Mode of Action of Chloramphenicol

Chloramphenicol has been shown to exert a strong inhibitory action on microbial protein synthesis (Gale and Folkes, 1953; Maxwell and Nickel, 1954; Wisseman et al., 1954). Hence, the hypotheses that the antibiotic may act simply as the antagonist of one amino acid or another are especially attractive (Mentzer et al., 1950; Woolley, 1950; Bergmann and Sicher, 1952; Molho and Molho-Lacroix, 1952). Nevertheless, examination of the evidence in support of this proposed mechanism of action reveals that it is derived from experiments in which reversal of growth inhibition by the respective substances was generally minimal and was demonstrable only under special circumstances. Moreover, results of this kind are not limited to selected amino acids; indeed, a wide variety of other substances has also been found to yield comparable degrees of reversal of growth inhibition (Smith, 1952; Swenseid et al., 1952; Foster and Pittillo, 1953; Roblin, 1954; Hitchings and Elion, 1955). Although it is conceivable that a complex relationship of antagonists exists here, as in the case of the sulfonamides (Work and Work, 1948), it is also possible that these minimal "reversal effects" may be due to indirect factors which influence bacterial growth in some other manner and whose relation to chloramphenicol inhibition is more non-specific in nature than is that usually included in the concept of antimetabolites as discussed by Work and Work (1948), Martin (1951) and Woolley (1952). The structural relation of chloramphenicol to the aromatic amino acids phenylalanine, tryosine and tryptophan in particular, bears reconsideration in discussing this problem. When the formula is written in the conventional two-dimensional projection (see A, figure 1), there is a suggestion of a resemblance to the aromatic amino acids or to D-serine. However, the x-ray diffraction studies of Dunitz (1952) on crystalline chloramphenicol and bromamphenicol indicate that in this physical state, a second six membered ring may be formed by a hydrogen bond in the propanediol moiety, giving the molecule a much different appearance (see B, figure 1). It is, of course, possible that this ring does not actually exist in solution or under the conditions of antibiotic action, but compounds in which a similar but more stable ring structure has been substituted actually have been synthesized and possess antibiotic activity (U. S. Patent, 1952; Taylor, 1953). Formula C in figure 1 illustrates one of these compounds. An extensive discussion of the structural and configurational factors which contribute to the antibiotic activity of chloramphenicol is published elsewhere (Hahn et al., 1956). Furthermore, the theoretical relation of phenylalanine to chloramphenicol requires the assumption of four distinct substitutions (Woolley, 1950). This multiplicity of alterations might change the properties of the resulting compound to such an extent that it could no longer function as an antimetabolite of phenylalanine. In any case, the structural basis for relating chloramphenicol to the aromatic amino acids in an antimetabolite relationship would appear less well-founded than formerly supposed. The present report is an outgrowth of studies which have been continued intermittently since our first reported failure to detect reversal of growth inhibition by a variety of natural substances (Wisseman et al., 1954). The results are presented at this time because of their bearing on hypotheses concerning the mechanism by which protein synthesis is inhibited by chloramphenicol. In the course of these studies there have been many variations in conditions of testing, including different strains of organisms, different media and small as well as large inocula. The particular results selected for inclusion in this report were obtained under conditions designed to eliminate in so far as possible the influence of non-specific factors. In our experience