Retro-1 Analogues Differentially Affect OligonucleotideDelivery and Toxin TraffickingBing Yang,[a] Xin Ming,[a] Hajer Abdelkafi,[b] Valerie Pons,[b] Aurelien Michau,[c] Daniel Gillet,[c]

Jean-Christophe Cintrat,[b] Julien Barbier,[b] and Rudy Juliano*[a]

Retro-1 is a small molecule that displays two important biolog-ical activities : First, it blocks the actions of certain toxins by al-tering their intracellular trafficking. Second, it enhances the ac-tivity of oligonucleotides by releasing them from entrapmentin endosomes. This raises the question of whether the two ac-tions involve the same cellular target. Herein we report the ef-fects of several Retro-1 analogues on both toxins and oligonu-cleotides. We found analogues that affect toxins but not oligo-nucleotides and vice-versa, while Retro-1 is the only compoundthat affects both. This indicates that the molecular target(s) in-volved in the two processes are distinct.

For more than two decades investigators have sought to har-ness the precision implicit in the Watson–Crick base pairing ofantisense oligonucleotides, siRNA, and splice switching oligo-nucleotides (SSOs) to the therapy of disease.[1] However, oligo-nucleotide therapeutics has thus far succeeded to only a limit-ed degree despite massive investments in the chemistry andformulation of these molecules and despite initial advances inthe clinic.[2] A major limitation is the poor delivery of oligonu-cleotides to their intracellular sites of action in the cytosol ornucleus because of pharmacologically non-productive trappingin endosomal compartments.[3] Various lipid or polymeric nano-carriers have been used to enhance oligonucleotide uptakeand to promote release from endosomes.[4] However, the cat-ionic nanocarriers typically used present problems in terms oftoxicity[5] and biodistribution.[6]

Recently we have pursued an alternative approach that in-volves the use of small molecules to modulate the processingof oligonucleotides through the endosomal machinery. Thuswe have shown that the compound Retro-1 selectively releasesoligonucleotides from Rab 7/9 positive late endosomes leadingto a substantial enhancement of oligonucleotide pharmacolog-ical effects both in cell culture and in vivo.[7] Retro-1 is

a member of a group of compounds that were initially discov-ered in a screen for inhibitors of bacterial and plant toxins.[8]

Retro-1 and some of its analogues inhibit the actions of certaintoxins such as Shiga and ricin by blocking their requisite trans-port through the retrograde intracellular trafficking pathway.[9]

In addition to Retro-1, recent studies have identified otherclasses of compounds that act to increase oligonucleotide ef-fects by causing their release from endosomes,[10] suggestingthat this approach may be an important general tool for oligo-nucleotide pharmacology.

The fact that Retro-1 affects both toxin trafficking and oligo-nucleotide release from endosomes raises the question ofwhether the two effects involve the same or different targetswithin the cell. The observation that the effect on oligonucleo-tides requires a considerably higher concentration than theeffect on toxins suggests that more than one cellular targetmay be involved. To address this issue we have examineda number of Retro-1 analogues for their actions on both toxinsand oligonucleotides. We found compounds that affectedtoxins but not oligonucleotides and vice-versa, with Retro-1 being the only compound discovered thus far that could sig-nificantly affect both. This suggests that the molecular target(s)involved in enhancing oligonucleotide effectiveness are differ-ent from those involved in blockade of toxin trafficking.

A series of Retro compound analogues were tested for theirability to enhance the effect of a SSO in correcting splicing ofa luciferase reporter containing an aberrant intron.[7, 11] The ini-tial screening for oligonucleotide-related effects involved ap-proximately 50 compounds and was performed in 96-wellplates. Many of the tested compounds either failed to providesignificant induction of luciferase or clearly had substantial tox-icity or both. From the initial set, six compounds were chosenfor further testing. The structures of these compounds areshown in Figure 1. The compounds were tested for luciferaseinduction with correction for cell protein using an assay in 24-well plates, and were also tested for cytotoxicity.

As shown in Figure 2 a the compounds Retro-1 and HA061provided substantial enhancements of the effect of a SSO onluciferase induction, while compounds VP173, VP174 andVP184 provided modest but clear-cut enhancements. The com-pounds were effective in the concentration range of ~60–120 mm. In contrast, HA229 did not produce luciferase levelsthat were significantly different from baseline. In several repeatexperiments HA061 was consistently more effective thanRetro-1 while HA229 was inactive. When tested for cytotoxicity,all the compounds except VP184 displayed negligible toxicityover the concentration range tested for luciferase induction

[a] B. Yang, Dr. X. Ming, Dr. R. JulianoDivision of Molecular Pharmaceutics, UNC Eshelman School of Pharmacy,University of North Carolina, Chapel Hill, NC 27599 (USA)E-mail : arjay@med.unc.edu

[b] H. Abdelkafi, V. Pons, Dr. J.-C. Cintrat, Dr. J. BarbierService de Chimie Bio-organique et Marquage (SCBM), IBITECS, CEA, LabExLERMIT, Universit� Paris-Saclay, 91191, Gif-sur-Yvette (France)

[c] A. Michau, Dr. D. GilletService d’Ing�nierie Mol�culaire des Prot�ines (SIMOPRO), IBITECS, CEA,LabEx LERMIT, Universit� Paris-Saclay, 91191, Gif-sur-Yvette (France)

The ORCID identification number(s) for the author(s) of this article canbe found under http://dx.doi.org/10.1002/cmdc.201600463.

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(Figure 2 b). However, VP184 displayed considerable toxicity inthe concentration range where luciferase induction effectswere observed.

The compounds were also tested for their ability to relieveprotein synthesis inhibition caused by Shiga toxin.[8a] Com-pounds HA229, VP184 and Retro-1 substantially diminishedtoxin action, with HA229 being especially effective, whileVP173, VP174 and HA061 were without effect on toxins(Figure 3).

Thus the Retro analogues include compounds that affecttoxins but not oligonucleotides, such as HA229, and othercompounds that affect oligonucleotides but not toxins, suchas HA061, VP173 and VP174. Retro-1 is the only compound wehave identified thus far that has both actions at nontoxic con-centrations. While VP184 affects toxins it only affects oligonu-cleotides at concentrations that manifest substantial cytotoxici-ty. These results clearly indicate that toxin trafficking and en-hancement of oligonucleotide effects involve distinct targets,as there are molecules that exert one effect but not the other.

We had previously determined that Retro-1 enhances SSOactions by allowing release of the oligonucleotide from non-productive entrapment in endosomes thus providing access tothe cytosol and nucleus.[7] Therefore, we expected that ana-logues that enhanced oligonucleotide actions would cause oli-gonucleotides to escape from endosomes and reach the nu-cleus while those that failed to influence oligonucleotide ac-tions would not. Using confocal microscopy, we examined theeffects of the compounds on subcellular distribution of a fluo-rescent oligonucleotide (Figure 4 a,b). In the control cells theoligonucleotide was located within cytosolic vesicles, presuma-bly endosomes. As expected, Retro-1 caused partial redistribu-tion of the oligonucleotide from endosomes to the nucleus.Surprisingly, however, there was less evidence of nuclear locali-zation in cells treated with HA061; some faint nuclear fluores-cence was observed in a few cells, but this was clearly lessthan in the case of Retro-1.

Because the fluor-labeled oligonucleotide provided a relative-ly weak signal, we chose to further study effects of the com-pounds on endosomes by using a highly fluorescentAlexa 488–dextran as an endosomal marker, as we have previ-ously described.[10] As shown in Figure 4 c–j, in the control cellsthe dextran was present in intracellular vesicles with no evi-dence of accumulation in the cytosol or nucleus. In contrast,Retro-1 clearly caused partial but substantial redistribution ofthe dextran into the cytosol and nucleus. As expected therewas no release of dextran with the inactive compound HA229.However, we also observed only a minor redistribution usingHA061, clearly less than that observed with Retro-1.

An alternative explanation for the strong effect of HA061 onluciferase induction would be a direct action on RNA transcrip-tion or splicing. However, when we delivered the SSO by elec-troporation, thus bypassing all endocytotic mechanisms and al-lowing the oligonucleotide to directly access the cytosol andnucleus, there was no significant enhancement by HA061 overthe diluent control (Figure 5). This indicates that HA061 doesnot directly affect transcription or splicing. These observationsindicate that certain compounds, such as HA061, can enhance

Figure 1. Structures of Retro-1 and derivatives.

Figure 2. Retro analogues: enhancing effects on oligonucleotides and cyto-toxicity. a) Luciferase induction: HeLa Luc705 cells were preloaded overnightwith 100 nm SSO623 by incubation in complete medium. Cells were rinsedand then exposed to various concentrations of the test compounds for 2 hin DMEM + 1 % FBS. After removal of the compounds, cells were further in-cubated for 4 h, and luciferase activity and cell protein content were deter-mined. As a negative control a mismatched version of SSO623 (100 nm) wasalso tested at 90 mm for each analogue tested. Values are the mean�SEM ofn = 3 replicates ; results with mismatched oligonucleotide were at baselinelevels and are not shown. b) Cytotoxicity : Cells were exposed to compoundsunder the same conditions as in panel (a). The incubation was continued to24 h, and the viable cell number determined by Alamar Blue assay. Data nor-malized to control untreated cells : 100 %. Values are the mean�SEM ofn = 3 replicates. Differences in luciferase activity at 120 mm HA061, Retro-1,VP174, and VP173 versus the inactive compound HA229 are significant atthe 95 % level or greater, as determined by the Student t-test.

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delivery of oligonucleotide to nuclear splicesosomes withoutcausing an overall redistribution of oligonucleotide that isreadily detectable by fluorescence microscopy.

These studies demonstrate that the actions of Retro-typecompounds on toxins and on oligonucleotides involve differ-ent molecular targets. At nontoxic concentrations, one subsetof compounds influences toxin trafficking but not the actionsof oligonucleotides, while another subset has the converseeffect. This distinction implies that optimization of these com-pounds as toxin blockers and as oligonucleotide enhancersshould proceed along different synthetic chemistry pathways.Because their effective concentrations are in the 100 mm range,at present the Retro compounds are not sufficiently potent asoligonucleotide enhancers to warrant their evaluation in thera-peutic settings and thus additional structure–activity studieswill be needed to further develop these compounds. The cur-rent studies indicate that such oligonucleotide enhancingRetro analogues can be developed without impacting the vitalretrograde trafficking pathway.

A surprising sidelight to our studies concerns the apparentdifferences in the actions of Retro-1 and HA061. The enhancingeffect of Retro-1 is paralleled by an obvious redistribution ofoligonucleotide from endosomes to the nucleus. Most likelythis is via initial release to the cytosol, as seen with the fluores-cent dextran, followed by nuclear uptake. With HA061 a strongoligonucleotide enhancing effect is seen with only subtlechanges in visible oligonucleotide redistribution. This indicatesthe presence of an alternative trafficking pathway from endo-somes to the nuclear splicing machinery that is quite efficient.However, at this point we cannot suggest a definitive mecha-nism for this pathway.

Experimental Section

Synthesis : Strategies for the synthesis of Retro compounds havebeen described elsewhere.[8b]

Cellular assays of effect and toxicity : Effects of SSOs were testedusing HeLa cells stably transfected with a reporter cassette com-prised of the coding sequence of firefly luciferase interrupted byan abnormal intron (HeLa Luc705). Delivery of an appropriate SSOto the cell nucleus results in corrected splicing and increased ex-pression of the reporter.[7, 11] All oligonucleotide assays were donein the absence of any transfection agents other than the Retro ana-logues being tested. Cytotoxicity of the Retro compounds wasmonitored using the Alamar Blue assay.[12] Cell protein determina-tions were carried out with the BCA assay (Pierce) as per the sup-plier’s directions. The various Retro analogues were dissolved inDMSO at 30 mm and added directly to the cells in culture. Assayswere done in 96- or 24-well formats. The SSO used was termedSSO623 and was a 2’-O-methylphosphorothioate, whose sequencehas been described elsewhere.[7]

Intoxication assays with Shiga toxin (Stx): HeLa cells were main-tained at 37 8C under 5 % CO2 in Dulbecco’s modified Eagle’smedium (DMEM, Invitrogen) supplemented with 10 % fetal bovineserum (FBS), 4.5 g L

�1 glucose, 100 U mL�1 penicillin, 100 mg mL�1

streptomycin, 4 mm glutamine, 5 mm pyruvate. The cells wereplated at a density of 50 000 cells per well in 96-well Cytostar-TTM

scintillating microplates (PerkinElmer) with scintillator incorporatedinto the polystyrene plastic. After incubation with either 30 mm

Retro compound (or 0.1 % DMSO) for 4 h at 37 8C, cells were chal-lenged with increasing doses of Stx in the continued presence ofcompounds. After incubation for 20 h, the medium was removedand replaced with DMEM without leucine (Eurobio) containing10 % FBS, 2 mm l-glutamine, 0.1 mm non-essential amino acids,1 % penicillin/streptomycin supplemented by 0.5 mCi mL�1

[14C]leucine. The cells were grown for an additional 7 h at 37 8C in

Figure 3. Evaluation of protective activity toward Shiga toxin (Stx): HeLa cells were incubated for 4 h with test compound (30 mm, *) or carrier only (DMSO,*) before the addition of Stx at the indicated concentrations for 20 min. The media were removed and replaced with DMEM containing [14C]leucine at0.5 mCi mL�1 for 7 h before counting. Data points represent the mean�SEM of duplicates of a representative experiment.

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an atmosphere of 5 % CO2 and 95 % air. Protein biosynthesis wasthen determined by measuring the incorporation of radiolabeledleucine into cells using a Wallac 1450 MicroBeta liquid scintillationcounter (PerkinElmer). The mean percentage of protein biosynthe-sis was determined and normalized from duplicate wells. All valuesare expressed as means�SEM, and data were fitted with Prismver. 5 software (GraphPad Inc. , San Diego, CA, USA).

Confocal microscopy : Visualization of effects on endosomes in-volved the use of Alexa 488 labeled 10 000 Da dextran (Life Tech-nologies) or a TAMRA-labeled version of SSO623.[7] Cells wereimaged on an Olympus FV1000 MPE laser scanning confocal micro-scope with environmental chamber to maintain 37 8C, 40 % humidi-ty and 5 % CO2; images were collected with a 60 � oil immersionlens. Nuclear to cytoplasmic ratios of fluorescence were quantitat-ed using NIH Image J.

Acknowledgements

This work was supported by US National Institutes of Health(NIH) grant R01A151964 to R.L.J. and by the Joint Ministerial Pro-gram of R&D against CBRNE Risks, Agence Nationale de la Re-

Figure 4. a) Effects on the subcellular distribution of oligonucleotide: Hela Luc705 cells (50 000) were seeded into glass-bottom dishes and then incubatedovernight with 1 mm TAMRA-623 oligonucleotide. After removal of the oligonucleotide, cells were treated for 2 h in medium + 10 % FBS with 120 mm Retro-1 or HA061 and then rinsed. Live cells were observed using a confocal microscope with environmental stage. Yellow arrows indicate cells with distinct nuclearfluorescence. b) Nuclear/cytosolic intensity ratios of the TAMRA oligonucleotide. Values are the mean�SEM of n = 6 replicates. c)–j) Effects on endosome per-meability : Hela cells (50 000) were seeded into glass-bottom dishes and briefly incubated at 37 8C in DMEM + 10 % FBS for attachment. Alexa 488–dextran(10 kDa, 200 mg mL�1) was added to the medium, and the cells were incubated for 24 h and then rinsed. Cells were then placed in medium + 10 % FBS andtreated with 140 mm test compounds for 2 h (or left as untreated controls). The compounds were removed, the cells were further incubated for 24 h inDMEM + 10 % FBS, and live cells imaged with an Olympus FV1200 confocal microscope at 37 8C. Both fluorescence and DIC/fluorescence overlap images areshown. (c,d) untreated control, (e,f) Retro-1 treatment, (g,h) HA061 treatment, (i,j) HA229 treatment. Results shown are typical of three independent assays.

Figure 5. Electroporation of the SSO. HeLa Luc705 cells were electroporatedwith various amounts of SSO623 or with mismatched oligonucleotide(MM623). The cells were cultured for 3 h to allow cell attachment. Cells werethen treated for 2 h with 120 mm Retro-1 or HA061 or with DMSO diluent.After removal of the compounds, cells were incubated for an additional 4 hand then luciferase activity and cell protein content were determined.

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cherche (ANR, France) (grant Anti-HUS ANR-14-CE16-0004), LabExLERMIT (grant R3 RetroLeishma), the Conseil R�gional d’�le deFrance (grant from the DIM Malinf initiative 140101 and CEA) toD.G. , J.C.C. , and J.B.

Keywords: endocytosis · oligonucleotides · Retro-1 · toxins ·trafficking

[1] a) C. F. Bennett, E. E. Swayze, Annu. Rev. Pharmacol. Toxicol. 2013, 50,259 – 293; b) J. K. Watts, D. R. Corey, J. Pathol. 2011, 226, 365 – 379;c) J. C. Burnett, J. J. Rossi, Chem. Biol. 2012, 19, 60 – 71; d) R. L. Juliano,Nucleic Acids Res. 2016, 44, 6518 – 6548.

[2] M. T. Tse, Nat. Rev. Drug Discovery 2013, 12, 179.[3] a) J. Gilleron, W. Querbes, A. Zeigerer, A. Borodovsky, G. Marsico, U.

Schubert, K. Manygoats, S. Seifert, C. Andree, M. Stoter, H. Epstein-Barash, L. Zhang, V. Koteliansky, K. Fitzgerald, E. Fava, M. Bickle, Y. Ka-laidzidis, A. Akinc, M. Maier, M. Zerial, Nat. Biotechnol. 2013, 31, 638 –646; b) A. K. Varkouhi, M. Scholte, G. Storm, H. J. Haisma, J. ControlledRelease 2011, 151, 220 – 228.

[4] a) R. Kanasty, J. R. Dorkin, A. Vegas, D. Anderson, Nat. Mater. 2013, 12,967 – 977; b) J. Li, Y. Wang, Y. Zhu, D. Oupicky, J. Controlled Release 2013,172, 589 – 600.

[5] S. Akhtar, Expert Opin. Drug Metab. Toxicol. 2010, 6, 1347 – 1362.[6] R. Juliano, J. Bauman, H. Kang, X. Ming, Mol. Pharmaceutics 2009, 6,

686 – 695.[7] X. Ming, K. Carver, M. Fisher, R. Noel, J. C. Cintrat, D. Gillet, J. Barbier, C.

Cao, J. Bauman, R. L. Juliano, Nucleic Acids Res. 2013, 41, 3673 – 3687.[8] a) B. Stechmann, S. K. Bai, E. Gobbo, R. Lopez, G. Merer, S. Pinchard, L.

Panigai, D. Tenza, G. Raposo, B. Beaumelle, D. Sauvaire, D. Gillet, L. Jo-hannes, J. Barbier, Cell 2010, 141, 231 – 242; b) H. Abdelkafi, A. Michau,A. Clerget, D. A. Buisson, L. Johannes, D. Gillet, J. Barbier, J. C. Cintrat,ChemMedChem 2015, 10, 1153 – 1156.

[9] L. Johannes, C. Wunder, Traffic 2011, 12, 956 – 962.[10] B. Yang, X. Ming, C. Cao, B. Laing, A. Yuan, M. A. Porter, E. A. Hull-Ryde,

J. Maddry, M. Suto, W. P. Janzen, R. L. Juliano, Nucleic Acids Res. 2015,43, 1987 – 1996.

[11] M. R. Alam, V. Dixit, H. Kang, Z. B. Li, X. Chen, J. Trejo, M. Fisher, R. L. Ju-liano, Nucleic Acids Res. 2008, 36, 2764 – 2776.

[12] X. Ming, W. Ju, H. Wu, R. R. Tidwell, J. E. Hall, D. R. Thakker, Drug Metab.Dispos. 2009, 37, 424 – 430.

Received: September 9, 2016

Revised: October 12, 2016

Published online on && &&, 0000

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B. Yang, X. Ming, H. Abdelkafi, V. Pons,A. Michau, D. Gillet, J.-C. Cintrat,J. Barbier, R. Juliano*

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Retro-1 Analogues Differentially AffectOligonucleotide Delivery and ToxinTrafficking

Clarity in Retro-spect: Both oligonucle-otides (red ribbons) and toxins (orangesquares) enter cells by endocytosis andtraffic to early endosomes (EE). Oligostraffic to late endosomes (LE) andthence to lysosomes (LY), where theyare degraded. Toxins traffic via the Ret-romer complex (R) to the trans Golgi(TG) to become active. Retro-1 blockstoxin trafficking and also releases oligosfrom late endosomes.

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