A pre-catalytic non-covalent step governs DNA polymerase ß fidelity.

DNA polymerase ß (pol ß) selects the correct deoxyribonucleoside triphosphate for incorporation into the DNA polymer. Mistakes made by pol ß lead to mutations, some of which occur within specific sequence contexts to generate mutation hotspots. The adenomatous polyposis coli (APC) gene is mutated within specific sequence contexts in colorectal carcinomas but the underlying mechanism is not fully understood. In previous work, we demonstrated that a somatic colon cancer variant of pol ß, K289M, misincorporates deoxynucleotides at significantly increased frequencies over wild-type pol ß within a mutation hotspot that is present several times within the APC gene. Kinetic studies provide evidence that the rate-determining step of pol ß catalysis is phosphodiester bond formation and suggest that substrate selection is governed at this step. Remarkably, we show that, unlike WT, a pre-catalytic step in the K289M pol ß kinetic pathway becomes slower than phosphodiester bond formation with the APC DNA sequence but not with a different DNA substrate. Based on our studies, we propose that pre-catalytic conformational changes are of critical importance for DNA polymerase fidelity within specific DNA sequence contexts.

DNA Polymerase ß Cancer-Associated Variant I260M Exhibits Nonspecific Selectivity toward the ß-γ Bridging Group of the Incoming dNTP.

The hydrophobic hinge region of DNA polymerase ß (pol ß) is located between the fingers and palm subdomains. The hydrophobicity of the hinge region is important for maintaining the geometry of the binding pocket and for the selectivity of the enzyme. Various cancer-associated pol ß variants in the hinge region have reduced fidelity resulting from a decreased discrimination at the level of dNTP binding. Specifically, I260M, a prostate cancer-associated variant of pol ß, has been shown to have a reduced discrimination during dNTP binding and also during nucleotidyl transfer. To test whether fidelity of the I260M variant is dependent on leaving group chemistry, we employed a toolkit comprising dNTP bisphosphonate analogues modified at the ß-γ bridging methylene to modulate leaving group (pCXYp mimicking PPi) basicity. Construction of linear free energy relationship plots for the dependence of log(kpol) on leaving group pKa4 revealed that I260M catalyzes dNMP incorporation with a marked negative dependence on leaving group basicity, consistent with a chemical transition state, during both correct and incorrect incorporation. Additionally, we provide evidence that I260M fidelity is altered in the presence of some of the analogues, possibly resulting from a lack of coordination between the fingers and palm subdomains in the presence of the I260M mutation.

Evolution of an amino acid based prodrug approach: stay tuned.

Certain acyclic nucleoside phosphonates (ANPs) such as (S)-HPMPC (cidofovir, Vistide) and (S)-HPMPA have been shown to be active against a broad spectrum of DNA and retroviruses. However, their poor absorption as well as their toxicity limit the utilization of these therapeutics in the clinic. Nucleoside phosphonates are poorly absorbed primarily due to the presence of the phosphonic acid group, which ionizes at physiological pH. When dosed intravenously they display dose-limiting nephrotoxicity due to their accumulation in the kidney. To overcome these limitations, nucleoside phosphonate prodrug strategies have taken center stage in the development pathway and a number of different approaches are at various stages of development. Our efforts have focused on the development of ANP prodrugs in which a benign amino acid promoiety masks a phosphonate P-OH via a hydroxyl side chain. The design of these prodrugs incorporates multiple chemical groups (the P-X-C linkage, the amino acid stereochemistry, the C-terminal and N-terminal functional groups) that can be tuned to modify absorption, pharmacokinetic and efficacy properties with the goal of improving overall prodrug performance.

Structure of cyclic nucleoside phosphonate ester prodrugs: an inquiry.

The configuration at phosphorus in cyclic (S)-HPMPC (1, cidofovir) and (S)-HPMPA (2) phenyl ester (5 and 6, respectively) diastereomers ((R(p))-5, (R(p))-6, (S(p))-6) was determined by X-ray crystallography and correlated to their (1)H and (31)P NMR spectra in solution. (R(p))-5 and (R(p))-6 have chair conformations with the nucleobase substituent equatorial and the P-OPh axial. Perhaps surprisingly, (S(p))-6 is (a, a) in the crystal and exists largely as an equilibrium of (a, a)/(e, e) conformers in chloroform or acetonitrile.

Serine side chain-linked peptidomimetic conjugates of cyclic HPMPC and HPMPA: synthesis and interaction with hPEPT1.

Cidofovir (HPMPC), a broad spectrum antiviral agent, cannot be administered orally due to ionization of its phosphonic acid group at physiological pH. One prodrug approach involves conversion to the cyclic form (cHPMPC, 1) and esterification by the side chain hydroxyl group of a peptidomimetic serine. Transport studies in a rat model have shown enhanced levels of total cidofovir species in the plasma after oral dosing with L-Val-L-Ser-OMe cHPMPC, 2a. To explore the possibility that 2a and its three L/D stereoisomers 2b-d undergo active transport mediated by the peptide-specific intestinal transporter PEPT1, we performed radiotracer uptake and electrophysiology experiments applying the two-electrode voltage clamp technique in Xenopus laevis oocytes overexpressing human PEPT1 (hPEPT1, SLC15A1). 2a-d did not induce inward currents, indicating that they are not transported, but the stereoisomers with an L-configuration at the N-terminal valine (2a and 2b) potently inhibited transport of the hPEPT1 substrate glycylsarcosine (Gly-Sar). A "reversed" dipeptide conjugate, L-Ser-L-Ala-OiPr cHPMPC (4), also did not exhibit detectable transport, but completely abolished the Gly-Sar signal, suggesting that affinity of the transporter for these prodrugs is not impaired by a proximate linkage to the drug in the N-terminal amino acid of the dipeptide. Single amino acid conjugates of cHPMPC (3a and 3b) or cHPMPA (5, 6a and 6b) were not transported and only weakly inhibited Gly-Sar transport. The known hPEPT1 prodrug substrate valacyclovir (7) and its L-Val-L-Val dipeptide analogue (8) were used to verify coupled transport by the oocyte model. The results indicate that the previously observed enhanced oral bioavailability of 2a relative to the parent drug is unlikely to be due to active transport by hPEPT1. Syntheses of the novel compounds 2b-d and 3-6 are described, including a convenient solid-phase method to prepare 5, 6a and 6b.

Approaches to tyrosine-linked peptidomimetic prodrugs of (S)-HPMP-based acyclic nucleoside phosphonates.

Synthetic approaches to a new class of tyrosine-linked prodrugs of two 3-hydroxy-2-(phosphonomethoxypropyl) (HPMP) nucleotide analogues ((S)-HPMPC and (S)-HPMPA) are outlined.

Tyrosine-based 1-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine and -adenine ((S)-HPMPC and (S)-HPMPA) prodrugs: synthesis, stability, antiviral activity, and in vivo transport studies.

Eight novel single amino acid (6-11) and dipeptide (12, 13) tyrosine P-O esters of cyclic cidofovir ((S)-cHPMPC, 4) and its cyclic adenine analogue ((S)-cHPMPA, 3) were synthesized and evaluated as prodrugs. In vitro IC(50) values for the prodrugs (<0.1-50 µM) vs vaccinia, cowpox, human cytomegalovirus, and herpes simplex type 1 virus were compared to those for the parent drugs ((S)-HPMPC, 2; (S)-HPMPA, 1; IC(50) 0.3-35 µM); there was no cytoxicity with KB or HFF cells at ≤100 µM. The prodrugs exhibited a wide range of half-lives in rat intestinal homogenate at pH 6.5 (<30-1732 min) with differences of 3-10× between phostonate diastereomers. The tyrosine alkylamide derivatives of 3 and 4 were the most stable. (l)-Tyr-NH-i-Bu cHPMPA (11) was converted in rat or mouse plasma solely to two active metabolites and had significantly enhanced oral bioavailability vs parent drug 1 in a mouse model (39% vs <5%).

Synthesis of peptidomimetic conjugates of cyclic nucleoside phosphonates.

Cyclic nucleoside phosphonates connected through a P-O-C linkage to a promoiety represent a class of prodrugs designed to overcome the low oral bioavailability of parent antiviral acyclic nucleoside phosphonates. In our prodrug approach, a nontoxic promoiety, such as an amino acid or dipeptide, is conjugated to the cyclic form of the parent drug by esterification of the phosphonic acid moiety with an alcoholic amino acid side chain (Ser, Tyr, and Thr) or a glycol linker. For the biological evaluation and investigation of the pharmacokinetic profiles of these modified nucleoside phosphonates, a reliable synthetic procedure that allows preparation of sufficient amount of potential prodrugs is needed. This unit provides a procedure for synthesizing peptidomimetic conjugates of two broad-spectrum antiviral acyclic nucleoside phosphonates: (S)-HPMPC and (S)-HPMPA. Two alternate strategies allowing synthesizing selected amino acid, dipeptide, or ethylene glycol-linked amino acid prodrugs of (S)-HPMPC and (S)-HPMPA in solution and using a solid-phase approach are presented.