Target validation of the inosine monophosphate dehydrogenase (IMPDH) gene in Cryptosporidium using Phylomer® peptides

Abstract

• Yielded a relatively high functional hit rate (~17% i.e. 2/12 Phylomer ® peptides tested).• Successfully shown that TAT can deliver therapeutic Phylomer cargoes inside cells.• Opens up new therapeutic opportunities. Cryptosporidiosis, a gastroenteric disease characterised mainly by diarrheal illnesses in humans and mammals is caused by infection with the protozoan parasite Cryptosporidium. Treatment options for cryptosporidiosis are limited, with the current therapeutic nitazoxanide, only partly efficacious in immunocompetent individuals. The parasite lacks de novo purine synthesis, and is exclusively dependant on purine salvage from its host. Inhibition of the inosine 5' monophosphate dehydrogenase (IMPDH), a purine salvage enzyme that is essential for DNA synthesis, thereby offers a potential drug target against this parasite. In the present study, a yeast-two-hybrid system was used to identify Phylomer peptides within a library constructed from the genomes of 25 phylogenetically diverse bacteria that targeted the IMPDH of Cryptosporidium parvum (IMPcp) and Cryptosporidium hominis (IMPch). We identified 38 unique interacting Phylomers, of which, 12 were synthesised and screened against C. parvum in vitro. Two Phylomers exhibited significant growth inhibition (81.2-83.8% inhibition; P < 0.05), one of which consistently 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 exhibited positive interactions with IMPcp and IMPch during primary and recapitulation yeast two-hybrid screening and did not interact with either of the human IMPDH proteins. The present study highlightsthe potential of Phylomer peptides as target validation tools for Cryptosporidium and other organisms and diseases because of their ability to bind with high affinity to target proteins and disrupt function. G R A P H I C A L A B S T R A C T

Cryptosporidium in humans and animals—a one health approach to prophylaxis

“…To date, the best studied drug target is the bacterial‐derived inosine 5′‐monophosphate dehydrogenase (IMPDH) gene, as Cryptosporidium does not contain guanine salvage enzymes and is totally dependent on this enzyme to convert adenosine salvaged from the host into guanine nucleotides . This coupled with the parasite's high metabolic demand for nucleotides due to the complicated life cycle of this parasite make IMPDH an important drug target …”

“…[73][74][75][76] This coupled with the parasite's high metabolic demand for nucleotides due to the complicated life cycle of this parasite make IMPDH an important drug target. [77][78][79][80][81][82][83][84][85] Other drug targets include long-chain fatty acyl-coenzyme A synthetases (LC-ACS), which are essential in fatty acid metabolism, 68 and a recent study reported good efficacy of the ACS inhibitor triacsin C against cryptosporidial infection in mice. 86 A parasite cysteine protease inhibitor was also effective in vitro and in an animal model.…”

Treatment of Cryptosporidium: What We Know, Gaps, and the Way Forward

“…Another study investigated the use of Phylomer ® peptides to inhibit Cp IMPDH functions. These studies were conducted in vitro and identified two out of twelve peptides with anti-cryptosporidium functions [59]. …”

Drug Development Against the Major Diarrhea-Causing Parasites of the Small Intestine, Cryptosporidium and Giardia

“…As an alternative strategy to conventional hit identification and lead optimization of small “drug-like” molecules, peptides have been explored as IMPDH inhibitors. In particular, phylomer peptides, which represent naturally stable protein segments from phylogenetically diverse bacterial genomes with an evolutionarily optimized ability to bind protein surfaces ( Watt, 2009 ), have been screened for inhibitory activity against C. parvum IMPDH ( Jefferies et al, 2015 ). Peptides that interact with IMPDH were identified in a yeast two hybrid screen, and then tested for parasite inhibition in vitro .…”

“…Peptides that interact with IMPDH were identified in a yeast two hybrid screen, and then tested for parasite inhibition in vitro . The best peptides suppressed C. parvum growth with 50% efficiency at 8–46 μM, but had only negligible cytotoxicity in human cells ( Jefferies et al, 2015 ). Although the in vivo efficacy of these peptides remains to be established, the data underline that IMPDH is a valid pharmacological target for parasite inhibition.…”