Use of a 2-aminothiazole to Treat Chronic Wasting Disease in Transgenic Mice

Abstract

Treatment with the 2-aminothiazole IND24 extended the survival of mice infected with mouse-adapted scrapie but also resulted in the emergence of a drug-resistant prion strain. Here, we determined whether IND24 extended the survival of transgenic mice infected with prions that caused scrapie in sheep or prions that caused chronic wasting disease (CWD; hereafter “CWD prions”) in deer, using 2 isolates for each disease. IND24 doubled the incubation times for mice infected with CWD prions but had no effect on the survival of those infected with scrapie prions. Biochemical, neuropathologic, and cell culture analyses were used to characterize prion strain properties following treatment, and results indicated that the CWD prions were not altered by IND24, regardless of survival extension. These results suggest that IND24 may be a viable candidate for treating CWD in infected captive cervid populations and raise questions about why some prion strains develop drug resistance whereas others do not.

chronic wasting disease, therapeutic, prion disease, transgenic mice, survival extension

Prion diseases are a class of protein-based, invariably fatal, neurodegenerative disorders that are increasingly recognized to underlie the majority of neurodegenerative diseases [1, 2]. In these diseases, an endogenous protein misfolds into a stable, aggregation-prone conformation that promotes the self-templated conversion of additional copies of the particular conformation. There are currently no pharmacologic treatments that halt or even slow the progress of any neurodegenerative disease.

Chronic wasting disease (CWD) is the only prion disease known to spread in free-ranging animals, infecting deer, elk, and moose. CWD was first identified in Colorado in 1967, and cases have since been reported across the United States, in Canada, and in captive animals in South Korea. The CWD epidemic in deer and elk in North America continues to spread, causing great concern about the implications for human health. Although there is no evidence that prions that cause CWD (hereafter, “CWD prions”) can directly infect humans [3], CWD prions can be transmitted to other species [4–8]. The range of susceptible hosts can expand as a result of the adaption of CWD prions to a new species [4], and CWD prions remain infectious when bound to soil [9]. With no known treatment, the possibility that a CWD-derived prion strain could eventually become infectious to humans cannot be ignored.

Prion strains are defined by several characteristics, including the incubation period of the disease, the banding pattern of partially digested prion proteins on an immunoblot, the pattern of neuropathology in infected animals, the ability to infect naive animals (serial passage of the prions), the ability to infect cell culture, and resistance to treatment with antiprion compounds in cell culture. We previously demonstrated that IND24, a 2-aminothiazole, dramatically extended the survival of wild-type mice infected with the mouse-adapted scrapie strain RML [10]. However, treatment with IND24 changed the biochemical properties of the disease-causing prion protein isoform (PrP^Sc) and neuropathologic patterns of the disease. Prions from the brains of mice treated with IND24 were able to infect CAD5 cells but were no longer responsive to subsequent treatment with IND24, suggesting the emergence of drug resistance. The novel, IND24-resistant prion strain was named “RML[IND24]” to denote that these prions were produced by treating RML-infected mice with IND24. Previously, drug resistance had been suggested in prion-infected mice treated with quinacrine [11], and spontaneous resistance to swainsonine was described in prion-infected cell culture [12].

In an earlier study, we reported that IND24 at 210 mg/kg/day significantly extended the survival times of transgenic (Tg) mice expressing elk prion protein (PrP) [10]. Here, we investigated the efficacy of IND24 against prions from sheep with scrapie (hereafter, “scrapie prions”) and a second CWD prion isolate, obtained from a white-tailed deer (WTD). Despite being ineffective in scrapie prion–infected Tg mice, IND24 successfully extended the incubation period for both CWD isolates, approximately doubling the survival times, even at a much lower dose (50 mg/kg/day). In contrast to characteristics of the RML strain, the properties of CWD prions were not changed by treatment with IND24, as judged by cell culture studies and by biochemical and neuropathologic analyses. The reasons why some but not other prion strains develop resistance to IND24 remain to be determined. Our findings argue that IND24 may be useful in treating CWD prion–infected cervids and provide important insights for developing effective therapeutics for human prion diseases.

MATERIALS AND METHODS

Materials

RK13 cells expressing elk PrP (RK21) were a gift from Dr Glenn Telling (Colorado State University). Dulbecco's modified Eagle's medium (DMEM) high glucose 1× with 4.5 g/L D-glucose and L-glutamine and without sodium pyruvate, fetal bovine serum (FBS), penicillin-streptomycin (pen-strep; each at 10 000 units/mL), GlutaMAX, dithiothreitol (DTT; 0.5 M 10×), 4× loading buffer, and proteinase K (PK) were purchased from Invitrogen. Enzyme-free cell dissociation buffer was purchased from Millipore. IND24 was synthesized by ChemVeda (Hyderabad, India). SSBP/1 and CH1641 scrapie isolates were a gift from Nora Hunter and Jim Foster (University of Edinburgh).

Animal Husbandry and Tissue Preparation

All protocols were approved by the University of California San Francisco Animal Care and Use Committee. All mice were bred in our facility. Tg mice expressing elk PrP [Tg(ElkPrP:M132)12584; hereafter, “Tg(ElkPrP) mice”] and Tg mice expressing sheep PrP [Tg(OvPrP:V136)14882; hereafter, “Tg(OvPrP) mice”] have been previously reported [13, 14]. Female mice were used for all experiments, owing to problems observed with long-term dosing of male mice [15]. Tg(ElkPrP) mice were inoculated intracerebrally in the right parietal cortex with 30 µL of 1% brain homogenate containing CWD prions from an elk or WTD with a clinical diagnosis of CWD, respectively denoted “Elk1” and “WTD1” in a previous report [13]. Tg(OvPrP) mice were inoculated intracerebrally with 30 µL of 1% of the SSBP/1 or CH1641 brain homogenate isolates. Mice were observed twice per week for signs of neurologic disease and received a diagnosis of prion disease when they exhibited at least 2 characteristic neurologic signs, as described elsewhere [16]. Upon diagnosis, mice were euthanized and their brains collected. The left hemisphere was snap frozen on dry ice and then stored at −80°C for biochemical analysis; the right hemisphere was fixed for pathologic analysis.

Long-term Dosing of Compounds

Dosing was performed as previously reported [15]. Briefly, compounds were dissolved in 100% PEG400 and stored at 4°C until use. Samples were diluted to make final dosing concentrations of 0.125% PEG400 in a complete rodent liquid diet (Bio Serv, product no. F1256SP). For vehicle (V)–treated mice, pure PEG400 was diluted appropriately into the previously prepared rodent liquid diet. The amount of food served was based on the number of animals in each cage and replaced by new batches every 2–4 days. Doses are indicated in the text for each experiment.

Immunoblotting

For immunoblot analysis, brain homogenate samples were digested with 20 µg/mL PK at 37°C for 1 hour. Pellets from 500 µL of PK-digested samples were collected and resuspended with sodium dodecyl sulfate (SDS) sample running buffer and 0.1 M DTT. Samples were heated to 100°C for 10 minutes and run in a 4%–12% Tris-glycine SDS gel (Invitrogen). The gel was transferred to a polyvinylidene fluoride membrane, using an iBlot (Invitrogen), and the membrane was blocked with 5% milk for 30 minutes. The membranes were subsequently incubated overnight with horseradish peroxidase–conjugated HuM-P Fab and washed 3 times with Tris-buffered saline with Tween-20 for 10 minutes before developing with an enhanced chemiluminescence reagent (Invitrogen).

Bands on immunoblots were quantified using ImageJ 1.46r [17]. The total intensity for an area encompassing each band was measured, and average background was subtracted. Normalized intensities were totaled for each lane. For glycoform proportions, a ratio was calculated for each normalized band intensity, compared with the sum of the 3 isoforms, and these ratios were averaged across 3–9 samples.

Pathology

Brains were immersion fixed in 10% (vol/vol) buffered formalin. Fixed samples were paraffin embedded, sectioned, and stained with hematoxylin and eosin or processed by immunohistochemical staining for PrP with the HuM-P Fab antibody, as described previously [18]. Images were taken at 20 times and 40 times original magnification, using the SpotFlex camera and program on the Leica DM-IRB microscope. Neuropathologic analyses were performed on 3–9 mice per experimental condition. PrP^Sc plaque intensities were determined by a visual assessment of the intensity of immunohistologic staining, multiplied by the area of the brain region occupied by staining, and summed for each cross-sectional brain region. The intensity of immunostaining was given a value of 1, for barely detectable; 2, for mild; 3, for moderate; and 4, for strongly intensive. PrP^Sc accumulation was evaluated by a single rater (S. J. D.).

Cell Culture

Cells were infected with CWD prions as previously described [19]. All cell lines were maintained at 37°C in their respective media in 100-mm plates and fed with fresh medium every 2–3 days. RK21 cells were maintained in DMEM supplemented with 1% GlutaMAX, 100 units pen-strep, and 10% FBS. Dividing cells were plated at 10% confluency and split at a 1:10 ratio when they became confluent. Cells were incubated with IND24 at the doses indicated in the main text over 5 days with refeeding. IND24 was diluted in DMSO, with a 50-µL final volume in 10 mL of medium. At the end of the 5-day treatment period, lysis buffer (100 mM Tris/HCl, pH 8.0; 150 mM NaCl; 0.5% NP-40; and 0.5% sodium deoxycholate) was added to cells, and protein concentrations were measured using a bicinchoninic acid protein assay kit (Fisher, Rockford, Illinois). Protein extracts were normalized to 1 mg/mL total protein with lysis buffer prior to PK digestion. Lysates were digested with 20 µg/mL PK at 37°C for 1 hour. The reaction was stopped with phenylmethylsulfonyl fluoride (2 mM final concentration), and samples were subjected to immunoblot analysis.

Calculation of Survival and Statistical Analysis

Survival indexes were calculated to compare survival extension between experiments by dividing the mean survival time of compound-treated mice by the mean survival time of vehicle-treated control mice on the first passage, multiplied by 100. Data are mean values ± standard error of the mean, unless otherwise indicated. All significance calculations were performed with the Student t test, using Prism 5 (GraphPad Software, La Jolla, California).

RESULTS

Treatment With IND24 Did Not Extend Survival Times Among Tg(OvPrP) Mice Infected With Sheep Scrapie Isolates

We previously reported that administering 210 mg/kg/day IND24 doubled the incubation time in Tg mice infected with 2 independent mouse-passaged scrapie strains (RML and ME7) but did not extend survival times among Tg mice expressing human PrP and infected with prions causing human Creutzfeldt-Jakob disease, indicating that the efficacy of the compound was dependent on prion strain [10]. To examine the efficacy of IND24 on other naturally occurring prions, we treated Tg(OvPrP) mice infected with either of 2 distinct sheep scrapie isolates (SSBP/1 and CH1641) with 210 mg/kg/day IND24. We found no extension in survival times, compared with vehicle-treated mice (Figure 1A and 1C and Table 1). Prions from treated mice are identified by the prion isolate with the treatment appended (eg, “SSBP/1[IND24]”). Immunoblotting of PrP^Sc in the brains of the ill mice indicated no change in the molecular weight or the glycoform ratio (Figure 1B and 1D and Supplementary Table 1), indicating that the biochemical properties of the sheep scrapie isolates were not altered as a result of treatment.

Table 1.

Incubation Periods of Transgenic (Tg) Mice Infected With 3 Different Naturally Occurring Prion Isolates and Treated With IND24

Prion Inoculum	Tg Mouse	Treatment	Dose, mg/kg/d	Incubation Period, d, Mean ± SEM	No. of Ill Mice/No. Observed	Survival Index, Mean ± SEM
SSBP/1	Tg(OvPrP)	Vehicle	…	57 ± 2	9/9	100a
SSBP/1	Tg(OvPrP)	IND24	210	62 ± 2	9/9	109 ± 5
CH1641	Tg(OvPrP)	Vehicle	…	169 ± 4	8/8	100a
CH1641	Tg(OvPrP)	IND24	210	172 ± 8	7/7	101 ± 5
WTD CWD	Tg(ElkPrP)	Vehicle	…	137 ± 4	8/8	100a
WTD CWD	Tg(ElkPrP)	IND24	210	216 ± 6	8/8	158 ± 5

Abbreviations: CWD, chronic wasting disease; SEM, standard error of the mean; Tg(ElkPrP), Tg mice expressing elk prion protein; Tg(OvPrP), Tg mice expressing ovine prion protein; WTD, white-tailed deer.

^a Control experiment was used to normalize survival index.

Figure 1.

Incubation periods of transgenic (Tg) mice inoculated with natural prion isolates and treated with vehicle (V; red) or IND24 (green). Kaplan–Meier survival curves of Tg mice expressing ovine prion protein that were inoculated with the sheep scrapie isolate SSBP/1 (IND24, n = 9; V, n = 9; A) or the sheep scrapie isolate CH1641 (IND24, n = 7; V, n = 8; C) and of Tg mice expressing elk prion protein that were inoculated with white-tailed deer (WTD) CWD prions (IND24, n = 8; V, n = 8; E). Immunoblots show proteinase K–resistant prion protein in the brains of animals with terminal disease that were infected with SSBP/1 (B), CH1641 (D), or WTD CWD prions (F) and treated with vehicle or IND24. PrP^Sc was probed using horseradish peroxidase–conjugated HuM-P Fab. Molecular weight markers of migrated protein standards represent 30 and 20 kDa.

IND24 Treatment Was Efficacious Against WTD CWD

Since IND24 was previously shown to be effective against elk CWD prions, we investigated whether IND24 would extend the survival of mice infected with a WTD CWD isolate. To eliminate any variation due to differences in the host PrP^C sequence, a WTD CWD isolate was inoculated into Tg(ElkPrP) mice, the Tg line used for elk CWD studies. Infected mice received 210 mg/kg/day IND24 or vehicle. Tg(ElkPrP) mice treated with IND24 survived 216 ± 6 days after inoculation, a substantial extension in the survival duration as compared to that for mice treated with vehicle (137 ± 4 days after inoculation; Table 1 and Figure 1E), producing a survival index of 158 ± 5. This extension is considerably lower than that observed for Tg(ElkPrP) mice inoculated with elk CWD prions and treated at the same IND24 dose (survival index, 200 ± 6; [10]), suggesting that strain differences may exist between the WTD and elk CWD isolates, given that the prions were propagated within the same Tg host line.

Alternative IND24 Dosing Regimens Extended Survival Times Among Tg(ElkPrP) Mice

Treating Tg(ElkPrP) mice with 210 mg/kg/day IND24 doubled the mean survival time among the mice following inoculation with elk CWD prions [10]. To determine whether this high dose was necessary to achieve that magnitude of extension in the survival duration, we tested 2 additional dosing paradigms: 210 mg/kg/day for the first 138 days after inoculation, followed by 50 mg/kg/day; and 50 mg/kg/day, beginning 1 day after inoculation. The animals were treated continuously until they succumbed to disease. Treating elk CWD prion–infected Tg(ElkPrP) mice with 50 mg/kg/day IND24 resulted in a survival index of 205 ± 4 (Table 2 and Figure 2A), demonstrating that much lower doses of IND24 are sufficient to achieve this survival extension. However, treating first with 210 mg/kg/day and then 50 mg/kg/day yielded an unexpected biphasic survival curve: the first 4 mice to show signs of prion disease had an incubation period of 200 ± 3 days after inoculation, and the last 5 had an incubation period of 274 ± 8 days after inoculation (survival index, 185 ± 6 and 254 ± 10, respectively). Considering all mice together gave a survival index of 223 ± 14.

Table 2.

Incubation Periods of Transgenic Mice Expressing Elk Prion Protein That Were Infected With Elk Chronic Wasting Disease (CWD) Prions or Passaged Elk CWD Prions

Prion Inoculum	Treatment	Dose, mg/kg/d	Incubation Period, d, Mean ± SEM	No. of Ill Mice/No. Observed	Survival Index, Mean ± SEM
Elk CWD	Vehicle	…	108 ± 3	9/9	100a,b
Elk CWD	IND24	210	237 ± 0	6/6	220 ± 3b
Elk CWD	IND24	50	221 ± 3	8/8	205 ± 4
Elk CWD	IND24	210 and then 50	241 ± 14	9/9	223 ± 14
Elk CWD[V]	None	…	95 ± 0	8/8	88 ± 3
Elk CWD[IND24]	None	…	111 ± 2	8/8	103 ± 3

Abbreviation: SEM, standard error of the mean.

^a Control experiment used to normalize survival index.

^b Data were previously reported elsewhere [10].

Figure 2.

Biochemical and neuropathologic characterization of transgenic (Tg) mice inoculated with elk chronic wasting disease (CWD) prions and treated with IND24. A, Kaplan–Meier survival curves of Tg mice expressing elk prion protein (Tg[ElkPrP]) that were inoculated with elk CWD prions and treated with 3 different dosing regimens of IND24: 210 mg/kg/day from day 1 (dashed green; n = 8), as previously reported [10]; 210 mg/kg/day for 138 days, followed by 50 mg/kg/day (solid green; n = 9); or 50 mg/kg/day (dotted green; n = 8). Survival for mice treated with (V; solid red; n = 9), as previously reported [10], is also shown. B, Proteinase K–resistant PrP from the brains of terminal animals that were infected with elk CWD prions and treated with IND24 (green) or vehicle (red), as indicated. PrP^Sc was probed using horseradish peroxidase–conjugated HuM-P Fab. Molecular weight markers of migrated protein standards represent 30 and 20 kDa. C–H, Neuropathologic analysis of brain sections from terminal animals that were infected with elk CWD prions and treated with IND24 (C–E) or V (F–H). Immunohistochemical staining for PrP^Sc was performed using HuM-P Fab. Regions are caudate nucleus/septum (CN/S; C and F), hippocampus/thalamus (Hp/Th; D and G), and pons/cerebellum (P/Cb; E and H). Black bars represent 50 µM and also apply to the respective micrograph below each image. I, Quantification of plaque intensity (arbitrary units [AU]) in brain slices of Tg(ElkPrP) mice inoculated with elk CWD prions and treated with IND24 (green) or vehicle (red).

Treament With IND24 Did Not Alter the Characteristics of PrP^Sc in the Brains of Tg(ElkPrP) Mice

To determine whether the prion strain characteristics were changed by IND24, as previously observed [10], we characterized elk CWD[IND24], elk CWD[V], WTD CWD[IND24], and WTD CWD[V] prions from infected Tg(ElkPrP) mice. We observed no differences in the biochemical properties or neuropathologic deposition of PrP between IND24- or vehicle-treated terminal Tg(ElkPrP) mice. The molecular weights of the PK-resistant PrP bands in IND24- or vehicle-treated samples were identical when assayed by Western blot (Figures 1F and 2B). The ratio of mono- and diglycosylated PrP^Sc was not significantly different between IND24 and V recipients (Supplementary Table 1). We found no differences in the morphology (Figure 2C–H) or intensity (Figure 2I) of PrP^Sc deposited in the brains of Tg(ElkPrP) mice treated with IND24 or V following infection with elk CWD or WTD CWD (Supplementary Figure 1). Vacuolation was sparse and not significantly different between treatments (data not shown).

Treatment With IND24 Did Not Alter the CWD Infectivity Profile or Drug Susceptibility

To determine whether IND24 altered CWD prion infectivity, we prepared brain homogenates from elk CWD prion–infected Tg(ElkPrP) mice treated with IND24 or V; these samples are respectively denoted “elk CWD[IND24]” and “elk CWD[V].” We used elk CWD[IND24] and elk CWD[V] to infect RK13 cells expressing ElkPrP (RK21). Previously, RK21 cells were infected with elk CWD isolates, resulting in RKE cells [20]. Our infection attempts with CWD[IND24] and CWD[V] were successful (6 of 6 for both inocula); we denote these newly infected cells “RK21-CWD[IND24]” and “RK21-CWD[V],” respectively. We found no significant difference between the ratios of mono- to diglycosylated PrP^Sc in the resultant infected cell lines (Supplementary Table 1). To determine whether the CWD prions remained susceptible to IND24, we treated RK21-CWD[IND24], RK21-CWD[V], and RKE cells with 0–10 µM of IND24 (Figure 3). For all 3 cell lines, levels of CWD prions were dramatically reduced by IND24 at doses of >0.32 µM. The measured half-maximal effective concentrations varied between the 3 prion infections (mean ± standard deviation for 4 replicates, 1.7 ± 0.4 µM for RKE, 3.3 ± 3.4 µM for RK21-CWD[V], and 1.4 ± 0.1 µM for RK21-CWD[IND24]), but these variations were not statistically significant (P = .3–.5, by the Student t test).

Figure 3.

Infected RK13 cells expressing elk prion protein (RK21) and treated with IND24. Proteinase K–resistant PrP from RK13-ElkPrP cells infected with elk chronic wasting disease (CWD) prions (top) [20] and with CWD prions from the brains of transgenic mice expressing ElkPrP and treated with vehicle (V; middle) or IND24 (bottom). Doses of IND24 are indicated in µM. PrP^Sc was probed using horseradish peroxidase–conjugated HuM-P Fab. Molecular weight markers of migrated protein standards represent 30 and 20 kDa for each blot.

As reported previously, RML[IND24] could also be distinguished from RML[V] by an increased incubation period when used to inoculate naive mice [10]. To determine the impact of IND24 on the incubation period of passaged elk CWD prions, we inoculated elk CWD[V] and elk CWD[IND24] samples into Tg(ElkPrP) mice. The respective incubation periods were 95 ± 0 days after inoculation and 111 ± 2 days after inoculation (Table 2 and Figure 4A). Although the incubation periods for individual inoculation experiments are highly reproducible, repeated experiments using the same inoculum have demonstrated that the survival time for any individual experiment can vary by as much as 10% of the mean incubation period [21]. Thus, it is unlikely that the observed incubation periods for CWD[IND24] and CWD[V] reflect a difference between these 2 inocula. PrP^Sc in the brains of these mice had banding patterns and glycoform ratios similar to those for their respective inocula (Figure 4B). Taken together, the biochemical, neuropathologic, cell culture infection, and passaging results suggest that IND24 did not alter the strain properties of elk CWD prions in mice despite significantly extending survival times.

Figure 4.

Passage of CWD[IND24]. A, Kaplan–Meier survival curves of transgenic mice expressing elk prion protein that were inoculated with CWD[V] (pink; n = 8) or CWD[IND24] (light green; n = 8) and given no treatment, compared with findings from the previously reported CWD[V] experiment (red; n = 9; [10]) and CWD[IND24] (dark green; n = 9). The circles indicate the incubation periods of animals used to derive the inocula. B, Proteinase K–resistant prion protein from the brains of terminal animals infected with CWD[V] or CWD[IND24]. PrP^Sc was probed using horseradish peroxidase–conjugated HuM-P Fab. Molecular weight markers of migrated protein standards represent 30 and 20 kDa.

DISCUSSION

Increasing evidence argues that the most common neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and various tauopathies are the result of self-propagating misfolded proteins, or “prions” [1, 2]. Similar to antibiotic-resistant pathogenic bacteria [22], drug-resistant prions have emerged as a result of efficacious treatment [10]. Drug resistance represents a serious challenge to the development of therapies for neurodegenerative diseases. The observation that IND24 can extend the survival times of CWD-infected mice without altering the prion strain properties increases its potential utility as a therapeutic compound for cervids. Although IND24 did not cure the infected animals, it did not generate a drug-resistant prion strain. Moreover, IND24 doubled the survival time at one fourth the previously reported dose (survival index, 205 ± 4 for 50 mg/kg/day treatment). IND24 extended survival times in mice infected with CWD isolates from 2 different species, suggesting that IND24 may be broadly able to reduce the propagation of CWD prions. It is therefore plausible to use IND24, or a related compound, as a response to infection or a prophylactic treatment in captive cervid populations. Whether prophylactic treatment will reduce the likelihood of infection with CWD remains to be determined. It is also undetermined whether treated cervids will continue to shed infectious CWD prions while remaining asymptomatic. If they do, prophylactic treatment would not halt environmental contamination with prions.

The inability of IND24 to extend survival times in Tg(OvPrP) mice infected with either of the sheep scrapie isolates SSBP/1 or CH1641 is somewhat surprising, since IND24 extended the survival of wild-type mice infected with either of 2 mouse-adapted scrapie strains, RML or ME7 [10], with RML having been derived from the original SSBP/1 pool [23]. IND24 also failed to extend the survival times of susceptible Tg mice infected with human sporadic Creutzfeldt-Jakob disease–associated prions. However, given the absence of efficacy against SSBP/1 and CH1641 sheep scrapie isolates, it is also possible that differences in the primary sequence of OvPrP played a role. These results highlight the strain specificity of antiprion therapeutics, an observation that has been made with a variety of compounds [24]. By comparing the various properties of PrP^Sc strains against which IND24 is effective, we may be able to determine similarities in strain characteristics that predict efficacy.

In contrast to the emergence of drug resistance with RML prions observed previously [10], IND24 administration did not alter the strain properties or drug susceptibility of CWD prions. The previously observed IND24 resistance may have arisen as a result of an interaction between the compound and the prion, resulting in a change of conformation (ie, a conformational “mutation” [25]), and/or because of the selection of a preexisting IND24-resistant variant of RML that propagates more slowly than the dominant IND24-susceptible strain. Alternatively, IND24 may interact with CWD and RML prions differently, either because of the conformation or the sequence of the protein. Still another possibility, the CWD isolates may not produce off-path variants that are resistant to IND24, allowing the mice to survive longer because of the incomplete inhibition of prion propagation.

The observation that a hybrid dosing scheme resulted in a survival curve with a wider, possibly biphasic distribution may suggest the appearance of 2 strains. Biochemical and neuropathologic analysis of the “early” and “late” groups did not show any obvious differences. However, changing the dosing paradigm may have introduced meaningful variation into prion strain propagation that will only be apparent on serial passaging in the presence and absence of IND24. Alternatively, these differences may reflect the interanimal variation that is sometimes observed in prion inoculation experiments.

That IND24-treated mice eventually succumbed to prion disease suggests that a cocktail of compounds will be necessary to halt disease progression. To identify additional compounds with anti-CWD prion properties, high-throughput screening will be assisted by the facile infectibility and the rapid replication of RK13-ElkPrP cells [20]. However, quinacrine treatment of infected RKE cells led to increased accumulation and altered the strain properties of CWD prions, whereas treating CWD-infected mice did not [26]. Quinacrine treatment of RML-infected N2a cells and mice also produced conflicting results [11, 27–29]. Scrapie prion–infected N2a cells nonetheless led to the identification of 2-aminothiazoles as potent antiprion compounds [30], which were optimized using the same cell assay [31, 32] and showed efficacy in mice [10]. New anti-CWD compounds could be tested using mouse models to establish the predictive quality of CWD-infected RK13 cells.

In this work, we have demonstrated that efficacious treatment with IND24 does not invariably generate drug-resistant prions. Although we do not yet understand the specific features that allow one prion strain to become resistant and another to remain susceptible, or why some prion infections respond to treatment and others do not, these results serve as a foundation for exploring those questions. Moreover, IND24 is the first compound with demonstrated efficacy against a natural prion disease in Tg mice, providing a unique opportunity to test the translational value of these models. The underlying assumption in these studies, and in every screen for an antiprion compound that extends the survival times of infected mice, is that efficacy with antiprion compounds in Tg mouse models will translate to efficacy against natural prion diseases. Unlike treatments for human diseases, use of IND24 to treat deer or elk can be conducted in a controlled inoculation experiment. Successfully treating CWD prion–infected deer or elk would validate this approach to drug discovery and increase our confidence that, when we eventually find antiprion compounds for human diseases by using Tg mouse models, they will translate to treating human disease.

Supplementary Data

Supplementary materials are available at The Journal of Infectious Diseases online (http://jid.oxfordjournals.org). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.

Notes

Financial support. This work was supported by the National Institutes of Health (grants AG021601, AG002132, AG010770, AG031220, and NS080630), the Glenn Foundation for Medical Research, and the Sherman Fairchild Foundation.

Potential conflicts of interest. All authors: No reported conflicts.

All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.

Author notes

Presented in part: The Hilary Koprowski, MD, Symposium, Philadelphia, Pennsylvania, 27 June 2014.