Novel inhibitors of neurotropic alphavirus replication that improve host survival in a mouse model of acute viral encephalitis

Abstract

Arboviral encephalitis is a potentially devastating human disease with no approved therapies that target virus replication. We previously discovered a novel class of thieno[3,2-b]pyrrole-based inhibitors active against neurotropic alphaviruses such as western equine encephalitis virus (WEEV) in cultured cells. In this report, we describe initial development of these novel antiviral compounds, including bioisosteric replacement of the 4H-thieno[3,2-b]pyrrole core with indole to improve metabolic stability and the introduction of chirality to assess target enantioselectivity. Selected modifications enhanced antiviral activity while maintaining low cytotoxicity, increased stability to microsomal metabolism, and also revealed striking enantiospecific activity in cultured cells. Furthermore, we demonstrate improved outcomes (both symptoms and survival) following treatment with indole analogue 9h (CCG-203926) in an in vivo mouse model of alphaviral encephalitis that closely correlate with the enantiospecific in vitro antiviral activity. These results represent a substantial advancement in the early preclinical development of a promising class of novel antiviral drugs against virulent neurotropic alphaviruses.

Fig. 1

Structure of HTS lead 1

Figure 2

Neuronal cell infection studies. Cultured human BE(2)-C neuronal cells were simultaneously infected with WEEV or NSV and incubated with the indicated compounds at a final concentration of 25 µM. Cell viability (A) and virus titers (B) were determined 24 h post-infection by MTT and plaque assays, respectively. Ribavirin (R) was used as the positive control for both assays. *p < 0.05 compared to DMSO (—) controls.

Figure 3

Clinical effects of the indole enantiomers, 9g and 9h, in mice with acute NSV encephalomyelitis. Cohorts of infected mice (n=13/group) were otherwise not manipulated or were treated with 9g or 9h (30 mg/kg/dose) or with a vehicle control by intraperitoneal injection every 12 hours beginning 12 hours after virus challenge and continuing for the next 7 days. (A) Survival differences between drug- and vehicle-treated animals were measured using a log-rank (Mantel-Cox) test. (B) Similarly, the proportion of mice that either developed mild or no hind limb paralysis following NSV challenge was determined in each group, and differences between drug- and vehicle-treated animals determined by a log-rank (Mantel-Cox) test.

Figure 4

Virological effects of the indole enantiomers, 9g and 9h, in mice with acute NSV encephalomyelitis. Cohorts of NSV-infected mice were treated with 9g or 9h (30 mg/kg/dose) or with a vehicle control by intraperitoneal injection every 12 hours beginning 12 hours after virus challenge. At 24-hour intervals, viral titers were measured in quadruplicate brain (A) and spinal cord (B) tissue samples from each group by plaque titration assay. Statistical differences (*p<0.05) in tissue viral loads were determined by an unpaired Student’s t-test compared to vehicle-treated controls.

Figure 5

Effects of the indole enantiomers, 9g and 9h, on neuronal survival in the brains of NSV-infected mice. The anatomically-defined hippocampus, a site of heavy NSV infection, was chosen for further study. (A) Representative fluorojade staining of damaged hippocampal neurons in the brains of NSV-infected mice. NisslRed was first used to label neurons in sections through the hippocampus, while fluorojade staining (arrows) was then performed to identify those cells undergoing active degeneration. (B) The proportion of fluorojade-positive hippocampal neurons was determined in quadruplicate slides prepared from 3 mice in each group. Statistical differences (*p<0.05) in the number of labeled (injured) neurons were determined by an unpaired Student’s t-test compared to vehicle-treated control animals.

Figure 6

Effects of the indole enantiomers, 9g and 9h, on neuronal survival in the spinal cords of NSV-infected mice. Silver staining of tissue sections prepared from the lumbar spinal column was used to identify the ventral nerve roots and the motor neuron axons they carry. (A) A representative specimen from an uninfected animal is shown, with the L4 and L5 ventral nerve roots identified (marked with an *). (B) Quantification of axonal density in these lumbar ventral nerve roots shows relative neuronal sparing in 9h-treated animals compared to vehicle- or 9g-treated controls. Statistical differences (*p<0.05) were determined using unpaired Student’s t-tests.

Scheme 1

Preparation of Thieno[3,2-b]pyrrole Analogs of 1a ^aReagents and Conditions: (a) K₂CO₃, DMF, R¹Cl or Ph₃P, DIAD, R¹OH, THF. (b) KOH, aq EtOH, 40 °C. (c) ethyl isonipecotate, EDC, HOBt, DIEA, DCM, RT. (d) HNR²R³, EDC, HOBt, DIEA, RT.

Scheme 2

Preparation of Indole Analogs of 1a ^aReagents and Conditions: (a) K₂CO₃, DMF, R¹Cl, 60 °C. (b) LiOH, aq THF, 60 °C. (c) ethyl isonipecotate, EDC, HOBt, DIEA, DMF, RT. (d) HNR²R³, EDC, HOBt, DIEA, RT.