Activation Pathway of a Nucleoside Analog Inhibiting Respiratory

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Activation Pathway of a Nucleoside Analog Inhibiting RSV Polymerase Paul C. Jordan, Sarah K. Stevens, Yuen Tam, Ryan P. Pemberton, Shuvam Chaudhuri, Antitsa D. Stoycheva, Natalia Dyatkina, Guangyi Wang, Julian A. Symons, Jerome Deval, and Leo Beigelman ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b00788 • Publication Date (Web): 14 Nov 2016 Downloaded from http://pubs.acs.org on November 19, 2016

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ACS Chemical Biology

Activation Pathway of a Nucleoside Analog Inhibiting RSV Polymerase Paul C. Jordan,1 Sarah K. Stevens,1 Yuen Tam,1 Ryan P. Pemberton,1 Shuvam Chaudhuri,1 Antitsa D. Stoycheva,1 Natalia Dyatkina,1 Guangyi Wang,1 Julian A. Symons,1 Jerome Deval,*,1,§ and Leo Beigelman1,§ 1

Alios BioPharma, Inc., part of the Janssen Pharmaceutical Companies, South San Francisco, CA, USA *To whom correspondence should be addressed: [email protected] § These authors contributed equally to this work. ABSTRACT 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 understand 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. INTRODUCTION Human respiratory syncytial virus (RSV) is a single-stranded, negative-sense RNA virus and is a member of the family Pneumoviridae (previously a subfamily within the Paramyxoviridae) in the Mononegavirales order.1 RSV is a significant cause of respiratory infections in the elderly and a significant contributor to infant mortality from viral respiratory disease.2, 3 The 15 kb RNA genome of RSV encodes for 11 proteins. Of these proteins, F (fusion protein), P (phosphoprotein), and L (large protein) are emerging antiviral targets.2 The L protein contains the RNA-dependent RNA-polymerase and the RNA capping domains responsible for the catalytic functions associated with viral genome replication and transcription. Current therapeutic options for RSV infections include palivizumab, a monoclonal antibody approved only for prophylaxis in high-risk patients and ribavirin, a small molecule with cytotoxic effects and limited efficacy.4, 5 The small treatment landscape underscores the pressing need for new antiviral therapies and has catalyzed the discovery and development of new therapeutics. Potential therapies under current clinical development target the inhibition of the F protein or the RNA-dependent RNA-polymerase domain of the L protein.6, 7 ALS-8176 is a first-in-class nucleoside analog under evaluation in infants and adults for the treatment of RSV.6, 8, 9 ALS-8176 is the 3′,5′ bisisobutyrate prodrug of 2'F-4'ClCH2-cytidine (ALS-8112), aimed to

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maximize oral bioavailability. Both ALS-8176 and ALS-8112 have previously been shown to inhibit in vitro and in vivo RSV replication.8, 10 In an adult human challenge study, ALS-8176 has shown efficacy against RSV infection.6 The 5'-triphosphate form of ALS-8112 (ALS-8112– TP) is the active form of the drug and selectively inhibits RSV polymerase through chain termination of RNA synthesis. The mechanism of conversion of ALS-8112 to ALS-8112-TP by human kinases is currently unknown. Further understanding of the nucleoside analog phosphorylation pathway, including bottlenecks in its efficiency, is key to optimizing the biological properties of potential nucleoside analog therapies.11, 12 A central goal of this work was to identify the kinases involved in converting ALS-8112 to its active 5'-triphosphate form. We hypothesized that deoxycytidine kinase (dCK), uridine-cytidine kinase 1 (UCK-1), and uridine-cytidine kinase 2 (UCK-2) are the likely kinases involved in the first phosphorylation step.13 These enzymes, along with several additional kinases, are involved in the salvage and de novo pathway of nucleosides.14 dCK phosphorylates a broad range of nucleosides and their analogs to their monophosphate forms.15 Using enzymatic assays and selective dCK inhibitors, we identified dCK as a significant contributor but not the rate-limiting step in ALS-8112 activation. Using a series of 2'- and 4'-modified nucleosides, we also developed a structure-activity profile using enzymatic assays to understand which chemical features account for the improved catalytic efficiency of ALS-8112 for dCK. The crystal structure of dCK in complex with deoxycytidine16 provided insights into key protein-substrate interactions. Finally, previous work demonstrated that human uridine monophosphate (UMP)–cytidine monophosphate (CMP) kinase (YMPK) and nucleoside diphosphate kinase (NDPK) are the primary kinases for the remaining two phosphorylation steps.17-19 This prompted us to probe the catalytic efficiency of YMPK and NDPK to show that phosphorylation by YMPK is the rate-limiting step in ALS-8112 activation. RESULTS AND DISCUSSION Phosphorylation of ALS-8112 by dCK, UCK-1, and UCK-2. The main enzymes implicated in the first phosphorylation step of cytidine analogs are dCK, UCK-1, and UCK-2.20 To understand the enzymes responsible for the first phosphorylation step of ALS-8112, we used a biochemical assay consisting of recombinant kinase (either dCK, UCK-1, or UCK-2) with the coupled enzymes pyruvate kinase, and the NADH-dependent lactate dehydrogenase (LDH). The NADH dependence of LDH allows for convenient reaction monitoring at 340 nm (Supplementary Figure 1). The steady-state kinetics of human dCK using natural cytidine, deoxycytidine, and ALS8112 (2'-fluoro-4'-chloromethyl cytidine) as substrates were measured. Cytidine and deoxycytidine were phosphorylated by dCK with reaction efficiencies (kcat/Km) of 3.1×10-3 and 3.5×10-3 µM-1s-1, respectively, consistent with previously reported values under similar assay conditions (Table 1).21 In contrast to cytidine, ALS-8112 was phosphorylated by dCK with an efficiency of 9.7×10-3 µM-1s-1, representing a 3-fold increase in efficiency compared with natural cytidine (Table 1). We cloned, expressed in Escherichia coli, and purified UCK-1 and UCK-2 to determine whether these kinases, in addition to dCK, also contribute to the first phosphorylation step. Cytidine was phosphorylated with efficiencies of 2.7×10-3 and 1.6×10-1 µM-1s-1 for UCK-1 and UCK-2, respectively, consistent with literature reports for these enzymes under similar reaction conditions (Table 1).21 Deoxycytidine was also tested as a substrate for UCK-1 and UCK-2 and was phosphorylated at efficiencies at undetectable levels in our assay, also consistent with


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literature reports (Table 1).13, 21 ALS-8112 was phosphorylated by UCK-1 and UCK-2 at low relative efficiencies: 3.2×10-5 and 1.1×10-4 µM-1s-1, respectively. These values for UCK-1 and UCK-2 represent relative reaction efficiencies compared with cytidine of 10% and 1%, respectively, which confirm that dCK is the primary enzyme responsible for converting ALS8112 to its monophosphate form. dCK Inhibitors Antagonize Phosphorylation and Antiviral Activity of ALS-8112. Our biochemical phosphorylation assays identify dCK as the significant contributor to ALS-8112 phosphorylation. This prompted us to further validate the role of dCK in cell-based studies using selective dCK inhibitors. We hypothesized that, if dCK is the major kinase responsible for ALS-8112-monophosphate (MP) formation, selective dCK inhibitors would reduce ALS-8112MP and ALS-8112-TP levels in cells. We chose two previously identified, selective dCK inhibitors and designated them as inhibitors 1 and 2 (Figure 1A).22, 23 Half-maximal inhibitory concentration (IC50) values for these inhibitors have been previously reported using both cell-based and biochemical techniques.22, 23 We validated these inhibitors in our enzymatic kinase assay using ALS-8112 as a substrate, demonstrating that inhibitors 1 and 2 prevented the formation of ALS-8112-MP by dCK, with half-maximal inhibitory concentration (IC50) values of 5.9 and 33 µM, respectively (Figure 1B). We also examined the effects of these inhibitors on UCK-1 and UCK-2 in biochemical assays. Importantly, inhibitors 1 and 2 did not inhibit UCK-1 or UCK-2 in enzymatic assays, verifying their selectivity for dCK (Supplementary Figure 2). Previous work has shown that UCKL1, an enzyme with sequence homology to UCK-1 and UCK-2 may play potential roles in the ribonucleotide salvage pathway and cell proliferation and survival.24, 25 Its exact biological function has not been fully elucidated and is not associated with the phosphorylation of anticancer or antiviral nucleoside analogs. Therefore, it was not examined in our biochemical or cell-based assays. In an effort to understand the effect of these dCK inhibitors on intracellular ALS-8112 phosphorylation, A549 cells were treated for 24 hours under three conditions: ALS-8112 alone, ALS-8112 + inhibitor 1, or ALS-8112 + inhibitor 2. Intracellular monophosphate, and triphosphate levels were measured using LC/MS/MS and quantitated using synthetic standards (Figure 1C and 1D; Supplementary Figure 3). Unsuccessful attempts were made to monitor intracellular ALS-8112-DP, possibly due to poor stability of this analyte and/or other unknown technical limitations. Levels of ALS-8112-MP and ALS-8112-TP were 621 ± 75 pmol ALS8112-MP/million cells and 145 ± 17 pmol ALS-8112-TP/million cells. These results contrast with those of cells treated with both ALS-8112 and dCK inhibitor. On average, cells treated with ALS-8112 + dCK inhibitor exhibited a 17-fold reduction in intracellular nucleoside monophosphate (NMP) and an 8.3-fold reduction in NTP compared with cells treated with ALS8112 alone (Figure 1E). Importantly, inhibitor 1 and 2 did not decrease the intracellular levels of ALS-8112, ruling out any possibility that these compounds might interfere with nucleoside transport (Figure 1E). In a similar fashion, we analyzed the effect of these dCK inhibitors on RSV replication in the presence of ALS-8112 at a non-toxic concentration of 10 µM (Supplementary Figure 4). 8, 9 RSV-RFP (red fluorescent protein) infected HEp-2 cells were treated with DMSO, ALS-8112 alone, ALS-8112 + inhibitor 1, or ALS-8112 + inhibitor 2. Fluorescence micrographs of infected cells (Figure 2A) show an 86% reduction of red fluorescence in ALS-8112-treated cells compared with RSV-RFP-infected cells without drug, indicative of strong antiviral activity. Dosing of ALS-8112 with either dCK inhibitor 1 or 2 showed the rescue of RSV-RFP infection



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by 123% and 90%, respectively (Figure 2B). Collectively, these biochemical and cellular results show that dCK inhibitors antagonize the phosphorylation and antiviral activity of ALS-8112, which confirms the essential role of dCK as the main kinase involved in the first step of ALS8112 phosphorylation. Profiling dCK Recognition of 2'- and 4'-Modified Nucleosides. ALS-8112 is a 2'fluoro-4'-chloromethyl-modified cytidine analog, but it is not clear which structural features contribute to its enhanced catalytic efficiency as a substrate for dCK. We examined the substrate efficiency of 2'- and 4'-substituted cytidine analogs on dCK activity to develop a structure– activity relationship for dCK. Our expectation was that this would help elucidate which structural features drive the catalytic efficiency of ALS-8112 (see Table 2). Our studies revealed that the 2'-fluorine in the arabino configuration caused an approximately 4-fold reduction in kcat/Km in comparison to cytidine, while the 2'-fluorine in the ribo configuration (2'-FdC) or 2',2'difluoro (gemcitabine) increased kcat/Km of 6- and 4-fold, respectively (Figure 3A). This indicates dCK significantly prefers 2'-ribo-fluoro and the 2'-ribo-fluoro of gemcitabine may compensate for the negative effects of the second 2'-arabino-fluorine substituent. ALS-8112 is differentiated from other cytidine analogs through its 4'-chloromethyl moiety. This prompted us to examine 2'-fluoro substituted nucleosides (monosubstituted in the ribo or arabino configuration or di-substituted) with a 4'-chloromethyl (see Figure 3). Our biochemical assays showed that ALS-8112 and compound 1 (2', 2'-difluoro-4'-chloromethyl cytidine) caused approximately 1- to 3-fold increases in kcat/Km compared with cytidine while compound 2 (2'-fluoro-4'-chloromethyl arabinocytidine), containing a 2'-arabino-fluorine caused a decrease in kcat/Km (Figure 3B). Compound 3 (4'-chloromethyl-cytidine) caused an approximately 10-fold decrease in kcat/Km compared with cytidine. The combined results of compounds 1-3 suggest the 2'-fluoro (mono- or di-subsituted) combined with the 4'-chloromethyl is important for the enhanced substrate specificity of ALS-8112. The effects of 2'-fluoro versus other less polar substitutions on cytidine-based analogs was important for developing a comprehensive structure–activity relationship. We also examined the dCK substrate efficiency of 2'-C-MeC and compound 4 (2'-C-Me-4'-chloromethyl cytidine), both of which contain a 2'-CMe substitution but differ through a 4'-chloromethyl.21 2'-C-MeC is the parent nucleoside of valopicitabine, a prodrug inhibitor of HCV; previous work has demonstrated it is a poor substrate for dCK.21, 26 In our assay system, both compound 4 and 2'-C-MeC were poor substrates for dCK suggesting dCK prefers the smaller 2'-fluorine over the bulky 2'-methyl group. Additionally, our data show arabinocytidine is similar in efficiency to cytidine while compound 5 (4'-chloromethyl-arabinocytidine) demonstrated a 2.3-fold decrease in kcat/Km (Supplementary Figure 5). Importantly, these data indicate the enhancement of ALS-8112 as a dCK substrate is driven by a combination of the 2' and 4' substitutions. Computational Analysis of 2'- and 4'-Modified Nucleosides Bound to dCK. We utilized the known structure of dCK in complex with deoxycytidine (PDB ID 1P61) 16 to create a computer model of the dCK substrate binding site (Figure 4A), and carried out docking calculations on a set of five dCK substrates, including ALS-8112. The goal of this modeling work was to provide structure-based information to address the differences in dCK recognition observed in the biochemical assay. The selected compounds span nearly three log units in biochemically determined substrate efficiencies (Tables 1 and 4). Docked binding modes were compared to deoxycytidine in the X-ray reference structure by computing root mean square deviations (RMSDs) of the ligands’ “cores” (base heavy atoms, C1', C4', O4', C5', and O5') to the corresponding atoms of deoxycytidine in the structure (PDB ID 1P61).


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Deoxycytidine, cytidine, ALS-8112, and compound 3, whose biochemical substrate efficiencies are within ~10-fold, had core RMSD values in the range of 0.2 Å (Table 5). These four compounds recapitulate the hydrogen bonding interactions with Glu53, Glu197, Tyr86, Asp133, Gln97, H2O303, and H2O323 observed in the deoxycytidine X-ray binding mode16 (Figure 4A). In contrast, compound 4, with a dCK substrate efficiency nearly three logs lower than the four other compounds, has about a 2-fold higher core RMSD (0.4 Å). This higher core RMSD is primarily driven by a substantial displacement of O5' (0.85 Å) compared to the deoxycytidine X-ray binding conformation, resulting in loss of the hydrogen bond to H2O323 - the crystallographically resolved, ordered water molecule that is anticipated to facilitate catalytic attack during installation of the monophosphate moiety. The side chain of Ile30 prevents the bulky 2'-Me substituent in compound 4 from adopting an equatorial conformation. This results in an unfavorable Van der Waals interaction between the 2'-Me group of compound 4 and the side chain of Arg128 (Figure 4B), close intra-ligand contacts, and an overall poor predicted binding to the dCK substrate site. Overall, these results support the biochemical observation that 2'-C-MeC and compound 4 are poor substrates of dCK. Phosphorylation of ALS-8112-MP by YMPK. Another goal was to develop the full activation scheme of ALS-8112, prompting an investigation of the second and third phosphorylation steps. Human UMP-CMP kinase (YMPK) phosphorylates CMP, UMP, and dCMP using ATP as a phosphate donor.17, 19 Earlier reports have shown that phosphorylation of cytidine analogs by YMPK is not the rate-limiting step and we hypothesized that ALS-8112, a cytidine analog, would follow similar trends.21 Human YMPK was cloned, expressed in E. coli, and purified using standard techniques. We examined the steady-state kinetics of YMPK using coupled pyruvate kinase/LDH as before. The kcat/Km of the natural substrates for YMPK, CMP and UMP, were 0.15 and 0.24 µM-1s-1, respectively (Table 3). The Km for these two substrates was 107 ± 26 µM and 323 ± 20 µM, which are within the range of previous literature reports.19, 21 Interestingly, ALS-8112-MP was converted to ALS-8112-diphosphate (DP) at a catalytic efficiency of 2.7×10-4 µM-1s-1, representing a reaction efficiency of 0.2% compared with CMP, indicating that ALS-8112-MP is a poor substrate for YMPK. Phosphorylation of ALS-8112-DP by NDPK. The conversion of ALS-8112-DP to ALS-8112-TP was studied using human NDP kinase (NDPK) that was cloned, expressed in E. coli, and purified using standard techniques. Previous reports have shown pyruvate kinase, a component of the coupling enzyme used in the biochemical assay, shows broad substrate specificity for nucleoside diphosphates.21, 27-29 To account for this parameter, the background reaction was subtracted for the CDP reaction but it was not measurable for ALS-8112-DP. The steady-state kinetics for NDPK were determined and are shown in Table 4. The kcat/Km of CDP, a natural substrate for NDPK, was 0.33 µM-1 s-1 while the Km was 358 ± 49 µM. These results are consistent with literature reports of the enzyme under similar assay conditions.21 ALS-8112DP was converted to ALS-8112-TP with a catalytic efficiency of 1.1×10-2 µM-1 s-1. Compared with the natural substrate of NDPK, cytidine diphosphate, kcat (119 ± 5 vs. 6.7 ± 0.3 s-1) was reduced while Km (358 ± 49 vs. 631 ± 61 µM) increased. However, the efficiency of this reaction is greater than both dCK and YMPK and consistent with a literature report for a similar cytidine analog (Figure 5A).21 Conclusions. We describe the previously unknown metabolic activation of ALS-8112, the parent cytidine of a first-in-class nucleoside inhibitor of respiratory syncytial virus (RSV) replication currently under clinical evaluation in infants and in the elderly. Results of our biochemical assays indicate that phosphorylation by dCK and NDPK are the most catalytically



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efficient in the metabolic activation of ALS-8112. For many nucleoside analogs, monophosphate formation is the main rate-limiting step in the activation process.20, 21 As shown here, the activation pathway of ALS-8112 is unlike that of other antiviral cytidine analogs because the second phosphorylation step limits the formation of triphosphate, the active antiviral species (Figure 5B). This result is reminiscent of the kinase activation pathway of AZT, an antiHIV thymidine analogs that was also poorly phosphorylated in the second kinase step.30, 31 Although the 2'-fluoro, 4'-chloromethyl chemical moieties of ALS-8112 seem to have a positive impact on dCK recognition, they also make it a poor substrate for YMPK. Our ability to rationalize substrate efficiency differences would benefit from additional computational investigations that further explore the behavior of these systems at the molecular level and extend our analysis beyond the initial substrate binding event. Studies such as the one presented here rely on the comparative biochemical analysis between modified nucleosides. We propose that the structure-activity relationships elucidated by these comparative biochemical analyses presented here provide new insights into current drug discovery efforts by further emphasizing the need to develop diphosphate or triphosphate prodrugs instead of monophosphate prodrugs, as previously described for deoxyribonucleosides.32, 33 This could represent an important paradigm shift and an exciting new approach in the way antiviral nucleoside analogs are designed. ACKNOWLEDGEMENTS We thank M. Moore from Emory University for providing the recombinant RSV-RFP clone. We acknowledge M. Prhavc and M. Fitzgerald for their careful review of the manuscript. SUPPORTING INFORMATION The Supporting Information is available free of charge on the ACS Publications website at DOI: METHODS Compounds ALS-8112, ALS-8112-MP, and ALS-8112-TP, and its derivatives were synthesized at Alios BioPharma according to previously reported procedures.8, 34 All other compounds were purchased from Sigma-Aldrich unless described otherwise. Enzymes Deoxycytidine kinase (dCK) was purchased from NovoCIB. All other proteins were cloned, expressed, and purified at Biozilla, LLC. The gene encoding for UCK-1, designed with an Nterminal MGHHHHHHHHGG tag was synthesized, cloned into a pET11 expression vector and transformed into BL21(DE3) E. coli cells. Bacteria were grown in LB media and gene expression was induced with IPTG at 16°C for 16 hours. The culture was centrifuged, the cellpellet was resuspended in lysis buffer, lysed by sonication with lysozyme treatment and a freezethaw cycle followed by centrifugation at 20K×g for 30 min. The soluble protein fraction was purified by Co-IMAC, followed by Superdex 200. The gene encoding for UCK-2 bearing an Nterminal MGHHHHHHHHGG tag was synthesized, cloned into a pET11 expression vector and the plasmid was transformed into BL21(DE3) E. coli cells. Bacteria were grown in Terrific Broth at 30°C for 4 hours, and UCK-2 was purified as described for UCK-1. The gene encoding for 6xHIS-YMPK was synthesized with the inclusion of a C-terminal SSHHHHHHHH tag, cloned into a pET11 expression vector and the plasmid was transformed into BL21(DE3) E. coli cells. Bacteria were grown in Terrific Broth at 30°C for 4 hours. The culture was centrifuged,


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the cell-pellet was resuspended in lysis buffer, lysed by sonication with lysozyme treatment and a freeze-thaw cycle followed by centrifugation at 20K×g for 30 min. The soluble protein fraction was purified by Co-IMAC. The gene encoding for NDPK was synthesized bearing an N-terminal MGHHHHHHHHGG tag. The protein was produced as described for UCK-1. Enzyme Kinetics The phosphorylation of nucleosides, nucleoside MP, and nucleoside DP was monitored using appropriate kinase with a coupled enzyme, consisting of pyruvate kinase and NADH-dependent LDH. The concentration of dCK and UCK-1 used was 100 to 250 nM, depending on the substrate. The concentration of UCK-2 used was between 4 and 423 nM, depending on the substrate. The concentration of YMPK used was between 11 and 325 nM, depending on the substrate. The concentration of NDPK was 10 and 40 nM, depending on substrate. The depletion of NADH was monitored at 340 nm using a SpectraMax Plate Reader. All reactions except NDPK were conducted at 37°C in 50 mM Tris-HCl pH 8, 10 mM MgCl2, 100 mM KCl, 5 mM ATP, 500 µM NADH, 500 µM phosphoenolpyruvate, 5 mM DTT, pyruvate kinase (6-10 U/mL), and LDH (9-14 U/mL). For NDPK, the ATP concentration was 1 mM, pyruvate kinase was (0.4-0.6 U/mL), and LDH (0.6-0.9 U/mL). All concentrations listed are the final concentrations. All reagents were obtained from Sigma-Aldrich, with the exception of KCl and Tris-HCl which were purchased from Ambion. dCK Inhibition Studies Inhibitor 1 (2-thio-2'-deoxycytidine) and inhibitor 2 (2-(sulfanyl)pyrimidine-4,6-diamine) were dissolved in DMSO to a final concentration of 10 mM. For cell-based assays, HEp-2 cells were plated at 5×104 cells/well in a 96-well plate (Corning) in complete growth media at 37°C and, 5% CO2. Eight hours after plating, cells were treated with 10 µM ALS-8112, 10 µM ALS-8112 + 10 µM inhibitor 1, 10 µM ALS-8112 + 10 µM inhibitor 2, or DMSO only. Twenty-four hours after compound treatment, cells were infected with RSVRFPpSynk-AZ at a multiplicity of infection of 0.1 and incubated for 72 hours as previously described.9 Following the 3-day incubation, cells were photographed with a confocal Olympus IX71 with the red fluorescent filter. Cells were then lysed in the 96-well plates with RIPA buffer (Thermo Fisher) and fluorescence quantitated using a Victor5 (Perkin Elmer) with an excitation of 560 nm and emission of 615 nm. For enzymatic assays, IC50 values for inhibitors 1 and 2 were determined using recombinant dCK with ALS-8112 (300 µM) as a substrate under reactions as previously described. Measurement of NMP/NTP Formation ALS-8112-MP and ALS-8112-TP formation was measured in A549 (human lung epithelial) cells purchased from American Type Culture Collection. The cells were maintained and cultured at Alios BioPharma. The cells were plated in 6-well plates at 0.5 million cells/well with their corresponding media and incubated overnight in a cell culture incubator at 37°C, 5% CO2, before use. In the experiment, 10 µM of either dCK inhibitor 1 or 2 was added to each well and dosed with 50 µM of ALS-8112. The incubation was carried out for 24 hours at 37°C, 5% CO2. At the end of the incubation, the medium was removed and the cells were washed twice with cold 0.9% sodium chloride in water. The cells were lysed with methanol/water (70%/30%, v/v) and the extracted supernatant was dried and reconstituted in 1 mM ammonium phosphate before



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LC/MS/MS analysis to determine the corresponding ALS-8112-MP and ALS-8112-TP. The concentrations of the ALS-8112-MP and ALS-8112-TP were normalized by the number of cells and reported as pmol/million cells. Computational Modeling Ligand conformational space was sampled exhaustively prior to docking using the Macrocycle Conformational Sampling routine as implemented in MacroModel 35: MD simulation cycles and large-scale low-mode search steps were set to 5000; torsional angles were sampled exhaustively; structures within 10 kcal/mol of the determined global minima were saved and were coarsegrained into clusters using an RMSD cutoff of 0.75 Å. The resulting ensembles of ligand conformers were used as input for docking calculations with Glide.36-38 Docking calculations were carried out at the SP precision level, with 1.0 Å RMSD core constraints in place for the heavy atoms of the cytosine base, C1', C4', O4', C5', and O5', using the deoxycytidine conformation in PDB 1P61 as the reference core molecule. The computational protocol was validated with deoxycytidine, for which in silico results were compared with its X-ray binding mode in PDB 1P61. For each entry in the input conformer libraries, up to 20 docked poses were collected, resulting in 1740 explored binding modes for the five investigated compounds. For each explored compound, docked binding modes were sorted by core root mean square deviation (RMSD; base heavy atoms, C1', C4', O4', C5', and O5') with respect to the corresponding atoms in the reference X-ray binding conformation of deoxycytidine. A separate RMSD was calculated for the position of the O5' atom. The binding modes with the lowest core RMSD values were then selected for further analysis. Per-residue SP Glide scores outline a rough trend that the bulky 4'-chloromethyl substitutions in ALS-8112 and compound 3 navigate steric interactions with the neighboring Leu82 side chain more successfully than in compound 4, which bears the additional 2'-Me bulk. Van der Waals interactions with the side chain of Val55 are of the same order of magnitude for all explored compounds presenting a 4'-chloromethyl group. FIGURE CAPTIONS Figure 1. dCK Inhibitors 1 and 2 Antagonize the Phosphorylation and Antiviral Activity of ALS-8112. (A) Chemical structures of ALS-8112, inhibitor 1 and inhibitor 2. (B) Inhibition of recombinant dCK by inhibitors 1 and 2. (C) The raw LC/MS/MS chromatograms for the detection of ALS-8112, ALS-8112-monophosphate (MP) and ALS-8112-triphosphate (TP) in the absence (-) or presence (+) of two known dCK inhibitors. (D) Quantification of intracellular ALS-8112, ALS-8112-MP and ALS-8112-TP in A549 cells with or without dCK inhibitors. Figure 2. dCK Inhibitors 1 and 2 Rescue RSV-RFP Infected HEp-2 Cells. (A) Fluorescence microphotographs of HEp-2 infected with RSV-RFP treated with DMSO, ALS-8112 alone, ALS-8112 + inhibitor 1, or ALS-8112 + inhibitor 2. (B) The relative fluorescence was quantified in reference to RSV-RFP. Figure 3. Comparing the dCK Substrate Efficiency of 2'- and 4'-Modified Nucleosides. (A) The dCK substrate efficiency (efficiency is defined as kcat/Km) was measured for 2'-modified cytidine in addition to 2'- and 4'-modified cytidine. (B) The efficiencies of 2'-C-MeC, compound 3, and compound 4 as dCK substrates were compared with natural cytidine.


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Figure 4. The Predicted Binding Mode of ALS-8112 in the dCK Catalytic Site and Site-Specific Interaction Energies for Selected dCK Substrates. (A) The crystal structure of dCK in complex with deoxycytidine16 provides insight into the key protein-ligand interactions associated with efficient ligand binding to the enzyme. Inspection of the structure reveals that, upon binding to dCK (shown in gray), deoxycytidine (shown in yellow) is anchored in place by four distinct hydrogen-bonding networks: the 5'OH group of the ligand engages the side chains of Glu53 and a crystallographically resolved water molecule H2O323; the 3'OH group straddles the side chains of Glu197 and Tyr86; the base interacts with the side chains of Asp133 and Gln97, as well as a second crystallographically resolved water molecule H2O303; finally - H2O303 is hydrogen-bonded back to Tyr86 and Tyr204, completing the efficient set of polar interactions that characterize the system. ALS-8112 (shown in cyan) successfully recapitulates the hydrogen bonding interactions with Glu53, Glu197, Tyr86, Asp133, Gln97, H2O303, and H2O323 observed for deoxycytidine. Two water molecules coordinating ligand binding, as well as key protein side chains that interact with ALS-8112 are shown in gray carbons, white hydrogens, red oxygen atoms. Hydrogen bonds are shown as cyan dashed lines. (B) Per-residue, Glide SP interaction energies (kcal/mol) with three key protein side chains in the dCK catalytic site that impact substrate binding: Arg128, Leu82 and Val55, for five dCK substrates. Figure 5. The Metabolic Activation of ALS-8112. (A) A scheme showing the overall substrate efficiency of ALS-8112 (reported as kcat/Km) for dCK, YMPK, and NDPK in its conversion to ALS-8112-TP. (B) The reaction efficiencies are compared with another cytidine analog with antiviral properties, PSI-6130.21

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Table 1 – Steady-State Kinetics for dCK, UCK-1, and UCK-2 kcat (s-1)

Km (µM)

kcat/Km (µM-1 s-1) % Efficiency

1.5 ± 0.01

486 ± 15

3.1×10-3

100

deoxycytidine 8.3×10-2 ± 3.5×10-3 ALS-8112 2.6 ± 0.5

24 ± 6 265 ± 58

-3

3.5×10 9.7×10-3

113 316

cytidine

1382 ± 60

2.7×10-3

100

>1000

N.D.

177 ± 90

3.2×10-5

1.2

229 ± 81

0.16

100

>10,000

N.D.

Enzyme Substrate cytidine dCK UCK-1

UCK-2

3.7 ± 0.05

deoxycytidine

Activation Pathway of a Nucleoside Analog Inhibiting Respiratory

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