Resistance to DL-alpha-difluoromethylornithine by clinical isolates of Trypanosoma brucei rhodesiense. Role of S-adenosylmethionine.

The ornithine decarboxylase (ODC) inhibitor DL-alpha-difluoromethylornithine (DFMO) has emerged as a new treatment for West African sleeping sickness but is less effective against East African sleeping sickness. We examined uncloned clinical isolates of Trypanosoma brucei rhodesiense, agent of the disease in East Africa, which were refractory to DFMO in laboratory infections, for characteristics that would explain their resistance. None of the isolates were from patients treated with DFMO. Two isolates took up [3H]DFMO at 50-70% lower rates than drug-sensitive strains but ODC activities, Ki values for DFMO, spermidine and spermine uptake rates, polyamine content and inhibition of polyamine metabolism by DFMO were statistically (P < 0.05) similar between sensitive and refractory isolates. One cloned strain, continuously passaged in vivo under DFMO pressure and included for comparison, had > 85% lower ODC activity and up to 14-fold higher putrescine uptake rates than sensitive controls. A statistically important trend was the metabolism of S-adenosylmethionine (AdoMet): activities of AdoMet synthetase and AdoMet decarboxylase were 2- to 5-fold and 3- to 40-fold lower in resistant strains, respectively, while intracellular AdoMet pools (AdoMet + decarboxylated AdoMet) that were > 60-fold elevated in sensitive strains during DFMO treatment, increased only 9-fold in refractory isolates. The extreme elevation of the AdoMet pool in sensitive isolates from 0.7 to 44 nmol/mg protein and an intracellular pool concentration of approximately 5 mM may lead to an imbalance in methylation of proteins or other cell constituents as a consequence of DFMO action. These studies indicate that the metabolism of AdoMet is altered significantly in DFMO refractory isolates and suggest that differences in AdoMet metabolism may be responsible for increased tolerance to DFMO.

S-adenosylmethionine synthetase in bloodstream Trypanosoma brucei.

S-adenosylmethionine synthetase was studied from bloodstream forms of Trypanosoma brucei brucei, the agent of African sleeping sickness. Two isoforms of the enzyme were evident from Eadie Hofstee and Hanes-Woolf plots of varying ATP or methionine concentrations. In the range 10-250 microM the Km for methionine was 20 microM, and this changed to 200 microM for the range 0.5-5.0 mM. In the range 10-250 microM the Km for ATP was 53 microM, and this changed to 1.75 mM for the range 0.5-5.0 mM. The trypanosome enzyme had a molecular weight of 145 kDa determined by agarose gel filtration. Methionine analogs including selenomethionine, L-2-amino-4-methoxy-cis but-3-enoic acid and ethionine acted as competitive inhibitors of methionine and as weak substrates when tested in the absence of methionine with [14C]ATP. The enzyme was not inducible in procyclic trypomastigotes in vitro, and the enzyme half-life was > 6 h. T. b. brucei AdoMet synthetase was inhibited by AdoMet (Ki 240 microM). The relative insensitivity of the trypanosome enzyme to control by product inhibition indicates it is markedly different from mammalian isoforms of the enzyme which are highly sensitive to AdoMet. Since trypanosomes treated with the ornithine decarboxylase antagonist DL-alpha-difluoromethylornithine accumulate AdoMet and dcAdoMet (final concentration approximately 5 mM), this enzyme may be the critical drug target linking inhibition of polyamine synthesis to disruption of AdoMet metabolism.