Inhibition of Ricin by an RNA Stem-Loop Containing a Ribo-Oxocarbenium Mimic

Full text of DOI:10.1021/ja9600385

J. Am. Chem. Soc. 1996, 118, 3067-3068
3067
Inhibition of Ricin by an RNA Stem-Loop
Containing a Ribo-Oxocarbenium Mimic
Xiang-Yang Chen, Todd M. Link, and Vern L. Schramm*
Department of Biochemistry
Albert Einstein College of Medicine of
YeshiVa UniVersity, 1300 Morris Park AVenue
Bronx, New York 10461
ReceiVed January 4, 1996
Ricin is a cytotoxic heterodimeric protein isolated from castor
beans.1 It is one of the most toxic substances, with a single
ricin molecule being lethal for eukaryotic cells.2 Ricin’s toxic
nature is derived from its catalytic subunit, ricin toxin A-chain
(RTA), which inactivates ribosomes and thereby destroys protein
synthesis. Inactivation results from hydrolysis of a single
N-ribosidic bond at adenosine 4324 in 28S rRNA.3 It is
postulated that adenine depurination occurs through an oxocar-
benium ion transition state (Figure 1), similar to that for AMP
nucleosidase.4 The depurination site is located in a highly
conserved stem-loop region, which has been established as a
GA4324GA tetraloop.5 NMR structural studies indicate that
stem-loop oligonucleotides with the GAGA tetraloop motif fold
compactly in solution with an unusual base interaction between
the first G and second A in the tetraloop. This G‚A mismatch
creates a sharp turn between the first G and first A in the
phosphodiester backbone, exposing both the ribosyl moiety and
the unpaired adenine base for catalytic attack by RTA.6 The
GAGA tetraloop is essential for ricin enzymatic activity, while
the sequence of the stem is only important to maintain stem-
loop structure identity. However, RNA structures without stems
have been shown to be poor substrates for RTA.7 The minimal
substrate for ricin is reported to be an RNA 10-mer with the
GAGA tetraloop.
It is of interest to obtain specific inhibitors of RTA due to
its use in immunochemotherapy. Hydrolysis of the N-ribosidic
bond of adenosine by nucleoside hydrolase from Crithidia
fasciculata is chemically similar to that catalyzed by the RTA
reaction.8 Nucleoside hydrolase stabilizes an oxocarbenium-
ion transition state, characterized by protonation of the leaving
group and distortion of the ribose toward the oxocarbenium.9
Phenyliminoribitol was synthesized as a transition state inhibitor
of this enzyme and inhibits with a Kd of 30 nM.10,11 We
proposed that ricin A-chain may also be subject to inhibition
Figure 1. Proposed oxocarbenium-ion transition state of the ricin
A-chain N-ribohydrolase reaction and a proposed inhibitor for incor-
poration into the site for depurination by ricin A-chain.
by phenyliminoribitol when placed in the suitable RNA context.
In this communication, we report an approach to site-specific
incorporation of phenyliminoribitol into stem-loop RNA at the
RTA depurination site by chemical solid-phase RNA synthesis
and describe its inhibitory properties.
Phenyliminoribitol (1, Scheme 1)11 was protected by treatment
with excess trifluoroacetic anhydride in dry pyridine, and
subsequent O-hydrolysis in methanol containing two drops of
concentrated ammonium hydroxide.12 N-trifluoroacetylated 2
was protected with the dimethoxytrityl (DMT) group using
DMTCl.13 Silylation of 3 with tert-butyldimethylsilyl chloride
(TBDMSCl) gave a mixture of 2′- and 3′-silyl ethers in a ratio
of 1:2, and attempts to improve the selectivity were unsuccess-
ful.14 The desired 2′-silyl ether 4 was isolated by normal-phase
HPLC (1% ethyl acetate in methylene chloride) or flash
chromatography (methylene chloride:hexane ) 8:1, Rf ) 0.49
in CH2Cl2), and its structure was assigned by COSY NMR.15
Phosphitylation of 4 yielded crude phosphoramidite 5.16 With-
out further purification, 5 was subsequently incorporated into
the oligonucleotide, CGCGC GXGA GCGCG (X-14, Figure
1), by standard solid-phase RNA synthesis.17 Equivalent DMT
release was observed during reactions of G, C, and 5. The
oligonucleotide was deprotected,17 purified by DEAE ion-
exchange HPLC, eluting with 0-1.2 M ammonium acetate in
20% acetonitrile, and desalted on a G-10 Sephadex column in
H2O.18 The presence of phenyliminoribitol was verified by
digesting X-14 with snake venom phosphodiesterase and calf
intestinal alkaline phosphatase followed by reversed-phase
HPLC analysis. In addition, pXpGp was synthesized according
to the same procedure.20
* Author to whom correspondence should be addressed: telephone (718)
430-2813; FAX (718) 430-8565; email vern@aecom.yu.edu.
(1) (a) Olsnes, S.; Pihl, A. Biochemistry 1973, 12, 3121-3126. (b)
Barbieri, L.; Battelli, M. G.; Stirpe, F. Biochim. Biophys. Acta 1993, 1154,
237-282.
(2) Olsnes, S.; Pihl, A. In Molecular Action of Toxins and Viruses; Cohen,
P., van Heyningen, S., Eds.; Elsevier Biomedical: New York, 1982; pp
51-105.
(3) Endo, Y.; Tsurugi, K. J. Biol. Chem. 1987, 262, 8128-8130.
(4) (a) Mentch, F.; Parkin, D. W.; Schramm, V. L. Biochemistry 1987,
26, 921-930. (b) Lord, J. M.; Roberts, L. M.; Robertus, J. D. FASEB J.
1994, 8, 201-208.
(5) (a) Endo, Y.; Tsurugi, K. J. Biol. Chem. 1988, 263, 8735-8739. (b)
Endo, Y.; Gluck, A.; Wool, I. G. J. Mol. Biol. 1991, 221, 193-207. (c)
Gluck, A.; Endo, Y.; Wool, I. G. Nucleic Acids Res. 1994, 22, 321-324.
(6) (a) Szewczak, A. A.; Moore, P. B.; Chan, Y.-L.; Wool, I. G. Proc.
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1993, 21, 5670-5678.
(7) Gluck, A.; Endo, Y.; Wool, I. G. J. Mol. Biol. 1992, 226, 411-424.
(8) Parkin, D. W.; Horenstein, B. A.; Abdulah, D. R.; Estupinan, B.;
Schramm, V. L. J. Biol. Chem. 1991, 266, 20658-20665.
(9) Horenstein, B. A.; Parkin, D. W.; Estupinan, B.; Schramm, V. L.
Biochemistry 1991, 30, 10788-10795.
(10) Horenstein, B. A.; Schramm, V. L. Biochemistry 1993, 32, 9917-
9925.
A synthetic oligonucleotide 10-mer (A-10, Figure 1) was
synthesized as a RTA substrate. Samples were injected onto a
(12) (a) Sharma, R. A.; Bobek, M.; Bloch, A. J. Org. Chem. 1974, 17,
466-468. (b) The Fmoc group has also been used to block pyrrolidine
nitrogen for solid-phase synthesis. See: Scharer, O. D.; Ortholand, J.-Y.;
Ganesan, A.; Ezaz-Nikpay, K.; Verdine, G. L. J. Am. Chem. Soc. 1995,
117, 6623-6624.
(13) Oligonucleotides and Analogues: A Practical Approach; Eckstein,
F., Ed.; IRL: Oxford, 1991.
(14) Hakimelahi, G. H.; Proba, Z. A.; Ogilvie, K. K. Can. J. Chem. 1982,
60, 1106-1113.
(15) Also see: Altmann, K.-H.; Freier, S. M.; Pieles, U.; Winkler, T.
Angew. Chem., Int. Ed. Engl. 1994, 33. 1654-1657.
(16) Scaringe, S. A.; Francklyn, C.; Usman, N. Nucleic Acids Res. 1990,
18, 5433-5441.
(17) Protocols for Oligonucleotides and Analogs: Synthesis and Proper-
ties; Agrawal, S., Ed.; Methods in Molecular Biology 20; Humana: New
Jersey, 1993.
(18) Sproat, B.; Colonna, F.; Mullah, B.; Tsou, D.; Andrus, A.; Hampel,
A.; Vinayak, R. Nucleosides Nucleotides 1995, 14, 255-273.
(19) Allerson, C. R.; Verdine, G. L. Chem. Biol. 1995, 2, 667-675.
(20) Phosphorylation of XpG was achieved by automated chemical
synthesis. Electrosprary mass spectrometry gave the expected m/z ) 714.
(11) Horenstein, B. A.; Zabinski, R. F.; Schramm, V. L. Tetrahedron
Lett. 1993, 34, 7213-7216.
0002-7863/96/1518-3067$12.00/0 © 1996 American Chemical Society

3068 J. Am. Chem. Soc., Vol. 118, No. 12, 1996
Communications to the Editor
Scheme 1a
a Conditions: (a) (i) (CF3CO)2O, py; (ii) NH4OH, MeOH, 84%; (b)
DMTCl, Et3N, DMAP, py, 81%; (c) TBDMSCl, AgClO4, py, THF,
31%; (d) 2-cyanoethyl N,N-diisopropylchlorophosphoramidite, 2,4,6-
collidine, N-methylimidazole, CH2Cl2.
reversed-phase C18 analytic column with isocratic elution in
50 mM ammonium acetate (pH 5.0) containing 10% methanol,
at a flow rate of 1 mL/min. The adenine peak was compared
to adenine standards treated in the same way. Km and kcat of
stem-loop RNA A-10 were determined to be 6.2 ( 0.7 µM
and 1.7 ( 0.1 min-1, respectively (Figure 2, upper), similar to
those recently reported for a 19-base stem-loop RNA.19
Addition of the stem-loop X-14 at 4 µM caused 75%
inhibition of RTA using 10 µM A-10 as substrate. The
inhibition data (Figure 2, lower) gave a good fit to the expression
V ) (kcatA)/(Km(1 + I/Ki) + A), which assumes that A-10 and
X-14 compete for the catalytic site. The inhibition constant
(Ki) is 0.73 ( 0.02 µM. Neither phenyliminoribitol (X) nor
pXpGp exhibited significant inhibition at concentrations of 200
and 100 µM, respectively. Thus, the inhibitor is most efficient
in the stem-loop structure.
The mechanism of action of RTA hydrolysis has been
proposed by X-ray crystal structural analysis.21ab In the crystal
structure, the adenine ring π-stacks between tyrosines 80 and
123, while the proposed oxocarbenium ion could be stabilized
by ion pairing to Glu 177. When phenyliminoribitol is
positioned at the targeted adenosine site, it would fit into the
proposed structure by hydrogen bonding between nitrogen on
the azasugar ring with Glu 177 and π-stacking of phenyl ring
between Tyr 80 and 123 at the RTA active site, similar to the
interactions proposed for 1 with nucleoside hydrolase.22 There-
fore, good binding is achieved between X-14 and RTA.
Although the binding of X-14 to RTA is 10-fold better than
for the substrate, it is weaker than expected for an efficient
transition state inhibitor.23 Leaving-group interactions are
known to be important in considering transition state interactions
of related N-ribohydrolases24 and will need to be considered in
improved inhibitor design for RTA.
Figure 2. Substrate saturation of RTA by A-10 (upper panel).
Reactions were in 50 µL of buffer (10 mM KCl, 1 mM EDTA, and 10
mM Tris-HCl (pH 7.5)) containing 0.2 µM RTA and the indicated
concentrations of A-10. After incubation at 37 °C for 10 min, the
released adenine was analyzed by HPLC. The rate of adenine release
was linear with time to approximately 30 min. The line was drawn
from a fit of the experimental data to the equation for a rectangular
hyperbola to obtain values of Km and kcat. The lower panel shows the
inhibiton of RTA by X-14 using 10 µM stem-loop A-10 as substrate.
Reactions were in 50 µL of the same buffer containing 1.2 µM RTA,
10 µM A-10, and the indicated concentrations of X-14. After incubation
at 37 °C for 30 min, the released adenine was analyzed by HPLC. The
line is the best fit of the data to the equation for competitive inhibition
(see text).
In summary, we have incorporated a purine N-glycohydrolase
inhibitor into stem-loop RNA and developed a convenient assay
for screening inhibitors with RTA. Stem loop RNA with an
oxocarbenium-ion mimic at the RTA depurination site exhibits
promising submicromolar inhibition activity for the hydrolytic
action of RTA. Further study of the RTA depurination
mechanism is being conducted to develop the next generation
of inhibitors for ricin and its potential chemotherapeutic
applications.
Acknowledgment. This work was supported by the U.S. Army
Medical Research and Development Command under Contract No.
DAMD 17-93-C-3051. The views, opinions, and/or findings contained
in this report are those of the authors and should not be construed as
an official Department of the Army position, policy, or decision unless
so designated by other documentation. The authors thank Dr. Jon
Robertus, University of Texas, Austin, for the generous supply of ricin
A-chain.
(21) (a) Monzingo, A. F.; Robertus, J. D. J. Mol. Biol. 1992, 227, 1136-
1145. (b) Weston, S. A.; Tucker, A. D.; Thatcher, D. R.; Derbyshire, D. J.;
Pauptit, R. A. J. Mol. Biol. 1994, 244, 410-422.
(22) Parkin, D. W.; Schramm, V. L. Biochemistry 1995, 34, 13961-
13966.
(23) Schramm, V. L.; Horenstein, B. A.; Kline, P. C. J. Biol. Chem.
1994, 269, 18259-18262.
JA9600385
(24) Mazzella, L. J.; Parkin, D. W.; Tyler, P. C.; Furneaux, R. H.;
Schramm, V. L. J. Am. Chem. Soc., in press.