Enhancing the Antiviral Efficacy of RNA-Dependent RNA Polymerase Inhibition by Combination with Modulators of Pyrimidine Metabolism

Article

Enhancing the Antiviral Efﬁcacy of RNA-Dependent

RNA Polymerase Inhibition by Combination with

Modulators of Pyrimidine Metabolism

Graphical Abstract

Authors

Qi Liu, Amita Gupta,

Ayse Okesli-Armlovich, ...,

Jan E. Carette, Michael C. Bassik,

Chaitan Khosla

Correspondence

khosla@stanford.edu

In Brief

Many RNA virus infections lack suitable

treatments. Liu et al. identiﬁed a host-

targeting antiviral strategy of modulating

pyrimidine metabolism with

cyclopentenyl uracil, an inhibitor of

pyrimidine salvage, and GSK983, an

inhibitor of de novo biosynthesis. This

combination also increased the potency

of an RNA-dependent RNA polymerase

inhibitor, against dengue virus.

Highlights

d

Cyclopentenyl uracil and analogs inhibit pyrimidine salvage

in vitro and in cells

A host-targeting antiviral strategy combining DHODH

inhibitors with CPU is explored

The strategy rescues the antiviral efﬁcacy of a DHODH

inhibitor in 20 mM uridine

Modulating pyrimidine metabolism boosts the antiviral

activity of an RdRp inhibitor

Liu et al., 2020, Cell Chemical Biology 27, 1–10

June 18, 2020 ª 2020 Elsevier Ltd.

https://doi.org/10.1016/j.chembiol.2020.05.002

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Please cite this article in press as: Liu et al., Enhancing the Antiviral Efﬁcacy of RNA-Dependent RNA Polymerase Inhibition by Combination with Mod-

ulators of Pyrimidine Metabolism, Cell Chemical Biology (2020), https://doi.org/10.1016/j.chembiol.2020.05.002

ll

Article

Enhancing the Antiviral Efﬁcacy of RNA-Dependent

RNA Polymerase Inhibition by Combination

with Modulators of Pyrimidine Metabolism

Qi Liu,¹^,²^,⁶^,⁹Amita Gupta,

2,3,9

Ayse Okesli-Armlovich,

1,2,7

Wenjie Qiao, Curt R. Fischer, Mark Smith, Jan E. Carette,

4

2,8

2

2,4

2,5

1,2,3,10,

Michael C. Bassik, and Chaitan Khosla

*

1

Department of Chemistry, Stanford University, Stanford, CA 94305, USA

²Stanford Chemistry, Engineering and Medicine for Human Health (ChEM-H), Stanford University, Stanford, CA 94305, USA

³Department of Chemical Engineering, Stanford University, Stanford, CA 94305, USA

⁴Department of Microbiology and Immunology, Stanford University, Stanford, CA 94305, USA

⁵Department of Genetics, Stanford University, Stanford, CA 94305, USA

⁶Present address: Abbvie, Sunnyvale, CA 94085, USA

⁷Present address: Gilead Sciences, Foster City, CA 94404, USA

⁸Present address: Octant, Emeryville, CA 94608, USA

⁹These authors contributed equally

¹⁰Lead Contact

*Correspondence: khosla@stanford.edu

https://doi.org/10.1016/j.chembiol.2020.05.002

SUMMARY

Genome-wide analysis of the mode of action of GSK983, a potent antiviral agent, led to the identiﬁcation of

dihydroorotate dehydrogenase as its target along with the discovery that genetic knockdown of pyrimidine

salvage sensitized cells to GSK983. Because GSK983 is an ineffective antiviral in the presence of physiolog-

ical uridine concentrations, we explored combining GSK983 with pyrimidine salvage inhibitors. We synthe-

sized and evaluated analogs of cyclopentenyl uracil (CPU), an inhibitor of uridine salvage. We found that

CPU was converted into its triphosphate in cells. When combined with GSK983, CPU resulted in large drops

in cellular UTP and CTP pools. Consequently, CPU-GSK983 suppressed dengue virus replication in the pres-

ence of physiological concentrations of uridine. In addition, the CPU-GSK983 combination markedly

enhanced the effect of RNA-dependent RNA polymerase (RdRp) inhibition on viral infection. Our ﬁndings

highlight a new host-targeting strategy for potentiating the antiviral activity of RdRp inhibitors.

INTRODUCTION

we focus on devising a host-targeting antiviral approach for

the treatment of RNA viruses, which cause many serious dis-

Signiﬁcant progress in the development of antiviral drugs has eases such as viral hepatitis, inﬂuenza, Ebola, dengue, and

been achieved by targeting viral proteins with small molecules COVID-19.

(

Jordheim et al., 2013; Lou et al., 2014). For example,

In mammalian cells, pyrimidine biosynthesis is a tightly regu-

compounds like aciclovir and zidovudine block viral reverse tran- lated metabolic process. Two complementary pathways—de

scriptase to treat herpes simplex virus and HIV infections, novo biosynthesis and pyrimidine salvage—are responsible for

respectively, and RNA-dependent RNA polymerase (RdRp) in- producing UTP and CTP for host as well as viral RNA synthesis

hibitors such as dasabuvir and sofosbuvir are used to treat hep- (Figure 1). De novo pyrimidine biosynthesis is a resource-inten-

atitis C virus infections (Nov a´ kov a´ et al., 2018). More recently, sive process. In contrast, salvage occurs via phosphorylation

the broad-spectrum RdRp inhibitor prodrug remdesivir (Gordon of UMP and CMP derived from intracellular RNA degradation

et al., 2020) has received U.S. Food and Drug Administration or via facilitated transport and phosphorylation of extracellular

Emergency Use Authorization for the treatment of coronavirus uridine, whose plasma concentration is tightly controlled in the

disease 2019 (COVID-19). Meanwhile, targeting host proteins low micromolar range (Traut, 1994). Recently, we discovered

required for viral propagation is emerging as an attractive alter- that GSK983, a broad-spectrum antiviral agent ﬁrst reported in

native that may circumvent the emergence of resistance (Beker- 2009 (Harvey et al., 2009), is a potent inhibitor of dihydroorotate

man and Einav, 2015). For example, maraviroc inhibits the hu- dehydrogenase (DHODH), a rate-limiting step in de novo pyrim-

man chemokine receptor CCR5, and is therefore used to treat idine biosynthesis (Deans et al., 2016). In the course of those un-

multidrug-resistant HIV (Lieberman-Blum et al., 2008). More biased genome-wide studies, we also found that knockdown of

recently, pyrimidine biosynthesis has emerged as a potential uridine/cytidine kinase 2 (UCK2) and uridine monophosphate/

host-targeting strategy for antivirals (Okesli et al., 2017). Here, cytidine monophosphate kinase 1 (CMPK1) in the pyrimidine

Cell Chemical Biology 27, 1–10, June 18, 2020 ª 2020 Elsevier Ltd.

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Article

Figure 1. De Novo and Salvage Biosynthesis

of Pyrimidine Nucleotides for Host and Viral

RNA Synthesis

GSK983 is a DHODH inhibitor. Genes that sensitize

cells to GSK983 are highlighted in yellow boxes.

Reactions shown with blue arrows comprise the de

novo biosynthetic pathway, whereas those with

red arrows comprise the salvage pathway. CAD,

carbamoyl-phosphate synthetase 2, aspartate

transcarbamylase, and dihydroorotase; CDA,

cytidine deaminase; CMPK1, uridine mono-

phosphate/cytidine monophosphate kinase 1;

CTPS, CTP synthetase; DHODH, dihydroorotate

dehydrogenase; NDPK, nucleoside diphosphate

kinase; UCK2, uridine/cytidine kinase 2; UMPS,

uridine monophosphate synthase.

salvage pathway strongly sensitized cells to growth inhibition by (2a–2d) (Racine et al., 2014) furnished the carbon skeleton of

GSK983 (Deans et al., 2016). This ﬁnding was consistent with the C-5 analogs. Removal of acetal, benzoyl, and tert-butyldiphenyl

observation that most DHODH inhibitors lack antiviral efﬁcacy silyl (TBDPS) groups then delivered CPU (4a) and analogs

in vivo despite high potency in vitro presumably due to salvage 4b–4d.

metabolism of circulating uridine by virus-infected cells (Bonavia Next, we turned to synthesis of CPU analogs modiﬁed at the

0

et al., 2011; Cheung et al., 2017; Grandin et al., 2016; Smee et al., C5 position of the cyclopentenyl moiety (Figure 2B). Selective

2

012; Wang et al., 2011; Xiong et al., 2020). deprotection of TBDPS with tetra-n-butylammonium ﬂuoride re-

To restore the antiviral efﬁcacy of GSK983 in the presence of vealed the primary hydroxyl group (Song et al., 2001), which was

extracellular uridine, we therefore sought to inhibit pyrimidine then methylated in the presence of Ag

0

2

O (Francisco et al., 2001)

salvage. Cyclopentenyl uridine (CPU) is a carbocyclic analog of to afford the protected 5 -methoxy-CPU. Alternatively, the

uridine that has been shown to inhibit human UCK2 (Lim et al., alcohol could be converted to a terminal ﬂuoride with diethyla-

1

984). To our surprise, we learned that the antiviral activity of mino sulfurotriﬂuoride (Moon et al., 2004), resulting in protected

0

CPU is due to its remarkable ability to deplete intracellular pyrim- 5 -ﬂuoro CPU. Mesylation or acetylation of 5, followed by NaN

3

idine nucleotide pools via inhibition of salvage biosynthesis substitution (Schaudt and Blechert, 2003) or Pd(OH)

2

/C-cata-

pathways. Our ﬁndings led us to redirect our search for a funda- lyzed hydrogenation (Bianco et al., 1989) afforded the protected

0

mentally new type of combination chemotherapy for RNA vi- 5 -azido- and 5 -deoxy-CPU, respectively. The resulting pro-

tected intermediates were treated with methanolic ammonia

ruses, as described below.

and/or HCl to provide analogs 6,7, 9, and 11.

RESULTS

Diversity-Oriented Syntheses of CPU Analogs

Enzymatic Analysis of CPU Analogs Against UCK2

Our search for lead inhibitors of pyrimidine salvage was inspired and CMPK1

by earlier reports on the biological activity of CPU and cyclopen- In order to guide our SAR studies, we developed an enzymatic

tenyl cytosine (CPC), which were shown to block uridine salvage assay to evaluate the inhibitory effect of each CPU analog

in vitro and in vivo (Cysyk et al., 1995; Lim et al., 1984; Moyer against recombinant human UCK2, which was expressed and

et al., 1985; Schimmel et al., 2007). In these and other reports, puriﬁed in Escherichia coli. A continuous assay system was opti-

CPU was found to be remarkably well tolerated, whereas CPC mized by coupling UCK2 activity to the pyruvate kinase reaction,

was considerably more cytotoxic (Blaney et al., 1992; Ford which in turn was coupled to lactate dehydrogenase (Tomoike

et al., 1991; Song et al., 2001). This contrast is likely due to down- et al., 2017). Overall reaction progress was continuously moni-

stream inhibition of the CTP synthetase by CPC (Schimmel tored by detecting the UV absorption change at 340 nm, which

et al., 2007).

Due to the high toxicity of CPC, we undertook structure-activ-

was correlated with the ATP consumption by UCK2.

The effect of 250 mM of each CPU analog on UCK2 activity was

ity relationship (SAR) analysis of CPU wherein uracil nucleobase evaluated in the presence of 50 mM uridine. CPU and 5-F-CPU

was maintained intact or only modiﬁed at the C5 position. Mean- showed much higher ATP consumption compared with other an-

0

while, the C-5 substituent of CPU was modiﬁed because this is alogs (Figure 3A) even after the 50 mM uridine was presumably

the site of UCK2-catalyzed phosphorylation. In order to rapidly exhausted, suggesting these two compounds were substrates

access both nucleobase and carbocyclic moiety analogs, we im- of UCK2. We then individually measured the steady-state kinetic

plemented a diversity-oriented synthetic approach featuring a parameters of UCK2 using uridine, CPU, and 5-F-CPU as sub-

Mitsunobu reaction as the strategic transformation (Choi strates. Their K

M

values were 86, 25, and 41 mM, and their kcat/K

M

5

et al., 2012). values were 2.6310 , 1.5310 , and 7.3310

5

4

ꢀ1

s

,M

(Fig-

We ﬁrst synthesized CPU analogs with nucleobase modiﬁca- ure 3B). As predicted by the relative magnitude of these kinetic

tions (Figure 2A). Mitsunobu reactions between the common parameters, addition of CPU or 5-F-CPU to an assay mixture

cyclopentenyl moiety (1) (Choi et al., 2004) and the benzoyl containing UCK2, uridine, and ATP resulted in a dose-dependent

protected uracil, C(5)-ﬂuoro-uracil, C(5)-iodo-uracil or thymine decrease in the rate of UMP synthesis (Figure 3C).

2

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Article

Figure 2. Diversity-Oriented Syntheses of

CPU Analogs

(

A) Synthesis of CPU (4a) and uracil moiety analogs

b–4d.

B) Modiﬁcation of the C5 position to furnish

carbocyclic moiety analogs 6, 7, 9, and 11. (a)

DEAD, PPh , THF; (b) NH , MeOH; (c) HCl, THF; (d)

TBAF, THF; (e) Ag O, MeI, acetone; (f) DAST,

CH Cl ; (g) MsCl, Et N, CH Cl ; (h) NaN , DMF; (i)

AcCl, CH Cl ; (j) Pd(OH) , cyclohexene, ethanol.

4

0

(

3

2

3

2

3

2

Ac, acetyl; Bz, benzoyl; DAST, diethylaminosulfur

triﬂuoride; DEAD, diethyl azodicarboxylate; Ms,

methanesulfonyl; TBAF, tetra-n-butylammonium

ﬂuoride; TBDPS, tert-butyldiphenyl silyl; THF,

tetrahydrofuran.

washing (Figure S3) (Martano et al.,

2014). LC-MS/MS of nucleotides (Table

S1 for MS/MS transitions of selected pre-

cursors) was performed using a dynamic

multiple reaction monitoring (dMRM)

method (Sartain, 2016). Addition of micro-

molar concentrations of medronic acid

into the mobile phase markedly increased

the sensitivity for detecting di- and tri-nu-

cleotides by this method (Hsiao

et al., 2018).

With optimized sampling and the anal-

ysis method in hand, pyrimidine nucleo-

tide levels were measured in cells

cultured with either CPU, 5-F-CPU, or

0

5

-F-CPU in combination with 1 mM

While CPU and 5-F-CPU were the only UCK2 substrates iden- GSK983 and 5 mM uridine for 6 h (Figure 4A). Inclusion of CPU

tiﬁed from our panel of carbocyclic nucleoside analogs, some of or 5-F-CPU led to large decreases in UTP and CTP levels

the other agents had measurable inhibitory activity against this compared with cells treated with GSK983 alone; these effects

enzyme (Figure 3D). The K

0

i

values of the two most potent grew more pronounced at 12 h (Figure S4). CPU depleted UTP

0

competitive inhibitors, 5 -F-CPU and 5 -deoxy-CPU, were and CTP concentrations more strongly than 5-F-CPU. Triphos-

phates of both CPU and 5-F-CPU could be detected by LC-

1

70 mM and 230 mM, respectively (Figures 3E and 3F).

Given that CPU and 5-F-CPU could be phosphorylated by MS/MS (Figure 4B), suggesting that nucleoside diphosphate

UCK2, we sought to establish whether the corresponding mono- kinase, a mammalian enzyme known to have broad substrate

phosphates were substrates of human CMPK1. For this scope (Lascu and Gonin, 2000), could convert CPU-DP and

purpose, human CMPK1 was expressed in E. coli and puriﬁed. 5-F-CPU-DP into their corresponding nucleoside triphosphate

We then used recombinant UCK2 to synthesize CMP, CPU- analogs. Inclusion of CPU or 5-F-CPU also led to moderate de-

MP, and 5-F-CPU-MP and conﬁrmed their identities by liquid creases in UMP, CMP, UDP, and CDP levels compared with cells

chromatography tandem mass spectrometry (LC-MS/MS) (Fig- treated with GSK983 alone at both 6 h and 12 h (Figure S4).

0

ure S1). The results shown in Figure 3G demonstrate that both

Despite its UCK2 inhibitory activity in vitro, 5 -F-CPU did not

CPU-MP and 5-F-CPU-MP are substrates of CMPK1, but that deplete intracellular nucleotide levels and even appeared to in-

the former is a better substrate. Identities of their corresponding crease levels of some intracellular nucleotides (Figures 4A and

products, CPU-DP and 5-F-CPU-DP, were also conﬁrmed by S4). This contrast suggests that the activity of CPU and 5-F-

LC-MS/MS (Figure S2).

CPU is derived primarily from their ﬂux through pyrimidine meta-

bolism pathways and not from their inhibition of UCK2.

LC-MS/MS Analysis of Intracellular Nucleotides

To understand the metabolic implications of the above biochem- Antiviral and Cytotoxic Activities of Selected CPU

ical ﬁndings, we developed an LC-MS/MS-based assay that Analogs

0

measures the effects of CPU, 5-F-CPU, and 5 -F-CPU on intra- From the above enzymological and metabolic data, we hypoth-

cellular pyrimidine nucleotide levels. To minimize the perturba- esized that the combination of CPU and GSK983 would be more

tion of sample quenching and analysis on the physiological effective at inhibiting dengue virus replication than any other

concentrations of these metabolites, we adapted an established combination. Using an infectious clone of dengue serotype 2

protocol for growing cells on glass coverslips to facilitate rapid (DENV-2) strain 16681 engineered to express a luciferase

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Figure 3. Enzymatic Analysis of CPU Ana-

logs

(

A) CPU and 5-F-CPU are UCK2 substrates, as

observed by addition of 250 mM of each CPU

analog to a UCK2 reaction mixture that also con-

tains 50 mM uridine. All other analogs tested are not

UCK2 substrates.

(

B) Steady-state kinetic analysis of uridine, CPU,

and 5-F-CPU as UCK2 substrates. Error bars

represent ±SD of three replicates.

(

C) Comparative ATP consumption by UCK2 in the

presence of 50 mM uridine and varying concen-

trations of CPU (blue bars) or 5-F-CPU (red bars).

Error bars represent ±SD of three replicates.

(

D) Initial velocities calculated from the data shown

in (A).

E and F) Lineweaver-Burk analysis of 5 -F-CPU (E)

0

(

0

and 5 -deoxy-CPU (F) as UCK2 inhibitors.

G) Comparative activity of CMPK1 on CMP, CPU-

(

MP, and 5-F-CPU-MP. An authentic standard of

CMP (std) was tested alongside a UCK2-synthe-

sized sample of the same compound (syn).

Error bars represent ±SD of three replicates. See

also Figures S1 and S2.

However, only CPU appeared to have a

workable therapeutic index at this con-

centration and time point.

To further examine the efﬁcacy and

cytotoxicity of CPU, we conducted a

more comprehensive checkerboard anal-

ysis of the CPU-GSK983 combination.

While neither GSK983 nor CPU was effec-

tive as a single agent in physiological

uridine levels, the combination of both

molecules

successfully

suppressed

dengue virus infection (Figure 5B). For

example, combining 0.2 mM GSK983

and 250 mM CPU inhibited ꢁ50% of virus

replication (Figure 5B). At a CPU dose of

1

mM, virus replication was suppressed

almost completely. Notably, the combina-

tion treatment had much less effect on

A549 cell growth, suggesting that combi-

nations of GSK983 and CPU could be se-

lective for inhibition of virus but not host

replication.

To further demonstrate that intracellular

nucleotide depletion is correlated with

reporter (Marceau et al., 2016), the efﬁcacy and cytotoxicity of antiviral activity, we also tested the effects of combining

each combination treatment was tested in infected or uninfected GSK983 with a broad inhibitor of nucleoside transport, dipyrida-

cultures of the A549 lung carcinoma cell line (Figure 5A). In all as- mole (DPY). This drug signiﬁcantly suppressed intracellular

says, culture medium was supplemented with 20 mM uridine to nucleotide levels (Figure S4) and consequently inhibited viral

mimic physiological plasma concentrations. In the presence of replication (Figure S5).

2

0 mM exogenous uridine, 1 mM of the de novo pyrimidine syn-

thesis inhibitor GSK983 alone did not signiﬁcantly inhibit viral Improving the Therapeutic Index of RdRp Inhibitor

infection or cellular viability (Figure 5A). In line with our original R1479 by Combination Treatment Targeting Pyrimidine

hypothesis, combination of GSK983 with either 500 mM CPU Biosynthesis

and 5-F-CPU caused signiﬁcant reductions in viral infection un- Given that the CPU-GSK983 combination was able to selectively

like the other cyclopentenyl compounds tested (Figure 5A). inhibit viral infection through modulation of intracellular

4

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dose-dependent inhibition of dengue virus replication with a half-

maximal effective concentration (EC50) of ꢁ32 mM (Figure S6A). In-

hibition of either de novo or salvage pyrimidine biosynthesis alone

did not potentiate the antiviral activity of R1479 or cause additional

cytotoxicity (Figure S6A). In contrast, inhibition of both the de novo

and salvage pathways markedly enhanced the potency of R1479

(Figure 6A). In the presence of 250 mM CPU, the EC50 of R1479

was lowered to ꢁ12 mM. Notably, such a three-component

regimen had minimal impact on the cytotoxicity proﬁle of R1479

(Figure 6B). Regarding other analogs, 5-F-CPU-GSK983 was

able to slightly potentiate the antiviral activity of R1479, whereas

0

5

-F-CPU appeared to actually decrease antiviral efﬁcacy of

R1479 (Figures S6B and S6C).

As robust intracellular phosphorylation is critical for both

R1479 and CPU activity, we conﬁrmed that competition between

these two compounds for intracellular kinases at the rate-limiting

mono-phosphorylation step (Ford et al., 1991; Klumpp et al.,

0

2008) should be minimal. Despite its 2 -a-OH group, R1479 is

known to be solely phosphorylated by deoxycytidine kinase

dCK) (Klumpp et al., 2008) and was indeed not a UCK2 substrate

(

in our hands (Figure S7). Similarly, cyclopentenyl compounds are

substrates of UCK2 and not dCK (Ford et al., 1991).

To gain insight into the mechanism underlying the improve-

ment of R1479 efﬁcacy with CPU, we deployed a replicon assay

that bypasses viral entry by electroporation of a reporter DENV-2

RNA construct (Alvarez et al., 2005; Marceau et al., 2016). Such

replicon systems include the coding region needed for viral RNA

translation and replication, while lacking the structural genes

necessary for the production of viral particles. Consequently,

viral genome replication is examined independently of viral entry

and particle assembly (Kato and Hishiki, 2016). In this assay (Fig-

ure 7), the EC50 of R1479 alone was ꢁ90 mM. Inhibition of de

novo pyrimidine biosynthesis alone with GSK983 did not alter

this value (ꢁ96 mM), whereas addition of 250 mM CPU shifted it

to ꢁ56 mM. Targeting both the de novo and salvage pathways

with a combination of 1 mM GSK and 250 mM CPU lowered the

EC50 of R1479 by over 4-fold to ꢁ19 mM. These results suggest

that intracellular nucleotide depletion is the major driver of CPU’s

antiviral activity. Together, these ﬁndings highlight the potential

of combining modulators of pyrimidine metabolism with inhibi-

tors of viral genome replication.

Figure 4. LC-MS/MS Analysis of Intracellular Nucleotides

A) LC-MS/MS analysis of intracellular uridine and cytidine nucleotide levels

after 6 h treatments. In all assays, the culture medium was supplemented with

mM uridine and 1 mM GSK983. Error bars represent ±SD of three biological

(

5

replicates with statistical comparisons with the untreated control denoted for

UTP levels (blue) and CTP levels (red) as not signiﬁcant (n.s.), *p < 0.05, **p <

0

.01, ***p < 0.001, and ****p < 0.0001 as calculated by one-way ANOVA,

Dunnett’s test.

B) LC-MS/MS detection of mono-, di-, and triphosphates of CPU and 5-F-

(

CPU in cells. The targeted multiple reaction monitoring (MRM) transitions were

CPU-MP (319/79), 5-F-CPU-MP (337/79), CPU-DP (399/159), 5-F-CPU-

DP (417/159), CPU-TP (479/159), and 5-F-CPU-TP (497/159) as detailed

in Table S1.

See also Figures S6 and S7.

interactions with multiple host and viral enzymes will likely be SIGNIFICANCE

useful for future SAR around CPU as this compound remained

the most efﬁcacious in our panel. In addition, efforts to develop RNA virus infections cause serious diseases such as viral

speciﬁc inhibitors of UCK2 are needed. We previously identiﬁed hepatitis, inﬂuenza, Ebola, dengue, and coronavirus disease

such inhibitors through a high-throughput screen (Okesli-Armlo- 2019, yet many of them lack suitable antiviral treatments. We

vich et al., 2019); however, these inhibitors appeared to lack identiﬁed a host-targeting antiviral strategy of modulating

sufﬁcient potency to synergize with GSK983 in antiviral models pyrimidine metabolism with analogs of cyclopentenyl uracil,

(

data not shown).

an inhibitor of pyrimidine salvage, and GSK983, an inhibitor

CPU is known to be safe in vitro (Blaney et al., 1992; Ford et al., of de novo biosynthesis. This combination therapy also

991; Song et al., 2001) and does not show appreciable toxicity markedly increased the potency of R1479, an RNA-depen-

1

in mice (Cysyk et al., 1995) or non-human primates (Blaney et al., dent RNA polymerase (RdRp) inhibitor, against dengue virus

990). While the safety proﬁle of GSK983 remains less clear, replication. At efﬁcacious drug doses, the effect on the

1

other DHODH inhibitors such as US Food and Drug Administra- growth rates of uninfected cells was minimal. In light of the

tion-approved arthritis drugs leﬂunomide and teriﬂunomide and

the clinical-stage anti-cancer compound brequinar have been

extensively studied in humans and are generally tolerated (Aly

et al., 2017). We note that the potential immunosuppressive ef-

fects of DHODH inhibitors may be viewed as helpful (Xiong

et al., 2020) or harmful (Bonavia et al., 2011) in the treatment of

viral infection. Meanwhile, many RdRp inhibitors have been eval-

uated in clinical trials against dengue. While a preliminary study

combining CPU with a de novo synthesis inhibitor in vivo resulted

in toxicity (Cysyk et al., 1995), there is precedent for combining

inhibitors of de novo pyrimidine synthesis with nucleoside trans-

port inhibitors in humans (Casper et al., 1991). With careful dose

Figure 7. Examining Viral Replication with Combinations of RdRp In-

optimization and an eye toward immune effects, our results sug-

hibitor R1479 with CPU-GSK983 Treatment

gest that a combination strategy targeting both host pyrimidine

Luminescence of luciferase-expressing DENV-2 replicon RNA in A549 cells at

biosynthesis and viral RdRp holds promise as a potential therapy

48 h. In all assays, the culture medium was supplemented with 20 mM uridine

and 1 mM GSK983. Error bars represent ±SD of three replicates.

against RNA viruses.

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Article

growing interest in RdRp inhibitors as antiviral agents, our

ﬁndings shine light on a promising way to enhance their clin-

ical utility by combining them with modulators of mamma-

lian pyrimidine metabolism.

DECLARATION OF INTERESTS

A patent has been ﬁled on this work by the Stanford University Ofﬁce of Tech-

nology Licensing on behalf of Q.L, A.G, and C.K. with reference label S19-283.

M.S. is a paid consultant for Riboscience LLD and Advisory Board Member for

Tranquis Therapeutics and Angarus Therapeutics. The other authors declare

no competing interests.

STAR+METHODS

Received: September 19, 2019

Revised: March 12, 2020

Accepted: May 4, 2020

Detailed methods are provided in the online version of this paper

and include the following:

Published: May 21, 2020

d KEY RESOURCES TABLE

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0

B Materials Availability

Role of RNA structures present at the 3 UTR of dengue virus on translation,

RNA synthesis, and viral replication. Virology 339 , 200–212.

B Data and Code Availability

Aly, L., Hemmer, B., and Korn, T. (2017). From leﬂunomide to teriﬂunomide:

drug development and immunosuppressive oral drugs in the treatment of mul-

tiple sclerosis. Curr. Neuropharmacol. 15 , 874–891.

d EXPERIMENTAL MODELS AND SUBJECT DETAILS

B Mammalian Cell Culture

B Bacterial Cell Culture

Bekerman, E., and Einav, S. (2015). Combating emerging viral threats. Science

B DENV-Luc Reporter Virus Generation

B pDENV-Luc Replicon Generation

d METHOD DETAILS

3

48 , 282–283.

Bianco, A., Passacantilli, P., and Righi, G. (1989). Mild hydrogenolysis process

by catalytic transfer hydrogenation. Tetrahedron Lett 30 , 1405–1408.

B Synthesis and Characterization of CPU Analogs

B Puriﬁcation of Recombinant Human UCK2

B Puriﬁcation of Recombinant Human CMPK1

B In Vitro Enzyme Activity Assays with UCK2

B Enzymatic Synthesis of CMP, CPU-MP, and 5-F-

CPU-MP

Blaney, S.M., Balis, F.M., Heideman, R.L., McCully, C., Murphy, R.F., Poplack,

D.G., Hegedus, L., and Kelley, J.A. (1990). Pharmacokinetics and metabolism

of cyclopentenyl cytosine in nonhuman primates. Cancer Res. 50 , 7915–7919.

Blaney, S.M., Balis, F.M., Grem, J., Cole, D.E., Adamson, P.C., and Poplack,

D.G. (1992). Modulation of the cytotoxic effect of cyclopentenylcytosine by

its primary metabolite, cyclopentenyluridine. Cancer Res. 52 , 3503–3505.

B In Vitro Enzyme Activity Assays with CMPK1

B Sample Preparation for LC-MS/MS Analysis

B LC-MS/MS Analysis

Boldescu, V., Behnam, M.A.M., Vasilakis, N., and Klein, C.D. (2017). Broad-

spectrum agents for ﬂaviviral infections: dengue, Zika and beyond. Nat. Rev.

Drug Discov. 16 , 565–586.

B DENV-2 Luciferase Reporter Assays

B Replicon Assays

Bonavia, A., Franti, M., Keaney, E.P., Kuhen, K., Seepersaud, M., Radetich, B.,

Shao, J., Honda, A., Dewhurst, J., Balabanis, K., et al. (2011). Identiﬁcation of

broad-spectrum antiviral compounds and assessment of the druggability of

their target for efﬁcacy against respiratory syncytial virus (RSV). Proc. Natl.

Acad. Sci. U S A 108 , 6739–6744.

d QUANTIFICATION AND STATISTICAL ANALYSIS

Casper, E.S., Baselga, J., Smart, T.B., Magill, G.B., Markman, M., and

Ranhosky, A. (1991). A phase II trial of PALA+dipyridamole in patients with

advanced soft-tissue sarcoma. Cancer Chemother. Pharmacol. 28 , 51–54.

SUPPLEMENTAL INFORMATION

Supplemental Information can be found online at https://doi.org/10.1016/j.

chembiol.2020.05.002 .

Chen, Y.-L., Abdul Ghafar, N., Karuna, R., Fu, Y., Lim, S.P., Schul, W., Gu, F.,

Herve, M., Yokohama, F., Wang, G., et al. (2014). Activation of peripheral blood

mononuclear cells by dengue virus infection depotentiates balapiravir. J. Virol.

8

8 , 1740–1747.

ACKNOWLEDGMENTS

Cheung, N.N., Lai, K.K., Dai, J., Kok, K.H., Chen, H., Chan, K.H., Yuen, K.Y.,

and Tsun Kao, R.Y. (2017). Broad-spectrum inhibition of common respiratory

RNA viruses by a pyrimidine synthesis inhibitor with involvement of the host

antiviral response. J. Gen. Virol. 98 , 946–954.

The authors wish to thank Roberto Mateo, David Constant, and Khanh Nguyen

for helpful discussions and technical advice. The authors also thank Maryline

Dong and Nielson Weng for feedback on this manuscript. This research was

supported by a grant from the National Institutes of Health (1U19 AI109662

from the National Institute of Allergy and Infectious Diseases) to C.K.,

M.C.B., and J.E.C. Also, J.E.C. acknowledges NIH (R01 AI141970) for ﬁnancial

support. A.G. is supported by a National Science Foundation Graduate

Research Fellowship, a Stanford ChEM-H Chemistry/Biology Interface Pre-

doctoral Training Program Fellowship, and an American Research College Sci-

entists (ARCS) Fellowship.

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AUTHOR CONTRIBUTIONS

Q.L, A.G., and C.K. designed the research; Q.L., A.G., and W.Q. performed the

research investigations; A.O.-A. and C.R.F. aided resource and method develop-

ment. J.E.C., M.C.B., and C.K. provided supervision and acquired funding. Q.L.,

A.G., and C.K. wrote the paper (original draft) which was reviewed by all authors.

M.S provided advice.

¨

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ulators of Pyrimidine Metabolism, Cell Chemical Biology (2020), https://doi.org/10.1016/j.chembiol.2020.05.002

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10 Cell Chemical Biology 27, 1–10, June 18, 2020

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ulators of Pyrimidine Metabolism, Cell Chemical Biology (2020), https://doi.org/10.1016/j.chembiol.2020.05.002

ll

Article

STAR+METHODS

KEY RESOURCES TABLE

REAGENTS OR RESOURCES

Bacterial and Virus Strains

E. coli BL21(DE3)

SOURCE

IDENTIFIER

Thermo Scientiﬁc

Cat#EC0014

N/A

pDENV-Luc infectious clone

pDENV-Luc replicon

Marceau et al. (2016)

N/A

Chemicals, Peptides and Recombinant Proteins

DMEM

Gibco

Cat#11995065

Cat#25300054

Cat#15140122

Cat#25030081

Cat#SH30396.03

Cat#E0399

Trypsin/EDTA

Gibco

Penicillin-Streptomycin

Glutamine

Gibco

Fetal Bovine Serum

HyClone

Electroporation Buffer for replicon

XbaI Restriction Enzyme

Teknova

New England BioLabs

EMD Millipore

Cat#R0145S

Lactate dehydrogenase, porcine

heart (LDH)

Cat#427211

Pyruvate kinase, rabbit muscle type III (PK)

Sigma-Aldrich

Cat#P9136

Cat#0220

Adenosine triphosphate disodium

trihydrate (ATP)

VMR Life Science

b-Nicotinamide adenine dinucleotide,

Santa Cruz

Cat# sc-205762A

Cat#P7127

reduced disodium salt hydrate (NADH)

Phosphoenolpyruvate monopotassium

salt (PEP)

Sigma-Aldrich

Cytidine 5’-monophospahte disodium

salt (CMP)

Cat#C1006

Uridine

Sigma-Aldrich

Cat#U3750

Cat#BP97235

Cat#R0392

LB Broth

Fisher BioReagents

Thermo Fisher Scientiﬁc

Isopropyl-1-thio-b-D-galacto-

pyranoside (IPTG)

R1479

MedChemExpress

MP Biomedicals

Chem Impex

Cat#HY-10444

Cat#0215372205

Cat#50494709

Cat#216143

Dipyridamole (DPY)

D-ribose

Tetrabutylammonium ﬂuoride solution

Sigma-Aldrich

(

TBAF), 1.0M

Diethyl azodicarboxylate solution (DEAD),

0 wt.%

Sigma-Aldrich

Cat#563110

4

Triphenylphosphine (PPh

Uracil

3

)

Sigma-Aldrich

AK Scientiﬁc

Cat#T84409

Cat#U0750

Cat#F6627

Cat#J91030

Cat#K165

5

-Fluorouracil

Thymine

-Iodouracil

Ammonia, ca. 7N solution in methanol

Silver (I) oxide (Ag O)

5

AK Scientiﬁc

Sigma-Aldrich

EMD Millipore

Oakwood

Cat#499145

Cat#226831

Cat#IX0185

Cat#002323

Cat#471259

Cat#04885

Cat#114189

2

Methyl iodide (MeI)

Diethylaminosulfur triﬂuoride (DAST)

Methanesulfonyl chloride (MsCl)

Sigma-Aldrich

Thermo Fisher Scientiﬁc

Sigma-Aldrich

Triethylamine (NEt

3

)

Acetyl chloride (AcCl)

(Continued on next page)

Cell Chemical Biology 27, 1–10.e1–e9, June 18, 2020 e1

Please cite this article in press as: Liu et al., Enhancing the Antiviral Efﬁcacy of RNA-Dependent RNA Polymerase Inhibition by Combination with Mod-

ulators of Pyrimidine Metabolism, Cell Chemical Biology (2020), https://doi.org/10.1016/j.chembiol.2020.05.002

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Article

Continued

REAGENTS OR RESOURCES

Palladium hydroxide on carbon

SOURCE

IDENTIFIER

Acros Organic

Cat#199620100

[

2

Pd(OH) /C], 5 wt.%

Cyclohexene

Sigma-Aldrich

Cat#29240

Sodium azide (NaN

3

)

Sigma-Aldrich

Cat#71289

4-(2-Hydroxyethyl)piperazine-1-

Fisher BioReagents

Cat#BP310-100

ethanesulfonic acid (HEPES)

Tris(hydroxymethyl)aminomethane

base (Tris)

Fisher BioReagents

Cat#BP152-1

Dithiothreitol (DTT)

Fisher BioReagents

Fisher Chemical

Agilent

Cat#BP172-25

Cat#M33-500

Cat#P217-500

Cat#5191-3940

Magnesium chloride (MgCl

Potassium chloride (KCl)

2

)

Medronic acid (InﬁnityLab Deactivator

Additive)

1

5

Adenosine

N

5

5’-monophosphate

Sigma-Aldrich

Cat#662658

Cat#662666

disodium salt

1

5

Uridine

sodium salt

N 5’-monophosphate

2

Critical Commercial Assays

MEGAscript T7 Transcription Kit

Invitrogen

Cat#AM1334

Cat#S1404S

m7G(5’)ppp(5’)G RNA Cap Structure

Analog

New England BioLabs

CellTiter-Glo

Promega

Cat#G7570

Cat#E2710

Cat#E2820

Renilla-Glo

Renilla

Experimental Models: Cell Lines

A549

ATCC

Cat#CCL-185

Cat#CRL-1586

Vero E6

Recombinant DNA

pET21a-UCK2 expression plasmid

pET28-CMPK1 expression plasmid

Okesli-Armlovich et al. (2019)

Deans et al. (2016)

N/A

Software

Prism 8.0

GraphPad

PerkinElmer

Mestrelab

https://www.graphpad.com/scientiﬁc-

software/prism/

ChemDraw Professional 17.1

MNova 12.0

https://www.perkinelmer.com/category/

chemdraw

https://mestrelab.com/download_ﬁle/

mnova-12-0-0/

Other

Centrifugal Filter Unit

Hi Trap Q anion exchange column

Amicon

Cat#UFC900308

Cat#17115301

GE Healthcare Life Sciences

RESOURCE AVAILABILITY

Lead Contact

Further information and requests for resources and reagents should be directed to and will be fulﬁlled by the lead contact, Chaitan

Khosla (Khosla@stanford.edu )

Materials Availability

This study did not generate any unique biological reagents. Synthesis of CPU and related chemicals is detailed within the manuscript.

Data and Code Availability

This study did not generate any unique datasets or code not available in the manuscript ﬁles.

e2 Cell Chemical Biology 27, 1–10.e1–e9, June 18, 2020

Please cite this article in press as: Liu et al., Enhancing the Antiviral Efﬁcacy of RNA-Dependent RNA Polymerase Inhibition by Combination with Mod-

ulators of Pyrimidine Metabolism, Cell Chemical Biology (2020), https://doi.org/10.1016/j.chembiol.2020.05.002

ll

Article

EXPERIMENTAL MODELS AND SUBJECT DETAILS

Mammalian Cell Culture

All mammalian cell lines were maintained in a humidiﬁed incubator (37 C, 5% CO

ꢂ

2

). A549 cells (male) and Vero E6 cells (female) were

obtained from the American Type Culture Collection and cultured in DMEM supplemented with 10% FBS, penicillin/streptomycin,

and L-glutamine. Upon reaching 50-75% conﬂuence, cells were detached from the growth surface using a trypsin/EDTA solution

prior to analysis. Cells were maintained in logarithmic growth during all biological assays.

Bacterial Cell Culture

BL21(DE3) cells were obtained from ThermoFisher Scientiﬁc. Cells were cultured in LB Broth at 37 C with agitation or as required for

recombinant protein production.

ꢂ

DENV-Luc Reporter Virus Generation

The design of the pDENV-Luc infectious clone derived from dengue serotype 2 (DENV-2) strain 16681 was detailed previously (Mar-

ceau et al., 2016). The plasmid was linearized with XbaI and in vitro transcribed into the genomic RNA of DENV-Luc virus using the T7

Megascript Kit (Ambion) in the presence of m7G(5’)ppp(5’)G RNA Cap Structure Analog (NEB). 5 mg DENV-Luc RNA was electropo-

6

rated into 2X10 Vero E6 cells. The transfected cells were resuspended in DMEM with 10% FBS and 100 U/ml penicillin-streptomycin

ꢂ

and transferred into a T-175 ﬂask incubated at 37 C with 5% CO

2

. Supernatants were collected and replenished with fresh medium

ꢂ

every 24 h from day 17 to 24 post-transfection, pooled together, clariﬁed by centrifugation and stored in aliquots at ꢀ80 C. The

amount of infectious DENV-Luc virus in the stock was titrated using the TCID50 assay and calculated by the Spearman & K a€ rber al-

gorithm as described previously (Hierholzer and Killington, 1996).

pDENV-Luc Replicon Generation

The plasmid pDENV-Luc replicon containing a Renilla luciferase expressing DENV subgenomic replicon was described previously

(

Marceau et al., 2016). For in vitro transcription, 10 ug of plasmid DNA was linearized using the XbaI restriction enzyme. Replicon

0

RNA was generated using the MEGAscript T7 Transcription Kit in the presence of 5mM m7G(5 )ppp(5 )G RNA Cap Structure

Analogue. For assays, two million A549 cells were washed twice with PBS and re-suspended in electroporation buffer (Teknova,

E0399), mixed with 4 ug of puriﬁed replicon RNA, and electroporated using Bio-Rad Gene Pulser Xcell electroporator using square

wave protocol. Electroporated cells were resuspended in pre-warmed culture medium without antibiotics and seeded as required.

METHOD DETAILS

Synthesis and Characterization of CPU Analogs

Unless otherwise noted, all reactions were performed under an argon atmosphere in ﬂame- or oven- dried glassware. Reaction mix-

tures were stirred using Teﬂon-coated magnetic stirrer bars and monitored by thin layer silica gel chromatography (TLC) using

0

.25 mm silica gel 60F plates with ﬂuorescent indicator from Merck. Plates were visualized under UV or treated by KMnO

with gentle heating. Products were puriﬁed on an AnaLogix IntelliFlash 280 Flash column chromatography system using the solvent

gradients indicated. Anhydrous tetrahydrofuran (THF), dichloromethane (CH Cl ), dimethylformamide (DMF), acetone, and Dimethyl-

sulfoxide (DMSO) were obtained from Acros Organics. Diethyl ether (Et O), ethyl acetate (EtOAc), hexanes, and methanol (MeOH)

4

stain

2

were from Fisher Scientiﬁc. DMSO used in bioassays and to prepare biological samples was from Fisher BioReagents. All other re-

agents were from commercial suppliers and were used as received without additional puriﬁcation. Samples prepared for biological

evaluation were puriﬁed via preparative HPLC in a water/acetonitrile (MeCN) gradient containing 0.1% (v/v) triﬂuoroacetic acid (TFA)

using an Agilent 1260 Inﬁnity system equipped with an Agilent Prep-C18 column (21.2 x 250 mm).

1

13

1

13

NMR spectra were measured on a Varian INOVA 500 ( H at 500 MHz, C at 125 MHz), a Varian 400 ( H at 400 MHz, C at 100

1

13

1

MHz), or a Varian INOVA 600 MHz ( H at 600 MHz, C at 150 MHz) magnetic resonance spectrometer, as noted. H chemical shifts

were reported relative to the residual solvent peak (CDCl = 7.26 ppm; CD OD = 3.31 ppm) as follows: chemical shift (d) [multiplicity

s = singlet, brs = broad singlet, d = doublet, t = triplet, q = quartet, dd = doublet of doublet, m = multiplet), coupling constant(s) in Hz,

3

(

1

3

13

integration]. C chemical shifts were reported relative to the residual deuterated solvent C signals (CDCl

3 3

= 77.16 ppm, CD OD =

4

9.00 ppm) and rounded to one decimal places. Infrared spectra were recorded on a Nicolet iS50 FT/IR Spectrometer at the Stanford

-1

Nano Share Facilities and were reported in wavenumbers (cm ). Optical rotation data were obtained using a JASCO DIP were re-

ported as [a]20D (c = grams/100 mL, solvent), where D indicates the sodium D line (589 nm). High resolution mass spectra were ob-

tained on an Agilent 6545 QTOF mass spectrometer at the Metabolic Chemistry Analysis Center at Stanford University. The MNova

and ChemDraw softwares aided analytical chemistry analysis.

3

-benzoyl-1-((3aS,4R,6aR)-6-(((tert-butyldiphenylsilyl)oxy)methyl)-2,2-dimethyl-3a,6a-dihydro-4H-cyclopenta[d][1,3]

dioxol-4-yl)pyrimidine-2,4(1H,3H)-dione (3a)

Compound 1 was prepared in 10 steps from D-ribose according to literature procedures (Choi et al., 2004). To a suspension of 1

(

440 mg, 1.04 mmol, 1 equiv.), 2a (270 mg, 1.25 mmol, 1.2 equiv.), and PPh

3

(410 mg, 1.56 mmol, 1.5 equiv.) in THF (12 mL) at

C was added DEAD solution in toluene (720 mL, 1.5 equiv., 40 w% in toluene). After the addition of DEAD, the reaction mixture

ꢂ

0

turned from a white suspension to a yellow solution, which was slowly warmed up to room temperature and stirred overnight. The

Cell Chemical Biology 27, 1–10.e1–e9, June 18, 2020 e3

Please cite this article in press as: Liu et al., Enhancing the Antiviral Efﬁcacy of RNA-Dependent RNA Polymerase Inhibition by Combination with Mod-

ulators of Pyrimidine Metabolism, Cell Chemical Biology (2020), https://doi.org/10.1016/j.chembiol.2020.05.002

ll

Article

reaction mixture was then concentrated, loaded onto a 12g SiO

2

ﬂash cartridge, and puriﬁed with a linear gradient of 20-40% EtOAc

-1

in hexanes to afford 3a (446 mg, 0.72 mmol, 69%) as white powder. [a]20D–17.0 (c 1.3, MeOH); IR (ﬁlm, cm ): 3071, 2930, 2857,

1

748, 1705, 1667, 1441, 1429, 1372, 1234, 1112, 905, 732, 704. H NMR (400 MHz, CDCl

m, 6H), 7.53 – 7.47 (m, 2H), 7.45 – 7.42 (m, 2H), 7.41 – 7.34 (m, 4H), 6.91 (d, J = 8.1 Hz, 1H), 5.77 (d, J = 8.1 Hz, 1H), 5.67 (s, 1H),

.37 (s, 1H), 5.08 (d, J = 5.8 Hz, 1H), 4.60 (d, J = 5.8 Hz, 1H), 4.47 (d, J = 16.4 Hz, 1H), 4.41 (d, J = 16.7 Hz, 1H), 1.34 (s, 3H), 1.29

3

) d 7.95 (d, J = 7.4 Hz, 1H), 7.71 – 7.61

(

5

1

3

(

s, 3H), 1.10 (s, 9H). C NMR (100 MHz, CDCl

3

) d 168.8, 162.3, 153.6, 149.8, 141.1, 135.6, 135.6, 135.3, 133.3, 133.1, 131.6,

1

30.7, 130.1, 129.3, 128.0, 128.0, 120.8, 112.9, 102.4, 84.6, 83.4, 68.4, 61.4, 27.3, 27.0, 25.9, 19.4. HRMS (ESI) m/z 623.2574

+

[

(M+H) ; calcd for C36

H

39

N

2

O

6

Si : 623.2572].

-((1R,4R,5S)-4,5-dihydroxy-3-(hydroxym ethyl)cyclopent-2-en-1-yl)pyrimidine-2,4(1H,3H)-dione (4a)

solution in methanol. After 1 h, the reaction solvent was removed

atmosphere and the resulting solid was further dried under high vacuum. The reaction crude was then treated with

0 mL HCl in 300 mL THF and stirred overnight. Excess NaHCO was added to neutralize the reaction, followed by the addition of 1 mL

MeOH. The resulting suspension was ﬁltered through a short Celite pad, rinsed with MeOH, and concentrated. The resulting crude

was then resuspended with 1 mL H O and subjected to HPLC puriﬁcation with a linear gradient of 5-20% MeCN (0.1% TFA) in H

0.1% TFA) on a prep C18 column (Agilent 10 prep-C18 250 3 21.1 mm). Fractions containing desired product was then combined

1

To compound 3a (30 mg, 0.048 mmol) was added 0.5 mL 7N NH

under positive N

3

2

3

2

O

(

-1

and lyophilized to afford the ﬁnal product 4a (4.6 mg, 0.019 mmol, 40%) as white powder. [a]20D– 62.8 (c 3.2, MeOH); IR (ﬁlm, cm ):

1

3

1

349 (br), 1667, 1465, 1390, 1258, 1202, 1114. H NMR (500 MHz, CD

3

OD) d 7.42 (d, J = 7.9 Hz, 1H), 5.71 (s, 1H), 5.49 (s, 1H), 4.90 (s,

13

H), 4.52 (d, J = 5.9, 1.2 Hz, 1H), 4.33 – 4.19 (m, 2H), 4.04 (t, J = 5.6 Hz, 1H). C NMR (125 MHz, CD

3

OD) d 166.4, 153.1, 152.1, 143.6,

+

25.5, 102.8, 78.4, 74.0, 67.4, 60.3. HRMS (ESI) m/z 263.0644 [(M+Na) ; calcd for C10

In a similar manner, compound 1 (360 mg, 0.85 mmol, 1 equiv.) was reacted with 2b (238 mg, 1.02 mmol, 1.2 equiv.) to afford in-

termediate 3b as white powder (288 mg, 0.45 mmol, 53%). Intermediate 3b (32 mg, 0.05 mmol) was then deprotected to afford 4b

7.7 mg, 0.03 mmol, 60%) as white powder.

-benzoyl-1-((3aS,4R,6aR)-6-(((tert-butyldiphenylsilyl)oxy)methyl)-2,2-dimethyl-3a,6a-dihydro-4H-cyclopenta[d][1,3]

dioxol-4-yl)-5-ﬂuoropyrimidine-2,4(1H,3H)-dione (3b)

12 2 5

H N NaO : 263.0638 ].

(

3

-1

[

a]20D– 12.9 (c 1.4, CHCl

3

); IR (ﬁlm, cm ): 3072, 2932, 2857, 1753, 1712, 1665, 1662, 1448, 1373, 1234, 1106, 1088, 732, 702, 687.

) d 7.97 – 7.92 (m, 2H), 7.73 – 7.63 (m, 5H), 7.55 – 7.49 (m, 2H), 7.48 – 7.42 (m, 2H), 7.42 – 7.35 (m, 4H), 7.06

d, J = 5.8 Hz, 1H), 5.68 (brs, 1H), 5.41 (brs, 1H), 5.08 (d, J = 5.8 Hz, 1H), 4.59 (dd, J = 12.1, 6.4 Hz, 1H), 4.54 – 4.35 (m, 2H), 1.34 (s, 3H),

1

H NMR (400 MHz, CDCl

3

(

1

3

1

.29 (s, 3H), 1.11 (s, 9H). C NMR (100 MHz, CDCl

3

) d 167.3, 154.3, 148.3, 141.4, 139.0, 135.6, 135.6, 133.2, 133.0, 131.2, 130.8,

19

1

30.2, 130.2, 129.4, 129.1, 128.0, 125.5, 125.1, 120.4, 113.0, 84.6, 83.2, 68.3, 61.4, 27.3, 27.0, 25.9, 19.4. F NMR (376 MHz, CDCl

3

)

+

d -163.44 (d, J = 5.7 Hz). HRMS (ESI) m/z 663.2323 [(M+Na) ; calcd for C36

-((1R,4R,5S)-4,5-dihydroxy-3-(hydroxymethyl)cyclopent-2-en-1-yl)-5-ﬂuoropyrimidine-2,4(1H,3H)-dione (4b)

2 6

H37FN O SiNa :663.2297].

1

-1

1

[

a]20D– 87.8 (c 0.45, MeOH); IR (ﬁlm, cm ): 3375 (br), 1696, 1660, 1386, 1242, 1115, 1011. H NMR (400 MHz, CD

.6 Hz, 1H), 5.68 (q, J = 1.8 Hz, 1H), 5.49 (brs, 1H), 4.52 (d, J = 5.6 Hz, 1H), 4.26 (d, J = 2.2 Hz, 2H), 4.03 (t, J = 5.5 Hz, 1H). C NMR

100 MHz, CD OD) d 159.54 (d, J = 26.1 Hz), 152.64, 151.74, 142.07 (d, J = 233.0 Hz), 127.39 (d, J = 33.7 Hz), 125.24, 78.17, 73.94,

3

OD) d 7.60 (d, J =

13

6

(

3

1

9

+

6

2

3 2 5

7.73, 60.26. F NMR (376 MHz, CD OD) d -168.53 (dd, J = 6.8, 1.7 Hz). HRMS (ESI) m/z 259.0722 [(M+H) ; calcd for C10H12FN O :

59.0725].

In a similar manner, compound 1 (245 mg, 0.58 mmol, 1 equiv.) was reacted with 2c (238 mg,0.70 mmol, 1.2 equiv.) to afford in-

termediate 3c (160 mg, 0.21mmol, 36%) as white powder. Intermediate 3c (40 mg, 0.053 mmol) was then deprotected to afford 4c

9.6 mg, 0.026 mmol, 49%) as white powder.

-benzoyl-1-((3aS,4R,6aR)-6-(((tert-butyldiphenylsilyl)oxy)methyl)-2,2-dimethyl-3a,6a-dihydro-4H-cyclopenta[d][1,3]

dioxol-4-yl)-5-iodopyrimidine-2,4(1H,3H)-dione (3c)

(

3

-

1

[

a]20D– 45.1 (c 1.3, CHCl

CDCl ) d 7.93 – 7.90 (m, 1H), 7.72 – 7.63 (m, 5H), 7.54 – 7.48 (m, 3H), 7.45 – 7.37 (m, 6H), 5.72 (brs, 1H), 5.37 (s, 1H), 5.13 (d, J =

.8 Hz, 1H), 4.62 (d, J = 5.8 Hz, 1H), 4.51 – 4.34 (m, 2H), 1.34 (s, 3H), 1.29 (s, 3H), 1.09 (s, 9H). C NMR (100 MHz, CDCl

d 167.8, 159.1, 154.1, 149.5, 145.8, 135.6, 135.4, 133.1, 133.1, 131.1, 130.7, 130.1, 129.4, 128.0, 128.0, 120.6, 113.0, 84.6, 83.4,

3

); IR (ﬁlm, cm ): 3071, 2932, 2857, 1749, 1703, 1666, 1608, 1420, 1233, 1112, 703. H NMR (400 MHz,

3

13

5

3

)

+

6

9.2, 68.1, 61.4, 27.3, 27.0, 25.9, 19.5. HRMS (ESI) m/z 749.1538 [(M+H) ; calcd for C36

H38IN

2

O

6

Si :749.1538].

1

-((1R,4R,5S)-4,5-dihydroxy-3-(hydroxymethyl)cyclopent-2-en-1-yl)-5-iodopyrimidine-2,4(1H,3H)-dione (4c).

-1

1

[

a]20D–104.0 (c 0.6, MeOH); IR (ﬁlm, cm ): 3370 (br), 1682, 1608, 1424, 1260, 1111. H NMR (400 MHz, CD

3

OD) d 7.78 (s, 1H), 5.70

13

(

q, J = 1.8 Hz, 1H), 5.47 (brs, 1H), 4.52 (d, J = 5.5 Hz, 1H), 4.33-4.20 (m, J = 2.1 Hz, 2H), 4.04 (t, J = 5.7 Hz, 1H). C NMR (100 MHz,

+

3 2 5

CD OD) d 163.0, 152.8, 152.4, 148.0, 125.5, 78.6, 73.9, 68.6, 67.9, 60.0.HRMS (ESI) m/z 366.9782 [(M+H) ; calcd for C10H12IN O :

3

66.9785].

In a similar manner, compound 1 (113 mg, 0.27 mmol, 1 equiv.) was reacted with 2d (74 mg, 0.32 mmol, 1.2 equiv.) to afford in-

termediate 3d as white powder (100 mg, 0.16 mmol, 59%). Intermediate 3d (30 mg, 0.047 mmol) was then deprotected to afford

d (4.3 mg, 0.017 mmol, 36%) as white powder.

-benzoyl-1-((3aS,4R,6aR)-6-(((tert-butyldiphenylsilyl)oxy)methyl)-2,2-dimethyl-3a,6a-dihydro-4H-cyclopenta[d][1,3]

dioxol-4-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (3d)

4

3

-1

1

[

a]20D– 27.8 (c 1.2, CHCl

CDCl ) d 7.97 – 7.89 (m, 2H), 7.71 – 7.61 (m, 5H), 7.53 – 7.46 (m, 3H), 7.45 – 7.42 (m, 1H), 7.40 – 7.35 (m, 4H), 6.90 (brs, 1H), 5.73 (s, 1H),

.37 (s, 1H), 5.13 (d, J = 5.8 Hz, 1H), 4.65 (dd, J = 16.3, 4.7 Hz, 1H), 4.47 – 4.37 (m, 2H), 1.96 (s, 3H), 1.34 (s, 3H), 1.29 (s, 3H), 1.09 (s,

3

); IR (ﬁlm, cm ): 3071, 2932, 2857, 1748, 1699, 1656, 1429, 1371, 1235, 1111, 732, 702. H NMR (500 MHz,

3

5

e4 Cell Chemical Biology 27, 1–10.e1–e9, June 18, 2020

Please cite this article in press as: Liu et al., Enhancing the Antiviral Efﬁcacy of RNA-Dependent RNA Polymerase Inhibition by Combination with Mod-

ulators of Pyrimidine Metabolism, Cell Chemical Biology (2020), https://doi.org/10.1016/j.chembiol.2020.05.002

ll

Article

1

3

9

1

6

3

H). C NMR (125 MHz, CDCl ) d 169.1, 163.0, 152.9, 149.8, 137.2, 135.6, 135.1, 133.2, 133.1, 131.7, 130.6, 130.1, 129.3, 128.0,

+

41 2 6

21.3, 112.7, 111.0, 84.6, 83.5, 68.3, 61.4, 27.3, 27.0, 25.9, 19.5, 12.8. HRMS (ESI) m/z 637.2734 [(M+H) ; calcd for C37H N O Si :

37.2728].

-((1R,4R,5S)-4,5-dihydroxy-3-(hydroxymethyl)cyclopent-2-en-1-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (4d)

1

-1

1

[a]20D– 78.5 (c 0.38 , MeOH); IR (ﬁlm, cm ): 3371, 1683, 1476, 1261, 1205, 1114, 1016. H NMR (400 MHz, CD

3

OD) d 7.23 (d, J =

1

.2 Hz, 1H), 5.68 (q, J = 1.8 Hz, 1H), 5.48 (brs, 1H), 4.52 (d, J = 5.9 Hz, 1H), 4.26 (q, J = 2.5 Hz, 2H), 4.04 (t, J = 5.6 Hz, 1H), 1.87

1

3

(

3

d, J = 1.2 Hz, 3H). C NMR (100 MHz, CD OD) d 166.5, 153.3, 151.8, 139.2, 126.0, 111.8, 78.3, 74.0, 67.2, 60.3, 12.3. HRMS

+

ESI) m/z 277.0799 [(M+Na) ; calcd for C11

-benzoyl-1-((3aS,4R,6aR)-6-(hydroxymethyl)-2,2-dimethyl-3a,6a-dihydro-4H-cyclopenta[d][1,3]dioxol-4-yl)

pyrimidine-2,4(1H,3H)-dione (5)

To a solution of 3a (300 mg, 0.48 mmol, 1 equiv.) in THF (5 mL) was added TBAF (580 mL, 0.58 mmol, 1.2 equiv., 1 M in THF) at 0 C.

After 2 h, the reaction mixture was quenched with 5 mL of saturated NH OH solution and extracted with 5 3 5 mL EtOAc. The com-

bined organic layers were dried over anhydrous Na SO , concentrated, loaded onto a 4 g SiO ﬂash cartridge, and puriﬁed with a

linear gradient 80-95% EtOAc in hexanes to afford the free primary alcohol 5 (160 mg, 0.42 mmol, 87%) as white powder.

14 2 5

H N O Na : 277.0795].

3

ꢂ

4

2

4

2

-1

1

[

a]20D– 4.9 (c 4.5, CHCl

NMR (500 MHz, CDCl ) d 7.93 (d, J = 8.4 Hz, 2H), 7.70 – 7.61 (m, 1H), 7.50 (dd, J = 8.2, 7.4 Hz, 2H), 7.16 (d, J = 8.0 Hz, 1H), 5.81

d, J = 8.0 Hz, 1H), 5.64 (s, 1H), 5.30 (s, 1H), 5.25 (d, J = 5.8 Hz, 1H), 4.70 (d, J = 5.8 Hz, 1H), 4.43 (d, J = 15.9 Hz, 1H), 4.38 (d,

3

); IR (ﬁlm, cm ): 3473 (br), 3088, 2988, 2933, 1746, 1702, 1665, 1443, 1374, 1239, 1179, 1239, 1059. H

3

(

1

3

J = 15.9 Hz, 1H), 1.43 (s, 3H), 1.34 (s, 3H). C NMR (125 MHz, CDCl ) d 168.8, 162.2, 152.5, 149.7, 141.6, 135.3, 131.5, 130.7,

+

1

21 2 6

29.3, 121.7, 113.0, 102.5, 84.3, 84.0, 69.2, 60.2, 27.3, 25.8. HRMS (ESI) m/z 385.1391 [(M+H), calcd for C20H N O : 385.1394].

1

-((1R,4R,5S)-4,5-dihydroxy-3-(methoxymethyl)cyclopent-2-en-1-yl)pyrimidine-2,4(1H,3H)-dione (6)

O (23 mg, 0.1 mmol, 2 equiv.) and MeI (31 mL,

.5 mmol, 10 equiv.). The reaction mixture was stirred at room temperature for 24 h, ﬁltered through Celite pad, and concentrated in

vacuo to afford a residue oil 17 mg. In a similar manner as the synthesis of 3a from 4a, the crude was treated with 0.5 mL 7N NH in

methanol, followed by 25 mL HCl in 250 mL of THF to furnish the analog 6 (2.5 mg, 0.01 mmol, 20% for three steps). [a]20D–64.7 (c

To a solution of 5 (20 mg, 0.05 mmol, 1 equiv.) in dry acetone (0.5 mL) was added Ag

2

0

3

-

1

0

8

1

.25, MeOH); IR (ﬁlm, cm ): 3369 (br), 2921, 2851, 1682, 1469, 1410, 1262, 1204, 1096. H NMR (600 MHz, CD

3

OD) d 7.40 (d, J =

.0 Hz, 1H), 5.72 (dd, J = 3.8, 1.7 Hz, 1H), 5.69 (d, J = 8.0 Hz, 1H), 5.51 – 5.43 (m, 1H), 4.50 (d, J = 5.8 Hz, 1H), 4.14 (ddd, J =

13

4.1, 2.5, 2.4 Hz, 1H), 4.08 (ddd, J = 14.2, 2.2, 1.8 Hz, 1H), 4.05 (dd, J = 5.7, 5.5 Hz, 1H), 3.40 (s, 3H). C NMR (100 MHz,

+

3

CD OD) d 166.4, 153.0, 148.8, 143.7, 127.5, 102.8, 78.1, 74.0, 70.3, 67.6, 59.0. HRMS (ESI) m/z 277.0799 [(M+Na) ; calcd for

+

Na : 277.0795].

C

11

H

14

N

2

O

5

3

-benzoyl-1-((3aS,4R,6aR)-6-(ﬂuoromethyl)-2,2-dimethyl-3a,6a-dihydro-4H-cyclopenta[d][1,3]dioxol-4-yl)

pyrimidine-2,4(1H,3H)-dione (M1)

ꢂ

To alcohol 5 (50 mg, 0.13 mmol, 1 equiv.) in 1 mL CH

00 mL, prepared by diluting 100 mL of DAST with CH

2

Cl

2

at –78 C was added a stock solution of DAST (0.15 mmol, 1.2 equiv.,

to 1 mL). The reaction mixture was warmed up to room temperature

2

and stirred overnight before quenching with 1 mL saturated NaHCO

The combined organic layers were dried over anhydrous Na SO , concentrated, loaded onto a 4 g SiO

with a linear gradient 20-50% EtOAc in hexanes to afford the intermediate M1 (27 mg, 0.070 mmol, 54%) as white powder. [a]20D–

3

. The resulting mixture was then extracted with 333 mL EtOAc.

2

4

2

ﬂash cartridge, and puriﬁed

-

1

CDCl3) d 7.93 (d, J = 8.1 Hz, 2H), 7.66 (t, J = 7.5 Hz, 1H), 7.51 (dd, J = 7.91, 7.13 Hz, 2H), 7.13 (d, J = 8.1 Hz, 1H), 5.83 (d, J = 8.1 Hz, 1H),

2.8 (c 2.4, CHCl

3

); IR (ﬁlm, cm ): 3100, 2989, 2937, 1746, 1704, 1667, 1599, 1441, 1374, 1239, 1179, 1060, 989. H NMR (600 MHz,

13

5

.72 (s, 1H), 5.32 (s, 1H), 5.26 (d, J = 5.9 Hz, 1H), 5.14 (s, 1H), 5.07 (s, 1H), 4.73 (d, J = 5.7 Hz, 1H), 1.43 (s, 3H), 1.34 (s, 3H). C NMR

125 MHz, CDCl ) d 168.7, 162.2, 149.6, 141.5, 135.4, 131.5, 130.7, 129.4, 123.2, 113.2, 102.7, 95.7, 84.1, 83.2, 80.5 (d, J = 149 Hz),

(

3

1

9

+

7

3

8.8, 77.4, 77.2, 76.9, 69.2, 27.3, 25.8. F NMR (376 MHz, CDCl ) d – 224.4 (t, J = 46.5). HRMS (ESI) m/z 387.1366 [(M+H) ; calcd for

+

C

20

H20FN

2

O

5

: 387.1351].

-((1R,4R,5S)-3-(ﬂuoromethyl)-4,5-dihydroxycyclopent-2-en-1-yl)pyrimidine-2,4(1H,3H)-dione (7)

In a similar manner as the synthesis of 4a from 3a, compound M1 (13 mg, 0.034 mmol, 1 equiv.) was treated with 0.5 mL 7N NH

methanol, followed by 25 mL HCl in 250 mL THF to furnish the analog 7 (4.4 mg, 0.018 mmol, 53% for two steps) as white powder.

1

3

in

-1

1

[

a]20D– 86.0 (c 0.22, MeOH); IR (ﬁlm, cm ): 3365 (br), 2920, 2852, 1675, 1466, 1389, 1265, 1203, 1115. H NMR (600 MHz,

CD OD) d 7.42 (d, J = 7.9 Hz, 1H), 5.85 (brs, 1H), 5.70 (d, J = 8.0 Hz, 1H), 5.48 (brs, 1H), 5.15 – 4.99 (m, 2H), 4.57 (d, J = 6.0 Hz,

H), 4.10 (dd, J = 6.7, 6.0 Hz, 1H). C NMR (150 MHz, CD

3

1

3

1

3

OD) d 166.2, 152.8, 147.2, 143.6, 128.2, 102.8, 80.5 (d, J = 149 Hz),

Cell Chemical Biology 27, 1–10.e1–e9, June 18, 2020 e5

Please cite this article in press as: Liu et al., Enhancing the Antiviral Efﬁcacy of RNA-Dependent RNA Polymerase Inhibition by Combination with Mod-

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1

9

+

7

3

7.6, 73.1, 67.4. F NMR (376 MHz, CD OD) d – 224.9 (t, J = 46.8 Hz). HRMS (ESI) m/z 265.0592 [(M+Na) ; calcd for

+

C

10

H

11FN

2

O

4

Na : 265.0595].

(3aS,4R,6aR)-4-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,2-dimethyl-3a,6a-dihydro-4H-cyclopenta[d]

1,3]dioxol-6-yl)methyl methanesulfonate (8)

ꢂ

2 3

To a solution of 5 (60 mg, 0.16 mmol, 1 equiv.) in anhydrous CH Cl (2 mL) was added Et N (44 mL, 0.32 mmol, 2 equiv.) at 0 C, fol-

(

[

2

lowed by the dropwise addition of 0.2 mL MsCl stock solution (0.26 mmol, 1.6 equiv.; Stock solution was prepared by diluting 0.1 mL

ꢂ

MsCl with CH

extracted with 3 3 3 mL CH

linear gradient 50-95% EtOAc in hexanes to afford the compound 8 (53 mg, 0.11 mmol, 72%) as pale yellow oil. [a]20D – 35.3 (c 2.8,

2

Cl

2

to 1 mL). After 20 min at 0 C, the reaction mixture was quenched with 2 mL saturated aqueous solution of NH

4

Cl,

2

Cl

2

, dried over anhydrous Na

2

SO

4

, concentrated, loaded on a 4g SiO

2

ﬂash cartridge, and puriﬁed with a

-1

1

3 3

CHCl ); IR (ﬁlm, cm ): 2989, 2937, 1745, 1702, 1661, 1597, 1440, 1354, 1235, 1174, 1087, 959, 906, 729. H NMR (500 MHz, CDCl )

d 7.92 (d, J = 7.2 Hz, 2H), 7.66 (dd, J = 8.0, 7.7 Hz, 1H), 7.50 (d, J = 7.8 Hz, 2H), 7.19 (d, J = 8.1 Hz, 1H), 5.83 – 5.67 (m, 2H), 5.30 (d, J =

13

4

.8 Hz, 2H), 4.98 (d, J = 14.0 Hz, 1H), 4.83 (d, J = 14.0 Hz, 1H), 4.71 (d, J = 5.8 Hz, 1H), 3.04 (s, 3H), 1.41 (s, 3H), 1.33 (s, 3H). C NMR

(

125 MHz, CDCl ) d 168.7, 162.1, 149.6, 146.3, 141.8, 135.4, 131.4, 130.6, 129.4, 125.9, 113.1, 102.7, 83.8, 83.1, 77.4, 77.2, 76.9,

3

+

6

23 2 8

8.7, 65.0, 38.1, 27.3, 25.7. HRMS (ESI) m/z 463.1185 [(M+H) ; calcd for C21H N O S : 463.1170].

1

-((3aS,4R,6aR)-6-(azidomethyl)-2,2-dimethyl-3a,6a-dihydro-4H-cyclopenta[d][1,3]dioxol-4-yl)pyrimidine-

,4(1H,3H)-dione (M2)

2

To the mesylate intermediate 8 (26 mg, 0.056 mmol, 1 equiv.) was added DMF (3 mL) and NaN

3

(80 mg, 1.23 mmol, 22 equiv.). The

Cl. The

O, washed with brine, and concentrated under reduced pressure. The crude material was

ﬂash cartridge with a linear gradient 40-95% EtOAc in hexanes to afford the azide intermediate S2 (14 mg,

ꢂ

resulting suspension was heated to 100 C and stirred for 18 hours, followed by the addition of 3 mL saturated solution of NH

4

organic layer was extracted with 3 3 5 Et

puriﬁed through a 4g SiO

2

-1

0

1

5

.046 mmol, 82%) as brown oil. [a]20D– 34.0 (c 0.7, CHCl

3

); IR (ﬁlm, cm ): 3197, 3059, 2988, 2929, 2102, 1687, 1456, 1380, 1243,

1

082, 1058. H NMR (500 MHz, CDCl

.22 (d, J = 5.7 Hz, 1H), 4.65 (d, J = 5.8 Hz, 1H), 4.13 (d, J = 16.0 Hz, 1H), 4.04 (d, J = 16.0 Hz, 1H), 1.44 (s, 3H), 1.35 (s, 3H). C NMR

) d 162.8, 150.5, 150.1, 141.2, 122.1, 112.6, 102.3, 86.2, 84.4, 68.0, 27.4, 26.0, 14.6. HRMS (ESI) m/z 306.1195

3

) d 8.78 (brs, 1H), 7.02 (d, J = 8.0 Hz, 1H), 5.71 (d, J = 8.0 Hz, 1H), 5.64 (brs, 1H), 5.31 (brs, 1H),

13

(

125 MHz, CDCl

3

+

[

(M+H) ; calcd for C13

-((1R,4R,5S)-3-(azidomethyl)-4,5-dihydroxycyclopent-2-en-1-yl)pyrimidine-2,4(1H,3H)-dione (9)

To intermediate S2 (6 mg, 0.023 mmol, 1 equiv.) was added THF (250 mL) and concentrated HCl (25 mL). The reaction mixture was

stirred at room temperature for 5 h. An excess amount of NaHCO was added to neutralize the reaction, followed by the addition of

mL MeOH. The resulting suspension was then ﬁltered through a short Celite pad, rinsed with 2 3 2 mL MeOH, and concentrated.

16 5 4

H N O : 306.1197].

1

3

1

2

The resulting crude was resuspended with 1 mL H O and subjected to HPLC puriﬁcation with a linear gradient of 5-20% MeCN (0.1%

TFA) in H

2

O (0.01% TFA) on a prep C18 column (Agilent 10 prep-C18 250 3 21.1 mm). Fractions containing the desired product were

then combined and lyophilized to afford the ﬁnal product 9 (3.6 mg, 0.014 mmol, 59%) as white powder. [a]20D– 50.0 (c 0.36, MeOH);

-

1

3

IR (ﬁlm, cm ): 3358 (br), 2921, 2104, 1683, 1467, 1393, 1261, 1205, 1116. H NMR (400 MHz, CD OD) d 7.39 (d, J = 8.0, 0.6 Hz, 1H),

13

5

MHz, CD

.81 (dd, J = 3.2, 1.9 Hz, 1H), 5.70 (d, J = 8.0 Hz, 1H), 5.52 – 5.40 (m, 1H), 4.52 (d, J = 5.8 Hz, 1H), 4.14 – 3.97 (m, 3H). C NMR (100

+

3

OD) d 166.4, 153.0, 146.7, 143.7, 128.5, 102.9, 77.9, 74.5, 67.9, 50.4. HRMS (ESI) m/z 288.0697 [(M+Na) ; calcd for

+

Na : 288.0703].

C

10

H

11

N

5

O

4

(

(3aS,4R,6aR)-4-(3-benzoyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,2-dimethyl-3a,6a-dihydro-4H-cyclopenta[d]

1,3]dioxol-6-yl)methyl acetate (10)

Triethylamine (66 uL, 0.48mmol, 3 equiv.) and acetyl chloride (AcCl) (17 mL, 0.24 mmol, 1.5 equiv.) were sequentially added to a so-

[

ꢂ

lution of 5 (60 mg, 0.16 mmol, 1 equiv.) in CH

stirred for another hour. A saturated solution of NH

washed with H O, and concentrated under reduced pressure. The crude material was puriﬁed through a 4g SiO

with a linear gradient 65-95% EtOAc in hexanes to afford the intermediate 10 (56 mg, 0.13 mmol, 82%) as colorless oil. [a]20D–

2

Cl

2

(2 mL) at 0 C. The reaction mixture was then warmed up to room temperature and

4

Cl (2 mL) was added. The organic layer was extracted with 3 3 3 mL CH

2 2

Cl ,

2

ﬂash cartridge

-

1

6

2.8 (c 1.3, CHCl

NMR (400 MHz, CDCl

d, J = 8.1 Hz, 1H), 5.61 (brs, 1H), 5.34 (brs, 1H), 5.23 (d, J = 5.7 Hz, 1H), 4.79 (qt, J = 15.0, 1.8 Hz, 2H), 4.67 (d, J = 5.3 Hz, 1H),

3

); IR (ﬁlm, cm ): 3080, 2988, 2935, 1742, 1703, 1663, 1599, 1440, 1372, 1236, 1179, 1236, 1058, 904, 731. H

3

) d 7.93 (d, J = 7.6 Hz, 2H), 7.78 – 7.61 (m, 1H), 7.50 (dd, J = 8.1, 7.7 Hz, 2H), 7.10 (d, J = 8.1 Hz, 1H), 5.81

(

1

3

2

3

.12 (s, 3H), 1.42 (s, 3H), 1.34 (s, 3H). C NMR (100 MHz, CDCl ) d 170.7, 168.7, 162.2, 149.7, 148.3, 141.2, 135.3, 131.5, 130.7,

e6 Cell Chemical Biology 27, 1–10.e1–e9, June 18, 2020

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1

29.3, 123.2, 113.1, 102.6, 84.1, 83.7, 68.6, 60.9, 27.3, 25.9, 20.9. HRMS (ESI) m/z 427.1508 [(M+H) ; calcd for C22

H

23

N

2

O

7

:

4

27.1500].

1

-((1R,4R,5S)-4,5-dihydroxy-3-methylcyclopent-2-en-1-yl)pyrimidine-2,4(1H,3H)-dione (11)

To a solution of the allylic acetate 10 (56 mg, 0.13 mmol, 1 equiv.) in 95% ethanol (4 mL) and cyclohexene (2 mL) was added 20 w%

Pd(OH) on carbon (20 mg, 1:3 catalyst substrate by weight). The resulting suspension was stirred under reﬂux overnight. The reac-

2

tion mixture was then ﬁltered, concentrated, and dried under high vacuum to afford 20 mg crude material as pale-yellow oil. In a

similar manner as the preparation of 9, 6 mg of the crude product was treated with 25 mL HCl in 250 mL THF to afford the ﬁnal product

-

1

4

1

1 (8 mg, 0.037 mmol, 28% for two steps) as white solid. [a]20D–78.0 (c 0.24, MeOH); IR (ﬁlm, cm ): 3364 (br), 2921, 2851, 1680,

1

468, 1393, 1251, 1203, 1116. H NMR (400 MHz, CD

3

OD) d 7.39 (d, J = 8.0 Hz, 1H), 5.67 (d, J = 8.0 Hz, 1H), 5.52 – 5.35 (m, 2H),

13

.37 (d, J = 5.8 Hz, 1H), 3.99 (dd, J = 5.9, 5.4 Hz, 1H), 1.88 (s, 3H). C NMR (100 MHz, MeOH) d 166.4, 153.1, 148.9, 143.5,

+

12 2 4

25.6, 102.7, 78.2, 77.2, 68.1, 15.1. HRMS (ESI) m/z 247.0685 [(M+Na) ; calcd for C10H N O Na: 247.0689].

Puriﬁcation of Recombinant Human UCK2

Previously, human UCK2 was cloned into the pET21a(+) vector resulting in an expression vector for C-terminally 63His-tagged UCK2

(

sion plasmid, recovered and plated onto selective LB agar plates (carbenicillin) overnight at 37 C. A single colony was used to inoc-

Okesli-Armlovich et al., 2019). As previously described, E. coli BL21(DE3) cells were electroporated with this pET21a-UCK2 expres-

ꢂ

ulate a 30 mL starter culture in LB Broth with carbenicillin and grown at 37 C overnight. The next day, this starter culture was used to

inoculate 1 L LB Broth with carbenicillin and grown, with agitation, at 37 C. When cells reached an optical density (OD600) of 0.7,

protein expression was induced with 0.5 mM isopropyl b-D-1-thiogalactopyranoside (IPTG) and cells were shaken for an additional

ꢂ

15 h at 18 C. Cell pellets were collected by centrifugation, ﬂash-frozen and stored at -80 C as previously detailed (Okesli-Armlovich

et al., 2019).

Pellets were thawed and resuspended in lysis buffer containing Tris (40 mM pH 7.5), NaCl (10 mM), imidazole (10 mM), dithiothreitol

DTT) (1 mM) and glycerol (10% v/v). Cells were lysed by sonication and centrifuged at 25,000g for 1 h. The supernatant was incu-

(

bated with a slurry of Ni-NTA resin for 1 h at 4 C prior to column loading. The nickel column wash buffer was identical to lysis buffer

with each wash step introducing increasing imidazole concentrations (10 mM, 50 mM, 250 mM). After SDS-PAGE conﬁrmation of

ꢂ

¨

protein fractions, the protein was further puriﬁed using an Akta Pure 25 FPLC with a 5 mL Hi Trap Q anion exchange column. Protein

was eluted on a linear gradient from FPLC Buffer A (50 mM Tris-HCl pH 8, 1mM DTT, 10% glycerol) to FPLC buffer B (50 mM Tris-HCl

pH 8, 1 mM DTT, 10% glycerol with 500 mM NaCl) from 2% to 100% Buffer B. Eluates containing recombinant UCK2 were concen-

trated and exchanged into storage buffer (50 mM Tris-HCl pH 7.5, 10% glycerol) using a centrifugal ﬁlter with a 3 KDa cut-off

ꢂ

(

Amicon). Enzyme was aliquoted, ﬂash-frozen and kept at -80 C.

Puriﬁcation of Recombinant Human CMPK1

Previously, human CMPK1 was cloned into the pET28 vector resulting in an expression vector for N-terminally 63His-tagged CMPK1

(

and single colony transformants were used to inoculate a 5 mL starter culture in LB Broth with kanamycin and grown at 37 C overnight.

Deans et al., 2016). As previously described, E. coli BL21(DE3) cells were transformed with this pET28-CMPK1 expression plasmid,

ꢂ

The next day, this starter culture was used to inoculate 1 L LB Broth with kanamycin and grown, with agitation, at 37 C until an OD600 of

ꢂ

ꢁ

0.65 was reached (Deans et al., 2016). Cell pellets were harvested by centrifugation and stored at -80 C (Deans et al., 2016).

Pellets were thawed and resuspended in lysis buffer containing Tris (40 mM pH 7.5), NaCl (10 mM), NaF (5 mM) and DTT (1 mM).

Cells were lysed by sonication and supernatant was clariﬁed at 25,000g for 1 h. This supernatant was incubated with a slurry of Ni-

ꢂ

NTA resin for 1 h at 4 C prior to column loading. The nickel column wash buffer was identical to the lysis buffer with each wash step

introducing increasing imidazole (0 mM, 10 mM, 40 mM, 200 mM) (Deans et al., 2016). After conﬁrming Ni-NTA wash eluates by SDS-

PAGE, relevant protein fractions were diluted as needed, in FPLC Buffer AA (50 mM Tris-HCl pH 8.0, 1 mM DTT) as previously detailed

¨

Deans et al., 2016). The protein was further puriﬁed using an Akta Pure 25 FPLC with a Hi Trap Q anion exchange column. Protein was

(

eluted using a linear gradient from 0 to 95% FPLC Buffer BB (50 mM Tris-HCl pH 8.0, 1 mM DTT, 500 mM NaCl) under established

conditions (Deans et al., 2016). Fractions containing CMPK1 were concentrated and exchanged into storage buffer (50 mM Tris-HCl

pH 7.5, 10% glycerol) using a 10 kDa ﬁlter as previously detailed (Deans et al., 2016). Enzyme was then aliquoted, ﬂash-frozen and

ꢂ

kept at -80 C.

In Vitro Enzyme Activity Assays with UCK2

To continuously monitor reaction progress spectrophotometrically, ATP hydrolysis was coupled to NADH oxidation via pyruvate ki-

nase (PK) and lactate dehydrogenase (LDH) (Tomoike et al., 2017). Reactions were conducted at room temperature in 100 mL in 96-

well plates (Greiner Bio-One, UV-Star, Half Area). Mixtures contained 20 mM HEPES pH 7.2, 100 mM KCl, 2 mM MgCl

2

, 300 mM ATP,

0

-500 mM uridine, 0-500 mM CPU analogs, 10 nM UCK2 (unless otherwise noted), 1 mM phosphoenolpyruvate, 500 mM NADH and 20

units/mL of PK and LDH. Progress was monitored in the linear region using a Biotek Synergy HT and kinetic and inhibition constants

were determined using GraphPad Prism.

Enzymatic Synthesis of CMP, CPU-MP, and 5-F-CPU-MP

Reactions were conducted at room temperature in 100 mL in Eppendorf tubes. Mixtures contained 20 mM HEPES pH 7.2, 100 mM

KCl, 2 mM MgCl

2

, 2.5 mM ATP, 5 mM substrates (cytidine, CPU and 5-F-CPU), and 2 mM UCK2. After gently mixing for 24 h on a

Cell Chemical Biology 27, 1–10.e1–e9, June 18, 2020 e7

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ꢂ

rocking shaker, the reaction mixtures were heated for 3 minutes at 95 C to denature the UCK2 enzyme. Formation of CPU-MP and

5

-F-CPU-MP were conﬁrmed by LC-MS/MS (Figure S1).

In Vitro Enzyme Activity Assays with CMPK1

In a similar manner as the UCK2 assay, ATP hydrolysis was coupled to NADH oxidation via PK and LDH to continuously monitor re-

action progress spectrophotometrically. Reactions were conducted at room temperature in 50 mL in 96-well plates (Greiner Bio-One,

UV-Star, Half Area). The CMPK1 assay buffer contained 50 mM Tris-HCl pH 7.5, 50 mM KCl, 5 mM MgCl

mM PEP and20 units/mL of PK and LDH. To 39.5 mL of CMPK1 assay buffer, 5 mL were added of 0-1 mM stock solutions of sub-

strates (CMP from the commercial vendor and denatured UCK2 reaction mixtures containing newly synthesized CMP, CPU-MP and

-F-CPUMP) in UCK2 assay buffer. After UV-readouts at 340 nm stabilized (ꢁ 5 minutes), a mixture of ATP and CMPK1 in CMPK1

assay buffer was added to a ﬁnal reaction volume of 50 mL, a ﬁnal ATP concentration of 500 mM and a ﬁnal CMPK1 concentration of

0-100 nM. Progress was monitored in the linear region using a Biotek Synergy HT, and kinetic constants were determined using

2

, 2 mM DTT, 500 mM NADH,

1

5

2

GraphPad Prism. Generation of CPU-DP and 5-F-CPU-DP was conﬁrmed by LC-MS/MS (Figure S2).

Sample Preparation for LC-MS/MS Analysis

A549 cells were plated overnight at 80,000 cells/well in 24-well plates in complete DMEM. The next day, cells were treated with

dipyridamole or 250 mM or 500 mM CPU, 5-F-CPU or 5’-F-CPU in DMEM additionally supplemented with 1 mM GSK983 and 5 mM

uridine. Six or twelve hours after drug addition, the cell culture medium was removed and the whole plate was rapidly rinsed twice

ꢂ

by dipping vertically into a beaker containing 37 C Milli-Q water. The plate was then placed on dry ice, followed by the addition of

15

ꢂ

0

.5 mL –20 C lysis buffer (MeCN:MeOH:H

2

O = 2:2:1) containing 0.5 M formic acid and 450 nM uridine

2

N monophosphate sodium

salt (Sigma Aldrich). The solution was then sonicated for 3 3 5 minutes on ice for the metabolite extraction. Subsequently, the sample

was frozen with liquid nitrogen, freeze-dried, re-suspended with 150 mL Milli-Q water, and ﬁnally ﬁltered through a MultiScreen 96-

well ﬁlter plate prior to LC-MS analysis.

LC-MS/MS Analysis

LC-MS/MS was performed on an Agilent 1290 inﬁnity II LC system tandem with 6470 triple quad mass spectrometer using ion pairing

ꢂ

chromatography. LC separation was performed at 40 C with a solvent ﬂow rate of 0.25 mL/min on a Zorbax RRHD Extended-C18

2

column (2.1 3 150 mm, 1.8 mm). Buffer A contained 97% H O, 3% MeOH, 5 mM tributylamine, 5.5 mM Acetic acid and 1 mM medronic

acid with pH=5.0. Buffer B contains ca. 100% MeOH, 5 mM tributylamine, 5.5 mM acetic acid and 10 mM medronic acid with pH = 7.0

The initial mobile phase composition was 100% solvent A, and the gradient (%B) after sample injection was as follows: 0% at 2.5 min,

ꢂ

2

0% at 7.5 min, 45% at 14 min, 99% at 20 to 23 min, and 0% from 23.1 to 27.1 min. The autosampler temperature was set to 4 C and

the sample was injected at 5 mL. Samples were measured in the negative ESI mode with capillary voltage at ꢀ3.5kV. Further source

ꢂ

settings were as follows: gas temperature, 250 C; gas ﬂow, 13 L/min; nebulizer, 35 psi; sheath gas temperature, 325 C; sheath gas

ﬂow, 12 L/min; nozzle voltage, 500 V; and delta EMV, – 200. Acquisition was performed in dynamic multiple reaction monitoring

(

dMRM) mode with fragmentation pattern and retention time setting as Table S1.

DENV-2 Luciferase Reporter Assays

ꢂ

A549 cells were plated overnight at 5,000 cells/well in 96-well plates in complete DMEM and incubated for 24h at 37 C. Cells were

then treated with pyrimidine de novo synthesis inhibitors, and/or salvage inhibitors, and/or R1479 in DMEM supplemented with 20 mM

uridine. Four hours after drug addition, cells were infected with DENV-2 virus at a MOI = 0.04. After 48 h or 72 h, DENV-Luc replication

was monitored by the production of Renilla luciferase, which was measured using the Renilla-Glo Luciferase Assay System (Prom-

ega) according to the speciﬁcations of the manufacturer.

For the accompanying cell viability assay, A549 cells were seeded at 5,000 cells/well in 96-well plates in complete DMEM and incu-

ꢂ

bated for 24 h at 37 C. Cell were then treated with pyrimidine de novo synthesis inhibitors, and/or salvage inhibitors, and/or R1479 in

DMEM supplemented with 20 mM uridine. Following 48 h or 72 h treatment, cell viability was monitored via ATP levels, which were

measured using the CellTiter-Glo Luciferase Assay System (Promega) according to the speciﬁcations of the manufacturer.

For viral tests of the CPU-GSK983 combination (Figure 5B), A549 cells were plated overnight at 20,000 cells/well in 24-well plates in

ꢂ

complete DMEM and incubated for 24 h at 37 C. Cell were then treated with pyrimidine de novo synthesis and/or salvage inhibitors in

DMEM supplemented with 20 mM uridine. Four hours after drug addition, cells were infected with the DENV-2 virus infectious clone at

a MOI = 0.01. After 48 h, DENV-Luc replication was monitored by the production of Renilla luciferase, which was measured using the

Renilla-Glo Luciferase Assay System (Promega) according to the speciﬁcations of the manufacturer.

For cell viability tests of the CPU-GSK983 combination (Figure 5B), A549 cells were seeded into 24-well plates at a density of

ꢂ

20,000 cells/well incubated for 24 h at 37 C. Cell were then treated with pyrimidine de novo synthesis and/or salvage inhibitors in

DMEM supplemented with 20 mM uridine. Following 48h treatment, cells were harvested, and the density of viable cells was deter-

mined by ﬂow cytometry (FSC/SSC) using a BD Accuri C6 Flow Cytometer.

Replicon Assays

A549 cells transfected with the pDENV-Luc replicon were plated at 15,000 cells/well in 96-well plates, and immediately treated with

R1479, and/or, pyrimidine de novo synthesis inhibitors, and/or salvage inhibitors in complete DMEM medium supplemented with

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2

0 mM uridine without antibiotics (Marceau et al, 2016). Forty-eight hours after drug treatment, cells were lysed and subjected to

Renilla luciferase detection, which was measured using the Renilla-Glo Luciferase Assay System (Promega) according to the spec-

iﬁcations of the manufacturer.

QUANTIFICATION AND STATISTICAL ANALYSIS

Statistical analyses were performed using GraphPad Prism. In Figure 3, kinetic constants were calculated using this software. In Fig-

ures 4A, 5A, and S4, comparisons to an untreated control were calculated using one-way ANOVA, Dunnett’s test as detailed in Fig-

ure legends. In Figures 5B, 6, 7, S5, and S6, the IC50 curves visualized and IC50 values provided were generated using a non-linear ﬁt

(

variable slope, four parameters, constrained to positive values). Additional experimental conditions are detailed in Figure captions.

Cell Chemical Biology 27, 1–10.e1–e9, June 18, 2020 e9

Article Doi

Enhancing the Antiviral Efficacy of RNA-Dependent RNA Polymerase Inhibition by Combination with Modulators of Pyrimidine Metabolism

DOI: 10.1016/j.chembiol.2020.05.002

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Article abstract of DOI:10.1016/j.chembiol.2020.05.002

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