The slow dissociation rate of K-1602 contributes to the enhanced inhibitory activity of this novel alkyl–aryl-bearing fluoroketolide

Figures & data

Figure 1. Chemical structures of macrolides erythromycin, tylosin, cethromycin, telithromycin, and Kosan-1602.

Figure 2. (A) Progress curve of puromycin reaction, in the absence of antibiotic (▴) and in the presence of 1 μM K-1602 (•) and tylosin 3 μM (○). (B) Puromycin reaction in the absence of antibiotic (bar 1) and in the presence of 5 μM K-1602 (bar 2), 5 μM Tylosin (bar 3), and 5 μM Tylosin in the simultaneous presence of K-1602 either at 5 μM (bar 4) or 20 μM (bar 5). (C) Protection of bases in domain V of 23S rRNA from DMS modification by bound antibiotics. Lanes are marked from the left as follows: U, A, G, C sequencing lanes. Lane 1, untreated 23S rRNA; lane 2, 23S rRNA modified by DMS in the absence of antibiotic; lanes 3–6, ribosomes were first preincubated for 10 min at 37 °C with erythromycin, telithromycin, K-1602 and tylosin, and then modified by DMS. All antibiotics were used at a final concentration equal to 50 μM.

Figure 3. (A) Progress curve of complex C inactivation by tylosin in the presence of K-1602. Complex C absorbed on a cellulose nitrate filter was exposed to a solution containing 3 μM tylosin plus K-1602 either 2 μM (•) or 8 μM (○). (B) Function of the apparent rate constant F versus K-1602 concentration in the simultaneous presence of tylosin. Tylosin concentration was either 3 μM (Δ) or 6 μM (•). The apparent rate constant F was calculated from the slope of the plots as (A).

Figure 4. (A) Inactivation of complex C by tylosin in the presence of 4 μM K-1602. (C*I)_∞ represents the inactivated complex C by tylosin at infinite time. (B) Variation of 1/(C*I)_∞ as a function of reverse of tylosin concentration. The K-1602 concentration remains constant and equal to 4 μM as in (A). (C*I) is presented as the ratio of total (C).

Figure 5. Determination of the dissociation rate constant (k₇) of C*A complex. Drug–ribosome complex (C*A) was prepared in the presence of 5 μM K-1602, and absorbed on a cellulose nitrate filter. Then after dilution, it was exposed to 10 μM tylosin for various time intervals, after which the remaining activity was titrated with puromycin. The k₇ value was estimated from the slope of the linear time plot.

Scheme 1. Kinetic model of puromycin reaction.

Scheme 2. Two step kinetic model of competition between K-1602 (A) and tylosin (I) for binding on the ribosomal complex (C).

Scheme 3. One step kinetic model of competition between K-1602 (A) and tylosin (I) for binding on the ribosomal complex (C).

Table 1. Kinetic equations for one-step and two-steps mechanisms of drug binding to ribosomes.

Function	One step mechanism	Two step mechanism
F
(C*I)∞

F represents the apparent rate constant of ribosomal complex C interaction with both antibiotics tylosin and K-1602, and (C*I)_∞ represents the product AcPhe-puromycin formed at infinite time of exposing the complex in defined antibiotic concentrations.