Inhibitory effects of (+)-spectaline and iso-6-spectaline from Senna spectabilis on the growth and ultrastructure of human-infective species Trypanosoma brucei rhodesiense bloodstream form.

In our ongoing work searching for new trypanocidal lead compounds from Malaysian plants, two known piperidine alkaloids (+)-spectaline (1) and iso-6-spectaline (2) were isolated from the leaves of Senna spectabilis (sin. Cassia spectabilis). Analysis of the 1H and 13C NMR spectra showed that 1 and 2 presented analytical and spectroscopic data in full agreement with those published in the literature. All compounds were screened in vitro against Trypanosoma brucei rhodesiense in comparison to the standard drug pentamidine. Compound 1 and 2 inhibited growth of T. b. rhodesiense with an IC50 value of 0.41 ± 0.01 µM and 0.71 ± 0.01 µM, without toxic effect on L6 cells with associated a selectivity index of 134.92 and 123.74, respectively. These data show that piperidine alkaloids constitute a class of natural products that feature a broad spectrum of biological activities, and are potential templates for the development of new trypanocidal drugs. To our knowledge, the compounds are being reported for the first time to have inhibitory effects on T. b. rhodesiense. The ultrastructural alterations in the trypanosome induced by 1 and 2, leading to programmed cell death were characterized using electron microscopy. These alterations include wrinkling of the trypanosome surface, formation of autophagic vacuoles, disorganization of kinetoplast, and swelling of the mitochondria. These findings evidence a possible autophagic cell death.

Development of resazurin-based assay in 384-well format for high throughput whole cell screening of Trypanosoma brucei rhodesiense strain STIB 900 for the identification of potential anti-trypanosomal agents.

To accelerate the discovery of novel leads for the treatment of Human African Trypanosomiasis (HAT), it is necessary to have a simple, robust and cost-effective assay to identify positive hits by high throughput whole cell screening. Most of the fluorescence assay was made in black plate however in this study the HTS assay developed in 384-well format using clear plate and black plate, for comparison. The HTS assay developed is simple, sensitive, reliable and reproducible in both types of plates. Assay robustness and reproducibility were determined under the optimized conditions in 384-well plate was well tolerated in the HTS assay, including percentage of coefficient of variation (% CV) of 4.68% and 4.74% in clear and black 384-well plate, signal-to-background ratio (S/B) of 12.75 in clear 384-well plate and 12.07 in black 384-well plate, Z' factor of 0.79 and 0.82 in clear 384-well plate and black 384-well plate, respectively and final concentration of 0.30% dimethylsulfoxide (DMSO) in both types of plate. Drug sensitivity was found to be comparable to the reported anti-trypanosomal assay in 96-well format. The reproducibility and sensitivity of this assay make it compliant to automated liquid handler use in HTS applications.

(+)-Spectaline and Iso-6-Spectaline Induce a Possible Cross-Talk between Autophagy and Apoptosis in Trypanosoma brucei rhodesiense.

In our previous study, two known piperidine alkaloids (+)-spectaline (1) and iso-6-spectaline (2) were isolated from the leaves of Senna spectabilis and showed no toxic effect on L6 cells. In view of the potential use of piperidine alkaloids in S. spectabilis for the treatment of sleeping sickness, further investigation on the cell death actions of the parasite after treatment with compound 1 and 2 suggested that the treated parasites died by a process of autophagy based on the characteristic morphological alterations observed in intracellular T. b. rhodesiense. In search for apoptosis, interestingly, trypanosomes treated with high concentration of compound 1 and 2 after 72 h significantly induced an early apoptosis-like programmed cell death (PCD) such as phosphatidylserine (PS) exposure, loss of mitochondrial membrane potential and caspases activation. No DNA laddering discriminated late apoptosis event. Taken together, these findings demonstrated the potential of compound 1 and 2 as a natural chemotherapeutic capable of inducing a possible cross-talk between autophagy and apoptosis in T. b. rhodesiense.