This journal is©The Royal Society of Chemistry 2017 Chem. Commun.

Cite this:DOI: 10.1039/c7cc00745k

SMI-Ribosome inactivating protein conjugatesselectively inhibit tumor cell growth†

Saumya Roy,a Jun Y. Axup,a Jane S. Forsyth,b Rajib K. Goswami, ‡a

Benjamin M. Hutchins,a Krishna M. Bajuri,a Stephanie A. Kazane,§a

Vaughn V. Smider,a Brunhilde H. Felding*b and Subhash C. Sinha ¶*a

Cell-targeting conjugates of Saporin 6, a ribosome inactivating

protein (RIP), were prepared using the Saporin Ala 157 Cys mutant,

a small molecule inhibitor (SMI) of integrins avb3/avb5, and a potent

cytotoxin, auristatin F (AF). The conjugates selectively and potently

inhibited proliferation of tumor cells expressing the target integrins.

We anticipate that the small molecule–RIP bioconjugate approach

can be broadly applied using other small molecule drugs.

Ribosome-inactivating proteins (RIPs) are extremely potenttoxins that act on both dividing and non-dividing cells througha medium sized (30 kDa) enzymatic ‘A’ chain as the toxindomain.1 They cause damage to ribosomes in an irreversiblemanner by removing one or more adenine residues from ribo-somal RNA (rRNA), thereby arresting protein synthesis. MostRIPs are classified as either type 1 or type 2 (RIP1 and RIP2).RIP1 toxins contain only the enzymatic ‘A’ chain, whereas RIP2toxins also possesses a cell binding lectin ‘B’ chain thatmediates its cellular uptake. It is anticipated that such toxinscould mediate strong therapeutic effects when they are targetedat tumor cells and are selectively taken up by such cells.2

Indeed, both RIP1 and the ‘A’ chain of RIP2, upon conjugationto cell-targeting agents, such as monoclonal antibodies (Abs) orgrowth factors, have shown promising results in animalmodels. Studies conducted with Ab–RIP or GF–RIP conjugates,however, did not progress beyond early Phase 1 Clinical Trials,because the conjugates generated immune responses and caused

side effects. In addition, the large molecular size of the conjugatesrestricted their penetration and distribution in solid tumors.3

In the meantime, there are indications that the side effects andimmunogenicity of a RIP conjugate are minimized throughPEGylation and by using humanized Abs.4 The molecular size ofa RIP conjugate is also reduced modestly by replacing a full lengthAb with an Ab fragment. Nonetheless, an alternative approach thatreduces the size further can offer RIP conjugates with superiorefficacies. With this notion, we develop a small molecule inhibitor(SMI)-directed approach for RIP delivery, which can afford SMI–RIPconjugates without increasing the molecular size of a RIP.

A SMI of a cell surface receptor mediates selective delivery ofboth the low molecular weight drug and the large proteins to targetcells, similarly to a classical monoclonal Ab. This SMI-directeddrug delivery approach can utilize potent SMIs of cancer-associatedtargets that are already available in large numbers.6 Earlier, wedeveloped SMI–cytotoxin conjugates, using a potent dual SMI ofintegrins avb3 and avb5, and monomethyl-auristatin (MMAE).MMAE and its analog, auristatin F (AF), are potent microtubuleinhibitors and vascular damaging agents (VDA),7 previously usedin antibody-drug conjugates. Integrins avb3 and avb5 are known tooverexpress in many human tumors and by endothelial cells liningthe vasculature in angiogenic tumors. Both integrins have beentargeted with several SMIs, Abs, SMI–drug, and SMI-bioconjugatesfor selective cancer therapy.8 Our studies with SMI–MMAE con-jugates have shown that the latter mediated a selective toxicity totumor cells expressing the target integrins.9 Separately, we prepareda series of chemically programmed Abs (cpAbs) by chemicalprograming of a monoclonal Ab, 38C2, using potent SMIs ofintegrins avb3, avb5, avb6, and a5b1. These cpAbs mediated a stronganti-tumor effect selectively to tumor cells expressing the respectiveintegrin(s).10 Along the same line, we have now prepared twoSMI–RIP5 conjugates using a RIP1 mutant, an ‘alanine 157 cysteine’(A157C) mutant of Saporin 6 (viz., Sap-C).11 Both conjugates aredirected towards integrins avb3 and avb5 using a potent and dualSMI 1 of these adhesion receptors (Fig. 1).12 The second conjugate,SMI(AF)–Sap, has SMI 1 and AF, 2,13 coupled to Sap through atrifunctional linker. We reasoned that AF could dissociate from the

a Department of Cell and Molecular Biology, The Scripps Research Institute, 10550

North Torrey Pines Road, La Jolla, CA 92037, USA. E-mail: ssinha@rockefeller.edub Department of Chemical Physiology, The Scripps Research Institute, 10550 North

Torrey Pines Road, La Jolla, CA 92037, USA. E-mail: brunie@scripps.edu

† Electronic supplementary information (ESI) available: Synthesis of compounds3 and 4, and production and evaluation of the SMI–Sap, SMI(AF)–Sap, and cpAb38C2-1. See DOI: 10.1039/c7cc00745k‡ Current address: Department of Organic Chemistry, Indian Association for theCultivation of Science, Jadavpur, Kolkata, India.§ Current address: Center for Therapeutic Innovation (CTI), Pfizer, Inc.,10770 Science Center Dr., San Diego, CA 92121, USA.¶ Current address: Laboratory of Molecular and Cellular Neuroscience,The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA.

Received 27th January 2017,Accepted 16th March 2017

DOI: 10.1039/c7cc00745k

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conjugate upon tumor-uptake, and enhance the cell-killing effectsof the SMI–Sap. Evaluation has shown that both the SMI–Sapand SMI(Sap)–AF conjugates mediated highly selective and strongtoxicity to tumor cells expressing the target integrins. Additionalconjugation of AF to Sap in the SMI(AF)–Sap conjugate did notfurther enhance the cytotoxicity of the already very potent SMI–Sapconjugate when measured in vitro, but might provide an advantagewhen targeting a tumor.

Our studies started with the synthesis of the two conjugates,SMI–Sap and SMI(AF)–Sap, which were prepared by reactingSap-C with compounds 3 and 4 as shown in Scheme 1 throughthe maleimide–thiol coupling reaction.14 Here, SMI 1 iscoupled to the maleimide function through a bi-functionallinker in compound 3, and through a tri-functional linkerhaving AF, 2, at the third end in compound 4. For the SMI–Sapand SMI(AF)–Sap production, the key intermediates 3 and 4 wereprepared starting with the readily available SMIs 5a or 5b in three

to five steps, (Scheme 1 and ESI,† Scheme S-1). Separately, Sap-Cwas produced in E. coli, and isolated from the culture by cationexchange chromatography and concentrating the eluants usingAmicon (10 kDa MCWO), as described.11 The residual materialunderwent a dithiothreitol-mediated dithiol reduction step, andwas further purified by size-exclusion chromatography. Subse-quently, Sap-C reacted with intermediates 3 and 4, and the crudeproducts were purified using FPLC to afford the desired SMI–Sapand SMI(AF)–Sap conjugates (Fig. 2).

To evaluate the selectivity and cytotoxic activity of the Sapconjugates, we chose M21 and M21-L human melanoma cellsand MDA-MB-435 human breast cancer cell variants. The M21and M21-L cell lines express b1 integrins, including a5b1, butthey differ from each other in the expression of av integrinswhile M21 cells express avb3 highly, avb5 integrin moderately,and avb8 integrin weakly, the M21-derived M21-L cells lack theav subunit gene and therefore do not express either of the targetintegrins.15 In the MDA-MB-435 cell model, the control cellsmoderately express the b3 integrin subunit gene which wasknocked down in the 435b3-minus variant for comparison.16

Fig. 1 Structure of a RIP1 mutant, Saporin 6 alanine 157 cysteine (A157C),and small the molecule inhibitor of integrins avb3 and avb5, as well as thepotent cytotoxin, auristatin F used here.

Scheme 1 Synthesis of Sap conjugates, SMI–Sap and SMI(AF)–Sap, using the Saporin 6 alanine 157 cysteine’ (Sap A157C) mutant and a small moleculeinhibitor (SMI) of avb3/avb5 integrins or the SMI–auristatin F (AF) heterodimer. Key: (a) TCEP buffer, purification using FPLC, (b) Cu-catalyzed alkyne–azidecoupling (Cu–AAC) reaction, (c) peptide coupling reaction.

Fig. 2 Analysis of Saporin and conjugates using gel, and ESI mass spectralanalysis.

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Both cell variants expressed low levels of avb5 and avb6, andmany of the b1 integrins.17 We confirmed the expression ofintegrins avb3 and avb5 in M21 cells and lack of av integrins inM21-L cells, as well as binding of compound 1-derived cpAb38C2-1 to avb3 and avb5 integrins using M21 and UCLA-P3 cells,the latter expressing avb5 but not avb3, as described previously(Fig. 3). We further analyzed and confirmed that 38C2-1 boundstrongly to OVCA433 cells and variants of the human coloncancer SW480 cell line, including puro control SW480, SW480b3, and SW480 b6 cells (see ESI†). OVCA433 cells expressintegrins avb5 and avb6, and the puro control SW480 cellsexpress avb5 integrin, whereas in SW480 b3 and SW480 b6 cells,the b3 and b6 integrins was experimentally over-expressed.18

These data suggest that SMI 1 is a broad inhibitor of several av

integrins, including avb3, avb5, and avb6. Because there was noappreciable binding to M21-L cells, we concluded that 38C2-1did not bind integrin a5b1 or other b1 integrins. We furtherargued that the two SMI–Sap and SMI(AF)–Sap conjugatesmight mediate toxicity to M21 cells through binding to theavb3 and avb5 integrins, to 435ScrB through all three avb3, avb5,and avb6 integrins, and to 435b3-minus cells through avb5 andavb6 in the absence of b3 integrin. We determined the selectivityand cytotoxic activity of our first SMI–Sap conjugate by incubatingM21 and M21-L cells with the test conjugates. Unconjugated SMI 1and the unmodified Sap-C were used for comparison. The resultsare shown in Fig. 4A and B. Evidently, the SMI–Sap conjugatemediated cell-death of M21 cells at a subnanomolar IC50, whereasboth inhibitor 1 and Sap-C were nontoxic up to 10 nM concen-tration (Fig. 4A). As shown in Fig. 4B, the SMI–Sap conjugate

Fig. 3 Flow cytometry analysis showing the avb3/avb5 integrin expressionlevel in M21, M21-L, and UCLA-P3 cells, and binding of cpAb 38C2-1,prepared by chemical programming of aldolase Ab 38C2 using a diketonederivative, 1a, of a potent avb3/avb5 integrin SMI, 1, to M21 and UCLA-P3tumor cells expressing the target integrins. APC labeled anti-mousepolyclonal Ab (at a 1 : 100 dilution, i.e., 10 mg mL�1 in FACS buffer) wasused to detect the binding of the control Abs (10 mg mL�1) and cpAb 38C2-1(20 mg mL�1) to cells. The y-axis gives the relative mean fluorescenceintensity on the linear scale, and the x-axis describes the cell line name.Key: Abs used to detect integrin expression on M21, M21-L, and UCLA-P3cells were: VNR1 (for avb3), P1F6 (avb5), 2077Z (avb6), 14E5 (avb8), P5D2 (b1),and P1D6 (a5).

Fig. 4 Effect of the SMI–Sap and SMI(AF)–Sap conjugates on cell viability and proliferation. SMI–Sap conjugate mediates cellular toxicity to M21 cells at asubnanomolar IC50, but not to M21-L cells (A and B). SMI 1 and Sap also show minimum toxicity to M21 cells at 20 nM concentration. SMI–Sap andSMI(AF)–Sap conjugates mediate strong toxicity (sub-nanomolar IC50) to M21 cells (C) but not to M21-L cells (D). SMI–Sap and SMI(AF)–Sap conjugatesare also active against MDA-MB-435 cell variants 435 b3� in which the b3 integrin subunit gene had been knocked down, as well as to 435 ScrB in whichb3 integrin expression had been restored by transduction with the b3 wild type gene (E and F). To measure the effects of the compounds and conjugateson tumor cell viability and proliferation, 5 � 1034 cells were plated onto 24 well plates and incubated with or without compounds and conjugates atvarious concentration. After 72 h, cells were harvested and counted. Live cells were identified and counted based on trypan blue exclusion. Cell-survivalassay was performed once, and there were three replicates.

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mediated toxicity in M21 cells, whereas the M21-L cells lacking thetarget integrins were not affected under the conditions tested(at the 20 nM SMI conjugate).

In subsequent experiments, we examined and compared thetwo SMI–Sap and SMI(AF)–Sap conjugates using M21 and M21-Lcells, and determined whether AF addition in the second con-jugate amplified the effects of Sap in the SMI(AF)–Sap conjugate.As shown in Fig. 4C and D, the SMI(AF)–Sap also mediated astrong and selective toxicity to M21 cells, but the effect on M21cells was not different from the SMI–Sap conjugate. These resultsdocument two major findings. First, the SMI clearly delivers targetspecificity and the constructs are stable until internalized by thetarget cells to enable cytotoxic activity. Second, the presence of Sapalone as the cytotoxic moiety of the conjugates is sufficient to fullyexecute target cell killing. Nevertheless, additional inclusion ofAF in the SMI(AF)–Sap conjugate might enhance desired effectswithin the complexity of a tumor where one type of cytotoxicmoiety might not be sufficient to achieve full target toxicity.Importantly, the unchanged efficacy between SMI–Sap andSMI(AF)–Sap in our models demonstrates that this doubleconjugate targets cells with equal selectivity and equal efficacy,providing strong evidence for stability and maintained target-ing potency of an SMI, even in a complex multi-functionalconjugate. Importantly, the in vitro data show that the SMI(AF)–Sap conjugate is not metabolized by the cells to release free AF,indicating that the selectivity is fully maintained with thisconjugate based targeting mechanism. In fact, using compound13, SMI(AF)-linker, we found that the free compound indeedcaused a low toxicity to M21 cells at 20 nM concentration. Thisunwanted effect is eliminated when the compound is included inthe stable conjugate and taken up by the targeted cells only.19

Finally, we examined the two SMI–Sap conjugates using theMDA-MB-435ScrB and 435b3-minus cell variants, and foundthat both conjugates mediated a strong cytotoxic effect (IC50

values of B5 nM) in these cells (Fig. 4E and F). These resultsindicate that on tumor cells that express multiple target integrinsrecognized by the SMI component of the drug conjugate, experi-mental knock-down of one of at least three av containing targetintegrins still left sufficient recognition sites on the tumor cells foreffective cell killing. This result is remarkable in view of the factthat avb3, the target experimentally reduced in this cell model, isthe major av integrin on MDA-MB-435 breast cancer cells. Thus,the threshold for target expression by tumor cells to be effectivelyeliminated by the SMI–drug conjugates is apparently remarkablylow. The result demonstrates that the conjugates are exclusivelyselective, and highly effective and the targeting mechanism is verysensitive.

In summary, we have shown for the first time that a RIP1mutant, such as Sap-6 A157C, reacts with an SMI or a hetero-dimer of two SMI drugs through thiol–maleimide coupling toafford bi- or tri-functional SMI–RIP1 conjugates. In this manner,we have coupled the Sap-6 A157C mutant to an SMI that targetsintegrins avb3 and avb5, as well as an integrin SMI–AF (AF as asecond SMI) heterodimer to afford SMI–Sap and the SMI(AF)–Sapconjugates, respectively. Both conjugates mediate strong and

selective toxicity to tumor cells expressing the target integrins,with the addition of AF conjugation to potentially convey tumorrelevant cytotoxic efficacy while maintaining exquisite selectivetargeting. We conclude that this SMI-directed strategy for RIPdelivery might be broadly applied using other RIPs and SMIs,while inclusion of the second SMI drug designed to neutralizeanother intracellular target through a noncleavable linker ispossible without compromising the selectivity and cellularuptake of the very potent SMI–RIP conjugates.

We thank Dr Peter G. Schultz for support in the preparationand isolation of saporin and its conjugates, and acknowledgethe support of grants from the NCI (R01CA120289 to SCS,R21CA198595 to BF, R01CA170140 to BF, R01CA170737 to BF)and from the BCRP CDMRP (BC 123479 to BF).

Notes and references1 J. Schrot, A. Weng and M. F. Melzig, Toxins, 2015, 7, 1556–1615.2 (a) L. Polito, M. Bortolotti, M. Pedrazzi and A. Bolognesi, Toxins,

2011, 3, 697–720; (b) R. Gilabert-Oriol, A. Weng, B. Mallinckrodt,M. F. Melzig, H. Fuchs and M. Thakur, Curr. Pharm. Des., 2014, 20,6584–6643.

3 S. Bagga, D. Seth and J. K. Batra, J. Biol. Chem., 2003, 278,4813–4820.

4 (a) C. A. Pennell and H. A. Erickson, Immunol. Res., 2002, 25,177–191; (b) J. C. Zheng, N. Lei, Q. C. He, W. Hu, J. G. Jin,Y. Meng, N. H. Deng, Y. F. Meng, C. J. Zhang and F. B. Shen,Immunopharmacol. Immunotoxicol., 2012, 34, 866–873; (c) M. C. Shin,J. Zhang, A. E. David, W. E. Trommer, Y. M. Kwon, K. A. Min,J. H. Kim and V. C. Yang, J. Controlled Release, 2013, 172, 169–178.

5 B. E. Collins, O. Blixt, S. Han, B. Duong, H. Li, J. K. Nathan, N. Bovinand J. C. Paulson, J. Immunol., 2006, 177, 2994–3003.

6 N. Krall, F. Pretto, W. Decurtins, G. J. Bernardes, C. T. Supuran andD. Neri, Angew. Chem., Int. Ed., 2014, 53, 4231–4235.

7 E. M. Prokopiou, P. A. Cooper, G. R. Pettit, M. C. Bibby andS. D. Shnyder, Mol. Med. Rep., 2010, 3, 309–313.

8 A. Dal Corso, L. Pignataro, L. Belvisi and C. Gennari, Curr. Top. Med.Chem., 2016, 16, 314–329.

9 Y. Liu, K. M. Bajjuri, C. Liu and S. C. Sinha, Mol. Pharmaceutics,2012, 9, 168–175.

10 (a) S. C. Sinha, S. Das, L.-S. Li, R. A. Lerner and C. F. Barbas III,Nat. Protoc., 2007, 2, 449–456; (b) C. Rader, Trends Biotechnol., 2014,32, 186–197; (c) Y. Liu, R. K. Goswami, C. Liu and S. C. Sinha, Mol.Pharmaceutics, 2015, 12, 2544–2550.

11 B. M. Hutchins, S. A. Kazane, K. Staflin, J. S. Forsyth, B. Felding-Habermann, V. V. Smider and P. G. Schultz, Chem. Biol., 2011, 18,299–303.

12 L. Seguin, J. S. Desgrosellier, S. M. Weis and D. A. Cheresh, TrendsCell Biol., 2015, 25, 234–240.

13 S. O. Doronina, T. D. Bovee, D. W. Meyer, J. B. Miyamoto, M. E.Anderson, C. A. Morris-Tilden and P. D. Senter, Bioconjugate Chem.,2008, 19, 1960–1963.

14 C. W. Scales, A. J. Convertine and C. L. McCormick, Biomacromolecules,2006, 7, 1389–1392.

15 B. Felding-Habermann, B. M. Mueller, C. A. Romerdahl andD. A. Cheresh, J. Clin. Invest., 1992, 89, 2018–2022.

16 M. Lorger, J. S. Krueger, M. O’Neal, K. Staflin and B. Felding-Habermann, Proc. Natl. Acad. Sci. U. S. A., 2009, 106, 10666–10671.

17 A. Taherian, X. Li, Y. Liu and T. A. Haas, BMC Cancer, 2011, 11, 293.18 P. H. Weinreb, K. J. Simon, P. Rayhorn, W. J. Yang, D. R. Leone,

B. M. Dolinski, B. R. Pearse, Y. Yokota, H. Kawakatsu, A. Atakilit,D. Sheppard and S. M. Violette, J. Biol. Chem., 2004, 279,17875–17877.

19 J. Y. Axup, K. M. Bajjuri, M. Ritland, B. M. Hutchins, C. H. Kim,S. A. Kazane, R. Halder, J. S. Forsyth, A. F. Santidrian, K. Stafin,Y. Liu, H. Tran, A. J. Seller, S. L. Biroc, A. Szydlik, J. Pinkstaff, F. Tian,S. C. Sinha, B. Felding-Habermann, V. V. Smider and P. G. Schultz,Proc. Natl. Acad. Sci. U. S. A., 2012, 109, 16101–16106.

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