Rational design of nitrofuran derivatives: Synthesis and valuation as inhibitors of Trypanosoma cruzi trypanothione reductase.

The rational design and synthesis of a series of 5-nitro-2-furoic acid analogues are presented. The trypanocidal activity against epimastigote forms of Trypanosoma cruzi and the toxic effects on human HeLa cells were tested. Between all synthetic compounds, three of thirteen had an IC50 value in the range of Nfx, but compound 13 exhibited an improved effect with an IC50 of 1.0 ± 0.1 µM and a selective index of 70 in its toxicity against HeLa cells. We analyzed the activity of compounds 8, 12 and 13 to interfere in the central redox metabolic pathway in trypanosomatids, which is dependent of reduced trypanothione as the major pivotal thiol. The three compounds behaved as better inhibitors of trypanothione reductase than Nfx (Ki values of 118 µM, 61 µM and 68 µM for 8, 12 and 13, respectively, compared with 245 µM for Nfx), all following an uncompetitive enzyme inhibition pattern. Docking analysis predicted a binding of inhibitors to the enzyme-substrate complex with binding energy calculated in-silico that supports such molecular interaction.

Effects of tri-iodothyronine on alternative splicing events in the coding region of cytochrome P450 aromatase in immature rat Sertoli cells.

Transient postnatal hypothyroidism in male rats induces a prolonged proliferation of immature Sertoli cells. This change in Sertoli cell replication at young ages is coincident with enhanced and prolonged aromatase activity that leads to a marked increase in the conversion of androgens into estrogens. Both events are drastically inhibited by tri-iodothyronine (T(3)) replacement either in vivo or in vitro. This study, after the immunolocalization of aromatase in cultured rat Sertoli cells, examined the effects elicited by T(3) on this enzyme, by simultaneously investigating three functional levels of aromatase: mRNA expression, protein content, and enzymatic activity. The immunolocalization of cytochrome P450 aromatase (P450 arom) was shown in the cytoplasm of cultured Sertoli cells from 15- and 21-day-old rats. Western blot analysis revealed an enhancement of aromatase protein content upon stimulation with N(6),2'-O-dibutyryladenosine-3':5'-cyclic monophosphate ((Bu)(2)cAMP) that was clearly down-regulated by T(3). The presence of a functional P450 arom protein in purified Sertoli cells was confirmed by the measurement of [(3)H]H(2)O released after incubation with [1 beta-(3)H]androst-4-ene-3,17-dione. With 100 nM T3, a decrease in both P450 arom mRNA levels and aromatase activity was observed. The aromatase enzymatic activity was strongly stimulated by (Bu)(2)cAMP and markedly down-regulated by T(3). In contrast, the strong increase in aromatase mRNA upon (Bu)(2)cAMP stimulation was apparently unaffected by T(3) administration. This paper shows how the identification of an altered transcript induced by T(3) coding for putative truncated and inactive aromatase protein might explain such a decrease in aromatase activity in T(3)-treated cells. On the basis of these results, it is concluded that at least two mechanisms could be involved in the down-regulatory effect of T(3) on aromatase activity in prepuberal Sertoli cells. The first mechanism is linked to a possible direct modulatory role for T(3) in the regulation of the aromatase promoter, whilst the second one is represented by the induction of altered transcripts coding for truncated and inactive aromatase proteins.

Aromatase expression in prepuberal Sertoli cells: effect of thyroid hormone.

Aromatase activity has recently been assumed as a Sertoli cell functional maturation marker since it is maximally expressed in prepuberal age then it dramatically decreases at puberty and is virtually absent in adult age. Neonatal hypothyroidism is associated with a prolonged proliferation of Sertoli cells. This immature stage persists concomitantly with a dramatic enhancement of aromatase activity reversed by triiodothyronine (T3) either in vivo or in vitro administration. Therefore, in the present study, after immunolocalisation of aromatase in the cytoplasm of cultured Sertoli cells as well as in testis section, we investigate the regulatory effects of T3 in the same cells just at the age when aromatase activity is reported to be maximally expressed. In this aim, the effects of thyroid hormone have been evaluated in 2-weeks-old rats, in basal condition and upon stimulation with dibutyryl cyclic AMP [(Bu)(2)cAMP] by simultaneously analysing three functional levels of aromatase, mRNA expression; protein content; enzymatic activity. Western-blot analysis of Sertoli cell extracts revealed a protein, which co-migrated with a 55 kDa protein detected in human placenta used a positive control. The presence of a functional P450 aromatase protein in purified Sertoli cells was confirmed by the measurement [3H]H(2)O released after incubation with [1beta-(3)H]androst-4-3,17-dione. At the dose used, T3 down-regulates basal aromatase activity, while aromatase mRNA expression was apparently not inhibited. It is noteworthy that aromatase content pattern evaluated by Western blot analysis did not tightly parallel the aromatase activity pattern which clearly displays the inhibitory effects of T3, in basal condition ad upon (Bu)(2)cAMP stimulation, simulating FSH stimulation. The detection of mRNA altered transcript coding for putative protein lacking both aromatic and heme-binding regions upon T3 treatment and unable to convert androgens into estrogens, provides a reasonable explanation for the observed discrepancies between aromatase protein pattern, P450arom mRNA levels and aromatase activity. The authors conclude that although the altered transcript induced by prolonged exposure to T3 is a mechanism by which T3 may down regulate aromatase activity, it cannot be ruled out a direct effect of this hormone at the transcription levels since a recognisable emisite for potential TR(s) binding is located in the promoter region of aromatase gene. Thus a further investigation on T3 modulator role on aromatase gene promoter should be pursued even utilising higher doses of T3.