Inhibitor design for ribonuclease A: the binding of two 5′-phosphate uridine analogues

Abstract

PDB References: ribonuclease A, U5P complex, 3dxg, r3dxgsf; UDP complex, 3dxh, r3dxhsf. In the quest for the rational design of selective and potent inhibitors for members of the pancreatic ribonuclease A (RNase A) family of biomedical interest, the binding of uridine 5 0 -phosphate (U5P) and uridine 5 0 -diphosphate (UDP) to RNase A have been investigated using kinetic studies and X-ray crystallography. Both nucleotides are competitive inhibitors of the enzyme, with K i values of 4.0 and 0.65 mM, respectively. They bind to the active site of the enzyme by anchoring two molecules connected to each other by hydrogen bonds and van der Waals interactions. While the first of the inhibitor molecules binds with its nucleobase in the pyrimidinyl-binding subsite, the second is bound at the purine-preferring subsite. The unexpected binding of a pyrimidine at the purinebinding subsite has added new important elements to the rational design approach for the discovery of new potent inhibitors of the RNase A superfamily.

The crystal structure of ribonuclease A in complex with thymidine‐3′‐monophosphate provides further insight into ligand binding

“…2(A)]. Among other pyrimidines, this stabilizing interaction is also observed in the crystal structures of RNase A complexed with 2′‐CMP46, 3′‐CMP23, 3′‐UMP24, 5′‐UMP48, and uridine‐2′,3′‐vanadate49. Depending on its orientation, the protruding O5′ oxygen atom of the ribose moiety can sometimes interact with a water molecule that is hydrogen bonded with the carbonyl oxygen of Asp121 and/or with the terminal Nζ atom of Lys66.…”

“…Structures of RNase A complexed with the non‐natural pyrimidine ligands 2′‐CMP46, 5′‐UMP48, 5′‐UDP48, uridine vanadate49, 52, d(pT) 4 53 and pTppAp18 also yield interesting structural measures of comparison with respect to 3′‐TMP binding to RNase A. In all of these complexes, binding of the pyrimidine moiety to the B 1 R 1 P 1 subsite of RNase A is very similar to that of 3′‐TMP and does not appear to show significant structural variations to explain the vast array of ligand binding affinities.…”

Structure of bovine pancreatic ribonuclease complexed with uridine 5′-monophosphate at 1.60 Å resolution

“…Thus, the main U5P interactions are through the U5P N3Á Á ÁThr45 OG1 and U5P O2Á Á ÁThr45 N hydrogen bonds. Although they can accommodate pyrimidine bases [Raines, 1998; PDB codes 8rsa (Nachman et al, 1990), 1rta (Birdsall & McPherson, 1992), 3dxg and 3dxh (Tsirkone et al, 2009)], the purine-binding sites were vacant. In addition, the ribose ring interacts with the enzyme through an O2 0 Á Á ÁLys41 NZ hydrogen bond (2.61 and 2.98 Å in molecules A and B, respectively).…”

“…vanadate [PDB codes 6rsa (Borah et al, 1985) and 1ruv (Ladner et al, 1997)]. In fact, most pyrimidine nucleotide-RNase A structures have a similar Lys41 NZÁ Á ÁO2 0 interaction unless they are devoid of the O2 0 atom (PDB code 1w4p, Jenkins et al, 2005), the ribose pucker is 2 0 -endo or 3 0 -exo [PDB codes 1o0n (Leonidas et al, 2003) and 1rta (Birdsall & McPherson, 1992)] or Lys41 is directed away from the ribose ring (PDB code 3dxg; Tsirkone et al, 2009). The only other hydrogen-bonding interaction involving U5P is seen in molecule B, in which Lys66 appears to hydrogen bond to either orientation of the phosphate group, as shown in Fig.…”

“…One other structure of an RNase A-U5P complex has recently been reported (PDB code 3dxg; Tsirkone et al, 2009). It was obtained by soaking U5P into C2 crystals of RNase A, which have two molecules per asymmetric unit.…”