Comprehensive Study of Human Pathogenic Trypanosomatids: From Chemotherapies to Disease Ecology
The vector-borne diseases Leishmaniasis and Chagas disease, caused by the kinetoplastid parasites Leishmania spp. and Trypanosoma cruzi respectively, are among the most important parasitic diseases in the group of neglected tropical diseases. Even though the two diseases are endemic to the Americas, they affect millions of people worldwide. Leishmaniasis and Chagas disease cause a great array of symptoms and some of them can be fatal if left undiagnosed and untreated. Current treatment regimens are becoming less effective, vaccines are still not available, and diagnosis needs to be improved. Vector control has been responsible for a decrease of diseases in endemic areas. However, in non-endemic areas, vector control strategies are nonexistent due to an inadequate understanding of vector epidemiology and without proper monitoring and evaluation of vector and associated parasite. Therefore, there is an urgent need to evaluate different strategies for the treatment, diagnosis and prevention (vaccine prevention and vector control) of Leishmaniasis and Chagas disease. In this regard, the first part of this study, we evaluated two types of compounds, thiophene derivatives and methionine aminopeptidase 1 (MetAP1 inhibitors), for the treatment of cutaneous leishmaniasis, one of the clinical manifestations of leishmaniasis, caused by Leishmania major. The first class of compounds, thiophene derivatives, were synthesized and evaluated their parasitic activity, and potential mechanism of action (MOA). We developed a structure–activity relationship (SAR) study of the thiophene molecule 5A. Overall, eight thiophene derivatives of 5A were synthesized and purified by silica gel column chromatography. Of these eight analogs, the molecule 5D showed the highest in vitro activity against Leishmania major promastigotes (EC50 0.09 ± 0.02 μM), with an inhibition of the proliferation of intracellular amastigotes higher than 75% at only 0.63 μM and an excellent selective index. Moreover, the effect of 5D on L. major promastigotes was associated with generation of reactive oxygen species (ROS), and in silico docking studies suggested that 5D may play a role in inhibiting trypanothione reductase. In summary, the combined SAR study and the in vitro evaluation of 5A derivatives allowed the identification of the novel molecule 5D, which exhibited potent in vitro anti-leishmanial activity resulting in ROS production leading to cell death with no significant cytotoxicity towards mammalian cells. The second class of compounds, MetAP1 inhibitors, were validated as a chemotherapeutic target against CL infection. The in vitro antileishmanial activities of eight novel MetAP1 inhibitors (OJT001 to OJT008) were investigated. Three compounds, OJT006, OJT007, and OJT008, demonstrated potent antiproliferative effects in macrophages infected with L. major amastigotes and promastigotes at submicromolar concentrations, with no cytotoxicity against host cells. Importantly, the leishmanicidal effect in transgenic L. major promastigotes overexpressing MetAP1Lm was diminished by almost 10-fold in comparison to the effect in wild-type promastigotes. Furthermore, the in vivo activities of OJT006, OJT007, and OJT008 were investigated in L. major-infected BALB/c mice. In comparison to the footpad parasite load in the control group, OJT008 decreased the footpad parasite load significantly, by 86%, and exhibited no toxicity in treated mice. We propose thiophene derivative 5D and MetAP1 inhibitor OJT008 as potential chemotherapeutic candidates against CL infection caused by L. major. The second part, we aim to identify and characterized glycosylphosphatidylinositol-anchored proteins (GPI-AP) that display highly immunogenic terminal nonreducing α-galactopyranosyl (α-Gal)- containing glycotopes that can be used as biomarker and vaccine candidates. Here, GPI-AP were extracted from promastigotes (PR) and metacyclic promastigotes (MP) of L. major, L. braziliensis and L. guyanensis, and L. donovani which cause CL, MCL and VL respectively and analyzed by CL-ELISA, western blot and mass spectrometry. α-Gal containing GPI-AP were isolated by lectin affinity chromatography (LAC) with immobilized Bandeiraea simplicifolia isolectin 4 (IB4) and further fractionated by hydrophobic interaction chromatography with an octyl-Sepharose column (HIC-OS) in a propanol gradient. Results indicated the presence of α-Gal containing GPI-AP in L. major and two strain of L. braziliensis in both stages, PR and MP. Further analysis is needed to fully characterize α -gal containing GPI-AP in each Leishmania sample. Moreover, a ~33 kDa glycoprotein was found by western blot probed with IB4 and serum from CL patients. This glycoprotein needs to be further characterized by mass spectrometry and be tested as biomarker. The third and final part of this study, vector surveillance and epidemiology were implemented in the Southwestern region of U.S. where epidemiological studies are limited. We have determined the prevalence of T. cruzi in triatomines, feral cats and dogs, and wild animals, as well as the infecting parasite genotypes and the mammalian host bloodmeal sources of the triatomines at four different geographical sites in the U.S.-Mexico border, including El Paso County, Texas, and nearby cities in New Mexico. Using qualitative polymerase chain reaction to detect T. cruzi infections, we found 66.4% (n=225) of triatomines, 45.3% (n=95) of feral dogs, 39.2% (n=24) of feral cats, and 71.4% (n=7) of wild animals positive for T. cruzi. Over 95% of T. cruzi genotypes or discrete typing units (DTUs) identified were TcI and some TcIV. Furthermore, Triatoma rubida was the triatomine species most frequently (98.2%) collected in all samples analyzed. These findings suggest a high prevalence of T. cruzi infections among triatomines, and feral and wild animals in the studied sites. Therefore, our results underscore the urgent need for implementation of a systematic epidemiological surveillance program for T. cruzi infections in insect vectors, and feral and wild animals, and Chagas disease in the human population in the southwestern region of the United States.
Rodriguez, Felipe, "Comprehensive Study of Human Pathogenic Trypanosomatids: From Chemotherapies to Disease Ecology" (2021). ETD Collection for University of Texas, El Paso. AAI28776069.