Fig 1.
Structures of CAM, CLB, and the synthesized CAM dimers.
Abbreviations: CAM, chloramphenicol; CLB, chloramphenicol base.
Fig 2.
Synthesis of compounds studied in the present work.
Reagents and conditions: (i) malonic acid (for compound 1), fumaric acid (for compound 2), adipic acid (for compound 4), suberic acid (for compound 6), azelaic acid (for compound 7), 1,4-phenylenediacrylic acid (for compound 8), HBTU, iPr2NEt, DMF, 0°C then RT, 1–3 h; yield: 75% (1), 55% (2), 85% (4), 67% (6), 89% (7), and 83% (8); (ii) (a) glutaric anhydride, DMF, RT, 2h; (b) HBTU, iPr2NEt, DMF, 0°C then RT, 1 h; yield: 80% (3); (iii) terephthaloyl chloride, Et3N, DMF, 0°C then RT, 1h; yield: 80% (5). See also S1 Supplemental Procedures for details.
Fig 3.
AcPhe-puromycin synthesis in the presence or absence of compound 5.
(A) First-order time plots; complex C reacted at 25°C in buffer A, with (black) 400 μM puromycin or with a mixture containing 400 μM puromycin and compound 5 at concentrations of 4 μM (magenta), 8 μM (green), 15 μM (blue), and 30 μM (red). (B) Variation of the apparent equilibration rate constant, keq, as a function of compound 5 concentration (I). The reaction was carried out in buffer A, in the presence of puromycin at concentrations of 200 μM (red), 400 μM (black), or 2 mM (blue). The keq values were determined by non linear regression fitting of the kinetic data to Eq 2 [11]: (C) Kinetic model for the inhibition of the puromycin reaction by CAM dimers. Symbols: C, poly(U)-programmed 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.
Table 1.
Equilibrium and kinetic constants involved in the inhibition of AcPhe-puromycin synthesis by CAM dimersa.
Table 2.
Relative reactivity of nucleosides in the central loop of Domain V of 23S rRNA, when a CAM dimer (I) binds to E. coli ribosomes (R) in the initial (RI) and the final (R*I) binding sitesa.
Fig 4.
Binding positions of compounds 4 and 5 on the E. coli ribosome, as detected by Molecular Dynamics simulations.
Compounds 4 and 5 have been docked into the 50S ribosomal subunit, by positioning one of their CAM moieties within the CAM crystallographic pocket [4]. (A) Binding position of compound 5 (yellow); hydrogen bonding with residues of the catalytic center is shown by black dots. Other residues of 23S rRNA placed adjacently to the binding pocket of 5 are ignored for clarity. (B) Binding position of compound 4 (yellow).
Fig 5.
CAM dimer crosslinking at the entrance to the exit tunnel, upon UV-irradiation.
Ribosomes from E. coli were irradiated with 365 nm light for 30 min (panels A-C), in the absence (lane 1) or the presence of compound 4 (lanes 2 and 3) or compound 5 (lanes 5 and 6). The irradiation products were analyzed by probing with DMS (panels A and B) or CMCT (panel C) and primer extension, before (lanes 3 and 6) or after discharging from excess CAM dimer (lanes 2 and 5). Probing and primer extension analysis were also applied to non-treated ribosomes (lane 4). Numbering of nucleosides for the sequencing lanes is indicated at the left. (A) Analysis of the A2600-U2615 region of 23S rRNA. (B) Analysis of the C2055-A2065 region (entrance to the exit tunnel) of 23S rRNA. (C) Analysis of the A2500-U2506 region (PTase catalytic center) of 23S rRNA.
Table 3.
Determination of EC50 for CAM and CAM dimers, that indicates how much concentration of each compound is needed to produce 50% of the maximal inhibitory effect of that compounda.
Fig 6.
Kinetic analysis of the CAM acetyltransferase reaction using CAM or compounds 4 and 5 as substrates.
The reaction was carried out in 3 ml of 94 mM Tris/HCl pH 7.8, containing 0.083 mM 5,5’-dithio-bis(2-nitrobenzoic acid), 0.16 mM acetyl coenzyme A, 25 units CAM acetyltransferase, and either CAM (●), compound 4 (▲), or compound 5 (■) at the concentrations indicated. The product of the enzymatic reaction, coenzyme A, reacted with 5,5’-dithio-bis(2-nitrobenzoic acid) to yield 5-thio-2-nitrobenzoate which absorbs at 412 nm, with a micromolar extinction coefficient equal to 0.0136. The Vmax and Km values were determined by fitting the substrate concentrations [S] and the obtained ΔA412nm/min (Vo) values into equation V0 = Vmax[S]/(Km + [S]). The obtained Vmax values were divided by 0.0136 to convert their units in μM·min-1 (http://www.sigmaaldrich.com/technical-documents/protocols/biology/enzymatic-assay-of-chloramphenicol-acetyltransferase.html). The ratio Vmax/Km for each curve is given in parenthesis.
Fig 7.
Toxicity assays in human peripheral blood cells.
Peripheral blood was collected in EDTA-coated tubes from 5 healthy volunteers (age range: 25–30 years). Concentration was adjusted to 1.8×109 cells/L using RPMI-1640 medium containing 1% penicillin/streptomycin. Cells were cultured in triplicate in the presence or the absence of 30 or 60 μM CAM or compound 5, under a humidified 5% CO2 atmosphere for 5 days, at 37°C. Cultures were counted daily by a CELL-DYN 3700 Hematology Analyzer and values were expressed as a percentage of cells measured in controls.
Fig 8.
Toxicity assays in Jurkat cells.
Jurkat cells were adjusted to 1×109 cells/L in RPMI-1640 medium containing 1% Penicillin/Streptomycin and 10% fetal bovine serum. The cells were grown in triplicate in the presence or absence of compound 5 at the indicated concentrations for 4 days at 37°C, under a humidified 5% CO2 atmosphere. CAM was used as a reference compound. For cell necrosis and apoptosis assays, samples (106 cells) were collected daily and determined by flow cytometry. Apoptotic and necrotic cells were expressed as a percentage of total cells.