Incorporation of the carbocyclic analog of 2'-deoxyguanosine into the DNA of herpes simplex virus and of HEp-2 cells infected with herpes simplex virus.

The carbocyclic analog of 2'-deoxyguanosine (CdG) is active against herpes simplex virus (HSV), human cytomegalovirus, and human hepatitis-B virus. In order to understand the mechanism of action of this compound against HSV, we have evaluated (a) the incorporation of [3H]CdG into viral and host DNA in HEp-2 cells infected with HSV and (b) the interaction of the 5'-triphosphate of CdG (CdG-TP) with the HSV DNA polymerase and human DNA polymerases alpha, beta, and gamma (EC 2.7.7.7). Incubation of HSV-1-infected HEp-2 cells with [3H]CdG resulted in the incorporation of CdG into both the HSV and the host cell DNA. These results indicated that CdG-TP was used as a substrate for HSV DNA polymerase and for at least one of the cellular DNA polymerases. Degradation of both viral and host DNA with micrococcal nuclease and spleen phosphodiesterase indicated that CdG was incorporated primarily into internal positions in both DNAs. The viral DNA containing CdG sedimented in neutral and alkaline sucrose gradients in the same way as did viral DNA labeled with [3H]thymidine, indicating that the HSV DNA containing CdG was similar in size to untreated HSV DNA. CdG-TP was a competitive inhibitor of the incorporation of dGTP into DNA by the HSV DNA polymerase (Ki of 0.35 microM) and the human DNA polymerase alpha (Ki of 1 microM). CdG-TP was not a potent inhibitor of either DNA polymerase beta or gamma. Using DNA-sequencing technology, CdG-TP was found to be an efficient substrate for HSV DNA polymerase. Incorporation of CdG monophosphate (CdG-MP) into the DNA by HSV DNA polymerase did not interfere with subsequent chain extension. These results suggested that the antiviral activity of CdG was due to its incorporation into the DNA and subsequent disruption of viral functions. In contrast, CdG-TP was not as good as dGTP as a substrate for DNA synthesis by DNA polymerase alpha, and incorporation of CdG-MP by DNA polymerase alpha inhibited further DNA chain elongation.     

2'-Deoxy-6-thioguanosine 5'-triphosphate as a substrate for purified human DNA polymerases and calf thymus terminal deoxynucleotidyltransferase in vitro

2'-Deoxy-6-thioguanosine 5'-triphosphate (S6dGTP), a metabolite of the antileukemia agent 6-thioguanine, was evaluated as a substrate for purified human DNA polymerases. Using bacteriophage M13 single-strand DNA as a template, S6dGTP substituted efficiently for dGTP and stimulated DNA synthesis in reactions without dGTP, with DNA polymerases alpha, delta, and gamma from the human leukemia cell line K562. The apparent Km values for dGTP and S6dGTP were very similar, i.e., 1.2 microM each for polymerase alpha, 2.8 and 3.6 microM, respectively, for polymerase delta, and 0.8 microM each for polymerase gamma; however, the relative Vmax values for the modified nucleotide were 25-50% lower than those of the corresponding natural substrate. Using a highly sensitive electrophoretic assay of chain elongation across M13mp9 (+)-strand DNA by the aforementioned human DNA polymerases, S6dGTP was shown to be incorporated at the 3' end of the nascent growing DNA chain, and the patterns of chain extension with S6dGTP as substrate were identical to those obtained in the presence of dGTP. There were no major differences using S6dGTP in place of dGTP with these DNA polymerases; however, at higher concentrations (1-10 microM) the analog stimulated primer elongation in reactions without dATP, indicating some misincorporation at sites of S6G.T base pairs during DNA synthesis. Using p(dA)12-18 as the initiator for calf thymus terminal deoxynucleotidyltransferase, S6dGTP inhibited the incorporation of all four natural deoxyribonucleoside 5'-triphosphates into the primer, in a competitive manner. The apparent Ki values for the analog were 6-20 times lower than the Km values for the four endogenous substrates. As a substrate, S6dGTP was added to the 3'-hydroxyl termini of primer, although tailing efficiency with the analog was lower than that in the presence of the natural substrate. These findings indicate that S6dGTP is a relatively good substrate for several mammalian DNA polymerases, including terminal deoxynucleotidyltransferase.     

Forodesine (BCX-1777, Immucillin H)--a new purine nucleoside analogue: mechanism of action and potential clinical application

Recently a few new purine nucleoside analogues (PNA) have been synthesized and introduced into preclinical and clinical trials. The transition-state theory has led to the design of 9-deazanucleotide analogues that are purine nucleoside phosphorylase (PNP) inhibitors, termed immucillins. Among them the most promising results have been obtained with forodesine. Forodesine (BCX-1777, Immucillin H, 1-(9-deazahypoxanthin)-1,4-dideoxy-1,4-imino-D-ribitol) has carbon-carbon linkage between a cyclic amine moiety that replaces ribose and 9-deaza-hypixanthine. The drug is a novel T-cell selective immunosuppressive agent which in the presence of 2'-deoxyguanosine (dGuo) inhibits human lymphocyte proliferation activated by various agents such as interleukin-2 (IL-2), mixed lymphocyte reaction and phytohemagglutinin. In the mechanism of forodesine action two enzymes are involved: PNP and deoxycytidine kinase (dCK). PNP catalyzes the phosphorolysis of dGuo to guanine (Gu) and 2'-deoxyribose-1-phosphate, whereas dCK converts dGuo to deoxyguanosino-5'-monophosphate (dGMP) and finally to deoxyguanosino-5'-triphosphate (dGTP). The affinity of dGuo is higher for PNP than for dCK. Nevertheless, if PNP is blocked by forodesine, plasma dGuo is not cleaved to Gu, but instead it is intracellularly converted to dGTP by high dCK activity, which leads to inhibition of ribonucleotide reductase (RR), an enzyme required for DNA synthesis and cell replication, which eventually results in apoptosis. Forodesine is active in some experimental tumors in mice, however it could be used for the treatment of human T-cell proliferative disorders and it is undergoing phase II clinical trials for the treatment of T-cell non-Hodgkin's lymphoma, which includes cutaneous T-cell lymphoma (CTCL). Moreover, recent preclinical and clinical data showed activity of forodesine in B-cell acute lympholastic leukemia (ALL).     

Effects of overexpression and antisense RNA expression of Orf17, a MutT-type enzyme

The Escherichia coli Orf17 (NtpA, NudB) protein, a MutT-type enzyme, hydrolyzes oxidized deoxyribonucleotides, including 8-hydroxy-2'-deoxyadenosine 5'-triphosphate and 8-hydroxy-2'-deoxyguanosine 5'-triphosphate, in vitro. To examine its in vivo role(s) in bacteria, plasmid DNAs containing the orf17 gene in the sense and antisense orientations were introduced. When the Orf17 protein was overexpressed in mutT cells, the rpoB mutant frequency was decreased. On the other hand, similar effects were not observed when Orf17 was overexpressed in wild type and orf135 cells. Expression of the antisense RNA of the orf17 gene did not produce an obvious phenotype, such as increased mutant frequency and resistance to ionizing radiation. These results suggest that the role of the Orf17 protein is to back up the MutT function, and to assist in the elimination of 8-hydroxy-2'-deoxyguanosine nucleotides.     

Inhibition of herpes simplex virus-induced DNA polymerases and cellular DNA polymerase alpha by triphosphates of acyclic guanosine analogs

The triphosphates of the antiherpes acyclic guanosine analogs (R)- and (S)-enantiomers of 9-(3,4-dihydroxybutyl)guanine [BCVTP and (S)-DHBGTP], 9-(4-hydroxybutyl)guanine (HBGTP), and 9-(2-hydroxyethoxymethyl)guanine (ACVTP) were investigated for their effects on partially purified DNA polymerases of herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2) as well as cellular DNA polymerase alpha of calf thymus and Vero cells. The triphosphates of the four analogs were all competitive inhibitors when dGTP was the variable substrate with both the viral and the cellular DNA polymerases with activated calf thymus DNA or poly(dC)oligo(dG)12-18 as template. No inhibition was observed with deoxythymidine 5'-triphosphate as substrate and poly(dA)oligo(dT)12-18 as template. All analogs were preferential inhibitors of the viral DNA polymerases. Ordering the compounds according to their decreasing binding affinities, as reflected by their increasing inhibition constants for the viral DNA polymerases, gave ACVTP greater than HBGTP greater than BCVTP greater than (S)-DHBGTP. The DNA polymerase from the HSV-1 mutant, CI(101)P2C5, resistant to ACV, showed a stronger decrease in sensitivity for ACVTP and HBGTP than for BCVTP compared to the effects on DNA polymerase from the wild-type strain CI(101). The analogs were not able to support DNA synthesis in the absence of the competing substrate dGTP. A decrease in the ability of calf thymus DNA to serve as primer template for HSV-2 DNA polymerase was observed after preincubation with the triphosphates of the acyclic guanosine analogs. The analogs showed a progressive inhibition of the HSV-2 DNA polymerase activity with incubation time, and the inhibition could be reversed by high concentrations of dGTP both with and without addition of fresh enzyme or fresh template. However, no reversion was obtained when fresh enzyme or template was added if dGTP was omitted. The data indicate that these analogs inhibited the DNA polymerases by a similar mechanism and that the inhibition was reversible.     

(+-)-carbocyclic 5'-nor-2'-deoxyguanosine and related purine derivatives: synthesis and antiviral properties.

Beginning with 3-cyclopenten-1-ylamine hydrochloride, the 5'-nor derivatives of carbocyclic 2'-deoxyguanosine (2), 2'-deoxyadenosine (3), and 2,6-diaminopurine 2'-deoxyribofuranoside (4) have been prepared. These compounds were evaluated for antiviral potential versus herpes simplex virus, varicella-zoster virus, cytomegalovirus, vaccinia virus, vesicular stomatitis virus, and human immunodeficiency virus and found to lack activity. Also, compounds 2-4 were virtually nontoxic toward the host (human diploid fibroblast ESM and HEL) cells. These biological properties may be due to the inability of 2-4 to be phosphorylated to the requisite nucleotide level that is likely to be necessary for biological activity by correlation to carbocyclic 2'-deoxyguanosine (1), which possesses significant antiviral properties as a result of conversion to its 5'-triphosphate derivative.     

Inhibition of ribonucleotide reduction in CCRF-CEM cells by 2',2'-difluorodeoxycytidine

The new deoxycytidine analogue 2',2'-difluorodeoxycytidine (dFdC) is a specific inhibitor of DNA synthesis that has marked cytotoxicity and therapeutic activity. A 2-hr incubation with 0.1-10 microM dFdC decreased cellular viability 78-97%. This treatment reduced deoxynucleoside triphosphate pools, similar to the action of the ribonucleotide reductase inhibitor hydroxyurea. The most pronounced decrease occurred in the dCTP pool, quantitatively followed by the decrease of dATP, dGTP, and dTTP. In contrast, inhibition of DNA synthesis by arabinosylcytosine did not affect the dCTP level, whereas dATP, dGTP, and dTTP pools increased, but less than 2-fold. The incorporation of [5-3H]cytidine into the dCTP pool, a measure of ribonucleotide reductase activity in whole cells, was reduced to 3% of controls by 0.1 microM dFdC, but to only 40% by 0.1 microM ara-C. Each drug decreased incorporation of [5-3H]cytidine into DNA to a similar extent (greater than 94%), suggesting limitation by a reaction proximal to this step. The cellular concentration of dFdC 5'-diphosphate was 0.3 microM at 50% inhibition of the in situ activity of ribonucleotide reductase. Direct assays of partially purified ribonucleoside diphosphate reductase (EC 1.17.4.1) demonstrated 50% inhibition by 4 microM dFdC 5'-diphosphate; dFdC 5'-triphosphate was much less inhibitory. We conclude that dFdC 5'-diphosphate acts as an inhibitor of ribonucleoside diphosphate reductase.     

Mode of action of acyclovir triphosphate on herpesviral and cellular DNA polymerases

The effect of 5'-triphosphate of acyclovir (ACV) on DNA polymerases of two human herpes-viruses, herpes simplex virus type-1 (HSV-1) and Epstein-Barr virus (EBV) as well as human cellular DNA polymerases alpha and beta has been examined. Of the enzymes tested, HSV-1 DNA polymerase was the most sensitive to inhibition by acyclovir triphosphate (ACVTP). The EBV DNA polymerase and DNA polymerase beta were less sensitive. ACVTP inhibition was competitive with dGTP with Ki values of 0.03, 0.15, 9.8 and 11.9 microM for HSV-1 DNA polymerase, DNA polymerase alpha, EBV DNA polymerase and DNA polymerase beta, respectively. Substituting a synthetic primer template (dG) approximately 15 x (dC)n for activated DNA template did not alter the pattern of inhibition. In a time course experiment, addition of ACVTP instead of dGTP did not increase DNA synthesis and it appeared to act as a chain terminator in DNA replication catalyzed by either HSV-1 DNA polymerase or DNA polymerase alpha. Although EBV DNA polymerase was less sensitive to ACVTP inhibition, the nucleoside analog itself was inhibitory to EB virus production by P3HR1 cell line as determined by a reduction in the percentage of cells expressing virus capsid antigen (VCA). On day 4, ACV at 10 and 25 micrograms/ml reduced the cell growth by 10% and 32%, respectively, while it reduced the VCA-positive cells by 80% and 84%, respectively. These results indicate that inhibition of EBV DNA polymerase activity by ACVTP may not be the primary mechanism responsible for ACV inhibition of EBV replication.     

Inactivation of O(6)-alkylguanine-DNA alkyltransferase by folate esters of O(6)-benzyl-2'-deoxyguanosine and of O(6)-[4-(hydroxymethyl)benzyl]guanine

O6-Alkylguanine-DNA alkyltransferase (alkyltransferase) provides an important source of resistance to some cancer chemotherapeutic alkylating agents. Folate ester derivatives of O6-benzyl-2'-deoxyguanosine and of O6-[4-(hydroxymethyl)benzyl]guanine were synthesized and tested for their ability to inactivate human alkyltransferase. Inactivation of alkyltransferase by the gamma-folate ester of O6-[4-(hydroxymethyl)benzyl]guanine was similar to that of the parent base. The gamma-folate esters of O6-benzyl-2'-deoxyguanosine were more potent alkyltransferase inactivators than the parent nucleoside. The 3'-ester was considerably more potent than the 5'-ester and was more than an order of magnitude more active than O6-benzylguanine, which is currently in clinical trials to enhance therapy with alkylating agents. They were also able to sensitize human tumor cells to killing by 1,3-bis(2-chloroethyl)-1-nitrosourea, with O6-benzyl-3'-O-(gamma-folyl)-2'-deoxyguanosine being most active. These compounds provide a new class of highly water-soluble alkyltransferase inactivators and form the basis to construct more tumor-specific and potent compounds targeting this DNA repair protein.     

Absence of correlation between telomerase activity and hepatic neoplasia in B6C3F1 mice.

Telomeres are the physical ends of eukaryotic chromosomes, which maintain chromosome stability and are progressively shortened with aging in somatic cells. The enzyme telomerase elongates telometric DNA and while not usually detectable in human somatic cells is expressed in most human tumors. The present study was conducted to determine if telomerase activity is a marker for spontaneous hepatic neoplastic changes in B6C3F1 mice, a strain frequently used in rodent carcinogenicity studies. Telomerase activity was generally higher in microscopically normal liver tissue from 8-week-old compared to aged mice (110-week-old); however, telomerase activity was not consistently increased in hepatocellular adenomas and carcinomas. It is proposed that, while elevated telomerase activity may modulate human tumor development, modulation of telomerase activity is not a feature of hepatic tumors in B6C3F1 mice and therefore is unlikely to have utility as a molecular marker for hepatic neoplasia in this mouse strain.     

Purine nucleoside phosphorylase inhibitor BCX-1777 (Immucillin-H)--a novel potent and orally active immunosuppressive agent.

Patients with purine nucleoside phosphorylase (PNP) deficiency present a selective T-cell immunodeficiency. Inhibitors of PNP are, therefore, of interest as potential T-cell selective immunosuppressive agents. BCX-1777 is a potent inhibitor of PNP from various species including human, mouse, rat, monkey and dog, with IC50 values ranging from 0.48 to 1.57 nM. BCX-1777, in the presence of 2'-deoxyguanosine (dGuo, 3-10 microM), inhibits human lymphocyte proliferation activated by various agents such as interleukin-2 (IL-2), mixed lymphocyte reaction (MLR) and phytohemagglutinin (PHA) (IC50 values < 0.1-0.38 microM). BCX-1777 is a 10-100-fold more potent inhibitor of human lymphocyte proliferation than other known PNP inhibitors like PD141955 and BCX-34. Nucleotide analysis of human lymphocytes indicate that inhibition of proliferation by BCX-1777 correlates with dGTP levels in the cells. BCX-1777 has excellent oral bioavailability (63%) in mice. At a single dose of 10 mg/kg in mice, BCX-1777 elevates dGuo to approximately 5 microM. BCX-1777 was not effective in mouse T-cell models such as delayed type hypersensitivity (DTH) and splenomegaly because mouse T-cells do not accumulate dGTP as do human T-cells. However, in the human peripheral blood lymphocyte severe combined immunodeficiency (hu-PBL-SCID) mouse model, BCX-1777 was effective in prolonging the life span 2-fold or more. This is the first known example of a PNP inhibitor that elevates dGuo in mice similar to the levels observed in PNP-deficient patients. Furthermore, these dGuo levels are also required for in vitro T-cell inhibition by BCX-1777. Thus, BCX-1777 represents a novel class of selective immunosuppressive agents that could have therapeutic utility in various T-cell disorders.     

Metabolism and DNA interaction of 2',3'-didehydro-2',3'-dideoxythymidine in human bone marrow cells.

2',3'-Didehydro-2',3'-dideoxythymidine (D4T) is a potent inhibitor of human immunodeficiency virus (HIV), with low hematological toxicity. In the present study, the cellular pharmacology of D4T was investigated in human bone marrow cells (BMC), in an attempt to understand the mechanism of the observed low bone marrow toxicity. After exposure of human BMC to 10 microM [3H]D4T for 24 hr, D4T-5'-triphosphate (D4T-TP) was the predominant metabolite, reaching a concentration of 0.3 pmol/10(6) cells. The D4T-5'-monophosphate levels were slightly lower, whereas the D4T-5'-diphosphate levels were about 6-fold lower than those of D4T-TP at 24 hr. Nucleic acids of human BMC exposed to 10 microM [3H]D4T for 24 hr were purified and analyzed by cesium sulfate density gradient centrifugation. No radioactivity was detected in the RNA region, whereas a limited amount was associated with the DNA region. The amount of label incorporated into DNA correlated with the extracellular D4T concentration and the length of incubation time. Enzymatic hydrolysis of radiolabeled DNA and subsequent analysis by high performance liquid chromatography demonstrated incorporation of both D4T and thymidine (dThd) into DNA. Degradation of D4T to thymine and subsequent formation of labeled dThd was also detected in human BMC. Pulse (24 hr)-chase (48 hr) experiments with 10 microM [3H]D4T demonstrated that the amount of radiolabel from D4T in DNA decreased over time during the chase. Under similar conditions, [3H]3'-azido-3'-deoxythymidine (AZT) incorporated into DNA of human BMC did not decrease during the chase. Although D4T-TP standard was demonstrated to be unstable at 37 degrees and neutral pH, D4T was much more stable in solution when incorporated into newly synthesized DNA isolated from human BMC, suggesting that enzymatic excision may be the mechanism for D4T removal from DNA. In summary, although higher concentrations of D4T-TP, compared with AZT-5'-triphosphate, are observed in human BMC, after exposure of cells to similar extracellular concentrations of parent drug, steady state levels of D4T incorporated into DNA are 10-50-fold lower, compared with AZT. Competition with dTTP formed by D4T metabolism and excision of D4T from DNA may be responsible, in part, for these effects. This study further demonstrates that incorporation of 2',3'-dideoxynucleosides into nuclear DNA of human BMC may be related to the ability of these anti-HIV agents to induce hematological side effects.     

Cellular pharmacology of 3'-azido-3'-deoxythymidine with evidence of incorporation into DNA of human bone marrow cells

We previously demonstrated that 3'-azido-3'-deoxythymidine (AZT) inhibits growth proliferation of human bone marrow progenitor cells in vitro [Antimicrob. Agents Chemother. 31:452-454 (1987)]. The present study evaluates the effect of toxic concentrations of AZT on possible sites of toxicity in human bone marrow cells. Exposure of cells over a 6-hr period to AZT concentrations between 0.5 and 50 microM resulted in a decreased incorporation of tritiated deoxyguanosine into DNA. Unchanged AZT and its phosphorylated metabolites accumulated within cells after exposure to 10 microM [3H]AZT. 3'-Azido-3'-deoxythymidine-5'-monophosphate was the predominant metabolite, reaching a concentration of 49.2 +/- 14.1 pmol/10(6) cells after 48 hr, and a continuous increase was observed in all phosphorylated derivative levels between 2 and 48 hr of incubation. Using a highly sensitive and specific DNA polymerase assay, endogenous deoxyribonucleotide pool size(s) were analyzed for 48 hr after incubation of cells with a pharmacologically relevant concentration of 10 microM AZT. After a 6-hr exposure, 2'-deoxycytidine-5'-triphosphate and 2'-deoxythymidine-5'-triphosphate pools represented approximately 86 and 70% of the control values; levels returned to normal after 24 hr and remained subsequently unchanged. Nucleic acids of human bone marrow cells exposed for 24 hr to 10 microM [3H]AZT were purified and analyzed by cesium sulfate density gradient. No radioactivity was detected in the RNA region, whereas a significant amount was associated with the DNA region. Hydrolysis of radiolabeled DNA and subsequent analysis by high performance liquid chromatography demonstrated specific incorporation of AZT into DNA. In additional studies, the amount of AZT incorporated into DNA was correlated with the initial extracellular AZT concentration. In particular, a significant relationship (p less than 0.0001) between the level of AZT incorporated into DNA and the inhibition of clonal growth was observed at concentrations of AZT between 1 and 25 microM (IC50 and IC85 for human bone marrow cells). In summary, these studies demonstrate that AZT is incorporated into DNA of human bone marrow cells and suggest that incorporation of AZT into DNA may be one mechanism responsible for AZT-induced bone marrow toxicity. In contrast, imbalance of deoxyribonucleotide pools by AZT appears unlikely to be associated with inhibition of DNA synthesis and toxicity in human bone marrow cells.     

Modulation of deoxycytidylate deaminase in intact human leukemia cells. Action of 2',2'-difluorodeoxycytidine.

Cellular metabolism studies had demonstrated previously that low cellular concentrations of 2',2'-difluorodeoxycytidine (dFdC) nucleotides are eliminated by deoxycytidylate deaminase (dCMPD), whereas dCMPD activity is inhibited at high cellular dFdC nucleotide levels (Heinemann et al., Cancer Res 52: 533-539, 1992). An assay for measuring dCMPD activity in intact human leukemia cells has now been developed to permit investigations of the interactions of dFdC nucleotides with dCMPD in intact cells in which the regulated nature of this enzyme was not disrupted. Using [14C]dCyd as the substrate, radioactivity that accumulated in dTTP was quantitated after high-pressure liquid chromotography by a radioactive flow detector. The assay was first characterized using either the dCMPD inhibitor tetrahydrodeoxyuridine (H4dUrd) which directly inhibits dCMPD, or thymidine and 5-fluoro-2'-deoxyuridine (FdUrd) which indirectly inhibit and activate dCMPD, respectively, by affecting the cellular dCTP:dTTP value. Measured by this in situ assay, there was a strong correlation between dCMPD activity and dCTP:dTTP levels. Consistent with previous studies using partially purified enzyme, incubation of cells with dFdC resulted in a concentration-dependent inhibition of dCMPD in situ. The mechanism of modulation of dCMPD by dFdC, however, was clearly different from that of thymidine and FdUrd. In addition to the effect of dFdC on cellular dCTP:dTTP, our findings also suggested an additional inhibitory mechanism, possibly a direct interaction between dCMPD and dFdC 5'-triphosphate. Thus, results obtained using this direct assay of dCMPD in intact cells support the hypothesis that dCMPD is inhibited by nucleotides of dFdC.     

Novel pyrazolo[3,4-d]pyrimidine-based inhibitors of Staphlococcus aureus DNA polymerase III: design,synthesis, and biological evaluation

6-Anilinopyrazolo[3,4-d]pyrimidin-4-ones are novel dGTP analogues that inhibit the replication-specific enzyme DNA polymerase III (DNA pol III) of Staphlococcus aureus and other Gram-positive (Gr+) bacteria. To enhance the potential of these inhibitors as antimicrobial agents, a structure-activity relationship was developed involving substitutions at the 2, 4, and pyrazolo NH positions. All of the new inhibitors were tested for their ability to inhibit S. aureus DNA pol III and the growth of several other Gr+ bacteria in culture. 2-Anilino groups with small hydrophobic groups in the meta or para position enhanced both antipolymerase and antimicrobial activity. 2-Benzyl-substituted inhibitors were substantially less active. Substitution in the 4-position by oxygen gave the optimal activity, whereas substitution at the pyrazolo NH was not tolerated. These pyrazolo[3,4-d]pyrimidine derivatives represent a novel class of antimicrobials with promising activities against Gr+ bacteria.