Aminoglycoside antibiotics constitute an important class of clinically useful drugs which are imperiled by the emergence of resistant organisms. Aminoglycoside resistance in the clinics is primarily due to the presence of modifying enzymes which N-acetylate, O-adenylate or O-phosphorylate the antibiotics. The latter family of enzymes are termed the aminoglycoside phosphotransferases or kinases and are the subject of this review. There are seven classes of aminoglycoside phosphotransferases (APH(3'), APH(2''), APH(3'off'), APH(6), APH(9), APH(4), APH(7'')) and many isozymes in each class, and although there is very little overall general sequence homology among these enzymes, certain signature residues and sequences are common. The recent determination of the three-dimensional structure of the broad spectrum aminoglycoside kinase APH(3')-IIIa complexed with the product ADP, in addition to mechanistic and mutagenic studies on this and related enzymes, has added a great deal to our understanding of this class of antibiotic resistance enzyme. In particular, the revelation of structural and mechanistic similarities between APHs and Ser/Thr and Tyr kinases has set the stage for future inhibition studies which could prove important in reversing aminoglycoside resistance.

The work in the Brown laboratory has two long-range objectives. Both are derived from an interest in the replication of the genome of Gram-positive eubacteria. One objective is to gain a deeper understanding of the structure and function of DNA polymerase III, the unique species of DNA polymerase which is essential for chromosome replication. The second objective, the one from which this thesis is derived, is to determine whether a selective inhibitor of this DNA polymerase can serve as a basis for producing a new generation of clinically useful Gram-positive-selective antimicrobial agents. The polymerase III-specific inhibitor prototypes investigated in this work are members of a family of simple 6-substituted uracils. The following members of this family, TMAU and EMAU, were used as platforms for the manipulation of the N3 atom (arrow), the only ring component which could be substituted without severe reduction of inhibitory activity. The N3 position was substituted with a series of alkyl groups of increasing size. The resulting structure-activity relationships at the level of the polymerase was consistent with the presence of an N3-specific subdomain within the inhibitor binding site which could accommodate a wide variety of substituents. Although specific alkyl substituents at N3 also significantly enhanced the antibacterial potency of TMAU and EMAU, the respective compounds were found to have insufficient aqueous solubility for successful application in in vivo infection. To increase aqueous solubility, the N3 atom of the EMAU platform was substituted with selected hydroxy- and methoxyalkyl groups. The latter agents retained both anti-polymerase and antibacterial activity, and, as expected, they displayed a combination of lipid and aqueous solubility favorable to efficacy in in vivo infection. Two of the agents, N3-hydroxypropyl- and N3-methoxypropyl-EMAU were examined for their ability to protect mice from lethal staphylococcal infection. Both were found to be active in this model. In sum, the results of this work demonstrated, for the first time, that: (1) the eubacterial replication-specific DNA polymerase III is a valid target for antibiotic development, and (2) the N3-substituted 6-anilinouracil platform has strong potential as a basis for novel antibiotics useful against Gram-positive bacterial infection.

The 6-anilinouracils are novel dGTP analogs that selectively inhibit the replication-specific DNA polymerase III of gram-positive eubacteria. Two specific derivatives, IMAU (6-[3'-iodo-4'-methylanilino]uracil) and EMAU (6-[3'-ethyl-4'-methylanilino]uracil), were substituted with either a hydroxybutyl (HB) or a methoxybutyl (MB) group at their N3 positions to produce four agents: HB-EMAU, MB-EMAU, HB-IMAU, and MB-IMAU. These four new agents inhibited Staphylococcus aureus, coagulase-negative staphylococci, Enterococcus faecalis, and Enterococcus faecium. Time-kill assays and broth dilution testing confirmed bactericidal activity. These anilinouracil derivatives represent a novel class of antimicrobials with promising activities against gram-positive bacteria that are resistant to currently available agents, validating replication-specific DNA polymerase III as a new target for antimicrobial development.

The IL-1 receptor/Toll-like receptor superfamily comprises a diverse family of cell surface receptors defined by a characteristic conserved sequence in their cytosolic regions, termed the Toll/IL-1 receptor domain, which function in inflammation and host defence against microbial pathogens. Members include receptors for the proinflammatory cytokines IL-1 and IL-18 and Toll-like receptors 2 and 4, which are involved in host responses to Gram-positive and Gram-negative bacteria, respectively. Signalling pathways activated by these receptors are conserved and the superfamily represents a pan-genomic system involved in the host response to infection and injury.