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CMX157 is a lipid (1-0-hexadecyloxypropyl) conjugate of the acyclic nucleotide analog tenofovir (TFV) with activity against both wild-type and antiretroviral drug-resistant HIV strains, including multidrug nucleoside/nucleotide analog-resistant viruses. CMX157 was consistently >300-fold more active than tenofovir against multiple viruses in several different cell systems. CMX157 was active against all major subtypes of HIV-1 and HIV-2 in fresh human peripheral blood mononuclear cells (PBMCs) and against all HIV-1 strains evaluated in monocyte-derived macrophages, with 50% effective concentrations (EC(50)s) ranging between 0.20 and 7.2 nM. The lower CMX157 EC(50)s can be attributed to better cellular uptake of CMX157, resulting in higher intracellular levels of the active antiviral anabolite, TFV-diphosphate (TFV-PP), inside target cells. CMX157 produced >30-fold higher levels of TFV-PP in human PBMCs exposed to physiologically relevant concentrations of the compounds than did TFV. Unlike conventional prodrugs, including TFV disoproxil fumarate (Viread), CMX157 remains intact in plasma, facilitating uptake by target cells and decreasing relative systemic exposure to TFV. There was no detectable antagonism with CMX157 in combination with any marketed antiretroviral drug, and it possessed an excellent in vitro cytotoxicity profile. CMX157 is a promising clinical candidate to treat wild-type and antiretroviral drug-resistant HIV, including strains that fail to respond to all currently available nucleoside/nucleotide reverse transcriptase inhibitors.

9-R-[2-(Phosphonomethoxy)propyl]-adenine (tenofovir) is an acyclic nucleoside phosphonate with antiviral activity against human immunodeficiency virus type 1 (HIV-1) and hepatitis B virus (HBV). Tenofovir is not orally bioavailable but becomes orally active against HIV-1 infection as the disoproxil ester (tenofovir disoproxil fumarate [Viread]). We have developed an alternative strategy for promoting the oral availability of nucleoside phosphonate analogs which involves esterification with a lipid to form a lysolecithin mimic. This mimic can utilize natural lysolecithin uptake pathways in the gut, resulting in high oral availability. Since the mimic is not subject to cleavage in the plasma by nonspecific esterases, it remains intact in the circulation and facilitates uptake by target cells. Significant drops in apparent antiviral 50% effective concentrations (EC(50)s) of up to 3 logs have been observed in comparison with non-lipid-conjugated parent compounds in target cells. We have applied this technology to tenofovir with the goal of increasing oral availability, decreasing the apparent EC(50), and decreasing the potential for nephrotoxicity by reducing the exposure of the kidney to the free dianionic tenofovir. Here we report that, in vitro, the hexadecyloxypropyl ester of tenofovir, CMX157, is 267-fold more active than tenofovir against HIV-1 and 4.5-fold more active against HBV. CMX157 is orally available and has no apparent toxicity when given orally to rats for 7 days at doses of 10, 30, or 100 mg/kg/day. Consequently, CMX157 represents a second-generation tenofovir analog which may have an improved clinical profile.

A new synthesis of (-)-DAPD, suitable for large scale development, is described.

HIV encodes an RNA directed DNA polymerase (reverse transcriptase, RT) that is an essential enzyme in the viral replication cycle. This enzyme catalyzes the synthesis of double stranded proviral DNA from single stranded genomic RNA via a bireactant-biproduct mechanism. The functional enzyme purified from virus particles is a complex consisting of two polypeptides of molecular weight 66,000 and 51,000. Two of the four classes of currently approved anti-HIV drugs, the nucleoside reverse transcriptase inhibitors (NRTIs) and the non-nucleoside reverse transcriptase inhibitors (NNRTIs), act by inhibiting this enzyme. In this review each step of DNA synthesis catalyzed by the RT is described and the mechanism of inhibition of catalysis and termination of DNA synthesis by NRTIs is detailed. The individual steps in the catalytic cycle and the effects that the NRTIs have on them have been examined using transient kinetic analysis. The impact of stereoisomerism and resistance mutations on the rate of NRTI triphosphate incorporation (k(pol)), binding in the catalytic complex (K(d)) and the overall efficiency of incorporation (k(pol)/K(d)) are summarized for lamivudine, coviracil and zalcitabine. The results provide insight into the molecular forces and structural features that make these molecules effective inhibitors.

A new synthesis of 2'-deoxy-2'-fluoro-5-methyl-beta-L-arabinofuranosyl uracil (13, L-FMAU) was achieved in 10 steps from L-arabinose.

A novel series of HIV protease inhibitors containing cyclic P1/P2 scaffolds has been synthesized and evaluated for biological activity. The trans 3,5-dibenzyl-2-oxo pyrrolidinone ring system resulted in a 50 pM enzyme inhibitor against HIV protease in vitro when combined with an indanolamine derived P'-backbone. This compound also shows comparable activity to currently marketed drugs in the MT-4 cell-based antiviral assay.

Substrate and product specificity studies were used to develop inhibitors of the cytosolic 5'-nucleotidase I (c-N-I) from myocardium. As measured by Vmax/Km, c-N-I preferred pyrimidine 2'-deoxyribonucleotides as substrates with thymidine monophosphate (TMP) being the most efficient. In product inhibition studies, thymidine inhibited noncompetitively and inorganic phosphate inhibited competitively, consistent with an ordered release of nucleoside prior to phosphate. Mirroring nucleotide substrate specificities, pyrimidine nucleosides were more potent product inhibitors than purine nucleosides. Thus, pyrimidine nucleotide and nucleoside analogues were developed as inhibitors. Phosphonate analogues of TMP were synthesized by a novel method. The most potent was the 5'-phosphonate of 3'-deoxythymidine (ddT) (apparent Ki value of 63 nM). In addition, pyrimidine nucleoside analogues were inhibitors with 5-ethynyl-2',3'-dideoxyuridine being the most potent (apparent Ki value of 3.7 microM). The most potent nucleotide and nucleoside inhibitor were both greater than 1000-fold more potent inhibiting c-N-I than the cytosolic 5'-nucleotidase II. The nucleoside analogue was also greater than 1000-fold more potent against c-N-I than the membrane ecto-5'-nucleotidase (e-N). Because the phosphonate analogues measurably inhibited e-N (apparent Ki values of 6-12 microM), the selectivity of the phosphonates for c-N-I versus e-N was less (40-200-fold). Because of the high selectivity for c-N-I versus both of the other 5'-nucleotidases, the nucleoside inhibitors of c-N-I may be useful biochemical tools in discerning the role that c-N-I plays in generating adenosine within myocardium.

Rat liver homogenates catalyzed the elimination of fluoride from (R,S)-alpha-fluoro-beta-alanine. The substrate specificity and physical properties of the defluorinating enzyme were similar to those of mitochondrial L-alanine-glyoxylate aminotransferase II (EC 2.6.1.44, AlaAT-II). Furthermore, AlaAT-II activity, measured with L-alanine and glyoxylate as substrates, copurified with the alpha-fluoro-beta-alanine-defluorinating enzyme. The NH2-terminal sequence (18 residues) of the enzyme did not show significant sequence similarity with any of the proteins currently listed in GenBank. The purified enzyme catalyzed the transamination of L-alanine (Ala) and glyoxylate (glyx) at pH 8.5 by a ping-pong mechanism with kinetic parameters of kcat = 17 sec-1, KL-Ala = 3.2 mM, and Kglyx = 0.3 mM, respectively. The kinetic parameters for the defluorination of (R)-alpha-fluoro-beta-alanine and (S)-alpha-fluoro-beta-alanine were kcat = 6.2 and 2.6 min-1, respectively, and Km = 2.7 and 0.88 mM, respectively. L-Alanine potently inhibited the defluorination reaction with an apparent Ki of 0.024 mM. (R,S)-alpha-Fluoro-beta-alanine converted the optical spectrum of the enzyme-bound cofactor from the pyridoxal form to the pyridoxamino form, which indicated that this cofactor may participate in the defluorination reaction. The product of the enzymatic reaction, malonic semialdehyde, reacted nonenzymatically with (R,S)-alpha-fluoro-beta-alanine to form an adduct that was detected spectrally. AlaAT-II was not inactivated during dehalogenation of (R,S)-alpha-fluoro-beta-alanine but was inactivated completely during dehalogenation of beta-chloro-L-alanine.

The biologically active isomer of 5-fluoro-5,6-dihydrouracil [(R)-5-fluoro-5,6-dihydrouracil, R-FUH2] was synthesized to study the kinetics of its enzymatic oxidation and hydrolysis by homogeneous dihydropyrimidine dehydrogenase (DPDase) and dihydropyrimidine aminohydrolase (DPHase), respectively. DPDase catalyzed the slow oxidation of R-FUH2 at pH 8 and 37 degrees with a Km of 210 microM and a kcat of 0.026 sec-1 at a saturating concentration of NADP+. The catalytic efficiency (kcat/Km) of DPDase for R-FUH2 was 1/14th of that for 5,6-dihydrouracil (UH2). In the opposite direction, DPDase catalyzed the reduction of 5-fluorouracil (FU) with a Km of 0.70 microM and a kcat of 3 sec-1 at a saturating concentration of NADPH. Thus, DPDase catalyzed the reduction of FU 30,000-fold more efficiently than the oxidation of R-FUH2. In contrast to the slow oxidation of R-FUH2 by DPDase, R-FUH2 was hydrolyzed very efficiently by DPHase with a Km of 130 microM and a kcat of 126 sec-1. The catalytic efficiency of DPHase for the hydrolysis of R-FUH2 was approximately twice that for the hydrolysis of UH2. Because R-FUH2 is hydrolysis of R-FUH2 was approximately twice that for the hydrolysis of UH2. Because R-FUH2 is hydrolyzed considerably more efficiently than it is oxidized and because the activity of DPHase was 250- to 500-fold greater than that of DPDase in bovine and rat liver, the hydrolytic pathway should predominate in vivo.

5-Ethynyluracil is a potent mechanism-based inactivator of dihydropyrimidine dehydrogenase (DPD, EC 1.3.1.2) in vitro (Porter et al., J Biol Chem 267: 5236-5242, 1992) and in vivo (Spector et al., Biochem Pharmacol, 46: 2243-2248, 1993. 5-Ethynyl-2(1H)-pyrimidinone was rapidly oxidized to 5-ethynyluracil by aldehyde oxidase. The substrate efficiency (kcat/Km) was 60-fold greater than that for N-methylnicotinamide. In contrast, xanthine oxidase oxidized 5-ethynyl-2(1H)-pyrimidinone to 5-ethynyluracil with a substrate efficiency that was only 0.02% that of xanthine. Because 5-ethynyl-2(1H)-pyrimidinone did not itself inactivate purified DPD in vitro and aldehyde oxidase is predominately found in liver, we hypothesized that 5-ethynyl-2(1H)-pyrimidinone could be a liver-specific inactivator of DPD. We found that 5-ethynyl-2(1H)-pyrimidinone administered orally to rats at 2 micrograms/kg inactivated DPD in all tissues studied. Although 5-ethynyl-2(1H)-pyrimidinone produced slightly less inactivation than 5-ethynyluracil, the two compounds showed fairly similar patterns of inactivation of DPD in these tissues. At doses of 20 micrograms/kg, however, 5-ethynyl-2-pyrimidinone and 5-ethynyluracil produced equivalent inactivation of DPD. Thus, 5-ethynyl-2(1H)-pyrimidinone appeared to be an efficient, but not highly liver-selective prodrug of 5-ethynyluracil.

The varicella-zoster virus (VZV) thymidine kinase (TK) EC 2.7.2.21) catalyzes the phosphorylation of many anti-VZV nucleosides. Purified, bacterially expressed VZV TK was characterized with regard to N-terminal amino acid sequence, pI value, pH optimum, metal ion requirement, phosphate donor and acceptor specificity, and inhibition by dTTP. Initial velocities of thymidine phosphorylation with variable MgATP concentrations fit a two-site model with apparent Km values for MgATP of 0.10 and 900 microM. dTTP was a noncompetitive inhibitor of thymidine phosphorylation but was competitive with MgATP. Phosphate donor and acceptor specificities of the bacterially expressed enzyme were indistinguishable from those of VZV TK purified from infected cells. Detailed studies of the nucleoside specificity with the bacterially expressed enzyme showed that, for a given sugar moiety, thymine nucleosides were the most efficient substrates followed by nucleosides of cytosine, uracil, adenine, and with some exceptions, guanine. For a given pyrimidine or purine (except guanine), 2'-deoxyribonucleosides were the most efficient substrates, followed by arabinosides, ribonucleosides, 2',3'-dideoxyribonucleosides, and the acyclic moiety of acyclovir.