Dengue virus (DENV) is a mosquito-borne flavivirus that exists in four distinct serotypes and can cause severe disease and death. Antiviral drugs to specifically treat DENV infections are not currently available. In a new study, researchers at Novartis report the development of a small molecule that is effective against all four DENV serotypes in vitro and shows promising efficacy and safety in animal models.
The need for pan-serotype efficacy while maintaining favourable drug-like properties has presented a challenge for drug discovery efforts against DENV so far. In the current study, Moquin et al. undertook phenotypic screening, rather than a target-based approach, to ensure they identified broad-spectrum anti-DENV activity at an early stage in the drug development programme.
Following an initial screen of 1.5 million compounds for their ability to prevent cytopathic effects in cell lines infected with DENV serotype 2 (DENV2), the investigators selected the 160 most potent hits for further screening against DENV1–DENV4 in vitro. They also performed a counter screen against chikungunya virus to help improve selectivity for DENV and to avoid compounds that target host proteins. Further narrowing of the selected compounds on the basis of toxicity and specificity, followed by structure–activity relationship optimization, produced NITD-688.
This compound, a tetrahydrothienopyridine, showed high potency against all four DENV serotypes in vitro, as well as good solubility. Moreover, NITD-688 was considerably less effective against six other flaviviruses, indicating DENV-specific targeting.
Next, the researchers sought to determine the target of the NITD-688 class of compounds. Serial passaging of DENV2-infected cell lines in the presence of increasing drug concentrations for 13 weeks led to the development of resistance in 5 of 11 lines. By sequencing the viral RNA, the authors showed that resistance mutations map to nonstructural protein 4B (NS4B), suggesting this protein as the drug target. Moreover, the mutations occurred at residues of NS4B (in transmembrane domains TM4 and TM5) that are highly conserved among DENV serotypes but not in other flaviviruses.
NMR spectroscopy also confirmed that NITD-688 directly binds wild-type but not mutant NS4B.
Notably, resistance to NITD-688 compounds was hard to induce experimentally, and examination of publicly available DENV sequences revealed resistance mutations have low prevalence in the environment. These findings suggest a potentially low risk of development of resistance to NITD-688, although widespread use should be monitored for clinical resistance.
Progressing to in vivo studies, the researchers tested NITD-688 in a nonlethal mouse model of DENV2 viraemia. Oral treatment with the drug twice daily for 3 days, starting at the time of infection, produced a significant, dose-dependent decrease in viraemia. Importantly, delaying treatment initiation by 48 hours still resulted in substantial DENV2 reduction, suggesting the utility of NITD-688 in a clinically relevant, post-infection setting.
The compound was shown to have favourable pharmacokinetic properties and good oral bioavailability in mice, rats and dogs. Using scaling algorithms and physiology-based simulation, Moquin et al. identified 35 mg as the equivalent daily dose in humans needed to maintain a minimum concentration of three times the half-maximal effective concentration (EC50).
Testing of NITD-688 in an extensive panel of safety assays largely did not show off-target activity. Exceptions, which will require further investigation, included inhibition of the cytochrome P450 enzyme CYP3A4, the cardiac potassium channel hERG and cyclooxygenase 1 (COX1). The compound was well tolerated over 7 days in rats and dogs, although high doses were associated with liver toxicity. Encouragingly, studies in dogs did not find cardiac toxicity or gastrointestinal ulceration, which commonly result from inhibition of hERG and COX1, respectively.
“We will continue to study the mechanism of action of NITD-688 and its interaction with NS4B in viral inhibition,” says Feng Gu, one of the study leads. They also hope to advance NITD-688 into clinical trials, he adds.
This study identifies a promising preclinical candidate for the treatment of DENV infection and showcases the benefit of phenotypic approaches for addressing the challenge of pan-serotype activity.
Nature Reviews Drug Discovery 20, 263 (2021)