Demetres Leonidas - Academia.edu (original) (raw)

Papers by Demetres Leonidas

Research paper thumbnail of Toward Rational Design of Ribonuclease Inhibitors: High-Resolution Crystal Structure of a Ribonuclease A Complex with a Potent 3‘,5‘-Pyrophosphate-Linked Dinucleotide Inhibitor

Biochemistry, 1999

The crystal structure of ribonuclease A (RNase A) in complex with pdUppA-3'-p [5'... more The crystal structure of ribonuclease A (RNase A) in complex with pdUppA-3'-p [5'-phospho-2'-deoxyuridine-3'-pyrophosphate (P'-->5') adenosine 3'-phosphate] has been determined at 1.7 A resolution. This dinucleotide is the most potent low molecular weight inhibitor of RNase A reported to date (K(i) = 27 nM) and is also effective against two major nonpancreatic RNases: eosinophil-derived neurotoxin and RNase-4; in all cases, tight binding in large part derives from the unusual 3',5'-pyrophosphate internucleotide linkage [Russo, N., and Shapiro, R. (1999) J. Biol. Chem. 274, 14902-14908]. The design of pdUppA-3'-p was based on the crystal structure of RNase A complexed with 5'-diphosphoadenosine 3'-phosphate (ppA-3'-p) [Leonidas, D. D., Shapiro, R., Irons, L. I., Russo, N., and Acharya, K. R. (1997) Biochemistry 36, 5578-5588]. The adenosine of pdUppA-3'-p adopts an atypical syn conformation not observed for standard adenosine nucleotides bound to RNase A. This conformation, which allows extensive interactions with Asn 67, Gln 69, Asn 71, and His 119, is associated with the placement of the 5'-beta-phosphate of the adenylate, rather than alpha-phosphate, at the site where substrate phosphodiester bond cleavage occurs. The contacts of the deoxyuridine 5'-phosphate portion of pdUppA-3'-p appear to be responsible for the 9-fold increased affinity of this compound as compared to ppA-3'-p: the uracil base binds to Thr 45 in the same manner as previous pyrimidine inhibitors, and the terminal 5'-phosphate is positioned to form medium-range Coulombic interactions with Lys 66. The full potential benefit of these added interactions is not realized because of compensatory losses of hydrogen bonds of Lys 7 and Gln 11 with the terminal 3'-phosphate and the adenylate 5'-alpha-phosphate, which were not predicted by modeling. The results reported here have important implications for the design of improved inhibitors of RNase A and for the development of therapeutic agents to control the activities of RNase homologues such as eosinophil-derived neurotoxin and angiogenin that have roles in human pathologies.

Research paper thumbnail of Crystal Structure of Eosinophil Cationic Protein at 2.4 Å Resolution

Research paper thumbnail of Binding of the potential antitumour agent indirubin-5-sulphonate at the inhibitor site of rabbit muscle glycogen phosphorylase b: Comparison with ligand binding to pCDK2-cyclin A complex

European Journal of Biochemistry, 2004

The binding of indirubin-5-sulphonate (E226), a potential anti-tumour agent and a potent inhibito... more The binding of indirubin-5-sulphonate (E226), a potential anti-tumour agent and a potent inhibitor (IC50 = 35 nm) of cyclin-dependent kinase 2 (CDK2) and glycogen phosphorylase (GP) has been studied by kinetic and crystallographic methods. Kinetic analysis revealed that E226 is a moderate inhibitor of GPb (Ki = 13.8 ± 0.2 µm) and GPa (Ki = 57.8 ± 7.1 µm) and acts synergistically with glucose. To explore the molecular basis of E226 binding we have determined the crystal structure of the GPb/E226 complex at 2.3 Å resolution. Structure analysis shows clearly that E226 binds at the purine inhibitor site, where caffeine and flavopiridol also bind [Oikonomakos, N.G., Schnier, J.B., Zographos, S.E., Skamnaki, V.T., Tsitsanou, K.E. & Johnson, L.N. (2000) J. Biol. Chem.275, 34566–34573], by intercalating between the two aromatic rings of Phe285 and Tyr613. The mode of binding of E226 to GPb is similar, but not identical, to that of caffeine and flavopiridol. Comparative structural analyses of the GPb–E226, GPb–caffeine and GPb–flavopiridol complex structures reveal the structural basis of the differences in the potencies of the three inhibitors and indicate binding residues in the inhibitor site that can be exploited to obtain more potent inhibitors. Structural comparison of the GPb–E226 complex structure with the active pCDK2–cyclin A–E226 complex structure clearly shows the different binding modes of the ligand to GPb and CDK2; the more extensive interactions of E226 with the active site of CDK2 may explain its higher affinity towards the latter enzyme.

Research paper thumbnail of In the Search of Glycogen Phosphorylase Inhibitors: Synthesis of C-D-Glycopyranosylbenzo(hydro)quinones – Inhibition of and Binding to Glycogen Phosphorylase in the Crystal

European Journal of Organic Chemistry, 2007

Penta-O-acetyl-β-D-glycopyranoses and 1,4-dimethoxybenzene led selectively by electrophilic subst... more Penta-O-acetyl-β-D-glycopyranoses and 1,4-dimethoxybenzene led selectively by electrophilic substitution to C-β-D-glycopyranosyl-1,4-dimethoxybenzenes which were converted by simple and efficient reactions (oxidation, reduction and deacetylation) to the corresponding C-glycosylhydro- and C-glycosylbenzoquinones, with either an acetylated or deprotected sugar moiety. C-β-D-Glucosylbenzoquinone 19 and C-β-D-Glucosylhydroquinone 23 were found to be competitive inhibitors of rabbit muscle glycogen phosphorylase b (GPb), with respect to the substrate α-D-glucose-1-phosphate, with Ki values of 1.3 and 0.9 mM, respectively, whereas C-β-D-glucosylhydroquinone 17 was not effective up to a concentration of 8 mM. In order to elucidate the structural basis of inhibition, we determined the crystal structures of 19 and 23 in complex with GPb at a 2.03–2.05 Å resolution. The complex structures reveal that the inhibitors can be accommodated at the catalytic site at approximately the same position as α-D-glucose and stabilise the transition state conformation of the 280s loop by making several favourable contacts to Asp283 and Asn284 of this loop. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)

Research paper thumbnail of Binding of β- d-glucopyranosyl bismethoxyphosphoramidate to glycogen phosphorylase b: kinetic and crystallographic studies

Bioorganic & Medicinal Chemistry, 2005

In an attempt to identify a new lead molecule that would enable the design of inhibitors with enh... more In an attempt to identify a new lead molecule that would enable the design of inhibitors with enhanced affinity for glycogen phosphorylase (GP), beta-D-glucopyranosyl bismethoxyphosphoramidate (phosphoramidate), a glucosyl phosphate analogue, was tested for inhibition of the enzyme. Kinetic experiments showed that the compound was a weak competitive inhibitor of rabbit muscle GPb (with respect to alpha-D-glucose-1-phosphate (Glc-1-P)) with a Ki value of 5.9 (+/-0.1) mM. In order to elucidate the structural basis of inhibition, we determined the structure of GPb complexed with the phosphoramidate at 1.83 A resolution. The complex structure reveals that the inhibitor binds at the catalytic site and induces significant conformational changes in the vicinity of this site. In particular, the 280s loop (residues 282-287) shifts 0.4-4.3 A (main-chain atoms) to accommodate the phosphoramidate, but these conformational changes do not lead to increased contacts between the inhibitor and the protein that would improve ligand binding.

Research paper thumbnail of Naturally Occurring Pentacyclic Triterpenes as Inhibitors of Glycogen Phosphorylase: Synthesis, Structure−Activity Relationships, and X-ray Crystallographic Studies

Journal of Medicinal Chemistry, 2008

Twenty-five naturally occurring pentacyclic triterpenes, 15 of which were synthesized in this stu... more Twenty-five naturally occurring pentacyclic triterpenes, 15 of which were synthesized in this study, were biologically evaluated as inhibitors of rabbit muscle glycogen phosphorylase a (GPa). From SAR studies, the presence of a sugar moiety in triterpene saponins resulted in a markedly decreased activity ( 7, 18- 20) or no activity ( 21, 22). These saponins, however, might find their value as potential natural prodrugs which are much more water-soluble than their corresponding aglycones. To elucidate the mechanism of GP inhibition, we have determined the crystal structures of the GPb-asiatic acid and GPb-maslinic acid complexes. The X-ray analysis indicates that the inhibitors bind at the allosteric activator site, where the physiological activator AMP binds. Pentacyclic triterpenes represent a promising class of multiple-target antidiabetic agents that exert hypoglycemic effects, at least in part, through GP inhibition.

Research paper thumbnail of Structural Basis for the Carbohydrate Recognition of the Sclerotium rolfsii Lectin

Journal of Molecular Biology, 2007

The crystal structure of a novel fungal lectin from Sclerotium rolfsii (SRL) in its free form and... more The crystal structure of a novel fungal lectin from Sclerotium rolfsii (SRL) in its free form and in complex with N-acetyl-D-galactosamine (GalNAc) and N-acetyl-D -glucosamine (GlcNAc) has been determined at 1.1 Å, 2.0 Å, and 1.7 Å resolution, respectively. The protein structure is composed of two β-sheets, which consist of four and six β-strands, connected by two α-helices. Sequence and structural comparisons reveal that SRL is the third member of a newly identified family of fungal lectins, which includes lectins from Agaricus bisporus and Xerocomus chrysenteron that share a high degree of structural similarity and carbohydrate specificity. The data for the free SRL are the highest resolution data for any protein of this family. The crystal structures of the SRL in complex with two carbohydrates, GalNAc and GlcNAc, which differ only in the configuration of a single epimeric hydroxyl group, provide the structural basis for its carbohydrate specificity. SRL has two distinct carbohydrate-binding sites, a primary and a secondary. GalNAc binds at the primary site, whereas GlcNAc binds only at the secondary site. Thus, SRL has the ability to recognize and probably bind at the same time two different carbohydrate structures. Structural comparison to Agaricus bisporus lectin-carbohydrate complexes reveals that the primary site is also able to bind the Thomsen-Friedenreich antigen (Galβ1→3 GalNAc-α-glycan structures) whereas the secondary site cannot. The features of the molecular recognition at the two sites are described in detail.

Research paper thumbnail of High-resolution crystal structures of ribonuclease A complexed with adenylic and uridylic nucleotide inhibitors. Implications for structure-based design of ribonucleolytic inhibitors

Protein Science, 2009

The crystal structures of bovine pancreatic ribonuclease A (RNase A) in complex with 3′,5′-ADP, 2... more The crystal structures of bovine pancreatic ribonuclease A (RNase A) in complex with 3′,5′-ADP, 2′,5′-ADP, 5′-ADP, U-2′-p and U-3′-p have been determined at high resolution. The structures reveal that each inhibitor binds differently in the RNase A active site by anchoring a phosphate group in subsite P1. The most potent inhibitor of all five, 5′-ADP (Ki = 1.2 μM), adopts a syn conformation (in contrast to 3′,5′-ADP and 2′,5′-ADP, which adopt an anti), and it is the β- rather than the α-phosphate group that binds to P1. 3′,5′-ADP binds with the 5′-phosphate group in P1 and the adenosine in the B2 pocket. Two different binding modes are observed in the two RNase A molecules of the asymmetric unit for 2′,5′-ADP. This inhibitor binds with either the 3′ or the 5′ phosphate groups in subsite P1, and in each case, the adenosine binds in two different positions within the B2 subsite. The two uridilyl inhibitors bind similarly with the uridine moiety in the B1 subsite but the placement of a different phosphate group in P1 (2′ versus 3′) has significant implications on their potency against RNase A. Comparative structural analysis of the RNase A, eosinophil-derived neurotoxin (EDN), eosinophil cationic protein (ECP), and human angiogenin (Ang) complexes with these and other phosphonucleotide inhibitors provides a wealth of information for structure-based design of inhibitors specific for each RNase. These inhibitors could be developed to therapeutic agents that could control the biological activities of EDN, ECP, and ANG, which play key roles in human pathologies.

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

Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2009

PDB References: ribonuclease A, U5P complex, 3dxg, r3dxgsf; UDP complex, 3dxh, r3dxhsf. In the qu... more 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.

Research paper thumbnail of Binding of Phosphate and pyrophosphate ions at the active site of human angiogenin as revealed by X-ray crystallography

Protein Science, 2001

Human angiogenin (Ang) is an unusual homolog of bovine pancreatic RNase A that utilizes its ribon... more Human angiogenin (Ang) is an unusual homolog of bovine pancreatic RNase A that utilizes its ribonucleolytic activity to induce the formation of new blood vessels. The pyrimidine-binding site of Ang was shown previously to be blocked by glutamine 117, indicating that Ang must undergo a conformational change to bind and cleave RNA. The mechanism and nature of this change are not known, and no Ang-inhibitor complexes have been characterized structurally thus far. Here, we report crystal structures for the complexes of Ang with the inhibitors phosphate and pyrophosphate, and the structure of the complex of the superactive Ang variant Q117G with phosphate, all at 2.0 Å resolution. Phosphate binds to the catalytic site of both Ang and Q117G in essentially the same manner observed in the RNase A–phosphate complex, forming hydrogen bonds with the side chains of His 13, His 114, and Gln 12, and the main chain of Leu 115; it makes an additional interaction with the Lys 40 ammonium group in the Ang complex. One of the phosphate groups of pyrophosphate occupies a similar position. The other phosphate extends toward Gln 117, and lies within hydrogen-bonding distance from the side-chain amide of this residue as well as the imidazole group of His 13 and the main-chain oxygen of Leu 115. The pyrimidine site remains obstructed in all three complex structures, that is, binding to the catalytic center is not sufficient to trigger the conformational change required for catalytic activity, even in the absence of the Gln 117 side chain. The Ang–pyrophosphate complex structure suggests how nucleoside pyrophosphate inhibitors might bind to Ang; this information may be useful for the design of Ang antagonists as potential anti-angiogenic drugs.

Research paper thumbnail of The binding of IMP to Ribonuclease A

Febs Journal, 2005

The binding of inosine 5′ phosphate (IMP) to ribonuclease A has been studied by kinetic and X-ray... more The binding of inosine 5′ phosphate (IMP) to ribonuclease A has been studied by kinetic and X-ray crystallographic experiments at high (1.5 Å) resolution. IMP is a competitive inhibitor of the enzyme with respect to C>p and binds to the catalytic cleft by anchoring three IMP molecules in a novel binding mode. The three IMP molecules are connected to each other by hydrogen bond and van der Waals interactions and collectively occupy the B1R1P1B2P0P-1 region of the ribonucleolytic active site. One of the IMP molecules binds with its nucleobase in the outskirts of the B2 subsite and interacts with Glu111 while its phosphoryl group binds in P1. Another IMP molecule binds by following the retro-binding mode previously observed only for guanosines with its nucleobase at B1 and the phosphoryl group in P-1. The third IMP molecule binds in a novel mode towards the C-terminus. The RNase A–IMP complex provides structural evidence for the functional components of subsite P-1 while it further supports the role inferred by other studies to Asn71 as the primary structural determinant for the adenine specificity of the B2 subsite. Comparative structural analysis of the IMP and AMP complexes highlights key aspects of the specificity of the base binding subsites of RNase A and provides a structural explanation for their potencies. The binding of IMP suggests ways to develop more potent inhibitors of the pancreatic RNase superfamily using this nucleotide as the starting point.

Research paper thumbnail of Iminosugars as Potential Inhibitors of Glycogenolysis: Structural Insights into the Molecular Basis of Glycogen Phosphorylase Inhibition

Journal of Medicinal Chemistry, 2006

Iminosugars DAB (5), isofagomine (9), and several N-substituted derivatives have been identified ... more Iminosugars DAB (5), isofagomine (9), and several N-substituted derivatives have been identified as potent inhibitors of liver glycogen phosphorylase a (IC(50) = 0.4-1.2 microM) and of basal and glucagon-stimulated glycogenolysis (IC(50) = 1-3 microM). The X-ray structures of 5, 9, and its N-3-phenylpropyl analogue 8 in complex with rabbit muscle glycogen phosphorylase (GPb) shows that iminosugars bind tightly at the catalytic site in the presence of the substrate phosphate and induce conformational changes that characterize the R-state conformation of the enzyme. Charged nitrogen N1 is within hydrogen-bonding distance with the carbonyl oxygen of His377 (5) and in ionic contact with the substrate phosphate oxygen (8 and 9). Our findings suggest that the inhibitors function as oxocarbenium ion transition-state analogues. The conformational change to the R state provides an explanation for previous findings that 5, unlike inhibitors that favor the T state, promotes phosphorylation of GPb in hepatocytes with sequential inactivation of glycogen synthase.

Research paper thumbnail of Cleavage of 3‘,5‘-Pyrophosphate-Linked Dinucleotides by Ribonuclease A and Angiogenin

Biochemistry, 2001

Recently, 3′,5′-pyrophosphate-linked 2′-deoxyribodinucleotides were shown to be >100-fold more ef... more Recently, 3′,5′-pyrophosphate-linked 2′-deoxyribodinucleotides were shown to be >100-fold more effective inhibitors of RNase A superfamily enzymes than were the corresponding monophosphatelinked (i.e., standard) dinucleotides. Here, we have investigated two ribo analogues of these compounds, cytidine 3′-pyrophosphate (P′f5′) adenosine (CppA) and uridine 3′-pyrophosphate (P′f5′) adenosine (UppA), as potential substrates for RNase A and angiogenin. CppA and UppA are cleaved efficiently by RNase A, yielding as products 5′-AMP and cytidine or uridine cyclic 2′,3′-phosphate. The k cat /K m values are only 4-fold smaller than for the standard dinucleotides CpA and UpA, and the K m values (10-16 µM) are lower than those reported for any earlier small substrates (e.g., 500-700 µM for CpA and UpA). The k cat /K m value for CppA with angiogenin is also only severalfold smaller than for CpA, but the effect of lengthening the internucleotide linkage on K m is more modest. Ribonucleotide 3′,5′-pyrophosphate linkages were proposed previously to exist in nature as chemically labile intermediates in the pathway for the generation of cyclic 2′,3′-phosphate termini in various RNAs. We demonstrate that in fact they are relatively stable (t 1/2 > 15 days for uncatalyzed degradation of UppA at pH 6 and 25°C) and that cleavage in vivo is most likely enzymatic. Replacements of the RNase A catalytic residues His12 and His119 by alanine reduce activity toward UppA by ∼10 5 -and 10 3.3 -fold, respectively. Thus, both residues play important roles. His12 probably acts as a base catalyst in cleavage of UppA (as with RNA). However, the major function of His119 in RNA cleavage, protonation of the 5′-O leaving group, is not required for UppA cleavage because the pK a of the leaving group is much lower than that for RNA substrates. A crystal structure of the complex of RNase A with 2′-deoxyuridine 3′-pyrophosphate (P′f5′) adenosine (dUppA), determined at 1.7 Å resolution, together with models of the UppA complex based on this structure suggest that His119 contributes to UppA cleavage through a hydrogen bond with a nonbridging oxygen atom in the pyrophosphate and through π-π stacking with the six-membered ring of adenine.

Research paper thumbnail of Mapping the ribonucleolytic active site of bovine seminal ribonuclease. The binding of pyrimidinyl phosphonucleotide inhibitors

European Journal of Medicinal Chemistry, 2009

Bovine seminal ribonuclease (BS-RNase) is a 27kDa homodimeric enzyme and a member of the pancreat... more Bovine seminal ribonuclease (BS-RNase) is a 27kDa homodimeric enzyme and a member of the pancreatic RNase A superfamily. It is the only RNase with a quaternary structure and it is a mixture of two dimeric forms. In the most abundant form the active site is formed by the swapping of the N-terminal segments. BS-RNase is a potent antitumor agent with severe side effects such as aspermatogenicity, and immunosuppression. As a first step towards the design of potent inhibitors of this enzyme we mapped its active site through the study of the binding of uridine 2'-phosphate (U2'p), uridine 3'-phosphate (U3'p), uridine 5'-diphosphate (UDP), cytidine 3'-phosphate (C3'p), and cytidine 5-phosphate (C5'p), by kinetics, and X-ray crystallography. These phosphonucleotides are potent inhibitors with C3'p being the most potent with a K(i) value of 22 microM. Absorption, distribution, metabolism, and excretion pharmacokinetic property predictions reveal U2'p, U3'p, and C5'p as the most promising with respect to oral bioavailability. In vivo studies on the aspermatogenic effect have shown that C3'p and C5'p inhibit significantly this biological action of BS-RNase.

Research paper thumbnail of The binding of 3′- N-piperidine-4-carboxyl-3′-deoxy- ara-uridine to ribonuclease A in the crystal

Bioorganic & Medicinal Chemistry, 2006

The binding of a moderate inhibitor, 3 0 -N-piperidine-4-carboxyl-3 0 -deoxy-ara-uridine, to ribo... more The binding of a moderate inhibitor, 3 0 -N-piperidine-4-carboxyl-3 0 -deoxy-ara-uridine, to ribonuclease A has been studied by X-ray crystallography at 1.7 Å resolution. Two inhibitor molecules are bound in the central RNA binding cavity of RNase A exploiting interactions with residues from peripheral binding sites rather than from the active site of the enzyme. The uracyl moiety of the first inhibitor molecule occupies the purine-preferring site of RNase A, while the rest of the molecule projects to the solvent. The second inhibitor molecule binds with the carboxyl group at the pyrimidine recognition site and the uridine moiety exploits interactions with RNase A residues Lys66, His119 and Asp121. Comparative structural analysis of the 3 0 -N-piperidine-4-carboxyl-3 0 -deoxy-ara-uridine complex with other RNase A-ligand complexes provides a structural explanation of its potency. The crystal structure of the RNase A-3 0 -N-piperidine-4-carboxyl-3 0 -deoxy-ara-uridine complex provides evidence of a novel ligand-binding pattern in RNase A for 3 0 -N-aminonucleosides that was not anticipated by modelling studies, while it also suggests ways to improve the efficiency and selectivity of such compounds to develop pharmaceuticals against pathologies associated with RNase A homologues.

Research paper thumbnail of Crystallographic studies on two bioisosteric analogues, N-acetyl-β- d-glucopyranosylamine and N-trifluoroacetyl-β- d-glucopyranosylamine, potent inhibitors of muscle glycogen phosphorylase

Bioorganic & Medicinal Chemistry, 2006

Structure-based inhibitor design has led to the discovery of a number of potent inhibitors of gly... more Structure-based inhibitor design has led to the discovery of a number of potent inhibitors of glycogen phosphorylase b (GPb), N-acyl derivatives of beta-D-glucopyranosylamine, that bind at the catalytic site of the enzyme. The first good inhibitor in this class of compounds, N-acetyl-beta-D-glucopyranosylamine (NAG) (K(i) = 32 microM), has been previously characterized by biochemical, biological and crystallographic experiments at 2.3 angstroms resolution. Bioisosteric replacement of the acetyl group by trifluoroacetyl group resulted in an inhibitor, N-trifluoroacetyl-beta-D-glucopyranosylamine (NFAG), with a K(i) = 75 microM. To elucidate the structural basis of its reduced potency, we determined the ligand structure in complex with GPb at 1.8 angstroms resolution. To compare the binding mode of N-trifluoroacetyl derivative with that of the lead molecule, we also determined the structure of GPb-NAG complex at a higher resolution (1.9 angstroms). NFAG can be accommodated in the catalytic site of T-state GPb at approximately the same position as that of NAG and stabilize the T-state conformation of the 280 s loop by making several favourable contacts to Asn284 of this loop. The difference observed in the K(i) values of the two analogues can be interpreted in terms of subtle conformational changes of protein residues and shifts of water molecules in the vicinity of the catalytic site, variations in van der Waals interaction, and desolvation effects.

Research paper thumbnail of Binding of N-acetyl-N '-beta-d-glucopyranosyl urea and N-benzoyl-N '-beta-d-glucopyranosyl urea to glycogen phosphorylase b. Kinetic and crystallographic studies

European Journal of Biochemistry, 2002

Two substituted ureas of beta-D-glucose, N-acetyl-N&a... more Two substituted ureas of beta-D-glucose, N-acetyl-N'-beta-D-glucopyranosyl urea (Acurea) and N-benzoyl-N'-beta-D-glucopyranosyl urea (Bzurea), have been identified as inhibitors of glycogen phosphorylase, a potential target for therapeutic intervention in type 2 diabetes. To elucidate the structural basis of inhibition, we determined the structure of muscle glycogen phosphorylase b (GPb) complexed with the two compounds at 2.0 A and 1.8 A resolution, respectively. The structure of the GPb-Acurea complex reveals that the inhibitor can be accommodated in the catalytic site of T-state GPb with very little change in the tertiary structure. The glucopyranose moiety makes the standard hydrogen bonds and van der Waals contacts as observed in the GPb-glucose complex, while the acetyl urea moiety is in a favourable electrostatic environment and makes additional polar contacts with the protein. The structure of the GPb-Bzurea complex shows that Bzurea binds tightly at the catalytic site and induces substantial conformational changes in the vicinity of the catalytic site. In particular, the loop of the polypeptide chain containing residues 282-287 shifts 1.3-3.7 A (Calpha atoms) to accommodate Bzurea. Bzurea can also occupy the new allosteric site, some 33 A from the catalytic site, which is currently the target for the design of antidiabetic drugs.

Research paper thumbnail of Crystal structure of rabbit muscle glycogen phosphorylase a in complex with a potential hypoglycaemic drug at 2.0 Å resolution

Biochimica Et Biophysica Acta-proteins and Proteomics, 2003

CP320626 has been identified as a potent inhibitor, synergistic with glucose, of human liver glyc... more CP320626 has been identified as a potent inhibitor, synergistic with glucose, of human liver glycogen phosphorylase a (LGPa), a possible target for type 2 diabetes therapy. CP320626 is also a potent inhibitor of human muscle GPa. In order to elucidate the structural basis of the mechanism of CP320626 inhibition, the structures of T state rabbit muscle GPa (MGPa) in complex with glucose and in complex with both glucose and CP320626 were determined at 2.0 A resolution, and refined to crystallographic R values of 0.179 (R(free)=0.218) and 0.207 (R(free)=0.235), respectively. CP320626 binds at the new allosteric site, some 33 A from the catalytic site, where glucose binds. The binding of CP320626 to MGPa does not promote extensive conformational changes except for small shifts of the side chain atoms of residues R60, V64, and K191. Both CP320626 and glucose promote the less active T state, while structural comparisons of MGPa-glucose-CP320626 complex with LGPa complexed with a related compound (CP403700) and a glucose analogue inhibitor indicate that the residues of the new allosteric site, conserved in the two isozymes, show no significant differences in their positions.

Research paper thumbnail of The binding of β- and γ-cyclodextrins to glycogen phosphorylase b: Kinetic and crystallographic studies

Protein Science, 2003

A number of regulatory binding sites of glycogen phosphorylase (GP), such as the catalytic, the i... more A number of regulatory binding sites of glycogen phosphorylase (GP), such as the catalytic, the inhibitor, and the new allosteric sites are currently under investigation as targets for inhibition of hepatic glycogenolysis under high glucose concentrations; in some cases specific inhibitors are under evaluation in human clinical trials for therapeutic intervention in type 2 diabetes. In an attempt to investigate whether the storage site can be exploited as target for modulating hepatic glucose production, α-, β-, and γ-cyclodextrins were identified as moderate mixed-type competitive inhibitors of GPb (with respect to glycogen) with Ki values of 47.1, 14.1, and 7.4 mM, respectively. To elucidate the structural basis of inhibition, we determined the structure of GPb complexed with β- and γ-cyclodextrins at 1.94 Å and 2.3 Å resolution, respectively. The structures of the two complexes reveal that the inhibitors can be accommodated in the glycogen storage site of T-state GPb with very little change of the tertiary structure and provide a basis for understanding their potency and subsite specificity. Structural comparisons of the two complexes with GPb in complex with either maltopentaose (G5) or maltoheptaose (G7) show that β- andγ-cyclodextrins bind in a mode analogous to the G5 and G7 binding with only some differences imposed by their cyclic conformations. It appears that the binding energy for stabilization of enzyme complexes derives from hydrogen bonding and van der Waals contacts to protein residues. The binding of α-cyclodextrin and octakis (2,3,6-tri-O-methyl)-γ-cyclodextrin was also investigated, but none of them was bound in the crystal; moreover, the latter did not inhibit the phosphorylase reaction.

Research paper thumbnail of Crystallographic studies on acyl ureas, a new class of glycogen phosphorylase inhibitors, as potential antidiabetic drugs

Protein Science, 2005

Acyl ureas were discovered as a novel class of inhibitors for glycogen phosphorylase, a molecular... more Acyl ureas were discovered as a novel class of inhibitors for glycogen phosphorylase, a molecular target to control hyperglycemia in type 2 diabetics. This series is exemplified by 6-{2,6-Dichloro- 4-[3-(2-chloro-benzoyl)-ureido]-phenoxy}-hexanoic acid, which inhibits human liver glycogen phosphorylase a with an IC50 of 2.0 μM. Here we analyze four crystal structures of acyl urea derivatives in complex with rabbit muscle glycogen phosphorylase b to elucidate the mechanism of inhibition of these inhibitors. The structures were determined and refined to 2.26Å resolution and demonstrate that the inhibitors bind at the allosteric activator site, where the physiological activator AMP binds. Acyl ureas induce conformational changes in the vicinity of the allosteric site. Our findings suggest that acyl ureas inhibit glycogen phosphorylase by direct inhibition of AMP binding and by indirect inhibition of substrate binding through stabilization of the T′ state.

Research paper thumbnail of Toward Rational Design of Ribonuclease Inhibitors: High-Resolution Crystal Structure of a Ribonuclease A Complex with a Potent 3‘,5‘-Pyrophosphate-Linked Dinucleotide Inhibitor

Biochemistry, 1999

The crystal structure of ribonuclease A (RNase A) in complex with pdUppA-3'-p [5'... more The crystal structure of ribonuclease A (RNase A) in complex with pdUppA-3'-p [5'-phospho-2'-deoxyuridine-3'-pyrophosphate (P'-->5') adenosine 3'-phosphate] has been determined at 1.7 A resolution. This dinucleotide is the most potent low molecular weight inhibitor of RNase A reported to date (K(i) = 27 nM) and is also effective against two major nonpancreatic RNases: eosinophil-derived neurotoxin and RNase-4; in all cases, tight binding in large part derives from the unusual 3',5'-pyrophosphate internucleotide linkage [Russo, N., and Shapiro, R. (1999) J. Biol. Chem. 274, 14902-14908]. The design of pdUppA-3'-p was based on the crystal structure of RNase A complexed with 5'-diphosphoadenosine 3'-phosphate (ppA-3'-p) [Leonidas, D. D., Shapiro, R., Irons, L. I., Russo, N., and Acharya, K. R. (1997) Biochemistry 36, 5578-5588]. The adenosine of pdUppA-3'-p adopts an atypical syn conformation not observed for standard adenosine nucleotides bound to RNase A. This conformation, which allows extensive interactions with Asn 67, Gln 69, Asn 71, and His 119, is associated with the placement of the 5'-beta-phosphate of the adenylate, rather than alpha-phosphate, at the site where substrate phosphodiester bond cleavage occurs. The contacts of the deoxyuridine 5'-phosphate portion of pdUppA-3'-p appear to be responsible for the 9-fold increased affinity of this compound as compared to ppA-3'-p: the uracil base binds to Thr 45 in the same manner as previous pyrimidine inhibitors, and the terminal 5'-phosphate is positioned to form medium-range Coulombic interactions with Lys 66. The full potential benefit of these added interactions is not realized because of compensatory losses of hydrogen bonds of Lys 7 and Gln 11 with the terminal 3'-phosphate and the adenylate 5'-alpha-phosphate, which were not predicted by modeling. The results reported here have important implications for the design of improved inhibitors of RNase A and for the development of therapeutic agents to control the activities of RNase homologues such as eosinophil-derived neurotoxin and angiogenin that have roles in human pathologies.

Research paper thumbnail of Crystal Structure of Eosinophil Cationic Protein at 2.4 Å Resolution

Research paper thumbnail of Binding of the potential antitumour agent indirubin-5-sulphonate at the inhibitor site of rabbit muscle glycogen phosphorylase b: Comparison with ligand binding to pCDK2-cyclin A complex

European Journal of Biochemistry, 2004

The binding of indirubin-5-sulphonate (E226), a potential anti-tumour agent and a potent inhibito... more The binding of indirubin-5-sulphonate (E226), a potential anti-tumour agent and a potent inhibitor (IC50 = 35 nm) of cyclin-dependent kinase 2 (CDK2) and glycogen phosphorylase (GP) has been studied by kinetic and crystallographic methods. Kinetic analysis revealed that E226 is a moderate inhibitor of GPb (Ki = 13.8 ± 0.2 µm) and GPa (Ki = 57.8 ± 7.1 µm) and acts synergistically with glucose. To explore the molecular basis of E226 binding we have determined the crystal structure of the GPb/E226 complex at 2.3 Å resolution. Structure analysis shows clearly that E226 binds at the purine inhibitor site, where caffeine and flavopiridol also bind [Oikonomakos, N.G., Schnier, J.B., Zographos, S.E., Skamnaki, V.T., Tsitsanou, K.E. & Johnson, L.N. (2000) J. Biol. Chem.275, 34566–34573], by intercalating between the two aromatic rings of Phe285 and Tyr613. The mode of binding of E226 to GPb is similar, but not identical, to that of caffeine and flavopiridol. Comparative structural analyses of the GPb–E226, GPb–caffeine and GPb–flavopiridol complex structures reveal the structural basis of the differences in the potencies of the three inhibitors and indicate binding residues in the inhibitor site that can be exploited to obtain more potent inhibitors. Structural comparison of the GPb–E226 complex structure with the active pCDK2–cyclin A–E226 complex structure clearly shows the different binding modes of the ligand to GPb and CDK2; the more extensive interactions of E226 with the active site of CDK2 may explain its higher affinity towards the latter enzyme.

Research paper thumbnail of In the Search of Glycogen Phosphorylase Inhibitors: Synthesis of C-D-Glycopyranosylbenzo(hydro)quinones – Inhibition of and Binding to Glycogen Phosphorylase in the Crystal

European Journal of Organic Chemistry, 2007

Penta-O-acetyl-β-D-glycopyranoses and 1,4-dimethoxybenzene led selectively by electrophilic subst... more Penta-O-acetyl-β-D-glycopyranoses and 1,4-dimethoxybenzene led selectively by electrophilic substitution to C-β-D-glycopyranosyl-1,4-dimethoxybenzenes which were converted by simple and efficient reactions (oxidation, reduction and deacetylation) to the corresponding C-glycosylhydro- and C-glycosylbenzoquinones, with either an acetylated or deprotected sugar moiety. C-β-D-Glucosylbenzoquinone 19 and C-β-D-Glucosylhydroquinone 23 were found to be competitive inhibitors of rabbit muscle glycogen phosphorylase b (GPb), with respect to the substrate α-D-glucose-1-phosphate, with Ki values of 1.3 and 0.9 mM, respectively, whereas C-β-D-glucosylhydroquinone 17 was not effective up to a concentration of 8 mM. In order to elucidate the structural basis of inhibition, we determined the crystal structures of 19 and 23 in complex with GPb at a 2.03–2.05 Å resolution. The complex structures reveal that the inhibitors can be accommodated at the catalytic site at approximately the same position as α-D-glucose and stabilise the transition state conformation of the 280s loop by making several favourable contacts to Asp283 and Asn284 of this loop. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)

Research paper thumbnail of Binding of β- d-glucopyranosyl bismethoxyphosphoramidate to glycogen phosphorylase b: kinetic and crystallographic studies

Bioorganic & Medicinal Chemistry, 2005

In an attempt to identify a new lead molecule that would enable the design of inhibitors with enh... more In an attempt to identify a new lead molecule that would enable the design of inhibitors with enhanced affinity for glycogen phosphorylase (GP), beta-D-glucopyranosyl bismethoxyphosphoramidate (phosphoramidate), a glucosyl phosphate analogue, was tested for inhibition of the enzyme. Kinetic experiments showed that the compound was a weak competitive inhibitor of rabbit muscle GPb (with respect to alpha-D-glucose-1-phosphate (Glc-1-P)) with a Ki value of 5.9 (+/-0.1) mM. In order to elucidate the structural basis of inhibition, we determined the structure of GPb complexed with the phosphoramidate at 1.83 A resolution. The complex structure reveals that the inhibitor binds at the catalytic site and induces significant conformational changes in the vicinity of this site. In particular, the 280s loop (residues 282-287) shifts 0.4-4.3 A (main-chain atoms) to accommodate the phosphoramidate, but these conformational changes do not lead to increased contacts between the inhibitor and the protein that would improve ligand binding.

Research paper thumbnail of Naturally Occurring Pentacyclic Triterpenes as Inhibitors of Glycogen Phosphorylase: Synthesis, Structure−Activity Relationships, and X-ray Crystallographic Studies

Journal of Medicinal Chemistry, 2008

Twenty-five naturally occurring pentacyclic triterpenes, 15 of which were synthesized in this stu... more Twenty-five naturally occurring pentacyclic triterpenes, 15 of which were synthesized in this study, were biologically evaluated as inhibitors of rabbit muscle glycogen phosphorylase a (GPa). From SAR studies, the presence of a sugar moiety in triterpene saponins resulted in a markedly decreased activity ( 7, 18- 20) or no activity ( 21, 22). These saponins, however, might find their value as potential natural prodrugs which are much more water-soluble than their corresponding aglycones. To elucidate the mechanism of GP inhibition, we have determined the crystal structures of the GPb-asiatic acid and GPb-maslinic acid complexes. The X-ray analysis indicates that the inhibitors bind at the allosteric activator site, where the physiological activator AMP binds. Pentacyclic triterpenes represent a promising class of multiple-target antidiabetic agents that exert hypoglycemic effects, at least in part, through GP inhibition.

Research paper thumbnail of Structural Basis for the Carbohydrate Recognition of the Sclerotium rolfsii Lectin

Journal of Molecular Biology, 2007

The crystal structure of a novel fungal lectin from Sclerotium rolfsii (SRL) in its free form and... more The crystal structure of a novel fungal lectin from Sclerotium rolfsii (SRL) in its free form and in complex with N-acetyl-D-galactosamine (GalNAc) and N-acetyl-D -glucosamine (GlcNAc) has been determined at 1.1 Å, 2.0 Å, and 1.7 Å resolution, respectively. The protein structure is composed of two β-sheets, which consist of four and six β-strands, connected by two α-helices. Sequence and structural comparisons reveal that SRL is the third member of a newly identified family of fungal lectins, which includes lectins from Agaricus bisporus and Xerocomus chrysenteron that share a high degree of structural similarity and carbohydrate specificity. The data for the free SRL are the highest resolution data for any protein of this family. The crystal structures of the SRL in complex with two carbohydrates, GalNAc and GlcNAc, which differ only in the configuration of a single epimeric hydroxyl group, provide the structural basis for its carbohydrate specificity. SRL has two distinct carbohydrate-binding sites, a primary and a secondary. GalNAc binds at the primary site, whereas GlcNAc binds only at the secondary site. Thus, SRL has the ability to recognize and probably bind at the same time two different carbohydrate structures. Structural comparison to Agaricus bisporus lectin-carbohydrate complexes reveals that the primary site is also able to bind the Thomsen-Friedenreich antigen (Galβ1→3 GalNAc-α-glycan structures) whereas the secondary site cannot. The features of the molecular recognition at the two sites are described in detail.

Research paper thumbnail of High-resolution crystal structures of ribonuclease A complexed with adenylic and uridylic nucleotide inhibitors. Implications for structure-based design of ribonucleolytic inhibitors

Protein Science, 2009

The crystal structures of bovine pancreatic ribonuclease A (RNase A) in complex with 3′,5′-ADP, 2... more The crystal structures of bovine pancreatic ribonuclease A (RNase A) in complex with 3′,5′-ADP, 2′,5′-ADP, 5′-ADP, U-2′-p and U-3′-p have been determined at high resolution. The structures reveal that each inhibitor binds differently in the RNase A active site by anchoring a phosphate group in subsite P1. The most potent inhibitor of all five, 5′-ADP (Ki = 1.2 μM), adopts a syn conformation (in contrast to 3′,5′-ADP and 2′,5′-ADP, which adopt an anti), and it is the β- rather than the α-phosphate group that binds to P1. 3′,5′-ADP binds with the 5′-phosphate group in P1 and the adenosine in the B2 pocket. Two different binding modes are observed in the two RNase A molecules of the asymmetric unit for 2′,5′-ADP. This inhibitor binds with either the 3′ or the 5′ phosphate groups in subsite P1, and in each case, the adenosine binds in two different positions within the B2 subsite. The two uridilyl inhibitors bind similarly with the uridine moiety in the B1 subsite but the placement of a different phosphate group in P1 (2′ versus 3′) has significant implications on their potency against RNase A. Comparative structural analysis of the RNase A, eosinophil-derived neurotoxin (EDN), eosinophil cationic protein (ECP), and human angiogenin (Ang) complexes with these and other phosphonucleotide inhibitors provides a wealth of information for structure-based design of inhibitors specific for each RNase. These inhibitors could be developed to therapeutic agents that could control the biological activities of EDN, ECP, and ANG, which play key roles in human pathologies.

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

Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2009

PDB References: ribonuclease A, U5P complex, 3dxg, r3dxgsf; UDP complex, 3dxh, r3dxhsf. In the qu... more 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.

Research paper thumbnail of Binding of Phosphate and pyrophosphate ions at the active site of human angiogenin as revealed by X-ray crystallography

Protein Science, 2001

Human angiogenin (Ang) is an unusual homolog of bovine pancreatic RNase A that utilizes its ribon... more Human angiogenin (Ang) is an unusual homolog of bovine pancreatic RNase A that utilizes its ribonucleolytic activity to induce the formation of new blood vessels. The pyrimidine-binding site of Ang was shown previously to be blocked by glutamine 117, indicating that Ang must undergo a conformational change to bind and cleave RNA. The mechanism and nature of this change are not known, and no Ang-inhibitor complexes have been characterized structurally thus far. Here, we report crystal structures for the complexes of Ang with the inhibitors phosphate and pyrophosphate, and the structure of the complex of the superactive Ang variant Q117G with phosphate, all at 2.0 Å resolution. Phosphate binds to the catalytic site of both Ang and Q117G in essentially the same manner observed in the RNase A–phosphate complex, forming hydrogen bonds with the side chains of His 13, His 114, and Gln 12, and the main chain of Leu 115; it makes an additional interaction with the Lys 40 ammonium group in the Ang complex. One of the phosphate groups of pyrophosphate occupies a similar position. The other phosphate extends toward Gln 117, and lies within hydrogen-bonding distance from the side-chain amide of this residue as well as the imidazole group of His 13 and the main-chain oxygen of Leu 115. The pyrimidine site remains obstructed in all three complex structures, that is, binding to the catalytic center is not sufficient to trigger the conformational change required for catalytic activity, even in the absence of the Gln 117 side chain. The Ang–pyrophosphate complex structure suggests how nucleoside pyrophosphate inhibitors might bind to Ang; this information may be useful for the design of Ang antagonists as potential anti-angiogenic drugs.

Research paper thumbnail of The binding of IMP to Ribonuclease A

Febs Journal, 2005

The binding of inosine 5′ phosphate (IMP) to ribonuclease A has been studied by kinetic and X-ray... more The binding of inosine 5′ phosphate (IMP) to ribonuclease A has been studied by kinetic and X-ray crystallographic experiments at high (1.5 Å) resolution. IMP is a competitive inhibitor of the enzyme with respect to C>p and binds to the catalytic cleft by anchoring three IMP molecules in a novel binding mode. The three IMP molecules are connected to each other by hydrogen bond and van der Waals interactions and collectively occupy the B1R1P1B2P0P-1 region of the ribonucleolytic active site. One of the IMP molecules binds with its nucleobase in the outskirts of the B2 subsite and interacts with Glu111 while its phosphoryl group binds in P1. Another IMP molecule binds by following the retro-binding mode previously observed only for guanosines with its nucleobase at B1 and the phosphoryl group in P-1. The third IMP molecule binds in a novel mode towards the C-terminus. The RNase A–IMP complex provides structural evidence for the functional components of subsite P-1 while it further supports the role inferred by other studies to Asn71 as the primary structural determinant for the adenine specificity of the B2 subsite. Comparative structural analysis of the IMP and AMP complexes highlights key aspects of the specificity of the base binding subsites of RNase A and provides a structural explanation for their potencies. The binding of IMP suggests ways to develop more potent inhibitors of the pancreatic RNase superfamily using this nucleotide as the starting point.

Research paper thumbnail of Iminosugars as Potential Inhibitors of Glycogenolysis: Structural Insights into the Molecular Basis of Glycogen Phosphorylase Inhibition

Journal of Medicinal Chemistry, 2006

Iminosugars DAB (5), isofagomine (9), and several N-substituted derivatives have been identified ... more Iminosugars DAB (5), isofagomine (9), and several N-substituted derivatives have been identified as potent inhibitors of liver glycogen phosphorylase a (IC(50) = 0.4-1.2 microM) and of basal and glucagon-stimulated glycogenolysis (IC(50) = 1-3 microM). The X-ray structures of 5, 9, and its N-3-phenylpropyl analogue 8 in complex with rabbit muscle glycogen phosphorylase (GPb) shows that iminosugars bind tightly at the catalytic site in the presence of the substrate phosphate and induce conformational changes that characterize the R-state conformation of the enzyme. Charged nitrogen N1 is within hydrogen-bonding distance with the carbonyl oxygen of His377 (5) and in ionic contact with the substrate phosphate oxygen (8 and 9). Our findings suggest that the inhibitors function as oxocarbenium ion transition-state analogues. The conformational change to the R state provides an explanation for previous findings that 5, unlike inhibitors that favor the T state, promotes phosphorylation of GPb in hepatocytes with sequential inactivation of glycogen synthase.

Research paper thumbnail of Cleavage of 3‘,5‘-Pyrophosphate-Linked Dinucleotides by Ribonuclease A and Angiogenin

Biochemistry, 2001

Recently, 3′,5′-pyrophosphate-linked 2′-deoxyribodinucleotides were shown to be >100-fold more ef... more Recently, 3′,5′-pyrophosphate-linked 2′-deoxyribodinucleotides were shown to be >100-fold more effective inhibitors of RNase A superfamily enzymes than were the corresponding monophosphatelinked (i.e., standard) dinucleotides. Here, we have investigated two ribo analogues of these compounds, cytidine 3′-pyrophosphate (P′f5′) adenosine (CppA) and uridine 3′-pyrophosphate (P′f5′) adenosine (UppA), as potential substrates for RNase A and angiogenin. CppA and UppA are cleaved efficiently by RNase A, yielding as products 5′-AMP and cytidine or uridine cyclic 2′,3′-phosphate. The k cat /K m values are only 4-fold smaller than for the standard dinucleotides CpA and UpA, and the K m values (10-16 µM) are lower than those reported for any earlier small substrates (e.g., 500-700 µM for CpA and UpA). The k cat /K m value for CppA with angiogenin is also only severalfold smaller than for CpA, but the effect of lengthening the internucleotide linkage on K m is more modest. Ribonucleotide 3′,5′-pyrophosphate linkages were proposed previously to exist in nature as chemically labile intermediates in the pathway for the generation of cyclic 2′,3′-phosphate termini in various RNAs. We demonstrate that in fact they are relatively stable (t 1/2 > 15 days for uncatalyzed degradation of UppA at pH 6 and 25°C) and that cleavage in vivo is most likely enzymatic. Replacements of the RNase A catalytic residues His12 and His119 by alanine reduce activity toward UppA by ∼10 5 -and 10 3.3 -fold, respectively. Thus, both residues play important roles. His12 probably acts as a base catalyst in cleavage of UppA (as with RNA). However, the major function of His119 in RNA cleavage, protonation of the 5′-O leaving group, is not required for UppA cleavage because the pK a of the leaving group is much lower than that for RNA substrates. A crystal structure of the complex of RNase A with 2′-deoxyuridine 3′-pyrophosphate (P′f5′) adenosine (dUppA), determined at 1.7 Å resolution, together with models of the UppA complex based on this structure suggest that His119 contributes to UppA cleavage through a hydrogen bond with a nonbridging oxygen atom in the pyrophosphate and through π-π stacking with the six-membered ring of adenine.

Research paper thumbnail of Mapping the ribonucleolytic active site of bovine seminal ribonuclease. The binding of pyrimidinyl phosphonucleotide inhibitors

European Journal of Medicinal Chemistry, 2009

Bovine seminal ribonuclease (BS-RNase) is a 27kDa homodimeric enzyme and a member of the pancreat... more Bovine seminal ribonuclease (BS-RNase) is a 27kDa homodimeric enzyme and a member of the pancreatic RNase A superfamily. It is the only RNase with a quaternary structure and it is a mixture of two dimeric forms. In the most abundant form the active site is formed by the swapping of the N-terminal segments. BS-RNase is a potent antitumor agent with severe side effects such as aspermatogenicity, and immunosuppression. As a first step towards the design of potent inhibitors of this enzyme we mapped its active site through the study of the binding of uridine 2'-phosphate (U2'p), uridine 3'-phosphate (U3'p), uridine 5'-diphosphate (UDP), cytidine 3'-phosphate (C3'p), and cytidine 5-phosphate (C5'p), by kinetics, and X-ray crystallography. These phosphonucleotides are potent inhibitors with C3'p being the most potent with a K(i) value of 22 microM. Absorption, distribution, metabolism, and excretion pharmacokinetic property predictions reveal U2'p, U3'p, and C5'p as the most promising with respect to oral bioavailability. In vivo studies on the aspermatogenic effect have shown that C3'p and C5'p inhibit significantly this biological action of BS-RNase.

Research paper thumbnail of The binding of 3′- N-piperidine-4-carboxyl-3′-deoxy- ara-uridine to ribonuclease A in the crystal

Bioorganic & Medicinal Chemistry, 2006

The binding of a moderate inhibitor, 3 0 -N-piperidine-4-carboxyl-3 0 -deoxy-ara-uridine, to ribo... more The binding of a moderate inhibitor, 3 0 -N-piperidine-4-carboxyl-3 0 -deoxy-ara-uridine, to ribonuclease A has been studied by X-ray crystallography at 1.7 Å resolution. Two inhibitor molecules are bound in the central RNA binding cavity of RNase A exploiting interactions with residues from peripheral binding sites rather than from the active site of the enzyme. The uracyl moiety of the first inhibitor molecule occupies the purine-preferring site of RNase A, while the rest of the molecule projects to the solvent. The second inhibitor molecule binds with the carboxyl group at the pyrimidine recognition site and the uridine moiety exploits interactions with RNase A residues Lys66, His119 and Asp121. Comparative structural analysis of the 3 0 -N-piperidine-4-carboxyl-3 0 -deoxy-ara-uridine complex with other RNase A-ligand complexes provides a structural explanation of its potency. The crystal structure of the RNase A-3 0 -N-piperidine-4-carboxyl-3 0 -deoxy-ara-uridine complex provides evidence of a novel ligand-binding pattern in RNase A for 3 0 -N-aminonucleosides that was not anticipated by modelling studies, while it also suggests ways to improve the efficiency and selectivity of such compounds to develop pharmaceuticals against pathologies associated with RNase A homologues.

Research paper thumbnail of Crystallographic studies on two bioisosteric analogues, N-acetyl-β- d-glucopyranosylamine and N-trifluoroacetyl-β- d-glucopyranosylamine, potent inhibitors of muscle glycogen phosphorylase

Bioorganic & Medicinal Chemistry, 2006

Structure-based inhibitor design has led to the discovery of a number of potent inhibitors of gly... more Structure-based inhibitor design has led to the discovery of a number of potent inhibitors of glycogen phosphorylase b (GPb), N-acyl derivatives of beta-D-glucopyranosylamine, that bind at the catalytic site of the enzyme. The first good inhibitor in this class of compounds, N-acetyl-beta-D-glucopyranosylamine (NAG) (K(i) = 32 microM), has been previously characterized by biochemical, biological and crystallographic experiments at 2.3 angstroms resolution. Bioisosteric replacement of the acetyl group by trifluoroacetyl group resulted in an inhibitor, N-trifluoroacetyl-beta-D-glucopyranosylamine (NFAG), with a K(i) = 75 microM. To elucidate the structural basis of its reduced potency, we determined the ligand structure in complex with GPb at 1.8 angstroms resolution. To compare the binding mode of N-trifluoroacetyl derivative with that of the lead molecule, we also determined the structure of GPb-NAG complex at a higher resolution (1.9 angstroms). NFAG can be accommodated in the catalytic site of T-state GPb at approximately the same position as that of NAG and stabilize the T-state conformation of the 280 s loop by making several favourable contacts to Asn284 of this loop. The difference observed in the K(i) values of the two analogues can be interpreted in terms of subtle conformational changes of protein residues and shifts of water molecules in the vicinity of the catalytic site, variations in van der Waals interaction, and desolvation effects.

Research paper thumbnail of Binding of N-acetyl-N '-beta-d-glucopyranosyl urea and N-benzoyl-N '-beta-d-glucopyranosyl urea to glycogen phosphorylase b. Kinetic and crystallographic studies

European Journal of Biochemistry, 2002

Two substituted ureas of beta-D-glucose, N-acetyl-N&a... more Two substituted ureas of beta-D-glucose, N-acetyl-N'-beta-D-glucopyranosyl urea (Acurea) and N-benzoyl-N'-beta-D-glucopyranosyl urea (Bzurea), have been identified as inhibitors of glycogen phosphorylase, a potential target for therapeutic intervention in type 2 diabetes. To elucidate the structural basis of inhibition, we determined the structure of muscle glycogen phosphorylase b (GPb) complexed with the two compounds at 2.0 A and 1.8 A resolution, respectively. The structure of the GPb-Acurea complex reveals that the inhibitor can be accommodated in the catalytic site of T-state GPb with very little change in the tertiary structure. The glucopyranose moiety makes the standard hydrogen bonds and van der Waals contacts as observed in the GPb-glucose complex, while the acetyl urea moiety is in a favourable electrostatic environment and makes additional polar contacts with the protein. The structure of the GPb-Bzurea complex shows that Bzurea binds tightly at the catalytic site and induces substantial conformational changes in the vicinity of the catalytic site. In particular, the loop of the polypeptide chain containing residues 282-287 shifts 1.3-3.7 A (Calpha atoms) to accommodate Bzurea. Bzurea can also occupy the new allosteric site, some 33 A from the catalytic site, which is currently the target for the design of antidiabetic drugs.

Research paper thumbnail of Crystal structure of rabbit muscle glycogen phosphorylase a in complex with a potential hypoglycaemic drug at 2.0 Å resolution

Biochimica Et Biophysica Acta-proteins and Proteomics, 2003

CP320626 has been identified as a potent inhibitor, synergistic with glucose, of human liver glyc... more CP320626 has been identified as a potent inhibitor, synergistic with glucose, of human liver glycogen phosphorylase a (LGPa), a possible target for type 2 diabetes therapy. CP320626 is also a potent inhibitor of human muscle GPa. In order to elucidate the structural basis of the mechanism of CP320626 inhibition, the structures of T state rabbit muscle GPa (MGPa) in complex with glucose and in complex with both glucose and CP320626 were determined at 2.0 A resolution, and refined to crystallographic R values of 0.179 (R(free)=0.218) and 0.207 (R(free)=0.235), respectively. CP320626 binds at the new allosteric site, some 33 A from the catalytic site, where glucose binds. The binding of CP320626 to MGPa does not promote extensive conformational changes except for small shifts of the side chain atoms of residues R60, V64, and K191. Both CP320626 and glucose promote the less active T state, while structural comparisons of MGPa-glucose-CP320626 complex with LGPa complexed with a related compound (CP403700) and a glucose analogue inhibitor indicate that the residues of the new allosteric site, conserved in the two isozymes, show no significant differences in their positions.

Research paper thumbnail of The binding of β- and γ-cyclodextrins to glycogen phosphorylase b: Kinetic and crystallographic studies

Protein Science, 2003

A number of regulatory binding sites of glycogen phosphorylase (GP), such as the catalytic, the i... more A number of regulatory binding sites of glycogen phosphorylase (GP), such as the catalytic, the inhibitor, and the new allosteric sites are currently under investigation as targets for inhibition of hepatic glycogenolysis under high glucose concentrations; in some cases specific inhibitors are under evaluation in human clinical trials for therapeutic intervention in type 2 diabetes. In an attempt to investigate whether the storage site can be exploited as target for modulating hepatic glucose production, α-, β-, and γ-cyclodextrins were identified as moderate mixed-type competitive inhibitors of GPb (with respect to glycogen) with Ki values of 47.1, 14.1, and 7.4 mM, respectively. To elucidate the structural basis of inhibition, we determined the structure of GPb complexed with β- and γ-cyclodextrins at 1.94 Å and 2.3 Å resolution, respectively. The structures of the two complexes reveal that the inhibitors can be accommodated in the glycogen storage site of T-state GPb with very little change of the tertiary structure and provide a basis for understanding their potency and subsite specificity. Structural comparisons of the two complexes with GPb in complex with either maltopentaose (G5) or maltoheptaose (G7) show that β- andγ-cyclodextrins bind in a mode analogous to the G5 and G7 binding with only some differences imposed by their cyclic conformations. It appears that the binding energy for stabilization of enzyme complexes derives from hydrogen bonding and van der Waals contacts to protein residues. The binding of α-cyclodextrin and octakis (2,3,6-tri-O-methyl)-γ-cyclodextrin was also investigated, but none of them was bound in the crystal; moreover, the latter did not inhibit the phosphorylase reaction.

Research paper thumbnail of Crystallographic studies on acyl ureas, a new class of glycogen phosphorylase inhibitors, as potential antidiabetic drugs

Protein Science, 2005

Acyl ureas were discovered as a novel class of inhibitors for glycogen phosphorylase, a molecular... more Acyl ureas were discovered as a novel class of inhibitors for glycogen phosphorylase, a molecular target to control hyperglycemia in type 2 diabetics. This series is exemplified by 6-{2,6-Dichloro- 4-[3-(2-chloro-benzoyl)-ureido]-phenoxy}-hexanoic acid, which inhibits human liver glycogen phosphorylase a with an IC50 of 2.0 μM. Here we analyze four crystal structures of acyl urea derivatives in complex with rabbit muscle glycogen phosphorylase b to elucidate the mechanism of inhibition of these inhibitors. The structures were determined and refined to 2.26Å resolution and demonstrate that the inhibitors bind at the allosteric activator site, where the physiological activator AMP binds. Acyl ureas induce conformational changes in the vicinity of the allosteric site. Our findings suggest that acyl ureas inhibit glycogen phosphorylase by direct inhibition of AMP binding and by indirect inhibition of substrate binding through stabilization of the T′ state.