Potent Inhibition of Mammalian Ribonucleases by 3',5'-Pyrophosphate-linked Nucleotides*

Nello RussoDagger § and Robert ShapiroDagger parallel

From the Dagger  Center for Biochemical and Biophysical Sciences and Medicine and the  Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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' right-arrow 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
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ABSTRACT
INTRODUCTION
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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.


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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'-beta -phosphate of the inhibitor, rather than the 5'-alpha -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'-alpha -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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Fig. 2.   Crystal structure of the RNase A·ppAp complex (15). The inhibitor is indicated by thick lines, and RNase residues are indicated by thin lines. Only those RNase residues whose side chains form H bonds with ppAp are shown. The van der Waals surface of ppAp is indicated by stippling. 5'-PP and 3'-P indicate the 5'-pyrophosphate and the 3'-phosphate of ppAp, respectively.

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' right-arrow 5'-ester with adenosine 3'-phosphate (pdUppAp), and 5'-phosphothymidine 3'-pyrophosphate, P' right-arrow 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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Materials and General Procedures

RNase substrates, thymidine 3'-phosphate (Tp), 2'-deoxyuridine 3'-phosphate (dUp), ATP, thymidylyl-(3' right-arrow 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 (epsilon 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 alpha -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 alpha -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' right-arrow 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' right-arrow 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' right-arrow 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 epsilon 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' right-arrow 5')-adenosine and 0.25 mM adenylyl-(3' right-arrow 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' right-arrow 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' right-arrow 5')-adenosine (75 µM) and uridylyl-(3' right-arrow 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' right-arrow 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].

    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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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 alpha -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 alpha -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 beta -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 beta -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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Fig. 3.   Structure-based design of dUppAp and U2'ppAp. Inhibitors and RNase residues are indicated by thick and thin lines, respectively. Only the B1-P1 region of the active site is shown. 5'-PP indicates the 5'-pyrophosphate of ppAp; 2' and 3' mark the 2'- and 3'-carbons of the uridine ribose. The three panels show the modeled complexes of RNase A with ppAp and uridine (A), dUppAp (B), and U2'ppAp (C).

These observations suggested that a uridine attached through its 2' or 3' oxygen to the beta -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 beta -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 beta -phosphorus-O-3'C angle in the resultant dUppAp model and the beta -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 beta -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' right-arrow 5'-ester with adenosine 2',3'-cyclic phosphate and uridine 2'-pyrophosphate, P' right-arrow 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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Fig. 4.   Structures of pyrophosphate-linked nucleotides.


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Fig. 5.   Synthesis of dUppAp and U2'ppAp. The reaction conditions were as follows: A, morpholine and dicyclohexylcarbodiimide in 70% t-butyl alcohol at boiling temperature; B, dry pyridine at room temperature; C, RNase T2 in 60 mM Tris-Cl (pH 7.5) at 37 °C. Other details are provided in the text.

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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Table I
Inhibition of RNase A by various nucleotides and dinucleotides
Assays were performed in 0.2 M Mes (pH 5.9) or 0.2 M Hepes (pH 7.0) at 25 °C. Values of Ki for ppAp are from Ref. 14.

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' beta -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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Fig. 6.   RNase A·pdUppAp model. Each atom is represented by its van der Waals surface. The enzyme is white, the inhibitor gray.

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' beta -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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Table II
Inhibition of RNase-2 and RNase-4 by various dinucleotides
Assays were performed in 0.2 M Mes (pH 5.9) at 25 °C.

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 alpha -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 alpha -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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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.

parallel 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.

    ABBREVIATIONS

The abbreviations used are: ppAp, 5'-diphosphoadenosine 3'-phosphate; Tp, thymidine 3'-phosphate; dUp, 2'-deoxyuridine 3'-phosphate; TpdA, thymidylyl-(3' right-arrow 5')-2'-deoxyadenosine; UVan, uridine vanadate; FAB, fast atom bombardment; HRMS, high resolution mass spectrometry; UppAp, uridine 3'-pyrophosphate, P' right-arrow 5'-ester with adenosine 3'-phosphate; dUppAp, 2'-deoxyuridine 3'-pyrophosphate, P' right-arrow 5'-ester with adenosine 3'-phosphate; U2'ppAp, uridine 2'-pyrophosphate, P' right-arrow 5'-ester with adenosine 3'-phosphate; pdUppAp, 5'-phospho-2'-deoxyuridine 3'-pyrophosphate, P' right-arrow 5'-ester with adenosine 3'-phosphate; pTppAp, 5'-phosphothymidine 3'-pyrophosphate, P' right-arrow 5'-ester with adenosine 3'-phosphate; TppdA, thymidine 3'-pyro-phosphate, P' right-arrow 5'-ester with 2'-deoxyadenosine; Mes, 4-morpholineethanesulfonic acid; HPLC, high performance liquid chromatography.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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