Cancer‐Cell‐Specific Drug Delivery by a Tumor‐Homing CPP‐Gossypol Conjugate Employing a Tracelessly Cleavable Linker

Abstract Tumor‐targeted drug delivery is highly important for improving chemotherapy, as it reduces the dose of cytotoxic agents and minimizes the death of healthy tissues. Towards this goal, a conjugate was synthesized of gossypol and a MCF‐7 cancer cell specific CPP (cell penetrating peptide), thus providing a selective drug delivery system. Utilizing the aldehyde moiety of gossypol, the tumor homing CPP RLYMRYYSPTTRRYG was attached through a semi‐labile imine linker, which was cleaved in a traceless fashion under aqueous conditions and had a half‐life of approximately 10 hours. The conjugate killed MCF‐7 cells to a significantly greater extent than HeLa cells or healthy fibroblasts.

In memory of Prof. Dr.Carsten Schmuck1 968-2019 Abstract: Tumor-targeted drug delivery is highly important for improving chemotherapy,a si tr educes the dose of cytotoxic agentsa nd minimizes the deatho fh ealthy tissues.T owardst his goal, ac onjugate was synthesizedo f gossypol andaMCF-7 cancerc ell specific CPP (cell penetrating peptide), thus providing as elective drug delivery system.U tilizing the aldehydem oiety of gossypol,t he tumor homingC PP RLYMRYYSPTTRRYG was attached throughas emi-labile imine linker,w hich was cleaved in a traceless fashion under aqueous conditions and had a half-lifeo fa pproximately 10 hours. The conjugate killed MCF-7 cells to as ignificantly greatere xtentt han HeLa cells or healthyf ibroblasts.
To date, canceri so ne of the leadingc auses of globald eath due to the difficulties associated with tumor selective therapy, such as inefficientd rug accumulation, cancer cell heterogeneity,a nd drug resistance. [1] The nonspecifict oxicityo fa nticancer agents towardsh ealthyt issues is am ajor challengei nc onventional chemotherapeutic treatments. [2] Thus, targeted drug delivery,e nvisioned by Paul Ehrlich as a" magic bullet",i salong standing researcho bjective. [3] Towards this goal,s ignificant advancements were achieved by exploiting the advantages of different cancers pecific vectors, [4] such as antibodies, [5] aptamers, [6] folic acid derivatives [4,7] and cell penetrating peptides. [8] Tumor-homing peptides, which are small oligopeptides (3 to 15 residues)i dentified through sophisticated techniques (phaged isplay,m RNA display), are evolving as specific vectors for cancerc ells. [9] The designo fs uch targeted drugd elivery systems relies majorly on the conjugation of anticancer drugs to the vectorsv ia ac leavablel inker.S ynthetic organic chemistry aims towards developing suitably cleavable linkers, which release unmodified drugs over am ulti-hour timeframeu nder physiological conditions. [10] Compared to antibodiesa nd aptamers,t umor homing peptides are relatively easyt om odify chemically and can be conjugatedt od rugs through cleavable linkers. Therefore, conjugating novel anticancer agentst o tumorh oming peptides in order to understand and harness their therapeutic potential is of major interest. Gossypol (AT101), an aldehydec ontaining phenol derivedf rom the cottonp lant, was initially explored as am ale antifertility drug. It exhibited promisinga nticancer activities [11] towards various tumors through different mechanisms includingp roliferation inhibition and apoptosis induction. [12,13] Its antiproliferative effecti sc aused by regulating cyclineD 1, [14] autophagy, [15] and inhibition of aldehyded ehydrogenase. [16] Furthermore Gossypol is studied for combination therapy in addition with other therapeutic agentsa gainst glioblastoma, [17] pancreatic cancer cells [18] and non-small-cell lung carcinoma. [19] It has also shown cytotoxicity against breast cancer cells by inhibitingt he expression of mouse double minute 2( MDM2)a nd vascular endothelial growth factor (VEGF). [20] Currently gossypoli si np hase II clinical trials as an anticancer drug. However,g ossypol, as many other conventional anticancer drugs, faces an umber of obstacles including bad water solubility,p oor cellular uptake and al ack of selectivity.T herefore, reversible attachment of gossypol to av ector that enables cell membrane penetration, increases solubility and allows for addressing cancercells selectively,seemedh ighly advantageous.
In this report, we describet he synthesis of cancer cell line specific peptide-gossypol conjugates and their cytotoxic effects.The aldehyde group of gossypol was utilized for conjugation, and thiazolidine (1a)a sw ell as imine linkages (1b)w ere explored as tracelessc leavable linkers( Figure 1). The cancer cell line specific cell penetrating peptide(CPP), RLYMRYYSPTTR-RYG was developed by Matsushita and co-wokers. [9a] It is specific to MCF-7breast cancer cells and was chosen as gossypol also showeda nticancera ctivity on this cell line. This CPP is internalized into cells through ad ynamin-dependente ndocytic pathway.C onjugationo ft his CPP to gossypol increased solubility of the hydrophobic drug (in working buffers:D ulbecco's Modified Eagle's Medium( DMEM)a nd RPMI mediums upplemented with 10 %F BS, streptomycin sulfate (0.1 mg mL À1 ), penicillin (10 UmL À1 )a nd amphotericin B( 0.25 mgmL À1 ), at pH 7.4);t his makes the use of potentially harmful solubilizing agents superfluous.I nitial attempts to synthesize the thiazolidine linked conjugate 1a wereb ased on standard procedures from the literature, [24] which led to the formation of byproducts among which also wasi mine 1b.T he inseparable mixture of conjugates 1a and 1b obtained from the above reactione xhibited specific toxicity to MCF-7 cells compared to HeLa cells. Time-dependent cytotoxicity assays revealed that imine 1b was much more efficient in killing MCF-7 cells compared to HeLa cells, whereas the thiazolidine-linked conjugatew as inactive towards both cells at any time point. AH PLC-study of the cleavage processesr evealed uncontrolledd egradation of the thiazolidine linked conjugate 1a over time, which wasp robably caused by reactive oxygen species produced by gossypol. On the other hand, the imine-linked conjugate 1b,c leanly released gossypol over the course of multiple hours, which is an important prerequisite for targeted tumor delivery.
Initially,asuitable linker between gossypol and the tumorhomingc ell penetrating peptideR LYMRYYSPTTRRYGh ad to be selected, which allows for efficient intracellular release of the anticancer drug. Although several cleavable linkers were reported for different applications,m any of them are difficult to incorporate into peptideb ackbones, while others require specific cleavage conditions such as the involvement of enzymes, nucleophilic/basic or electrophilic/acidic reagents,r educing or oxidizinga gents, photoirradiation, organometallic and metal reagents. [21] Furthermore, these methods sometimes leave ar esidual moiety on the released cargo. [21] Anticancerd rugs such as doxorubicin or paclitaxel were linked through ester and amide bonds to peptides, but slow cleavageo ft hese bonds in the cellular environment limits the activity of the drug. [22] Disulfide linkages, which can be cleaved by glutathione in cells, were also reported. However,t his strategy is not tracelessa nd the releaseo ft he actived rug can be inhibited due to drug dimer formation through disulfide bonds. [23] The aldehyde functionality of gossypol opens up the possibility to insert a thiazolidine linkage( 1a). In an earlierr eport at hiazolidine was employedf or traceless releaseo fad rug from an antibodydrug conjugate. [24] An imine-based linkage through an Alamodifiedh oming peptidew as also explored (1b). Gossypol is known to form comparably stable conjugates with amines as well as small peptides, [25] as the resultingS chiff's base is stabilized by two cooperative intramolecular hydrogen bonds formed by the ortho-and meta-hydroxyl groups. [26] For this work the tumor homing peptides pecific to MCF-7 cells was synthesized by Fmoc-based solid-phase peptides ynthesis with ac ysteine residue attached at the N-terminus (Figure 2A). The methionine residue was substituted by its analogue,n orleucine, to avoid oxidation. Initially,t he ligation reaction was attempted between peptide 2a and an excess of gossypol to avoid reactiono ft he second aldehyde moiety. There- fore, the peptide was dissolved in 6 m Gn·HCl buffera tp H5, followed by its addition to five equivalents of gossypol in methanol and the solution was heateda t4 58Cf or two hours. Consumption of the peptidew as observed by HPLC analysis with emergence of two new peaks, corresponding to diastereomers formed by the axially chiral rac-gossypol and the chiral peptide ( Figure 2B). The peptide-gossypol conjugates were purified by preparative HPLC followed by evaporation of methanol and lyophilization.M asss pectrometry showed as imilar m/z pattern for both peaks, the main signal, however,w as 32 Da lesst han 1a,w hich corresponds to imine 1b (Figure 2C).
According to mass spectrometric analysis, the product obtained mainly contained 1b buta lso showedamass corresponding to 1a.T he cytotoxicity of both fractions was tested on MCF-7 cells as target cellsa nd HeLa cells as an egative control. Gossypoli tself was equally potent in killing both cell lines ( Figure S13, Supporting Information), while the peptide 2a alone was nontoxic for both ( Figure S13). Gratifyingly,aspecific toxicityt owards MCF-7 cells was observed for the CPP-Gossypol conjugate mixture.C ell viability for HeLa cells for mixture ( Figure 2E) was higher than forthe MCF-7 cells ( Figure 2D).
Having evidencef or the cell specific toxicityo fp eptide-gossypol conjugates of type 1,w et urned our attention towards deducing the structure of the active conjugate. To exclude that some of the products arose from the simultaneousc ondensation of the second aldehydef unctionality of gossypol with a nearbyA rg residue ( Figure S9, Supporting Information), we probedthe gossypol conjugation reactionwith amodel tripeptide (CRL) derived from the N-terminus of the peptide 2a. However, only the expectedt hiazolidine linked conjugate was formed ( Figure S10, Supporting Information) as confirmed by mass spectrometry analyses. We also performed al igatione xperiment between gossypol and Fmoc-Arg-OH under similar conditions to excludec ondensation of the guanidine moiety with the gossypol aldehyde. No conjugation product was observed as evident from analytical HPLC( Figure S11, Supporting Information).
The molecular mass loss of 32 Da compared to the parent Cys containing peptide 1a (FigureS12,S upporting Information) was attributed to desulfurization of the Cys residue to Ala. As reliable thiazolidine formation under thesec onditions had been described before for other aldehydes, [24] desulfurization during the ligation reactionw as most likely promoted by gossypol, which is knownt op roduce reactive oxygen species (ROS). [27] To confirmt he formation of the imine linked conjugate 1b through desulfurization, we synthesized 1b throughs imple ligation of gossypol andt he tumor homing peptided erivative 2b,w hich was modifiedw ith an alanine residue at the Nterminus instead of cysteine. The reaction was also conducted in MeOH and aqueous Gn·HCl-Buffer.I ta fforded the peptidedrug conjugate 1b with as table imine linkage as confirmedb y HPLC and mass spectrometry (Figure3,F igureS4). As in this study preparation of CPP-gossypolc onjugates of type 1 was conducted by SPPS, the available materialw as limited to sub mg amounts. Therefore, imine formation was confirmed by reaction of am odel tripeptide NH 2 -Ala-Arg-Leu-CONH 2 (ARL)a nd gossypol. The formation was monitored by HPLC/mass analyses ( Figure S5, Supporting Information) and time-dependent 1 HNMR experiments (MeOD/deuterated Gn·HClb uffer). The proton signal at d = 11.05 ppm, which corresponds to the aldehyde moiety,g radually disappeared and an ew signal at d = 9.91 ppm, corresponding to imine, appeared after three hours ( Figure S7, Supporting Information). The resulting imine was isolated and fully characterized. Furthermore its structure was confirmed by HSQC ( 1 H- 13 Ch eteronuclear single quantum coherence spectroscopy,F igure S7, Supporting Information). As mentioned above, the unusual stabilityo fs uch Schiff's bases resultingf rom nucleophilic addition of Gossypol with amines has been described before and can be attributed to stabilization through intramolecular hydrogen bonds as indicated in Figure 3. [25,26] The question remained, whether it was the thiazolidine conjugate 1a or its iminec ongener 1b,w hich was responsible for the encouraging selectivec ell toxicity observed in the initial experiments. To preventd esulfurization of the Cys residue during ligation of gossypolt op eptide 2a,t he reaction was repeated under an argon atmosphere at lower temperature (37 8C), which yielded ac lean sample of 1a after purification by HPLC (Figure 3c).
The cytotoxic effects of the two differently linked peptidedrug conjugates 1a and 1b were investigated in at ime-dependentc ytotoxicity assay.C ellv iabilities were studied after 12, 24 and 48 hours ( Figure S15, Supporting Information). As shown in Figure4,t he iminel inked conjugate 1b reduced the cell viability of MCF-7 cells to 26 % [28] after 48 h, whereas both HeLa cells (derived from cervical cancer)a nd Wi-38 cells (normalh uman fibroblasts) had am uch higher viability of 67 and 74 %, respectively.T his underlines the cancert ype selectiv-ity of this compound. In contrast the thiazolidinel inked conjugate 1a was inactivet ob oth MCF-7 and HeLa cells at any time point. Moreover,t he other diastereomeric conjugates separated by HPLC for both linkages showed as imilar trend of cytotoxicity, that is,t he imine linked conjugate of type 1b was potent to selectively kill the MCF-7 cells, whilet he thiazolidine linked conjugate of type 1a was not ( Figure S14, Supporting Information).
The time-dependent gossypol release of both, the active imine based conjugate 1b and the inactive thiazolidine based conjugate 1a were studied in 6 m Gn·HClb uffer at pH 7a nd 37 8Ct hrough timed ependentH PLC analysis. The imine linked conjugate 1b had ah alf-life of approximately 10 hours and cleanly released gossypol as well as the homing peptide 2b ( Figure 5). On the other hand, the thiazolidine linked conjugate 1a decomposed to several unidentified species after two hours ( Figure S17 A, SupportingI nformation). This observation is consistent with the observed inactivityo ft he thiazolidine linked conjugate in both cell lines.A ssuming as imilar stability for 1b in the cytoplasm, the measuredh alf-life of 10 ho ffers a sufficient period for intracellular accumulation of the peptidedrug conjugate 1b before the controlled release of the anticancer drug within MCF-7 cells. This leads to substantial cell death after 24 and 48 h.   Figure S17). Solventgradient for HPLC was 40-100 %Bi n20min;buffer Bw as MeOH with 0.1 %T FA and UV was measured at 220 nm. Integration of the peaks correspondingt ot he cleavedp eptide and the intact peptide-drugc onjugate revealed that 52 %o ft he conjugate was cleavedafter 10 hours.
In summary,w ehave developed ac ancer cell specific delivery system for gossypol by using simple Schiff's base ligation chemistry to generate as emi-labile conjugate of Gossypola nd ac ancer type specific cell penetrating peptidea savector. Imine formationb etween gossypol with the Alanine functionalized CPP 2b resulted in ap otent conjugate, whichk illed specifically MCF-7 breast-cancer cells. Furthermore, the solubility of gossypol was improved, which made handling of the conjugates for cellular studies very convenient, as potentially harmful solubilizing agentsl ike DMSO were not necessary.I mportantly,t he presented drug delivery strategy does not rely on any external stimulus to initiate drug releasea nd activation. A particular advantage of this imine linkagei st he convenient half-life of 10 hours in aqueous media. Hopefully theser esults accelerate the applicabilityo fg ossypol particularly in tumor targeted chemotherapy. In future researchc ell line-derived xenograft (CDX) mouse models should be established to evaluate the in vivo efficacy of the CPP-gossypol conjugate 1b.I na broader sense, the reported approach demonstrates that cellpenetrating peptidesw ith tumor homing properties can be easily ligated to gossypolw ithout the need for an additional linker.T herefore, it should be easy to expand the scope of this approacht oo ther cancer types, for which appropriate homing peptides can be identified.