Novel derivatives of brincidofovir and (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine inhibit orthopoxviruses and human adenoviruses more potently than brincidofovir

Abstract

Brincidofovir (BCV) and tecovirimat are the only two chemical drugs that have been approved to treat smallpox and can be requested for monkeypox (Mpox) treatment through a single-patient Emergency Investigational New Drug (EIND) application. Disappointedly, the efficacy of tecovirimat manifested in recent clinical trials is far from being satisfactory, while the clinical efficacy of BCV is still inconclusive. Given that monkeypox virus (MPXV), variola and other emerging orthopoxviruses are posing serious threats to global health, it is urgent to develop better therapeutics. In this study, we tested the antiviral effects of three novel prodrugs, which were designed based on previously reported parent drugs, either (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine ((S)-HPMPC, cidofovir) or (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine ((S)-HPMPA). We found that one of the (S)-HPMPA-based prodrugs, ODE-(S)-HPMPA formate, exhibited significantly better anti-orthopoxvirus activity than BCV both in vitro and in vivo, which also inhibited human adenovirus type 2 and type 21 more efficiently than BCV. Most strikingly, the EC₅₀ and EC₉₀ of ODE-(S)-HPMPA formate against MPXV were more than 40-fold lower than those of BCV. In contrast, we observed that the anti-herpes simplex virus type 1 (HSV-1) activities of the (S)-HPMPA-based prodrugs were less effective than those of the cidofovir-based prodrugs (BCV and BCV formate), especially in vivo. Moreover, we showed for the first time that cytidine and adenine analog combined therapies could provide mice with complete protection against lethal challenges of both vaccinia and HSV-1. Collectively, we propose that both the ODE-(S)-HPMPA formate and the BCV/ODE-(S)-HPMPA formate combination are worth further investigations for their potential clinical applications.

Fig. 1

In vitro cytotoxicity and in vivo pharmacokinetic analyses of BCV and the candidate compounds. a The molecular structures and the theoretical metabolic pathways of BCV and the newly synthesized prodrugs. After two steps of hydrolysis, BCV formate can be converted to the same active parent drug (CDV) with BCV. ODE-(S)-HPMPA formate and HDP-(S)-HPMPA formate also take two steps of hydrolysis to turn into (S)-HPMPA. b Comparisons of cytotoxicities between candidate compounds and BCV. Limited by the solubility of BCV formate, ODE-(S)-HPMPA formate and HDP-(S)-HPMPA formate, the highest concentration employed in the cytotoxicity assay was 40 μM. Data are presented as mean ± SD. c Comparisons of mitochondrial toxicity between candidate compounds and BCV. The solvent and CCCP served as negative and positive control, respectively. d, e Plasma concentrations of BCV, BCV formate, ODE-(S)-HPMPA formate and HDP-(S)-HPMPA formate, as well as their respective one-step hydrolytic metabolites CDV, BCV, ODE-(S)-HPMPA and HDP-(S)-HPMPA, were measured at various time points following a single oral gavage dose (3 mice per group, the mice were administered 10 ml/kg of each prodrug at a concentration of 7.12 µmol/ml). f, g Plasma concentrations of BCV, ODE-(S)-HPMPA, and HDP-(S)-HPMPA, as well as their hydrolytic metabolites (CDV and (S)-HPMPA) were measured at various time points following a single oral gavage dose (3 mice per group, the mice were administered 10 ml/kg of each prodrug at a concentration of 7.12 µmol/ml). Data are presented as mean ± SD. Statistical significance is indicated as follows: *p < 0.05; **p < 0.01; ****p < 0.0001

Fig. 2

In vitro evaluations of anti-vaccinia and anti-MPXV activities of BCV and the candidate compounds. Vero cells were infected with vaccinia Tiantan strain (a) or MPXV (b) and treated with the indicated concentrations of BCV or the candidate prodrugs. All compounds were tested in triplicated wells at each concentration. The solvent was used as the non-treated control. The antiviral effects were determined by calculating the reduction in plaque formation. Data are presented as mean ± SD

Fig. 3

ODE-(S)-HPMPA formate and HDP-(S)-HPMPA formate showed superior therapeutic efficacies against lethal Tiantan vaccinia infection in mice. a The schematic illustration of experimental design. b Weight loss and survival rates of mice after being intranasally infected with the vaccinia Tiantan strain. Mice received the solvent served as the non-treated control. Weight loss ≥30% of initial body weight was recorded as ethical death and was therefore not represented in the figure. The data of weight loss are presented as mean ± SD. Statistical significance is indicated as follows: *, $, #: p < 0.05; **, $$, ##: p < 0.01; ***, $$$, ###: p < 0.001; ****, $$$$, ####: p < 0.0001

Fig. 4

ODE-(S)-HPMPA formate and HDP-(S)-HPMPA formate more effectively reduced lung vaccinia virus titers and mitigated pathological severity compared to BCV and BCV formate. a The schematic illustration of experimental design. b–g Viral titers in the lung, spleen, kidney, brain, testis, and liver were measured by plaque assays at 3, 5, 7, 10, and 14 days post-infection, respectively. h Histopathological examination of lung tissue at 7 days post infection. Black outlines indicate areas of observed pathology, and the percentages of the lesion area relative to the total area of the tissue are denoted in red

Fig. 5

Evaluations of in vivo and in vitro anti-HSV-1 potencies of BCV and the candidate prodrugs. a Vero cells were infected with HSV-1 stain 17 and treated with indicated concentrations of BCV or the candidate compounds. All compounds were tested in triplicated wells at each concentration. The solvent was used as the non-treated control. The antiviral effects were determined by calculating the reduction in plaque formation. Data are presented as mean ± SD. b The schematic illustration of in vivo experiment design. c Weight loss and survival rates of mice after being intraperitoneally infected with HSV-1. Mice received the solvent served as the non-treated control. The data of weight loss are presented as mean ± SD. Statistical significance is indicated as follows: *, $, #: p < 0.05; **, $$, ##: p < 0.01; ***, $$$, ###: p < 0.001; ****, $$$$, ####: p < 0.0001

Fig. 6

In vitro evaluation of anti-hAdV activities of BCV and the candidate compounds. Vero cells were infected with either hAdV C2 (a) or hAdV B21 (b) and subsequently treated with indicated concentrations of BCV or the candidate prodrugs. All compounds were tested in quadruplicate at each concentration. The solvent was used as the non-treated control. Data are presented as mean ± SD