Staphylococcus aureus produces a golden carotenoid virulence factor called staphyloxanthin (STX), and we report here the inhibition of the enzyme, dehydrosqualene synthase (CrtM), responsible for the first committed step in STX biosynthesis. The most active compounds are halogen-substituted phosphonosulfonates, with Ki values as low as 5 nM against the enzyme and IC50 values for STX inhibition in S. aureus as low as 11 nM. There is, however, only a poor correlation (R2 = 0.27) between enzyme and cell pIC50 (= −log10 IC50) values. The ability to predict cell from enzyme data improves considerably (to R2 = 0.72) with addition of two more descriptors. We also investigated the activity of these compounds against human squalene synthase (SQS), as a counterscreen, finding several potent STX biosynthesis inhibitors with essentially no squalene synthase activity. These results open up the way to developing potent and selective inhibitors of an important virulence factor in S. aureus, a major human pathogen.

A series of azasterol derivatives, designed as potential inhibitors of the ⌬ 24 -sterol methyltransferase enzyme (24-SMT), were synthesized and evaluated for their activities against parasitic protozoa. Values in the nanomolar range were obtained for 50% effective dose against the Trypanosoma brucei subsp. rhodesiense bloodstream form cultured in vitro. In order to investigate the mode of action, Trypanosoma brucei subsp. brucei 24-SMT was cloned and overexpressed and compounds were assayed for inhibitory activity. None of the inhibitors tested appeared to be active against the enzyme. Sterol composition analysis showed that only cholestane type sterols are present in membranes of bloodstream forms while ergosterol is a major component of procyclic sterol extracts. Interestingly, Northern blot analysis showed the presence of 24-SMT mRNA in both the procyclic and the bloodstream forms of the parasite, although levels of mRNA were threefold lower in the latter. Likewise, Western blot analysis and activity determinations evidenced the existence of active enzyme in both forms of the parasite. We conclude that the designed compounds act at sites other than 24-SMT in Trypanosoma brucei.

This paper describes the design and evaluation of novel azasterols as potential compounds for the treatment of leishmaniasis and other diseases caused by trypanosomatid parasites. Azasterols are a known class of (S)-adenosyl-L-methionine: ⌬ 24 -sterol methyltransferase(24-SMT) inhibitors in fungi, plants, and some parasitic protozoa. The compounds prepared showed activity at micromolar and nanomolar concentrations when tested against Leishmania spp. and Trypanosoma spp. The enzymatic and sterol composition studies indicated that the most active compounds acted by inhibiting 24-SMT. The role of the free hydroxyl group at position 3 of the sterol nucleus was also probed. When an acetate was attached to the 3␤-OH, the compounds did not inhibit the enzyme but had an effect on parasite growth and the levels of sterols in the parasite, suggesting that the acetate group was removed in the organism. Thus, an acetate group on the 3␤-OH may have application as a prodrug. However, there may be an additional mode(s) of action for these acetate derivatives. These compounds were shown to have ultrastructural effects on Leishmania amazonensis promastigote membranes, including the plasma membrane, the mitochondrial membrane, and the endoplasmic reticulum. The compounds were also found to be active against the bloodstream form (trypomastigotes) of Trypanosoma brucei rhodesiense, a causative agent of African trypanosomiasis.