Peptide-Drug Conjugate: A Novel Drug Design

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REVIEW ARTICLE

Peptide-Drug Conjugate: A Novel Drug Design Approach Liang Maa, Chao Wanga,b, Zihao Hea, Biao Chengc, Ling Zhengd and Kun Huanga,b,* a

Tongji School of Pharmacy, Huazhong University of Science and Technology, Wuhan, China; bCenter for Biomedicine Research, Wuhan Institute of Biotechnology, Wuhan, China; cThe Central Hospital of Wuhan, Tongji Medical College; Huazhong University of Science and Technology; d Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan University, Wuhan, China Abstract: More than 100 years ago, German physician Paul Ehrlich first proposed the concept

ARTICLE HISTORY Received: August 29, 2016 Revised: March 15, 2017 Accepted: March 29, 2017 DOI: 10.2174/0929867324666170404142840

of selectively delivering “magic bullets” to tumors through targeting agents. The targeting therapy with antibody-drug conjugates (ADCs) and peptide-drug conjugate (PDCs), which are usually composed of monoclonal antibodies or peptides, toxic payloads and cleavage/noncleavage linkers, has been extensively studied for decades. The conjugates enable selective delivery of cytotoxic payloads to target cells, which results in improved efficacy, reduced systemic toxicity and improved pharmacokinetics (PK)/pharmacodynamics (PD) compared with traditional chemotherapy. PDC and ADC share similar concept, but with vastly different structures and properties. Humanized antibodies introduce high specificity and prolonged half-life, while small molecule weight peptides exhibit higher drug loading and enhanced tissue penetration capacity, and the flexible linear or cyclic peptides are also modified more easily. In this review, the principles of design, synthesis approaches and the latest advances of PDCs are summarized.

Keywords: Peptide-drug conjugates (PDCs), antibody-drug conjugates (ADCs), peptide, linker, payloads, drug design. 1. INTRODUCTION Nowadays, clinical drugs are usually divided into two categories: chemical drugs and biological drugs. Chemical drugs make up the majority of the therapeutic market today. On one hand, the small molecular weight makes chemical drugs cross cell membranes relatively easy and biologically stable when taken orally, with cost-effective as another advantage [1]. On the other hand, these small-molecular drugs continue to be plagued with the problems of non-specific toxicity, narrow therapeutic windows and increasing resistance rates [2]. Biological drugs, such as proteins, enzymes, nucleic acids, hormones, cytokines and monoclonal antibodies (mAbs), usually have high efficacy and exhibit strong specificity to certain tissues/cells [3-7]. *Address correspondence to this author at the Tongji School of Pharmacy, Huazhong University of Science and Technology, Wuhan, China; Center for Biomedicine Research, Wuhan Institute of Biotechnology, Wuhan, China; E-mail: [email protected]

0929-8673/17 $58.00+.00

In 1900, German physician Paul Ehrlich proposed the concept of selectively delivering “magic bullets” to tumors through a targeting agent [8]. Based on this concept, during the last decades, antibody-drug conjugates (ADCs) and peptide-drug conjugate (PDCs) have been extensively studied as new targeting therapies. The conjugates are usually composed of monoclonal antibodies or peptides, toxic payloads and cleavage/noncleavage linkers (Fig. 1) in order to take advantages of both cytotoxicity payloads and highly specific monoclonal antibodies/ligand peptides, while reducing adverse effects. PDC and ADC share similar concept, but their structure and properties are vastly different. ADCs are mainly composed of IG1, IG2 and IG4 antibodies, with a standard Y-shaped antibody structure and large molecule weight (usually more than 100 kDa). Apart from high specificity to corresponding antigens, prolonged blood circulation time and reduced renal clearance permit ADCs to be administered less frequently than conventional chemotherapeutics [9]. Early mAbs in © 2017 Bentham Science Publishers

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Fig. (1). Basic structures of PDCs.

ADCs were based on murine or chimeric antibodies that were modified to target human antigens, which were highly immunogenic and rapidly cleared from circulation [10]. With the evolution of antibody engineering, the latest generations of ADC predominantly use either humanized or fully human ADCs with the minimized immunogenicity [11].

agnostic radiology) in the market, with more PDCs under development (Table 1). Here, we summarize the peptides, payloads and linkers used in PDCs. The essential factors during the design, synthesis and applications of PDCs are also reviewed.

Compared with ADCs, PDCs are developed recently with relatively slow paces. PDCs are usually made up by linear or cyclic peptides, with a flexible structure and low molecule weight (usually only a few kDa) [12]. Although the targeting specificity and stability of PDCs may be not as good as ADCs, PDCs still possess their own advantages. For examples, lower molecule weight leads to higher drug loading and enhanced penetration into solid tissues [13-14]. PDCs may be more flexible in structure due to their short peptide nature, making modification and conjugation such as introducing non-natural amino acids and forming cyclic peptides much easier, which improve the targeting property and circular stability of PDCs [14]. In addition to cytotoxic payloads often seen in ADCs, some payloads in PDCs may act as the “driving agent” and exhibit targeting efficacy rather than toxicity, with details discussed in this review.

The peptides used in PDCs can generally be divided into two categories: cell penetrating peptides (CPPs) and cell targeting peptides (CTPs) (Tables 2 & 3). CPPs are able to transport drugs across cell membrane, whereas CTPs may specifically bind to receptors on the target cells. Since the peptide part of PDCs is usually degradable by enzymes in the digestive tract, PDCs are often administered through parenteral routes, through which PDCs drugs are transported via circulation system and then pass through the capillary wall to reach the target cells. For CPP-drug conjugates, the transmembrane transport is regarded as an energyindependent cellular process, some studies suggest that CPP-drug conjugates can directly cross the lipid bilayer [18], whereas endocytosis- or receptor-mediated energy-independent non-endocytic translocation pathway have also been reported (Fig. 2) [19]. On the other hand, for CTP-drug conjugates, the trans-membrane transport initiates on the binding of peptides with their membrane receptors that mediate the endocytosis of PDCs, in which the conjugate may travel through early and late endosomes and finally into the endolysosome (Fig. 2) [20-21]; in the end, the receptor is recycled to the surface of cell membrane [22].

With the fast technical developments lately, such as proteomics, systematic phage display and solid-phase peptide synthesis, more novel peptides have been identified or rationally designed, which greatly accelerate the development of PDCs [15-17]. Presently, there is only one PDC (111In-DTPA-D-Phe-1-octreotide for di-

2. PEPTIDES IN PDCs

Peptide-Drug Conjugate

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Table 1. The development phase of some PDCs. Name

Peptide

Molecule

Linker

Target

Indications

Development Phase

111In-DTPA-DPhe-1-octreotide

D-Phe-1octreotide

111In-DTPA

Amido bond

Somatostatin receptor 2

Diagnostic Radiology

Clinical

[195]

NGR-hTNF

NGR

hTNFα

Amido bond

Vesselsexpressingaminopeptidase N (CD13)

Elapsed ovarian cancer

Phase 2

[196]

Mipsagargin (G202)

β-Asp-Glu-γGlu-γ-Glu-γ-GluOH

8-O-(12aminododecanoyl)-8-Odebutanoyl thapsigargin

Amido bond

Prostate-specific membrane antigen (PSMA)

Advanced Adult Hepatocellular Carcinoma

Phase 2

[197]

CT2103

Poliglumex

Paclitaxel

Ester bond

Enriched in tumor for the lack of lymphatic drainage

Metastatic breast cancer

Phase 2

[198]

EC-145

Asp-Arg-AspAsp-Cys

Desacetylvinblastinemonohydrazide,

Folate receptor

Non Small Cell Lung Cancer

Phase 2

[199]

folic acid

Amido bond Disulfide bond

Ref

GRN1005

Angiopep-2

Paclitaxel

Ester bond

Low-density lipoprotein receptorrelated protein-1

Recurrent malignant glioma

Phase 1

[200]

EP-100

LHRH

CLIP71

Amido bond

LHRH receptor

LHRH-receptorexpressing solid tumors

Phase 1

[66]

AEZS-108(AN152)

[D-Lys6]LHRH

Doxorubicin

Ester bond

LHRH receptor

Urothelial Carcinoma

Phase 1

[65]

Asp-Asp-AspβDpr-Cys

Mitomycin-C, desacetyl vinblastine hydrazide,

Disulfide bond，Ami do bond

DNA

Gastrointestinal cancer

Phage 1

[201]

EC0225

folic acid DOTA-GABABBN

Bombesin(7-14)

DOTA

gaminobutyric acid

Gastrin-releasing peptide receptor

Diagnostic Radiology

Preclinical

[202]

MTX-YTA2

AcetylYTAIAWVKAFI RKLRK-amide

Methotrexate

Amido bond

Dihydrofolate reductase

Breast cancer

Cytotoxic T lymphocyte test

[169]

PTX-Tau

AcetylCGVQIVYKK

Paclitaxel

buSS

Tubulin

Ovarian cancer etc


[164]

CPT-Tau

AcCGVQIVYKK

Camptothecin

buSS

TopoisomeraseⅠ

Gastrointestinal cancer


[174]

C34-Chol

WMEWDREINNYTSLIHSLIEESQNQQEKNEQELL

Cholesterol

Gly-SerGly-Cys

Caveolin 1

HIV-1


[203]

2.1. Cell Penetrating Peptides (CPPs) Cell penetrating peptides are short peptides (up to 30 residues) that are able to cross cell membrane. Two common features of CPPs are positive charges and amphipathicity [23]. CPPs can be divided into three classes: protein derived CPPs, modified CPPs and designed CPPs [24]. Protein derived CPPs usually consist

of a minimal effective sequence of the parent translocation protein, and are also known as protein transduction domains or membrane translocation sequences [25]. Modified CPPs contain sequences mimicking the structures of known CPPs from different origin. Designed CPPs are usually chimeric peptides composed of a hydrophilic and a hydrophobic domain to form amphipathic helical structures [26].


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Table 2. Cell-penetration peptides used in PDCs.

Designed

Modified

Protein derived

Cell-penetrating Peptides

Sequence

Origin

Ref

TAT (48–60)

GRKKRRQRRRPPQ

Derived from naturally occurring proteins from HIV-TAT

[29]

Penetratin

RQIKIWFQNRRMKWKK-NH2

Derived from homeodomain transcription factor antennapaedia

[204]

Transportan

GWTLNSAGYLLGKINLKALAALAKKIL-NH2

Consisted of galanin at the N-terminus and mastoparan at the C-terminus

[45]

Transportan 10

AGYLLGKINLKALAALAKKIL-NH2

Deletion of 6 amino acids from the Nterminus of transportan

[45]

PepFect14

Stearyl-AGYLLGKLLOOLAAAALOOLL-NH2

Utilized ornithines and leucines instead of lysines and isoleucines of transportan 10

[205]

Polyarginine

Rn (n = 6–12)

Arginines are highly cationic, they strongly adsorb on the membrane surfaces via hydrogen bond-induced formations of the guanidino moieties in arginine with anionic phosphates, sulfates, and carboxylates of cellular components

[206]

Pep-1

KETWWETWWTEWSQPKKKRKV

Consists of three domains, a hydrophobic tryptophan-rich domain, a hydrophilic lysine-rich domain and a spacer domain

[47]

Table 3. Cell-targeting peptides used in PDCs. Cell Targeting Peptides

Sequence

Targets

Ref

Bombesin Receptors Bombesin Analog peptides

pEQRLGNQWAVGHLM-NH2

Overexpressed on various kinds of cancer cells. BN receptor Type 2 (also known as gastrinreleasing peptide receptor) is upregulated in breast, prostate, small cell lung, and pancreatic cancers.

[60]

GnRH Receptors GnRH (LHRH) Analog peptides

pEHWSYGLRPG-NH2

Receptors of LHRH are particularly overexpressed in cancer cells, related to the reproductive system, like prostate, breast, and ovarian cancer.

[63]

Somatostatin Receptors Somatostatin Analog Peptides

RGD Peptides

AGCKNFFWKTFTSC

The peptide somatostatin binds to five different receptor subtypes. The somatostatin receptor 2 and somatostatin receptor 5 are overexpressed in various kinds of cancers, like breast, lung, and neuroendocrine tumors.

RGD

αv integrins, Neuropilin-1

c(RGDfK)

αvintegrins are specifically expressed on the endothelium of tumor vessels. Neuropilin-1 is over-expressed on both angiogenic blood vessels and tumor cells

iRGD

[69]

[74]

Membrane-bound aminopeptidase P PEGA

CPGPEGAGC

The PEGA peptide accumulates in breast vasculature as well as in premalignant breast tissue and primary breast tumors.

Stearyl, Octadecanoyl; Ac,Acetyl; cya,Cyclosporin A; pE, Pyroglutamic acid; f, D-Phenylalanine

[84]



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Fig. (2). Mechanismes of PDCs transmembrane transport. Schematic of three types mechanism of PDCs transmembrane transport and intracellular cleavage. Targeting peptide/targeting small molecule of the PDCs bind to the membrane receptor and mediate endocytosis of the PDCs(target peptide may also be able to cause a change in downstream cell pathways), cell-penetrating peptides may translocate directly through the lipid bilayer enter the cell. The PDCs move to the cellular compartments with progressively higher enzyme concentration and lower acidity then the peptide and the small molecule drug are released into cytoplasm, nucleus or other organelles.

2.1.1. Transactivated-transcription (TAT) Protein TAT protein is an 86-residue transcription activating factor derived from the HIV-1 transactivatedtranscription domain [24]. In 1988, two groups independently found that extracellular TAT protein could transactivate the viral promoter into cytoplasm [27-28]. When co-incubating with cells, TAT is internalized and relocated to the nuclei where it transactivates the viral promoter [29-30]. TAT has been used to transport various agents into cells, for example, covalent attachment of cyclic TAT (cTAT) peptide to green fluorescent protein (GFP), delivering GFP into live cells with immediate cytosolic and nuclear availability [31]. TAT contains an N-terminal acidic region involved in transactivation, a cysteine rich region which is im-

portant for metal-linked dimerization in vivo and a highly basic region involved in nuclear and nucleolus localization and RNA binding [32]. Some TAT fragments, such as TAT(48-60) and TAT(47-57), also have the capacity to cross membrane [33]. Researchers conjugated small molecule payloads doxorubicin (DOX) or paclitaxel (PTX) with TAT(47-57); the IC50 of the resulted Dox-TAT/folate and PTX-TAT/folate micelles on KB cells were 0.172 µM and 0.043 µM, respectively, which was an order of magnitude lower than those of the micelles loaded with the drug only (3.873 µM for Dox-micelles and 0.790 µM for PTX-micelles) [34]. According to a recent report, the basic domain (49– 57) of TAT is essential for its cell internalization [35];


any deletion within this region reduces membrane translocation. Arthanari et al. conjugated siRNA with TAT(49-57) and a membrane lytic peptide LK15 to form a siRNA-TAT-LK15 conjugate. The conjugate showed higher efficacy and lower cytotoxicity than siRNA in K562 cell, which is difficult for transfection [36]. 2.1.2. Transportan Galanin is a 30-residue neuropeptide widely expressed in the brain, spinal cord, and gut [37]. Galanin receptors are expressed in the central nervous system, pancreas, and also in solid tumors [38]. Mastoparan, a peptide toxin from wasp venom, can interact with membranes as an amphipathic helix structure [39]. Transportan is a modified CPP with 27 residues, which includes 12 residues from galanin at its Nterminus and 14 residues from mastoparan at its Cterminus, connected with a lysine that helps labeling and cargo attachment [40]. The N-terminal 1-13 fragment of transportan is the smallest galanin receptor agonist and the C-terminal 14-27 fragment shows inhibitory effect on GTPase activity derived from mastoparan [41]. Transportan can receptor-independently cross the plasma membrane [42]. It has been used to transport siRNA into CHO cells, which decreased the luciferase levels by 63% within 24 h and remained stable for up to 3 days, which was significantly higher and longer-lasting compared to that delivered by lipofectamine [43]. Since the affinity for galanin receptor and the interaction with G-proteins could be a drawback for an ideal carrier peptide, some truncated derivatives of transportan have been synthesized [44-45]. Transportan 10, which deletes 6 residues from the N-terminus of Transportan, retained cell membrane penetration ability but lost the inhibitory capacity on GTPase [46]. Researchers conjugated Transportan 10 to a plasmid via a peptide nucleic acid (PNA) oligomer to generate a Transportan10-PNA conjugate, which showed an enhanced plasmid delivery capacity for up to 3.7-fold of that with standard protocol in Neuro-2a cells [37]. 2.1.3. Pep-1 Pep-1 is a designed CPP with 21 residues that consists of three domains: a hydrophobic tryptophan-rich domain (KETWWETWWTEW), which is attached to the cell membrane and hydrophobic/electronic interaction with other peptides or proteins; a hydrophilic lysine-rich domain (KKKRKV) derived from simian virus 40(SV-40) large T antigen, which improves the in-

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tracellular delivery and solubility of the peptide; and a proline-containing spacer that improves the flexibility and integrity of above two domains [47]. Pep-1 is acetylated at N-terminus and has a cysteamide group at C-terminus, both groups are essential for the stability and transduction of Pep-1 [48]. Previous researches have shown that Pep-1 strongly interacts with membrane lipids through its tryptophan-rich domain and then forms α−helical structure, thereby facilitating insertion into the membrane and initiation of the translocation process, the cysteamide group at the Cterminus is essential both for stabilization of the peptide and its interaction with lipids [49]. Moreover, Pep1 has been shown to be able to deliver multiple peptides or proteins into different cell lines [50]. Researchers conjugated Pep-1 with human epidermal growth factor (hEGF), and found dramatically increased transmembrane ability of the Pep-1-hEGF complex in COS-7 cells[51]. Moreover, without chemical covalent coupling, Pep-1 alone facilitated the transportation of green fluorescent protein (GFP) and β-galactosidase (βGal) into HS-68 cells and Cos-7 cells at concentrations one or two orders of magnitude lower [47]. 2.1.4. Other Cell Penetrating Peptides Many additional cell penetrating peptides have been studied to transport drugs into cell membrane, such as Herpes simplex virus type 1 (HSV-1) protein VP22, which is a 35 kDa inter-layer protein composed of 301 residues [52]. Elliott et al. showed that peptides and proteins of up to 27 kDa can be delivered to cells in the form of a VP22 fusion protein [53]. Haptides, a class of 19–21 residue peptides homologous to fibrinogen Cterminals, significantly enhanced doxorubicin (DOX) containing liposomes uptake by fibroblasts and endothelial cells [54]. SynB peptide, which is derived from the antimicrobial peptide protegrin-1(PG-1), was used to transport DOX through cellular membranes of the blood-brain barrier [55-56]. Sakuma et al. found that co-administration of insulin with L-octaarginine-linked GE-167 resulted in a 1.7-fold increase in the glucose reduction of experimental mice [57]. CPPs such as TAT provide an effective pathway for cell-impermeable compounds to reach intracellular targets (Fig. 3). However, low cell specificity is a major drawback of CPPs-mediated delivery, which hampers its extensive application [24]. Thus, cell targeting peptides make up to this defect. 2.2. Cell Targeting Peptides Peptides with specific binding activity to specific cells or tissues have been extensively reported. These



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Fig. (3). Structure of methotrexate. The molecular structure of methotrexate and folic acid are similar, so methotrexate can compete with folic acid in binding dihydrofolate reductase. The pterin portion of methotrexate, which has high affinity to dihydrofolate reductase, is the active site for its antimetabolite activity, any modification on pterin portion decreases the drug activity, whereas modification on the glutamic residue exhibits little influence on dihydrofolate reductase activity.

peptides (known as cell targeting peptides, CTPs) have the capacity to interact with receptors that are exclusively over-expressed by certain cells (Fig. 4). When therapeutic agents are conjugated with CTPs, they can be transported and enriched in target sites, which greatly reduce the side effects of these agents [58]. 2.2.1. Bombesin Analogs Bombesin (BN) is a 14-residue peptide originally isolated from the skin of the European fire-bellied toad (Bombinabombina). BN has two known homologs in mammals, neuromedin B and gastrin-releasing peptide [59]. Like gastrin-releasing peptide, BN can stimulate gastrin release from G cells. The level of BN receptor Type 2 (also known as gastrin-releasing peptide receptor 2, GRPR-2) is upregulated in the breast, prostate, small cell lung, and pancreatic cancers [60]. Therefore, BN analogs have been conjugated to different drugs for targeting cancer cells. In 2015, a BN analog (KGGCDFQWAV-βAla -HFNIe) was covalently connected with gold nanorods (GNR) and PEG [61]. It was found that the GNR-BNPEG conjugate was significantly accumulated in tumor tissues; the in vivo photo-thermal therapy showed a ca. 99% decrease in the average tumor growth for the GNR-BN-PEG treated group, which is much higher than that of the GNR-PEG group (ca. 50% decrease) [61]. Furthermore, some derivatives from the Cterminal sequence of bombesin, such as BN(7-14), BN receptor antagonist (RC-3095) and agonist (RC-3094), have been conjugated with small molecules. Nagy et al. conjugated RC3095 and RC-3094 with DOX and 2pyrrolino-DOX via a glutaric acid spacer. Both conjugates exhibited higher anticancer activity for cancer cells expressing BN receptors, including CFPAC-1

human pancreatic cancer cells, DMS-53 human lung cancer cells, PC-3 human prostate cancer cells and MKN-45 human gastric cancer cell lines, the IC50 of BN analog-DOX conjugates were 2-3 fold lower than that of DOX alone in these cancer cell lines [62]. 2.2.2. Luteinizing Hormone-releasing (LHRH) Analog Peptides

Hormone

Luteinizing hormone-releasing hormone (LHRH), also known as gonadotropin-releasing hormone (GnRH), is a trophic peptide hormone responsible for the release of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from the anterior pituitary. Receptors of LHRH are particularly overexpressed in cancer cells related to the reproductive system, such as prostate, breast, and ovarian cancer [63]. Dharap et al. used PEG as a linker to connect LHRH and camptothecin (CPT). A toxicity test performed on A2780 human ovarian carcinoma cells suggested the IC50 of the conjugate (CPT-PEG-LHRH) was decreased from nanomolar (for CPT and CPTPEG) to picomolar range (for CPT-PEG-LHRH conjugate), while the intake of the conjugate by A2780 cells was much higher than that of CPT and CPT-PEG [64]. AEZS-108, which is composed of [D-Lys6]LHRH and doxorubicin, has come to Clinical Phase I to treat castration- and taxane-resistant prostate cancer [65]; while another compound EP-100, which was generated by conjugating LHRH with a cationic membranedisrupting peptide CLIP71, has come to Clinical Phase I to treat solid tumors expressing LHRH-receptor [66]. 2.2.3. Somatostatin Analog Peptides Somatostatin (also known as growth hormone- inhibiting hormone, GHIH) is a peptide hormone that


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Fig. (4). The synthesis process of CPT-mal -Tau /CPT-buss-Tau. a. hydroxyl of CPT react with the carboxyl of 4-(pyridin-2yl-disulfanyl)butyrate（buSS）generate the ester bond under the catalysis of 4-dimethylaminopyridine(DMAP) and N,N'dicyclohexylcarbodiimide(DIC). Pure intermediates CPT-buSS is obtained by rapid silica gel chromatography. Then CPTbuSS and Tau peptide are dissolved in anaerobic and anhydrous DMF solvent, CPT-buSS-Tau is subsequently purified by reversed-phase high-performance liguidchromatography(RP-HPLC). b. Synthesis of CPT-mal is prepared via coupling CPT with 3-maleimidopropionic acid, the reaction conditions are the same as CPT-buSS. Michael addition of cysteine containing Tau peptide with the CPT-mal results in formation of the CPT-mal-Tau.

regulates the endocrine system and affects neurotransmission and cell proliferation via interacting with G protein-coupled somatostatin receptors, then inhibiting the release of secondary hormones [67]. Somatostatin binds to five different receptor subtypes, among which somatostatin receptor 2 and somatostatin receptor 5 are

overexpressed in varied cancers, including breast, lung, and neuroendocrine tumors [68]. Most peptide–drug conjugates from the somatostatin peptide family are octreotide analogs. Octreotide (fCFwKTCT, capital letters refer to L-amino acids, lower-cases refer to D-amino acids) is an octapeptide


that mimics natural somatostatin pharmacologically [69]. Huang et al. conjugated octreotide with paclitaxel through a succinic acid spacer [70]. The conjugate exhibited comparable toxicity with free paclitaxel on somatostatin receptor 2 and 5 expressing MCF-7 breast cancer cells. In contrast, no toxic effect was observed on somatostatin receptor-negative Chinese hamster ovary (CHO) cells for the conjugate, whereas paclitaxel alone exerted similar toxicities on both cell lines [70]. AN-162 (AEZS-124), consisting of doxorubicin linked through glutaric acid to the somatostatin octapeptide RC-121, is susceptible to hydrolysis by serum carboxylesterase enzymes, AN-162 significantly inhibited tumor growth compared with the control groups receiving mannitol or doxorubicin only [71]. 2.2.4. Arginine-glycine-Aspartic Acid (RGD) Peptides Arginine-glycine-aspartic acid (RGD) has become a popular tool for drug targeting since 1990s. A mass of studies have been made on transporting small molecules, peptides, proteins, nucleic acids and siRNAs with RGD peptides [72-73]. This tripeptide homes to tumor by binding to αv integrin, which is specifically expressed on the endothelium of tumor vessels [74]. It was further demonstrated that the constrained analogs, embedding the sequence RGD, have an even higher affinity for integrins. Therefore, the cyclic c(RGDfK), RGD4C and RGD10 are frequently used for generation of peptide–drug conjugates [75-78]. Recently, internalizing RGD (iRGD) has become a hotspot in tumor targeting and penetrating. The sequence of iRGD is CRGDK/RGPD/EC, in which the Cys between the C- and N-terminus are connected through a disulfide bond [79]. iRGD targets tumors by binding to αv integrins, after which iRGD is proteolytically cleaved to produce CRGDK/R. The truncated peptide loses much of the integrin-binding activity, but gains affinity for neuropilin-1 (NRP-1). The NRP-1 binding is also considered as tumor specific because the cleavage requires prior binding of the peptide to integrins [80]. iRGD has the capacity to increase the vascular and tissue permeability in a tumor-specific and NRP-1-dependent manner, making it easier for conjugated drugs to penetrate into tumor tissues [81]. Researchers conjugated iRGD and an amphipathic Damino acid-modified apoptotic peptide KLA to form a m(KLA)-iRGD conjugate, and found the cytotoxicity of m(KLA)-iRGD is significantly higher in NRP+ MDAMB-231 cells than in NRP- B16 cells, and m(KLA)-iRGD treatment significantly decreases tumor growth and metastasis [82]. Interestingly, unlike other CTPs, iRGD may help cargoes accumulation/ penetra-


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tion in tumor tissues even without covalent coupling. Kazuki et al. found that co-administration drugs with iRGD in mouse tumor models enhanced the efficacy of anti-cancer drugs while reducing their side effects. A small molecule (doxorubicin), nanoparticles (nabpaclitaxel and doxorubicin liposomes), and a monoclonal antibody (trastuzumab) were systemically coadministrated with iRGD, respectively [81]. Results showed iRGD enhances the tumor accumulation and penetration of compounds with different sizes and chemical properties. And co-administrating with iRGD improved the therapeutic index of drugs of various compositions[83]. 2.2.5. Other Cell Targeting Peptides There are several additional cell-targeting peptides studied as peptide–drug conjugates. PEGA (CPGPEGAGC) is a cyclic nonapeptide, which is accumulated in breast vasculature as well as in premalignant breast tissue and primary breast tumors [79]. It is thought to bind to the membrane-bound aminopeptidase P (APaseP), which is expressed at high levels in breast tissue [84-85]. Myrberg et al. found that when chlorambucil was conjugated with the PEGA-pVEC chimeric peptide, the conjugate mainly accumulated in blood vessels of breast tumor tissues with anticancer efficacy increased for more than 4 times [84]. The vasoactive intestinal peptide (VIP) which binds to the VIP receptors (VIPRs), are overexpressed in many types of cancer, such as colon cancer, breast cancer and endocrine tumors [86]. Peptides derived from intercellular adhesion molecule-1 (ICAM-1) is a promising class of cell adhesion peptides with the potential of targeting drugs to leukocytes [87]. cIBR, an ICAM1-derived cyclic peptide was conjugated to MTX (methotrexate) to form MTX-cIBR, which effectively inhibited the progression of rheumatoid arthritis in a rat adjuvant model [88]. Self-assembling peptides (SAPs) are a category of peptides which undergo spontaneous assembling into ordered structures [89-90]. SAPs contain amphipathic peptides, cyclic peptides, and ionic-complementary peptides [91-94]. SAPs can form hydrogels as scaffolds for drug delivery, which help to improve the solubility, stability and controlled release of drugs (including small molecule drugs and peitide/protein drugs) [95]. Cheetham AG et al. conjugated camptothecin with a βsheet forming peptide derived from the Tau protein through a biodegradable linker to form CPT-buSS-Tau. They introduced branching points at the N-terminal, which allows access to drug amphiphiles with 1, 2 or 4


drug molecules per conjugate and the drug loadings were 23%, 31% and 38%, respectively [96]. 3. PAYLOADS IN PDCs Many functional small molecules have strong pharmacological activities but also with drawbacks like poor solubility, poor selectivity and short half-life, which lead to side effects and multiple drug resistance and limit their therapeutic applications [97]. Conjugation with peptides may help to solve these problems and improve the applications of functional small molecules [1, 98]. Many functional molecules can be conjugated with peptides to generate PDCs, among which cytotoxic payloads and targeting payloads are most commonly used. 3.1. Cytotoxic Payloads Cytotoxic payloads used in peptide–drug conjugates are often classical chemotherapy drugs, like paclitaxel (PTX), doxorubicin (Dox), camptothecin (CPT), etc. They are classified as cytotoxic payloads with antitumor mechanisms through interfering with or blocking cell proliferation process; on the other hand, their poor tumor-specific targeting ability lead to both tumor and normal cells damage without high selectivity. Peptidedrug conjugates may enrich cytotoxic payloadss in tumor tissues while reduce drug distribution in normal tissues, as a result, reduce side effects and suppress multiple drug resistance. 3.1.1. Paclitaxel Paclitaxel is a widely used cytotoxic agent in treating various carcinomas. It can specifically bind to the β-tubulin subunit of microtubules to arrest mitosis and cause programmed cell death [99]. The efficiency for human metastatic breast cancer of paclitaxel is 53%, it also shows beneficial effects for ovarian cancer, esophageal cancer and lung cancer [100]. However, paclitaxel induces a variety of adverse effects in clinical applications, including blood toxicity, allergic reactions and neurotoxicity [101]. Due to its poor aqueous solubility (0.55-0.59 µg/ml in water at 25°C), paclitaxel has to be dissolved in Cremophor EL, a vehicle that often elicits hypersensitivity side effects [102]. Moreover, paclitaxel is more likely to elicit multidrug resistance than other chemotherapeutic agents because of Pgpmediated efflux [103-105]. Several peptide-paclitaxel conjugates have been reported to improve the solubility and overcome the multidrug resistance of paclitaxel [106-107]. Elena et al. attached an octaarginine transporter to the C2’ positions of paclitaxel via a bio-

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cleavable disulfide linker to get octaarginine-paclitaxel transporter conjugate, in which they [108-109]. They tested the efficacy of this conjugate in eleven paclitaxel sensitive or resistant cell lines, the octaargininepaclitaxel displayed higher ability in overcoming drug resistance than paclitaxel itself in all tested cell lines, the IC50 of paclitaxel in paclitaxel sensitive and paclitaxel resistant OVCA 433 cells were 14- and 47-fold higher than that of the octaarginine-paclitaxel [109]. Treating mouse model of ovarian cancer with octaarginine-paclitaxel or the control paclitaxel at the same dosage (10 mg/kg), the conjugate resulted in 4.8-fold complete response than paclitaxel [109]. This conjugate was more effective than paclitaxel itself because conjugating with octaarginine improved the water solubility and suppressed systemic distribution. Moreover, the conjugate releases paclitaxel intracellularly at a sustained rate, thus avoiding bolus effects associated with administration and uptake of paclitaxel alone [110]. 3.1.2. Doxorubicin Doxorubicin (Dox), a topoisomerase II inhibitor, is widely used in treating a variety of cancers, particularly for liver cancer chemotherapy in the form of doxorubicin-loaded drug-eluting beads (DEB) in the clinic [111]. However, due to the intrinsic P-glycoprotein overexpression and multidrug resistance (MDR) development, it is difficult to achieve a therapeutic intracellular concentration of Dox [110, 112]. Andrew et al. designed a Tat-Dox conjugate to improve the cellular uptake and cytotoxicity against drug-resistant cancer cells. In Dox-resistant KB-V1 cells, the drug resistance was partially overcome as 86% of cells were killed after treating with 50 µM Tat-Dox conjugate, in contrast, only 14% of cells were killed after 50 µM Dox treatment [113]. 3.1.3. Other Cytotoxic Payloads Camptothecin(CPT) and methotrexate(MTX) are also frequently used in generating peptide-drug conjugates. Lee et al. connected an RGD cyclic peptide, a CPT and a fluorescent reporter to form "compound 1", which was found to be uptaken by U87 cells and CPT was released within the endoplasmic reticulum [114]. MTX was introduced at positions 4 and 22 of [F(7),P(34)]-NPY which is able to deliver toxic agents specifically to breast cancer cells that overexpress the human Y1-receptor (hY1R). The conjugate exhibited higher potency than MTX on MTX-resistant cells [115]. Cytotoxic payloads with appropriate conjugating site are all potential candidates for generating PDCs.


3.2. Targeting Payloads Targeting payloads mainly refer to small molecules with specific receptor on the surface of targeting cells. There are many applications of targeting payloads, such as Gefitinib (an epidermal growth factor receptor tyrosine kinase inhibitor) and Glivec (a tyrosine kinase 3 inhibitor) [116-117]. In addition to therapeutic uses as tumor inhibitors, some targeting payloads, such as folic acid and cholesterol, may also be conjugated to the functional peptides to enhance the effects of the peptides [118-120]. 3.2.1. Folate Folate receptor (FR) is a tumor-associated protein, which is significantly overexpressed in most ovarian and other gynecological cancer cells [119]. Folate receptor can specifically bind folic acid or folic analogs, the latter are then taken up into the cytoplasm through endocytosis. Folate has been used as a targeting ligand for delivering therapeutic and imaging agents selectively to FR overexpressing cancer cells and activated macrophages, which had been successfully exemplified at both preclinical and clinical levels [121-123]. Folate is widely used as a molecular targeting ligand with multiple advantages, such as low molecular weight (441.4 Da), high stability, non-immunogenic property and high affinity to FR [123]. Didemnin B is a cyclic depsipeptide considered to be a potent protein synthesis inhibitor [124]. It is the first marine biological compound that had entered phaseⅡclinical trials as an anti-cancer drug for the treatment of ovarian, breast, cervical and lung cancers [125-128]. Unfortunately, the trail of didemnin B was later failed largely due to dose limiting toxicities and poor water solubility [129]. Walter et al. designed a folate-didemnin B conjugate against inflammatory diseases. This conjugate consists of folate, didemnin B and a releasable disulfide carbonate linker. In vitro cytotoxicity of folate-didemnin B was evaluated using XTT assay on RAW264.7 cells (a macrophage-like cell line which expresses FR). The IC50 in cytotoxity and TNF-α inhibition of folate-didemnin B were 13 and 5 nM, respectively, one to two magnitude lower than that of unconjugated didemnin B, which was 692 nM and 562 nM, respectively [130]. 3.2.2. Cholesterol Cholesterol is an indispensable ingredient of animal cell membranes. Cholesterol and sphingolipids are often laterally segregated in membrane microdomains known as “lipid rafts” [131]. A mass of trans-


11

membrane proteins and receptors are particularly enriched in lipid rafts. Membrane proteins and receptors have important biological functions in receiving external signals, being carrier protein material transport and channel proteins, catalyzing various zymoproteins [132]. Conjugating functional peptides with cholesterol can help peptides anchoring on the cholesterol-enriched lipid rafts to interfere with the normal function of transmembrane proteins [120]. The composition of the lipid membrane of HIV differs from that of host cell membrane with particularly enriched cholesterol and sphingomyelin [132]. C34, a 34-residue peptide, is a HIV-1 heptad repeat 2 peptide fusion inhibitor, which has the same anti-AIDS mechanism with Enfuvirtide (the first generation of HIV-1 heptad repeat 2 peptide fusion inhibitor on the market) [133]. C34 binds to a critical intermediate of the HIVcell membranes fusion process to disrupt the fusion of HIV and cellular membranes [134]. Antomello et al. conjugated cholesterol with C34 through a tetrapeptide (Gly-Ser-Gly-Cys) linker. C34-Chol showed dramatically increased antiviral potency with IC90 values 15- to 300-fold lower than Enfuvirtide [120] . Caveolin-1 is a cholesterol-binding membrane protein. Cholesterol is enriched in caveolin-1 protein where the virus-cell fusion occurs [135-136]. Conjugating with cholesterol effectively helps C34 peptide anchoring on the caveolin-1 protein [137]. The C34-Chol showed dramatically increased antiviral potency on a panel of primary isolates and the IC50 value of C34Chol is 15-fold lower than that of enfuvirtide. C34Chol and controls were incubated with P4-2/R5 cells at 37℃, free peptides were then removed by washing, the washing only induce a 7-fold shift in the IC50 of C34Chol compared with a 373-fold shift for C34 alone, suggesting high affinity of C34-Chol to the membrane raft compartments [120, 122], making C34-Chol the most potent HIV fusion inhibitor to date. 3.2.3. Other Targeting Payloads Hyaluronic acid (HA) is an essential glycosaminoglycan in human body. HA can easily bind to CD44 receptor, which is overexpressed in many malignant tumor cells, with high affinity [138-139]. It is also extensively employed in tumor-targeted imaging and drug delivery. Yu et al. conjugated HA with Fe3 O4 nanoparticles (NPs) to get Fe3O4-HA NPs, the conjugate exhibited enhanced targeting delivery and cellular uptaking of Fe3O4 nanoparticles [140]. Phenylboronic acid can readily form stable borate with sialic acid, the overexpressing of which is often related to tumor develop-


ment and metastasis [141]. Biotin receptors are also overexpressed on cancer cell surfaces of a variety of cancer cell lines [142]. These three molecules have emerged as new targets for tumor-targeting drug delivery. 3.3. Other Payloads Many other payloads apart from cytotoxic payloads and targeting payloads have been applied in designing PDCs, such as siRNAs and antisense oligonucleotides. siRNAs (short interfering RNA) is a class of double-stranded RNA molecules with 20-25 base pairs in length. Once siRNA enters the cell, it is incorporated into other proteins to form RNA-induced silencing complex (RISC) that contains the RNA endonuclease Ago2 to cleave its target mRNA [143-145]. Both CPPs and CTPs have been conjugated with siRNAs to enhance the delivery and targeting of siRNAs. Chiu et al. conjugated siRNA targeted CDK9 with TAT47-57 to form TAT-siRNA conjugate. Immunohistochemistry revealed the specific knockdown of SOD1, Casp3 and Casp9 after TAT-siRNA conjugate treatment [146]. Shuai H et al. added a cRGD peptide to a methoxymodified EGFR siRNA, the cRGD-EGFR siRNA significantly inhibited tumor growth, reduced EGFR expression and down-regulated mRNA and protein levels of EGFR in tumor tissues [147]. Antisense oligonucleotides (AONs) are single strands of DNA or RNA that are complementary to a chosen sequence. They bind to the messenger RNA (mRNA) transcribed from the target gene and shut it down [148]. AONs are widely used to interfere with biological processes in cells and are being developed in some cases as drugs [149]. Suzan M. et al. recently reported that phosphorodiamidate oligomer internalizing peptide (Pip) effectively delivered single stranded antisense splice-switching oligonucleotides (SSOs) and at dosages an order-of-magnitude lower than that required by naked SSOs in a mouse model to alleviate spinal muscular atrophy [150]. 4. LINKERS IN PDCs Linker is the bridge that connects peptides with payloads. A good linker should not affect the functionality of either peptide or payload. Low molecular weight, appropriate length, suitable stability and polarity are key requirements of linkers. Here we divide linker into two types: non-cleavage linkers and cleavage linkers, the latter includes subtypes such as pH sensitive linker, enzyme sensitive linker and redox sensitive linker. (Table 4) Their pros and cons are summarized below.

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4.1. Non-cleavage Linker Non-cleavage linker is the simplest linker for conjugation. Carbon chain, amide bond and ether bond are all common forms of non-cleavage linkers. They are very stable, which reduce the non-preferred drug release in blood and thus attenuate the risk of potential side effects [151]. Meanwhile, the conjugated drug is flexible to achieve proper target-binding conformation [136]. Since the length and polarity of the chain are important for pharmacokinetics and pharmacological properties, the number of atoms and the side chain need to be adjusted to provide suitable length and polarity [152]. When a payload is conjugated to a carbon chain, the stability of the drug is usually increased in vivo with extended half-life [151]. On the other hand, the good flexibility of carbon also reduces the hindrance and facilitates the binding of drug payload to its target [88]. The amide bond and ether bond are highly stable under different pH and hydrolases [151-152]. They can be used alone or as portion of a long linker. For PDCs targeting tumor cells, the linker enables specific intracellular release of the conjugated toxic payloads. Therefore the linker should be stable in bloodstream and interstitial, but clearable after delivery. Estradiol has been implicated as a steroid hormone for the therapy of obesity and type 2 diabetes [153]. However, gynecological and tumor-promoting drawback limited its clinical application [154]. Conjugating estradiol with GLP-1 peptide through an amide-ether linker improved the stability and targeting delivery of estradiol. This amide-ether conjugate was stable for over 120 h, and also devoid of adverse estrogen tumorpromoting effect. The conjugate did not lead to uterine hypertrophy compared with saline-treated group at a maximal dose of 4,000 µg per kg body weight in ovariectomized (OVX) lean mice, while another group, GLP-1-estradiol conjugate with a labile phenolic ester bond, was unstable intracellularly and led to a 2.5-fold higher uterine weight compared to the amide-ether conjugate treated mice, suggesting non-cleavable linker is better than cleavable linker in this estradiol-GLP-1 conjugate [151]. 4.2. pH Sensitive Linker pH sensitive linker, which is designed to achieve controlled release of anti-cancer drugs, has a pHsensitive bond that can be rapidly hydrolyzed by acid. Since tumor cells grow and metabolize rapidly, tumor vessels are often unable to provide sufficient nutrients and oxygen. Anaerobic glycolysis inside tumor cells



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Table 4. Linkers used in PDCs. Linker Type

Name

Structure

Ref.

Carbon chain

n

R1

[207]

R2

O

Amido bond Non-cleavage

[154]

R2 R1

NH

O

Ether bond

R1

[151]

R2

R1

Hydrazone bond pH sensitive

N

NH

R2

[155]

Acidic environment in endosomes

R2

[208]


R2

[159]


[70]

Reduction in endosomes by GSH

[157]

Cathepsin B

[1]

Cathepsin B

[1]

Cathepsin B

R3

Vinyl ether bond

R1

Acetal/Ketal bond

R1

O

O

O R

Redox sensitive

Disulfide bond

S

R1

O

Gly-Phe-Leu-Gly

Remark

R1

S

R2

O NH

NH

NH O

NH

R2

O

H3C CH3

Enzyme sensitive

O

Val-Cit(Cit=Citrulline)

R1

O

NH O

NH NH2 NH R 2 O

Phe-Lys

O N

HN

R2

R1 NH2

produces lactic acid, which cannot be fully discharged due to the lack of the interior tumor vascular, and thus cause acidic pH within tumor tissues [155]. The physiological pH is 7.4 while the pH within cancer issues and endosomes/lysosomes are 6.85-6.95 and 4.56.5, respectively [1]. Such pH variations make the pH sensitive linkers stable in circulatory system, upon reaching tumor microenvironment or intracellular organelles, they will be cleaved and release the payloads [156]. Nowadays, the most widely used pH-sensitive linker is hydrazone bond [155], which is stable under

neutral environment and easily hydrolyzed under weak acidic environment [157]. Neuropeptide Y (NPY) is a 36-residue peptide belongs to the pancreatic polypeptide family, NPY receptors are overexpressed in a number of neuroblastoma tumor and its derived cell lines [158]. Daunorubicin and DOX have been conjugated with NPY via two linkers with different stability: an acid-sensitive hydrazone bond at the 13-keto position of daunorubicin and a stable amide bond at the 3’amino position of daunorubicin and DOX. This conjugate was able to bind the receptor with high affinity, the daunorubicin-hydrazine-NPY conjugate showed


cytotoxic activity comparable to free daunorubicin [159]. The confocal laser scanning microscopy showed that the conjugate released daunorubicin close to nucleus, whereas the stable amide conjugate did not release drug at all [160]. 4.3. Enzyme Sensitive Linker Enzyme sensitive linker is a specific peptide sequence that can be utilized as a cleavable spacer between drug and carrier. Since proteases are usually not active extracellular due to the environment pH and the serum protease inhibitors [161]. These enzyme sensitive linkers hold good stability in plasma with a designed hydrolysis site. The location and expression level of the target enzymes are correlated to tumor progression and metastasis that allow targeted release of the active drug in tumor sites. Thus, enzymatic cleavage often proceeds with high specificity. Gly-Phe-Leu-Gly (GFLG) is an enzymatically degradable tetrapeptide which is specially cleaved by cathepsin B, the most important lysosomal cysteine proteinase [1, 162]. DOX has been conjugated to a TAT peptide (GRKKRRQRRRPPQ-NH2) through the GFLG linker, while a conjugate using an amide linker served as the control [163]. After 8 h incubation with cathepsin B (100 U/L), the control showed no noticeable release of DOX throughout the incubation period, whereas DOX-GFLG-TAT almost completely released DOX. For HepG2 liver cancer cells treated with two conjugates (15 µM each) for 2 hr, the control exhibited 95% viability while DOX-GFLG-TAT showed 73% viability [163]. 4.4. Redox Sensitive Linker Disulfide linker breaks down in the presence of glutathione (GSH), a reducing reagent that resides in cytosol with an intracellular concentration 1000-fold higher than that in plasma (typically 15 mM intracellular compared to 15 µM extracellular) [109, 164], which makes disulfide bond thermodynamically stable in plasma. In tumor cells, the abnormal state of blood flow leads to hypoxia, increases the activities of reducing enzyme and therefore increases the concentration of glutathione. Then disulfide linker is broken down to release the cytotoxic payload to tumor cells [165-166]. 5. OTHER ELEMENTS IN PDCs DESIGN AND SYNTHESIS When conjugating a cytotoxic payload with a peptide, modification sites of the cytotoxic payloads and peptides are the first problem needs to be considered.

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At the same time, drug loading and the synergistic effect between cytotoxic payloads also need to be considered. 5.1. Conjugation Sites 5.1.1. Drug Conjugation Site During modification, it is important to choose the right conjugation site to avoid losing drug activities, thus the conjugation site should be away from the functional groups. For example, methotrexate is an antifolate antineoplastic which mainly blocks tumor cell growth and reproduction through inhibition of dihydrofolate reductase [167]. The molecular structure of methotrexate and folic acid are very similar, so methotrexate can competitive with folic acid to bind dihydrofolate reductase, therefore, the binding of folic acid with dihydrofolate reductase is reduced, and the synthesis of tetrahydrofolic acid is inhibited. The pterin portion of methotrexate, which has high affinity to dihydrofolate reductase, is an essential structure for its antimetabolite activity, any modification on pterin portion decreases the drug activity, whereas modification on the glutamic residue exhibits little inhibition on dihydrofolate reductase activity (Fig. 3), thus the glutamic residue was selected as the conjugation site [168170]. 5.1.2. Peptide Conjugation Site Conjugating cargos with peptides affect the efficiency of cell penetration or cell targeting via influencing spatial structure and hydrophobicity of peptides [171]. Amido and carboxyl terminal are commonly used peptide conjugation sites. What’s more, side chains of lysine, arginine and cysteine are also good conjugation sites for PDC design. The structurefunction relationship of peptide is the basic requirement in choosing the conjugation site, the key function sites should not be chosen for conjugation. The N-terminus (GRCCK) of TAT peptide is responsible for nuclear localization, and these residues are also important for the interactions between TAT peptide and phospholipid-based membrane, so most cargos are conjugated at the C-terminus [30, 172]. Pep1 is a synthetic amphipathic peptide consisted of three domains: an N-terminal hydrophobic tryptophan-rich motif (KETWWETWWTEW), a spacer (SQP), and a hydrophilic lysine-rich domain (KKKRKV) at Cterminus, most cargos are conjugated at its C-terminus [51]. The sequence of iRGD is CRGDK/RGPD/EC, in which the Cys between the C- and N-terminus are connected through a disulfide bond. Cargoes are usually


conjugated with the -NH2 of cysteine residue at the Nterminus of iRGD [79]. For bombesin, LHRH and somatostatin, cargoes are conjugated at the lysine or cysteine at the N-terminus [61, 65, 68]. When choosing conjugation site, fully understanding the structureactivity relationship of the peptides and payloads are necessary to avoid impairing their activity. 5.2. Drug Loading Drug loading refers to the quality ratio of payloads in PDCs. It is needed for drugs to reach certain threshold concentrations in the target tissue(s) to get therapeutic effects [173]. Loading more payloads to carrier can also effectively improve drug concentration in target tissue(s) [174] . PDCs have higher drug loading capacity compared to other delivery system, such as ADCs, poly-carrier system and nanocarrier system [58]. As to ADCs, in order to prevent antibody aggregation or antigen recognition disturbance, the number of payloads conjugated to the peptide is usually between 2 to 4. Compared to the large molecule weight of antibody, the molecular weight of payloads portion is only 2-5% [175]. The drug load of widely used polymer carrier and nanocarrier per carrier is typically less than 10% [102, 176]. Ran et al conjugated taxol with a tumor lineagehoming CPP (RLYMRYYSPTT RRYG) to form Taxol–CPP, which has a high drug loading of 26.4% [177]. Cui et al. connected a short peptide VQIVYK derived from Tau protein with paclitaxel, which possessed a fixed 41% paclitaxel loading [164]. 5.3. Synergistic Effect Drugs are often used in combination for better outcome in clinical treatment. For examples, ranitidine and domperidone are used in combination against gastritis [178]; combination usage of the chemotherapeutic agent docetaxel and the hedgehog pathway inhibitor cyclopamine kill cancer stem cells and bulk tumor cells more effectively than either of drug alone [179]. The hedgehog pathway is not only involved in prostate cancer progression, but also critical in promoting prostate cancer stem cells self-renewal and growth [180]. Another example is thymidylate synthase inhibitor 5fluorouracil (5-Fu) combination with doxorubicin (DOX). 5-Fu was recently reported to selectively destroy tumor-associated myeloid derived suppressor cells and therefore promoted T cell-dependent antitumor responses [181]. 5-Fu and DOX together with other chemotherapeutics (e.g. cyclophosphamide or mitomycin-C) are often used for combination therapy


15

in clinic to treat breast and pancreatic carcinoma [182183]. DOX and 5-Fu were conjugated to a galectin-3 targeting peptide G3-C12 (ANTPCGPYTHDCPVKR) via a pH-sensitive hydrazone bond and an enzymatically degradable GFLG tetrapeptide spacer [181, 184]. In galectin-3 overexpressing PC-3 human prostate carcinoma cells, this conjugate exhibited comparable cytotoxicity to free DOX at high concentration by increasing cell internalization and exerting synergistic genotoxic effects of cell cycle arrest, caspase-3 activation and DNA damage. In mice bearing PC-3 tumor xenografts, this conjugate inhibited tumor growth to a greater extent (tumor inhibition of 81.6%) than DOX•HCl (40.5%) and 5-Fu(14.6%), demonstrating the potential of synergistic combination PDC therapeutics for cancer [184]. 6. EXAMPLES OF PDCs For most PDCs, peptides are synthesized with relatively consistent standard solid-phase peptide synthesis procedures. However, there are different procedures for payloads and linker conjugate formation and the linkerpeptide conjugation. The general design and synthesis processes of PDCs of two examples are discussed below. 6.1. Design and Synthesis of CPT-mal-Tau /CPTbuSS-Tau CPT is a cytotoxic quinoline alkaloid which inhibits the DNA enzyme topoisomerase I. High hydrophobicity induces poor solubility in tumor tissues, which limits the efficacy of CPT [185]. Several peptides have been conjugated with CPT to increase solubility, local efficacy while reduce the peripheral toxicity, such as c(RGDfK), SSTR2 specific backbone cyclic peptide 3207-86, integrin αvβ3 ligand ALOS-4 [77-78, 186187]. Cheetham AG et al. synthesized CPT-mal-Tau and CPT-buSS-Tau (Fig. 4 shows the synthetic route of two PDCs) in which CPT was conjugated with PHF6 (VQIVYK), a domain of the microtubule-binding protein Tau, with maleimide (mal, an unreducible linker) or buss (a reducible disulfylbutyrate linker) [174]. The 20-hydroxyl site of CPT was selected to bind with linker through an ester bond; the bound CPT is noncytotoxic before it is cleaved by esterases [188-189]. The solubility of the peptide was improved by adding a lysine to the hydrophilic side, a glycine and a cysteine to the hydrophobic side. CPT-mal-Tau and CPT-bussTau are prone to self-aggregate to form β-sheet struc-


ture, which makes the link stable in plasma for a long period of time and avoids CPT from being released in plasma. Upon reaching cells, CPT-mal-Tau and CPTbuss-Tau are transported into cells through endocytosis, then degraded by esterases and release CPT intracellular. The strategy increases the solubility and decreases the toxicities and doses of CPT. 6.2. Design and Synthesis of GLP-1-Estrogen Gut-derived hormone GLP-1 peptide is effective in treating type 2 diabetes. GLP-1 has a glucose concentration-dependent hypoglycemic effect and can reduce weight [190-191]. GLP-1 has the metabolic profile to influence incretin secretion in endocrine pancreas and to regulate satiety effects in the central nervous system (CNS) [192]. Estrogens are steroid compounds with a wide range of biological activities and have been used to treat obesity and type 2 diabetes [193]. Estrogens regulate energy and food intake through a role similar to leptin, but the gynecological and tumor-promoting actions limit their clinical application [194].

Ma et al.

Finan et al. designed a GLP-1-estrogen conjugate for combination therapy of type 2 diabetes [151]. Estrogen was conjugated to GLP-1 at the third position of 17α-estradiol through an ether bond to form a stabile conjugate (which was stable in plasma for over 120 h) or conjugated through an aromatic ester bond to form a labile conjugate (with an estimated half-life of ~1.5 h). Estrogen was enriched in target tissues and cells through GLP-1 receptor (GLP-1R)-mediated cellular targeting and intracellular delivery. The conjugate had superior efficacy than either of the individual hormones alone to treat obesity, hyperglycemia and dyslipidemia in mice [151]. Fig. (5) shows the different effects of labile and stable GLP-1-estrogen conjugates. Moreover, unlike the labile GLP-1-estrogen conjugate, the gynecological toxicity and oncogenicity of estrogen was significantly reduced in the GLP-1-estrogen conjugate, which was stable in the plasma for a long period, thus avoid estrogen from being released in plasma [151].

Fig. (5). Different effects of labiled and stable GLP-1-estrogen conjugate. The modified GLP-1 is conjugated with estrogen through phenolic ether bond to form a structure and physiological stable GLP-1-estrogen conjugation. This conjugation can remain stable in the plasma for a long period that avoiding estrogen been released in plasma, stable covalent attachment of estrogen to GLP-1 prevented the reproductive endocrine toxicity and oncogenicity. After arriving the organization of high GLP-1 receptor expression (endocrine pancreas and CNS, etc.), GLP-1 receptor(GLP-1 R) mediates the endocytosis of the conjugation and the CLP-1 R activation can promote the activation of downstream pathways to promote glucolipid metabolism, reduce weight, etc. Estrogen on the end can help the stable GLP-1-estrogen transport into nucleus. Selective activation of estrogen receptor in GLP-1-targeted tissues induces unparalleled efficacy to enhance the metabolic benefits of GLP-1 agonism.


CONCLUSION AND FUTURE PERSPECTIVES As the examples of “magic bullets”, ADCs and PDCs both show advantages and disadvantages; better understanding the actions of these molecules will lead to more rational selection of targeting therapeutics. With the recent advances in peptide chemistry, peptides exhibit good specificity and stability, presenting a good alternative choice as drug carriers in addition to antibodies, especially in treating solid tumors. With exhaustive screening and rational designing of peptides and linkers, together with extensive research on the molecular mechanism underlying cellular transport processes and drug release, PDCs are expected to be used extensively in clinic.

Current Medicinal Chemistry, 2017, Vol. 24, No. 00 [11]

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[13]

[14] [15]

CONFLICT OF INTEREST The authors confirm that this article content has no conflict of interest. ACKNOWLEDGEMENTS

[16]

Declared none. REFERENCES [1]

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[4]

[5] [6]

[7]

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[17]

[18]

[19] [20] [21]

[22] [23]

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