Squalene Synthase As a Target for Chagas Disease Therapeutics

Abstract

Trypanosomatid parasites are the causative agents of many neglected tropical diseases and there is currently considerable interest in targeting endogenous sterol biosynthesis in these organisms as a route to the development of novel anti-infective drugs. Here, we report the first x-ray crystallographic structures of the enzyme squalene synthase (SQS) from a trypanosomatid parasite, Trypanosoma cruzi, the causative agent of Chagas disease. We obtained five structures of T. cruzi SQS and eight structures of human SQS with four classes of inhibitors: the substrate-analog S-thiolo-farnesyl diphosphate, the quinuclidines E5700 and ER119884, several lipophilic bisphosphonates, and the thiocyanate WC-9, with the structures of the two very potent quinuclidines suggesting strategies for selective inhibitor development. We also show that the lipophilic bisphosphonates have low nM activity against T. cruzi and inhibit endogenous sterol biosynthesis and that E5700 acts synergistically with the azole drug, posaconazole. The determination of the structures of trypanosomatid and human SQS enzymes with a diverse set of inhibitors active in cells provides insights into SQS inhibition, of interest in the context of the development of drugs against Chagas disease.

Potent In Vitro Antiproliferative Synergism of Combinations of Ergosterol Biosynthesis Inhibitors against Leishmania amazonensis

“…The remarkable in vitro antiproliferative synergism against both proliferative stages of L. amazonensis of combinations of ITZ and POSA with E5700 observed in this work, and its correlate with the effects of the drugs on promastigote free sterol composition, confirms the notion that drugs acting at sequential steps of a metabolic pathway should have synergistic effects (25). We have recently reported similar synergistic effects of combinations of POSA and E5700 against T. cruzi intracellular amastigotes (53), but the FIC values obtained in the current study are, to the best of our knowledge, the lowest ever reported for the effects of drug combinations against any trypanosomatid parasite. Comparing the MIC and IC 50 s with those of miltefosine, a standard drug used to treat leishmaniasis, the combinations were very potent, as they reduced significantly the growth of L. amazonensis at subnanomolar concentrations.…”

SQ109, a New Drug Lead for Chagas Disease

“…cruzi CL strain overexpressing a tdTomato red fluorescent protein, performed basically as described earlier (33). To evaluate antiproliferative synergism against intracellular amastigotes, fractional inhibitory concentration indices (FICIs) were calculated as described by Hallander et al (34).…”

“…In addition, in previous work, we found that SQ109 was an inhibitor of the bacterial enzyme dehydrosqualene synthase, a squalene synthase homolog (26,27), and we reported (25) two X-ray crystallographic structures of SQ109 bound to S. aureus dehydrosqualene synthase (SaCrtM; PDB IDs 4EA1 and 4EA2). We also found that SQ109 was a modest (IC 50 , ϳ100 M) inhibitor of T. cruzi squalene synthase (25), whose structure we recently reported (33). Since SQS inhibition is of interest as a T. cruzi drug target, we next sought to determine how it binds, since clearly, as with amiodarone, targeting both sterol biosynthesis and the proton motive force (PMF) should lead to a potent inhibition of cell growth.…”

“…Moreover, the rapid (Ͻ24-h) onset of growth arrest by SQ109 appears incompatible with the notion that its primary mechanism of action is the blockade of de novo synthesis of 24-alkyl sterols, since the results of many studies indicate that the full proliferation arrest is associated only with the complete disappearance of mature (24-alkyl and 14-desmethyl) endogenous sterols. This means that for T. cruzi, whose generation time is 24 to 30 h, complete growth inhibition is observed only after 72 to 120 h of drug exposure, while for Leishmania spp., for which the generation time is 12 to 18 h, complete growth arrest in the presence of optimal drug concentration is seen at 36 to 72 h (33,(42)(43)(44)(45)(46)(47)(48)(49)(50). For the same reason, T. cruzi trypomastigotes, which are nonproliferative cells, are refractory to bona fide ergosterol biosynthesis inhibitors such as ketoconazole, posaconazole, terbinafine, azasterols, and aryl-quinuclidines in the nanomolar range of concentrations at which they are active against epimastigotes and intracellular amastigotes.…”

In Vitro and In Vivo Investigation of the Inhibition of Trypanosoma brucei Cell Growth by Lipophilic Bisphosphonates

“…For example, the T. brucei genome contains a sequence that appears to code for a long-chain prenyl synthase that is highly homologous to both Staphylococcus aureus heptaprenyl diphosphate synthase and TbFPPS, suggesting a possible target involved in quinone biosynthesis. Squalene synthase (36) is another potential target, since ergosterol (at low levels) is required for cell proliferation (8) and a T. cruzi squalene synthase is inhibited by lipophilic bisphosphonates (36). Also, some lipophilic bisphosphonates tar- get farnesyl protein transferases (37).…”