SUMMARY : N-tolyl-a-naphthylamine-8-sulphonic acid forms conjugates with protein which fluoresce when excited by ultraviolet light. When washed cell suspensions of Pseudmnonas aeruginosa are treated with N-tolyl-cc-naphthylamine-8-sulphonic acid no fluorescence develops, but when polymyxin is also added, fluorescence develops, thus demonstrating that the dye can penetrate to proteincontaining portions of the cells. This technique has been used to study the competition between polymyxin and certain cations for sites on the cells. From a comparison of the affinities of these cations for the polpyxin-combining groups of the cells with their ability to reverse the charge on certain colloids it is suggested that the polymyxin-combining loci of the cells may be polyphosphates.The addition of polymyxin to a washed cell suspension of a strain of Pseudomonm aeruginosa results in a leakage from the cells of pentose, phosphate and materials which have an absorption maximum a t 260 mp. (Newton, 1 9 5 3~) .

The pseudopeptide pyrrolidinedione antibiotics, such as moiramide B, have recently been discovered to target the multisubunit acetyl coenzyme A (acetyl-CoA) carboxylases of bacteria. In this paper, we describe synthetic variations of each moiety of the modularly composed pyrrolidinediones, providing insight into structure-activity relationships of biochemical target activity, in vitro potency, and in vivo efficacy. The novel derivatives showed highly improved activities against gram-positive bacteria compared to those of previously reported variants. The compounds exhibited a MIC 90 value of 0.1 g/ml against a broad spectrum of Staphylococcus aureus clinical isolates. No cross-resistance to antibiotics currently used in clinical practice was observed. Resistance mutations induced by pyrrolidinediones are exclusively located in the carboxyltransferase subunits of the bacterial acetyl-CoA carboxylase, indicating the identical mechanisms of action of all derivatives tested. Improvement of the physicochemical profile was achieved by salt formation, leading to aqueous solubilities of up to 5 g/liter. For the first time, the in vitro activity of this compound class was compared with its in vivo efficacy, demonstrating a path from compounds weakly active in vivo to agents with significant efficacy. In a murine model of S. aureus sepsis, the 100% effective dose of the best compound reported was 25 mg/kg of body weight, only fourfold higher than that of the comparator molecule linezolid. The obvious improvements achieved by chemical derivatization reflect the potential of this novel antibiotic compound class for future therapy.

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