Discovery of Antiamebic Compounds That Inhibit Cysteine Synthase From the Enteric Parasitic Protist Entamoeba histolytica by Screening of Microbial Secondary Metabolites

Abstract

Amebiasis is caused by infection with the protozoan parasite Entamoeba histolytica. Although metronidazole has been a drug of choice against amebiasis for decades, it shows side effects and low efficacy against asymptomatic cyst carriers. In addition, metronidazole resistance has been documented for bacteria and protozoa that share its targets, anaerobic energy metabolism. Therefore, drugs with new mode of action or targets are urgently needed. L-cysteine is the major thiol and an essential amino acid for proliferation and anti-oxidative defense of E. histolytica trophozoites. E. histolytica possesses the de novo L-cysteine biosynthetic pathway, consisting of two reactions catalyzed by serine acetyltransferase and cysteine synthase (CS, O-acetylserine sulfhydrylase). As the pathway is missing in humans, it is considered to be a rational drug target against amebiasis. In this study, we established a protocol to screen both a library of structurally known compounds and microbial culture extracts to discover compounds that target de novo cysteine biosynthesis of E. histolytica. The new screening system allowed us to identify the compounds that differentially affect the growth of the trophozoites in the cysteine-deprived medium compared to the cysteine-containing medium. A total of 431 structurally defined compounds of the Kitasato Natural Products Library and 6,900 microbial culture broth extracts were screened on the system described above. Five compounds, aspochalasin B, chaetoglobosin A, prochaetoglobosin III, cerulenin, and deoxyfrenolicin, from the Kitasato Natural Products Library, showed differential antiamebic activities in the cysteine-deprived medium when compared to the growth in the cysteine-containing medium. The selectivity of three cytochalasans apparently depends on their structural instability. Eleven microbial extracts showed selective antiamebic activities, and one fungal secondary metabolite, pencolide, was isolated. Pencolide showed cysteine deprivation-dependent antiamebic activity (7.6 times lower IC50 in the absence of cysteine than that in the presence of cysteine), although the IC50 value in the cysteine-deprived medium was rather high (283 μM). Pencolide also showed inhibitory activity against both CS1 and CS3 isoenzymes with comparable IC50 values (233 and 217 μM, respectively). These results indicated that antiamebic activity of pencolide is attributable to inhibition of CS. Cytotoxicity of pencolide was 6.7 times weaker against mammalian MRC-5 cell line than E. histotytica. Pencolide has the maleimide structure, which is easily attacked by Michael donors including the thiol moiety of cysteine. The cysteine-adducts of pencolide were detected by mass spectrometric analysis as predicted. As CS inhibition by the pencolide adducts was weak and their IC50 values to CS was comparable to that to the parasite in the cysteine-containing medium, the cysteine-adducts of pencolide likely contribute to toxicity of pencolide to the parasite in the cysteine-rich conditions. However, we cannot exclude a ...

Bioactivities and Future Perspectives of Chaetoglobosins

“…Compound 36 showed antiacetylcholinesterase activity and weak anticoagulant activity with PT at 16.8 s [10]. Mori et al also found compounds 1 and 51 showed antiamebic activities in the cysteine-deprived medium, in comparable to in the cysteine-containing medium [24]. In addition, compounds 66, 67, 68, 71, and 72 showed significant anti-HIV activities, with EC 50 values ranging from 0.11 to 0.55 μmol/L and selectivity index values ranging from 12.33 to 75.42 [11].…”

Combination of SAXS and Protein Painting Discloses the Three-Dimensional Organization of the Bacterial Cysteine Synthase Complex, a Potential Target for Enhancers of Antibiotic Action

“…Only four of these enzymes have been the target of significant medicinal chemistry efforts, either in enteric bacteria or in Mycobacterium tuberculosis : O -acetylserine sulfhydrylase isozymes A and B (CysK/CysM) [35,36,37,40,41], phosphoadenosine phosphosulphate reductase (CysH) [38], and serine acetyltransferase (CysE) [42]. Although medicinal chemistry campaigns have successfully led to the discovery of very potent enzyme inhibitors [17,29], the translation of these inhibitors to molecules with antibacterial activity requires more effort [37,38,40]. One important aspect of the cysteine metabolism is the ability of some enzymes of the pathway, namely CysK, CysE, ATP sulfurylase (CysD) and CysD-associated GTPase (CysN), to form complexes.…”

Revisiting Drug Development Against the Neglected Tropical Disease, Amebiasis

“…Although diacetyl kinamycin C and nanaomycin A have been found to be good inhibitors of CS, their use is not feasible for the treatment of amebiasis owing to their toxicity ( Mori et al., 2015 ). However, pencolide is a promising drug candidate with very low toxicity and better anitamoebic activity ( Mori et al., 2018 ). Hence, inhibiting the synthesis pathway of the endogenous reducing agent L-cysteine would hamper the survival of the parasite in the host gut.…”