s characterized by linearity. However, when the reaction proceeded in the presence of a CAM dimer, e.g. compound 5, the time plots were characterized by two unique features that distinguish them from a typical kinetic behavior. First, biphasic logarithmic time plots were obtained, with the second phase exhibiting stronger inhibition characteristics than the first one. Second, the slopes of both progress curves varied as a function of the inhibitor concentration. When analyzed by double Fig 3. AcPhe-puromycin synthesis in the presence or absence of compound 5. First-order time plots; ONO4059 price complex C reacted at 25C in buffer A, with 400 M puromycin or with a mixture containing 400 M puromycin and compound 5 at concentrations of 4 M, 8 M, 15 M, and 30 M. Variation of the apparent equilibration rate constant, keq, as a function of compound 5 concentration. The reaction was carried out in buffer A, in the presence of puromycin at concentrations of 200 M, 400 M, or 2 mM. The 
keq values were determined by non linear regression fitting of the kinetic data to Eq 2: Kinetic model for the inhibition of the puromycin reaction by CAM dimers. Symbols: C, polyprogrammed ribosomes from E. coli, bearing AcPhe-tRNAPhe at the P-site of the catalytic center and tRNAPhe at the E-site; I, CAM dimer; S, puromycin; C’, ribosomal complex not recycling; P, AcPhe-puromycin. See also S1 Fig. doi:10.1371/journal.pone.0134526.g003 5 / 22 Development of Chloramphenicol Homodimers reciprocal plotting, both phases exhibited characteristics of simple competitive PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19756382 inhibition. These kinetic results are consistent with compound 5 operating through an induced fit mechanism, in which the inhibitor first binds rapidly to complex C to form the encounter complex CI, which then undergoes a slow conformational change to produce a final, tighter complex C I. Corroborative evidence for the consistency of this model is provided by the hyperbolic shape of the equilibration plots, in which keq represents the apparent rate constant for the attainment of equilibrium among C, CI, and C I. If one-step mechanism of inhibition was applicable, keq should be a linear function of . Yet the apparent association rate constant, /Ki, was found to be 3104M1s-1 that is lower than the upper limit 106M-1s-1 set for the characterization of a compound as a slow-binding inhibitor. Because the isomerization constant kon/koff was calculated to be 3.6, the inhibition process was finally associated with high overall inhibition of peptide-bond formation. In addition, the slow koff rate provided prolonged residence time for compound 5 at the ribosome, a behavior potentially predicting good efficacy in vivo. Although a direct comparison is not accurate, compound 5 is ~10-fold more potent than homodimers of CAM previously synthesized by Berkov-Zrihen et al.Development of Chloramphenicol Homodimers Consistently, compounds 911 were almost inactive in inhibiting the growth of Staphylococcus aureus or E. coli cells, at concentrations up to 100 M. Taking into account that in a closed system, like the cell-free system used in our study, the inhibitory constant is an adequate metrics for differentiating compound potency, we used the Ki constant for ranking compounds 18; Ki by definition represents the overall inhibition constant engaged in both sequential reactions of the two-step mechanism shown in Fig 3C. PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19755711 Ki Ki koff CI kon koff CI C I 3 Accordingly, we estimated that compound 5 is 3-fold more potent than CAM. The rest of CA
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