Dominique Deville-Bonne

Structural basis for activation of α-boranophosphate nucleotide analogues targeting drug-resistant reverse transcriptase

AIDS chemotherapy is limited by inadequate intracellular concentrations of the active triphosphate form of nucleoside analogues, leading to incomplete inhibition of viral replication and the appearance of drug‐resistant virus. Drug activation by nucleoside diphosphate kinase and inhibition of HIV‐1 reverse transcriptase were studied comparatively. We synthesized analogues with a borano (BH3−) group on the α‐phosphate, and found that they are substrates for both enzymes. X‐ray structures of complexes with nucleotide diphosphate kinase provided a structural basis for their activation. The complex with d4T triphosphate displayed an intramolecular CH…O bond contributing to catalysis, and the Rp diastereoisomer of thymidine α‐boranotriphosphate bound like a normal substrate. Using α‐(Rp)‐boranophosphate derivatives of the clinically relevant compounds AZT and d4T, the presence of the α‐borano group improved both phosphorylation by nucleotide diphosphate kinase and inhibition of reverse transcription. Moreover, repair of blocked DNA chains by pyrophosphorolysis was reduced significantly in variant reverse transcriptases bearing substitutions found in drug‐resistant viruses. Thus, the α‐borano modification of analogues targeting reverse transcriptase may be of generic value in fighting viral drug resistance.

KINETICS OF CHEY PHOSPHORYLATION BY SMALL MOLECULE PHOSPHODONORS

The chemotaxis response regulator CheY can acquire phosphoryl groups either from its associated autophosphorylating protein kinase, CheA, or from small phosphodonor molecules such as acetyl phosphate. We report a stopped‐flow kinetic analysis of CheY phosphorylation by acetyl phosphate. The results show that CheY has a very low affinity for this phosphodonor (K s≫0.1 M), consistent with the conclusion that, whereas CheY provides catalytic functions for the phosphotransfer reaction, the CheA kinase may act simply to increase the effective phosphodonor concentration at the CheY active site.

Novel Antiviral C5-Substituted Pyrimidine Acyclic Nucleoside Phosphonates Selected as Human Thymidylate Kinase Substrates

Acyclic nucleoside phosphonates (ANPs) are at the cornerstone of DNA virus and retrovirus therapies. They reach their target, the viral DNA polymerase, after two phosphorylation steps catalyzed by cellular kinases. New pyrimidine ANPs have been synthesized with unsaturated acyclic side chains (prop-2-enyl-, but-2-enyl-, pent-2-enyl-) and different substituents at the C5 position of the uracil nucleobase. Several derivatives in the but-2-enyl- series 9d and 9e, with (E) but not with (Z) configuration, were efficient substrates for human thymidine monophosphate (TMP) kinase, but not for uridine monophosphate-cytosine monophosphate (UMP-CMP) kinase, which is in contrast to cidofovir. Human TMP kinase was successfully crystallized in a complex with phosphorylated (E)-thymidine-but-2-enyl phosphonate 9e and ADP. The bis-pivaloyloxymethyl (POM) esters of (E)-9d and (E)-9e were synthesized and shown to exert activity against herpes virus in vitro (IC(50) = 3 μM) and against varicella zoster virus in vitro (IC(50) = 0.19 μM), in contrast to the corresponding inactive (Z) derivatives. Thus, their antiviral activity correlates with their ability to act as thymidylate kinase substrates.

3′-Phosphorylated Nucleotides Are Tight Binding Inhibitors of Nucleoside Diphosphate Kinase Activity

Nucleoside diphosphate (NDP) kinase catalyzes the phosphorylation of ribo- and deoxyribonucleosides diphosphates into triphosphates. NDP kinase is also involved in malignant tumors and was shown to activate in vitro transcription of the c-myc oncogene by binding to its NHE sequence. The structure of the complex of NDP kinase with bound ADP shows that the nucleotide adopts a different conformation from that observed in other phosphokinases with an internal H bond between the 3′-OH and the β-O made free by the phosphate transfer. We use intrinsic protein fluorescence to investigate the inhibitory and binding potential of nucleotide analogues phosphorylated in 3′-OH position of the ribose to both wild type and F64W mutant NDP kinase from Dictyostelium discoideum. Due to their 3′-phosphate, 5′-phosphoadenosine 3′-phosphate (PAP) and adenosine 3′-phosphate 5′-phosphosulfate (PAPS) can be regarded as structural analogues of enzyme-bound ADP. TheK D of PAPS (10 μm) is three times lower than the K D of ADP. PAPS also acts as a competitive inhibitor toward natural substrates during catalysis, with a K I in agreement with binding data. The crystal structure of the binary complex between Dictyostelium NDP kinase and PAPS was solved at 2.8-Å resolution. It shows a new mode of nucleotide binding at the active site with the 3′-phosphate of PAPS located near the catalytic histidine, at the same position as the γ-phosphate in the transition state. The sulfate group is directed toward the protein surface. PAPS will be useful for the design of high affinity drugs targeted to NDP kinases.

Crystal structure of poxvirus thymidylate kinase: An unexpected dimerization has implications for antiviral therapy

Unlike most DNA viruses, poxviruses replicate in the cytoplasm of host cells. They encode enzymes needed for genome replication and transcription, including their own thymidine and thymidylate kinases. Some herpes viruses encode only 1 enzyme catalyzing both reactions, a peculiarity used for prodrug activation to obtain maximum specificity. We have solved the crystal structures of vaccinia virus thymidylate kinase bound to TDP or brivudin monophosphate. Although the viral and human enzymes have similar sequences (42% identity), they differ in their homodimeric association and active-site geometry. The vaccinia TMP kinase dimer arrangement is orthogonal and not antiparallel as in human enzyme. This different monomer orientation is related to the presence of a canal connecting the edge of the dimer interface to the TMP base binding pocket. Consequently, the pox enzyme accommodates nucleotides with bulkier bases, like brivudin monophosphate and dGMP; these are efficiently phosphorylated and stabilize the enzyme. The brivudin monophosphate-bound structure explains the structural basis for this specificity, opening the way to the rational development of specific antipox agents that may also be suitable for poxvirus TMP kinase gene-based chemotherapy of cancer.

Improving nucleoside diphosphate kinase for antiviral nucleotide analogs activation

Antiviral nucleoside analog therapies rely on their incorporation by viral DNA polymerases/reverse transcriptase leading to chain termination. The analogs (3′-deoxy-3′-azidothymidine (AZT), 2′,3′-didehydro-2′,3′-dideoxythymidine (d4T), and other dideoxynucleosides) are sequentially converted into triphosphate by cellular kinases of the nucleoside salvage pathway and are often poor substrates of these enzymes. Nucleoside diphosphate (NDP) kinase phosphorylates the diphosphate derivatives of the analogs with an efficiency some 104 lower than for its natural substrates. Kinetic and structural studies of Dictyosteliumand human NDP kinases show that the sugar 3′-OH, absent from all antiviral analogs, is required for catalysis. To improve the catalytic efficiency of NDP kinase on the analogs, we engineered several mutants with a protein OH group replacing the sugar 3′-OH. The substitution of Asn-115 in Ser and Leu-55 in His results in an NDP kinase mutant with an enhanced ability to phosphorylate antiviral derivatives. Transfection of the mutant enzyme in Escherichia coliresults in an increased sensitivity to AZT. An x-ray structure at 2.15-Å resolution of the Dictyostelium enzyme bearing the serine substitution in complex with theR p-α-borano-triphosphate derivative of AZT shows that the enhanced activity reflects an improved geometry of binding and a favorable interaction of the 3′-azido group with the engineered serine.

Role of nucleoside diphosphate kinase in the activation of anti-HIV nucleoside analogs.

Nucleoside analogs are currently used in antiretrovirus therapies. The best known example isAZT one of the first drug to be used for the treatment of AIDS. However, only the triphosphatederivatives of these compounds act as substrates of the viral reverse transcriptase. Since theydo not enter cells, nucleoside analogs are administered and phosphorylated by cellular kinases.The last step in this phosphorylation pathway is catalyzed by nucleoside diphosphate (NDP)kinase. The incorporation of the nucleoside triphosphates into nascent viral DNA chain resultsin termination of the elongation process. We have performed kinetics studies of thephosphorylation reaction by NDP kinase of dideoxynucleoside diphosphates such as 2′,3′-dideoxy-3′-azidothymidine diphosphate (AZT-DP) and 2′,3′-dideoxy-2′,3′-didehydrothymidinediphosphate (d4T-DP). We show that the catalytic efficiency is strongly decreased and, therefore,that the reaction step catalyzed by NDP kinase constitutes a bottleneck in the processingpathway of anti-HIV compounds. In addition, the affinity of the analogs in the absence ofcatalysis was determined using a catalytically inactive NDP kinase mutant, showing a reductionof affinity by a factor of 2 to 30, depending on the analog. The structure of NDP kinaseprovides a structural explanation for these results. Indeed, all nucleoside analogs acting aschain terminators must lack a 3′-OH in the nucleotide deoxyribose. Unfortunately, this samesubstitution is detrimental for their capacity to be phosphorylated by NDP kinase. This definesthe framework for the design of new nucleoside analogs with increased efficiency inantiretroviral therapies.

ACTIVATION OF ANTI-REVERSE TRANSCRIPTASE NUCLEOTIDE ANALOGS BY NUCLEOSIDE DIPHOSPHATE KINASE: IMPROVEMENT BY α-BORANOPHOSPHATE SUBSTITUTION

Nucleoside activation by nucleoside diphosphate kinase and inhibition of HIV-1 reverse transcriptase were studied comparatively for a new class of nucleoside analogs with a borano (BH− 3) or a thio (SH) group on the α-phosphate. Both the α-Rp-borano derivatives of AZT and d4T improved phosphorylation by NDP kinase, inhibition of reverse transcription as well as stability of α-borano monophosphate derivatives in terminated viral DNA chain.

Nucleotide affinity for a stable phosphorylated intermediate of nucleoside diphosphate kinase

Nucleoside diphosphate (NDP) kinase is transiently phosphorylated on a histidine of the active site during the catalytic cycle. In the presence of a nucleotide acceptor, the phosphohistidine bond is unstable and the phosphate is transferred to the acceptor in less than 1 msec. We describe the synthesis of an analog of the phosphoenzyme intermediate with an inactive mutant of NDP kinase in which the catalytic histidine is replaced by a cysteine. In two sequential disulfide exchange reactions, a thiophosphate group reacts with the thiol function of the cysteine that had previously reacted with dithionitrobenzoate (DTNB). The thiophosphoenzyme presents a 400,000‐fold increased stability in the presence of NDPs compared with the phosphoenzyme. The binding of NDP is studied at the steady state and presteady state. Data were analyzed according to a bimolecular association model. For the first time, the true equilibrium dissociation constants of NDP for the analog of the phosphoenzyme are determined in the absence of phosphotransfer, allowing a better understanding of the catalytic mechanism of the enzyme.

Adenosine phosphonoacetic acid is slowly metabolized by NDP kinase.

NDP kinase catalyzes the last step in the phosphorylation of nucleotides. It is also involved in the activation by cellular kinases of nucleoside analogs used in antiviral therapies. Adenosine phosphonoacetic acid, a close analog of ADP already proposed as an inhibitor of ribonucleotide reductase, was found to be a poor substrate for human NDP kinase, as well as a weak inhibitor with an equilibrium dissociation constant of 0.6 mM to be compared to 0.025 mM for ADP. The X-ray structure of a complex of adenosine phosphonoacetic acid and the NDP kinase from Dictyostelium was determined to 2.0 A resolution showing that the analog adopts a binding mode similar to ADP, but that no magnesium ion is present at the active site. As ACP may also interfere with other cellular kinases, its potential as a drug targeting NDP kinase or ribonucleotide reductase is likely to be limited due to strong side effects. The design of new molecules with a narrower specificity and a stronger affinity will benefit from the detailed knowledge of the complex ACP-NDP kinase.