Inhibition of cyclin-dependent kinase 1 by purines and pyrrolo[2,3-d]pyrimidines does not correlate with antiviral activity

Abstract

We have previously shown that a series of nonnucleoside pyrrolo[2,3-d]pyrimidines selectively inhibit the replication of herpes simplex virus type 1 (HSV-1) and human cytomegalovirus (HCMV). These compounds act at the immediate-early or early stage of HCMV replication and have antiviral properties somewhat similar to those of roscovitine and olomoucine, specific inhibitors of cyclin-dependent kinases (cdks). In the present study we examine the hypothesis that pyrrolo[2,3-d]pyrimidines exert their antiviral effects by inhibition of cellular cdks. Much higher concentrations of a panel of pyrrolo[2,3-d]pyrimidine nucleoside analogs with antiviral activity were required to inhibit recombinant cdk1/cyclin B compared to the submicromolar concentrations required to inhibit HCMV and HSV-1 replication. 4,6-Diamino-5-cyano-7-(2-phenylethyl)pyrrolo[2,3-d]pyrimidine (compound 1369) was the best inhibitor of cdk1 and cyclin B, with a 50% inhibitory concentration (IC(50); 14 microM) similar to that of roscovitine; it was competitive with respect to ATP (K(i) = 14 microM). The potency of compound 1369 against cdk1 and cyclin B was similar to its cytotoxicity (IC(50)s, 32 to 100 microM) but not its antiviral efficacy (IC(50)s, 0.02 to 0.3 microM). Thus, our results indicated the null hypothesis. In contrast, roscovitine was only weakly active against HSV-1 (IC(50), 38 microM) and HCMV (IC(50), 40 microM). These values were similar to those derived by cytotoxicity and cell growth inhibition assays, thereby suggesting that roscovitine is not a selective antiviral. Therefore, we propose that inhibition of cdk1 and cyclin B is not responsible for selective antiviral activity and that pyrrolo[2,3-d]pyrimidines constitute novel pharmacophores which compete with ATP to inhibit cdk1 and cyclin B.

FIG. 1.

Structures of the substituted purine and pyrrolo[2,3-d]pyrimidines used in this study. The compounds were purchased or were synthesized as described in Materials and Methods.

FIG. 2.

Effect of compound 1369 on cell growth. Cells (12,000 to 20,000 per well) were planted in 24-well plates and incubated with the indicated concentrations of compound 1369 or no drug. The number of cells per well was enumerated at 24-h intervals by use of a Coulter counter. Data are presented as the means ± standard deviations of duplicate experiments. Concentrations of compound 1369 of 0 μM (⋄), 0.1 μM (▪), 1.0 μM (▵),10 μM (•), and 30 μM (○) were tested.

FIG. 3.

Double-reciprocal plots of kinetic inhibitory data from assays of cdk1/cyclin B protein kinase with different concentrations of compound 1369. Enzyme activities were assayed in pentuplicate. The ATP concentration in the reaction mixture was varied from 24 to 160 μM; the concentration of histone H1 was kept constant at 1.2 μg/μl. The inset shows a secondary plot of the slopes from the primary plot against the concentration of compound 1369. The negative of the apparent inhibition constant (K_i) is shown with an arrow.

FIG. 4.

Effect of roscovitine on cell growth. Cells (40,000 to 50,000 per well) were planted in 24-well plates and incubated with the given concentrations of roscovitine, no drug, or an amount of dimethyl sulfoxide equivalent to the highest concentration tested. The number of cells per well was enumerated at 24-h intervals by use of a Coulter counter. Data are presented as the means ± standard deviations of duplicate experiments. Concentrations of roscovitine of 0 μM (♦), 3.7 μM (▪), 11 μM (▵), 33 μM (•), 100 μM(○) were tested. □, dimethyl sulfoxide control