1. Volume 13, Issue 3, Pages 297-307 (March 2006)
Dual Inhibition of Mycobacterial Fatty Acid Biosynthesis and Degradation by 2-Alkynoic Acids 
Hector R. Morbidoni, Catherine Vilchèze, Laurent Kremer, Robert Bittman, James C. Sacchettini, William R. Jacobs 
Chemistry & Biology 
Volume 13, Issue 3, Pages (March 2006)
DOI: /j.chembiol
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2. Figure 1
Effect of Hexadecynoic and Octadecynoic Acids on the Growth of M. smegmatis
(A and B) Early-log phase cultures of (A) M. bovis BCG and (B) M. smegmatis mc2155 were treated with DMSO solutions of 2-HA/OA (0.5–50 μg/ml) and 3-HA/OA (50 μg/ml) for M. smegmatis. The addition of DMSO did not affect culture viability. Aliquots were taken, diluted, and plated. CFUs were counted after 4 days of incubation for M. smegmatis or 3 weeks of incubation for M. bovis BCG at 37°C. For clarity, the data obtained for M. smegmatis treated with 2-HA (10 μg/ml and 25 μg/ml) and 2-OA (10 μg/ml) are not shown since they overlap with 2-OA (25 μg/ml). The data in (B) are representative of three independent experiments.
Chemistry & Biology  , DOI: ( /j.chembiol )
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3. Figure 2 2-HA and 2-OA Induce a KasA-Containing Complex
Mid-log phase cultures of M. smegmatis were treated with increasing concentrations of 2-HA (2.5–50 μg/ml) and 2-OA (1–50 μg/ml) for 30 min. INH (5 μg/ml) was used as a control for InhA inhibition. Crude lysates were loaded on a 12% acrylamide gel and transferred to a membrane for immunoblot analysis. For the detection of unbound KasA and the KasA-containing complex, the membranes were probed with rat anti-KasA antibodies and then incubated with anti-rat antibodies conjugated to alkaline phosphatase prior to detection.
Chemistry & Biology  , DOI: ( /j.chembiol )
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4. Figure 3
Dose-Response Effects of 2-HA, 3-HA, 2-OA, and 3-KHA on Fatty Acid and Mycolic Acid Biosynthesis
(A) The effect of 2-HA on the incorporation of [1-14C]-acetate was assayed by labeling M. smegmatis in the presence of increasing concentrations of 2-HA (10 μg/ml [lanes 1, 4, 7, and 10]; 25 μg/ml [lanes 2, 5, 8, and 11], or 50 μg/ml [lanes 3, 6, 9, and 12]) or in the absence of drug (lane 13). 10% of total counts were loaded in each lane.
(B) Mid-log phase cultures of M. bovis BCG were treated with 2-HA or 2-OA (0, 2.5, 5, and 10 μg/ml) and labeled with [1-14C]-acetate.
(C) Mid-log phase M. smegmatis cultures were treated with low concentrations of 2-HA or 2-OA and labeled with [1-14C]-acetate.
(D) Log-phase cultures of M. smegmatis were treated with 3-KHA (50 μg/ml, lanes 1, 6, and 11), 3-HA (10 μg/ml, lanes 2, 7, and 12; or 25 μg/ml, lanes 3, 8, and 13), or a combination of 3-KHA (40 μg/ml) and 3-HA (10 μg/ml [lanes 4, 9, and 14] or 25 μg/ml [lanes 5, 10, and 15]) and labeled with [1-14C]-acetate. Fatty acid methyl esters (FAMEs) and mycolic acid methyl esters (MAMEs) were extracted, analyzed, and detected by autoradiography as described in Experimental Procedures.
Chemistry & Biology  , DOI: ( /j.chembiol )
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5. Figure 4
2-HA Is Metabolized into Active Species that Inhibit Fatty Acid Biosynthesis
(A and B) M. smegmatis cultures treated with 0, 10, 25, or 50 μg/ml 2-HA were labeled with (A) [1-14C]-acetate or (B) [1-14C]-2-HA. [1-14C]-Labeled fatty acids were extracted, derivatized to the corresponding p-bromophenacyl esters, and analyzed by HPLC. The identification code and retention times for the principal p-bromophenacyl esters are as follows: (a) 10.9 min, C14:0; (b) 11.3 min, C16:1 cis-Δ9; (c) 18.0 min, C16:0; (d) 19.5 min, C18:1 cis-Δ9; (e) 23.6 min, C18:0; (f) 24.1 min, 10-methyl-C18:0; (g) 25.2 min, C20:0; (h) 27.4 min, C 22:0; (i) 29.8 min, C24:0; (j) 26.5 min, C22:1 trans-Δ2; (k) 28.8 min, C24:1 trans-Δ2; (l) 7.3 min, 3-hydroxyhexadecanoic acid; (m) 13.5 min, C16:1 trans-Δ3; (n) 7.5 min, 3-KHA; (o) 9.0 min, 3-HA; (x) 4.9 min, an unidentified compound.
Chemistry & Biology  , DOI: ( /j.chembiol )
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6. Figure 5 Metabolism of 2-HA Occurs via a Functional FASI System
(A and B) [1-14C]-Fatty acids obtained from (A) labeling M. smegmatis cultures with [1-14C]-2-HA, [1-14C]-palmitic acid, [1-14C]-oleic acid, [1-14C]-linoleic acid, or [1-14C]-linolenic acid or (B) after treatment of M. smegmatis cultures with cerulenin (5 μg/ml), 5-Cl-PZA (100 μg/ml), or TLM (50 μg/ml), followed by labeling with [1-14C]-2-HA, were analyzed by HPLC.
Chemistry & Biology  , DOI: ( /j.chembiol )
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7. Figure 6 Metabolism of 2-HA in M. smegmatis
The proposed pathway is based on the metabolites derived from [1-14C]-2-HA and detected by HPLC. The identification key for the compounds shown is as follows: (1) 2-HA, (2) 2-hexadecynoyl-CoA, (3) 3-ketohexadecanoyl-CoA, (4) 3-hydroxyhexadecanoyl-CoA, (5) 2-trans-hexadecenoyl-CoA, (6) hexadecanoyl-CoA, (7) hexadecanoyl-ACP, (8) 2,3-hexadecadienoyl-CoA, (9) 3-hexadecynoyl-CoA, (10) 3-hexadecenoyl-CoA.
Chemistry & Biology  , DOI: ( /j.chembiol )
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