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Current and future use of nucleo(s)tide prodrugs in the treatment of hepatitis C virus infection

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This review describes the current state of discovery of past most important nucleoside and nucleotide prodrugs in the treatment of hepatitis C virus infection as well as future potential drugs currently in discovery or clinical evaluation. I highlight first generation landmark prodrug compounds which have been the foundations of incremental improvements toward the discovery and approval milestone of Sofosbuvir. Sofosbuvir is the first nucleotide prodrug marketed for hepatitis C virus treatment and the backbone of current combination therapies. Since this approval, new nucleotide prodrugs using the same design of Sofosbuvir McGuigan prodrug have emerged, some of them progressing through advanced clinical trials and may become available as new incremental alternative hepatitis C virus treatments in the future. Although since Sofosbuvir success, only minimal design efforts have been invested in finding better liver targeted prodrugs, a few novel prodrugs are being studied and their different modes of activation may prove beneficial over the heart/liver targeting ratio to reduce potential drug–drug interaction in combination therapies and yield safer treatment to patients. Prodrugs have long been avoided as much as possible in the past by development teams due to their metabolism and kinetic characterization complexity, but with their current success in hepatitis C virus treatment, and the knowledge gained in this endeavor, should become a first choice in future tissue targeting drug discovery programs beyond the particular case of nucleos(t)ide analogs.
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SI: Advances in Antiviral Nucleoside Analogs and Their Prodrugs Antiviral Chemistry and Chemotherapy
Current and future use of nucleo(s)tide
prodrugs in the treatment of hepatitis C
virus infection
Cyril B Dousson
This review describes the current state of discovery of past most important nucleoside and nucleotide prodrugs in the
treatment of hepatitis C virus infection as well as future potential drugs currently in discovery or clinical evaluation.
I highlight first generation landmark prodrug compounds which have been the foundations of incremental improvements
toward the discovery and approval milestone of Sofosbuvir. Sofosbuvir is the first nucleotide prodrug marketed for
hepatitis C virus treatment and the backbone of current combination therapies. Since this approval, new nucleotide
prodrugs using the same design of Sofosbuvir McGuigan prodrug have emerged, some of them progressing through
advanced clinical trials and may become available as new incremental alternative hepatitis C virus treatments in the
future. Although since Sofosbuvir success, only minimal design efforts have been invested in finding better liver targeted
prodrugs, a few novel prodrugs are being studied and their different modes of activation may prove beneficial over the
heart/liver targeting ratio to reduce potential drug–drug interaction in combination therapies and yield safer treatment
to patients. Prodrugs have long been avoided as much as possible in the past by development teams due to their
metabolism and kinetic characterization complexity, but with their current success in hepatitis C virus treatment, and
the knowledge gained in this endeavor, should become a first choice in future tissue targeting drug discovery programs
beyond the particular case of nucleos(t)ide analogs.
Hepatitis C virus, nucleoside analogs, nucleotide analogs, prodrugs, NS5B
Date received: 21 September 2017. accepted: 13 December 2017
According to a recent report,
in 2015 globally, an esti-
mated 71 million people were living with chronic
hepatitis C infection accounting for 1% of the world
population, with only 20% knowing their infection
status. Mortality was still increasing and an estimated
1.75 million new HCV infections occurred worldwide
in 2015. Infection with HCV becomes chronic in most
infected persons and a person may be infected with
HCV for as long as 30 years or more before developing
any clinical symptoms of disease and 20% or more
develop life-threatening end-stage chronic liver disease,
such as cirrhosis or hepatocellular carcinoma. In 2015,
HCV led to 411,000 deaths.
The research for more effective HCV treatments has
developed and advanced significantly in the recent
years and the focus on direct-acting antiviral agents
(DAAs) and specially nucleotide prodrugs having a
broad genotypic coverage and high barrier to resistance
have emerged as the best promise for backbone com-
bination to eradicate HCV in the next decade.
Nucleo(s)tide prodrugs are pharmacologically inac-
tive modified analogs able to be transformed in vivo to
their parent nucleo(s)tide via metabolic or chemical
processes occurring in the body. For the purpose of
clarity, I will here use under the generic “prodrug”
term only “carrier prodrugs” (covalently bound chem-
ical entity releasing the “drug” by hydrolytic
cleavage at the target site) and not bioprecursors
Idenix, an MSD Company—Medicinal Chemistry Cap Gamma,
Montpellier, France
Corresponding author:
Cyril B Dousson, Idenix, an MSD Company—Medicinal Chemistry Cap
Gamma, 1682 rue de la Valsiere BP 50001, Montpellier 34189, France.
Antiviral Chemistry and Chemotherapy
2018, Vol. 26: 1–8
!The Author(s) 2018
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DOI: 10.1177/2040206618756430
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(chemical entity metabolized into the pharmacological-
ly active entity) as defined elsewhere.
This review will cover the different main current and
future prodrugging strategies used with the more sig-
nificant reported active nucleo(s)tides which landmark
the field of HCV. Comprehensive reviews of nucleos(t)
ide prodrugs have been reported elsewhere.
A-nucleoside prodrugging
Both nucleosides and nucleotides can be prodrugged
depending on the shortcoming properties one wants
to overcome.
Nucleoside prodrugging is performed on a nucleo-
side that can be efficiently metabolized to its active
triphosphate (TP) in order to overcome bioavailability
or tissue targeting shortcomings. The more common
oral bioavailability issues are usually due to lack of
permeation through biological membranes (lipophi-
licity is too low) and the prodrug design will mask or
counterbalance the polar functions of the parent
nucleoside (e.g. isobutyryl esters of Balapiravir),
or taking advantage of amino acid active transport
(e.g. L-valine ester of Valopicitabine);
solubility, which is less common with nucleoside
analogs, but can be mitigated by prodrugging with
polar or ionizable pro-moieties (e.g. valine esters);
Addressing tissue targeting topics can be more com-
plex as the required enzymatic pro-moiety cleavage in a
specific tissue can be different from one parent nucle-
oside analog to another as well as species dependent.
The first nucleoside prodrug evaluated in clinical
trials for HCV was NM283 from Idenix (Figure 1), a
30-L-valine ester of its parent nucleoside NM107 setting
the scene in HCV therapies with the most used 20C-Me
sugar modification. The valine ester substituent was
chosen to improve poor bioavailability of NM107
when given orally.
NM283 was shown in later clinical
phases to be not stable enough in the gastrointestinal
(GI) tract, leading to GI side effects and was
Other first generation prodrugs followed with
Balapiravir and Mericitabine, both tri- and di-isobu-
tyryl esters, respectively, of their corresponding nucle-
oside (Figure 1).
None of these first generation nucleoside prodrugs
led to sufficient clinical benefit to allow approval of a
simple nucleoside prodrug, because daily dose normal-
ized viral load reductions were too low (Table 1).
B-nucleotide prodrugging
On the other hand, nucleotide prodrugging is usually
performed to overcome 50-monophosphorylation prob-
lem or to improve liver targeting. As opposed to nucle-
oside prodrugging, the advantage in HCV activity of a
50-monophosphate prodrug can be demonstrated in cell
culture experiments as shown in Table 2.
1. SATE-phosphoramidate prodrugs
The first clinical proof of concept for such kind of
nucleotide prodrugs was reported by Idenix with the
discovery of IDX184 (Scheme 1).
Other nucleotide
prodrugs were then reported based on the 20C-Me well-
known sugar backbone with different prodrug moieties
giving various improvements over the parent
IDX184 is a benzylamine/“SATE” phosphorami-
date prodrug which benefits from a thioester enzymatic
cleavage liberating the corresponding carboxylic acid
and the 2-thioethyl side chain which undergoes self
emulating cleavage.
While ethylene sulfide was pro-
posed as a cleavage metabolite, it has been shown that
this metabolite was not found in vivo, but glutathione
adduct was instead formed.
The benzylamine phos-
phoramidic acid is further cleaved by a phosphorami-
dase to yield the 50-monophosphate.
metabolism by cellular kinases gives the active corre-
sponding TP (Scheme 1).
IDX184 improved dramatically the clinical dose effi-
ciency as over a two weeks once a day 100 mg dose
Figure 1. First clinical stage nucleoside prodrugs. (a) Valopicitabine (NM283), (b) Balapiravir (R1626), and (c) Mericitabine (R7128).
2Antiviral Chemistry and Chemotherapy 0(0)
treatment, HCV viral load reduced by 2.7 log
one of the highest viral load reduction efficiency per
gram of drug at 27 (Table 1). However, IDX184 suf-
fered from dose-limited absorption as seen in the dose
escalation nonlinearity Cmax.
IDX184 clinical devel-
opment was stopped as a consequence of BMS-986094
severe cardiac side effects, both compounds sharing the
same active Nuc-TP in vivo (vide infra).
2. McGuigan prodrugs
GS-7977 (Sofosbuvir) is a McGuigan phosphorami-
date prodrug (L-alanine/phenol) originally developed
by Pharmasset and is to date the only nucleotide pro-
drug which has received approval for HCV treatment
in December 2013.
After the clinical proof of concept
of GS-7977, other groups have used similar McGuigan
prodrug as exemplified by BMS-986094, AL-335,
ACH-3422, and MIV-802 to reduce the development
risk associated with the metabolites formed by the
pro-moieties (L-alanine and phenol). The cleavage
and release of these pro-moieties in vivo have been
well characterized for GS-7977 or other analogs bear-
ing the same prodrug stereochemistry.
The first step involves hydrolysis of the carboxylic
ester by cathepsin A (Cat A) and carboxylesterase 1
followed by an intramolecular cyclization of the car-
boxylate on the phosphorus atom, displacing the phe-
nolate and followed by water hydrolysis of the unstable
cyclized intermediate to yield the alanyl phosphorami-
dic acid metabolite which is further hydrolyzed by the
enzyme hHint 1 to the nucleoside-monophosphate
(NMP). In the case of GS-7977, this NMP is then phos-
phorylated by UMP-CMP kinase to its nucleoside-
diphosphate (NDP), and final phosphorylation
by Nucleoside DiPhosphate Kinase (NDPK) affords
its nucleoside-triphosphate (Scheme 2).
BMS-986094 is a McGuigan prodrug that was
designed to improve in vitro activity in the replicon
assay owing to an increase of the lipophilicity by
using a naphthol in place of the usual phenol, substitut-
ing the shorter isopropyl ester with a neopentyl and by
removing a hydrogen bond donor on the guanine base
with a 6 methoxy analog. These structural modifica-
tions improved the replicon EC
with activities as
low as 10 nM but with a cytotoxic value CC
7mM giving a selectivity index (toxicity/activity) of
700 (Table 2).
BMS-986094 phase II clinical trial
was stopped due to a fatal cardiac adverse effect that
was characterized further as a mitochondrial toxicity
mainly due to its TP and to a lesser extent to its
Table 1. Clinical dose efficiency of HCV nucleoside and nucleotide prodrugs.
Prodrug Daily dose
Viral load reduction
) at end
of treatment
Dose normalized
viral load reduction
(Valopicitabine) 800 mg 1.2 (2 wk treatment) 1.5
(Balapiravir) 3000 mg
(1500 mg bid)
1.2 (2 wk treatment) 0.4
(Mericitabine) 3000 mg
(1500 mg bid)
2.7 (2 wk treatment) 0.9
100 mg 2.7 (2 wk treatment) 27.0
GS-7977 (Sofosbuvir)
400 mg 4.7 (1 wk treatment) 11.8
100 mg 2.53 (1 wk treatment) 25.3
(Adafosbuvir) 800 mg 4.00 (1 wk treatment) 5.0
700 mg 3.4 (1 wk treatment) 4.9
IDX21437 (MK-3682/Uprifosbuvir)
300 mg 4.23 (1 wk treatment) 14.1
HCV: hepatitis C virus.
Genotype 1 patients.
Table 2. HCV activity in cell culture experiments.
Prodrug EC
(mM) SI
7.600 0.3 >100 >13
1.100 1.2 >1 000 >909
0.850 0.7 >100 >118
0.203 16 >75 >370
0.092 >1087 >100 >1087
0.144 67 >100 >694
0.010 580 7 700
0.160 36 >100 >613
0.075 NR >100 >1333
0.050 NR >25 >500
0.045 >1111 >100 >2222
56.800 1 >100 >2
HCV: hepatitis C virus; NR: Not reported.
Genotype 1b replicon assay.
Fold change EC
prodrug activity (improvement of the
prodrug versus parent nucleoside).
Dousson 3
prodrug moieties.
The effect of neopentyl ester
prodrug of BMS-986094 in place of the isopropyl
ester present in Sofosbuvir can be clearly seen in a
previous study by McGuigan et al., where these
two esters were synthesized with the same nucleoside
backbone and tested.
The isopropyl ester analog
of BMS-986094 proved to be over 14 times less
toxic in the Huh7 cells, so some of the toxicity of
BMS-986094 can be attributed to its neopentyl
ester modification. It has also been reported by
Deval et al., with another comparable pair of com-
pounds by making the BMS-986094-monophosphate
prodrug on Sofosbuvir nucleoside. The Sofosbuvir-
modified hybrid had an increase in the cell toxicity
assay of Huh7 and U937 cells compared to
(Sp isomer)
Adafosbuvir (AL-335)
(Sp isomer)
(Rp and Sp isomer mixture)
Sofosbuvir (GS-7977)
(Sp isomer)
otential structure from corres
references30, 31, 32
Figure 2. McGuigan phosphoramidate nucleotide prodrugs. (a) Sofosbuvir (GS-7977) (Sp isomer), (b) BMS-986094 (Rp and Sp
isomer mixture), (c) Adafosbuvir (AL-335) (Sp isomer), (d) ACH-3422*, and (e) MIV-802* (Sp isomer). *Potential structure from
Kalayanov et al.,
and Andersson.
(Rp and Sp isomer mixture)
Scheme 1. SATE IDX184 nucleotide prodrug and its proposed decomposition pathway.
4Antiviral Chemistry and Chemotherapy 0(0)
Three other McGuigan prodrugs still in clinical
development are AL-335, ACH-3422, and MIV-802
for which little preclinical data have been reported but
for which the HCV replicon activity is similar or slightly
better than Sofosbuvir (Table 2). The early virologic
load decrease in patients is much less efficient than
Sofosbuvir (Table 1) for the first two more advanced
candidates (AL-335 and ACH-3422), and it was recently
announced that AL-335 would not be developed further
in combination.
Although one cannot exclude that
MIV-802 or ACH-3422 could potentially progress fur-
ther in combination with other DAAs.
3. Cyclic phosphotriester (CPO) prodrugs
The 30,50-CPO prodrug structural unit shows possi-
ble significant improvements on the medicinal chemis-
try perspective, allowing smaller molecular weight and
therefore better ligand efficiency as well as lower
number of rotational bonds which, with the former
property, may both provide enhanced passive diffusion
through cell membrane. Both GS-0938 and IDX19368
(Figure 3) are actually double prodrugs as they bear the
ethoxy masking group on the 6-guanine base position
allowing a better solubility of these guanosine deriva-
tives. The in vivo metabolism was studied in the case of
GS-0938 and is described in Scheme 3. It involves a first
oxidative cleavage by cytochrome (CYP3A4), followed
by opening of the cyclic 30,50-phosphodiester (CPOH)
by phosphodiesterase, the last step being the hydrolysis
of the 6-ethoxy guanine prodrug by adenosine
deaminase-like protein 1.
4. D-amino acid based aryl-phosphoramidate prodrugs
IDX21437 is a D-amino acid phosphoramidate pro-
drug of the well-established HCV active 20-b-modified
ribonucleoside family. As seen in Table 2, it has a very
different profile in cell culture experiments, compared
to other clinical candidates, due to the unnatural amino
acid configuration part of its prodrug, giving a lack of
activity in the HCV replicon system and would there-
fore not be viewed by classical medicinal chemists as a
promising compound. But actually, this compound dis-
played an unexpectedly good in vivo profile in regards
to its ability to form high levels of its corresponding
active TP in animal liver, the target organ for HCV.
The metabolism of IDX21437 was reported and proved
to require a different enzymatic system for the initial
cleavage compared to McGuigan prodrug Cat A
involvement (Scheme 4).
The different enzymes
involved in the metabolism of D-amino acid phosphor-
amidate is supposed to be responsible for the better
liver to heart selectivity, as D-alanylate phosphoramidic
acid metabolite was not observed in heart cells.
Currently, IDX21437 (now MK-3682) is progressing
in phase II combination studies.
5. Other miscellaneous prodrugs
Other HCV nucleotide prodrugs were reported in
early discovery studies as CC-1845 from Cocrystal,
for which the structure is unknown but likely
(Sp isomer)
(Rp isomer)
Figure 3. Clinical and preclinical 30,50-CPO prodrug.
(a) GS-0938 (Rp isomer) and (b) IDX19368 (Sp isomer).
NMP Alanylate phosphoramidic acid
Cyclized intermediate
hHint 1
Scheme 2. McGuigan prodrug metabolism.
Dousson 5
a McGuigan prodrug of 20C-Me-2,6,-disubstituted
purine analog. However, recently the company has
declared that preclinical studies indicated higher than
acceptable toxicity and have now switched to a backup
compound CC-2850.
From the first nucleosides through the first generation
of their prodrugs to the second generation of nucleo-
tide prodrugs demonstrating increasing added value of
liver targeting in HCV, no new simple nucleosides or
their prodrugs would be further developed but favoring
their nucleotide prodrugs as can be seen by the latest
candidates in discovery or ongoing clinical evaluation.
With the knowledge gathered by the different metabo-
lism pathways of pro-moieties, future nucleotide pro-
drugs will be designed toward more elaborated and
tissue targeted drugs with single or multiple prodrugs
and possible combinations of the above well character-
ized and main classes of prodrugs as can be already
seen in recent patent applications in the HCV and
other disease areas. I can envision for the future of
HCV nucleos(t)ide drugs better liver targeting based
on more specific liver metabolism, compared to other
tissues as exemplified by IDX21437, rather than first
path metabolism effect as observed in the earlier
per os prodrug design. HCV nucleotide drug discovery
has been a tremendous scientific emulation for the last
15 years and will be able to serve as a foundation case
4. D-aminoacid based Ar
s (PON)
Scheme 3. CPO prodrug metabolism.
MK-3682 (IDX21437)
(D-AA; Rp isomer) carboxylate
cyclized intermediate
hHint 1
D-alanylate phosphoramidic acid
Scheme 4. D-amino acid phosphoramidate prodrug IDX21437 and its proposed metabolism.
6Antiviral Chemistry and Chemotherapy 0(0)
for other disease area nucleos(t)ide prodrug develop-
ment as well as, more broadly, prodrug targeting exam-
ple for other class of drugs in the future.
The author wishes to thank Dr Gilles Gosselin for his invalu-
able assistance in reviewing this manuscript.
Declaration of conflicting interests
The author is an employee of Idenix, an MSD company.
The author(s) received no financial support for the research,
authorship, and/or publication of this article.
Cyril B Dousson
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8Antiviral Chemistry and Chemotherapy 0(0)
... Various amino residues could be introduced without affecting the biological activity of the corresponding prodrugs indicating a less restrictive intracellular decomposition process than previously reported in the literature. One of the most promising SATE phosphoramidate diester derivative was IDX 184 (Fig. 2), a 2′-methylguanosine pronucleotide, which demonstrated to be effective in patients with chronic HCV infections in phase II clinical trials [9][10] . Unfortunately, severe cardiotoxicity complications observed during phase III trials of INX 189, a McGuigan ProTide sharing the same nucleoside analogue parent, led to the withdrawal of IDX 184 from further clinical developments 10 . ...
... One of the most promising SATE phosphoramidate diester derivative was IDX 184 (Fig. 2), a 2′-methylguanosine pronucleotide, which demonstrated to be effective in patients with chronic HCV infections in phase II clinical trials [9][10] . Unfortunately, severe cardiotoxicity complications observed during phase III trials of INX 189, a McGuigan ProTide sharing the same nucleoside analogue parent, led to the withdrawal of IDX 184 from further clinical developments 10 . ...
... AT-527, an orally available prodrug of a guanosine nucleotide analog carrying a 2 ′ -fluoro-2 ′ C-methyl modified ribose (Fig. 7A), was shown to act as a potent broad-spectrum anti-coronavirus inhibitor in a variety of cell lines by targeting the RdRp activity [94]. After oral dosing, AT-527 is converted by cellular enzymes to the active triphosphate metabolite, AT-9010, which is structurally similar to that of sofosbuvir, with the only difference being that it has a guanine base in place of uracil [95] (Fig. 7B). AT-511, the free base of AT-527, also had potent antiviral activity when tested in vitro against several human coronaviruses, including SARS-CoV-2 [94]. ...
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The highly transmissible variants of SARS-CoV-2, the causative pathogen of the COVID-19 pandemic, bring new waves of infection worldwide. Identification of effective therapeutic drugs to combat the COVID-19 pandemic is an urgent global need. RNA-dependent RNA polymerase (RdRp), an essential enzyme for viral RNA replication, is the most promising target for antiviral drug research since it has no counterpart in human cells and shows the highest conservation across coronaviruses. This review summarizes recent progress in studies of RdRp inhibitors, focusing on interactions between these inhibitors and the enzyme complex, based on structural analysis, and their effectiveness. In addition, we propose new possible strategies to address the shortcomings of current inhibitors, which may guide the development of novel efficient inhibitors to combat COVID-19.
... Finally, nucleosides that work by the pseudo-obligate mechanism also possess a 3'-hydroxyl, but due to substituents on adjacent carbons, which cause steric hinderance, lead to immediate cessation of the growing chain. [5] Examples of pseudo-obligate terminators include the 4'-modified nucleosides such as azvudine (also known as FNC) [15] , islatravir [16] , balapiravir [17] , and AL-335 [18] (Figure 6). ...
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Over the past two and a half years the world has seen a desperate scramble to find a treatment for SARS-CoV-2 and COVID. In that regard, nucleosides have long served as the cornerstone to antiviral treatments due to their resemblance to the naturally occurring nucleosides that are involved in numerous biological processes. Unlike other viruses however, it was found early on during the search for drugs to treat SARS-1 and later MERS, that the coronaviruses possess a unique repair enzyme, an exonuclease (ExoN)[3] which rendered nucleoside analogues useless, thus negating their use.[4] During the current outbreak however, as both well-known and new nucleoside analogues were investigated or reinvestigated as a possible cure for SARS-CoV-2, several novel and/or lesser-known mechanisms of action were uncovered. This review briefly describes these mechanisms.
... To mitigate high polarity and improve cell permeability, we initially prepared 7, compound 3's di-isobutyrate prodrug (Table 1), a strategy that has been used in clinical stage HCV antiviral agents balapiravir and mericitabine as well as newly approved molnupiravir. 29 While compound 7 showed antiviral activity, we had difficulty determining its EC 50 value most likely due to compound 7's low solubility in assay solutions. As a result, we synthesized the less lipophilic diacetate prodrug 8, which was more soluble and possessed moderate antiviral activity (EC 50 = 53 μM). ...
Taking advantage of the uniquely constricted active site of SARS-CoV-2 Nsp14 methyltransferase, we have designed bisubstrate inhibitors interacting with the SAM and RNA substrate binding pockets. Our efforts have led to nanomolar inhibitors including compounds 3 and 10. As a prototypic inhibitor, compound 3 also has an excellent selectivity profile over a panel of human methyltransferases. Remarkably, C-nucleoside 10 exhibits high antiviral activity and low cytotoxicity, leading to a therapeutic index (CC50/EC50) greater than 139. Furthermore, a brief metabolic profiling of these two compounds suggests that they are less likely to suffer from major metabolic liabilities. Moreover, computational docking studies point to protein-ligand interactions that can be exploited to enhance inhibitory activity. In short, discovery of inhibitor 10 clearly demonstrates that potent and selective anti-SARS-CoV-2 activity can be achieved by targeting the Nsp14 methyltransferase. Therefore, the current work strongly supports the continued pursuit of Nsp14 methyltransferase inhibitors as COVID-19 therapeutics.
... It is now in phase III and II clinical trials for the treatment of COVID-19 and hepatitis C virus (HCV) infections 8 , respectively 9 ( AT-527 and its active 5′-triphosphate AT-9010 (Fig. 1a, right) carries a 2′-fluoro-2′-C-methyl modified ribose, identical to that of the clinically relevant anti-HCV uracil prodrug Sofosbuvir 10 . ...
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The guanosine analog AT-527 represents a promising candidate against Severe Acute Respiratory Syndrome coronavirus type 2 (SARS-CoV-2). AT-527 recently entered phase III clinical trials for the treatment of COVID-19. Once in cells, AT-527 is converted into its triphosphate form, AT-9010, that presumably targets the viral RNA-dependent RNA polymerase (RdRp, nsp12), for incorporation into viral RNA. Here we report a 2.98 Å cryo-EM structure of the SARS-CoV-2 nsp12-nsp7-nsp82-RNA complex, showing AT-9010 bound at three sites of nsp12. In the RdRp active-site, one AT-9010 is incorporated at the 3′ end of the RNA product strand. Its modified ribose group (2′-fluoro, 2′-methyl) prevents correct alignment of the incoming NTP, in this case a second AT-9010, causing immediate termination of RNA synthesis. The third AT-9010 is bound to the N-terminal domain of nsp12 - known as the NiRAN. In contrast to native NTPs, AT-9010 is in a flipped orientation in the active-site, with its guanine base unexpectedly occupying a previously unnoticed cavity. AT-9010 outcompetes all native nucleotides for NiRAN binding, inhibiting its nucleotidyltransferase activity. The dual mechanism of action of AT-527 at both RdRp and NiRAN active sites represents a promising research avenue against COVID-19. The drug AT-527 targets the SARS-CoV-2 replication machinery. Here the authors use Cryo-EM to show how AT-527 inhibits SARS-CoV-2 polymerase by acting as an immediate RNA chain terminator and stably binding in a NiRAN active-site pocket; impeding an essential nucleotide-transfer activity.
... Because the included patients in this meta-analysis were mainly those who achieved SVR, viral clearance could not account for the different changes in LDL between SOF-based DAAs and non-SOF DAAs. As a nucleotide prodrug, SOF is converted to active compounds by enzymatic cleavage of phosphoramidate side chain, and then is decomposed to GS-060965, phenolate ion, and propan-2-yl 2-aminopropanoate in hepatocytes (Additional file 2: Fig. S2) [28]. Another drug with a similar structure and metabolic pathway to SOF is tenofovir alafenamide (TAF), a prodrug of tenofovir for the treatment of HIV and chronic hepatitis B. Several studies also reported an increased level of LDL with TAF. ...
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Background Previous studies reported worsened lipid profiles in patients infected with hepatitis C virus (HCV) during direct-acting antivirals (DAAs) treatment. This study aimed to investigate the effect of sofosbuvir (SOF)-based DAAs on changes in low-density lipoprotein (LDL) in HCV patients. Methods A systematic review of articles published before 31 May 2021 was conducted by searching MEDLINE, Cochrane Library, EMBASE, and CINAHL Plus. Eligible studies were those comparing SOF-based DAAs and non-SOF DAAs for HCV patients and providing numerical data for changes in LDL. Risk of Bias in Non-randomized Studies- of Interventions was used for assessing risk of bias, and meta-analysis was performed for changes in LDL. Results Six studies comprising 1248 patients were included, 848 patients treated with SOF-based DAAs and 400 patients with non-SOF DAAs vs. SOF-based DAAs group had significantly greater increases in LDL from baseline to week 4 than non-SOF DAAs group (P = 0.001). However, changes in LDL from baseline to the end of treatment (P = 0.060), to post-treatment week 12 (P = 0.263), and to post-treatment week 24 (P = 0.319) did not significantly differ between the two groups. Further comparison of SOF/ledipasvir with asunaprevir/daclatasvir revealed a similar trend in changes in LDL. Conclusions For HCV patients, SOF-based DAA regimens were associated with rapid and significant increases in LDL during the initial 4 weeks of treatment, and the changes did not sustain after the end of treatment. Potential mechanism might be related to the phosphoramidate side chain of SOF.
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Worsened lipid profiles were observed in chronic hepatitis C (CHC) patients during direct-acting antivirals (DAAs) treatment, among which combination drugs confounded the effect of individual ingredient on lipid. Tenofovir alafenamide (TAF) also worsened lipid profiles in HIV patients. Structural similarity between sofosbuvir (SOF) and TAF prompted us to investigate rapid increase in total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) in CHC patients treated with SOF-based DAAs. A retrospective study was performed to analyze 487 CHC patients receiving DAAs with SVR12. Relative risks on elevating TC and LDL-C were analyzed by logistic regression to determine SOF-based over non-SOF-based regimens. TC or LDL-C levels at baseline, week-4 and SVR12 were compared by Wilcoxon matched-pairs signed rank test. Week 4 or SVR12 to baseline ratios of serum TC or LDL-C between regimens were compared by Mann–Whitney's test. 487 patients were treated with Harvoni (SOF-based, 206 patients), Epclusa (SOF-based, 124 patients), Maviret (non-SOF-based, 122 patients), or Zepatier (non-SOF-based, 35 patients). At week 4 during drug treatment, Harvoni, Epclusa, and Maviret induced statistically significant elevation of TC and LDL-C, but Zepatier did not. SOF-based regimens had 2.72-fold higher relative risk (RR) causing 10% elevation of TC (95% CI 1.84–4.02, p < 0.001) and 2.04-fold higher RR causing 10% elevation of LDL-C (95% CI 1.39–3.01, p < 0.001) than non-SOF-based DAAs. SOF-based DAAs were associated with significantly larger amplitude of increases in TC and LDL-C than non-SOF-based DAAs during the initial 4 weeks of treatment, but the increases were not sustained to SVR12.
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Pyrophosphates have important functions in living systems and thus pyrophosphate-containing molecules and their more stable bisphosphonate analogues have the potential to be used as drugs for treating many diseases including cancer and viral infections. Both pyrophosphates and bisphosphonates are polyanionic at physiological pH and, whilst this is essential for their biological activity, it also limits their use as therapeutic agents. In particular, the high negative charge density of these compounds prohibits cell entry other than by endocytosis, prevents transcellular oral absorption and causes sequestration to bone. Therefore, prodrug strategies have been developed to temporarily disguise the charges of these compounds. This review examines the various systems that have been used to mask the phosphorus-containing moieties of pyrophosphates and bisphosphonates and also illustrates the utility of such prodrugs.
Arthropod-borne viruses (arboviruses) recently received global attention due to the (re-)emergence of several viruses. Global temperature changes have favored the spread of indigenous Culicoides and consequently the (re-)emergence of arbovirus infections in both hemispheres. In particular, the five human epidemic mosquito-borne arboviruses: Zika virus (ZIKV), yellow fever virus (YFV), dengue virus (DENV), West Nile virus (WNV), and chikungunya virus (CHIKV) are of special interest. Despite the morbidity and mortality caused by arbovirus infections worldwide, only a few vaccines are currently available. Furthermore, no specific antiviral treatments for any of these viruses have been approved yet. This chapter summarizes the novel advances toward antiviral drug discovery against emerging arboviruses.
Stereocontrolled introduction of a nitrogen atom at either C-2′ or C-3′ positions of nucleosides derived from uridine, 4-N-benzoylcytidine and adenosine was investigated. An efficient and rapid procedure was employed for creating new chiral centers at C-2′ and C-3′ positions using [3 De Clercq, E. Milestones in the Discovery of Antiviral Agents: Nucleosides and Nucleotides. Acta Pharm. Sin. B 2012, 2, 535–548. DOI: 10.1016/j.apsb.2012.10.001.[Crossref] , [Google Scholar],3 De Clercq, E. Milestones in the Discovery of Antiviral Agents: Nucleosides and Nucleotides. Acta Pharm. Sin. B 2012, 2, 535–548. DOI: 10.1016/j.apsb.2012.10.001.[Crossref] , [Google Scholar]]-sigmatropic aza-Claisen rearrangement of allyl thiocyanates under conventional and microwave conditions. Structure of isothiocyanate products was confirmed by 1-D and 2-D NMR spectral analyses including selective ¹H 1-D-NOE experiments.
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Recent cases of severe toxicity during clinical trials have been associated with antiviral ribonucleoside analogs (e.g. INX-08189 and balapiravir). Some have hypothesized that the active metabolites of toxic ribonucleoside analogs, the triphosphate forms, inadvertently target human mitochondrial RNA polymerase (POLRMT), thus inhibiting mitochondrial RNA transcription and protein synthesis. Others have proposed that the prodrug moiety released from the ribonucleoside analogs might instead cause toxicity. Here, we report the mitochondrial effects of several clinically relevant and structurally diverse ribonucleoside analogs including NITD-008, T-705 (favipiravir), R1479 (parent nucleoside of balapiravir), PSI-7851 (sofosbuvir), and INX-08189 (BMS-986094). We found that efficient substrates and chain terminators of POLRMT, such as the nucleoside triphosphate forms of R1479, NITD-008, and INX-08189, are likely to cause mitochondrial toxicity in cells, while weaker chain terminators and inhibitors of POLRMT such as T-705 ribonucleoside triphosphate do not elicit strong in vitro mitochondrial effects. Within a fixed 3’-deoxy or 2′-C-methyl ribose scaffold, changing the base moiety of nucleotides did not strongly affect their inhibition constant (Ki) against POLRMT. By swapping the nucleoside and prodrug moieties of PSI-7851 and INX-08189, we demonstrated that the cell-based toxicity of INX-08189 is mainly caused by the nucleoside component of the molecule. Taken together, these results show that diverse 2′ or 4′ mono-substituted ribonucleoside scaffolds cause mitochondrial toxicity. Given the unpredictable structure-activity relationship of this ribonucleoside liability, we propose a rapid and systematic in vitro screen combining cell-based and biochemical assays to identify the early potential for mitochondrial toxicity.
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Severe bradycardia/bradyarrhythmia following coadministration of the HCV-NS5B prodrug sofosbuvir with amiodarone was recently reported. Our previous preclinical in vivo experiments demonstrated that only certain HCV-NS5B prodrugs elicit bradycardia when combined with amiodarone. In this study, we evaluate the impact of HCV-NS5B prodrug phosphoramidate diastereochemistry (D-/L-alanine, R-/S-phosphoryl) in vitro and in vivo. Co-applied with amiodarone, L-ala,SP prodrugs increased beating rate and decreased beat amplitude in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), but D-ala,RP produgs, including MK-3682, did not. Stereochemical selectivity on emerging bradycardia was confirmed in vivo. Diastereomer pairs entered cells equally well, and there was no difference in intracellular accumulation of L-ala,SP metabolites ± amiodarone, but no D-ala,RP metabolites were detected. Cathepsin A (CatA) inhibitors attenuated L-ala,SP prodrug metabolite formation, yet exacerbated L-ala,SP + amiodarone effects, implicating the prodrugs in these effects. Experiments indicate that pharmacological effects and metabolic conversion to UTP analog are L-ala,SP prodrug-dependent in cardiomyocytes.
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Compound 1 with the structure I is an antiviral HCV protide, which is surprisingly soluble in ethanol, thereby facilitating the preparation of pharmaceutical formulations, such as adsorbed mesoporous carriers or SEDDS of Pouton Types III or IV.
The ProTide technology is a prodrug approach developed for the efficient intracellular delivery of nucleoside analogue monophosphates and monophosphonates. In this approach, the hydroxyls of the monophosphate or monophosphonate groups are masked by an aromatic group and an amino acid ester moiety, which are enzymatically cleaved-off inside cells to release the free nucleoside monophosphate and monophosphonate species. Structurally, this represents the current end-point of an extensive medicinal chemistry endeavour that spans almost three decades. It started from the masking of nucleoside monophosphate and monophosphonate groups by simple alkyl groups and evolved into the sophisticated ProTide system as known today. This technology has been extensively employed in drug discovery and it has already led to the discovery of two FDA-approved (antiviral) ProTides. In this work, we will review the development of the ProTide technology, its application in drug discovery and its role in the improvement of drug delivery and efficacy.
Human Histidine Triad Nucleotide Binding Protein 1 (hHint1) is classified as an efficient nucleoside phosphoramidase and acyl-adenosine monophosphate hydrolase. Human Hint1 has been shown to be essential for the metabolic activation of nucleotide antiviral pronucleotides (i.e. proTides), such as the FDA approved Hepatitis C drug, sofosbuvir. The active site of hHint1 comprises an ensemble of strictly conserved histidines, including nucleophilic His112. To structurally investigate the mechanism of hHint1 catalysis, we have designed and prepared nucleoside thiophosphoramidate substrates that are able to capture the transiently formed nucleotidylated-His112 intermediate (E*) using time-dependent crystallography. Utilizing a catalytically inactive hHint1 His112Asn enzyme variant and wild-type enzyme, the enzyme-substrate (ES1) and product (EP2) complexes were also co-crystallized, respectively, thus providing a structural map of the reaction trajectory. Based on these observations and the mechanistic necessity of proton transfers, proton inventory studies were carried out. Although we cannot completely exclude the possibility of more than one proton in flight, the results of these studies were consistent with the transfer of a single proton during formation of the intermediate. Interestingly, structural analysis revealed that the critical proton transfers required for intermediate formation and hydrolysis maybe mediated by a conserved active site water channel. Taken together, our results provide mechanistic insights underpinning histidine nucleophilic catalysis in general and hHint1 catalysis, in particular, thus aiding the design of future proTides and the elucidation of the natural function of Hint family of enzymes.
Objective: The review covers basic principles of the prodrug strategy applied to antiviral nucleoside drugs or drug candidates. Specific role of amino acids as promoieties is explained with respect to transport mechanisms, pharmacokinetics and a low toxicity of compounds. Synthetic approaches to the most important representatives (compounds under clinical investigations or available on the market) are described, including valacyclovir, valganciclovir, valomaciclovir stearate, valcyclopropavir, valtorcitabine, valopicitabine and several attempts to amino acid modifications of antiretroviral nucleosides. Method: A special attention is paid to acyclic nucleoside phosphonates, where the phosphonic acid residue is esterified with a side-chain hydroxyl group of appropriate amino acid (serine, tyrosine) which can be used as single amino acid or as a part of dipeptides further modified on the terminal carboxyl function. The most advantageous pharmacokinetic profile and the best oral bioavailability were found in tyrosinebased prodrugs. Results & conclusion: Studies were performed successfully on 1-(S)-[3-hydroxy-2-(phosphonomethoxy) propyl]cytosine (cidofovir), 9-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine and some (R)-2- (phosphonomethoxy)propyl and 2-(phosphonomethoxy)ethyl derivatives including adefovir.
The masking of nucleoside phosphate and phosphonate groups by an aryl motif and an amino acid ester, nowadays known as the 'ProTide' technology, has proven to be effective in the discovery of nucleotide therapeutics. Indeed, this technology, which was invented by Chris McGuigan in the early 1990s, has inspired the discovery of two FDA-approved antiviral nucleotide drugs, and many more are currently undergoing (pre)clinical development. The usefulness of this technology in the discovery of nucleotide therapeutics is showcased in this Highlight by discussing the ProTides development and the various ProTides that have reached clinical trials.