Synthesis and biological evaluation of novel flexible nucleoside analogues that inhibit flavivirus replication in vitro

Abstract

Flaviviruses, such as Dengue (DENV) and Zika (ZIKV) viruses, represent a severe health burden. There are currently no FDA-approved treatments, and vaccines against most flaviviruses are still lacking. We have developed several flexible analogues ("fleximers") of the FDA-approved nucleoside Acyclovir that exhibit activity against various RNA viruses, demonstrating their broad-spectrum potential. The current study reports activity against DENV and Yellow Fever Virus (YFV), particularly for compound 1. Studies to elucidate the mechanism of action suggest the flex-analogue triphosphates, especially 1-TP, inhibit DENV and ZIKV methyltransferases, and a secondary, albeit weak, effect on the DENV RNA-dependent RNA polymerase was observed at high concentrations. The results of these studies are reported herein.

Keywords: Acyclovir; Dengue; Flavivirus; Fleximers; Methyltransferase; Nucleoside; Yellow Fever; Zika.

Graphical abstract

Fig. 1

General structure of the flavivirus genome including 5′ and 3′ untranslated regions and the polyprotein processing of both the structural and nonstructural protein regions.,

Fig. 2

Conserved flavivirus 5′-cap structure., , , ,

Fig. 3

Early examples of antiflaviviral nucleoside inhibitors.

Fig. 4

Structure of proximal and distal guanosine fleximers compared to natural guanosine., , ,

Fig. 5

Structure of the target fleximer analogues compared to the parent analogue Acyclovir.

Scheme 1

Reagents and conditions: (a) 4,5-diiodoimidazole, BSA, TMSOTf, ACN, rt for 4 h then 80 °C for 18 h; (b) 30% EtOH, 5 eq Na₂SO₃, 120 °C, overnight.

Scheme 2

Reagents and conditions: (a) DIPEA, 10% Pd/C, H₂, rt, 4 h; (b) NBS, CHCl₃, rt, dark, 5 h; (c) Br₂, NaHCO₃, 50% MeOH, rt, 3 h; (d) pin₂B₂, KOAc, Pd(PPh₃)₄, DME, 90 °C, overnight; (e) 5, Pd(PPh₃)₄, NaHCO₃, 90 °C, 4 h; (f) 1 or 2, Ac₂O, DMAP, DMF, rt, 3 h.

Scheme 3

Reagents and conditions: (a) 1 or 2, tBuMgCl, THF, rt, overnight.

Scheme 4

Reagents and conditions: (a) i. 1 or 2, SalPCl, Pyr, 1,4-dioxane, rt, 45 min; ii. tributyl ammonium pyrophosphate, tributylamine, DMF, rt, 45 min; iii. I₂, H₂O, Pyr, rt, 30 min.

Fig. 6

Effect of increasing concentrations of 1-TP (2MR04) on DENV polymerase activity. An elongation complex was formed of DENV NS5 and a primer/template combination corresponding to the 5’ end of DENV2 genome and the 3’ end of the antigenome. Primer elongation reactions were done in presence of all NTPs or in absence of GTP to test the incorporation of 1-TP as a GTP analogue. Reaction mixtures were analyzed by denaturating PAGE and substrate (P10) and product bands were visualized by autoradiography.

Fig. 7

Percent inhibition of ZIKV and DENV MTase by series 1 and 2 (50 µM). None of the compounds inhibited human N7 MTase, suggesting that these analogues selectively inhibit the viral MTases.

Fig. 8

Predicted binding of A) 1-TP (carbon atoms in light blue) and B) 2-TP (carbon atoms in pink) to the GTP pocket of ZIKV NS5 MTase (PDB ID 5GOZ). Co-crystallized GTP is shown in light grey.

Fig. 9

Predicted binding of A) 1-TP (carbon atoms in light blue) and B) 2-TP (carbon atoms in pink) to the GTP pocket of DENV NS5 MTase (PDB ID 4V0R). Co-crystallized GTP is shown in light grey.

Fig. 10

Predicted binding of A) 1-TP (carbon atoms in light blue) and B) 2-TP (carbon atoms in pink) to the GTP pocket of YFV NS5 MTase (PDB ID 3EVD). Co-crystallized GTP is shown in light grey.

Fig. 11

Predicted binding of A) 1-TP (carbon atoms in ight blue) and B) 2-TP (carbon atoms in pink) to the GTP pocket of human mRNA cap guanine-N7 MTase GTP binding site (PDB ID 5E9W). GTP is shown in light grey.