Mechanism of anti-HIV action of masked alaninyl d4T-MP derivatives.

Abstract

So324 is a 2',3'-dideoxy-2',3'-didehydrothymidine-5'-monophosphate (d4T-MP) prodrug containing at the phosphate moiety a phenyl group and the methylester ofalanine linked to the phosphate through a phosphoramidate linkage. So324 has anti-HIV activity in human CEM, MT4, and monocyte/macrophage cells that is superior to that of d4T. In contrast to d4T, So324 is also able to inhibit HIV replication in thymidine kinase-deficient CEM cells. After uptake of So324 by intact human lymphocytes, d4T-MP is released and subsequently converted intracellularly to d4T-TP. In addition, accumulation of substantial amounts of a novel d4T derivative has been found. This d4T metabolite has been characterized as alaninyl d4T-MP. The latter metabolite accumulates at "13-to 200-fold higher levels than d4T-TP depending the experimental conditions. Alaninyl d4T-MP should be considered as an intra-and/or extracellular depot form of d4T and/or d4T-MP. These findings may explain the superior anti-retroviral activity of So324 over d4T in cell culture.2',3'-Dideoxynucleoside (ddN) analogues are potent inhibitors of HIV replication in cell culture. To date, five ddN analogues have been formally licensed for treatment of HIVinfected individuals [(i) zidovudine (azidothymidine; AZT), (ii) zalcitabine (dideoxycytidine; ddC), (iii) didanosine (dideoxyinosine; ddI), (iv) stavudine (didehydrodideoxythymidine; d4T), and (v) lamivudine (3'-thia-2',3'-dideoxycytidine; 3TC)]. The ddN analogues have to be converted to their corresponding 5'-triphosphate derivatives to act as inhibitors of the retroviral reverse transcriptase. However, the rate and extent of phosphorylation of the ddN analogues to their active 5'-triphosphate metabolite may be insufficient. In particular, the first activation (phosphorylation) step may be rate-limiting in that most ddN analogues show poor affinity for nucleoside kinases [i.e., thymidine kinase (TK), 2'-deoxycytidine kinase, adenosine kinase] or other cellular enzymes (i.e., 5'-nucleotidase) that play a crucial role in the conversion of the ddN analogues to their 5'-monophosphates (1-6). In efforts to circumvent the dependence of ddN analogues on activation by the nucleoside kinases, prodrugs of the 5'-monophosphate forms of several nucleoside analogues have been prepared. The phosphoramidate derivatives of azidothymidine and fluorothymidine (FLT), two good substrates for cytosol TK, have been found to exhibit marked antiviral activity in cell culture (7,8). We have recently focused on masked nucleoside phosphoramidate derivatives of nucleoside analogues [i.e., d4T and dideoxyuridine (ddU)] that have relatively poor affinity for cytosol TK (2,(9)(10)(11). In this study, we revealed that the phosphoramidate derivative of d4T, designated So324, not only proved able to deliver intracellularly the free 5'-monophosphate of d4T (d4T-MP) [leading to pronounced levels of the corresponding active d4T 5'-triphosphate (d4T-TP)], but also released a phosphoramidate derivative of d4T-MP. This latter metabolite markedly accumul...

Adenosine Monophosphoramidase Activity of Hint and Hnt1 Supports Function of Kin28, Ccl1, and Tfb3

“…Additionally, it has been appreciated for several years that nucleoside-based prodrugs, such as 2Ј,3Ј-dideoxy-2Ј,3Ј-didehydrothymidine (d4T) can be made more potent by conversion to a 5Ј phosphoramide. For example, d4T containing an ␣-phosphate linked to alanine methyl ester is readily intracellularly available and is converted to the triphosphate after hydrolysis of the nucleoside monophosphoramide by an unknown intracellular enzymatic activity (66). The apparent identity of Hint to nucleoside monophosphoramidate hydrolase (60) and the fact that Hint and Hnt1 liberate AMP from AMP alanine methyl ester suggest that Hint may be the enzyme responsible for phosphoramide prodrug maturation in vivo.…”

31P NMR and Genetic Analysis Establish hinT as the Only Escherchia coli Purine Nucleoside Phosphoramidase and as Essential for Growth under High Salt Conditions

“…Part of the reason for continued interest in phosphoramidates has been their demonstrated utility as prodrugs of antiviral and anticancer nucleoside monophosphates or as pronucleo-tides (21)(22)(23)(24)(25). Typically, therapeutic nucleosides must be converted to the corresponding mono-, di-, and triphosphates before exhibiting biological activity.…”

Improved antiviral activity of the aryloxymethoxyalaninyl phosphoramidate (APA) prodrug of abacavir (ABC) is due to the formation of markedly increased carbovir 5′‐triphosphate metabolite levels

“…Assessment of the cytotoxic effect was performed twice weekly by visual inspection and then by counting cells and by trypan blue dye exclusion at day 14 after the beginning of drug treatment. CEM and monocyte/macrophage cell cultures according to previously established procedures [11,17]. Briefly, CEM cells were seeded at 2-4 Â 10 5 cells/ml in RPMI-1640 culture medium supplemented with 10% fetal calf serum, 2 mM L L -glutamine, and 0.075% NaHCO 3 .…”

“…One of these approaches has proven very successful when applied on thymidine analogues such as d4T. Indeed, the aryloxymethoxyalaninyl phosphoramidate (APA) prodrug derivative of d4T proved highly successful in retaining anti-HIV activity in dThd kinase-deficient CEM cells, whereas the parent d4T compound had poor, if any, antiviral activity in this cell line [11,12]. However, when Abbreviations: CBV, carbovir; ABC, abacavir; CBV-MP, CBV-5 0 -monophosphate; CBV-DP, CBV-5 0 -diphosphate; CBV-TP, CBV-5 0 -triphosphate; ABC-MP, ABC-5 0 -monophosphate; ABC-DP, ABC-5 0 -diphosphate; ABC-TP, ABC-5 0 -triphosphate; Ala-ABC-MP, alaninyl-ABC-MP; pro-ABC-MP, aryloxymethoxyalaninyl phosphoramidate prodrug of ABC the same prodrug technology was applied on zidovudine (AZT), another TK-dependent nucleoside analogue, no significant antiviral activity was retained in TK-deficient CEM cells [12] as also the parent AZT compound failed to do so.…”

“…Examples of a relatively poor conversion rate by the activating enzyme other than ABC are stavudine (d4T) by thymidine kinase (TK) [7,8], zalcitabine (ddC) by 2 0 -deoxycytidine kinase [9,10], and didanosine (ddI) by 5 0 -nucleotidase [4,5]. Therefore, several attempts have been undertaken to deliver activated (phosphorylated) nucleoside analogues directly into the virusinfected target cells [11][12][13][14][15][16][17][18][19][20][21][22][23][24]. One of these approaches has proven very successful when applied on thymidine analogues such as d4T.…”