Evaluation of Modulators of cAMP-Response in Terms of Their Impact on Cell Cycle and Mitochondrial Activity of Leishmania donovani.

Amrita Saha,Arunima Biswas,Amit Vij,Pijush K Das,Anindita Bhattacharjee,Arijit Bhattacharya

doi:10.3389/fphar.2020.00782

Abstract

With the identification of novel cAMP binding effector molecules in Trypanosoma, the role of cAMP in kinetoplastida parasites gained an intriguing breakthrough. Despite earlier demonstrations of the role of cAMP in the survival of Leishmania during macrophage infection, there is essential need to specifically clarify the involvement of cAMP in various cellular processes in the parasite. In this context, we sought to gain a comprehensive understanding of the effect of cAMP analogs and cAMP-cyclic nucleotide phosphodiesterase (PDE) inhibitors on proliferation of log phase parasites. Administration of both hydrolyzable (8-pCPT-cAMP) and nonhydrolyzable analogs (Sp-8-pCPT-cAMPS) of cAMP resulted in a significant decrease of Leishmania proliferation. Among the various PDE inhibitors, etazolate was found to be potently antiproliferative. BrdU cell proliferation and K/N/F-enumeration microscopic study revealed that both cAMP analogs and selective PDE inhibitors resulted in significant cell cycle arrest at G1 phase with reduced S-phase population. Furthermore, careful examination of the ﬂagellar motility patterns revealed significantly reduced coordinated forward flagellar movement of the promastigotes with a concomitant decrease in cellular ATP levels. Alongside, 8-pCPT-cAMP and PDE inhibitors etazolate and trequinsin showed marked reduction in mitochondrial membrane potential. Treatment of etazolate at subcytotoxic concentration to infected macrophages significantly reduced parasite burden, and administration of etazolate to Leishmania-infected BALB/c mice showed reduced liver and spleen parasite burden. Collectively, these results imply involvement of cAMP in various crucial processes paving the avenue for developing potent antileishmanial agent.

Highlights

Leishmaniasis, defined as an array of diseases including selfhealing cutaneous leishmaniasis and fatal visceral leishmaniasis, is caused by various species of protozoan parasite Leishmania
While studying the resistance against PDE inhibitor cpdA and cpdB, a novel class of cAMP effectors, CARP was identified in T. brucei (Gould et al, 2013), indicating the existence of noncanonical cAMP effectors in kinetoplastida parasites
On the receptor/cAMP generator side of the pathway, an oxygen stimulated soluble adenylyl cyclase has been identified; since this gene is absent in Trypanosoma, this enzyme probably contributes to some cAMP-dependent function restricted to Leishmania

Summary

Introduction

Leishmaniasis, defined as an array of diseases including selfhealing cutaneous leishmaniasis and fatal visceral leishmaniasis, is caused by various species of protozoan parasite Leishmania. The isoform PDEC from T. cruzi was shown to be involved in osmoregulation, and its potential to be a drug target was validated by high throughput screening of a series of PDE inhibitors (Wang et al, 2012), which led to the identification of tetrahydrophthalazinone compound A (Cpd A) (Salmon et al, 2012). These observations underpin the possibilities of cAMP pathway as a candidate target for drug development (Wang et al, 2012)

Methods

Results

Conclusion