From the Center for Biochemical and Biophysical
Sciences and Medicine and the ¶ Department of Pathology, Harvard
Medical School, Boston, Massachusetts 02115
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ABSTRACT |
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Molecular modeling based on the crystal structure
of the complex of bovine pancreatic RNase A with the inhibitor
5'-diphosphoadenosine 3'-phosphate (ppAp) (Leonidas, D. D.,
Shapiro, R., Irons, L. I., Russo, N., and Acharya, K. R. (1997) Biochemistry 36, 5578-5588) was used to design new
inhibitors that extend into unoccupied regions of the enzyme active
site. These compounds are dinucleotides that contain an unusual
3',5'-pyrophosphate linkage and were synthesized in solution by a
combined chemical and enzymatic procedure. The most potent of them,
5'-phospho-2'-deoxyuridine 3'-pyrophosphate, P' Proteins in the mammalian pancreatic RNase superfamily are
noncytosolic endonucleases that degrade RNA through a two-step transphosphorolytic-hydrolytic reaction (1, 2). Some of these enzymes
appear to play a purely digestive role, whereas others exhibit potent
and unusual biological activities (3). Angiogenin, an RNase found
largely in plasma (4, 5), induces new blood vessel formation (6, 7) and
has been shown to play a critical role in the establishment of human
tumors in athymic mice (8). Two other RNases, eosinophil-derived
neurotoxin (also known as RNase-2, RNase Us, and liver
RNase) and eosinophil cationic protein, are neurotoxic and may produce
some of the symptoms of diseases associated with overproduction of
eosinophils (9). Bovine seminal RNase is endowed with antitumor,
immunosuppressive, and antispermatogenic activities (10). Abolition of
the ribonucleolytic activities of these enzymes by mutagenesis or
chemical modification also eliminates biological activity (10-13),
suggesting that the physiological effects in all cases involve cleavage
of RNA. This implies that inhibitors targeted to the active site might
suppress the biological actions of these proteins and hence be useful
for treatment of human diseases and for mechanistic studies.
Our initial efforts to design potent low molecular weight inhibitors of
the biologically active RNases have focused on bovine pancreatic RNase
A (14, 15) as a convenient model system. The ribonucleolytic center of
RNase A is constituted by multiple subsites that bind the phosphate,
base, and ribose components of its RNA substrate (1, 16) (Fig.
1). The most important of these are: (i)
the P1 site, where cleavage of the phosphodiester bond
occurs, (ii) the B1 site for binding the base whose ribose donates its 3' oxygen to the P1 phosphate, (iii) the
B2 site, which binds the base whose ribose donates its 5'
oxygen to the P1 phosphate, (iv) the P0 site,
which binds the 5'-phosphate of the B1 nucleotide, and (v)
the P2 site, which binds the 3'-phosphate of the
B2 nucleotide. The B1 site has a virtually
absolute specificity for pyrimidines. The B2 site can bind
all bases but strongly prefers purines.
5'-ester with
adenosine 3'-phosphate (pdUppAp), binds to RNase A with
Ki values of 27 and 220 nM at pH 5.9 and 7, respectively. These values are 6-9-fold lower than those for ppAp and 50-fold lower than that for the transition state analogue, uridine vanadate. pdUppAp has broad specificity; it is an effective inhibitor of at least two other members of the pancreatic RNase superfamily, human RNase-2 (eosinophil-derived neurotoxin) and RNase-4,
which share only 36-44% sequence identity with the pancreatic enzyme.
The potency of pdUppAp and the other inhibitors described here depends
critically on the extended internucleotide linkage; the pyrophosphate
group enhances dinucleotide binding to the three RNases by 2.1-2.9
orders of magnitude, as compared with a monophosphate. These data give
further insight into the organization of the catalytic centers of the
various RNases. Moreover, the new class of inhibitors provides a useful
means by which to probe the biological actions of these and other
related enzymes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Subsites of the ribonucleolytic center of
RNase A. 5'-N-p ... N-3' represents the RNA
substrate, and the arrow indicates the site at which it is
cleaved. B1 and B2 are
base-binding sites. P0, P1, and
P2 are phosphate-binding sites. The major residues
that constitute each subsite are also indicated.
The most effective small molecule inhibitor of RNase A identified
previously is 5'-diphosphoadenosine 3'-phosphate
(ppAp),1 with
Ki values of 0.24 and 1.3 µM at pH 5.9 and 7.0, respectively (14). The crystal structure of the RNase A·ppAp complex (15) has revealed that the inhibitor binds to the
P1-B2-P2 region of the active site
in a manner strikingly different from that expected on the basis of
earlier structures of RNase complexes (Fig.
2). Specifically, the 5'--phosphate of
the inhibitor, rather than the 5'-
-phosphate, is positioned at
P1, where it forms H bonds with the main chain N of Phe-120
and the side chains of the catalytic residues His-12, Lys-41, and
His-119. The 5'-
-phosphate H bonds to the side chains of Gln-11 in
P1 and Lys-7 in P2. The adenine moiety of ppAp,
in contrast to that of other RNase A inhibitors, adopts a
syn conformation; despite the ~180 ° rotation of the ring, it still makes extensive interactions with the amino acids that
normally constitute B2 (H bonds with Asn-71, Gln-69, and Asn-67; van der Waals contacts with these residues plus Cys-65, Ala-109, Glu-111, and Val-118; and stacking with the imidazole of
His-119). Finally, the 3'-phosphate H bonds with Lys-7.
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The present report describes the use of this structural information for
the design of derivatives of ppAp that are extended into the
B1 and P0 sites of RNase A. The most potent of
the new inhibitors, 5'-phospho-2'-deoxyuridine 3'-pyrophosphate, P' 5'-ester with adenosine 3'-phosphate (pdUppAp), and 5'-phosphothymidine 3'-pyrophosphate, P'
5'-ester with adenosine 3'-phosphate (pTppAp), bind to RNase A with Ki values of 27 and 41 nM, respectively, at pH 5.9. Importantly, these compounds
are stable and have broad specificity for members of the mammalian
RNase superfamily that share only 36-44% overall sequence identity
with RNase A. The tight binding of the new class of inhibitor to all of
the RNases is largely due to the unusual 3',5'-pyrophosphate linkage
between the two nucleotide components.
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EXPERIMENTAL PROCEDURES |
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Materials and General Procedures
RNase substrates, thymidine 3'-phosphate (Tp), 2'-deoxyuridine
3'-phosphate (dUp), ATP, thymidylyl-(3' 5')-2'-deoxyadenosine (TpdA), RNase T2 from Aspergillus oryzae, and nucleotide
pyrophosphatase from Crotalus adamanteus venom were obtained
from Sigma. RNase A and nuclease-free bovine serum albumin were
purchased from Worthington. Fetal bovine serum was from Life
Technologies, Inc. Human RNase-4 was purified from HT-29 adenocarcinoma
cells as described (17). Concentrations of RNase A preparations were
determined spectrophotometrically (
278 = 9800 M
1 cm
1; Ref. 18), and those of
RNases-2 and -4 were determined by amino acid analysis. Mass
spectrometry was performed by the Department of Chemistry and Chemical
Biology at Harvard University.
Molecular Modeling
Modeling was performed with the program QUANTA (Molecular
Simulations) implemented on a Silicon Graphics Indy workstation. Superposition of the RNase A·ppAp (Protein Data Bank code 1AFK, molecule I of the asymmetric unit) and RNase A·uridine vanadate (UVan; Protein Data Bank code 6RSA) complexes was achieved by aligning
the -carbons of Thr-45, Phe-120, and Ser-123 in the two structures.
Reconfiguration of the uridine ribose to 2'- and 3'-endo conformations
was based on the atomic coordinates of the thymidine and cytidine in
the complexes of RNase A with d(ApTpApA) (Protein Data Bank code 1RCN)
and cytidine 2'-phosphate (Protein Data Bank code 1ROB), respectively.
Superposition of the RNase A·pdUppAp model and the crystal structure
of human RNase-2 was performed by aligning the
-carbons of
Gln-11/14, His-12/15, Lys-41/38, Thr-45/42, Asn-71/70, and
His-119/129.
Synthesis of Pyrophosphate-linked Nucleotides
2'-Deoxyuridine 3'-Pyrophosphate, P' 5'-Ester with Adenosine
3'-phosphate (dUppAp)--
A mixture of 2',5'- and 3',5'-ADP
(morpholinium salt; 0.24 mmol total) was reacted with
dicyclohexylcarbodiimide (2.4 mmol) and morpholine (1.92 mmol) in 10 ml
of 70% t-butyl alcohol and 30% water (v/v) to yield
adenosine 2',3'-cyclic phosphate 5'-phosphomorpholidate, bis-(4-morpholine
N,N'-dicyclohexylcarboxamidinium) salt (19). 50 µmol of the morpholidate were then dried by five coevaporations with
anhydrous pyridine and reconstituted in 2 ml of pyridine. Separately,
75 µmol of the sodium salt of dUp were converted to the
tri-n-butylammonium salt by passage through a 0.7 × 1.5-cm column of SP-Sepharose resin that had been treated with 0.5 M tri-n-butylammonium acetate and then washed
with water. This material was lyophilized and then dried by five
coevaporations with pyridine, redissolved in 2 ml of pyridine, and
added to the morpholidate. After 24 h of incubation at room
temperature, the solvent was evaporated, and residual pyridine was
removed by three coevaporations with water. The sample was then
dissolved in 6 ml of 60 mM Tris-Cl (pH 7.5), containing 60 units of RNase T2, an enzyme that catalyzes the hydrolysis of
nucleoside and oligonucleotide 2',3'-cyclic phosphates specifically to
the 3'-phosphate (20). After 24 h of incubation at 37 °C, the
reaction mixture was loaded onto a QAE-Sepharose column (1.5 × 10 cm) that had been equilibrated with 0.1 M triethylammonium
bicarbonate (pH 7.3), and a 6-h linear gradient from 0.1 to 0.4 M triethylammonium bicarbonate was applied at a flow rate
of 1.5 ml/min. The main peak of absorbance at 280 nm was diluted with 2 volumes of water and lyophilized, and residual triethylammonium
bicarbonate was removed by three coevaporations with methanol. The
sample was then dissolved in 1 ml of methanol and precipitated by the
addition of 10 ml of dry ether. The precipitate was collected by
centrifugation, residual ether was evaporated, and the sample was
finally reconstituted in water. Fast atom bombardment (FAB) high
resolution mass spectrometry (HRMS) yielded an [M
H]
value of 716.0520 versus the calculated
value of 716.0519 for C19H26N7O17P3.
Uridine 2'-Pyrophosphate, P' 5'-Ester with Adenosine
3'-Phosphate (U2'ppAp)--
U2'ppAp was prepared by the same procedure
described for dUppAp, except that uridine 2'-phosphate was substituted
for dUp in the second chemical reaction. [M
H]
calculated for
C19H26N7O18P3
was 732.0468, and the value found by HRMS was 732.0469.
pdUppAp--
pdUppAp was prepared by incubating dUppAp (~30
µmol) with 360 units of T4 polynucleotide kinase (Promega) in 18 ml
of 80 mM Tris-Cl (pH 7.5), 10 mM
MgCl2, 5 mM dithiothreitol, 9 mM
ATP at 37 °C for 24 h. The final product was purified by anion
exchange chromatography as for dUppAp, except that the gradient was
from 0.25-0.5 M triethylammonium bicarbonate. [M H]
calculated for
C19H27N7O20P4
was 796.0182, and the value found by HRMS was 796.0184.
pTppAp--
pTppAp was prepared as for pdUppAp, except that Tp
was substituted for dUp. [M H]
calculated for
C20H29N7O20P4
was 810.0339, and the value found by HRMS was 810.0340.
Thymidine 3'-Pyrophosphate, P' 5'-Ester with
2'-Deoxyadenosine (TppdA)--
TppdA was prepared by reacting 100 µmol of 2'-deoxyadenosine 5'-phosphomorpholidate (Sigma) with 120 µmol of Tp (tri-n-butylammonium salt) in 3 ml of dry
pyridine for 24 h at room temperature. The product was purified on
QAE-Sepharose as for dUppAp, except that a gradient from 0.05-0.2
M triethylammonium bicarbonate was used. [M
H]
calculated for
C20H27N7O13P2
was 634.1064, and the value found by HRMS was 634.1063.
Physical Characterization of Pyrophosphate-linked Nucleotides
Purity was assessed by chromatography on a Mono Q column (HR5/5;
Amersham Pharmacia Biotech) with a 25-min linear gradient from 25-400
mM NaCl in Tris-Cl (pH 8) at a flow rate of 1.2 ml/min, recording the absorbance at 254 nm. Concentrations of inhibitor solutions were determined spectrophotometrically, using
260 values of 25,000 M
1
cm
1 for dUppAp, U2'ppAp, and pdUppAp, and 23,400 M
1 cm
1 for TppdA and pTppAp,
all of which were calculated from the values for mononucleotides listed
by Beaven et al. (21). Nucleotide pyrophosphatase digestions
were conducted in 50 µl of 50 mM Tris-Cl (pH 7.5), 50 mM NaCl, 1 mM MgCl2, containing 0.5 mM dinucleotide and 0.04 units of enzyme, at 25 °C for
1 h. The chemical stability of each pyrophosphate-linked
nucleotide was examined by performing Mono Q chromatography (as above)
on a 25-nmol aliquot that had been incubated in 50 µl of 20 mM Hepes (pH 7.0) at 25 °C for 3 days. The stability of
pTppAp in the presence of serum was examined by analyzing in the same
manner an 80-nmol aliquot that had been incubated in 40 µl of 10 mM Tris-Cl (pH 8.0) containing 10% fetal bovine serum at
37 °C for 48 h. RNase contamination of the final preparations
was assessed by incubating aliquots of each dinucleotide (15 nmol) in
30 µl of 0.2 M Mes (pH 5.9), containing 0.25 mM cytidylyl-(3'
5')-adenosine and 0.25 mM
adenylyl-(3'
5')-adenosine for 20 h at 25 °C and then using
C18 HPLC to measure any adenosine released (22).
Isolation of Human RNase-2
Human RNase-2 was purified from urine by a modification of the method used for isolation from placenta (23). All operations were performed at 4 °C unless specified otherwise. Urine (1 liter) was diluted 8-fold with water and drawn through a 400-ml filter cake of CM-52 cation exchange resin (Whatman) in a 9.4-cm diameter sintered glass funnel under vacuum at a flow rate of 2.5 liters/h. The resin was then washed with 2 liters of 20 mM disodium phosphate (pH 6.6) and transferred into a 2.5-cm diameter column. Additional phosphate buffer was passed through the column until the absorbance of the effluent at 280 nm was <0.05, and the column was then eluted with 1 M NaCl in the same buffer. Salt-eluted material was concentrated to <10 ml by ultrafiltration in an Amicon device equipped with a YM3 membrane, diluted 10-fold with 20 mM sodium acetate (pH 5.5), reconcentrated, and centrifuged at 15,600 × g for 15 min. The supernatant was then loaded onto a Mono S cation exchange column that had been equilibrated with 20 mM sodium acetate (pH 5.5). Elution was achieved with a 50-min linear gradient from 0 to 0.4 M NaCl in 20 mM sodium acetate (pH 5.5) at 0.8 ml/min at ambient temperature. RNase-2 eluted at 44 min. The amino acid composition of this material was indistinguishable from that expected from the sequence (24), indicating that the protein was >95% homogeneous. The absence of any significant contamination by pancreatic RNase was established by spectrophotometric assay (25). The RNase-2 preparation cleaved CpA >500-fold more rapidly than CpG; this preference is similar to that reported previously and is 25-fold larger than that for the pancreatic enzyme (23, 26).
Inhibition Kinetics
Inhibition of RNase A by all compounds except for TpdA was
assessed with a spectrophotometric method (25) as described previously (14). Assays were performed in 0.2 M Mes (pH 5.9) or 0.2 M Hepes (pH 7.0), containing 10 µg/ml bovine serum
albumin at 25 °C with 1.3-10 nM enzyme and the
substrate cytidylyl-(3' 5')-guanosine at a concentration (75 µM) well below Km. The
Ki values reported represent the
[I] intercepts
of plots of [E]/v0 versus [I],
where v0 is the initial reaction velocity and
[E] and [I] are the concentrations of enzyme and inhibitor,
respectively. Values of Ki for RNase-2 and RNase-4
with pyrophosphate-linked nucleotides were determined by the same
procedure, except that the substrates were cytidylyl-(3'
5')-adenosine (75 µM) and uridylyl-(3'
5')-adenosine
(55 µM), respectively, and the enzyme concentrations were
4-12 nM. The high concentrations of TpdA required to
produce significant inhibition of the three enzymes precluded the use
of the spectrophotometric assay to measure reaction velocities, and
inhibition was assessed by using an HPLC method (17, 22) to determine
the dependence of kcat/Km
values on [I]. Reaction conditions were as above, except that the
concentration of bovine serum albumin was 50 µg/ml, the substrate for
RNase-2 was uridylyl-(3'
5')-cytidine (100 µM), and
the concentrations of RNase A, RNase-2, and RNase-4 were 0.7, 1-2, and
50 nM, respectively. Values of Ki
represent the
[I] intercepts of plots of Km/kcat versus
[I].
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RESULTS AND DISCUSSION |
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Design of ppAp Derivatives Extended into the B1 Site of
RNase A--
The crystal structure of the RNase A·ppAp complex (15)
reveals that the B1 pyrimidine-binding site, a cleft formed
by the side chains of Val-43, Asn-44, Thr-45, Phe-120, and Ser-123, is unoccupied. Therefore, molecular modeling was used to explore how a
pyrimidine moiety could be linked to ppAp so that it would interact
with this site. Uracil was selected as the base because RNase-4, one of
the targets for the inhibitors under development (see below), strongly
prefers this pyrimidine over cytosine (17), and most of the other
mammalian RNases bind uridine and cytidine nucleotides with similar
affinity (1, 23, 27). Modeling was begun by superimposing the
B1 regions of the structures of the RNase A·ppAp and
RNase A·UVan (28) complexes. The positions of Thr-45, Phe-120, and
Ser-123 for the two RNases in the superposition are quite similar, with
deviations of <0.11 Å and 0.16-0.51 Å for -carbons and side
chain atoms, respectively. All three of these residues in the ppAp
complex are oriented such that they would be able to form the same
interactions with uracil as in the UVan complex, i.e. H
bonds with OG1 and NH of Thr-45, van der Waals contacts with the phenyl
ring of Phe-120, and a water-mediated H bond with OG of Ser-123. The
other active site components also aligned well (the root mean square
deviation for the positions of the
-carbons of Lys-7, His-12,
Lys-41, Asn-67, Gln-69, Asn-71, and His-119 in the two structures was
0.5 Å), suggesting that occupation of the B1 site will not
perturb the interactions with ppAp.
The next modeling step was to remove the vanadium atom, vanadate atoms
O1V, O2V, and O3V, and the RNase molecule of the RNase A·UVan
complex, leaving a single RNase molecule with ppAp bound to its
P1-B2-P2 region and uridine
positioned at the B1 site (Fig. 3A). In this model, the 2'-
and 3'-oxygens of uridine are 1.0 and 0.9 Å from the ppAp
-phosphate atoms O2B and O3B, respectively, indicating that only a
relatively minor adjustment would be required to connect the uridine
ribose with the phosphate. Because the puckering of the UVan-derived
ribose is atypical, we explored the effects of altering this
conformation to those more commonly found in complexes of RNase A with
pyrimidine nucleotides. Reconfiguration to C2'-endo followed
by rotation of the
-phosphate by 80 ° produced an excellent
alignment of O3' and O3B (Fig. 3B). Reconfiguration to
C3'-endo followed by a 20 ° rotation of the phosphate in
the opposite direction placed O2' in nearly perfect alignment with O2B
(Fig. 3C).
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These observations suggested that a uridine attached through its 2' or
3' oxygen to the -phosphate of ppAp would form interactions with the
B1 site of RNase A similar to those of UVan. Therefore, the
working structures were modified by removing O3B or O2B and then
joining the
-phosphorus to O3' or O2', producing modeled complexes
of RNase A with the compounds UppAp and U2'ppAp, respectively. The
RNase A·UppAp model was then altered further by removing the 2'-OH
group of the uridine ribose to prevent any possible RNase-catalyzed cleavage of the pyrophosphate linkage. The
-phosphorus-O-3'C angle
in the resultant dUppAp model and the
-phosphorus-O-2'C angle in the
theoretical U2'ppAp complex were similar to those in other
B1-P1 bound nucleotides, and no steric clashes
were observed. It should be noted that the pAp portions of dUppAp and
U2'ppAp in the models bind to RNase A in precisely the same manner as that of ppAp in the crystal structure (i.e. this portion of
the inhibitors was not moved during the modeling) but that the
interactions of the adenosine
-phosphate differ somewhat due to the
rotations of this group that were required. Thus, the lengths and/or
angles of some H bonds with P1 residues may be less
favorable than in the ppAp complex, where the phosphate was free to
orient optimally.
The preceding exercise suggested that the active site of RNase A can accommodate 2'(3'),5'-pyrophosphate-linked nucleotides and that dUppAp and U2'ppAp might bind to the enzyme more tightly than ppAp. The synthesis of these two nucleotides was therefore undertaken.
Preparation and Structural Characterization of dUppAp and
U2'ppAp--
A search of the structures of dUppAp and U2'ppAp (Fig.
4) in the Chemical Abstracts Service data
base revealed that these compounds had not been described previously.
Both were synthesized in solution by a combined chemical and enzymatic
procedure (Fig. 5) similar to that
reported for the preparation of ppAp (14). A mixture of 3',5'- and
2',5'-ADP (Fig. 5, I) was reacted with morpholine and
carbodiimide to activate the 5'-phosphate and to protect the
2'(3')-phosphate by converting it to a 2',3'-cyclic phosphate. The
product, adenosine 2',3'-cyclic phosphate 5'-phosphomorpholidate (Fig.
5, II), was then reacted with dUp or uridine 2'-phosphate (Fig. 5, III) to yield 2'-deoxyuridine 3'-pyrophosphate, P'
5'-ester with adenosine 2',3'-cyclic phosphate and uridine
2'-pyrophosphate, P'
5'-ester with adenosine 2',3'-cyclic
phosphate, respectively (Fig. 5, IV). The cyclic phosphates
were then hydrolyzed to form the 3'-phosphate isomers dUppAp and
U2'ppAp (Fig. 5, V) by incubation with RNase T2. These
products were purified to >98% and >92% homogeneity, respectively.
Both nucleotides eluted from a Mono Q column somewhat earlier than
ppAp, consistent with their lower charges. Digestion with nucleotide
pyrophosphatase in both cases yielded compounds that had the same
retention times as the expected products (adenosine 3',5'-diphosphate
and either dUp or uridine 2'-phosphate) during Mono Q chromatography.
Final confirmation of the proposed chemical structures was provided by
high resolution mass spectrometry. The chemical stability of the two
new compounds was also tested. Incubation of dUppAp for 3 days at
neutral pH at 25 °C resulted in no detectable breakdown, whereas
U2'ppAp had a half-life of about 5 days under the same conditions. Both
compounds were found to be free of any measurable contaminant
RNase.
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Testing of dUppAp and U2'ppAp for Inhibition of RNase A-- The effectiveness of the two inhibitors against RNase A was tested at two pH values, 5.9 and 7.0. The lower pH is close to the optimum for dinucleotide cleavage (29) and for binding standard nucleotides (30); the higher pH is within the physiological range and was used previously to study inhibition of RNase A by the putative transition state analogue UVan (Ki = 10 µM; Ref. 31). At pH 5.9, both dUppAp and U2'ppAp were about 2-fold more effective than ppAp (Table I). The potency of dUppAp decreased by a factor of 6 when the pH was raised to 7; this pH effect is similar to that measured with ppAp. The Ki value for dUppAp at pH 7 is 14-fold lower than that reported for UVan. Inhibition by U2'ppAp was affected much more markedly by the pH increase and became 70-fold less strong. Therefore, the design of additional inhibitors was based on the RNase·dUppAp model.
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The small improvement in binding for dUppAp as compared with ppAp can
potentially be attributed to the net sum of multiple opposing factors.
On the positive side are the enthalpic contributions of the
interactions of the added pyrimidine moiety at B1 and the decreased loss of entropy upon binding now that the adenosine 5'
-phosphate is conformationally restrained. Partially compensating for these are the less optimal interactions of the phosphate in P1 predicted to result from the same rotational restriction
as well as the lower charge of this phosphate in dUppAp and U2'ppAp, where it is involved in an ester linkage with the uridine ribose. Other
considerations may also come into play that are not apparent from the modeling.
Design, Preparation, and Testing of pdUppAp and pTppAp--
RNase
A has a kinetically demonstrable P0 site that is thought to
include Lys-66 (32-34). Therefore, molecular modeling was used to
examine whether a phosphate attached to the 5'-OH group of dUppAp
(generating pdUppAp (Fig. 4)) might interact with this residue and
thereby improve inhibitor binding. The shortest distance between a
5'-phosphate oxygen atom and NZ of Lys-66 that could be achieved by
orienting the phosphate in the RNase·pdUppAp model was 4.4 Å.
However, a slight reorientation of the lysine side chain, whose
position is not fixed by inter- or intramolecular interactions, brought
the two atoms within H bonding range (<3.3 Å) (Fig.
6 shows the final RNase A·pdUppAp
model). pdUppAp was prepared by phosphorylating dUppAp with T4
polynucleotide kinase and was purified to >98% homogeneity. Its
structure was confirmed by high resolution mass spectrometry and by
pyrophosphatase digestion, which yielded the expected products as
judged by Mono Q chromatography. No degradation products were detected
after incubation of this compound for 2 days at 25 °C and pH 7. The
Ki values for inhibition of RNase A at pH 5.9 and
7.0 were 27 and 220 nM, respectively (Table I), 4.8- and
3.2-fold lower, respectively, than those for dUppAp. The overall
improvement in Ki for pdUppAp compared with ppAp,
the starting molecule of this study, is about 9-fold.
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The functional effect of substituting thymidine for uridine in pdUppAp was also investigated; this replacement would facilitate bulk preparation of the present generation of inhibitors, as well as synthesis of related compounds, because thymidine derivatives are more readily available than are their 2'-deoxyuridine counterparts. Earlier kinetic studies (1, 32) had demonstrated that thymidine nucleotides bind to RNase A with Ki values comparable with those of uridine and cytidine inhibitors. Moreover, the thymine moiety in the crystal structure of the RNase A·d(ApTpApA) complex (33) forms the same interactions as uracil in the RNase A·pdUppAp model, and its 5-methyl group is oriented away from the B1 pocket. pTppAp (Fig. 4) was synthesized by the same procedure used for pdUppAp and was purified to >98% homogeneity. The chemical structure of the final product was confirmed by mass spectrometry and pyrophosphatase digestion. The inhibitory potency of pTppAp with RNase A was found to be nearly equivalent to that of pdUppAp (Table I).
Contribution of the Pyrophosphate Linkage to the Affinity of
Dinucleotides for RNase A--
The newly synthesized
dinucleotides, dUppAp, pdUppAp, and pTppAp, constitute a novel
class of RNase inhibitors characterized by the presence of a
3',5'-pyrophosphate linkage. The Ki values of RNase
A for these compounds are ~4 orders of magnitude lower than the
Ki reported previously for TpdA, a dinucleotide with
an ordinary phosphate linkage (35). Although part of this difference
can be attributed to the extra phosphates at the 3'- and 5'-ends of the
new inhibitors, much of it is likely to derive from the elongated
connection between the two nucleotide components: i.e. from
the additional interactions that the pyrophosphate can form or from the
altered (and possibly more favorable) orientation of the adenine ring
in B2 that is induced by placement of the adenosine 5'
-phosphate in P1. The contribution of the pyrophosphate to RNase binding was assessed directly by synthesizing TppdA (Fig. 4)
and comparing its inhibitory activity to that of TpdA. As shown in
Table I, the Ki for this compound with RNase A is 4.0 µM, 300-fold lower than that for TpdA. Thus a
pyrophosphate linkage substantially increases the affinity of
dinucleotides for this enzyme. To determine whether this unusual
structural feature enhances inhibition of mammalian RNases other than
RNase A, some of the new compounds were tested with RNase-2 and
RNase-4.
Inhibition of Human RNase-2-- RNase-2 is one of the most abundant RNases in humans and has been isolated from a wide variety of sources, including liver (36), placenta (23), eosinophils (9), and urine (27). The amino acid sequence of the human enzyme is 36% identical to that of RNase A (24). RNase-2 has nearly the same potency as RNase A with RNA substrates but is much less active than the pancreatic protein with dinucleotide substrates (23, 27). It exhibits a small preference for cytosine versus uracil in the B1 subsite and strong specificity for adenine versus guanine at B2, as judged by kcat/Km values for dinucleotide cleavage.
TppdA was found to inhibit RNase-2 130-fold more effectively than does TpdA (Table II), indicating that the pyrophosphate linkage improves affinity for this enzyme almost as greatly as with RNase A. However, the Ki values for the two compounds with RNase-2 are 16-35-fold higher than those measured with the pancreatic enzyme. The Ki difference for TpdA was not surprising in view of the earlier finding that RNase-2 has a 6-fold larger Km value for CpA than does RNase A (23). These differences presumably derive from variations in sequence and/or three-dimensional structure between the B1/P1/B2 regions of the active sites of the two proteins, where the inhibitors are expected to bind. The P1 residues Gln-11, His-12, Lys-41, and His-119 of RNase A are all maintained in RNase-2 (as Gln-14, His-15, Lys-38, and His-129), as are the B1 and B2 residues Thr-45, Asn-67, and Asn-71 (as Thr-42, Asn-65, and Asn-70). However, the B1 site components Phe-120 and Ser-123 are replaced by Leu-130 and Ile-133, and Gln-69 and Glu-111 of the RNase A B2 site are substituted by Arg-68 and Asp-112.
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The best of the RNase A inhibitors, pdUppAp and pTppAp, bound to
RNase-2 with Ki values of 0.18 and 0.46 µM, respectively, i.e. 300-770-fold lower
than that for TppdA. Thus, the relative effectiveness of these
nucleotides for RNase-2 versus RNase A was improved
significantly by addition of the 5' and 3' phosphates, suggesting that
the P0 and/or P2 sites of RNase-2 are even more efficient than those of RNase A. This seemed surprising because Lys-66
and Lys-7, the primary residues that constitute these two sites in
RNase A, are replaced nonconservatively in RNase-2 by Pro-63 and
Trp-10. To identify potential components of these subsites in RNase-2,
the crystal structure of RNase-2 (37) and the RNase A·pdUppAp model
were superimposed. The distances between the -carbons of analogous
active site residues (14/11, 15/12, 38/41, 42/45, and 129/119;
RNase-2/RNase A) in the superposition ranged from 0.2 to 0.6 Å, and
the side chains of these residues, with the exception of His-129/119,
were oriented almost identically in the two structures. His-119 adopts
two distinct conformations, A and B, in the structure of free RNase A
(38); the conformation of this residue in the ppAp complex and pdUppAp
model is A, whereas that of its counterpart in the free RNase-2
structure is B. Orientation B is incompatible with binding of a base in
the B2 site of RNase A, and it is likely that rotation of
His-129 to the A conformation would be required for binding of pdUppAp
(or standard dinucleotide substrates) to RNase-2.
No RNase-2 residue corresponds precisely to Lys-66 of RNase A in the
superposition. The OG atom of Ser-64, the RNase-2 residue whose
-carbon lies closest to that of Lys-66, is 4.6 Å from the phosphate
and could not move within H bonding distance without a reorientation of
the backbone. Beintema (39) proposed previously that Arg-132 of RNase-2
may functionally replace Lys-66 of RNase A in binding the
P0 phosphate, and the RNase-2·pdUppAp model suggests that
this is possible. The NH1 atom of Arg-132 is 6.1 Å from one of the
terminal 5'-phosphate oxygens in the model and could be brought close
enough to form a coulombic interaction or even an H bond by rotation of
the arginine side chain. On the other hand, the terminal 3'-phosphate
of the inhibitor does not appear to make any favorable contacts with
RNase-2. In fact, this group clashes with the side chain of Trp-7,
indicating that changes in enzyme and/or inhibitor conformation would
be required to allow binding. In light of these observations, it is
remarkable that the inhibition constants for pdUppAp and pTppAp with
RNase-2 are only 7-11-fold higher than those with RNase A.
Inhibition of RNase-4-- RNase-4 was originally isolated from human tumor cell-conditioned medium and plasma (17) and subsequently from porcine (40) and bovine (41) liver. The degree of amino acid sequence identity among the proteins from the three species (87-94%) is much higher than for other RNases (42). In addition, RNase-4 is unique among the mammalian pancreatic family enzymes in that it has nearly absolute specificity for uracil versus cytosine in its B1 site (17). These unusual features suggest that RNase-4 may have an important biological role other than general RNA catabolism (42). The identification of such a function might be facilitated by the availability of potent inhibitors.
The primary structure of human RNase-4 is 44% identical to that of RNase A, and most of the active site residues of RNase A (Gln-11, His-12, Lys-41, and His-119 in P1; Thr-45 and Phe-120 in B1; Asn-67, Asn-71 and Glu-111 in B2; and Lys-66 in P0) are conserved. However, the P2 site residue Lys-7 of RNase A is substituted in RNase-4 by Arg-7, and two B1 site components of RNase A, Val-43 and Ser-123, are not maintained. The valine is replaced by phenylalanine, and the serine is deleted (RNase-4 terminates at the preceding residue). Both of these differences have been shown to play a role in defining pyrimidine specificity (43, 44). A detailed comparison of the active sites of the two enzymes must await the determination of a three-dimensional structure for RNase-4.
As with RNase A and RNase-2, TppdA bound to RNase-4 much more tightly than did TpdA; indeed the difference was even larger (800-fold) with this enzyme (Table II). Thus the 3',5'-pyrophosphate linkage markedly increases inhibitory potency with RNases that have widely divergent amino acid sequences and active site compositions. The Ki values for both pdUppAp and pTppAp were ~65-fold lower than that for TppdA. This improvement is slightly less than that observed with RNase A (100-150-fold), suggesting that the combined P0 and P2 peripheral subsites of RNase-4 are almost as competent as those of RNase A. The affinities of pTppAp and pdUppAp for RNase-4 are only 5- and 10-fold, respectively, lower than for RNase A.
Stability of pTppAp in Serum-- The most effective inhibitors described in this study, pdUppAp and pTppAp, have free phosphates on their 5' and 3' ends and are therefore susceptible to dephosphorylation by phosphatases, which would markedly diminish their potency. This raised concern regarding whether these compounds and future derivatives of them would be sufficiently stable when used for inhibition of RNases in vivo or in cell culture. Therefore, we measured the rate of degradation of one of the nucleotides, pTppAp, in the presence of serum (see "Experimental Procedures"). Incubation for 46 h at 37 °C in 10% fetal bovine serum resulted in the loss of only 8.5% of the inhibitor. This suggests that phosphatases are unlikely to interfere with studies of these compounds in many biological systems.
Conclusions--
pdUppAp and pTppAp are the most potent low
molecular weight inhibitors of mammalian RNases described thus far.
They bind to RNase A 6-9-fold more tightly than does ppAp, the
starting molecule of this study, and ~50-fold more tightly than UVan
(31). They are also effective inhibitors of RNase-2 and RNase-4 and
should be useful for investigating the biological actions of these
enzymes. The high affinity of pdUppAp and pTppAp for the various RNases derives in large part from the unusual pyrophosphate linkage. The
terminal phosphate groups also play an important role with all three
enzymes, despite the relatively poor conservation of the subsites that
are thought to recognize them. Structural studies will be required to
gain a better understanding of the mode of interaction of these
inhibitors with the different RNases. Such studies can also provide a
basis for the design of new compounds that bind even more tightly or
that are targeted at specific RNases. Finally, we note that
pyrophosphate-linked nucleotides or their derivatives might also be
effective against other homologous RNases not tested here such as
angiogenin and eosinophil cationic protein.
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ACKNOWLEDGEMENTS |
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We thank Drs. Bert L. Vallee, James F. Riordan, John Shultz and Randall Dimond for helpful discussions, Dr. Daniel Strydom for amino acid analyses, and Jody Tversky for excellent technical assistance. We are also grateful to Dr. Michael N. G. James for providing the atomic coordinates of human RNase-2.
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FOOTNOTES |
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* This work was supported by the Endowment for Research in Human Biology, Inc. (Boston, MA) under a research agreement with Promega Corporation (Madison, WI).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: Dipartimento di Scienze della Vita, Seconda Università di Napoli, Via Arena 18, 81100 Caserta, Italy.
To whom correspondence should be addressed: Center for
Biochemical and Biophysical Sciences and Medicine, Harvard Medical School, Seeley G. Mudd Bldg., 250 Longwood Ave., Boston, MA 02115. Tel.: 617-432-4010; Fax: 617-566-3137; E-mail:
shapiro{at}ferret.med.harvard.edu.
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ABBREVIATIONS |
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The abbreviations used are:
ppAp, 5'-diphosphoadenosine 3'-phosphate;
Tp, thymidine 3'-phosphate;
dUp, 2'-deoxyuridine 3'-phosphate;
TpdA, thymidylyl-(3' 5')-2'-deoxyadenosine;
UVan, uridine vanadate;
FAB, fast atom
bombardment;
HRMS, high resolution mass spectrometry;
UppAp, uridine
3'-pyrophosphate, P'
5'-ester with adenosine 3'-phosphate;
dUppAp, 2'-deoxyuridine 3'-pyrophosphate, P'
5'-ester with adenosine
3'-phosphate;
U2'ppAp, uridine 2'-pyrophosphate, P'
5'-ester with
adenosine 3'-phosphate;
pdUppAp, 5'-phospho-2'-deoxyuridine
3'-pyrophosphate, P'
5'-ester with adenosine 3'-phosphate;
pTppAp, 5'-phosphothymidine 3'-pyrophosphate, P'
5'-ester with adenosine
3'-phosphate;
TppdA, thymidine 3'-pyro-phosphate, P'
5'-ester with 2'-deoxyadenosine;
Mes, 4-morpholineethanesulfonic
acid;
HPLC, high performance liquid chromatography.
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REFERENCES |
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