A classical genetic approach to identifying the target of a drug is to screen for a drug-resistant mutant and then identify the gene responsible for the resistance phenotype. There have been several earlier reports of the isolation of sfr
mutants or resistant strain variants of protozoan organisms that are normally sensitive to the drug (25–30
). An sfr
isolate of Leishmania
displayed impaired uptake of sinefungin compared with the wild-type strain (30
). The present study shows that impaired sinefungin uptake is the predominant route to spontaneous sinefungin-resistance of S. cerevisiae
, which is caused by a variety of loss-of-function mutations in the yeast high-affinity AdoMet transporter Sam3. To our knowledge, sinefungin resistance-conferring genetic changes in a free-living organism had not been assigned previously to specific genes.
was identified initially by Rouillon et al.
) as a gene required for growth of budding yeast on sulfur-free medium containing AdoMet as the sole sulfur source. The Sam3 protein consists of 12 putative membrane-spanning segments and is classified as a member of the amino acid permease superfamily. Sam3 is capable of discriminating structurally related sulfonium derivatives of methionine, insofar as Sam3 suffices to transport AdoMet, but not S
). The genetic evidence presented here indicates that Sam3 is the principle (if not the only) yeast transporter of sinefungin. Although sinefungin has a primary amine instead of the sulfonium center found in AdoMet, the two compounds are virtually isosteric and both have a positive charge on the amine/sulfonium groups. Thus, it is sensible that Sam3 would be able to transport sinefungin. A recent study suggests that Sam3 is also able to transport other positively charged compounds such as putrescine and spermidine (17
The Sam3 mutations that confer sinefungin-resistance also result in loss-of-function in AdoMet uptake, as gauged by impaired growth on AdoMet-containing B medium. Most of the sfr
mutants isolated in the screen are functionally null for Sam3 because of premature translation stops that remove three or more of the 12 predicted membrane-spanning segments. The Sam3 missense mutations that confer sinefungin-resistance are Q126P, S171R, E213K, and S476F. Gln126, Ser171 and Ser476 are located in predicted membrane spanning segments 2, 3 and 11, respectively. Glu213 is situated between the third and fourth membrane-spanning segments. All four of these Sam3 side chains are conserved in the yeast Mmp1 permease that transports S
Our findings that sinefungin's antifungal activity is contingent on its import by the yeast AdoMet permease resonate strongly with previous studies of AdoMet and sinefungin in protozoan parasites. Sinefungin-sensitive kinetoplastid parasites such as Leishmania
can import exogenous AdoMet. Inhibition of AdoMet uptake by sinefungin and direct assays of intracellular sinefungin accumulation suggest that kinetoplastids import sinefungin via
the AdoMet uptake pathway (19
). Resistance of Leishmania
to sinefungin is correlated with decreased uptake of both sinefungin and AdoMet (30
). To our knowledge, there has been no published report of the identification of the gene encoding the putative kinetoplastid AdoMet/sinefungin transporter.
To a first approximation, it appears that the anti-infective spectrum of sinefungin correlates with the presence in the susceptible organisms of an AdoMet transport system that accepts sinefungin as cargo. The acquisition of spontaneous sinefungin-resistance by inactivating mutations of the fungal AdoMet permease is a potential obstacle to sustained clinical efficacy of a sinefungin-based drug. However, this problem is less daunting if the parasite relies on AdoMet uptake for growth or persistence in the animal host, in which case mutations in the AdoMet transporter that affect sinefungin susceptibility would simultaneously diminish the virulence of the parasite. This is a plausible scenario for the fungal pathogen Pneumocystis carinii
, which has no detectable AdoMet synthase activity and is naturally auxotrophic for exogenous AdoMet taken up by a high-affinity transporter (18
). Sinefungin inhibits Pneumocystis
growth in culture (32
The fact that the sfr phenotype in yeast is dominated by sam3 mutations confounded our initial efforts to identify an intracellular methyltransferase target of sinefungin by screening haploid strains for drug-resistance. Because the sinefungin-sensitive trait of wild-type SAM3 is dominant in the presence of a sfr sam3 allele, it might be feasible to screen in diploid strains for non-Sam3 resistance-conferring changes. However, we observed the rapid emergence of sfr colonies in the middle of the zone of inhibition when drug was spotted on a lawn of the SAM3 sam3(sfr-1) diploid strain (data not shown). We surmise that gene conversion between homologous chromosomes resulted in transfer of the resistance mutation to the previously wild-type SAM3 locus. A potentially better way to seek resistance mutations in intracellular targets is to exploit the observation that a 2 µ SAM3 plasmid sensitizes yeast to AdoMet. The high copy number of the 2 µ plasmid makes it unlikely that an inactivating mutation in the chromosomal SAM3 gene, or any one of the plasmid-borne genes, will result in resistance in the presence of an excess of wild-type SAM3 alleles. In this background, non-Sam3 resistance-conferring mutations might be identified.
The observation that wild-type SAM3
cells become sfr
upon overexpression of AdoMet synthase plus Abd1 reinforces the earlier suggestion that RNA cap methylation is a principal intracellular event targeted by the drug (8
). It is sensible that simultaneously increasing the concentration of the enzyme responsible for AdoMet production and the level of the relevant methyltransferase enzyme target should generate greater resistance than either maneuver alone.