Abstract
Synthetic iridium-, rhodium-, and ruthenium-amino acid complexes with hydrophobic l-amino acids have antibiotic activity against Mycobacterium spp., including Mycobacterium bovis BCG and the rapidly growing species Mycobacterium abscessus and Mycobacterium chelonae. Concentrations of transition metal-amino acid complexes demonstrating hemolysis or cytotoxicity were 10- to 25-fold higher than were the MICs.
TEXT
A number of species of the environmental nontuberculous mycobacteria (NTM) are opportunistic human pathogens (1). There is considerable accumulated evidence that the prevalence and incidence of nontuberculous mycobacterial infection are increasing (2–5). The major NTM representatives include the slowly growing species Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacterium xenopi, and Mycobacterium malmoense (1) and the rapidly growing species Mycobacterium abscessus, Mycobacterium chelonae, and Mycobacterium fortuitum (6). A number of species, including Mycobacterium marinum (7), Mycobacterium ulcerans (8), and Mycobacterium haemophilum, cause dermal and subcutaneous infections in humans and animals (9). Because of the paucity of effective antimycobacterial antibiotics and relative antibiotic resistance of the mycobacteria, treatment of these infections is problematic (10). Thus, there continues to be a need for both systemic and topical antimycobacterial agents.
Transition metals, in combination with a variety of ligands, have been shown to exhibit cytotoxic or antibiotic activity (11). The transition metals, primarily ruthenium (Ru) (12) and platinum (13), have been linked with a variety of ligands with various degrees of cytotoxic or antibiotic activity, and antimycobacterial activity has been found with ruthenium complexes (14). To date, there has been no significant systematic study designed to elucidate the role of the transition metal (e.g., rhodium, iridium, and ruthenium) or ligand in antibiotic activity. Here we report the MICs, minimal bactericidal concentrations (MBCs), and hemolytic and cytotoxic activities of a group of 41 transition metal-amino acid complexes against strains of Mycobacterium.
The structures of the 4 classes of transition metal-α-amino acid complexes are illustrated in Fig. 1. Compounds of classes 1 and 2 are cyclopentadienyls (Cp*), with class 1 having an α-amino acid complexed with iridium (Ir) and class 2 an α-amino acid complexed with rhodium (Rh). Class 3 compounds are paracymenes, with the α-amino acid complexed with ruthenium (Ru). Class 4 compounds are cyclooctadienes (COD) with the α-amino acid complexed to rhodium. Separate publications detailing syntheses, characterizations, and crystal structures of the tested compounds either have been submitted or are in preparation. In all cases, a π-complexed metal chloride dimer is synthesized by the reaction with the π ligand (pentamethylcylopentadiene, phellandrene, or 1,5-cycloctadiene). Then, an amino acid is allowed to react with the π-complexed metal chloride dimer to form the final product. This simple, high-yield, two-step process is exemplified in Fig. 2 for the (η5-pentamethylcycylopentadienyl)(aminoacidato)chloroiridium complexes.
Fig 1.
Structure of transition metal-amino acid complexes. 1, (η5-pentamethylcyclopentadienyl)(aminoacidato)chloroiridium; 2, (η5-pentamethylcyclopentadienyl)(aminoacidato)chlororhodium; 3, (p-cymene)(aminoacidato)chlororuthenium; 4, (η4-1,5-cyclooctadiene)(aminoacidato)rhodium.
Fig 2.
Example of a synthetic scheme for the formation of the tested complexes.
A transition metal derivate of ethambutol was synthesized using the p-cymene-chlororuthenium starting material to form a complex that was tested against the mycobacteria. This was to determine what effect the coordination to the metal might have on the activity of a known antibacterial agent with activity against tuberculosis.
M. abscessus strain AAy-P-1, M. avium strain A5, M. intracellulare strain 1406T, M. chelonae strain EO-P-1, M. marinum strain ATCC 927, M. smegmatis strain VT 307, and M. bovis BCG strain ATCC 35745 (Connaught) were used in the study (15). The strains were grown in stages from a single isolated colony in 2 ml, then 1 ml of that culture into 10 ml, and finally in 50 ml of Middlebrook 7H9 (M7H9) broth (BD, Sparks, MD) containing 0.5% glycerol and 10% oleic acid–albumin and incubated with aeration (60 rpm) at 37°C or 30°C (M. marinum only) to the mid-log phase (3 to 7 days). MICs and MBCs of compounds dissolved in M7H9 broth medium were measured by broth microdilution in 96-well microtiter plates. A 2-fold dilution series of each compound in 50 μl of M7H9 was prepared and inoculated with 50 μl of a 1,000-fold dilution of each mid-log-phase mycobacterial culture (105 CFU/ml), plates were sealed and incubated at 37°C or 30°C, and turbidity (absorbance, 580 nm) was measured after 7 days incubation. The MIC was defined as the lowest concentration of compound completely inhibiting growth. MBC was measured by spreading 10 μl of the content of the wells on M7H10 agar, and the MBC was defined as the lowest concentration resulting in 99.9% reduced CFU/ml compared to the inoculum density. Hemolysis measurements were performed as described by Maisuria et al. (16). Cytotoxicity measurements of the transition metal-amino acid complexes were determined using Vero cells and the CellTiter 96 One Solution Cell Proliferation assay (Promega Corp., Madison, WI).
All 42 compounds were soluble in aqueous solution (lowest concentration > 3 mg/ml), and they did not decay or form precipitates. The MICs (Table 1) demonstrate that hydrophobic α-amino acids were more active than hydrophilic α-amino acids and that transition metals complexed with d-amino acids were inactive. In contrast to the cyclopentadienyl ring complexes with iridium or rhodium (classes 1 and 2), the paracymene-ruthenium complexes (class 3) lacked antimycobacterial activity, and the cyclooctadiene-rhodium complexes (class 4) had modest antimycobacterial activity (Table 1). Complexes 1 (l-pro), 1 (l-phe), 1 and 2 (l-val), and 1, 2, and 4 (l-phengly) were active against the rapidly growing mycobacteria (Table 1). Ethambutol complexed with ruthenium 3 (EMB) was weakly antimycobacterial for all three rapidly growing mycobacterial strains (Table 1). Along with a hydrophobic l-amino acid, the cyclopentadienyl ring appears to be essential for strong antimycobacterial activity. Minimal bactericidal concentrations (MBCs) were equal to the MICs for all the compounds and for the most active antimycobacterial transition metal-α-amino acid complexes, and the cytotoxic and hemolytic concentrations (>250 mg/ml) were 10-fold higher than the MIC/MBC values. Demonstration of broad-spectrum antimycobacterial activity of the transition metal-α-amino acid complexes in the absence of hemolytic and cytotoxic activity suggests that these compounds offer promise for their use in treating mycobacterial disease.
Table 1.
MICs of transition metal-amino acid complexes against mycobacterial strains
| Compounda | MIC (mg/ml) |
||||||
|---|---|---|---|---|---|---|---|
| M. smegmatis | M. avium | M. intracellulare | M. abscessus | M. marinum | M. bovis | M. chelonae | |
| 1 (l-gly) | 0.061 | >0.250 | >0.250 | 0.125 | >0.250 | NDb | 0.125 |
| 1 (l-pro) | 0.010 | 0.125 | 0.061 | 0.015 | >0.250 | 0.015 | 0.015 |
| 1 (l-ala) | 0.015 | >0.250 | 0.061 | 0.031 | >0.250 | 0.031 | 0.061 |
| 1 (l-phe) | 0.010 | >0.250 | 0.061 | 0.061 | >0.250 | 0.015 | 0.015 |
| 1 (l-phengly) | 0.005 | >0.250 | 0.015 | 0.031 | >0.250 | 0.010 | 0.010 |
| 1 (l-val) | 0.017 | >0.250 | >0.250 | 0.061 | >0.250 | 0.031 | 0.031 |
| 1 (l-ser) | >0.250 | >0.250 | ND | ND | ND | ND | ND |
| 1 (l-gln) | 0.061 | >0.250 | ND | ND | ND | ND | ND |
| 1 (d-val) | >0.250 | >0.250 | ND | ND | ND | ND | ND |
| 1 (d-pro) | >0.250 | >0.250 | >0.250 | >0.250 | >0.250 | >0.250 | >0.250 |
| 1 (l-leu) | 0.032 | >0.250 | 0.032 | 0.032 | >0.250 | ND | 0.032 |
| 1 (l-iso) | 0.015 | >0.250 | 0.032 | 0.032 | >0.250 | 0.015 | 0.032 |
| 1 (l-hyp) | >0.250 | >0.250 | ND | ND | ND | ND | ND |
| 1 (l-N-methylgly) | >0.250 | >0.250 | ND | ND | ND | ND | ND |
| 1 (l-N-methylpro) | >0.250 | >0.250 | ND | ND | ND | ND | ND |
| 2 (l-gly) | 0.061 | >0.250 | >0.250 | 0.061 | >0.250 | ND | 0.125 |
| 2 (l-pro) | 0.009 | >0.250 | 0.061 | 0.012 | >0.250 | 0.012 | 0.012 |
| 2 (l-ala) | 0.015 | >0.250 | 0.061 | 0.031 | >0.250 | 0.025 | 0.061 |
| 2 (l-phe) | 0.009 | >0.250 | 0.061 | 0.031 | >0.250 | 0.012 | 0.031 |
| 2 (l-phengly) | 0.007 | >0.250 | 0.015 | 0.015 | >0.250 | 0.007 | 0.015 |
| 2 (l-val) | 0.015 | >0.250 | >0.250 | 0.061 | >0.250 | 0.031 | 0.061 |
| 2 (l-ser) | >0.250 | >0.250 | >0.250 | >0.250 | >0.250 | ND | >0.250 |
| 2 (l-gln) | 0.032 | >0.250 | 0.032 | 0.032 | >0.250 | ND | 0.032 |
| 2 (d-val) | >0.250 | >0.250 | >0.250 | >0.250 | >0.250 | ND | >0.250 |
| 2 (d-pro) | >0.250 | >0.250 | >0.250 | >0.250 | >0.250 | ND | >0.250 |
| 2 (d-leu) | 0.021 | >0.250 | 0.061 | 0.061 | >0.250 | ND | 0.031 |
| 2 (l-iso) | 0.010 | >0.250 | 0.061 | 0.031 | >0.250 | ND | 0.031 |
| 2 (l-hyp) | >0.250 | >0.250 | >0.250 | >0.250 | >0.250 | ND | >0.250 |
| 2 (l-N-methylgly) | >0.250 | >0.250 | >0.250 | >0.250 | >0.250 | ND | >0.250 |
| 2 (l-N-methylpro) | >0.250 | >0.250 | >0.250 | >0.250 | >0.250 | ND | >0.250 |
| 3 (l-gly) | >0.250 | >0.250 | >0.250 | >0.250 | >0.250 | >0.250 | >0.250 |
| 3 (l-phe) | >0.250 | >0.250 | >0.250 | >0.250 | >0.250 | >0.250 | >0.250 |
| 3 (l-ala) | >0.250 | >0.250 | >0.250 | >0.250 | >0.250 | >0.250 | >0.250 |
| 3 (l-ser) | >0.250 | >0.250 | >0.250 | >0.250 | >0.250 | >0.250 | >0.250 |
| 3 (en) | 0.125 | >0.250 | 0.061 | 0.061 | >0.251 | ND | 0.061 |
| 3 (l-methyl en) | >0.250 | >0.250 | >0.250 | >0.250 | >0.250 | >0.250 | >0.250 |
| 3 (N,N-methyl en) | >0.250 | >0.250 | >0.250 | >0.250 | >0.250 | >0.250 | >0.250 |
| 3 (EMB) | 0.061 | >0.250 | >0.250 | 0.061 | >0.250 | ND | 0.061 |
| 4 (l-gly) | 0.125 | 0.061 | >0.250 | 0.061 | ND | ND | 0.125 |
| 4 (l-phenylgly) | 0.061 | 0.032 | >0.250 | 0.031 | ND | ND | 0.031 |
| 4 (l-val) | 0.125 | 0.125 | >0.250 | >0.250 | ND | ND | 0.061 |
| 4 (l-ala) | >0.250 | 0.125 | >0.250 | >0.250 | ND | ND | 0.061 |
ACKNOWLEDGMENTS
We acknowledge the guidance and technical assistance of Myra D. Williams with the preparation of cultures, and measurements of MICs, MBCs, hemolysis, and cytotoxicity and Alex Mai with synthesis of compounds and measurement of MICs.
Footnotes
Published ahead of print 6 May 2013
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