Unique mechanism of action of the thiourea drug isoxyl on Mycobacterium tuberculosis.
The thiourea isoxyl (thiocarlide; 4,4'-diisoamyloxydiphenylthiourea) is known to be an effective anti-tuberculosis drug, active against a range of multidrug-resistant strains of Mycobacterium tuberculosis and has been used clinically. Little was known of its mode of action. We now demonstrate that isoxyl results in a dose-dependent decrease in the synthesis of oleic and, consequently, tuberculostearic acid in M. tuberculosis with complete inhibition at 3 microg/ml. Synthesis of mycolic acid was also affected. The anti-bacterial effect of isoxyl was partially reversed by supplementing growth medium with oleic acid. The specificity of this inhibition pointed to a Delta9-stearoyl desaturase as the drug target. Development of a cell-free assay for Delta9-desaturase activity allowed direct demonstration of the inhibition of oleic acid synthesis by isoxyl. Interestingly, sterculic acid, a known inhibitor of Delta9-desaturases, emulated the effect of isoxyl on oleic acid synthesis but did not affect mycolic acid synthesis, demonstrating the lack of a relationship between the two effects of the drug. The three putative fatty acid desaturases in the M. tuberculosis genome, desA1, desA2, and desA3, were cloned and expressed in Mycobacterium bovis BCG. Cell-free assays and whole cell labeling demonstrated increased Delta9-desaturase activity and oleic acid synthesis only in the desA3-overexpressing strain and an increase in the minimal inhibitory concentration for isoxyl, indicating that DesA3 is the target of the drug. These results validate membrane-bound Delta9-desaturase, DesA3, as a new therapeutic target, and the thioureas as anti-tuberculosis drugs worthy of further development.
Phetsuksiri
Benjawan
B
Department of Microbiology, Pathology, and Immunology, Colorado State University, Fort Collins, Colorado 80523-1682, USA.
Jackson
Mary
M
Scherman
Hataichanok
H
McNeil
Michael
M
Besra
Gurdyal S
GS
Baulard
Alain R
AR
Slayden
Richard A
RA
DeBarber
Andrea E
AE
Barry
Clifton E
CE
Baird
Mark S
MS
Crick
Dean C
DC
Brennan
Patrick J
PJ
D000595:Amino Acid Sequence; D000995:Antitubercular Agents / Q000494:pharmacology*; D015153:Blotting, Western; D002462:Cell Membrane / Q000201:enzymology; D002474:Cell-Free System; D002849:Chromatography, Gas; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D044943:Fatty Acid Desaturases / Q000235:genetics* / Q000378:metabolism; D005227:Fatty Acids / Q000378:metabolism; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009163:Mycobacterium bovis / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000378:metabolism*; D009171:Mycolic Acids / Q000378:metabolism; D019301:Oleic Acid / Q000378:metabolism; D010670:Phenylthiourea / Q000031:analogs & derivatives* / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013229:Stearic Acids / Q000378:metabolism; D013230:Stearoyl-CoA Desaturase; D013997:Time Factors
D000995:Antitubercular Agents; D005227:Fatty Acids; D009171:Mycolic Acids; D011994:Recombinant Proteins; D013229:Stearic Acids; D019301:Oleic Acid; C100189:thiocarlide; C026082:tuberculostearic acid; D010670:Phenylthiourea; D044943:Fatty Acid Desaturases; D013230:Stearoyl-CoA Desaturase; C029627:delta-9 fatty acid desaturase