Characterization of Trypanosoma brucei dihydroorotate dehydrogenase as a possible drug target; structural, kinetic and RNAi studies.
Nucleotide biosynthesis pathways have been reported to be essential in some protozoan pathogens. Hence, we evaluated the essentiality of one enzyme in the pyrimidine biosynthetic pathway, dihydroorotate dehydrogenase (DHODH) from the eukaryotic parasite Trypanosoma brucei through gene knockdown studies. RNAi knockdown of DHODH expression in bloodstream form T. brucei did not inhibit growth in normal medium, but profoundly retarded growth in pyrimidine-depleted media or in the presence of the known pyrimidine uptake antagonist 5-fluorouracil (5-FU). These results have significant implications for the development of therapeutics to combat T. brucei infection. Specifically, a combination therapy including a T. brucei-specific DHODH inhibitor plus 5-FU may prove to be an effective therapeutic strategy. We also show that this trypanosomal enzyme is inhibited by known inhibitors of bacterial Class 1A DHODH, in distinction to the sensitivity of DHODH from human and other higher eukaryotes. This selectivity is supported by the crystal structure of the T. brucei enzyme, which is reported here at a resolution of 1.95 A. Additional research, guided by the crystal structure described herein, is needed to identify potent inhibitors of T. brucei DHODH.
Arakaki
Tracy L
TL
Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.
Buckner
Frederick S
FS
Gillespie
J Robert
JR
Malmquist
Nicholas A
NA
Phillips
Margaret A
MA
Kalyuzhniy
Oleksandr
O
Luft
Joseph R
JR
Detitta
George T
GT
Verlinde
Christophe L M J
CL
Van Voorhis
Wesley C
WC
Hol
Wim G J
WG
Merritt
Ethan A
EA
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D018360:Crystallography, X-Ray; D000090722:Dihydroorotate Dehydrogenase; D015195:Drug Design; D005472:Fluorouracil / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011743:Pyrimidines / Q000378:metabolism; D034622:RNA Interference; D017386:Sequence Homology, Amino Acid; D014346:Trypanosoma brucei brucei / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*
D000090722:Dihydroorotate Dehydrogenase; D015800:Protozoan Proteins; D011743:Pyrimidines; D044925:Oxidoreductases Acting on CH-CH Group Donors; C030986:pyrimidine; D005472:Fluorouracil