structure of a peptide-based radiopharmaceutical (Figure 1) typically contains the following components:The structure a peptide of a peptide-basedto act as the targeting radiopharmaceutical entity, a linker, (Figure a radionuclide1) typically containsbearing moiety, the following and a PETcomponents: radionuclide. a peptide The tolinker act asis thesometimes targeting an entity, optional a linker, component a radionuclide of a radiopharmaceutical bearing moiety, and that a PET is incorporatedradionuclide. to The facilitate linker the is sometimesconjugation an of optional the targeting component peptide of and a radiopharmaceutical the radionuclide bearing that is moiety,incorporated and/or to facilitateimprove the its conjugation pharmacokinetics of the targeting such peptideas by andincreasing the radionuclide metabolic bearing stability moiety, or manipulatingand/or improve biodistribution its pharmacokinetics [8–11]. The such linker as can by also increasing be used as metabolic a spacer stabilityto distance or bulky manipulating portions ofbiodistribution a radiopharmaceutical, [8–11]. The such linker as chelators can also from be th usede bioactive as a spacer portions, to to distance reduce bulkysteric interference portions of anda radiopharmaceutical, maintain high binding such affinity as chelators [12]. from the bioactive portions, to reduce steric interference and maintain high binding aﬃnity [12].

Radionuclide-bearing moiety Linker/Spacer PET radionuclide Targeting peptide Biological target in vivo

Peptide-based PET radiopharmaceutical

Figure 1. The structural components of a peptide-based positron emission tomography (PET) Figure 1. The structural components of a peptide-based positron emission tomography (PET) radiopharmaceuticals. radiopharmaceuticals. There are many advantages offered by utilizing peptides as the targeting entity in radiopharmaceuticals, There are many advantages offered by utilizing peptides as the targeting entity in especially in the field of oncology. These peptide-based radiopharmaceuticals can take the form of radiopharmaceuticals, especially in the field of oncology. These peptide-based radiopharmaceuticals radiopharmaceuticals for the diagnosis of diseases by imaging the biological target associated with the can take the form of radiopharmaceuticals for the diagnosis of diseases by imaging the biological disease in specific tissues, or as radiotherapeutics for the treatment of cancers by subjecting the tissue to target associated with the disease in specific tissues, or as radiotherapeutics for the treatment of localized ionizing radiation. Furthermore, the opportunity to exploit the same targeting agent with either cancers by subjecting the tissue to localized ionizing radiation. Furthermore, the opportunity to an imaging or therapeutic radionuclide has given rise to the field of ‘Theranostics’ in nuclear medicine [13]. exploit the same targeting agent with either an imaging or therapeutic radionuclide has given rise to A key advantage of peptide-receptor targeting peptide-based radiopharmaceuticals is the the field of ‘Theranostics’ in nuclear medicine [13]. higher density of peptide receptors expressed on tumor cells than in normal tissues, thus specific A key advantage of peptide-receptor targeting peptide-based radiopharmaceuticals is the higher receptor-binding radiolabeled peptides can be designed to enable the efficient visualization and density of peptide receptors expressed on tumor cells than in normal tissues, thus specific receptor- treatment of various tumors [14]. Due to the relatively small size of peptides compared to other binding radiolabeled peptides can be designed to enable the efficient visualization and treatment of biologics, such as proteins and antibodies, peptides often exhibit rapid pharmacokinetics, with the various tumors [14]. Due to the relatively small size of peptides compared to other biologics, such as ability to efficiently penetrate tumors, fast clearance from the bloodstream and non-target tissues, proteins and antibodies, peptides often exhibit rapid pharmacokinetics, with the ability to efficiently and are not immunogenic [5,14–16]. Peptides can also be readily synthesized using conventional penetrate tumors, fast clearance from the bloodstream and non-target tissues, and are not peptide synthesizers, and any desired modifications to the structure can be easily engineered by immunogenic [5,14–16]. Peptides can also be readily synthesized using conventional peptide making the appropriate changes to the peptide sequence during synthesis and/or by adding other synthesizers, and any desired modifications to the structure can be easily engineered by making the structural modifications after synthesis [9,17,18]. appropriate changes to the peptide sequence during synthesis and/or by adding other structural Consequently, the last 20 years have seen an explosive growth in the development of radiolabeled modifications after synthesis [9,17,18]. peptides for targeted diagnostic imaging and therapy. While radiolabeled peptides have been applied to

Molecules 2020, 25, x 3 of 24 Molecules 2020, 25, 2314 3 of 24 Consequently, the last 20 years have seen an explosive growth in the development of radiolabeled peptides for targeted diagnostic imaging and therapy. While radiolabeled peptides have beenvarious applied molecular to various imaging molecular modalities imaging that use modalitie nuclearsprobes, that use such nuclear as scintigraphy probes, such and assingle-photon scintigraphy andemission single-photon computed tomographyemission computed (SPECT), tomography the superior image(SPECT), quality the andsuperior quantitative image dataquality available and quantitativefrom PET have data resulted available in afrom signiﬁcant PET have amount resulted of research in a significant being devoted amount toof theresearch development being devoted of PET 68 ® toradiolabeled the development peptides of [ 19PET]. Recently, radiolab [eledGa]DOTATATE, peptides [19]. alsoRecently, known [68 asGa]DOTATATE, NETSPOT (Figure also2 known), was the as NETSPOTﬁrst radiolabeled® (Figure peptide 2), was for the PET first imaging radiolabeled to receive peptide regulatory for PET approval imaging from to thereceive Food regulatory and Drug approvalAdministration from the (FDA) Food [20 and]. Drug Administration (FDA) [20].

Figure 2. Structure of [ 68Ga]DOTATATEGa]DOTATATE (NETSPOT®).).

DespiteDespite the the advantages advantages offered offered by peptides, there there are several challenges i inherentnherent to to the the use of peptides in drug design and development, including for imaging and therapy. The most significant significant ofof these these is is that that unmodified unmodified peptides peptides often often possess possess prohibitively prohibitively short short half-lives half-lives in vivo,in vivo primarily, primarily due todue rapid to rapid proteolytic proteolytic degradation degradation in the in blood, the blood, liver, liver,and kidneys and kidneys by endogenous by endogenous proteases proteases [21]. This [21]. liabilityThis liability results results in a inshort a short duration duration of in of invivo vivo acactivity,tivity, poor poor bioavailability, bioavailability, and and has has significantly significantly limitedlimited their their application application in in drug drug development [22]. [22].

2.2. Challenges Challenges Faced Faced by by Peptide-Ba Peptide-Basedsed Radiopharmaceuticals Radiopharmaceuticals in In Vivo Vivo TheThe rate at which a drug is metabolized and re removedmoved from the body determines its biological half-lifehalf-life [[23].23]. In the casecase ofof radioactiveradioactive drugs, drugs, such such as as PET PET radiopharmaceuticals, radiopharmaceuticals, the the physical physical half-life half- lifeis determined is determined by the by incorporated the incorporated radionuclide radionuclide [23]. Thus, [23]. to Thus, achieve to optimal achieve results, optimal the results, localization the localizationprocess of a radiopharmaceuticalprocess of a radiopharmaceutical has to be fast has relative to be to fast its biologicalrelative toand its biological physical half-lives, and physical such half- that lives,the drug such will that localize the drug to thewill target localize with to adequate the target time with for adequate imaging time or therapy for imaging before or degrading therapy before below degradingan effective below activity an and effective/or concentration activity and/or [24]. Inconcentration the case of peptide-based[24]. In the case radiopharmaceuticals, of peptide-based radiopharmaceuticals,the two most significant the forms two ofmost degradation significantin forms vivo that of degradation impact the e ffiincacy vivo of that the impact drug are the loss efficacy of the ofradionuclide the drug are and loss degradation of the radionuclide of the conjugated and degradation peptide. The of the premature conjugated degradation peptide. of The peptide-based premature degradationradiopharmaceuticals of peptide-basedin vivo radiopharmaceuticalsis of pressing concern in since vivo these is of drugspressing are concern typically since administered these drugs in aredoses typically that are administered significantly smallerin doses than that conventional are significantly drugs smaller and are than therefore conventional especially drugs vulnerable and are to thereforehaving their especially efficacy vulnerable significantly to disrupted having their by any efficacy amount significantly of degradation disrupted in vivo. by any amount of degradation in vivo. 2.1. Loss of the Radionuclide 2.1. LossMany of the radiopharmaceuticals Radionuclide are labeled with radiometals (e.g., technetium-99m, gallium-68, and copper-64).Many radiopharmaceuticals Ensuring that the are radiometal labeled with is notradiometals lost from (e.g., the radiopharmaceuticaltechnetium-99m, gallium-68,in vivo isand of copper-64).critical concern Ensuring when that developing the radiometal these drugsis not lost as the from free the radiometal radiopharmaceutical may exhibit in high vivo toxicityis of critical and concerncause significant when developing damage tothese the bodydrugs [ 25as]. the In thefree caseradiometal of fluorine-18 may exhibit labeled high radiopharmaceuticals, toxicity and cause 18 significantin vivo radiodefluorination damage to the body results [25]. in In the the release case of of fluorine-18 free [ F]fluoride labeled ions radiopharmaceuticals, that can readily accumulate in vivo radiodefluorinationinto the calcium-rich results fluorophilic in the bones release of theof free body [18 [F]fluoride26]. Radiodefluorination ions that can readily and in vivoaccumulatemetabolism into 18 theof [ calcium-richF]radiopharmaceuticals fluorophilic bones also present of the majorbody [26]. challenges Radiodefluorination to imaging studies and in as vivo non-specific metabolism uptake of

Molecules 2020, 25, 2314 4 of 24 of free [18F]ﬂuoride ions and [18F]metabolites can lead to a degradation of the signal to noise ratio [27]. Further information on the radiodemetallation and radiodeﬂuorination of radiopharmaceuticals, and the strategies that have been applied to mitigate these issues, are beyond the scope of this review but have been thoroughly reviewed elsewhere [28–30].

2.2. Degradation of the Peptide The major challenge of using peptides in the active component of a pharmaceutical is that naturally occurring peptides are usually rapidly degraded in vivo [21,22]. Compared to other biologics, such as proteins and antibodies, peptides are generally more susceptible to enzymatic and/or chemical degradation. One of the key reasons a peptide sequence can also be susceptible to proteolytic degradation is due to its backbone containing a recognition motif for an endogenous protease [31]. In addition to proteolytic degradation, peptide bonds can also undergo spontaneous degradation under physiological conditions when particularly labile sequence motifs are present [32]. In peptide-based radiopharmaceuticals, degradation of the conjugated peptide will significantly disrupt its ability to localize to the target tissue, and the subsequent radioactive metabolites will undergo non-specific binding to other tissues and/or be rapidly cleared from the body. In the case of radiopharmaceuticals, this will reduce imaging sensitivity due to increasing background radiation [33]. In radiotherapeutics, this can result in insufficient irradiation of the target tissue while increasing the irradiation of non-target tissues [34,35]. Another point to consider is that amide bonds are often used to conjugate the radionuclide bearing moiety to the biomolecule or linker and these bonds are also susceptible to the same degradation pathways as peptide bonds via proteases and amidases [36]. As a result of these challenges, most peptides for use in radiopharmaceuticals are synthetically modified to minimize metabolic degradation in vivo [15,37].

3. Increasing the In Vivo Stability of Peptide-Based Radiopharmaceuticals The peptide amide bond represents the central repeating structural element of peptides and proteins. This bond possesses partial double bond character, which is one of the key attributes that contributes to the rigidity of peptide chains. Its ability to form hydrogen bonds also makes the peptide bond play a crucial role in its recognition by and interactions with other proteins. Normally, the peptide amide bond is stable to hydrolysis, requiring harsh conditions involving concentrated acids or bases at increased temperatures [38,39]. However, the peptide bond can be readily cleaved under mild conditions at or even below room temperature in the presence of an appropriate protease or peptidase [39,40]. Peptidases can either be classiﬁed as exopeptidases, which speciﬁcally hydrolyze the C- or N-termini of a peptide, or as endopeptidases which are capable of hydrolyzing amide bonds within a peptide [41,42]. Mechanistically, hydrolysis of an amide bond with a peptidase occurs via a nucleophilic attack at the carbonyl carbon of the amide bond (Figure3), with its pathway dependent on the amino acids present in the peptidase’s active site [43]. Peptidases that have nucleophilic amino acids residues such as cysteine and serine in their active site, can attack the carbonyl carbon of the amide/peptide bond and form an acylated enzyme (as a thioester for cysteine or ester for serine), which is more vulnerable to attack by water than the original peptide bond (Figure3A). Often these peptidases also have histidine and aspartic acid or glutamic acid within the active site, as a ‘catalytic triad’ [43,44]. With peptidases that have amino acid residues such as aspartate/aspartic acid or glutamate/glutamic acid (but no serine or cysteine) in their active site, these residues directly assist a water molecule in its nucleophilic attack of the carbonyl carbon of the amide bond, leading to direct hydrolysis of the amide bond (Figure3B) [43]. Molecules 2020, 25, x 5 of 24

Molecules 2020, 25, 2314 5 of 24 Molecules 2020, 25, x 5 of 24

Figure 3. General mechanisms of hydrolysis via a peptidase (A) with nucleophilic amino acids; (B) withFigure acidic 3. General amino mechanismsacid residues of [43,44]. hydrolysis via a peptidase (A) with nucleophilic amino acids; (B) with acidicFigure amino 3. General acid residues mechanisms [43,44 of]. hydrolysis via a peptidase (A) with nucleophilic amino acids; (B) with acidic amino acid residues [43,44]. Several strategies have been developed to synthesize peptide analogues in which vulnerable Several strategies have been developed to synthesize peptide analogues in which vulnerable peptide Severalbonds arestrategies either have modified been developedor obscured to syntsuchhesize that peptidethey are analogues no longer in targetedwhich vulnerable by proteolytic peptide bonds are either modified or obscured such that they are no longer targeted by proteolytic enzymes.peptide The bonds goal are of either this modifiedis to generate or obscured metabolically such that theystable are peptide no longer analogues targeted by that proteolytic maintain the biologicalenzymes.enzymes. activity The The goalgoal of theof of this original this is isto to generatepeptide. generate metabolically This metabolically review stableis focused stablepeptide on peptide analogues strategies analogues that to enhancemaintain that metabolicthe maintain stabilitythebiological biological of PET activity activity peptide-based of the of original the original radiopharmaceuticals. peptide. peptide. This review This is focused reviewIt includes on is strategies focused modifications to on enhance strategies of metabolicthe toC- enhance and/or Nmetabolic-termini,stability introduction stabilityof PET peptide-based of PET of D- peptide-based or other radiopharmaceuticals. unnatural radiopharmaceuticals. amino Itacids, includes backbone modifications It includesmodification, of modifications the PEGylationC- and/or of and the alkylC- andN -termini,chain/or N incorporation,-termini, introduction introduction of cyclizat D- or otherion, of unnatural and D- or peptide other amino unnaturalbond acids, substitutibackbone amino modification,on. acids, While backbone some PEGylation of the modification, andexamples discussedPEGylationalkyl chain in andthis incorporation, alkylreview chain have cyclizat incorporation, only ion,been and applied peptide cyclization, to bond SPECT substituti and radiopharmaceuti peptideon. While bond some substitution.cals, of theradiotherapeutics, examples While some discussed in this review have only been applied to SPECT radiopharmaceuticals, radiotherapeutics, orof non-radioactive the examples discussed peptide-based in this pharmaceuticals, review have only the been strategies applied are to applicable SPECT radiopharmaceuticals, also to PET peptide- or non-radioactive peptide-based pharmaceuticals, the strategies are applicable also to PET peptide- basedradiotherapeutics, radiopharmaceuticals. or non-radioactive peptide-based pharmaceuticals, the strategies are applicable also based radiopharmaceuticals. to PET peptide-based radiopharmaceuticals.

3.1. 3.1.D-Amino D-Amino Acids Acids 3.1. d-Amino Acids ApartApart from from glycine, glycine, allall aminoamino acidsacids possesspossess ch chiralityirality and and can cantherefore therefore exist existin either in either Apart from glycine, all amino acids possess chirality and can therefore exist in either levorotatory levorotatorylevorotatory (L )( Lor) or dextrorotatory dextrorotatory ((D) forms (Figure (Figure 4). 4). However, However, nature nature has provenhas proven itself itselfremarkably remarkably (L) or dextrorotatory (D) forms (Figure4). However, nature has proven itself remarkably homochiral homochiralhomochiral and and most most amino amino acids acids are found in in their their L -formL-form in mammalianin mammalian systems. systems. D-Amino D-Amino acids acids and most amino acids are found in their L-form in mammalian systems. d-Amino acids can be found can canbe foundbe found in innature nature (e.g (e.g.,. ,in in somesome speciesspecies of of frogs frogs and and bacteria), bacteria), but butthese these cases cases are exceedingly are exceedingly in naturerare [45]. (e.g., in some species of frogs and bacteria), but these cases are exceedingly rare [45]. rare [45].

Figure 4. (a) Structure of an L-amino acid. (b) Structure of a D-amino acid. FigureFigure 4. 4. ((aa)) Structure Structure of of an an L L-amino-amino acid. acid. ( (bb)) Structure Structure of of a a Dd-amino-amino acid. acid. The fundamental differences in chirality between L- and D-amino acids means that peptides built from D-amino acids are not recognized by many proteins, including proteases [46]. The result of this TheThe fundamental fundamental differences diﬀerences in in chirality chirality between between L- andL- and D-aminod-amino acids acids means means that peptides that peptides built lack of recognition is that while most L-peptides are vulnerable to enzymatic degradation in vivo [22], frombuilt D from-aminod-amino acids are acids not are recognized not recognized by many by manyproteins, proteins, including including proteases proteases [46]. The [46 result]. The of result this analogous D-peptides are highly resistant to degradation by proteases and have low immunogenicity of this lack of recognition is that whileL most L-peptides are vulnerable to enzymatic degradation lack[47,48]. of recognition The simple is that substitution while most of all -peptides L-amino acids are vulnerable in a peptide to with enzymatic D-amino degradation acids, however, in vivo is [22], analogousin vivogenerally[22 D],-peptides an analogous ineffective ared highly-peptidesstrategy resistant as arethe highlyresulting to degradatio resistant changesn by toin degradationpeptideproteases conformation and by have proteases low and immunogenicity side and chain have low [47,48].immunogenicityorientation The simple can [ 47prevent substitution,48]. Thethe simplecorrect of all substitutionbinding L-amino geomet acids ofry all andinL-amino a thuspeptide destroy acids with in target a D peptide-amino binding withacids, [49–52].d -however,amino A acids, is generallyhowever,common isan solution generally ineffective to anthis inestrategy issueﬀective is retro-in as strategy theversion, resulting as thewhich resultingchanges constitutes in changes peptide reversing in peptideconformation the D-peptide’s conformation and sequence side and chain side orientationchain(Figure orientation 5).can This prevent can approach prevent the correcthas the pr correctoven binding successful binding geomet geometryinry increasing and andthus th thusdestroye biological destroy target targetactivity binding binding of some[49–52]. [ 49–52 A]. commonA common solution solution to this to issue this issueis retro-in is retro-inversion,version, which consti whichtutes constitutes reversing reversing the D-peptide’s the d-peptide’s sequence (Figuresequence 5). (Figure This approach5). This approachhas proven has successful proven successful in increasing in increasing the biological the biological activity of activity some of some unstructured d-peptides by restoring the native L-amino acid side chain angles [53,54].

Molecules 2020, 25, 2314x 6 of 24 Molecules 2020, 25, x 6 of 24 unstructured D-peptides by restoring the native L-amino acid side chain angles [53,54]. However, in structuredHowever,unstructured in peptides, structured D-peptides retro-inversion peptides, by restoring retro-inversion is thenot native enough is L not-amino to enough overcome acidto side overcome the chain conformational angles the conformational [53,54]. changes However, changes caused in structured peptides, retro-inversion is not enough to overcome the conformational changes caused bycaused the introduction by the introduction of D-amino of d-amino acids [46]. acids For [46 example,]. For example, left-handed left-handed helices helices in D-peptides in d-peptides remain remain left- handedleft-handedby the even introduction even after aftersequence of sequenceD-amino reversal acids reversal in [46]. retro-inversion, inFor retro-inversion, example, left-handedwhile while helices helices helices in L-peptides in in D-peptidesL-peptides are remainalways are always left-right- handed even after sequence reversal in retro-inversion, while helices in L-peptides are always right- handed;right-handed; this difference this difference results results in a significant in a significant decrea decreasese in the in binding the binding efficiency efficiency of peptides of peptides to their to handed; this difference results in a significant decrease in the binding efficiency of peptides to their biologicaltheir biological target target [51,55]. [51 ,55]. biological target [51,55].

FigureFigure 5.FigureStructure 5. 5.Structure Structure of an ofL of -peptidean an L -peptideL-peptide and itsand andd-peptide its its DD-peptide-peptide analogue analogue and d andand-retro-inverso-peptide D D-retro-inverso-peptide-retro-inverso-peptide analogue. analogue.analogue. Within the Protein Data Bank (PDB), approximately 62% of protein–protein interactions involve helicalWithin structuresWithin the the Protein [ 56Protein]. Furthermore, Data Data Bank Bank (PDB), approximately(PDB), approximat approximat 80%ely of 62% peptide ofof protein–proteiprotein–protei drugs approvednn interactions interactions by the FDA involve involve contain helicalhelical structures structures [[56].57 [56].]. DueFurthermore, Furthermore, to this, retro-inversion approximatapproximatelyely may 80% not of bepeptidepeptide able to drugsdrugs be applied approved approved to mostby by the clinicallythe FDA FDA containinterestingcontain helical helical peptides structures structures to correct [57]. [57]. forDue Due the to to introduction this,this, retro-inversionretro-inversion of d-amino maymay acids. notnot bebe However, able able to to be thebe applied applied benefits to to omostff mostered clinicallybyclinicallyd-amino interesting interesting acids can peptides alsopeptides be conferredto to correct correct for tofor athe the peptide introductionintroduction without ofof substituting DD-amino-amino acids. acids. every Howe Howe aminover,ver, the acid the benefits benefits with its offeredd-aminooffered by acid by D-amino D equivalent.-amino acids acids For can can example, also also be be conf substitutingconferrederred toto theaa peptideN-terminal withoutwithoutL-amino su substitutingbstituting acid of every mostevery proteinsamino amino acid withacid D L withthewith corresponding its itsD-amino -amino acidd acid-amino equivalent. equivalent. acid can For For significantly example,example, substituting increasesubstitutingin vivo thethe NstabilityN-terminal-terminal by preventingL-amino-amino acid acid recognition of of most most proteins with the corresponding D-amino acid can significantly increase in vivo stability by proteinsof the N-terminus with the of corresponding the protein by proteasesD-amino [acid58]. can significantly increase in vivo stability by preventingpreventingResearch recognition recognition conducted of of bythe the Donna N N-terminus-terminus et al. showedof of the the proteinprotein that the by L-histidine proteasesproteases [58]. [58]. and L-cysteine residues in the Research conducted by Donna et al. showed that the L-histidine and L-cysteine residues in the α5β1Research integrin inhibitorconducted peptide by Donna Ac-PHSCN-NH et al. showed2 (PHSCN) that the could L-histidine be replaced and L with-cysteine their dresidues-stereoisomers in the α5β1 integrin inhibitor peptide Ac-PHSCN-NH2 (PHSCN) could be replaced with their αto5β give1 integrin the mixed inhibitor chirality peptide peptide Ac-PhScN-NHAc-PHSCN-NH22 (PhScN)(PHSCN) (Figure could6)[ be59 ].replaced They found with that their the D-stereoisomers to give the mixed chirality peptide Ac-PhScN-NH2 (PhScN) (Figure 6) [59]. They Dmixed-stereoisomers chirality to peptide give the PhScN mixe showedd chirality significantly peptide Ac-PhScN-NH improved potency2 (PhScN) as an(Figure inhibitor 6) [59]. of αThey5β1 found that the mixed chirality peptide PhScN showed significantly improved potency as an inhibitor foundintegrin-mediated that the mixed invasion chirality of peptide naturally PhScN occurring showed basement significantly membranes improvedin vitro potency, with as IC an50 valuesinhibitor of of α5β1 integrin-mediated invasion of naturally occurring basement membranes in vitro, with IC50 0.097 pg/mL and 0.113 pg/mL for DU 145 and PC-3 cells, respectively, compared to 2600 pg/mL and of αvalues5β1 integrin-mediated of 0.097 pg/mL and invasion 0.113 pg/mL of naturally for DU 145occurring and PC-3 basement cells, respectively, membranes compared in vitro, towith 2600 IC 50 16,627 pg/mL for the unmodified analogue PHSCN [59]. Donna et al. proposed that the inclusion of valuespg/mL of and0.097 16,627 pg/mL pg/mL and for0.113 the pg/mL unmodified for DU analogue 145 and PHSCN PC-3 cells, [59]. Donnarespectively, et al. proposed compared that to the 2600 the d-amino acids, d-histidine, and d-cysteine in the PhScN peptide could greatly increase systemic pg/mLinclusion and 16,627of the D pg/mL-amino acids,for the D -histidine,unmodified and analogue D-cysteine PHSCN in the PhScN [59]. Donnapeptide et could al. proposed greatly increase that the inclusionstabilitysystemic of of the stabilitythe peptides D-amino of the comparedacids, peptides D-histidine, tocompared the unmodified and to D the-cysteine unmodified analogue in the PhScNanalogue due to thepeptide due resistance to could the resistance thesegreatly unnatural increase these systemicaminounnatural acids stability showamino of againstacids the peptidesshow endoproteinases against compared endoproteinases [59 to]. the unmodified [59]. analogue due to the resistance these unnatural amino acids show against endoproteinases [59].

FigureFigure 6. 6.Structures Structures of of PHSCN PHSCN and and PhScN PhScN peptides,peptides, with the D D-amino-amino acids acids highlighted highlighted in inred red [59]. [59 ]. Figure 6. Structures of PHSCN and PhScN peptides, with the D-amino acids highlighted in red [59]. OneOne of of the the best-known best-known examples examples ofof targetedtargeted peptidepeptide modification modiﬁcation for for increasing increasing the the metabolic metabolic stabilitystability of of a peptidea peptide is is octreotide octreotide (Figure (Figure7 );7); a a peptide-based peptide-based drugdrug developed from from the the endogenous endogenous One of the best-known examples of targeted peptide modification for increasing the metabolic hormonehormone somatostatin somatostatin [60 [60].]. Somatostatin Somatostatin (Figure (Figure7 7)) is is a a 1414 aminoamino acid long long cyclic cyclic peptide peptide that that inhibits inhibits stability of a peptide is octreotide (Figure 7); a peptide-based drug developed from the endogenous thethe secretion secretion of of a growtha growth hormone. hormone. Somatostatin Somatostatin receptorsreceptors are are also also found found in in high high concentrations concentrations in in hormone somatostatin [60]. Somatostatin (Figure 7) is a 14 amino acid long cyclic peptide that inhibits variousvarious neuroendocrine neuroendocrine tumors tumors [[60,61].60,61]. The The clinical clinical application application of somatostatin of somatostatin is severely is severely limited limited by the secretion of a growth hormone. Somatostatin receptors are also found in high concentrations in byits its inin vivo vivo half-lifehalf-life ofof only only 1–2 1–2 min min in human in human plasma plasma due to due rapid torapid enzymatic enzymatic degradation degradation [60]. This [60 ]. various neuroendocrine tumors [60,61]. The clinical application of somatostatin is severely limited by Thislimitation limitation spurred spurred the the development development of ofsomatostatin somatostatin analogues analogues with with more more useful useful properties, properties, its in vivo half-life of only 1–2 min in human plasma due to rapid enzymatic degradation [60]. This including octreotide, in which the somatostatin amino acid sequence was shortened from 14 to 8 amino limitation spurred the development of somatostatin analogues with more useful properties,

Molecules 2020, 25, 2314 7 of 24 Molecules 2020, 25, x 7 of 24 acids,including the l -tryptophanoctreotide, in residue which the was somatostatin replaced with aminod-tryptophan, acid sequence and was the shortenedN-terminal froml-amino 14 to acid8 wasamino replaced acids, withthe L a-tryptophand-amino acid residue [60]. was These replaced modifications with D-tryptophan, increased and the inthe vivo N-terminalhalf-life L-amino to 1.5 h in acid was replaced with a D-amino acid [60]. These modifications increased the in vivo half-life to 1.5 human plasma, while maintaining a high binding affinity for the somatostatin receptor subtype SSTR2, h in human plasma, while maintaining a high binding affinity for the somatostatin receptor subtype with an IC50 value of 0.56 nM for octreotide compared to 0.23 nM for the native somatostatin [60,62]. SSTR2, with an IC50 value of 0.56 nM for octreotide compared to 0.23 nM for the native somatostatin The improved half-life and maintained potency offered by octreotide and derivatives thereof led [60,62]. The improved half-life and maintained potency offered by octreotide and derivatives thereof to their radiolabeling with a variety of radioisotopes for use in the diagnosis and treatment of led to their radiolabeling with a variety of radioisotopes for use in the diagnosis and treatment of variousvarious neuroendocrine neuroendocrine tumors, tumors, includingincluding indium-111 for for SPECT SPECT imaging imaging [61,63], [61,63 carbon-11], carbon-11 [64] [64 and] and gallium-68gallium-68 [65 [65]] for for PET PET imaging, imaging, andand yttrium-90 [61] [61] and and lutetium-177 lutetium-177 [66] [66 for] for radiotherapy. radiotherapy.

Figure 7. Structures of somatostatin and octreotide, with the D-amino acid modifications on octreotide highlightedFigure 7. Structures in red. of somatostatin and octreotide, with the D-amino acid modifications on octreotide highlighted in red. Radiopharmaceuticals based upon the minigastrin peptide have been developed for the purposes of imagingRadiopharmaceuticals and therapy to target based cholecystokinin upon the miniga 2 receptorsstrin peptide overexpressed have been indeveloped a variety offor thyroid, the lung,purposes and ovarian of imaging tumors and [therapy67]. However, to target the cholecys clinicaltokinin utility 2 of receptors early minigastrin overexpressed radiopharmaceuticals in a variety of wasthyroid, compromised lung, and due ovarian to high kidneytumors retention, [67]. However, whichcan the cause clinical nephrotoxicity utility of [early67,68 ].minigastrin For example, theradiopharmaceuticals minigastrin radiopharmaceutical was compromised [111In-DOTA]MG0 due to high (Figure kidney8A) retention, showed awhich kidney can retention cause of 111 approximatelynephrotoxicity 127% [67,68]. ID For/g [ 67example,]. To address the minigastrin the issue radiopharmaceutical of kidney retention, [ newIn-DOTA]MG0 minigastrin analogues(Figure were8A) developedshowed a kidney with a decreasedretention of number approximately of glutamic 127% acid ID/g residues [67]. To to address reduce thethe negativeissue of kidney charge on retention, new minigastrin analogues were developed with a decreased number of glutamic acid the peptide [68]. A minigastrin analogue with the five glutamic acid residues in the linker deleted residues to reduce the negative charge on the peptide [68]. A minigastrin analogue with the five from the sequence of [111In-DOTA]MG0 had a significantly reduced kidney retention of approximately glutamic acid residues in the linker deleted from the sequence of [111In-DOTA]MG0 had a 0.3% ID/g [69], but it also had a decreased metabolic stability in human serum, with a mean half-life significantly reduced kidney retention of approximately 0.3% ID/g [69], but it also had a decreased of only 2 h compared to 72.6 h for [111In-DOTA]MG0, which ultimately reduced its suitability for metabolic stability in human serum, with a mean half-life of only 2 h compared to 72.6 h for [111In- clinicalDOTA]MG0, use [69 ].which Kolenic-Petial ultimately etreduced al. found its suitability that the half-life for clinical of these use [69]. minigastrin Kolenic-Petial analogues et al. found could be improvedthat the half-life by inserting of these a linker minigastrin of non-ionic analoguesd-amino could acids be improved into the by structure inserting [68 a]. linker The authorsof non-ionic further demonstratedD-amino acids the into metabolic the structure stability [68].of The the authors minigastrin further analogue demonstrated could the be improvedmetabolic bystability increasing of the the lengthminigastrin of the linker,analogue with could a linker be improved comprised by of increasing six D-glutamine the length residues of the proving linker, to with be optimal a linker and resultingcomprised in aof half-life six D-glutamine in human residues serumof proving approximately to be optimal 495 h and (Figure resulting8B) [68 in]. a half-life in human serum of approximately 495 h (Figure 8B) [68].

Molecules 2020, 25, 2314 8 of 24 Molecules 2020, 25, x 8 of 24

FigureFigure 8. 8.(A )(A Minigastrin) Minigastrin analogue analogue [111In-DOTA]MG0 [111In-DOTA]MG0 with thewith ﬁve the L-glutamic five L-glutamic acids linker acids highlighted linker inhighlighted red [67]; (B )in minigastrin red [67]; (B peptide) minigastrin radiopharmaceutical peptide radiopharmaceuti developedcal by developed Kolenic-Petial by Kolenic-Petial et al., with the et six d-aminoal., with acids the six linker D-amino highlighted acids linker in red highlighted [68]. in red [68].

WhileWhile the the research research ofof Kolenic-PetailKolenic-Petail et al al.. [68] [68] discussed discussed above above utilized utilized indium-111 indium-111 for for SPECT SPECT imaging,imaging, the the beneﬁts benefits achieved achieved from from utilizing utilizingd-amino D-amino acids acids could could be easily be easily carried carried over over to PET to studies,PET forstudies, example, for example, by chelating by chelating a PET radiometal a PET radiometal such as gallium-68such as gallium-68 in place in of place indium-111 of indium-111 or by utilizing or by d-aminoutilizing acids D-amino in the acids linker in the between linker abetween peptide a andpeptide a radioﬂuorinated and a radiofluorinated prosthetic prosthetic group. group.

3.2.3.2.β -Aminoβ-Amino Acids Acids ββ-Amino-Amino acids acids areare widelywidely found as as biologically biologically active active natural natural products products produced produced by by plants plants [70], [70 ], microorganismsmicroorganisms [71 [71],], and and marine marine organisms organisms [72], but[72] relatively, but relatively few β-amino few β-amino acids are acids found are in mammalianfound in systemsmammalian [73]. Thesystems incorporation [73]. The ofincorporation single or multiple of singleβ-amino or multiple acids intoβ-amino peptides acids is into of increasing peptides interestis of inincreasing the pharmaceutical interest in fieldthe pharmaceutical as they can enhance field asin they vivo canmetabolic enhance stabilityin vivo metabolic [74–78] and stability potency [74–78] [74,79 ]. Thisand has potency been attributed [74,79]. toThis their has different been electronicattributed environments to their different and backbone electronic/side environments chain configurations, and backbone/side chain configurations, compared to their α-amino acid analogues, decreasing compared to their α-amino acid analogues, decreasing recognition by proteases [80]. recognition by proteases [80]. Garayoa et al. showed that the incorporation of a β-alanine-β-alanine (βAla-βAla) linker into Garayoa et al. showed that the incorporation of a β-alanine-β-alanine (βAla-βAla) linker into a a human tumor targeting bombesin peptide radiopharmaceutical (Figure9) resulted in a two-fold human tumor targeting bombesin peptide radiopharmaceutical (Figure 9) resulted in a two-fold increase in metabolic stability against proteolytic degradation and no decrease in receptor affinity increase in metabolic stability against proteolytic degradation and no decrease in receptor affinity when compared to the unmodified structure (sans linker) in studies performed using in vitro tumor when compared to the unmodified structure (sans linker) in studies performed using in vitro tumor cellcell cultures cultures [81 [81].]. However, However, when when the the studies studies were were performed performed in in human human plasma, plasma, the the analogue analogue with with the β β Ala–the βAla–Alaβ linkerAla linker showed showed decreased decreased metabolic metabolic stability stability with with a half-lifea half-life of of 10 10 h h compared compared to to 16 16 h h for thefor unmodified the unmodified analogue analogue [81]. [81].

Molecules 2020,, 25,, 2314x 9 of 24 Molecules 2020, 25, x 9 of 24

Molecules 2020, 25, x 9 of 24

FigureFigure 9. 9. Bombesin-basedBombesin-based 9. Bombesin-based peptide peptide radiopharmaceutical radiopharmaceutical radiopharmaceutical investigated investigated investigated by by Garayoa Garayoa by Garayoa et al et., withal., et with the al., β theAla– with βAla– the βAlaAla– βlinkerβAlaAla linker linkerhighlighted highlighted highlighted in inred red in[81]. [81]. red [81]. Figure 9. Bombesin-based peptide radiopharmaceutical investigated by Garayoa et al., with the βAla– Further research by Garayoa et al. investigated alternative β-amino acid linkers (Figure 10) for FurtherβAla linker researchresearch highlighted byby Garayoa Garayoa in red et[81]. et al. al investigated. investigated alternative alternativeβ-amino β-amino acid acid linkers linkers (Figure (Figure 10) 10) for thefor samethe bombesin same bombesin peptide peptide radiopharmaceutical radiopharmaceutical as shown as shown above above (Figure (Figure9), with9), with a focus a focus on onβ β-amino-amino acids the sameacids bombesin that could peptidehold a charge radiopharmaceutical once incorporated as into shown the final above structure (Figure of the9), withradiopharmaceutical a focus on β-amino that couldFurther hold research a charge by once Garayoa incorporated et al. investigated into the alternative final structure β-amino of the acid radiopharmaceutical linkers (Figure 10) for [ 17]. acids[17]. that The could linkers hold were a charge constructed once incorporatedfrom a combination into theof two final or structurethree of the of selected the radiopharmaceutical β-amino acids the same bombesin peptide radiopharmaceutical as shown above (Figure 9), with a focusβ on β-amino The[17]. linkersinThe sequence. linkers were Itwereconstructed was constructedfound that from the from abombesin combination a combination analogue of twomodified of two or three or with three ofβ3-homoglutamic the of the selected selected acid-amino β-amino (Figure acids acids in acids that could hold a charge once incorporated into the final structure3 of the radiopharmaceutical sequence.in sequence.10d) in It the wasIt linker,was found found with that one that the single the bombesin bombesinnegative analogue charge analogue, showed modified modified a significant with withβ -homoglutamic increase β3-homoglutamic in tumor acid uptake acid (Figure and (Figure 10d) [17]. The linkers were constructed from a combination of two or three of the selected β-amino acids in10d) the intumor-to-tissue linker, the linker, with with oneratio, one single but single did negative not negative increase charge, charge metabolic showed, showed stability a significanta comparedsignificant increaseto increase the previously in in tumor tumor developed uptakeuptake andand in sequence. It was found that the bombesin analogue modified with β3-homoglutamic acid (Figure tumor-to-tissueradiopharmaceutical ratio, but (Figure did not 9) [17]. increase metabolic stability compared to the previously developed 10d) in the linker, with one single negative charge, showed a significant increase in tumor uptake and radiopharmaceutical (Figure9)[17]. radiopharmaceuticaltumor-to-tissue ratio, (Figure but did 9) not[17]. increase metabolic stability compared to the previously developed radiopharmaceutical (Figure 9) [17].

Figure 10. Alternate β-amino acids (a) β-alanine; (b) β3-homoserine; (c) β3-homolysine; and (d) β3-homoglutamic acid investigated by Garayoa et al. for use as linkers [17].

Figure 10.10. Alternate β-amino acids ((a)) ββ-alanine;-alanine; ((bb)) ββ33-homoserine; ( c) β3-homolysine;-homolysine; and and ( d) In a similar study carried out by Popp et al., a single3 β-alanine amino 3acid, with a methylated β3-homoglutamic-homoglutamicFigure 10. Alternate acid acid investigated investigatedβ-amino acids by by Garayoa( Garayoaa) β-alanine; et et al al. .( forb for) βuse use-homoserine; as as linkers linkers [17]. [(17c) ].β -homolysine; and (d) nitrogen,β3-homoglutamic was used acidas a investigatedlinker in a statine-basedby Garayoa et alGRPr-antagonist. for use as linkers radiopharmaceutical [17]. (Figure 11) designed to target receptors on the surface of several human tumors [82]. The introduction of the N- In a similarsimilar studystudy carriedcarried out byby PoppPopp etet al.,al., aa singlesingle ββ-alanine-alanine aminoamino acid,acid, withwith aa methylatedmethylated methylatedIn a similar β-alanine study didcarried not disrupt out by thePopp binding et al., affinitya single and β-alanine presented amino a similar acid, in with vivo a stabilitymethylated in nitrogen, was used as a linkerlinker inin aa statine-basedstatine-based GRPr-antagonistGRPr-antagonist radiopharmaceutical (Figure 11)11) nitrogen,mice compared was used to as the a linkerunmodified in a statine-based compound, GRPr-antagonistwith approximately radiopharmaceutical 50% of the activity (Figure in the 11) designed to target target receptors receptors on on the the surface surface of of se severalveral human human tumors tumors [82]. [82 ].The The introduction introduction of the of theN- designedbloodstream to target representing receptors intact on the co mpoundsurface of 15 se minveral post human injection tumors in the [82]. case The of both introduction compounds of the[82]. N - N-methylated β-alanine did not disrupt the binding aﬃnity and presented a similar in vivo stability methylatedmethylated β-alanine β-alanine did did not not disrupt disrupt the the bindingbinding affinity and and presented presented a similara similar in invivo vivo stability stability in in in mice compared to the unmodiﬁed compound, with approximately 50% of the activity in the micemice compared compared to tothe the unmodified unmodified compound,compound, with approximatelyapproximately 50% 50% of ofthe the activity activity in thein the bloodstreambloodstream representing representing intact intact compoundco compoundmpound 1515 minmin post injection injection in in the the case case of ofboth both compounds compounds [82]. [82]. [ 82].

Figure 11. Statine-based GRPr antagonist radiopharmaceutical investigated by Popp et al., with the N-methylated β-alanine linker highlighted in red [82].

FigureFigure 11. 11.Statine-based Statine-based GRPr GRPr antagonist antagonist radiopharmaceuticalradiopharmaceutical investigated investigated by byPopp Popp et al., et al.,with with the the Figure 11. Statine-based GRPr antagonist radiopharmaceutical investigated by Popp et al., with the N-methylatedN-methylatedβ-alanine β-alanine linker linker highlighted highlighted inin redred [[82].82]. N-methylated β-alanine linker highlighted in red [82].

Molecules 2020, 25, 2314 10 of 24

Molecules 2020, 25, x 10 of 24 Molecules 2020, 25, x 10 of 24 MoleculesThe research 2020, 25, x undertaken by Garayoa et al. [17,81] and Popp et al. [82] aimed to demonstrate10 of 24 that unnaturalTheβ research-amino acidsundertaken could beby introducedGarayoa et al. into [17,81] the structure and Popp of et peptide-based al. [82] aimed radiopharmaceuticalsto demonstrate that The research undertaken by Garayoa et al. [17,81] and Popp et al. [82] aimed to demonstrate that tounnatural increase metabolicβ-amino-amino stability.acidsacids couldcould While neitherbebe introducedintroduced author achievedintointo thethe a significantstructurestructure increaseofof peptide-basedpeptide-based in metabolic unnatural β-amino acids could be introduced into the structure of peptide-based stability,radiopharmaceuticalsradiopharmaceuticals they did produce toto compounds increaseincrease metabolicmetabolic that maintained stability.stability. metabolicWhileWhile neitherneither stability authorauthor and achievedachieved binding aaaffi significantsignificantnity close to radiopharmaceuticals to increase metabolic stability. While neither author achieved a significant theincreaseincrease unmodified inin metabolicmetabolic analogue. stability,stability, This indicates theythey diddid produceproduce that there compoundscompounds is room for thatthat improvement maintainedmaintained formetabolicmetabolic future stabilitystability peptide-based andand increasebinding affinityin metabolic close stability,to the unmodified they did produce analogue. compounds This indicates that that maintained there is roommetabolic for improvement stability and radiopharmaceuticalsbinding affinity close that to the incorporate unmodified unnatural analogue.β This-amino indicates acids. that there is room for improvement bindingfor future affinity peptide-based close to the radiopharmac unmodifiedeuticals analogue. that This incorporate indicates unnatural that there β is-amino room foracids. improvement forWhile future thepeptide-based research of radiopharmac Garayoa eteuticals al. only that usedincorporate the SPECT unnatural radionuclide β-amino acids. technetium-99m, for futureWhile peptide-based the research radiopharmacof Garayoa eteuticals al. only that us edincorporate the SPECT unnatural radionuclide β-amino technetium-99m, acids. it it shouldWhile be apparentthe research that of the Garayoa introduction et al. only of β -aminoused the acids SPECT can radionuclide easily be applied technetium-99m, to PET studies it shouldWhile be apparentthe research that ofthe Garayoa introduction et al. ofonly β-amino used theacids SPECT can easilyradionuclide be applied technetium-99m, to PET studies it should be apparent that the introduction of β-amino acids can easilyβ be applied to PET studies through,should forbe example,apparent chelatingthat the introduction PET radiometals of β-amino or incorporating acids can easily-amino be applied acids into to peptide-basedPET studies through,through, forfor example,example, chelatingchelating PET radiometals or incorporating β-amino-amino acidsacids intointo peptide-basedpeptide-based PETthrough, radiopharmaceuticals. for example, chelating Despite PET theradiometals potential or benefits incorporating to pharmacological β-amino acids properties,into peptide-based there are PET radiopharmaceuticals. Despite the potential benefits to pharmacologicall properties,properties, therethere areare currentlyPET radiopharmaceuticals. no examples in the Despite literature the ofpotentialβ-amino benefits acids to being pharmacologica applied tol improve properties, the there metabolic are currently no examples in the literature of β-amino-amino acidsacids beingbeing appliedapplied toto improveimprove thethe metabolicmetabolic stabilitycurrently of peptide-basedno examples in fluorine-18 the literature labeled of β-amino radiopharmaceuticals. acids being applied Interestingly, to improve the metabolic research by stability of peptide-based fluorine-18 labeled radiopharmaceuticals. Interestingly, the research by Schjoeth-Eskensenstability of peptide-based et al. successfully fluorine-18 demonstratedlabeled radiopharmaceuticals. the radiolabeling Interestingly, of the α-carbon the research of β-alanine by Schjoeth-Eskensen et al.. successfullysuccessfully demonstrateddemonstrated thethe radiolabelingradiolabeling ofof thethe α-carbon-carbon ofof β-alanine-alanine withSchjoeth-Eskensen fluorine-18 (Figure et al. successfully12), but no demonstrated stability studies the radiolabeling or subsequent of the conjugation α-carbon of to β-alanine a peptide with fluorine-18 (Figure 12), but no stability studies or subsequent conjugation to a peptide were werewith performed fluorine-18 [83 (Figure]. 12), but no stability studies or subsequent conjugation to a peptide were performed [83]. performed [83].

Figure 12. Fluorine-18 labeled β-alanine synthesized by Schjoeth-Eskensen et al. [83]. FigureFigure 12. 12.Fluorine-18 Fluorine-18 labeled labeledβ β-alanine-alanine synthesized by by Schjoeth-Eskensen Schjoeth-Eskensen et etal. al. [83] [83. ]. Figure 12. Fluorine-18 labeled β-alanine synthesized by Schjoeth-Eskensen et al. [83]. 3.3. N-Methylation 3.3.. N-Methylation 3.3. N-Methylation Peptides can be modified through N-methylation (also known as N-alkylation). This method Peptides can be modified through N-methylation-methylation (also(also knownknown asas N-alkylation).-alkylation). ThisThis methodmethod Peptides can be modified through N-methylation (also known as N-alkylation). This method constitutesconstitutes substituting substituting one one or or more more NHNH groupsgroups inin aa peptidepeptide backbone backbone with with NN-methyl-methyl-methyl substituentssubstituents substituents constitutes substituting one or more NH groups in a peptide backbone with N-methyl substituents (Figure(Figure(Figure 13 13).).13).N N-Methylation-Methylation-Methylation cancancan conferconferconfer seve severalseveralral benefitsbenefits benefits comparedcompared compared toto to theirtheir their unmodifiedunmodified unmodified analogues,analogues, analogues, (Figure 13). N-Methylation can confer several benefits compared to their unmodified analogues, includingincludingincluding enhanced enhancedenhanced protease proteaseprotease resistance, resistance,resistance, membrane membranemembrane permeability,permeability,permeability, andand biologicalbiological biological activityactivity activity [84].[84]. [84 ]. including enhanced protease resistance, membrane permeability, and biological activity [84].

Figure 13. Structures of natural compared to N-methylated amino acids. FigureFigure 13. 13.Structures Structures ofof naturalnatural comparedcompared to NN-methylated-methylated amino amino acids. acids. Figure 13. Structures of natural compared to N-methylated amino acids. N-MethylationN-Methylation-Methylation of ofof a peptide aa peptidepeptide backbone backbackbone replaces replaces a hydrogen a hydrogen bond bond donor donordonor (NH) (NH)(NH) with withwith a potential aa potentialpotential steric stericN clash-Methylation (NCH3), of thus a peptide eliminating backbone some replaces inter- aand hydrogen intramolecular bond donor hydrogen (NH) with bonds a potential [85–87]. clashsteric (NCH clash3), thus(NCH eliminating3), thus eliminating some inter- some and intramolecularinter- and intramolecular hydrogen bonds hydrogen [85–87 bonds]. N-Methylation [85–87]. steric clash (NCH3), thus eliminating some inter- and intramolecular hydrogen bonds [85–87]. canN also-Methylation-Methylation greatly alter cancan also thealso conformation greatlygreatly alteralter thethe of conformation theconformation entire peptide. ofof thethe This entireentire occurs peptide.peptide. because ThisThis occursoccurs the N-methyl becausebecause groupthethe N-Methylation-methyl group can will also influence greatly thealter conformational the conformation flexibility of the entireof both peptide. the peptide This occurs backbone because and the willN-methyl inﬂuence group theconformational will influence the ﬂexibility conformational of both flexibility the peptide of both backbone the peptide and the backbone side chains and ofthe the Nside-methyl chains group of the will residues influence close the to theconformational N-methyl amino flexibility acids. of Of both partic theular peptide note, thebackbone energy and barrier the residuesside chains close of to the the residuesN-methyl close amino to the acids. N-methyl Of particular amino acids. note, Of the partic energyular barriernote, the between energy barrier the trans sidebetween chains the of thetrans residues and cis close peptide to the bondN-methyl conformations amino acids. (Fig Of urepartic 14)ular is note, greatly the energyreduced barrier and andbetweencis peptide the bondtrans conformationsand cis peptide (Figure bond 14conformations) is greatly reduced (Figure and 14) consequently is greatly reduced the cis peptide and between the trans and cis peptide bond conformations (Figure 14) is greatly reduced and consequently the cis peptidepeptide bondbond conformationconformation bebecomes readily accessible [88]. bondconsequently conformation the cis becomes peptide readily bond conformation accessible [88 be].comes readily accessible [88].

Figure 14. Comparison of the trans and cis conformations of N-methylated peptide bonds. Figure 14. Comparison of the trans and cis conformations of N-methylated peptide bonds. FigureFigure 14. 14.Comparison Comparison of of thethetrans trans andand cis conformations of of NN-methylated-methylated peptide peptide bonds. bonds.

Molecules 2020, 25, x 11 of 24 Molecules 2020, 25, 2314 11 of 24

With the overall conformational change that can occur with N-methylated peptides, and the changeWith in H-bonding the overall capacity conformational and steric change features, that N can-methylation occur with oftenN-methylated leads to a peptides,decreased andaffinity the changeof the peptide in H-bonding for the capacityactive site and of steric proteases features, andN therefore-methylation increased often metabolic leads to a decreasedstability. It a ffihasnity also of thebeen peptide found forthat the N-methylation active site of proteasesof an amide and bond therefore adjacent increased to the metabolic cleavage stability.site can confer It has alsoa greater been foundresistance that againstN-methylation enzymatic of an degradation amide bond than adjacent N-methylation to the cleavage of the site amide can conferbond at a greaterthe cleavage resistance site againstitself [89]. enzymatic This behavior degradation may thanresultN -methylationfrom N-methylation of the amide making bond the at thecis cleavageconformation site itself readily [89]. Thisaccessible behavior and then may becoming result from theN preferred-methylation confor makingmation the of cistheconformation peptide in vivo. readily For example, accessible the and cis thenconformation becoming may the preferredresult in portions conformation of the of pept the peptideide beingin positioned vivo. For example, such that the theycis conformationare now less mayaccessible result to in proteolytic portions ofactivity the peptide or simply being no long positioneder fit into such the thatenzyme they binding are now site, less thus accessible increasing to proteolyticthe metabolic activity stability or simply [88]. no However, longer fit intothese the st enzymeructuralbinding changes site, may thus also increasing disrupt the intra- metabolic and stabilityintermolecular [88]. However, hydrogen these bonds structural that may changes be important may also disruptfor the intra-stabilization and intermolecular of biologically hydrogen active bondsconformations that may and be important for target for receptor the stabilization recognition of biologically[90]. Therefore, active the conformations use of N-methylation and for target for receptorincreasing recognition metabolic [ 90stability]. Therefore, must be the balanced use of N -methylationagainst maintaining for increasing biological metabolic activity stability against must the bedesired balanced target against receptor maintaining [91,92]. biological activity against the desired target receptor [91,92]. The endothelin peptides are potentpotent vasoconstrictors.vasoconstrictors. It It has has been been found that highly potent antagonists ofof endothelinendothelin receptors receptors can can be be developed developed from from the theC-terminal C-terminal hexapeptide hexapeptide of endothelin of endothelin [88]. However,[88]. However, these compoundsthese compounds are generally are generally unstable towardsunstable enzymatic towards proteolysisenzymatic andproteolysis consequently and haveconsequently relatively have short relatively half-lives, short which half-lives, reduces which their reduces utility for their clinical utility use for [ 88clinical]. Wayne use [88]. et al. Wayne found thatet al. the foundN-methylation that the N-methylation of a single isoleucine of a single residue isoleucine in the residue sequence in the of sequence these previously of these previously developed endothelindeveloped endothelin receptor antagonists receptor antagonists could significantly could significantly improve metabolic improve metabolic stability [ 88stability]. For example,[88]. For Nexample,-methylation N-methylation of the amide of the bond amide between bond between the Ile19 theand Ile Ile19 20andresidues Ile20 residues increased increa thesed half-life the half-life in rat intestinalin rat intestinal perfusate perfusate from 10.6 from min 10.6 to min 538 minto 538 [88 min]. This [88]. modification This modification also enhanced also enhanced receptor receptor binding abindingffinity fromaffinity an ICfrom50 of an 40 IC nM50 of in 40 the nM case in the of the case unmodified of the unmodified compound compound down to down 10 nM to [88 10]. nM [88]. N-Methylation hashas also also been been eff ectiveeffective when when combined combined with with other other peptide peptide modifications modifications to further to modulatefurther modulate the properties the properties of a peptide-based of a peptide-based radiopharmaceuticals. radiopharmaceuticals. For example, For asexample, previously as previously discussed indiscussed Section 3.2in Section, Popp et3.2., al. Popp successfully et al. successfully incorporated incorporated an N-methylated an N-methylatedβ-alanine residueβ-alanine as aresidue linker as in a statine-basedlinker in a statine-based GRPr antagonist GRPr antagonist radiopharmaceuticals radiopharmaceuticals [82]. [82].

3.4. PEGylation and Alkyl Linkers PEGylation defines defines the the process process of oflinking linking one one or more or more polyethylene polyethylene glycol glycol (PEG) (PEG)polymer polymer chains chains(Figure(Figure 15) to a 15peptide,) to a peptide, protein, protein,or non-peptide or non-peptide molecule. molecule. PEG possesses PEG possessesuseful properties, useful properties, including includinghigh solubility high in solubility water and in water many and organic many solven organicts, non-toxicity, solvents, non-toxicity, and non-immunogenicity, and non-immunogenicity, and has andbeen hasapproved been approved by the byFDA the FDAfor human for human use use[93]. [93 ].Many Many PEGyla PEGylatedted peptide-basedpeptide-based radiopharmaceuticals havehave been been developed developed and shownand shown to possess to possess improved impr pharmacokineticoved pharmacokinetic properties comparedproperties tocompared their unmodified to their unmodified analogues, analogue includings, increasedincluding receptorincreased binding receptor a ffibindingnity, increased affinity, tumorincreased uptake, tumor and uptake, decreased and decreased kidney uptake kidney [94 –upta102].ke Other [94–102]. potential Other improvements potential improvements that have beenthat achievedhave been thoughachieved PEGylation though PEGylation include longerinclude circulatory longer circulatory times, increasedtimes, increased aqueous aqueous solubility solubility [103], and[103], increased and increased metabolic metabolic stability stability by creatingby creating steric steric hinderance hinderance that that shields shields the the moleculemolecule from proteases [[104].104]. PEGylation PEGylation also also increases increases the the molecular mass and size of peptides, which further increases body-residence timetime duedue toto decreaseddecreased kidneykidney excretionexcretion [[105].105]. The length and shape of PEGs (linear, branched,branched, oror dendritic)dendritic) have beenbeen shownshown to influenceinfluence thethe pharmacologicalpharmacological properties of the PEGylated peptides and proteins, with branched PEG structures often most eeffective.ffective. This has been postulated to be due to both a greatergreater degreedegree ofof stericsteric hinderancehinderance against proteases and possessing additional sites for conjugation with the target peptidepeptide oror proteinprotein [[106].106].

Figure 15. Polyethylene glycol (PEG) structure. The advantages oﬀered by PEGylation has seen it applied to a variety of peptide-based The advantages offered by PEGylation has seen it applied to a variety of peptide-based radiopharmaceuticals. For example, Hausner et al. found that the PEGylation of a radiopharmaceuticals radiopharmaceuticals. For example, Hausner et al. found that the PEGylation of a

Molecules 2020, 25, x 12 of 24 radiopharmaceuticalsMolecules 2020,, 2525,, 2314x derived from the αvβ6 integrin targeting peptide A20FMDV2, at both12 of the 24 C- and N-termini, greatly improves its pharmacokinetic properties [107]. When evaluated in mice, the radiopharmaceuticals derived from the αvβ6 integrin targeting peptide A20FMDV2, at both the C- PEGylatedderived peptide from the showedα β integrin good targeting metabolic peptide stabi A20FMDV2,lity with approximately at both the C- and 80%N-termini, of the compound greatly and N-termini, greatlyv 6 improves its pharmacokinetic properties [107]. When evaluated in mice, the remainingimproves intact its pharmacokinetic after incubation properties in mouse [107 serum]. When for evaluated 1 h. The in mice,modified the PEGylated compound peptide also showed showed a PEGylated peptide showed good metabolic stability with approximately 80% of the compound good metabolicv stability6 with approximately 80% of the compound remaining intact after incubation in higherremaining uptake inintact α β after-expressing incubation tumors in mouse compared serum forto the1 h. unmoThe modifieddified peptide, compound with also a tumorshowed uptake a mouse serum for 1 h. The modified compound also showed a higher uptake in αvβ6-expressing tumors in BxPC-3higher cellsuptake of in4.7% αvβ 6ID/g-expressing compared tumors to compared0.69% ID/g to theat 1 unmo h fordified the unmodified peptide, with peptide a tumor (Figureuptake 16) compared to the unmodified peptide, with a tumor uptake in BxPC-3 cells of 4.7% ID/g compared to [107].in BxPC-3 cells of 4.7% ID/g compared to 0.69% ID/g at 1 h for the unmodified peptide (Figure 16) 0.69% ID/g at 1 h for the unmodified peptide (Figure 16)[107]. [107].

Figure 16. Structures of (a) [18F]FBA-A20FMDV2 and (b) the bi-terminally PEGylated [18F]FBA-PEG28- 18 18 18 18 Figure 16. StructuresStructures of of(a) ( a[)[F]FBA-A20FMDV2F]FBA-A20FMDV2 and and(b) the (b) bi-terminally the bi-terminally PEGylated PEGylated [ F]FBA-PEG [ F]FBA-28- A20FMDV2-PEG28 [107]. PEGA20FMDV2-PEG28-A20FMDV2-PEG28 [107].28 [107].

The Theinclusion inclusion of large of of large large PEG PEG PEG groups groups groups has has has also also also beenbeen been echoed echoed by by Dapp byDapp Dapp and and andco-workers co-workers co-workers [108,109], [108,109], [108, 109who], who usedwhoused a 5 usedKDaa 5 KDa aPEG 5 KDaPEG group PEGgroup in group inthe the inlinker linker the linkerof of aa bombesin ofbombesin a bombesin peptidepeptide peptide radiotherapeutic radiotherapeutic radiotherapeutic (Figure (Figure (Figure 17). 17). The17). The metabolicThemetabolic metabolic stability stability stability of theof the compound of compound the compound was was assessed wasassessed assessed in vitroin vitro byby incubating incubatingby incubating it init itinhuman in human human serum serum serum for fivefor five daysfivedays [109]. days [109]. The [109 The inclusion]. inclusion The inclusion of ofthe the PEG of PEG the into into PEG the the into linkerlinker the linker resulted resulted in in an an inincrease increase an increase in inthe the inamount theamount amount of intact of ofintact radiopharmaceuticalsintactradiopharmaceuticals radiopharmaceuticals remaining remaining remaining after after the afterthe five-d thefive-d five-dayay incubation, incubation, from from from 14% 14%14% with withwith the the the unmodifiedunmodified unmodified compoundcompound to 52% to 52% with withwith the thethe modified modifiedmodified compound compoundcompound [[109].[109].109].

Figure 17. Structure of (a) unmodified and (b) modified bombesin peptide radiotherapeutics

investigated by Dapp et al., with the PEG linker modification highlighted in red [109]. FigureFigure 17. 17.StructureStructure of of(a) (aunmodified) unmodified and and ( (bb)) modifiedmodified bombesinbombesin peptide peptide radiotherapeutics radiotherapeutics investigatedinvestigatedIn another by example,Dapp by Dapp et al., et Wu al., with et with al. the theincorporated PEG PEG linker linker modification modificationa mini-PEG highlighted (threehighlighted ethylene in redin red oxide [109 [109].]. units) spacer into 18 18 [ F]FB-E[c(RGDyK)]2 to produce [ F]FB-mini-PEG-E[c(RGDyK)]2 (Figure 18). This In another example, Wu et al. incorporated a mini-PEG (three ethylene oxide units) spacer into radiopharmaceuticalIn18 another example, showed Wu et greateral. 18incorporated radiolabeling a mi yield,ni-PEG reduced (three renal ethylene excretion, oxide and units) similar spacer tumor into [ F]FB-E[c(RGDyK)]2 to produce [ F]FB-mini-PEG-E[c(RGDyK)]2 (Figure 18). This radiopharmaceutical [18F]FB-E[c(RGDyK)]uptake compared to theto non-PEGylatedproduce analogue[18F]FB-mini-PEG-E[c(RGDyK)] (Figure 18) [110]. This example(Figure also demonstrated 18). This showed greater radiolabeling2 yield, reduced renal excretion, and similar tumor2 uptake compared to the that long PEG chains are not always necessary to significantly improve the properties of peptide- radiopharmaceuticalnon-PEGylated analogue showed (Figure greater 18) [radiolabeling110]. This example yield, also reduced demonstrated renal that excretion, long PEG and chains similar are not tumor based radiopharmaceuticals. uptakealways compared necessary to tothe significantly non-PEGylated improve analogue the properties (Figure of peptide-based 18) [110]. This radiopharmaceuticals. example also demonstrated that long PEG chains are not always necessary to significantly improve the properties of peptide- based radiopharmaceuticals.

Molecules 2020, 25, 2314 13 of 24

Molecules 2020, 25, x 13 of 24

Figure 18. Structures18 of [18F]FB-E[c(RGDyK)] and [18F]FB-mini-PEG-E[c(RGDyK)]18 [110]. Figure 18. Structures of [ F]FB-E[c(RGDyK)]22 and [ F]FB-mini-PEG-E[c(RGDyK)]2 2 [110].

Work conductedWork conducted by the by Maeckethe Maecke group group [111[111]] also investigated investigated the theeffect e ffofect PEGylation of PEGylation on the on the previously developed bombesin analogue DOTA-Bombesin (7–14). Initial studies using a PEG4 chain previously developed bombesin analogue DOTA-Bombesin (7–14). Initial studies using a PEG as a linker between the DOTA chelator and the peptide with gallium-68 or lutetium-177 as the 4 chain asradionuclide a linker between (Figure the19) found DOTA that chelator the modified and thepeptide-based peptide with radiopharmaceutical gallium-68 or lutetium-177had superior as the radionuclidepharmacokinetic (Figure 19 properties) found thatin the the PC-3 modified tumor-bearing peptide-based mouse model radiopharmaceutical than previously developed had superior pharmacokineticanalogues [111]. properties In a subsequent in the PC-3study, tumor-bearingthe Maecke group mousesynthesized model a series than of four previously lutetium-177 developed analoguesradiolabeled [111]. In statine-based a subsequent bombesin study, antagonist the Maecke radiopharmaceuticals group synthesized with aPEG series chains of fourof different lutetium-177 lengths (2, 4, 6, or 12 PEG units) as the linker between the DOTA chelator and the peptide analogue radiolabeled statine-based bombesin antagonist radiopharmaceuticals with PEG chains of different (Figure 19) [112]. The metabolic stability of these compounds was then assessed in human serum, lengthsand (2, 4,it was 6, or found 12 PEG that the units) half-life as the increased linker as between PEG chain the length DOTA increased chelator up to andthe PEG the6 linker peptide (half- analogue (Figure 19life)[ of112 PEG].2 The= 246metabolic h, PEG4 = 407 stability h, and PEG of these6 = 584 compounds h), but began wasto decrease then assessedwith the PEG in12 human linker serum, and it was(half-life found of 407 that h) the[112]. half-life increased as PEG chain length increased up to the PEG6 linker (half-life of PEG2 = 246 h, PEG4 = 407 h, and PEG6 = 584 h), but began to decrease with the PEG12 linker (half-life of 407 h) [112]. Molecules 2020, 25, x 14 of 24

Figure 19. Bombesin-based peptide radiopharmaceutical investigated by the Maecke group, with Figure 19. Bombesin-based peptide radiopharmaceutical investigated by the Maecke group, with PEG4 chain linkerPEG4 andchain alternative linker and alternative PEG chain PEG lengths chain lengths highlighted highlighted in red in red [111 [111,112].,112].

In a similar study by Bacher et al., the incorporation of a PEG3 linker into a different bombesin- based radiopharmaceutical was explored as a method to increase its metabolic stability (Figure 20) [113]. In vitro stability assays conducted in human serum found that incorporation of the PEG3 linker led to a 9% increase in stability compared to the compound not containing a linker [113]. In the same study, Bacher et al. also explored the potential stabilizing effects of other alkyl chains as linkers, including 4-amino-1-carboxymethyl-piperidine and 6-aminohexanoic acid (Figure 20) [113]. However, these analogues were found to decrease metabolic stability by 20% and 7%, respectively, when compared to the radiopharmaceutical without any linker. In contrast to this, it was found that when the 4-amino-1-carboxylmethyl-piperdine linker was incorporated into a dimer of the bombesin- based radiopharmaceutical (Figure 21), there was a 52% increase in stability compared to the dimer without any linker [113]. The incorporation of the 6-aminohexanoic acid linker into the dimer resulted in decreased metabolic stability of the radiopharmaceutical [113].

Figure 20. Bombesin-based radiopharmaceuticals investigated by Bacher et al., with the linker location and linkers highlighted in red [113].

Molecules 2020, 25, x 14 of 24

Molecules 2020, 25, 2314 14 of 24

Figure 19. Bombesin-based peptide radiopharmaceutical investigated by the Maecke group, with In a similar study by Bacher et al., the incorporation of a PEG3 linker into a diﬀerent bombesin-based PEG4 chain linker and alternative PEG chain lengths highlighted in red [111,112]. radiopharmaceutical was explored as a method to increase its metabolic stability (Figure 20)[113].

In vitro stabilityIn a similar assays study conducted by Bacher et in al., human the incorporation serum found of a PEG that3 linker incorporation into a different of bombesin- the PEG3 linker led to abased 9% increaseradiopharmaceutical in stability was compared explored as to a themethod compound to increase not its metabolic containing stability a linker (Figure [113 20)]. In the same study,[113]. In Bacher vitro stability et al. assays also conducted explored in the human potential serum found stabilizing that incorporation eﬀects of of other the PEG alkyl3 linker chains as linkers,led including to a 9% increase 4-amino-1-carboxymethyl-piperidine in stability compared to the compound and not 6-aminohexanoiccontaining a linker [113]. acid In (Figure the same 20 )[113]. study, Bacher et al. also explored the potential stabilizing effects of other alkyl chains as linkers, However, these analogues were found to decrease metabolic stability by 20% and 7%, respectively, including 4-amino-1-carboxymethyl-piperidine and 6-aminohexanoic acid (Figure 20) [113]. when comparedHowever, these to the analogues radiopharmaceutical were found to decrease without metabolic any linker.stability Inby contrast20% and 7%, to respectively, this, it was found that whenwhen the compared 4-amino-1-carboxylmethyl-piperdine to the radiopharmaceutical without any linker linker. was In contrast incorporated to this, it was into found a dimer that of the bombesin-basedwhen the 4-amino-1-carboxylmethyl-piperdine radiopharmaceutical (Figure 21), linker there was was incorporated a 52% increase into a dimer in stability of the bombesin- compared to the dimer withoutbased radiopharmaceutical any linker [113]. (Figure The incorporation 21), there was a of 52 the% increase 6-aminohexanoic in stability compared acid linker to the into dimer the dimer resultedwithout in decreased any linker metabolic [113]. The incorporation stability of the of the radiopharmaceutical 6-aminohexanoic acid linker [113]. into the dimer resulted in decreased metabolic stability of the radiopharmaceutical [113].

Figure 20.FigureBombesin-based 20. Bombesin-based radiopharmaceuticals radiopharmaceuticals investigated investigated by by Bacher Bacheret et al., al., with with the the linker linker location and linkerslocation highlighted and linkers in highlighted red [113]. in red [113].

Figure 21. Bombesin-based dimer radiopharmaceutical investigated by Bacher et al., with 4-amino-1- carboxylmethyl-piperidine linkers highlighted in red [113].

Molecules 2020, 25, x 15 of 24

O S NH O O N O O N O R H N O N N O O O 68Ga O S N H N N NH2 O

O O

N S NH NH O O O O H H H H R= N N N NH2 N N N N N H H H H O O O O

H2N O

MoleculesFigure2020 21., 25 ,Bombesin-based 2314 dimer radiopharmaceutical investigated by Bacher et al., with 4-amino-1-15 of 24 carboxylmethyl-piperidine linkers highlighted in red [113].

While beyond the scope of this review, it isis interestinginteresting to notenote thatthat PEGylationPEGylation has also been successfully applied to antibody fragments for the use as PET radiopharmaceuticals, with promising results [[114,115].114,115].

3.5. Peptide Cyclization Cyclization of a peptide sequence has been foundfound to enhanceenhance stability against proteolytic degradation [[116].116]. Cyclization is usually achieved by linking the C-terminus to the N-terminus of the peptide backbone [[117].117]. However, it can also be advantageous to to cyclize cyclize peptides peptides by by linking linking the the CC-- or N-terminus-terminus to to a a side side chain chain or or linking linking one one side side chai chainn to toanother another side side chain chain [118]. [118 Depending]. Depending on the on thedesired desired cyclization cyclization site, site,cyclic cyclic peptides peptides can be can arranged be arranged in several in several ways, ways,including including head-to-tail, head-to-tail, head- head-to-sideto-side chain, chain, tail-to-side tail-to-side chain, chain, and side and chain-to-side side chain-to-side chain chain(Figure (Figure 22) [119]. 22)[ 119].

Figure 22. DiDifferentﬀerent cyclizationcyclization arrangements: ( a) head-to-tail chain; ( b) head-to-side chain; (c) side chain-to-side chain; (d)) tail-to-sidetail-to-side chain.chain.

A distinct advantage of backbone cyclization over other cyclization methods is that cyclization is A distinct advantage of backbone cyclization over other cyclization methods is that cyclization performed between backbone atoms, leaving the side chains that are usually essential for biological is performed between backbone atoms, leaving the side chains that are usually essential for biological activity untouched [84]. activity untouched [84]. Cyclization of a peptide through the formation of an amide bond between the C- and N-terminus can prevent degradation of the peptide by exopeptidases [60]. Cyclization of a linear peptide can also be used to increase the peptides’ structural rigidity. This can increase metabolic stability by locking the peptide into a conformation that is less susceptible towards proteolytic enzymes (conformational constraints and/or selective molecular recognition). It can also be used to increase biological activity by locking the peptide into a more biologically active conformation [118,120,121]. Cyclization has been applied to the RGD (Arg-Gly-Asp) peptide sequence that can be used to target the αvβ3-integrin receptors, which are overexpressed on the surface of various tumor cells [33]. In cyclic RGD systems, the RGD peptide sequence is flanked by other amino acids to form a ring system that presents the RGD sequence in a specific conformation. Cyclic RGD systems are more potent, specific, and resistant to proteolysis than their linear analogues [122]. Consequently, cyclic RGD systems have been widely used in the development of various peptide-based PET radiopharmaceuticals [19,123].

3.6. Substitution of Amides with Sulfonamides Another strategy that has been applied to increase the metabolic stability of peptide-based drugs is to substitute one or more amide groups in the backbone of a peptide with sulfonamide groups (Figure 23). Incorporation of the sulfonamide moiety into drugs is well known, as exempliﬁed with the classical antibacterial sulfa drugs, and the sulfonamide group is inherently more stable than amides in mammalian systems [124]. Molecules 2020, 25, x 16 of 24 Molecules 2020, 25, x 16 of 24 Cyclization of a peptide through the formation of an amide bond between the C- and N-terminus can preventCyclization degradation of a peptide of the through peptide the by formation exopeptida of anses amide [60]. Cyclization bond between of a thelinear C- andpeptide N-terminus can also canbe usedprevent to increase degradation the peptides’ of the peptide structural by exopeptida rigidity. Thisses [60]. can Cyclizationincrease metabolic of a linear stability peptide by can locking also bethe used peptide to increase into a conformation the peptides’ that structural is less susceptiblerigidity. This towards can increase proteolytic metabolic enzymes stability (conformational by locking theconstraints peptide intoand/or a conformation selective molecular that is lessrecognition). susceptible It towardscan also beproteolytic used to increase enzymes biological (conformational activity constraintsby locking theand/or peptide selective into moleculara more biologically recognition). active It canconformation also be used [118,120,121]. to increase biological activity by lockingCyclization the peptide has been into applieda more biologicallyto the RGD active(Arg-Gly-Asp) conformation peptide [118,120,121]. sequence that can be used to targetCyclization the αvβ3-integrin has been receptors, applied whichto the areRGD overexpressed (Arg-Gly-Asp) on peptidethe surface sequence of vari ousthat tumor can be cells used [33]. to targetIn cyclic the RGDαvβ3-integrin systems, receptors, the RGD whichpeptide are sequence overexpressed is flanked on the by surface other aminoof vari ousacids tumor to form cells a [33].ring Insystem cyclic that RGD presents systems, the the RGD RGD sequence peptide insequence a specific is flankedconformation. by other Cyclic amino RGD acids systems to form are a morering systempotent, thatspecific, presents and resistantthe RGD tosequence proteolysis in a thanspec ifictheir conformation. linear analogues Cyclic [122]. RGD Consequently, systems are morecyclic potent,RGD systemsspecific, andhave resistant been widely to proteolysis used inthan th etheir development linear analogues of various [122]. Consequently,peptide-based cyclic PET RGDradiopharmaceuticals systems have been[19,123]. widely used in the development of various peptide-based PET radiopharmaceuticals [19,123]. 3.6. Substitution of Amides with Sulfonamides 3.6. Substitution of Amides with Sulfonamides Another strategy that has been applied to increase the metabolic stability of peptide-based drugs is toAnother substitute strategy one or that more has am beenide applied groups to in increase the backbone the metabolic of a peptide stability with of peptide-basedsulfonamide groups drugs is(Figure to substitute 23). Incorporation one or more of amtheide sulfonamide groups in moietythe backbone into drugs of a ispeptide well known, with sulfonamide as exemplified groups with (Figurethe classical 23). Incorporation antibacterial ofsulfa the drugs,sulfonamide and the moiety sulfonamide into drugs group is well is inherentlyknown, as exemplifiedmore stable withthan Moleculestheamides classical 2020in mammalian, 25antibacterial, 2314 systems sulfa [124].drugs, and the sulfonamide group is inherently more stable16 than of 24 amides in mammalian systems [124].

Figure 23. Structure of a regular peptides compared to sulfonamide analogue. Figure 23. StructureStructure of a regular peptides compared to sulfonamide analogue. Sulfonamide groups contain a tetrahedral achiral sulfur atom directly bound to two electronegativeSulfonamide oxygen groupsgroups atoms. contain contain These a tetrahedral a featurestetrahedral achiral with respect sulfurachiral atom tosulfur geometry directly atom bound and directly electronic to two bound electronegative environment to two oxygenelectronegativestrongly atoms. resemble These oxygen the features transitionatoms. with These respectstate features of to peptide geometry with bond respect and hydrolysis electronic to geometry environment (Figure and 24) electronic strongly[125]. Consequently, environment resemble the transitionstronglypeptides resemble statecontaining of peptide the sulfonamide transition bond hydrolysis statesubstitutions of (Figure peptide 24have) bond [125 been]. Consequently,hydrolysis investigated (Figure peptides as transition 24) containing [125]. state Consequently, sulfonamideisosteres for substitutionspeptidesthe use as containing protease have been inhibitorssulfonamide investigated [126]. substitutions as transition have state isosteresbeen investigated for the use as as transition protease inhibitors state isosteres [126]. for the use as protease inhibitors [126].

FigureFigure 24.24. ((A)) Peptide Peptide bond bond structure. structure. (B ()B Transition) Transition state state for for the the hydr hydrolysisolysis of the of thepeptide peptide bond. bond. (Figure(CC)) SulfonamideSulfonamide 24. (A) Peptide bondbond asbond as a a suggested suggested structure. transition transition(B) Transition state state isostere.state isostere. for the hydrolysis of the peptide bond. (C) Sulfonamide bond as a suggested transition state isostere. WhenWhen compared to to the the amide amide moiety, moiety, the the sulfonamide sulfonamide group group is a stronger is a stronger hydrogen hydrogen bond donor bond donor[127]When and [127 the]compared and sulfonamide the sulfonamide to the amideN-H is N-Hmoiety, more is moreacidic the sulfonamide acidicthan the than amide group the amide N-H, is a stronger N-H,but a butweaker hydrogen a weaker hydrogen bond hydrogen donor bond bond[127]acceptor and acceptor [127–129].the sulfonamide [127– Furthermore,129]. Furthermore, N-H is the more hydrogen the acidic hydrogen thanbond the accepting bond amide accepting N-H,character but character ofa weakerthe sulfonamide of thehydrogen sulfonamide moiety bond moietyacceptoris split isbetween [127–129]. split between two Furthermore, accepting two accepting sites the duehydrogen sites to duethe bondtwo to the sulfonamide accepting two sulfonamide character oxygens. oxygens. of These the sulfonamide factors These factorscan moietyimpact can impactisthe split native between the nativehydrogen two hydrogen accepting bonding bonding sitesnetwork due network toof the the of two thepeptide sulfonamide peptide and and disrupt disruptoxygens. the the Theseformation formation factors of of can secondarysecondary impact structures.thestructures. native InInhydrogen contrastcontrast tobondingto thethe relativelyrelatively network rigidrigid of amide amidthe epeptide peptidepeptide and bond,bond, disrupt thethe sulfonamidesulfonamide the formation bondbond ofisis moremoresecondary freelyfreely rotatablestructures.rotatable andand In thecontrastthecis cis––transtrans to theisomerism isomerism relatively is is rigid notnot observedobservedamide peptide [[127,130].127,130 bond,]. This the greatersulfonamide rotational bond freedomfreedom is more allows allowsfreely forrotatablefor thethe sulfonamidesulfonamide and the cis– trans oxygensoxygens isomerism toto assumeassume is not aa observed varietyvariety ofof [127,130]. positions,positions, This wherewhere greater oneone rotational oxygenoxygen occupiesoccupies freedom aaallows ciscis oror transfortrans the orientationorientation sulfonamide withwith oxygens respectrespect to toto assume thethe amideamide a variet N-H,N-H,y while whileof positions, thethe otherother where oxygenoxygen one isis oxygen inin neitherneither occupies aa ciscis nornor a cis transtrans or position.trans orientation This can with impede respect theformation to the amide of secondary N-H, while structures the other by oxygen preventing is in the neither proper a alignmentcis nor trans of hydrogen bonds [127]. These potential disruptions to secondary structure formation have been found to have a greater effect on α-helices and a lesser effect on β-sheets [127]. The replacement of one or more amide bonds along a peptide backbone with sulfonamides has been successfully applied to develop peptidosulfonamide peptide analogues that display increased stability towards proteases compared to their unmodified analogues while also maintaining satisfactory biological activity [127,128,131]. The most common method of applying this strategy is to identify the preferred protease cleavage sites on a peptide and substitute the amides at those locations with sulfonamides. However, it has also been found that the substitution of amides close to cleavage sites can also increase metabolic stability [131]. This may be due to an effect similar to that seen in N-methylation where the substitution of the native amide bond with a more flexible bond, in this case a sulfonamide, allows the peptide to take a conformation that prevents proteases accessing the cleavage site [88,90]. The synthesis of a peptide in which all amides in the sequence are substituted with sulfonamides would lead to a peptidosulfonamide oligomer. However, this approach is not wise as α-amino sulfonamides are prone to fragmentation, releasing SO2 [132]. This has been addressed by using β-aminosulfonamides, which are more stable than their α-amino analogues (Figure 25)[127]. Molecules 2020, 25, x 17 of 24 position. This can impede the formation of secondary structures by preventing the proper alignment of hydrogen bonds [127]. These potential disruptions to secondary structure formation have been found to have a greater effect on α-helices and a lesser effect on β-sheets [127]. The replacement of one or more amide bonds along a peptide backbone with sulfonamides has been successfully applied to develop peptidosulfonamide peptide analogues that display increased stability towards proteases compared to their unmodified analogues while also maintaining satisfactory biological activity [127,128,131]. The most common method of applying this strategy is to identify the preferred protease cleavage sites on a peptide and substitute the amides at those locations with sulfonamides. However, it has also been found that the substitution of amides close to cleavage sites can also increase metabolic stability [131]. This may be due to an effect similar to that seen in N-methylation where the substitution of the native amide bond with a more flexible bond, in this case a sulfonamide, allows the peptide to take a conformation that prevents proteases accessing the cleavage site [88,90]. The synthesis of a peptide in which all amides in the sequence are substituted with sulfonamides would lead to a peptidosulfonamide oligomer. However, this approach is not wise as α-amino sulfonamides are prone to fragmentation, releasing SO [132]. This has been addressed by using sulfonamidesMolecules 2020, 25 ,are 2314 prone to fragmentation, releasing SO2 [132]. This has been addressed by 17using of 24 β-aminosulfonamides, which are more stable than their α-amino analogues (Figure 25) [127].

Figure 25.25.(a ) Structure(a) Structure of α-peptidosulfonamide- of α-peptidosulfonamide-α-peptide hybrid.α-peptide (b) Structure hybrid. of β(-aminosulfonamide-b) Structure of βα-aminosulfonamide--peptide hybrid. α-peptide hybrid.

The substitution of the amide moiety with sulf sulfonamidesonamides is starting to be explored in the development of peptide-based radiopharmaceuticals, including including for for linking linking of of the peptide to the N targeting moiety. For example, common amine-reactiveamine-reactive prosthetic groups such as N-succinimidyl-succinimidyl 18 18 18 18 4-[18F]fluorobenzoateF]fluorobenzoate ([ ([18F]SFB)F]SFB) and and 4-[ 4-[18F]fluorobenzoicF]fluorobenzoic acid acid ([ ([18F]FBA)F]FBA) are are used used to to label peptides through the formation of amide bonds with primary amine residues (e.g., N-terminus-terminus or or lysine) lysine) present in the the peptide peptide backbone backbone [133,134]. [133,134]. While While this this method method of oflabeling labeling peptides peptides has has proven proven to be to convenient,be convenient, the thesusceptibility susceptibility of ofthe the resulting resulting amide amide bonds bonds to to hydrolysis hydrolysis inin vivo vivo isis a potential vulnerability [36,135]. [36,135]. Löser Löser et etal. al.sought sought to explore to explore this by this comparing by comparing the metabolic the metabolic stability stability of the fluorinatedof the fluorinated amide, amide,N-(4-fluorophenyl)-fluoroacetanilide,N-(4-fluorophenyl)-fluoroacetanilide, and the and fluorinated the fluorinated sulfonamide, sulfonamide, N-(4- fluorophenyl)-3-fluoropropane-1-sulfonamideN-(4-fluorophenyl)-3-fluoropropane-1-sulfonamide (Figure (Figure 26) 26[36].)[36 The]. The metabolic metabolic stability stability of of both compounds were tested, and after 120 min of incubati incubationon in pig liver esterase (the porcine homologue of carboxylesterase),carboxylesterase), 95%95% of of the theN -(4-fluorophenyl)-3-fluoropropane-1-sulfonamideN-(4-fluorophenyl)-3-fluoropropane-1-sulfonamide compared compared to only to only20% of20%N-(4-fluorophenyl)-fluoroacetanilide of N-(4-fluorophenyl)-fluoroacetanilide remained remained intact intact [36]. While [36]. While the compounds the compounds in this in study this werestudy not were complete not complete structural structural analogues analogues of each of other, each this other, research this research provides provides evidence evidence of the potential of the potentialbenefits of benefits substituting of substituting amide for amide sulfonamide for sulfonamide bonds in radiopharmaceuticals.bonds in radiopharmaceuticals.

Figure 26. StructuresStructures of of ( (aa)) NN-(4-fluorophenyl)-fluoroacetanilide-(4-fluorophenyl)-fluoroacetanilide and and ( (bb)) N-(4-fluorophenyl)-3--(4-fluorophenyl)-3- fluoropropane-1-sulfonamidefluoropropane-1-sulfonamide [[36].36].

4. Conclusions

The success of peptide-based PET radiopharmaceuticals, such as NETSPOT®, has sparked renewed interest in the development of new PET radiolabeled peptides for targeting diseases in the body. The applicability of new peptide-based radiopharmaceuticals will be influenced to a large extent by their in vivo stability as the inherently poor in vivo stability of natural peptides is one of the biggest challenges in the development of peptide-based radiopharmaceuticals, especially as degradation of the peptide can lead to non-specific binding. There have been several strategies developed to avoid this by modifying natural peptides to enhance their metabolic stability and sometimes other pharmacological properties such as receptor affinity. Effective strategies have included modification of the C- and/or N-termini, introduction of D- or other unnatural amino acids, backbone modification, PEGylation and alkyl chain incorporation, cyclization and peptide bond substitution. It has also been found that by applying more than one of these modifications in tandem on the same peptide, the different modifications can often work in concert to further enhance metabolic stability. However, no one approach fits all peptides and the decision of which strategy to apply must be made on a case-by-case basis. Consequently, it is rare to find individual studies where several different strategies have been applied to the same peptide to compare their efficacies. While some of the examples discussed in this Molecules 2020, 25, 2314 18 of 24 review have only been applied to SPECT radiopharmaceuticals, radiotherapeutics, or non-radioactive peptide-based pharmaceuticals, the strategies could still be applied to PET radiopharmaceuticals. This could be achieved through simple substitution of a SPECT radiometal with a PET radiometal or by using the modification in the linker between a peptide and the radionuclide bearing moiety in a PET radiopharmaceutical. With the use of the discussed strategies to ensure in vivo stability, the number of successful peptide-based PET radiopharmaceuticals will continue to grow, and their clinical use will continue to expand.

Author Contributions: Conceptualization, B.J.E., A.T.K., A.K., L.M., and J.F.J.; writing—original draft preparation, B.J.E. and A.T.K.; writing—review and editing, B.J.E., A.T.K., A.K., L.M., and J.F.J.; supervision, L.M., A.K., and J.F.J.; project administration, J.F.J. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Macquarie University, including Macquarie University research training scholarships for B.J.E. and A.T.K. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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