Ring-Expanded (“Fat”) Nucleoside and Nucleotide Analogues Exhibit Potent in Vitro Activity against Flaviviridae NTPases/Helicases, Including Those of the West Nile Virus, Hepatitis C Virus, and Japanese Encephalitis Virus

Abstract

A series of ring-expanded ("fat") heterocycles, nucleoside and nucleotide analogues (RENs) containing the imidazo[4,5-e][1,3]diazepine ring system (9, 14, 15, 18, 24-26, 28, 31, and 33) and imidazo[4,5-e][1,2,4]triazepine ring systems (30b, 30c, 32, and 34), have been synthesized as potential inhibitors of NTPases/helicases of Flaviviridae, including the West Nile virus (WNV), hepatitis C virus (HCV), and Japanese encephalitis virus (JEV). An amino-terminal truncated form of human enzyme Suv3(delta1-159) was also included in the study so as to assess the selectivity of RENs against the viral enzymes. The analogues of RENs included structural variations at position 1 of the heterocyclic base and contained changes in both the type of sugar moieties (ribo, 2'-deoxyribo, and acyclic sugars) and the mode of attachment (alpha versus beta anomeric configuration) of those sugars to the heterocyclic base. The target RENs were biochemically screened separately against the helicase and ATPase activities of the viral NTPases/helicases. A number of RENs inhibited the viral helicase activity with IC50 values that ranged in micromolar concentrations and exhibited differential selectivity between the viral enzymes. In view of the observed tight complex between some nucleosides and RNA and/or DNA substrates of a helicase, the mechanism of action of RENs might involve their interaction with the appropriate substrate through binding to the major or minor groove of the double helix. The REN-5'-triphosphates, on the other hand, did not influence the above unwinding reaction, but instead exerted the inhibitory effect on the ATPase activity of the enzymes. The activity was found to be highly dependent upon the low concentration levels of the substrate ATP. At concentrations >500 microM of RENs and the ATP concentrations >10 times the Km value of the enzyme, a significant activation of NTPase activity was observed. This activating effect underwent further dramatic enhancement (>1000%) by further increases in ATP concentration in the reaction mixture. A tentative mechanistic model has been proposed to explain the observed results, which includes an additional allosteric binding site on the viral NTPases/helicases that can be occupied by nucleoside/nucleotide-type molecules such as RENs.

Understanding Helicases as a Means of Virus Control

“…Such compounds would not competitively inhibit ATP hydrolysis, but would modulate ATP hydrolysis and/or unwinding by binding an allosteric site. Several such compounds recently have been reported [186][187][188]. Representatives are shown along with HMC-HO4 in Fig.…”

“…[189] for a review), have also recently been examined as potential inhibitors of the WNV, JEV, and HCV helicases [187,188]. At least five different RENs inhibit WNV, one inhibits JEV, but none inhibit HCV helicase catalyzed DNA or RNA unwinding.…”

RNA helicases in infection and disease

“…220,221 Ring-expanded nucleoside and nucleotide analogs have been shown to selectively inhibit HCV, West Nile Virus, and Japanese encephalitis virus NS3 helicases in vitro without affecting the activity of a control host helicase. 222,223 Interestingly, different derivatives of these compounds showed differential effects on the three viral helicases tested, suggesting that selective inhibition is possible despite their structural similarity. The mechanism of inhibition by these and other nucleosides may involve an unidentified allosteric nucleoside binding site on the helicases.…”

Tropolone and its Derivatives as Inhibitors of the Helicase Activity of Hepatitis C Virus Nucleotide Triphosphatase/helicase

“…Surprisingly, at K m values and higher ATP concentrations, no inhibition and even activation was observed (Borowski et al, 2000;Zhang et al, 2003). This suggests that all these compounds interact with the ATP-binding site and with an undefined allosteric site.…”