Enzyme-catalyzed therapeutic activation (ECTA) is a novel prodrug strategy to overcome drug resistance resulting from enzyme overexpression. ␤-Lactamase overexpression is a common mechanism of bacterial resistance to ␤-lactam antibiotics. We present here the results for one of the ␤-lactamase ECTA compounds, NB2001, which consists of the antibacterial agent triclosan in a prodrug form with a cephalosporin scaffold. Unlike conventional ␤-lactam antibiotics, where hydrolysis of the ␤-lactam ring inactivates the antibiotic, hydrolysis of NB2001 by ␤-lactamase releases triclosan. Evidence supporting the proposed mechanism is as follows. (i) NB2001 is a substrate for TEM-1 ␤-lactamase, forming triclosan with a second-order rate constant (k cat /K m ) of greater than 77,000 M ؊1 s ؊1 .(ii) Triclosan is detected in NB2001-treated, ␤-lactamase-producing Escherichia coli but not in E. coli that does not express ␤-lactamase. (iii) NB2001 activity against ␤-lactamaseproducing E. coli is decreased in the presence of the ␤-lactamase inhibitor clavulanic acid. NB2001 was similar to or more potent than reference antibiotics against clinical isolates of Staphylococcus aureus (including MRSA), Staphylococcus epidermidis, Streptococcus pneumoniae, vancomycin-resistant Enterococcus faecalis, Moraxella catarrhalis and Haemophilus influenzae. NB2001 is also active against Klebsiella pneumoniae, Enterobacter aerogenes, and Enterobacter cloacae. The results indicate that NB2001 is a potent, broad-spectrum antibacterial agent and demonstrate the potential of ECTA in overcoming ␤-lactamase-mediated resistance.

Two potent antibacterial agents designed to undergo enzyme-catalyzed therapeutic activation were evaluated for their mechanisms of action. The compounds, NB2001 and NB2030, contain a cephalosporin with a thienyl (NB2001) or a tetrazole (NB2030) ring at the C-7 position and are linked to the antibacterial triclosan at the C-3 position. The compounds exploit ␤-lactamases to release triclosan through hydrolysis of the ␤-lactam ring. Like cephalothin, NB2001 and NB2030 were hydrolyzed by class A ␤-lactamases (Escherichia coli TEM-1 and, to a lesser degree, Staphylococcus aureus PC1) and class C ␤-lactamases (Enterobacter cloacae P99 and E. coli AmpC) with comparable catalytic efficiencies (k cat /K m ). They also bound to the penicillin-binding proteins of S. aureus and E. coli, but with reduced affinities relative to that of cephalothin. Accordingly, they produced a cell morphology in E. coli consistent with the toxophore rather than the ␤-lactam being responsible for antibacterial activity. In biochemical assays, they inhibited the triclosan target enoyl reductase (FabI), with 50% inhibitory concentrations being markedly reduced relative to that of free triclosan. The transport of NB2001, NB2030, and triclosan was rapid, with significant accumulation of triclosan in both S. aureus and E. coli. Taken together, the results suggest that NB2001 and NB2030 act primarily as triclosan prodrugs in S. aureus and E. coli.The ubiquitous occurrence of ␤-lactamases in bacteria and their association with clinical resistance to ␤-lactams have allowed strong interest in these enzymes to be sustained (3, 26). ␤-Lactamases are periplasmic in gram-negative bacteria, while they are exocellular in gram-positive bacteria. They are conveniently divided into four classes (classes A, B, C, and D) on the basis of amino acid sequence homologies (Ambler classification) (1, 15). Of these classes, classes A and C are of the greatest clinical significance. ␤-Lactamase inhibitors, such as clavulanic acid and the penicillanic acid sulfones, mostly target class A enzymes; and their use in combination with older compounds has restored the broad-spectrum activities of older compounds, such as amoxicillin (18,22,25,26). Inhibitors that target class C and class B ␤-lactamases have yet to reach clinical development.Exploiting common ␤-lactamases to generate novel antibacterials is a strategy originally used by O'Callaghan et al. (24) and subsequently by Mobashery and Johnston (20) and other investigators (6,10,16). It has been part of NewBiotics' general enzyme-catalyzed therapeutic activation (ECTA) prodrug approach that harnesses unique enzymes in bacteria to achieve selective release of cytotoxic agents from substrate-like molecules (16; M. V.

Tumor cell resistance to fluoropyrimidines and other inhibitors of thymidylate synthase (TS) is a serious problem often associated with increased intracellular TS. Clinically, another problem that arises from the use of TS inhibitors is toxicity, which develops, in part, because normal cells may be adversely affected by doses of inhibitor that do not impact tumor cells. To circumvent this problem, we have devised a new strategy called enzyme-catalyzed therapeutic activation (ECTA), which takes advantage of overexpressed TS to enzymatically generate cytotoxic moieties preferentially in tumor cells. We show herein that tumor cells expressing elevated levels of TS are preferentially sensitive to NB1011, a phosphoramidate derivative of (E)-5-(2-bromovinyl)-2'-deoxyuridine. We find support for the proposed mechanism of NB1011 in the following results: 1) positive relationship between TS protein level and sensitivity to NB1011 in engineered HT1080 tumor cells, designed to express defined levels of TS protein; 2) NB1011 activity is enhanced on tumor cells which express endogenous elevated TS; 3) cytotoxicity of NB1011 is blocked by raltitrexed (Tomudex); 4) NB1011 selection of TS-overexpressing MCF7TDX tumor cells results in recovery of cell populations and clones with diminished TS levels (and restored sensitivity to raltitrexed). A preliminary comparison of TS mRNA levels in multiple normal tissues versus colon tumor samples suggests that selective tumor cytotoxicity of NB1011 may be possible in the clinical setting. Because NB1011 cytotoxicity is dependent upon activation by TS, its proposed mechanism of action is distinct from current TS-targeted drugs, which require inhibition of TS to be effective.