Inhibitors of DNA Polymerase III as Novel Antimicrobial Agents against Gram-Positive Eubacteria

Abstract

6-Anilinouracils are selective inhibitors of DNA polymerase III, the enzyme required for the replication of chromosomal DNA in gram-positive bacteria (N. C. Brown, L. W. Dudycz, and G. E. Wright, Drugs Exp. Clin. Res. 12:555–564, 1986). A new class of 6-anilinouracils based on N-3 alkyl substitution of the uracil ring was synthesized and analyzed for activity as inhibitors of the gram-positive bacterial DNA polymerase III and the growth of gram-positive bacterial pathogens. Favorable in vitro properties of N-3-alkyl derivatives prompted the synthesis of derivatives in which the R group at N-3 was replaced with more-hydrophilic methoxyalkyl and hydroxyalkyl groups. These hydroxyalkyl and methoxyalkyl derivatives displayed Ki values in the range from 0.4 to 2.8 μM against relevant gram-positive bacterial DNA polymerase IIIs and antimicrobial activity with MICs in the range from 0.5 to 15 μg/ml against a broad spectrum of gram-positive bacteria, including methicillin-resistant staphylococci and vancomycin-resistant enterococci. Two of these hydrophilic derivatives displayed protective activity in a simple mouse model of lethal staphylococcal infection.

7-Alkyl-N2-substituted-3-deazaguanines. Synthesis, DNA polymerase III inhibition and antibacterial activity

“…Early pol IIIC inhibitors were simple AU (6-anilinouracil) derivatives, which have two key structural features: a) a substituted pyrimidine ring that permits base pairing to a pyrimidine in the DNA template; b) a planar aryl ring at the 6-NH group. 3,4 Through its base-pairing domain, an AU molecule forms Watson-Crick-like hydrogen bonds with an unapposed cytosine residue in the template strand just distal to the DNA primer terminus; consequently its action is competitive with dGTP. Simultaneously, the aryl domain binds an aryl-specific “receptor” near the enzyme’s active site, causing the formation of an inactive ternary complex of inhibitor, DNA and pol IIIC (Figure 1).…”

“…7 HB-EMAU ( 1 ) is an effective Gram+ antibacterial with MIC values of 2.5–5 µg/ml against various organisms, but inactive against the Gram- bacterium E. coli . 4,7 Ciprofloxacin potently inhibited growth of all organisms with the exception of MRSA 1090, a methicillin-resistant strain of S. aureus with cross-resistance to the fluoroquinolones. Vancomycin was active against all Gram+ organisms except VRE, the vancomycin-resistant strain of E. faecalis , and linezolid was active against all Gram+ organisms.…”

DNA replication proteins as potential targets for antimicrobials in drug-resistant bacterial pathogens

“…The AU class of PolIII inhibitors, which includes HPUra, is composed of a uracil-containing base-pairing domain that binds the DNA at cytosine bases and an aryl domain that determines the selectivity and affinity for PolC 46 . AUs competitively inhibit PolC with respect to dGTP through simultaneous binding to the cytosine of the DNA strand and near the active site of PolC, resulting in a ternary inactive complex of AU inhibitor, DNA and PolC 46 , 47 . HPUra served as a scaffold for the development of numerous AUs with a broad range of antimicrobial and pharmacological properties.…”

“…HPUra served as a scaffold for the development of numerous AUs with a broad range of antimicrobial and pharmacological properties. Two promising AUs, 6-(3-ethyl-4-methylanilino) uracil (EMAU) and 6-([3,4-trimethylene]anilino) uracil (TMAU), were highly active against PolC in vitro , but required optimization to increase activity against various Gram-positive bacteria, including MRSA 46 , 48–50 . Improvement of solubility of AUs compromises antimicrobial activity, but allowed the production of compounds that could be delivered intravenously rather than subcutaneously in animal models of infection 46 , 50–52 .…”

“…Two promising AUs, 6-(3-ethyl-4-methylanilino) uracil (EMAU) and 6-([3,4-trimethylene]anilino) uracil (TMAU), were highly active against PolC in vitro , but required optimization to increase activity against various Gram-positive bacteria, including MRSA 46 , 48–50 . Improvement of solubility of AUs compromises antimicrobial activity, but allowed the production of compounds that could be delivered intravenously rather than subcutaneously in animal models of infection 46 , 50–52 . The frequencies of mutations leading to AU resistance ranged from 3.6 × 10 − 10 to 1.2 × 10 − 8 , comparable to the frequency of ciprofloxacin resistance 53 .…”