Theileria equi isolates vary in susceptibility to imidocarb dipropionate but demonstrate uniform in vitro susceptibility to a bumped kinase inhibitor

Abstract

BackgroundThe apicomplexan hemoparasite Theileria equi is a causative agent of equine piroplasmosis, eradicated from the United States in 1988. However, recent outbreaks have sparked renewed interest in treatment options for infected horses. Imidocarb dipropionate is the current drug of choice, however variation in clinical response to therapy has been observed.MethodsWe quantified the in vitro susceptibility of two T. equi isolates and a lab generated variant to both imidocarb dipropionate and a bumped kinase inhibitor compound 1294. We also evaluated the capacity of in vitro imidocarb dipropionate exposure to decrease susceptibility to that drug. The efficacy of imidocarb dipropionate for clearing infection in four T. equi infected ponies was also assessed.ResultsWe observed an almost four-fold difference in imidocarb dipropionate susceptibility between two distinct isolates of T. equi. Four ponies infected with the less susceptible USDA Florida strain failed to clear the parasite despite two rounds of treatment. Importantly, a further 15-fold decrease in susceptibility was produced in this strain by continuous in vitro imidocarb dipropionate exposure. Despite a demonstrated difference in imidocarb dipropionate susceptibility, there was no difference in the susceptibility of two T. equi isolates to bumped kinase inhibitor 1294.ConclusionsThe observed variation in imidocarb dipropionate susceptibility, further reduction in susceptibility caused by drug exposure in vitro, and failure to clear T. equi infection in vivo, raises concern for the emergence of drug resistance in clinical cases undergoing treatment. Bumped kinase inhibitors may be effective as alternative drugs for the treatment of resistant T. equi parasites.

Extended-spectrum antiprotozoal bumped kinase inhibitors: A review

“…Imidocarb dipropionate, a dicationic diamidine of the carbanilide series of antiprotozoal compounds, has been effective against some of the T. equi strains causing infections in U.S. horses (Ueti, et al, 2012), making it the current drug of choice. However, variable responses to imidocarb treatment occur in both natural and experimental T. equi infection (Butler, et al, 2008, Frerichs, et al, 1973, Grause, et al, 2013, Knowles, 1996, Kumar, et al, 2003, Kumar, et al, 2009, Kuttler, et al, 1987, Ueti, et al, 2012), and we have recently documented treatment failure associated with in vitro imidocarb resistance (Hines, et al, 2015). Moreover, acute adverse effects are common due to the anticholinesterase activity of the drug (Tecles and Cerón, 2001).…”

“…This selective difference in activity is sufficiently great to offer hope that further chemical elaboration, in particular of the R2 group, may allow distinguishing selectively between threonine-gatekeeper kinases of parasite and host. This extends the range of parasitic diseases potentially treatable with BKI drugs to include those caused by Plasmodium (Chapman, et al, 2013), Eimeria (Keyloun, et al, 2014), Theileria (Hines, et al, 2015), and Babesia (Pedroni, et al, 2016), all of which express threonine gatekeeper CDPKs. A validation of CDPK(s) as the sole molecular target(s) that cause inhibition of growth and proliferation of an apicomplexan parasite cells when exposed to BKIs will still need chemical genetic determination as demonstrated in T. gondii.…”

“…Because of the documented effectiveness of BKIs against other apicomplexans (reviewed herein), we recently evaluated the in vitro susceptibility of several T. equi isolates to both imidocarb dipropionate and to BKI-1294 (Hines, et al, 2015). Similar to other apicomplexans, T. equi CDPK1 has a small glycine gatekeeper residue within the ATP-binding region (Hines, et al, 2015).…”

“…Similar to other apicomplexans, T. equi CDPK1 has a small glycine gatekeeper residue within the ATP-binding region (Hines, et al, 2015). Despite a difference in imidocarb susceptibility amongst isolates (EC 50 : 6.4 - 414 nM), we observed uniform susceptibility to BKI-1294, albeit at a considerably higher IC 50 of approximately 4.0 μM.…”

Selective inhibition of Sarcocystis neurona calcium-dependent protein kinase 1 for equine protozoal myeloencephalitis therapy

“…By inhibiting certain highly conserved apicomplexan parasite CDPKs, bumped kinase inhibitors (BKIs) have shown broad ranging inhibitory effects in both in vitro and in vivo models of infections with Toxoplasma gondii , Cryptosporidium parvum , Plasmodium spp., Neospora caninum and Theileria equi (Murphy et al, 2010; Ojo et al, 2010, 2014a, b; Johnson et al, 2012; Castellanos-Gonzalez et al, 2013; Doggett et al, 2014; Hines et al, 2015; Huang et al, 2015; Vidadala et al, 2016). We have shown that inhibition of apicomplexan CDPK1 and subsequent interference with mammalian host cell invasion by BKIs was due to the atypical glycine “gatekeeper” residue in the ATP-binding site of the kinase as well as the overall topology of the ATP-binding site.…”