Antibacterial activity of rifamycins for M. smegmatis with comparison of oxidation and binding to tear lipocalin

Abstract

A mutant of Mycobacterium smegmatis is a potential class I model substitute for Mycobacterium tuberculosis. Because not all of the rifamycins have been tested in this organism, we determined bactericidal profiles for the 6 major rifamycin derivatives. The profiles closely mirrored those established for M. tuberculosis. Rifalazil was confirmed to be the most potent rifamycin. Because the tuberculous granuloma presents a harshly oxidizing environment we explored the effects of oxidation on rifamycins. Mass spectrometry confirmed that three of the six major rifamycins showed autoxidation in the presence of trace metals. Oxidation could be monitored by distinctive changes including isosbestic points in the ultraviolet-visible spectrum. Oxidation of rifamycins abrogated anti-mycobacterial activity in M. smegmatis. Protection from autoxidation was conferred by binding susceptible rifamycins to tear lipocalin, a promiscuous lipophilic protein. Rifalazil was not susceptible to autoxidation but was insoluble in aqueous solution. Solubility was enhanced when complexed to tear lipocalin and was accompanied by a spectral red shift. The positive solvatochromism was consistent with robust molecular interaction and binding. Other rifamycins also formed a complex with lipocalin, albeit to a lesser extent. Protection from oxidation and enhancement of solubility with protein binding may have implications for delivery of select rifamycin derivatives.

Keywords: Lipocalin-1; Mycobacteria smegmatis; Oxidation; Rifabutin (PubChem CID: 6323490); Rifalazil; Rifalazil (PubChem CID: 6540558); Rifampin (PubChem CID: 24871024); Rifamycin; Rifamycin S (PubChem CID: 6436726); Rifamycin SV (PubChem CID: 6324616); Rifapentine (PubChem CID: 6323497); Rifaximin (PubChem CID: 6436173); Tear lipocalin.

Fig. 1

Absorbance spectra of six rifamycin drugs at pH 7.3. Rifampin (15 μM) (), rifapentine (15 μM) (), rifamycin SV (15 μM) (), rifalazil (15 μM) (————), rifaximin (15 μM) (), and rifabutin (30 μM) (). Inset (A, B): Base chemical structures of all six rifamycin drugs (left) and unique groups (right). The hydroxyls at positions 1 and 4 may be oxidized to form the hydroquinone. The sole hydroxyl of the benzoxanino moiety is a candidate for oxidation on rifalazil. Hydroxyls are not available at position 1 and 4 for autoxidation for rifabutin or rifalazil.

Fig. 2

Autoxidation of rifamycin drugs at pH 7.3 after various times in phosphate buffer. (a) 1 min, (b) 20 min, (c) 40 min, (d) 80 min, (e) 120 min, (f) 180 min, (g) 24 h. Absorbance spectra of (A) rifampin (15 μM), (B) rifapentine (15 μM), (C) rifamycin SV (15 μM). (D) Mass spectrum of oxidized rifapentine shows predicted monoisotopic m/z of 825.5, 897.2 and 913.1 for the M+H, M+Na and M+K ions.

Fig. 3

A-D. Oxidation of rifamycin drugs in the presence of tear lipocalin. For comparison, autoxidation of rifamycin drugs alone (-●-), protected with tear lipocalin (100 μM) (-■-), and in the presence of the divalent ion chelator EDTA (5 mM) (-△-). (A) rifampin (15 μM), (B) rifapentine (15 μM), (C) rifamycin SV (15 μM). D-F. Addition of K₃Fe(CN)₆ to rifamycin drugs protected with tear lipocalin after 180 min at pH 7.3 in phosphate buffer. Absorbance spectra of rifamycin drugs and tear lipocalin (100 μM) after 1 min (————), 180 min (-- -- --), and 180 min with K₃Fe(CN)₆ (90 μM) (^…). (E) rifampin (15 μM), (F) rifapentine (15 μM), (G) rifamycin SV (15 μM), (H) rifalazil (15 μM).

Fig. 4

A. Dissociation constants for rifamycins. *Estimated by gel filtration experiments (profiles shown in Figure S1). B. Ligand binding curve for rifalazil and tear lipocalin determined by spectrophotometric measurement of the soluble complex. The experiment was performed at pH 7.4 in PBS-buffer. The fitting curve is indicated (————).

Fig. 5

Red shift (solvatochromism) in absorbance spectra taken at successive 2 minute intervals from A to W of rifalazil (30 μM) added to tear lipocalin (100 μM), in 10 mM sodium phosphate, pH 7.3 Inset: Kinetics of solvatochromism, baseline corrected absorbance at 635 nm plotted (black circles) over 40 minutes fits a sigmoidal function (solid line).

Fig. 6

Influence of antibiotics. Reduced versus oxidized rifamycin drugs. M. smegmatis mc²155 was incubated at pH 7.4 in PBS-buffer with rifamycin drugs (10 μg/ml) for 30 min. Thereafter 150 μl 7H9-G-TW was added and incubated for 48 h. (A) rifampin, (B) rifapentine, (C) rifamycin SV, ( Error bars indicate SD, n = 6.)