A retro-inverso TAT-like peptide designed to deliver cysteamine to cells

Article abstract of DOI:10.1016/j.bmcl.2010.07.046

A retro-inverso, TAT-like peptide wherein lysine residues are replaced with cysteine residues bearing a disulfide-linked cysteamine group is found to engage in thiol-disulfide exchange with cysteine. These peptides are transported into cells and localize to lysosomes. Cellular uptake is enhanced in peptides bearing two cysteamine groups over those with one or none, by factors of approximately 1.5 and 12, respectively.

Full text of DOI:10.1016/j.bmcl.2010.07.046

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6321–6323
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A retro-inverso TAT-like peptide designed to deliver cysteamine to cells
Jongdoo Lim a, Jean-Philippe Pellois b, Eric E. Simanek a,
⇑
a Department of Chemistry Texas A&M University, College Station, TX 77843, USA
b Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 25 February 2010
Revised 9 July 2010
Accepted 12 July 2010
Available online 15 July 2010
A retro-inverso, TAT-like peptide wherein lysine residues are replaced with cysteine residues bearing a
disulfide-linked cysteamine group is found to engage in thiol–disulfide exchange with cysteine. These
peptides are transported into cells and localize to lysosomes. Cellular uptake is enhanced in peptides
bearing two cysteamine groups over those with one or none, by factors of approximately 1.5 and 12,
respectively.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Drug delivery
TAT peptide
Thiol–disulfide exchange
Cysteamine
Cystinosis
Cell uptake
Rare diseases
Cystinosis, a lysosomal storage disorder resulting from a defec-
tive cystine transporter, offers a compelling model for drug deliv-
ery vehicles. Cystinosis results from the accumulation of cystine
(cysteine disulfide) in lysosomes after proteolysis because of failed
lysosomal transport.1–5 This accumulation leads to intracellular
crystallization and oftentimes, renal failure.6 Cystinosis is usually
treated with large doses of cysteamine, 2-aminoethanethiol. Upon
disulfide bond formation with cysteine, the cysteine–cysteamine
molecule acts as a lysine isostere. This mimic can be effectively
transported out of lysosomes by a lysine transporter.
tide (1), its ability to engage in thiol-disulfide exchange with cys-
teine, and its ability to translocate into cells when charged with
cysteamine. This peptide and the analogues used in this study
are shown in Chart 1.
The peptides described derive from the TAT-sequence16,17 and
incorporate
D
-amino acids using the retro-inverso strategy18 to
provide materials that should be less susceptible to proteolysis
YGRKKRRQRRR
(derived from the HIV-1 TAT protein)
Treatment of cystinosis with cysteamine bitartrate (Cystagon™)
was approved by the FDA in 1994 and significantly increases life
expectancy by retarding the onset of thyroid and renal failure.7,8
Cysteamine therapy, however, has limitations. Side effects of the
drug include nausea and vomiting, difficulty in walking, and odor.
Pediatric resistance to oral therapy has led to the investigation of
other strategies for drug delivery including catheterization, en-
zyme replacement therapy, and cell transplantation.9–11 This situ-
ation has also inspired the design of new pro-drugs including
phosphocysteamine,12 amino acid derivatives of cysteamine or
cystamine,13 and lipophilic derivatives of cysteamine.14
RRRQRRCcystCcystRGY
1
TMR-RRRQRRCcystCcystRGY
2
TMR-RRRQRRCcystCRGY
3
TMR-RRRQRRCCcyst RGY
4
Opportunities for enhancing treatment by facilitating delivery
of this rapidly-cleared, charged small molecule might be realized
by integrating the drug into a more bioavailable agent.15 Here we
investigate cysteamine delivery to lysosomes using a TAT-like pep-
TMR-RRRQRRCCRGY
5
Chart 1. The TAT-like peptides described in this study. All amino acids of 1–5 are D-
configuration. TMR is 5(6)-carboxytetramethylrhodamine. The designation ‘Ccyst’
refers to the cysteamine disulfide of cysteine. Isomers 3 and 4 are arbitrarily
assigned.
⇑
Corresponding author.
E-mail address: simanek@tamu.edu (E.E. Simanek).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.07.046

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J. Lim et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6321–6323
Figure 1. HPLC traces of a thiol–disulfide exchange reaction of peptide 1 in 1 mM
cysteine (aq) after 30 min (blue line), 6 h (red line), and 24 h (green line),
respectively.
than the corresponding peptide comprising L-amino acids. Wender
has shown that retro-inverso peptides gain entry to cells in a se-
quence-dependent manner.19 Two observations by Wender are
exploited in the design. First, the retro-inverso TAT is more effec-
tively transported than the natural analog. Second, lysine residues
which are replaced here with cysteine–cysteamine disulfides ap-
pear to be the least important for effective trafficking.
Figure 3. Average fluorescence intensity of 50 cells incubated with the TAT-like
peptides.
and a cysteine–cysteamine complex begin to appear after 30 min
of reaction (blue trace). The level of a cysteine–cysteamine com-
plex, the critical product, was significantly higher than that of cys-
tine at 6 h (red trace). The release of cysteamine is also observed in
the reaction. Peptide 1 yielded a peptide with an intramolecular
disulfide bond as the major product which eluted uniquely at
26.6 min by HPLC and was confirmed by MALDI-TOF mass spec-
trometry. The results encouragingly show that the peptide carrying
cysteamine can productively engage in thiol–disulfide exchange to
provide the desired mixed disulfide cysteine–cysteamine interme-
diate. The equilibrium between 1 and free cysteine is complex with
many species readily formulated hypothetically. In addition to 1
and its two isomeric single thiol/single cysteamine adducts, its
fully reduced form, and its intra-disulfide form, cysteine can ex-
change to form an additional five adducts. Indeed, following the
course of the equilibrium reaction between peptide and cysteine
by HPLC reveals a rich diversity of compounds which will be pur-
sued in future studies.
To obtain the peptides, solid phase peptide synthesis was exe-
cuted on using Fmoc-protected D-amino acids. Side chain protect-
ing groups included t-butyl (Tyr), trityl (Gln and Cys), and
2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Arg). Upon
cleavage from the resin and isolation, the crude peptide was re-
acted with 2-(2-pyridyditho)ethylamine to provide peptide (1)
charged with two cysteamine groups. The resulting product was
purified by HPLC and characterized by 1D and 2D NMR spectros-
copy and mass spectrometry. Installation of a fluorescent dye,
5(6)-carboxytetramethylrhodamine (TMR) was accomplished on
solid phase. The TMR-labeled peptides were cleaved from the resin,
isolated, and reacted with the pyridylthio-activated cysteamine to
provide the desired peptide bearing two cysteamine molecules (2)
and the side products bearing one cysteamine molecule (3 and 4 as
isomers arbitrarily assigned), and no cysteamine molecules (5),
respectively. HPLC and MALDI-TOF mass spectrometry was used
to identify these products.
In order to investigate the thiol–disulfide exchange reaction,
For this construct to productively participate in drug delivery,
cellular uptake is critical. We examined the ability of these pep-
tides to enter cells using fluorescence microscopy. Compound 2
peptide 1 (at 0.36 mM) was incubated in 1 mM L-cysteine solution
(aq). The reaction was analyzed by HPLC at 30 min, 6 h, and 24 h,
respectively. As shown in Figure 1, cystine (the Cys-Cys disulfide)
Figure 2. Confocal fluorescence images of a representative HeLa cell expressing Cerulean Lamp-1 (pseudocolored green) after treatment with compound 2 (pseudocolored
red). Colocalization of 2 and late endosomes/lysosomes is illustrated by the presence of the yellow color in the red/green overlay image.

J. Lim et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6321–6323
6323
(1 lM) was incubated for 1 h with cells transiently expressing
References and notes
Lamp-1 Cerulean, a blue fluorescent protein marker of late endo-
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in fresh media for two additional hours for the compound to accu-
mulate in lysosomes. Figure 2 shows a clear colocalization between
the TMR label and lysosomes as expected.20
Incubation of cells with 2–4 and 5 show an interesting trend
(Fig. 3). Of the peptides, peptide 2 is endocytosed at the highest le-
vel. The partly reduced peptides 3 and 4 shows markedly less up-
take, and little uptake is seen for the full reduced molecule, 5.
The drug delivery strategy proposed is designed to take advan-
tage of the equilibrium resulting from thiol–disulfide exchange.
Physiological conditions and disease state productively reinforce
this design and may offer an opportunity to use peptides like 1
in a catalytic role. For this peptide to be a therapeutic phase trans-
fer catalyst, it must enter cells, release cysteamine, be excreted, be
recharged with cysteamine, and be recycled. These elements will
be explored and reported in due course.
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The NIH (EES R01 NIGMS 64560) is thanked for support. Dr.
Bruce Barshop is thanked for helpful discussions.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.07.046.