Site-specific folate conjugation to a cytotoxic protein

Article abstract of DOI:10.1016/j.bmcl.2011.04.081

Conjugation to folic acid is known to enhance the uptake of molecules by human cells that over-produce folate receptors. Variants of bovine pancreatic ribonuclease (RNase A) that have attenuated affinity for the endogenous ribonuclease inhibitor protein (

Full text of DOI:10.1016/j.bmcl.2011.04.081

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5029–5032
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Site-specific folate conjugation to a cytotoxic protein
Bryan D. Smith a, , Joshua J. Higgin b,à, Ronald T. Raines a,b,
⇑
a Department of Biochemistry, University of Wisconsin–Madison, 433 Babcock Drive, Madison, WI 53706, USA
b Department of Chemistry, University of Wisconsin–Madison, 1101 University Avenue, Madison, WI 53706, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Conjugation to folic acid is known to enhance the uptake of molecules by human cells that over-produce
folate receptors. Variants of bovine pancreatic ribonuclease (RNase A) that have attenuated affinity for
the endogenous ribonuclease inhibitor protein (RI) are toxic to mammalian cells. Here, the random acyl-
ation of amino groups in wild-type RNase A with folic acid is shown to decrease its catalytic activity dra-
Received 23 March 2011
Revised 15 April 2011
Accepted 19 April 2011
Available online 24 April 2011
matically, presumably because of the alteration to a key active-site residue, Lys41. To effect site-specific
a
coupling, Nd-bromoacetyl-N -pteroyl-
L-ornithine, which is a folate analogue with an electrophilic bromo-
Keywords:
Bioconjugation
Cancer
Cytotoxin
Folic acid
acetamido group, was synthesized and used to S-alkylate Cys88 of the G88C variant of RNase A. The pen-
dant folate moiety does not decrease enzymatic activity, enables RI-evasion, and endows toxicity for
cancer cells that over-produce the folate receptor. These data reveal a propitious means for targeting pro-
teins and other molecules to cancer cells.
Ó 2011 Elsevier Ltd. All rights reserved.
Ribonuclease
Ribonuclease inhibitor
Secretory ribonucleases such as bovine pancreatic ribonuclease
(RNase A1; EC 3.1.27.5) can enter cells and evince a cytotoxic enzy-
matic activity.2–4 This attribute is rare amongst mammalian pro-
teins. In response, cells have evolved a femtomolar proteinaceous
antagonist, the ribonuclease inhibitor (RI), to mitigate damage
from secretory ribonucleases that enter the cytosol.5,6 Although
wild-type RNase A is not cytotoxic, variants that evade RI display
notable cytotoxic activity.7,8
The toxicity of ribonucleases is limited by the amount of en-
zyme that reaches the cytosol.9,10 For example, microinjection
experiments show that RNase A is ꢀ106-fold more toxic when in-
jected than when added to the extracellular medium.11 Increasing
the positive charge on RNase A by mutagenesis or chemical modi-
fication enhances its internalization and cytotoxicity.12–14 We
sought a more precise means to deliver ribonucleolytic activity
to the RNA of cells, especially those related to human disease.
Folic acid (vitamin B9) mediates much of one-carbon metabo-
lism, including the synthesis, repair, and methylation of DNA.
Receptors for folic acid are over-produced on the surface of many
types of cancer cells, such as those derived from ovarian, endome-
trial, and brain carcinomas.15,16 Attachment of folate to small
molecules, liposomes, and proteins can enhance their cellular
internalization.17 We reasoned that the conjugation of folate to a
ribonuclease could enhance its binding and uptake by cells that
over-produce the folate receptor, endowing toxic activity towards
those cells.
Historically, the coupling of folate to proteins has relied on non-
specific acylation reactions that lead to a heterogeneous mixture of
conjugates.18 Such random conjugation can affect adversely the
activity of a toxin.19 Herein, we describe the design, synthesis,
and use of a new folate analogue equipped for facile attachment
to a sulfhydryl group, as is found in the side chain of a cysteine res-
idue. We find that coupling a single folate moiety can render wild-
type RNase A into a cytotoxin.
We designed folate analogue 1 for site-specific conjugation to a
free cysteine residue in a protein. Our synthetic route to analogue 1
utilized the clever method of Fuchs and co-workers to excise an
activated pteroyl group from folic acid.20 We then availed solid-
phase chemistry to ease purification and avoid confounding
insolubility while reinstalling the
electrophilic bromoacetamido group in place of the nonessen-
tial21,17
-carboxyl group. We synthesized pteroyl azide in four
steps according to the method of Fuchs and co-workers (Fig. 1).20
Next, we coupled the pteroyl azide to -ornithine on a resin.
Finally, we N-acylated the resin-bound pteroyl- -ornithine with
a-carboxyl group along with an
c
L
L
bromoacetylbromide. Cleavage from the resin provided folate
analogue 1.
We prepared ribonuclease–folate conjugates in two distinct
ways: random and site-specific. For random conjugation, folate it-
self was activated with the water-soluble carbodiimide 1-ethyl-3-
⇑
Corresponding author. Tel.: +1 608 262 8588; fax: +1 608 890 2583.
E-mail address: rtraines@wisc.edu (R.T. Raines).
Current address: Deciphera Pharmaceuticals, LLC, 643 Massachusetts Street,
Suite 200, Lawrence, KS 66044, USA.
à
Current address: Research Triangle Institute, 3040 East Cornwallis Road, Research
(3-dimethylaminopropyl)carbodiimide) (EDC). Then,
a slight
Triangle Park, NC 27709, USA.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.04.081

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B. D. Smith et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5029–5032
Figure 2. Mass spectra of random and site-specific ribonuclease–folate conjugates.
(A) Mass spectrum of K41R/G88R–folate, which was prepared by the random
conjugation of folic acid. (n = 1, m/z 14,250; expected: 14,232). (B) Mass spectrum of
G88C–folate, which was prepared by site-specific conjugation of folate analogue 1
(m/z 14,205; expected: 14,195). Major peaks are labeled with the number of folate
moieties, n. Minor peaks are photochemically generated adducts of the matrix (3,5-
dimethoxy-4-hydroxycinnamic acid) with the proteins.
a
Figure 1. Scheme for the synthesis of Nd-bromoacetyl-N -pteroyl-
L-ornithine (1).
Pteroyl azide was synthesized from folic acid in four steps as described
previously.20
Table 1
Enzymatic activity of RNase A variants and folate conjugates
Protein/conjugate
kcat/KM (106 MÀ1 sÀ1
)
a
excess of the activated folate was added to RNase A variants to
acylate primary amino groups randomly with folate. In addition
to the randomness in their site(s) of conjugation, conjugates pro-
duced by this method were found to be heterogeneous in the num-
ber of folates conjugated per protein (Fig. 2). The catalytic activity
of secretory ribonucleases is necessary for their cytotoxicity.23
RNase A has amino groups in the side chain of 10 lysine residues
and at its N-terminus. Lys7, Lys41, and Lys66 are active-site resi-
dues that are important for catalysis.24,25 The side-chain amino
group of Lys41 has a low pKa value26 and thus suffers especially ra-
pid acylation. Its modification can reduce catalytic proficiency by
105-fold.24 Indeed, we found that RNase A subjected to random
acylation by folic acid retains only 0.4% of its catalytic activity
(Table 1). Hence, we also attached folate randomly to K41R RNase
A, wherein the guanidino group in the side chain of Arg41 retains a
positive charge but acts, in effect, as a protecting group that pre-
cludes acylation by an activated folate. The K41R variant has 56-
fold less catalytic activity than does wild-type RNase A, but retains
54% of its catalytic activity upon conjugation (Table 1). Accord-
ingly, the catalytic activity of K41R–folate is 2-fold greater than
that of wild-type-folate. Finally, we attached folate randomly to
K41R/G88R RNase A. Gly88 is at the RI–RNase A interface (Fig. 3).
The G88R substitution diminishes the affinity for RI by 104-fold.27
As with the K41R variant, K41R/G88R RNase A suffers only a mod-
est decrease in enzymatic activity upon folate conjugation
(Table 1).
Wild-type
Wild-type-folate
3.0 0.2
0.013 0.006
K41R
0.054 0.003
0.029 0.006
0.055 0.003
0.044 0.005
K41R–folate
K41R/G88R
K41R/G88R–folate
G88R
G88C–folate
A19C–folate
1.9 0.2
4.3 0.7
3.9 0.4
a
Values of kcat/KM
(
SE) were determined for catalysis of 6-FAM–dArUdAdA–
6-TAMRA cleavage in 0.05 M MES–NaOH buffer, pH 6.0, containing NaCl (0.10 M) at
25 °C.22
In contrast to lysine, cysteine is rare in proteins.28 Moreover,
cysteine residues are often engaged in disulfide bonds, which pro-
tect them from alkylation. For example, RNase A has eight cysteine
residues that form four disulfide bonds. Accordingly, a cysteine res-
idue installed by site-directed mutagenesis in RNase A would dis-
play the only enzymic sulfhydryl group.
For site-specific conjugation, we used folate analogue 1 to
S-alkylate a cysteine installed as residue 19 or 88 in RNase A. The
folate installed at residue 88 served a dual purpose—target the
conjugate to a cell-surface folate receptor and endow evasion of
cytosolic RI. Unlike residue 88, residue 19 is distal from the RI–
RNase A interface (Fig. 3), and installing folate there serves as a

B. D. Smith et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5029–5032
5031
and G88C–folate (which is a site-specific conjugate) inhibit DNA
synthesis, with IC50 = 11 and 9 M, respectively. No other conju-
gate exhibited a detectable effect on DNA synthesis at a concentra-
tion of 25 M. These data are in gratifying agreement with the
l
l
results shown in Figure 4—only those folate conjugates that have
weakened affinity for RI diminish cell proliferation. Apparently, en-
hanced cellular internalization mediated by a pendant folate moi-
ety must be accompanied by RI-evasion to enable a ribonuclease to
be cytotoxic. The ramifications of this duality are the basis for on-
going work in our laboratory.
In summary, we put forth a new folate analogue, 1, that can be
used to install a folate moiety at a designated site in a protein. Un-
like random folate conjugation, site-specific folate conjugation
need not compromise protein function. Importantly, site-specific
conjugation allows for the preparation of a homogeneous toxin,
which facilitates molecular characterization and optimization.
We anticipate that folate analogue 1 could be used to create useful
site-specific folate conjugates of a variety of cytotoxins.
Acknowledgments
This Letter is dedicated to Professor Carolyn R. Bertozzi on
the occasion of her winning the 2011 Tetrahedron Young Inves-
tigator Award in Bioorganic and Medicinal Chemistry. This work
was supported by grant R01 CA073808 (NIH). The Biophysics
Instrumentation Facility was established at the University of
Wisconsin–Madison with Grants BIR-9512577 (NSF) and S10
RR13790 (NIH). B.D.S. was supported by Biotechnology Training
Grant T32 GM008349 (NIH). We are grateful to P. A. Leland, K.
E. Staniszewski, and K. A. Dickson for help with conjugation
and cytotoxicity protocols, and for contributive discussions. We
also thank J. E. Lee, S. M. Fuchs, M. B. Soellner, and L. D. Lavis
for advice.
Figure 3. Three-dimensional structure of the complex between RNase A (blue) and
RI (red). The location of the three residues substituted herein are shown explicitly.
Images were made with PyMol (Delano Scientific) and Protein Data Bank entry
1dfj.29
Supplementary data
Supplementary data (experimental procedures and molecular
characterization) associated with this article can be found, in the
online version, at doi:10.1016/j.bmcl.2011.04.081.
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