Suramin interacts with the positively charged region surrounding the 5-fold axis of the EV-A71 capsid and inhibits multiple enterovirus A

Abstract

Suramin was previously shown to bind to the EV-A71 capsid through its naphthalenetrisulfonic acid groups, thereby reducing virus-cell binding and inhibiting viral replication. Here, we identify VP1-145 as the critical amino acid that accounts for the differential sensitivity of EVA-71 viruses to suramin. A single Q or G to E substitution at VP1-145 results in an approximately 30-fold shift of IC₅₀ or IC₉₀ values reproducing the inhibition profile observed with field isolates expressing either the 145Q or E mutation. Our data support the conclusion that suramin binds to the positively charged region surrounding the 5-fold axis of the capsid and consequently blocks the virus attachment and entry into host cells. In order to assess the antiviral-spectrum of suramin, we analyzed 18 representative enteroviruses: A (n = 7), B (n = 5), C (n = 5) and D (n = 1). We show that suramin potency is restricted to enterovirus A species. Clinical development of suramin is further supported by pharmacokinetic data demonstrating bioactive plasma levels after a single dose intramuscular administration in macaques. Altogether, our findings support the clinical development of suramin as a novel entry inhibitor for the treatment of enterovirus A infections.

Figure 1. VP1-145 is responsible for the sensitivity of EV-A71 to suramin.

(A) The inhibitions of suramin against the EV-A71 Fuyang 573, SH12-276 and 145 mutant viruses were measured using quantitative RT-PCR of the genome RNA. Vero cells were infected with different EV-A71 isolates or mutants at a MOI of 0.01 in the presence of 0–1000 μM suramin. After 46–48 hours post-infection, viral RNA was extracted from culture supernatants and quantified by quantitative RT-PCR. (B) The inhibition of suramin against EV-A71 Fuyang 573, SH12-276 and 145 mutant viruses was measured using a plaque assay. Vero cells were infected with EV-A71 in 12-well plates with 20–40 PFU/well in 0–256 μM suramin. The plaques were stained and counted after incubation for 7 days in 0.8% CMC-DMEM containing 0–256 μM suramin. The experiments were conducted in triplicate.

Figure 2. Suramin inhibition against HEVs.

(A) CV-A2, (B) CV-A3, (C) CV-A10, (D) CV-A12 and (E) CV-A16 are HEV-A. (F) CV-A9, (G) ECHO20 and (H) ECHO25 are HEV-B. (A,B,D,F,G,H). Inhibition was evaluated by quantitative RT-PCR. Vero cells were infected with enteroviruses at a MOI of 0.1 in 0–1000 μM suramin. The culture supernatant was collected at 46–48 hours post-infection, the RNA was extracted and the genome copy was determined by quantitative RT-PCR. (C) Inhibition against CV-A10 was tested using a plaque assay. Vero cells were infected in 12-well plates with 25 PFU/well in 0–100 μM suramin. The plaques were stained and counted after incubation for 7 days in 0.8% CMC-DMEM containing 0–100 μM suramin. (E) Inhibition against CV-A16 was evaluated in RD cells by CPE-based TCID₅₀ titration according to a previous study. For CV-A2, 3 and 12, each suramin concentration has only one read. Tests on CV-A10, CV-A16, CV-A9, ECHO20 and ECHO25 were performed in duplicate.

Figure 3. Suramin potency is correlated with HEV genotype.

The phylogenetics assay was performed on VP1 protein sequences from representative HEVs using ClustalW, and the results were visualized using MEGA5. The red branch indicates HEV-A, the blue branch indicates HEV-B, the green branch indicates HEV-C and the black branch indicates HEV-D. Viruses labeled with red dots can be inhibited by suramin, and viruses labeled with black dots are resistant to suramin. Suramin potency was not examined in the present study for the viruses without a circle.

Figure 4. Suramin PK after IM injection.

Three male adult cynomolgus monkeys were used to observe the plasma suramin distribution and metabolism. After a single dose injection of 46.5 mg/kg suramin in the muscle, the plasma suramin concentration was monitored by LC-MS/MS from 15 min until 168 hours.