Antitumor activity of ribonuclease multimers created by site-specific covalent tethering

Article abstract of DOI:10.1021/bc100292x

Site-specific cross-linking can generate homogeneous multimeric proteins of defined valency. Pancreatic-type ribonucleases are an especially attractive target, as their natural dimers can enter mammalian cells, evade the cytosolic ribonuclease inhibitor (

Full text of DOI:10.1021/bc100292x

NIH Public Access
Author Manuscript
Bioconjug Chem. Author manuscript; available in PMC 2011 September 15.
Published in final edited form as:
Bioconjug Chem. 2010 September 15; 21(9): 1691–1702. doi:10.1021/bc100292x.
Antitumor Activity of Ribonuclease Multimers Created by Site-
Specific Covalent Tethering
†,‡
§
§
∥
Thomas J. Rutkoski , John A. Kink , Laura E. Strong , Christine I. Schilling , and Ronald
T. Raines
†,#,*
†Department of Biochemistry, University of Wisconsin–Madison, Madison, Wisconsin 53706, USA
§Quintessence Biosciences, Inc., 505 South Rosa Road, Madison, Wisconsin 53719, USA
∥Institut für Organische Chemie, Universität Karlsruhe (TH), Fritz-Haber Weg 6, D-76131
Karlsruhe, Germany
#Department of Chemistry, University of Wisconsin–Madison, Madison, Wisconsin 53706, USA
Abstract
Site-specific cross-linking can generate homogeneous multimeric proteins of defined valency.
Pancreatic-type ribonucleases are an especially attractive target, as their natural dimers can enter
mammalian cells, evade the cytosolic ribonuclease inhibitor (RI), and exert their toxic
ribonucleolytic activity. Here, we report on the use of eight distinct thiol-reactive cross-linking
reagents to produce dimeric and trimeric conjugates of four pancreatic-type ribonucleases. Both
the site of conjugation and, to a lesser extent, the propinquity of the monomers within the
conjugate modulate affinity for RI, and hence cytotoxicity. Still, the cytotoxicity of the multimers
is confounded in vitro by their increased hydrodynamic radius, which attenuates cytosolic entry. A
monomeric RI-evasive variant of bovine pancreatic ribonuclease (RNase A) inhibits the growth of
human prostate and lung tumors in mice. An RI-evasive trimeric conjugate inhibits tumor growth
at a lower dose and with a less frequent administration than does the monomer. This effect is
attributable to an enhanced persistence of the trimers in circulation. On a molecular basis, the
trimer is ~300-fold more efficacious and as well tolerated as erlotinib, which is in clinical use for
the treatment of lung cancer. These data encourage the development of mammalian ribonucleases
for the treatment of human cancers.
INTRODUCTION
Bovine seminal ribonuclease (BS-RNase1) and several amphibian ribonucleases, including
®
ranpirnase (Onconase ; ONC), exhibit innate antitumoral activity (1-3). ONC has
demonstrated responses in patients with breast, non-small cell lung, and esophageal cancer,
*Corresponding author at Department of Biochemistry, University of Wisconsin–Madison, 433 Babcock Drive, Madison, WI
53706-1544. Tel: 608-262-8588. Fax: 608-262-3453. rtraines@wisc.edu..
‡
Current address: Deciphera Pharmaceuticals, LLC, 643 Massachusetts Street, Suite 200, Lawrence, KS 66044.
Supporting Information Available: Figures S1 and S2. This material is available free of charge via the internet at http://pubs.acs.org.
1
Abbreviations: BSA, bovine serum albumin; BS-RNase, bovine seminal ribonuclease; DMF, dimethylformamide; DPBS, Dulbecco's
phosphate-buffered saline; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); DTT, dithiothreitol; EDTA, ethylenediamine tetraacetic acid; 6-
FAM, 6-carboxyfluorescein; FBS, fetal bovine serum; GSH, reduced glutathione; GSSG, oxidized glutathione; i.p., intraperitoneal;
MALDI–TOF, matrix-assisted laser desorption ionization–time-of-flight; MOPS, 4-morpholinepropanesulfonic acid; NEM, N-
®
ethylmaleimide; NTB, 2-nitro-5-thiobenzoic acid; ONC, ranpirnase or Onconase (a registered trademark of Tamir Biotechnology);
p.o., per os or by mouth; qd, quaque die or once daily; RI, ribonuclease inhibitor; RNase A, bovine pancreatic ribonuclease; PAGE,
poly(acrylamide) gel electrophoresis; SDS, sodium dodecyl sulfate; 6-TAMRA, 6-carboxytetramethylrhodamine; TGI, tumor growth
inhibition; Tris, tris(hydroxymethyl)aminomethane; Z, net molecular charge: arginine + lysine − aspartate − glutamate −
pyroglutamate.

Rutkoski et al.
Page 2
and unresectable malignant mesothelioma (4-6). Although these two homologues of bovine
pancreatic ribonuclease (RNase A (7)) differ dramatically in both primary and quaternary
structure (8,9), they both evade inhibition by ribonuclease inhibitor (RI (10)), a protein that
resides in the cytosol of mammalian cells. BS-RNase is a natural dimer that evades RI by
steric repulsion (11); whereas ONC is a small monomer whose truncated surface loops
cannot interact intimately with RI (12). The ability to retain ribonucleolytic activity in the
presence of RI is a primary determinant of the cytotoxicity of BS-RNase, ONC, and
engineered ribonucleases (13).
Prior to the discovery of BS-RNase (14), lyophilization of solutions of RNase A from
aqueous acetic acid was found to yield dimers and higher order oligomers of RNase A (15).
These oligomers of RNase A—as large as nonameric (16)—formed by domain swapping of
the N- or C-termini, or both (17), and displayed antitumoral activity concomitant with their
degree of oligomerization: tetramers > trimers > dimers (18). Tandem dimers of RNase A
are likewise toxic to cancer cells (19).
In contrast to domain-swapping, covalent cross-linking does not allow for monomer
dissociation (20,21). Dimers of RNase A formed by cross-linking with amine-reactive imido
esters, inhibit tumor cell proliferation in vitro (22), exhibit antitumoral activity in mice
(23,24), and have enhanced persistence in the circulation of rats (25). Cross-linked trimers
of RNase A generated with a dimethyl suberimidate linker displayed higher toxicity towads
a cervical carcinoma cell line than did cross-linked dimers (26).
Lysine is a common residue. RNase A contains 10 lysine residues, as well as an N-terminal
amino group. Accordingly, a disadvantage of using amine-reactive reagents to cross-link
RNase A is the heterogeneity of the ensuing products. Moreover, the amino group in the
side-chain of Lys41 has a low pK of ~8.8 (27) and is thus especially reactive. Its
a
5
modification can reduce catalytic proficiency by 10 -fold (28) and obviate cytotoxicity (29).
Previously, we used dibromobimane (1) (Figure 1) to prepare a BS-RNase dimer by forming
thioether bonds at a particular cysteine residue (31). We found this dimer to be toxic to
cancer cells in vitro, but did not determine its affinity for RI. Guided by the structure of the
RI·RNase A complex (Figure 2), Youle and coworkers generated homogeneous
homodimeric conjugates of both human pancreatic ribonuclease (RNase 1) and human
eosinophil-derived neurotoxin (RNase 2) by installing a cysteine at a residue that contacts RI
and reacting its thiol with 1,6-bis(maleimido)hexane (2) (Figure 1) (33). The resulting
conjugates were less sensitive to inhibition by RI, and were cytotoxic. The thiol-reactive
linker used by Youle and coworkers was, however, long and flexible, inserting 12 atoms and
9 rotatable bonds between the sulfur atoms of the nascent cysteine residues. We reasoned
that greater RI-evasion, and thus greater cytotoxicity, could be achieved by using linkers that
introduced fewer degrees of freedom between the ribonuclease moieties. In addition, we
were inspired by the increased cytotoxicity of RNase A trimers, formed by either
noncovalent, domain-swapping (18) or non-specific cross-linking (26).
Here, we describe the semisynthesis and characterization of 13 dimeric and trimeric
ribonuclease conjugates, derived from cross-linkers 1–8 and RNase A, BS-RNase, RNase 1,
and ONC. We show that the choice of cross-linker as well as the site and valency of cross-
linking affect the toxic activity toward cancer cells. The results have important implications
for the development of ribonucleases as cancer chemotherapeutic agents.
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Rutkoski et al.
Page 3
MATERIALS AND METHODS
Materials
Protein production and characterization—E. coli BL21(DE3) cells, and pET22b(+)
and pET27b(+) plasmids were from Novagen (Madison, WI). K-562 (derived from a
continuous human chronic myelogenous leukemia line), DU 145 (human prostate
carcinoma), and A549 (human non-small cell lung carcinoma) cells and were from the
American Type Culture Collection (ATCC; Manassas, VA). Cell culture medium,
Dulbecco's phosphate-buffered saline (DPBS), antibiotics, trypsin, and fetal bovine serum
3
(FBS) were from Invitrogen (Carlsbad, CA). [methyl- H]Thymidine (6.7 Ci/mmol) was
from PerkinElmer (Boston, MA). Oligonucleotides for site-directed mutagenesis and
sequencing, and the ribonuclease substrates, 6-FAM–dArUdAdA–6-TAMRA and 6-FAM–
dArUdGdA–6-TAMRA, were from Integrated DNA Technologies (Coralville, IA).
1,6-Bis(maleimido)hexane (2), 1,2-bis(maleimido)ethane (3), tris(N-maleimidoethyl)amine
(8), N-ethylmaleimide were from Thermo Fisher Scientific (Rockford, IL). 1,2-
Bis(maleimido)benzene (4), 1,3-bis(maleimido)benzene (5), and 1,4-bis(maleimido)benzene
(6) were from Sigma–Aldrich. Dibromobimane (1) was from Molecular Probes (Eugene,
OR).
FPLC HiLoad 26/60 Superdex G75 and G200 gel-filtration columns, HiTrap Desalting, SP,
and Q columns were from GE Healthcare (Uppsala, Sweden). Gel-filtration standards, SDS–
PAGE molecular-weight standards, and pre-cast gels for poly(acrylamide) gel
electrophoresis were from BioRad (Hercules, CA). Flat-bottom, black polystyrene (PS),
Costar assay plates (96-well) with non-binding surface (NBS) were from Corning Life
Sciences (Acton, MA). Molecular biology-grade bovine serum albumin (BSA) was obtained
as a 20 mg/mL solution from Sigma–Aldrich (St. Louis, MO). Fluorometric enzymatic
activity measurements were made using 4.5-mL methacrylate cuvettes from VWR (West
Chester, PA). All other chemicals used were of commercial reagent grade or better, and
were used without further purification.
Chemical synthesis—DMF was drawn from a Baker CYCLE-TAINER solvent delivery
system. All other reagents were from Sigma–Aldrich or Fisher Scientific (Hanover Park,
IL), and used without further purification. Thin-layer chromatography was performed using
aluminum-backed plates coated with silica gel containing F phosphor and visualized by
254
UV illumination or staining with I , ceric ammonium molybdate, or phosphomolybdic acid.
2
NMR spectra were obtained with a Bruker DMX-400 Avance spectrometer at the National
Magnetic Resonance Facility at Madison (NMRFAM).
®
Xenograft studies—Erlotinib hydrochloride (Tarceva ) was from Genentech (South San
Francisco, CA); docetaxel and cisplatin were from Sigma–Aldrich. Homozygous (nu/nu)
male Balb/c (−/−) mice were from Harlan Sprague Dawley (Indianapolis, IN).
Instrumentation
3
[methyl- H]Thymidine incorporation into K-562 and A549 genomic DNA was quantitated
by scintillation counting using a Microbeta TriLux liquid scintillation and luminescence
counter (PerkinElmer, Wellesley, MA). The molecular mass of ribonuclease variants and
multimeric conjugates was confirmed by MALDI–TOF mass spectrometry using 3,5-
dimethoxy-4-hydroxycinnamic acid as a matrix and a Voyager-DE-PRO Biospectrometry
Workstation (Applied Biosystems, Foster City, CA) in the Biophysics Instrumentation
Facility at the University of Wisconsin–Madison. Cuvette-scale fluorescence measurements
were made with a QuantaMaster1 photon-counting fluorometer equipped with sample
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Rutkoski et al.
Page 4
stirring (Photon Technology International, South Brunswick, NJ). Fluorescence-based
activity inhibition assays, competition binding assays performed in 96-well plate format, and
cell-viability assays utilizing CellTiter-Blue were read with an EnVison 2100 microplate
reader (Perkin–Elmer, Waltham, MA) in the Keck Center for Chemical Genomics at the
University of Wisconsin-Madison. Thermal denaturation data were acquired using a Cary
400 Bio double-beam spectrophotometer equipped with a Cary temperature controller
(Varian, Palo Alto, CA) in the Biophysics Instrumentation Facility.
Synthesis of 1,3,5-tris(maleimido)benzene (7)
Linker 7 was designed to be a trimeric version of phenylene linkers 4–6, and to be more
compact than known linker 1,3,5-tris(N-maleimidomethyl)benzene (34). Linker 7 was
synthesized by the reaction shown in Scheme 1. First, 1,3,5-triaminobenzene (9) was
synthesized by hydrogenation of phloroglucinol trioxime with Raney nickel, as described
previously (35). Then, maleic anhydride (271 mg, 2.76 mmol) was dissolved in DMF (0.6
mL) under Ar(g). Compound 9 (98 mg, 0.78 mmol) in DMF (0.5 mL) was added dropwise,
giving rise to a white precipitate. Acetic anhydride (0.7 mL, 7.41 mmol), a catalytic amount
of Co(OAc) , and triethylamine (40 μL, 0.29 mmol) were added, and the resulting brown
2
solution was stirred for 20 h at 50 °C, protected from light. The reaction mixture was poured
into ice water, and the resulting dark brown precipitate was collected by filtration. The solid
was washed with water and Et O, and dried under high vacuum (≤1 mm Hg). Linker 7 was
2
isolated as a dark brown powder and used without further purification (286 mg, 98%
1
13
yield). H NMR (400 MHz, DMSO-d ) β (ppm): 7.43 (s, 3H), 7.21 (s, 6H). C NMR (100
6
MHz, DMSO-d ) δ (ppm): 169.50, 134.82, 132.26, 124.12.
6
Design of Cysteine-Containing Ribonuclease Variants
RNase A—We sought to test the hypothesis that greater evasion of RI would be attainable
by tethering multiple ribonuclease monomers intimately via a residue within the RI–
ribonuclease interface (36). In RNase A, residue 88 was chosen for conjugation (Figure 2),
as single amino-acid substitutions there were known to be highly destabilizing to the
RI·RNase A complex (37,38). Residue 88 is a glycine, and its substitution with cysteine
does not alter the net charge of the protein. Multimers of G88C were made with linkers 1–8
(Figure 1). Residue 19 of RNase A was chosen as a representative site outside of the RI–
RNase A interface (Figure 2). Small molecules had been attached there without any
detectable reduction in affinity for RI (39,40). A trimer of A19C was made with linker 8.
Trimeric conjugates were also prepared with D38R/R39D/N67R/G88C RNase A, which is a
variant of RI-evasive D38R/R39D/N67R/G88R RNase A (38), using linker 8.
BS-RNase—BS-RNase And RNase A have high sequence identity (83%) and structural
similarity (41). Accordingly, amino-acid substitutions in BS-RNase analogous to those that
enabled RNase A to evade RI were also effective in endowing monomeric (that is, C31A/
C32A) variants of BS-RNase with the ability to evade RI (42). Accordingly, we reasoned
that tethering at residue 88 with linker 8 would enable BS-RNase to evade RI.
RNase 1—Although no single amino-acid substitution in the β4–β5 loop of RNase 1 has
been shown to be as effective as the G88R substitution in RNase A in effecting RI-evasion,
the importance of this loop in mediating contacts with RI is well established (43-45). Youle
and coworkers demonstrated the utility of the G89C substitution to create RNase 1 dimers
that evade RI (33). We choose that same substitution and made trimers using linker 8.
ONC—Linker 8 was used to tether ONC via residue 72 in its β4–β5 loop, which is truncated
relative to that in mammalian homologues (9). The ensuing (S72C ONC) –8 trimer, like the
3
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Rutkoski et al.
Page 5
D38R/R39D/N67R/G88C RNase A trimer, was composed of RI-evasive monomers and thus
revealed the effect of molecular size, apart from RI evasion, on cytotoxicity.
Production of Ribonucleases and RI
cDNA encoding ribonuclease variants was generated by Quickchange site-directed
mutagenesis (Stratagene, La Jolla, CA) using plasmid pBXR (RNase A) (46), pSR1 (BS-
RNase) (47), pHP-RNase (RNase 1) (44), pONC (ONC) (37), or pET27b(+) containing
cDNA encoding D38R/R39D/N67R/G88R RNase A (38). Plasmid pHP-RNase was
modified to install the missing codon for the C-terminal threonine residue (Thr128), which
establishes native RNase 1 (48). RNase A, RNase 1, ONC, and G88R RNase A were
produced as described previously (37,38,44). Free cysteine-containing variants of RNase A
(A19C, G88C, and D38R/R39D/N67R/G88C), BS-RNase (C31A/C32A/G88C), RNase 1
(G89C), and ONC (S72C), were prepared in a similar manner, but with several alterations.
The protein solution containing dissolved inclusion bodies was diluted by 10-fold with a
thoroughly degassed solution of acetic acid (20 mM), subjected to centrifugation to remove
precipitate, and dialyzed overnight against aqueous acetic acid (20 mM) that had been
purged with N (g) or Ar(g). Ribonucleases were allowed to refold for ≥3 days at 4 °C
2
following slow dilution into 0.10 M Tris–HCl buffer at pH 8.0, containing NaCl (0.10 M),
arginine (0.5 M), ethylenediamine tetraacetic acid (EDTA; 10 mM), reduced glutathione
(GSH; 1.0 mM), and oxidized glutathione (GSSG; 0.2 mM). For free-cysteine containing
variants of RNase 1, the concentrations of GSH and GSSG used were 3.0 mM and 0.6 mM,
L
-
respectively. The refolding solution was purged with N (g) or Ar(g) prior to the addition of
2
denatured protein to prevent the oxidation of the free cysteines. Following purification by
gel-filtration chromatography, the free thiol groups of the unpaired cysteine residues were
protected from inadvertent air oxidation by reaction with 5,5′-dithiobis(2-nitrobenzoic acid)
(DTNB) as described previously (28). The resulting NTB-protected ribonucleases were
applied to a HiTrap SP cation-exchange column, eluted with a linear gradient of NaCl
(0.15–0.40 M) in 50 mM NaOAc buffer at pH 5.0, and stored at 4 °C until subsequent
modification with thiol-reactive compounds. Protein concentration (excluding NTB-
protected ribonucleases) was determined either by using a bicinchoninic acid-based kit
(Thermo Fisher Scientific) (49) and measuring absorbance at 570 nm in a 96-well plate
using an ELx800 microplate reader (Bio-Tek Instruments, Winooski, VT) or by UV
−1
−1
spectroscopy using an extinction coefficient of ε = 0.72 (mg/mL) cm for RNase A
278
−1
−1
and its variants (50), ε = 0.465 (mg/mL) cm for C31A/C32A/G88C BS-RNase (51),
278
−1
−1
ε
= 0.53 (mg/mL) cm for RNase 1 and its variants (44), and ε = 0.87 (mg/
280
280
−1
−1
mL) cm for ONC and its variants (37).
Human RI was produced and purified as described previously (38,45,52) and stored in
DPBS containing dithiothreitol (DTT; 10 mM). Freshly prepared RI was assumed to be
100% active (i.e., fully reduced) for up to one month following purification and was not
used beyond this time without re-purification.
After their purification, ribonucleases and RI migrated as single bands during SDS–PAGE
(e.g., Figure S1). The identity and integrity of purified ribonuclease variants was confirmed
by MALDI–TOF mass spectrometry.
Semisynthesis of Ribonuclease Dimers and Trimers
The pH of the solution containing NTB-protected ribonucleases in HiTrap SP elution buffer
was adjusted from 5.0 to ~8.0 by the addition of either 10× DPBS (to 10% v/v) or 1.0 M
Tris–HCl buffer at pH 8.0 (to 8–10% v/v). Deprotection by the addition of DTT (5–10
equiv) resulted in the generation of the yellow 2-nitro-5-thiobenzoate anion (53).
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Rutkoski et al.
Page 6
Ribonucleases were separated from small molecules using a HiTrap desalting column that
had been equilibrated with DPBS containing EDTA (1 mM).
Semisynthetic dimers—Stock solutions (20–100 mM) of cross-linkers 3–6 and 1 were
prepared in DMF, and 0.6 equiv were added to a solution containing a deprotected
ribonuclease (50–250 μM). This sub-stoichiometric amount of linker was found to produce
the desired dimeric (or trimeric) conjugates in high yield, as reported previously for the
dimerization of C31S BS-RNase (54). A 0.6–5.0-fold molar excess of linker favored the
production of a bis(maleimide)-derivatized ribonuclease monomer; a >10-fold molar excess
of linker resulted in the undesirable nonspecific modification of the ε-amino group of lysine
3
residues, despite the maleimido group being >10 -fold more reactive with thiols than amines
at pH 7 (55). Conjugation reaction mixtures were protected from light, and reactions were
allowed to proceed at room temperature for 2 h or overnight at 4 °C. Reactions were
terminated by the addition of DTT (5 equiv versus ribonuclease). The resulting crude
reaction mixture was applied to a HiLoad 26/60 G75 Superdex gel-filtration column, and
eluted with 50 mM NaOAc buffer at pH 5.0 containing NaCl (0.10 M) and NaN (0.05% w/
3
v). Fractions were analyzed by SDS–PAGE to distinguish monomeric and multimeric
species, and then combined to yield the final desired conjugate at ≥95% homogeneity.
Typical yields for the production of homodimers and homotrimers were 70% and 50%,
respectively. Semisynthetic variants were dialyzed exhaustively against DPBS prior to
further biochemical characterization.
Semisynthetic trimers—Trimeric ribonuclease conjugates were synthesized in the
manner described above for dimeric conjugates, except that the tris(maleimide)-containing
linkers in DMF were added to DPBS containing ribonucleases (50–250 μM) at 0.4 molar
equivalents (8) or 2.0 molar equivalents (7).
Modification of Ribonucleases with N-Ethylmaleimide
To enable comparisons of G88C RNase A and G89C RNase 1 to the wild-type enzymes,
these variants were modified with N-ethylmaleimide (NEM) under conditions similar to
those used for the semisynthesis of dimers and trimers, except that NEM (5 equiv from a
100 mM stock solution in DMF) was added to the deprotected protein that had been desalted
into DPBS. The reaction mixture was protected from light, and the reaction was allowed to
proceed overnight at 4 °C and then quenched by the addition of DTT (final concentration of
1 mM). The reaction mixture was then diluted 10-fold with NaOAc buffer (50 mM) at pH
5.0, applied to a HiTrap SP cation-exchange column, and eluted with a linear gradient of
NaCl (0–0.4 M). The masses of the resulting ribonuclease–NEM conjugates were confirmed
by MALDI–TOF mass spectrometry.
Biochemical Characterization of Ribonuclease Dimers and Trimers
The succinimidyl thioether formed by conjugation of a thiol and maleimide is susceptible to
a low rate of spontaneous hydrolysis (56). The products are isomeric succinamic acid
thioethers that bear an additional carboxylate group, which could obfuscate our analyses.
Accordingly, ribonuclease conjugates were used within 3 months of their semisynthesis.
Ribonucleolytic activity—The enzymatic activity of ribonucleases was determined by
assaying their ability to catalyze the cleavage of the hypersensitive fluorogenic substrate 6-
FAM–dArUdAdA–6-TAMRA (20 nM) (λ = 493 nm; λ = 515 nm) (57). Assays were
ex
em
carried out at ambient temperature in DPBS (2.0 mL) containing BSA (0.1 mg/mL) to
prevent the non-specific binding of ribonuclease to the cuvette. Values of k /K were
cat
M
obtained with the equation:
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Rutkoski et al.
Page 7
(1)
where ΔI/Δt is the initial reaction velocity for cleavage of 6-FAM–dArUdAdA–6-TAMRA
upon addition of ribonuclease (E) to the cuvette, and I and I are, respectively, the
0
max
fluorescence intensities prior to enzyme addition and following the complete cleavage of
substrate by addition of excess RNase A. Activity values for ONC and its conjugates were
determined likewise using the substrate 6-FAM–dArUdGdA–6-TAMRA (50 nM) (58).
Binding by RI—The affinity of ribonucleases for RI was determined directly by using a
fluorescence-based competition assay, as described previously (52). For the unlabeled
competing multimeric ribonuclease conjugates, the molar concentration used was that of the
constituent active sites and not that of the entire conjugate.
Inhibition of ribonucleolytic activity by RI—Twofold serial dilutions of RI (2 μM–1
pM) were prepared in 10 mM MOPS buffer at pH 7.1 containing NaCl (0.138 M), BSA (0.1
mg/mL), and DTT (5 mM) using Eppendorf Protein LoBind tubes. These RI-containing
serial dilutions were delivered (50 μL/well) to a 96-well plate. Ribonucleases and their
conjugates were diluted in the same buffer and added to the RI-containing wells (50 μL/
well; typical final working concentration: 15 pM active site). Plates were incubated for at
least 30 min at ambient temperature prior to the addition of fluorogenic substrate (6-FAM–
dArUdAdA–6-TAMRA; final concentration: 50 nM) to initiate the assay. The plate was
shaken briefly (30 s), and the fluorescence intensity was measured at 30-s intervals for 5 min
on a microplate reader using a FITC filter set (excitation at 485 nm with a 14-nm bandwidth;
emission at 535 nm with a 25-nm bandwidth; dichroic mirror cutoff at 505 nm). An identical
quantity of 6-FAM–dArUdAdA–6-TAMRA was cleaved with an excess amount of
ribonuclease (>10 nM), and the final fluorescence was measured to ensure that <10% of the
total substrate was degraded over the course of the activity assays. Additionally, an aliquot
of the untreated substrate was assayed at the end of the experiment to confirm that it had not
degraded appreciably during the course of the experiment, which would be indicative of
ribonuclease contamination. Initial slope values were obtained from the data points by linear
regression. Relative activities were determined by comparison to a control containing no RI.
Relative activity plots were fitted by nonlinear regression to a sigmoidal variable-slope
curve using the equation:
(2)
In eq 2, h is the slope of the curve, and F and F correspond to the maximum relative
T
B
activities at both low and high concentrations of RI, respectively.
Gel-filtration analysis—Ribonucleases (1 mL of a 1 mg/mL solution in gel-filtration
buffer) were applied to a HiLoad 26/60 Superdex G200 gel-filtration column and eluted with
NaOAc buffer (50 mM) at pH 5.0 containing NaCl (0.10 M) and NaN (0.05% w/v) at a
3
flow rate of 4 mL/min. Gel-filtration standards were prepared and separated by
chromatography using the same column.
Conformational stability—The conformational stability of dimers and trimers of RNase
A was determined by measuring their T value, which is the temperature at the midpoint of
m
the transition between the folded and unfolded states. Protein was dialyzed exhaustively
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Rutkoski et al.
Page 8
against DPBS, and diluted to a concentration of ~25 μM in DPBS. Assays were performed
by slow incremental heating (0.15 °C/min from 25–80 °C), and monitoring of the
absorbance at 287 nm, which decreases upon RNase A denaturation (59,60). Data were
collected and analyzed with the program THERMAL from Varian Analytical Instruments
(Walnut Creek, CA), which fitted the data to a two-state process and enabled the
determination of the value of T (61).
m
Inhibition of Tumor-cell Proliferation in Vitro
Pancreatic-type ribonucleases can inhibit tumor-cell proliferation by evoking apoptosis (62).
This cytotoxic activity was assessed by measuring the effect of ribonucleases on the
3
incorporation of [methyl- H]thymidine into the nascent DNA of K-562 cells, as described
previously (37). Cells were grown in Roswell Park Memorial Institute 1640 medium
(Mediatech, Herndon, VA). Cell-proliferation assays were performed at least three times in
triplicate. Each data point represents the mean (± SE) of three or more experimental values.
Values of IC , which is the concentration of ribonucleases that decreases cell proliferation
50
to 50%, were calculated by fitting the data using nonlinear regression to a sigmoidal dose–
response curve, eq 3.
(3)
3
In eq 3, y is the DNA synthesis following a 4-h [methyl- H]thymidine pulse, and h is the
slope of the curve.
Inhibition of Tumor-cell Proliferation in Vivo
Trimeric ribonucleases were tested for their ability to suppress the growth of human tumors
implanted into the flanks of nude mice. DU 145 cells (prostate cancer) and A549 cells (non-
small cell lung cancer) were selected for both their ability to proliferate in mice and their
low rate of spontaneous regression. Moreover, each line represents a clinically relevant
target that is used often in the testing of new chemotherapeutic agents.
DU 145 cells were grown in Dulbecco's modified Eagle's medium (ATCC) containing FBS
(10% v/v); A549 cells were grown in F12K medium (ATCC) containing FBS (10% v/v).
Cells were implanted into the right rear flank of 5–6 week old male homozygous (nu/nu)
3
nude mice. Tumors were allowed to grow to a volume of ≥75 mm before the initiation of
treatment. All the test compounds were diluted in vehicle (DPBS), and one set of animals
was treated with vehicle alone on the dosing schedule with greatest frequency. All
treatments were administered either by intraperitoneal injection (i.p.) or orally with a gavage
needle (p.o.); the volume of administered solution was based upon the body weight of the
animal (10 μL/g). Treatment with all agents was ongoing throughout the entire experiment.
(G88C RNase A) –8 was used to treat the DU 145 and A549 xenograft models. Mice in the
3
6
A549 xenograft model were each implanted with 4 × 10 cells; treatment was administered
at a dose of 25 mg/kg (i.p., 1×wk). Mice in the DU 145 xenograft model each received 3 ×
6
10 cells; again, treatment was administered at a dose of 25 mg/kg (i.p., 1×wk). Mice in the
A549 xenograft model were also treated with (G89C RNase 1) –8 at doses of 7.5 and 75
3
mg/kg (i.p., 1×wk).
Mice treated with trimeric RNase A or RNase 1 were compared with mice treated with
monomeric K7A/D38R/R39D/G88R RNase A at doses of 5, 15, and 45 mg/kg (i.p., qd×5)
and 75 mg/kg (i.p., 1×wk), and monomeric ONC at a dose of 5 mg/kg (i.p., 1×wk), as well
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Rutkoski et al.
Page 9
as the approved chemotherapeutic agents docetaxel (15 mg/kg; i.p., 1×wk), cisplatin (6 mg/
kg; i.p., 1×wk), and erlotinib (100 mg/kg; p.o., 2×wk).
3
Tumor size was measured twice weekly using calipers, and tumor volume (mm ) was
estimated by using the formula for a spheroid (eq 4):
(4)
(5)
The percent tumor growth inhibition (% TGI) was calculated with eq 5:
RESULTS
Conformational Stability
To maintain its biological activity, a ribonuclease must maintain its native tertiary structure
(63). To assess whether the covalent tethering of ribonucleases into dimers or trimers
compromises their conformational stability, we determined the T values of G88C RNase
m
A–NEM, (G88C RNase A) –3 and (G88C RNase A) –8 (Table 1). All three had T values
2
3
m
nearly indistinguishable from that of RNase A (Figure S2; Table 1). Thus, multimerization
did not appear to be deleterious to the conformational stability of these conjugates. No
evidence of cooperative unfolding was observed for dimeric or trimeric conjugates.
Ribonucleolytic Activity
Site-specific covalent tethering of mammalian ribonucleases had a negligible effect on their
ribonucleolytic activity (Table 1). Most multimeric conjugates as well as G88C RNase A–
NEM exhibited k /K values (per active site) that were within error of that of RNase A.
cat
M
Exceptions were (G88C RNase A) –1, (G88C RNase A) –2, and trimeric conjugates of
2
2
G88C RNase A prepared with either of the homotrifunctional linkers (7 or 8), which
displayed ~2-fold increased activity per active site toward the 6-FAM–dArUdAdA–6-
TAMRA substrate. The S72C ONC trimer displayed the largest change in activity upon
trimerization, as its k /K value per active site was nearly 7-fold higher than that of ONC.
cat
M
Affinity for Ribonuclease Inhibitor
Dimeric and trimeric ribonuclease conjugates could conceivably be bound by more than one
RI molecule. In addition, the initial binding of one RI molecule to the conjugate is likely to
influence the likelihood of subsequent binding events or preclude subsequent RI-binding
entirely. The fluorescence-based competition assay used herein did not report on the
stoichiometry of RI-binding or determine microscopic values of K . Hence, only values of
d
apparent K (K ′) are reported for the various conjugates.
d
d
The K = 1.8 nM obtained for G88R RNase A (Table 1) was in gratifying agreement with
d
values obtained previously (38,52). G88C RNase A–NEM exhibited a nearly equivalent
affinity for RI, having a K = 1.4 nM. Among the dimeric conjugates, (G88C RNase A) –4
d
2
had the lowest affinity for RI, with K ′ = 18 nM. With the exception of (G88C RNase A) –2
d
2
(K ′ = 2.6 nM), the remaining dimeric conjugates of G88C RNase A all had similar affinity
d
for RI, with K ′ = 8.2–14 nM. Each trimeric conjugate of G88C RNase A displayed a
d
significantly different affinity for RI. Those prepared with the novel tris(maleimide) linker 7
were substantially more evasive than those prepared with 8, with K ′ = 39 and 17 nM,
d
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Rutkoski et al.
Page 10
respectively. In general, trimeric conjugates of G88C RNase A had a lower affinity for RI
than did dimers of G88C RNase A. (BS-RNase) –8 trimeric conjugates were the most RI-
3
evasive of the conjugates assayed, having K ′ = 84 nM. Trimers of A19C RNase A, which
d
are tethered outside of the RI interface, possessed an affinity for RI that was sufficiently
high to preclude quantitation in our assay (K ′ <1.4 nM).
d
A novel assay was developed to determine the stoichiometry of RI-binding. The ability of
the ribonuclease conjugates to cleave a fluorogenic substrate following pre-incubation with
varying concentrations of RI was assayed. Apparent K ′ values were obtained by applying eq
i
2 to the data in Figure 3.
The G88R and K31A/D38R/R39D/N67R/G88R variants of RNase A had K ′ = 41.4 pM and
i
103 nM, respectively. (G88C RNase A) –8 exhibited K ′ = 0.94 nM, which is 23-fold higher
3
i
than that of G88R RNase A. Near physiological concentrations of RI, the fractional activity
of the trimeric conjugate reached a plateau at ~0.31. This value is consistent with one of the
three active sites in the trimer remaining inaccessible to micromolar concentrations of RI.
(G88C RNase A) –7 behaved in a nearly identical manner to (G88C RNase A) –8 (data not
3
3
shown). Conversely, (A19C RNase A) –8, which differs from (G88C RNase A) –8 only in
3
3
that it is restrictively tethered through a residue outside of the interface with RI, is inhibited
tightly and completely by RI with K ′ = 17 pM. This value is close to the concentration of
i
enzymic active sites present in the assay solution (15 pM), and thus merely represents an
upper limit for the K ′ value (65).
i
(G88C RNase A) –3 was more readily inhibited by RI than was (G88C RNase A) –8, with
2
3
K ′ = 0.40 nM. Interestingly, the fractional activity of this dimer also reached a plateau at
i
micromolar concentrations of RI, though the significance of its relative activity (~0.11) is
obvious. Nonetheless, we conclude that trimers in the cytosol would retain a larger amount
of unfettered ribonuclease activity than would dimers.
Cytotoxicity in Vitro
The toxicity of each ribonuclease was measured toward the K-562 human leukemia cell line.
IC values are given in Table 1 on both a conjugate and an active-site basis, and were
50
derived by applying eq 3 to the data in Figure 4. The results and fitted curves in Figure 4 are
based upon the concentration of the entire conjugate, not just the active sites, and those will
be the IC values discussed herein unless indicated otherwise. ONC and G88R RNase A
50
displayed IC values similar to those reported previously (37,38). (A19C RNase) –8
50
3
exhibited no cytotoxic activity, even at concentrations of 20 μM (Figure 4A). This lack of
cytotoxicity is consistent with its preserved sensitivity to RI, and underscores the importance
of constraining the ribonucleases by tethering within the RI–RNase A interface. G88C
RNase A–NEM (IC = 28 μM; Figure 4A) was nearly 10-fold less cytotoxic than G88R
50
RNase A. This substantial decrease in cytotoxic activity was surprising because of their
nearly identical affinities for RI. (G88C RNase A) –3 (IC = 11 μM) was 3-fold less toxic
2
50
to K-562 cells than was G88R RNase A (Figure 4A) despite its substantially greater
resistance to inhibition by RI in vitro (Figure 3).
The differential effects on cytotoxicity of linking two molecules of G88C RNase A either
ortho-, meta-, or para- to each other about a phenyl ring is shown in Figure 4B.
Surprisingly, the relatively subtle chemical differences led to detectable and reproducible
differences in cytotoxicity. The IC values exhibited by (G88C RNase A) –4, (G88C
50
2
RNase A) –5, and (G88C RNase A) –6 were 10, 14, and 27 μM, respectively, consistent
2
2
with our hypothesis that more restrictive tethering endows greater cytotoxicity.
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Page 11
Three homologous mammalian ribonucleases, RNase A, RNase 1, and BS-RNase, were
tethered with linker 8 through analogous residues within their β4–β5 surface loop. The
resulting trimers were tested for their ability to inhibit the proliferation of K-562 cells
(Figure 4C). As reported previously (44), RNase 1 had no effect on the proliferation of
K-562 cells. Per conjugate molecule, the (G88C RNase A) –8 conjugate (IC = 8.6 μM)
3
50
was more cytotoxic than any of the dimeric conjugates of RNase A linked through the same
residue. (G89C RNase 1) –8 (IC = 1.5 μM) and (C31A/C32A/G88C BS-RNase) –8 (IC
50
3
50
3
= 0.78 μM) were even more cytotoxic than the analogous RNase A trimer.
ONC and D38R/R39D/N67R/G88R RNase A are monomeric ribonucleases that are highly
cytotoxic (IC = 0.27 and 0.19 μM, respectively) because they are insensitive to RI (38).
50
The cytotoxic activities of trimeric conjugates of these two ribonucleases are shown in
Figure 4D. Both (S72C ONC) –8 (IC = 0.79 μM) and (D38R/R39D/N67R/G88C RNase
3
50
A) –8 (IC = 1.0 μM) are less cytotoxic than their monomers. On a per active-site basis, the
3
50
cytotoxic activity of ONC and D38R/R39D/N67R/G88R RNase A is reduced by 10- and 17-
fold, respectively, upon trimer formation.
Xenograft Mouse Studies
DU 145 prostate carcinoma cells are known to be sensitive to RI-evasive variants of RNase
A (38). To establish an effective dose and administration schedule, mice bearing DU 145
prostate carcinoma tumors were treated with K7A/D38R/R39D/G88R RNase A and ONC. A
dose of 15 mg/kg (i.p., qd×5) was identified as being optimal for eliciting the maximum
inhibition of tumor growth (TGI = 83%; Figure 5A) while displaying minimal off-target
effects as monitored by change in body weight (Figure 5A inset). Indeed, this dose of K7A/
D38R/R39D/G88R RNase A inhibits the growth of DU 145 tumors in mice as well as the
effective dose of ONC (5 mg/kg; i.p., 1×wk) (Figure 5B). Although the mice treated with the
RNase A variant received 15-fold more ribonuclease (by mass) on a weekly basis than did
those treated with ONC, the mammalian ribonuclease seems to be better tolerated, as
indicated by the large decrease in body weight of the animals in the ONC treatment group
during the course of the experiment (Figure 5B inset).
The importance of frequent administration of K7A/D38R/R39D/G88R RNase A to achieve
maximal tumor growth inhibition is shown in Figure 5C. These mice received 75 mg/kg
(1×wk) instead of the previous 15 mg/kg (qd×5). Although equivalent on a mass/week basis,
once-weekly administration of K7A/D38R/R39D/G88R RNase A was dramatically less
effective at inhibiting tumor growth (TGI = 15% versus 83%).
Next, mice were treated with a trimeric ribonuclease: (G88C RNase A) –8. A dose of 25
3
mg/kg (1×wk) of (G88C RNase A) –8, which is a third of the ribonuclease by mass that was
3
used in the experiments depicted in Figure 5C, was administered to mice bearing DU 145
tumors. This dose led to a dramatic inhibition of tumor growth (TGI = 75%; Figure 5D),
despite the infrequent administration.
Xenograft studies were continued with A549 lung adenocarcinoma cells, which are less
sensitive than DU 145 cells to RI-evasive variants of RNase A (38). A dose of 25 mg/kg
(1×wk) of (G88C RNase A) –8 inhibited the growth of these tumors substantially (TGI =
3
68%; Figure 6A). Photographs of mice from the vehicle and treatment groups upon
completion of the experiment show the large reduction in the size of the tumors in treated
animals (Figure 6B).
The human conjugate, (G89C RNase 1) –8, was also administered to mice bearing A549
3
tumors. Although a dose of 7.5 mg/kg (1×wk) inhibited tumor growth only slightly (TGI =
30%) (Figure 6C), a 10-fold higher dose of 75 mg/kg (1×wk) inhibited the growth of these
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Page 12
tumors at a high level (TGI = 79%) that was indistinguishable from that of a 100 mg/kg
(2×wk) dose of erlotinib hydrochloride. This tyrosine kinase inhibitor was approved in 2004
for the treatment of non-small cell lung cancer (66). It is noteworthy that a single molecule
of the human ribonuclease conjugate achieved the same efficacy as ~300 molecules of
erlotinib.
Hydrodynamic Radius
The hydrodynamic radius of RNase A conjugates was assessed by gel-filtration
chromatography. As expected, the radii decreased in the order trimer > dimers > monomers
(Figure 7), suggesting that trimers persist longest in plasma circulation.
DISCUSSION
Thiol-reactive cross-linking reagents have found broad utility in a wide range of applications
(55,67,68). For example, these reagents have been used as “molecular rulers” to probe the
structure of large macromolecular complexes (69-74). Inspired by dimeric linkers 4–6, we
synthesized the novel trimeric linker 7 (Scheme 1), expecting its phenyl scaffold to afford an
even more constrained linkage than tren linker 8.
Preservation of Ribonucleolytic Activity
The enzymatic activity of RNase A is known to diminish upon non-specific cross-linking
with diimido esters (21). In contrast, site-specific tethering did not have a deleterious effect
on the catalytic activity of any of the ribonucleases herein (Table 1). Indeed, (S72C ONC) –
3
8 exhibited increased activity. The low catalytic activity of ONC is thought to originate in its
low affinity for substrate (58). The increased charge density could accelerate the rate of
substrate association.
Linkage Position
Site-specific tethering can allow or disallow a biomolecular interaction. We prepared two
trimeric conjugates of RNase A, (A19C RNase A) –8 and (G88C RNase A) –8, that differ
3
3
only at their site of linkage (Figure 2). Residue 19 lies within the α1–α2 loop of RNase A.
Tethering three ribonuclease moieties there can readily accommodate the binding of three
molecules of RI with high affinity (K ′ ≤ 17 pM; Figure 3). This preservation of sensitivity
i
to RI is corroborated by the results of the competition binding assay (K ′ ≤ 1.4 nM; Table
d
1). In marked contrast, one active site of (G88C RNase A) –8 remains free to degrade
3
cellular RNA in the presence of micromolar RI (Figure 3). This evasion of RI endows
(G88C RNase A) –8 with toxicity for K-562 cells (Figure 4). This result is consistent with
3
the report of Yamada and coworkers, who spliced human basic fibroblast growth factor
(bFGF) into human pancreatic ribonuclease (RNase 1) as a separate domain through
insertional fusion (75). RI-evasion, and thus efficacy as an immunotoxin, was greater when
the bFGF graft was between residues 89 and 90, as opposed to residues 19 and 20, which are
structurally analogous to the linkage positions explored in this work (H. Tada, personal
communication).
Linker Conformation
A motivation of our work was to characterize ribonuclease multimers that were tethered
more restrictively than those prepared with linker 2. All of the bifunctional reagents tested
yielded dimeric ribonuclease conjugates that were indeed more RI-evasive than (G88C
RNase A) –2 (Table 1). Moreover, all but one of the reagents (6) yielded ribonuclease
2
dimers that were more effective at inhibiting the proliferation of K-562 cells than was
(G88C RNase A) –2. The novel linker 7 likewise yielded more RI-evasive trimers (~2-fold)
2
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Rutkoski et al.
Page 13
than those prepared with its more flexible congener, 8. Surprisingly, the greater ability of
(G88C RNase A) –7 to evade RI did not translate into greater cytotoxicity. The amino group
3
of linker 8 is likely to be protonated at physiological pH values, giving this trimeric
conjugate a net charge of +13 compared to +12 for (G88C RNase A) –7. The cationicity of
3
ribonucleases has been correlated with their efficiency of internalization and therefore
cytotoxicity (76-79), and could contribute to the greater than expected cytotoxicity of (G88C
RNase A) –8 relative to (G88C RNase A) –7.
3
3
Net Molecular Charge and Cytotoxicity
RNase A (Z = +4), RNase 1 (Z = +6), and BS-RNase (Z = +9) are highly cationic proteins
with >80% amino-acid sequence identity and comparable ribonucleolytic activity. Yet,
(G88C RNase A) –8, (G89C RNase 1) –8, and (C31A/C32A/G88C BS-RNase) –8 possess
3
3
3
significantly different net charges: Z = +13, +19, and +28, respectively. A correlation of net
charge with cytotoxicity (76-79) is apparent in the trimeric ribonuclease conjugates, which
have IC values of 8.6, 1.5, and 0.78 μM, respectively (Table 1; Figure 4C).
50
Molecular Size and Cytotoxicity
The hydrodynamic radius of the ribonuclease conjugates changes in a predictable manner
(Figure 7). Previously, we showed that the quaternary structure of BS-RNase was a liability
to its cytotoxic activity, as monomeric variants of BS-RNase that evade RI are more
cytotoxic than are dimers of the same variants (42). Likewise, trimerization of D38R/R39D/
N67R/G88R RNase A and ONC, which evade RI as monomers, reduces their cytotoxicity
by 10- and 17-fold, respectively (Table 1). What is the basis for this decrease? The cellular
uptake of fluorescent latex beads of discrete size was found to correlate inversely with size
(80). Although these beads were 50–500 nm in diameter and thus larger than our conjugates,
our data suggest that the correlation between cellular uptake and molecular size extends to
smaller entities as well.
Molecular Size and Circulation
The glomerular capillary wall acts as a molecular sieve, removing small proteins (e.g.,
proteohormones) from plasma circulation but retaining large ones (e.g., albumin and IgG
antibodies). In both mice and rats, the half-life of intravenously administered monomeric
ribonucleases is 2–5 min (23,25,81), which is similar to the glomerular filtration rate
(81,82). For a particular molecule, this rate is determined largely by molecular size; only
when molecular size approaches that of the filtration pore do other attributes, such as
molecular charge, have an appreciable influence (83-85). Given the small size of monomeric
ribonucleases (Figure 7), their rapid clearance is not surprising (86,87). Dimeric
ribonucleases exhibit a longer half-life in circulating plasma (23,25,81). Trimeric
ribonuclease conjugates such as (G88C RNase A) –8 are likely to persist even longer, as
3
they are similar in size to albumin. Such greater plasma retention could contribute to the
superior ability of trimeric ribonuclease conjugates to reduce tumor growth in vivo (Figures
5 and 6).
Conclusions
Using eight different thiol-reactive homobifunctional linkers (six dimeric and two trimeric),
we have demonstrated that the cytotoxic activity of ribonuclease multimers correlates with
how well they evade RI. Importantly, the catalytic activity and conformational stability are
preserved in tethered ribonucleases. Finally, trimeric conjugates of G88C RNase A and
G89C RNase 1 prepared with linker 8 were as effective as highly evasive monomeric
variants of RNase A at inhibiting tumor growth in tumor-bearing mouse models, but could
be administered less frequently and at a lower dose. Thus, this work demonstrates the
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Rutkoski et al.
Page 14
potential of non-reducible, covalent conjugates of mammalian ribonucleases as human
cancer therapeutics.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
This work was supported by grant R01 CA073808 (NIH). We are grateful to L. D. Lavis for advice on the synthesis
of linker 7, G. A. Ellis for the production and purification of RI, and J. Kalia, B. D. Smith, R. J. Johnson, G. A.
Ellis, L. D. Lavis, and R. F. Turcotte for contributive discussions. T.J.R. was supported by Biotechnology Training
Grant 08349 (NIH) and a William R. & Dorothy E. Sullivan Wisconsin Distinguished Graduate Fellowship. The
Biophysics Instrumentation Facility was established with grants BIR-9512577 (NSF) and S10 RR13790 (NIH). The
Keck Center for Chemical Genomics was established with a grant from the W.M. Keck Foundation. NMRFAM
was supported by grant P41 RR02301 (NIH).
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SCHEME 1.
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Figure 1.
Structures of thiol-reactive linkers used to tether ribonucleases into dimers and trimers. The
distances are between sulfur atoms in dimeric conjugates as calculated with stochastic
molecular dynamics simulations (30).
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Figure 2.
Structure of the porcine RI·RNase A complex showing the location of residues 19 and 88
(yellow), which were replaced with cysteine in variants of RNase A (blue). The image was
created with the program PyMOL (DeLano Scientific, South San Francisco, CA) using
protein data bank entry 1dfj (32).
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Figure 3.
Relative enzymatic activity of ribonucleases after pre-incubation with increasing
concentrations of RI. Data points are the mean (± SE) from three separate experiments, and
were fitted with eq 2. G88R RNase A (◆), K31A/D38R/R39D/N67R/G88R RNase A (●),
(G88C RNase A) –3 (▼), (G88C RNase A) –8 (▲), and (A19C RNase A) –8 (■).
2
3
3
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Figure 4.
Effect of ribonucleases on the proliferation of K-562 cells. The incorporation of
3
[methyl- H]thymidine into cellular DNA was used to monitor the proliferation of K-562
cells in the presence of ribonucleases. Data points are the mean (± SE) from at least three
separate experiments carried out in triplicate, and were fitted with eq 3. Values of IC are
50
listed in Table 1. For dimeric and trimeric conjugates, the concentration is of the molecule,
not the constitutive active sites. Each panel depicts a different theme; data for RNase A (○),
G88R RNase A (□), and ONC (▽) are repeated for comparison. (A) Tethering within or
outside of the RI-ribonuclease interface. (B) Analogous dimeric conjugates made with
bis(maleimido)benzene linkers 4–6. (C) Analogous trimeric conjugates of RNase A (Z =
+13), RNase 1 (Z = +19), and BS-RNase (Z = +28). (D) Trimeric conjugates of evasive
monomers, or made with linker 7.
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Figure 5.
Effect of K7A/D38R/R39D/G88R RNase A and (G88C RNase A) –8 on the tumor volume
3
and body weight (insets) of Balb c(−/−) mice in xenograft models bearing human DU 145
prostate tumors. Data points are the mean (± SE) for n mice; % TGI values in parentheses
were calculated with eq 5. Vehicle control (○; n = 7). (A) K7A/D38R/R39D/G88R RNase A
(■, 5 mg/kg; ▲, 15 mg/kg; ◆, 45 mg/kg; i.p., qd×5; n = 7). (B) K7A/D38R/R39D/G88R
RNase A (●, 15 mg/kg; i.p., qd×5; n = 7), ONC (□, 5 mg/kg; i.p., 1×wk; n = 7). (C) K7A/
D38R/R39D/G88R RNase A (●, 75 mg/kg; i.p., 1×wk; n = 7) and docetaxel (□, 15 mg/kg;
i.p., 1×wk, n = 7, though 4 mice died during the course of the experiment such that n = 6 on
day 28, n = 5 on day 35, n = 4 on day 49, and n = 3 on day 63). (D) (G88C RNase A) –8
3
(▲, 25 mg/kg; i.p., 1×wk, n = 7).
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Figure 6.
Effect of trimeric ribonuclease conjugates on tumor volume and body weight (insets) of
Balb c(−/−) mice in xenograft models bearing human A549 lung tumors. Data points are the
mean (± SE) for n mice; % TGI values in parentheses were calculated with eq 5. Vehicle
control (○; n = 7). (A) (G88C RNase A) –8 (▲, 25 mg/kg; i.p., 1×wk, n = 7) and cisplatin
3
(□, 6 mg/kg; i.p., 1×wk, n = 7). (B) Photographs of representative mice treated with vehicle
(left) or (G88C RNase A) –8 (right) on day 70 of the experiment in panel A. (C) (G89C
3
RNase 1) –8 (▼, 7.5 mg/kg; ▲, 75 mg/kg; i.p., 1×wk, n = 7) and erlotinib (□, 100 mg/kg;
3
p.o., 2×wk, n = 7).
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Figure 7.
Elution profile of monomeric, dimeric, and trimeric ribonucleases from gel-filtration
chromatography.
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