Cloning and functional complementation of ten Schistosoma mansoni phosphodiesterases expressed in the mammalian host stages

Abstract

Only a single drug against schistosomiasis is currently available and new drug development is urgently required but very few drug targets have been validated and characterised. However, regulatory systems including cyclic nucleotide metabolism are emerging as primary candidates for drug discovery. Here, we report the cloning of ten cyclic nucleotide phosphodiesterase (PDE) genes of S. mansoni, out of a total of 11 identified in its genome. We classify these PDEs by homology to human PDEs. Male worms displayed higher expression levels for all PDEs, in mature and juvenile worms, and schistosomula. Several functional complementation approaches were used to characterise these genes. We constructed a Trypanosoma brucei cell line in which expression of a cAMP-degrading PDE complements the deletion of TbrPDEB1/B2. Inhibitor screens of these cells expressing only either SmPDE4A, TbrPDEB1 or TbrPDEB2, identified highly potent inhibitors of the S. mansoni enzyme that elevated the cellular cAMP concentration. We further expressed most of the cloned SmPDEs in two pde1Δ/pde2Δ strains of Saccharomyces cerevisiae and some also in a specialised strain of Schizosacharomyces pombe. Five PDEs, SmPDE1, SmPDE4A, SmPDE8, SmPDE9A and SmPDE11 successfully complemented the S. cerevisiae strains, and SmPDE7var also complemented to a lesser degree, in liquid culture. SmPDE4A, SmPDE8 and SmPDE11 were further assessed in S. pombe for hydrolysis of cAMP and cGMP; SmPDE11 displayed considerable preferrence for cGMP over cAMP. These results and tools enable the pursuit of a rigorous drug discovery program based on inhibitors of S. mansoni PDEs.

To Target or Not to Target Schistosoma mansoni Cyclic Nucleotide Phosphodiesterase 4A?

“…After cloning the SmPDE4a gene [ 26 ], we set out to characterize this parasite PDE biochemically, pharmacologically, as well as structurally to enable a structure-based drug design program aiming to target SmPDE4A. To this end, two 6×His-tagged SmPDE4a constructs were generated, PCR amplified and cloned into pOPINF encoding a cleavable N -terminal 6×His tag.…”

“…Within the EU-funded consortium PDE4NPD aiming at investigating the potential of parasite PDEs as drug targets, we decided at its start in 2014 to embark on a PDE target discovery program for S. mansoni [ 25 ]. Munday et al [ 26 ] identified 11 different PDE open reading frames in the S. mansoni genome and cloned 10 of these SmPDEs (ranging from 517 to 1081 amino acids) from cDNA. Moreover, using different expression systems (yeast, T. brucei ), enzymatic activity was documented for six of the SmPDEs [ 26 ], making these parasite PDEs attractive new drug targets.…”

“…Munday et al [ 26 ] identified 11 different PDE open reading frames in the S. mansoni genome and cloned 10 of these SmPDEs (ranging from 517 to 1081 amino acids) from cDNA. Moreover, using different expression systems (yeast, T. brucei ), enzymatic activity was documented for six of the SmPDEs [ 26 ], making these parasite PDEs attractive new drug targets.…”

Use of a Fission Yeast Platform to Identify and Characterize Small Molecule PDE Inhibitors

“…Previously, we expressed in S. pombe PDE4, PDE8, and PDE11 family members from the blood fluke Schistosoma mansoni , whose genome includes members of all 11 Class I PDE families ( Vyas et al, 2021 ). 5FOA growth assays were used to determine the relative ability of these enzymes to hydrolyze cAMP and cGMP as shown by an increase in amount of exogenous cNMP required to confer 5FOA R growth ( Munday et al, 2020 ). However, an even more fertile area of research could involve the treatment of infections by parasitic nematodes due to the existence of the well-established model organism nematode Caenorhabditis elegans ( Corsi et al, 2015 ), whose genome includes only six Class I PDE genes.…”