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Recent cases of severe toxicity during clinical trials have been associated with antiviral ribonucleoside analogs (e.g. INX-08189 and balapiravir). Some have hypothesized that the active metabolites of toxic ribonucleoside analogs, the triphosphate forms, inadvertently target human mitochondrial RNA polymerase (POLRMT), thus inhibiting mitochondrial RNA transcription and protein synthesis. Others have proposed that the prodrug moiety released from the ribonucleoside analogs might instead cause toxicity. Here, we report the mitochondrial effects of several clinically relevant and structurally diverse ribonucleoside analogs including NITD-008, T-705 (favipiravir), R1479 (parent nucleoside of balapiravir), PSI-7851 (sofosbuvir), and INX-08189 (BMS-986094). We found that efficient substrates and chain terminators of POLRMT, such as the nucleoside triphosphate forms of R1479, NITD-008, and INX-08189, are likely to cause mitochondrial toxicity in cells, while weaker chain terminators and inhibitors of POLRMT such as T-705 ribonucleoside triphosphate do not elicit strong in vitro mitochondrial effects. Within a fixed 3’-deoxy or 2′-C-methyl ribose scaffold, changing the base moiety of nucleotides did not strongly affect their inhibition constant (Ki) against POLRMT. By swapping the nucleoside and prodrug moieties of PSI-7851 and INX-08189, we demonstrated that the cell-based toxicity of INX-08189 is mainly caused by the nucleoside component of the molecule. Taken together, these results show that diverse 2′ or 4′ mono-substituted ribonucleoside scaffolds cause mitochondrial toxicity. Given the unpredictable structure-activity relationship of this ribonucleoside liability, we propose a rapid and systematic in vitro screen combining cell-based and biochemical assays to identify the early potential for mitochondrial toxicity.

Human respiratory syncytial virus (RSV) is a negative-sense RNA virus and a significant cause of respiratory infection in infants and the elderly. No effective vaccines or antiviral therapies are available for the treatment of RSV. ALS-8176 is a first-in-class nucleoside prodrug inhibitor of RSV replication currently under clinical evaluation. ALS-8112, the parent molecule of ALS-8176, undergoes intracellular phosphorylation, yielding the active 5'-triphosphate metabolite. The host kinases responsible for this conversion are not known. Therefore, elucidation of the ALS-8112 activation pathway is key to further understanding its conversion mechanism, particularly given its potent antiviral effects. Here, we have identified the activation pathway of ALS-8112 and show it is unlike other antiviral cytidine analogs. The first step, driven by deoxycytidine kinase (dCK), is highly efficient, while the second step limits the formation of the active 5'-triphosphate species. ALS-8112 is a 2'- and 4'-modified nucleoside analog, prompting us to investigate dCK recognition of other 2'- and 4'-modified nucleosides. Our biochemical approach along with computational modeling contributes to an enhanced structure-activity profile for dCK. These results highlight an exciting potential to optimize nucleoside analogs based on the second activation step and increased attention toward nucleoside diphosphate and triphosphate prodrugs in drug discovery.

Norovirus (NoV) is a positive-sense single-stranded RNA virus that causes acute gastroenteritis and is responsible for 200,000 deaths per year worldwide. No effective vaccine or treatment is available. Recent studies have shown that the nucleoside analogs favipiravir (T-705) and 2′- C -methyl-cytidine (2CM-C) inhibit NoV replication in vitro and in animal models, but their precise mechanism of action is unknown. We evaluated the molecular interactions between nucleoside triphosphates and NoV RNA-dependent RNA polymerase (NoVpol), the enzyme responsible for replication and transcription of NoV genomic RNA. We found that T-705 ribonucleoside triphosphate (RTP) and 2CM-C triphosphate (2CM-CTP) equally inhibited human and mouse NoVpol activities at concentrations resulting in 50% of maximum inhibition (IC 50 s) in the low micromolar range. 2CM-CTP inhibited the viral polymerases by competing directly with natural CTP during primer elongation, whereas T-705 RTP competed mostly with ATP and GTP at the initiation and elongation steps. Incorporation of 2CM-CTP into viral RNA blocked subsequent RNA synthesis, whereas T-705 RTP did not cause immediate chain termination of NoVpol. 2CM-CTP and T-705 RTP displayed low levels of enzyme selectivity, as they were both recognized as substrates by human mitochondrial RNA polymerase. The level of discrimination by the human enzyme was increased with a novel analog of T-705 RTP containing a 2′- C -methyl substitution. Collectively, our data suggest that 2CM-C inhibits replication of NoV by acting as a classic chain terminator, while T-705 may inhibit the virus by multiple mechanisms of action. Understanding the precise mechanism of action of anti-NoV compounds could provide a rational basis for optimizing their inhibition potencies and selectivities.

ALS-8112 is the parent molecule of ALS-8176, a first-in-class nucleoside analog prodrug effective in the clinic against respiratory syncytial virus (RSV) infection. The antiviral activity of ALS-8112 is mediated by its 5'-triphosphate metabolite (ALS-8112-TP, or 2'F-4'ClCH2-cytidine triphosphate) inhibiting the RNA polymerase activity of the RSV L-P protein complex through RNA chain termination. Four amino acid mutations in the RNA-dependent RNA polymerase (RdRp) domain of L (QUAD: M628L, A789V, L795I, and I796V) confer in vitro resistance to ALS-8112-TP by increasing its discrimination relative to natural CTP. In this study, we show that the QUAD mutations specifically recognize the ClCH2 group of ALS-8112-TP. Among the four mutations, A789V conferred the greatest resistance phenotype, which was consistent with its putative position in the active site of the RdRp domain. AZ-27, a non-nucleoside inhibitor of RSV, also inhibited the RdRp activity, with decreased inhibition potency in the presence of the Y1631H mutation. The QUAD mutations had no effect on the antiviral activity of AZ-27, and the Y1631H mutation did not significantly increase the discrimination of ALS-8112-TP. Combining ALS-8112 with AZ-27 in vitro resulted in significant synergistic inhibition of RSV replication. Overall, this is the first mechanistic study showing a lack of cross-resistance between mutations selected by different classes of RSV polymerase inhibitors acting in synergy, opening the door to future potential combination therapies targeting different regions of the L protein.

Successful gene-targeting reagents must be functional under physiological conditions and must bind chromosomal target sequences embedded in chromatin. Triple helix-forming oligonucleotides (TFOs) recognize and bind specific sequences via the major groove of duplex DNA and may have potential for gene targeting in vivo. We have constructed chemically modified, psoralen-linked TFOs that mediate site-specific mutagenesis of a chromosomal gene in living cells. Here we show that targeting efficiency is sensitive to the biology of the cell, specifically, cell cycle status. Targeted mutagenesis was variable across the cycle with the greatest activity in S phase. This was the result of differential TFO binding as measured by cross-link formation. Targeted cross-linking was low in quiescent cells but substantially enhanced in S phase cells with adducts in approximately 20-30% of target sequences. 75-80% of adducts were repaired faithfully, whereas the remaining adducts were converted into mutations (>5% mutation frequency). Clones with mutations could be recovered by direct screening of colonies chosen at random. These results demonstrate high frequency target binding and target mutagenesis by TFOs in living cells. Successful protocols for TFO-mediated manipulation of chromosomal sequences are likely to reflect a combination of appropriate oligonucleotide chemistry and manipulation of the cell biology.

Ribonucleotide analog inhibitors of the RNA-dependent RNA polymerase of hepatitis C virus (HCV) represent one of the most exciting recent developments in HCV antiviral therapy. Although it is well established that these molecules cause chain termination by competing at the triphosphate level with natural nucleotides for incorporation into elongating RNA, strategies to rationally optimize antiviral potency based on enzyme kinetics remain elusive. In this study, we used the isolated HCV polymerase elongation complex to determine the pre-steady-state kinetics of incorporation of 2′F-2′C-Me-UTP, the active metabolite of the anti-HCV drug sofosbuvir. 2′F-2′C-Me-UTP was efficiently incorporated by HCV polymerase with apparent K d (equilibrium constant) and k pol (rate of nucleotide incorporation at saturating nucleotide concentration) values of 113 ± 28 μM and 0.67 ± 0.05 s −1 , respectively, giving an overall substrate efficiency ( k pol / K d ) of 0.0059 ± 0.0015 μM −1 s −1 . We also measured the substrate efficiency of other UTP analogs and found that substitutions at the 2′ position on the ribose can greatly affect their level of incorporation, with a rank order of OH > F > NH 2 > F-C-Me > C-Me > N 3 > ara. However, the efficiency of chain termination following the incorporation of UMP analogs followed a different order, with only 2′F-2′C-Me-, 2′C-Me-, and 2′ara-UTP causing complete and immediate chain termination. The chain termination profile of the 2′-modified nucleotides explains the apparent lack of correlation observed across all molecules between substrate efficiency at the single-nucleotide level and their overall inhibition potency. To our knowledge, these results provide the first attempt to use pre-steady-state kinetics to uncover the mechanism of action of 2′-modified NTP analogs against HCV polymerase.

The results obtained in our laboratory on investigating of substrate properties of a large number of compounds towards different DNA polymerases have been summarized. On the basis of systematic analysis a directed synthesis of nucleotides with antiviral properties was performed.

In our search for improved therapeutic agents against HCV we synthesized 7-deaza-7-ethynyl-2'-C-methyladenosine (1) and its 2'-deoxy-2'-fluoro analogue 2. The corresponding nucleoside triphosphates were efficient chain terminators of the HCV NS5b polymerase with IC(50)'s of 0.75 microM and 0.4 microM respectively. However, only the ribo-nucleoside 1 exhibited activity in a Huh7 cell based replicon assay with an EC(50) of 0.09 microM. In order to overcome the lack of activity of the fluoro analogue 2 we synthesised several phosphoroamidate prodrugs.

Several 2'-deoxythymidine 5'-triphosphate and 3'-azido-2', 3'-dideoxythymidine 5'-triphosphate analogs containing a hydrophobic phosphonate group instead of the gamma-phosphate were synthesized and evaluated as substrates for human immunodeficiency virus (HIV) and avian myeloblastosis virus reverse transcriptases, human placental DNA polymerases alpha and beta, and calf thymus terminal deoxynucleotidyl transferase. They were efficiently incorporated into the DNA chain by the retroviral enzymes but were not utilized by the mammalian ones. Also, some gamma-ester and gamma-amide derivatives of dTTP and 3'-azido-2',3'-dideoxythymidine 5'-triphosphate (AZTTP) were synthesized and studied. They proved to be substrates for both the retroviral and mammalian enzymes under study. The Km values for incorporation of the dTTP derivatives into the DNA chain were close to those for dTTP and AZTTP. The Km for the AZTTP derivatives were one order of magnitude greater than those for dTTP and AZTTP. The results obtained indicate that HIV and avian myeloblastosis virus reverse transcriptases have no sterical obstacles for binding the triphosphate fragment bearing a bulky substituent at the gamma-position. Modification of the gamma-phosphate in AZTTP increased the selectivity of HIV reverse transcriptase inhibition versus DNA polymerase alpha. gamma-Methylphosphonate and gamma-phenylphosphonate were dephosphorylated in human serum much less rapidly than AZTTP. Besides, they were shown to be markedly more hydrophobic than AZTTP. Thus, replacement of the gamma-phosphate in AZTTP with gamma-phosphonate markedly alters its substrate properties toward some cellular DNA polymerases and blood dephosphorylating enzymes but does not change its substrate activity with respect to HIV reverse transcriptase.

The substrate properties of four carbocyclic D and L nucleoside 5'-triphosphate analogs toward HIV and AMV reverse transcriptases and terminal deoxynucleotidyl transferase were evaluated. The compounds of the D-beta and L-beta series were found to be terminating substrates for these enzymes, while the derivatives of the D-alpha and L-alpha series were recognized only by terminal deoxynucleotidyl transferase, suggesting that for the template-independent enzyme the mutual orientation of the two fragments is of no significance. A hypothesis for binding of nucleotides to the DNA polymerase active center was proposed.

2′-3′-Dideoxy-2′,3′-dehydrothymidine 5′-triphosphate (dddTTP) shows termination substrate properties in the DNA synthesis catalyzed by E. coli DNA polymerase I KF, rat liver DNA polymerase β, reverse transcriptases of avian myeloblastosis virus and Raus sarcoma virus and calf thymus terminal deoxynucleotidyl transferase. This implies that the mononucleotide residue of dddTTP incorporates into 3′-termini of newly synthesized DNA chains. However, dddTTP has no influence on the DNA synthesis catalyzed by calf thymus DNA polymerase α. In the case of some DNA polymerases dddTTP was one order of magnitude more effective in comparison with the other known termination substrates.