Purine analogues cytotoxicity

It seems certain that the effect of SAB on the cytotoxicity and DNA repair of cells treated with monofunctional alkylating agents is mediated via an inhibition of ADP-ribosyl transferase activity (7, 8). Nevertheless, the pleiotropic effects of SAB could result in the modulation of the cytotoxicity of other dmgs independent of ADP-ribosyl transferase inhibition. In this paper, we describe the cytotoxic effects of SAB on a range of purine base and nucleoside analogues. The data lead us to hypothesize that SAB modulates purine analogue cytotoxicity by mechanism(s) independent of DNA repair inhibition, but involving effects on de novo purine biosynthesis and nucleoside transport. 

Is the enhancement of purine analogue cytotoxicity obtained with 3AB caused by an inhibition of ADP-ribosyl transferase activity and its consequential effect on DNA repair The following observations suggest not (i). 3AB is required during the first cell cycle of 6TG administration, although it has been shown that DNA strand breaks do not appear until the 2nd G2 phase and are associated exclusively with the nascent strand (11). 

Little quantitative work has appeared on the determination of the rate of conversion of the various purine analogues to their nucleotides with highly purified phosphoribosyltransferases from mammalian cells or any other source, but there would appear to be a rough correlation between the cytotoxicity of these analogues and their ability to serve as substrates (the Kj values for a number of purines and purine analogues have been determined [45a, 85a] but this value is not a measure of conversion to nucleotide). Table 2.1 lists a number of purine analogues and an estimate of their ability to serve as substrates for the phosphoribosyltransferases. 

Table 2.3. CORRELATION OI THE CYTOTOXICITY OF CERTAIN PURINE ANALOGUES AND THEIR RIBONUCLEOSIDES WITH THEIR INHIBITION OF FGAR...

A comparison of EDso values for feedback inhibition and for growth inhibition in H.Ep.-2 cells in culture is shown in Table 2.3. It is readily apparent that for most of the purine analogues listed, the correlation between feedback inhibition and cytotoxicity is good. The few discrepancies may be due to the fact that these particular compounds are not feedback inhibitors, but metabolism (or lack of it) of the analogue in question may be important. In comparing these ED50 values, the difference in experimental conditions for cytotoxicity determination (long... 

Much information on the mechanism of action and cross-resistance of purine analogues has been obtained in bacteria, some of which are quite sensitive to certain of these compounds in vitro. There is a great deal of variation in response of the various bacteria to a particular agent and of a particular bacterium to the various cytotoxic purine analogues. Some, if not most, of these differences are probably due to differences in the anabolism of the various compounds. Despite the fact that certain purine analogues have quite a spectrum of antibacterial activity in vitro, none has been useful in the treatment of bacterial infections in vivo because their toxicity is not selective—the metabolic events whose blockade is responsible for their antibacterial activity are also blocked in mammalian cells and thus inhibition of bacterial growth can only be attained at the cost of prohibitive host toxicity. In contrast, the sulpha drugs and antibiotics such as penicillin act on metabolic events peculiar to bacteria. 

An alternative mechanism of SAB action could involve its known effects on de novo purine biosynthesis (1, S) and/or nucleoside transport (5). The combined inhibitory effects of SAB and purine analogues on purine biosynthesis could result in sufficient depletion of intracellular nucleotide pools to result in enhanced cellular cytotoxicity. In addition, these effects would lead to an increased bioavailability of 5-phosphoribosyl-l-pyrophosphate (PRPP), the first enzymic product in the de novo pathway. Increased PRPP levels would enhance the activity of hypoxanthine phosphoribosyl transferase, leading to increased salvage of purine analogues. 

In conclusion, SAB enhances the cytotoxicity of a range of purine analogues by a mechanism which is probably independent of ADP-ribosyl transferase inhibition. The DBF values obtained are comparable or greater than those obtained with a monofunctional alkylating agent, DMS. Furthermore, the purine analogues are of major clinical value and these data indicate a role for the benzamides in chemotherapy. 

Growth of L. donovani in completely defined medium has facilitated the isolation of clonal variants resistant to cytotoxic purine analogues In these experiments 10 promastigotes were plated under selective conditions, either 5uM formycin b or luM tubercidin, and from one to about fifty resistant colonies appeared per plate. The isolation of such clonal drug-resistant variants from large populations of parasites is a necessary prelude to the genetic dissection of the purine salvage pathways unique to these parasites. 

Cytotoxic agents Since proliferation of cells is essential for the immune response, agents that inhibit DNA synthesis have been used as immunosuppressive agents for many years. The first were used in the 1960s, particularly to prevent rejection of a transplanted organ, for example purine and pyrimidine analogues. These agents are not now used in autoimmune diseases but are stiU used in cancer chemotherapy (Chapter 21). 

Antiviral and antitumor. Purine nucleosides analogues have been investigated as antitumor and antiviral agents. Bioisosteric derivatives of nucle-obases have been proposed, hi particular, as the sulphur atoms is analogous to a - CH = CH - group because of its steric and electronic properties, different imidazo[4,5-d]isothiazoles 292 (R = H, Me R = H, SMe, SBu) have been synthesised. All compounds were cytotoxic at micromolar concentrations, but showed no antiviral activity on human cytomegalovirus and herpes simplex virus type 1 [96]. The nucleoside analogue 12 was also prepared, but showed none antiviral activity nor cytotoxicity [10]. 

The Pt bis chelates of some S- and Se-containing nucleosides or nucleotides have been synthesized and evaluated for antitumour activity in both in vitro and in vivo systems. The increased effectiveness of the trans chelate of selenoguanine is believed to be due to its slow dissociation and consequent release of the ligand into the blood stream. The delayed cytotoxicity observed for the Se compound was not obtained with the S analogue. The cw-diammineplatinum(II) 1 1 chelates of thio- and seleno-purines also have been prepared and evaluated against L1210 cells in mice. The Se chelate is active, with its stability affected by mouse serum, although the reason for the tatter is unclear. ... 

An alternative mechanism may involve an inhibition of nucleoside transport. It has been shown that S-acetylamidobenzamide inhibits adenosine transport, and we have shown that SAB inhibits thymidine transport (unpublished results). This would explain the differential effect of SAB on purine base and nucleoside cytotoxicity. Grem and Fischer (17) observed that nucleoside transport inhibitors enhanced the cytotoxicity of 5 fluorouracil by preventing efflux of 5 -fluorodeoxyuridine. Since purine base analogues can be interconverted to the nucleoside derivatives, a similar mechanism could apply for the potentiation of 6TG by SAB. However in this case, we might expect to see an enhancement of 6TG cytotoxicity by dipyridamole, which is not seen. 

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