Synthesis, activity and structure-activity relationship of noroviral protease inhibitors

Article abstract of DOI:10.1039/c3md00219e

The protease of norovirus, an important human pathogen, is essential for the viral replication and, therefore, represents a potential drug target. A series of tripeptide-based inhibitors of the protease were designed, synthesized and tested, among which several potent inhibitors were identified with K _i values as low as 75 nM. The structure-activity relationships of these inhibitors are discussed.

Full text of DOI:10.1039/c3md00219e

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Synthesis, activity and structure–activity relationship of
noroviral protease inhibitors†
Cite this: Med. Chem. Commun., 2013,
4, 1354
Lisheng Deng,a Zana Muhaxhiri,b Mary K. Estes,c Timothy Palzkill,ac B. V. Venkataram
b
a
*
Prasad and Yongcheng Song
Received 29th July 2013
The protease of norovirus, an important human pathogen, is essential for the viral replication and,
therefore, represents a potential drug target. A series of tripeptide-based inhibitors of the protease
were designed, synthesized and tested, among which several potent inhibitors were identified with Ki
values as low as 75 nM. The structure–activity relationships of these inhibitors are discussed.
Accepted 2nd September 2013
DOI: 10.1039/c3md00219e
www.rsc.org/medchemcomm
To date, there have been no therapeutics or vaccines for the viral
infections and there is therefore a pressing need to nd effective
Introduction
Norovirus is the leading cause of epidemic acute gastroenteritis
in humans. Using PCR, more than 90% of outbreaks of
nonbacterial gastroenteritis in the US are caused by nor-
oviruses.1 Similar data are reported from the Netherlands and
other countries in Europe.2–4 Norwalk virus (GI.1) in the gen-
ogroup I is the prototype of this group of viruses, while gen-
ogroup II viruses, such as Houston virus (GII.4), are now more
prevalent.5 According to a recent CDC (Centers for Disease
Control and Prevention) survey, noroviral infections account for
21 million cases of gastroenteritis annually in the US, among
which ꢀ2 million require outpatient treatment and ꢀ70 000
hospitalization.6 While illness caused by norovirus is generally
short-lived, mild, and self-healing in healthy people, it can be
severe and sometimes fatal, especially among young children,
the elderly and immunocompromised individuals.7 Each year
noroviruses are responsible for the deaths of ꢀ218 000 children
less than 5 years old in developing countries.8 In developed
countries, these viruses have been associated with deaths in the
elderly in nursing homes, and are important pathogens causing
chronic and sometimes severe illness in intestinal, solid organ
and renal transplant patients.9–11 In addition, noroviruses are
classied as class B biodefense agents in the US because of their
high stability, extremely low infectious dose, and high rate of
susceptibility in all age groups of humans, making them a
potential threat to food and water supplies or the environment.
countermeasures for noroviruses.
Theoretically, the best way to prevent a virus infection is by
vaccination. While a noroviral virus-like particle vaccine is
being developed, the broad-spectrum efficacy of this against the
multiple types and emergence of new types of noroviruses
remains to be determined,12 Even if vaccines are effective,
antiviral drugs, would be useful to treat epidemics and chronic
infections, such as acyclovir and oseltamivir used to treat
herpes and inuenza virus infections, respectively. These
compounds have a broad antiviral activity because they target
an essential viral protein, e.g., protease or neuraminidase,
which oen exhibits a high homology among the different
serotypes of a viral family.
The genome of Norwalk virus (NV) consists of three open
reading frames (ORFs). As shown in Fig. 1a and b, ORF1
encodes a 200 kDa polyprotein, which is cleaved by a protease
(NVPro) into six non-structural proteins, including NVPro
itself.13 Because its function is essential for viral replication,
NVPro represents a potential drug target for intervention.
Our previous X-ray crystallographic study revealed NVPro is a
chymotrypsin-like protease with the residues Cys139, His30 and
Glu54 as the catalytic triad.14 A mechanism for the enzyme was
proposed based on the structural information, as shown in
Fig. 1c. Cys139 functions as a nucleophile, His30 as a general
base, and Glu54 is used to facilitate the correct orientation of
the His30 imidazole ring. A series of peptidomimetic inhibitors
of NVPro were recently reported, with the best compound in the
series having an IC50 value of 870 nM (Fig. 1d).15 We also
determined the X-ray crystal structures of three tripeptide
aldehyde inhibitors 1–3 (Fig. 1d) in complex with NVPro.16 Here,
we report the design, synthesis and activity of this series of
tripeptide based inhibitors against two noroviral proteases from
Norwalk and Houston viruses. Structure–activity relationships
(SAR) of these compounds are also discussed.
aDepartment of Pharmacology, Baylor College of Medicine, 1 Baylor Plaza, Houston,
Texas 77030, USA. E-mail: ysong@bcm.edu; Fax: +1 713-798-3145; Tel: +1 713-798-
7415
bVerna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor
College of Medicine, 1 Baylor Plaza, Houston, Texas 77030, USA
cDepartment of Molecular Virology and Microbiology, Baylor College of Medicine, 1
Baylor Plaza, Houston, Texas 77030, USA
† Electronic supplementary information (ESI) available: Detailed experimental
section. See DOI: 10.1039/c3md00219e
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Fig. 1 (A) The polyprotein encoded by NV ORF1 is cleaved by NVPro into 6 functional proteins; (B) the peptide sequences of the five cleavage sites, with those
highlighted in red being used for our inhibitor design; (C) a proposed mechanism of NVPro; (D) structures and activities of NVPro inhibitors.
(Asn-Phe-Gln(diMe)) and 4 (His-Leu-Gln(diMe)) possess low mM
inhibition (Ki: 1.6 and 1.9 mM, respectively). Compound 6
having a sequence of Val-Pro-Gln(diMe) exhibits the weakest
Results and discussion
Inhibitor design and SAR
Based on the mechanism of NVPro, we rst synthesized ve tri-
peptide aldehyde compounds 2, 4–7, as shown in Table 1. This
design exploited the P3–P1 sequences of the ve cleavage sites for
NVPro (Fig. 1b) with the P1 amino acid being modied. The
aldehyde functionality is to attract a possible nucleophilic attack
by the –SH of Cys139 to form a covalent bond with the enzyme to
increase the binding affinity. It is noted that since this covalent
bond is reversible with rapid kinetics, these aldehyde compounds
behave as the competitive inhibitors of NVPro, similar to those of
rhinovirus 3C protease.17 The Gln side chain was chosen because
the negatively charged Glu side chain could impair the cell
membrane permeability of these compounds. In addition, two
Me groups were used to mask the d-CONH2 to avoid its cycliza-
tion with the aldehyde to form a six-membered hemiglutaminal
species, as shown below in Scheme 1.
Similar inhibitor design has been applied to target the
proteases of other RNA viruses such as rhinovirus and picona-
virus.17–19 These ve compounds were tested for their inhibitory
activities against recombinant NVPro using our previously
reported method14 and the results are shown in Table 1. All
these ve initial compounds were found to be NVPro inhibitors
with their Ki values ranging from 0.31–8.2 mM. Compound 5
with a P3–P1 sequence of Gln-Leu-Gln(diMe) was found to be
the strongest among these ve, having a Ki value of 310 nM.
Compound 2 with a sequence of Ala-Leu-Gln(diMe) is also a
potent inhibitor with a Ki of 590 nM, while compounds 7
activity with a Ki value of 8.2 mM among this series.
Since compounds 2, 4 and 5 contain a P2 Leu residue, we
next synthesized compound 1 with a P2 Phe as well as 8 with a
P2 Pro to compare their activities with those of compounds 7
and 5 with the same P2 residue, respectively. Compound 1 (Z-
Leu-Phe-Gln(diMe)-CHO) was found to be a potent inhibitor of
NVPro with a Ki value of 120 nM (Table 1), being ꢀ3–5-fold more
active than compounds 5 and 2 with the natural substrate
sequences. On the other hand, compound 8 with a P3–P2
sequence of Leu-Pro is a very weak inhibitor (Ki ¼ 46.1 mM).
The activity data from this small set of tripeptide aldehyde
inhibitors suggest Leu or Phe is a more favorable P2 residue
than Pro. For example, 1 is 380-fold more active than 8 with the
same P3 and P1 residues, while the potencies of all inhibitors
with P2 ¼ Leu or Phe (i.e., 1, 2, 4, 5 and 7) fall within a relatively
narrow range of 0.1–1.9 mM. Our crystallographic studies show
the P2 Leu or Phe side chain is deeply inserted into the hydro-
phobic S2 pocket of the protein well dened by I109, Q110,
R112, and V114. However, the cyclic structure of Pro could
restrict it from having deep contacts with the S2 pocket. Our
SAR analysis further conrms that the van der Waals interac-
tions between the inhibitor P2 and the protein S2 signicantly
affect the binding affinity. The second SAR is that the P3 residue
seems to play a less important role, ne-tuning the inhibitory
activity. For compounds 5, 2 and 4 with the same P2/P1,
changing P3 residue from polar Gln to small/hydrophobic Ala as
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Table 1 Structures and inhibitory activity (Ki) of NVPro inhibitors
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more favorable hydrophobic interaction between the cyclopropyl
with the S1 pocket of NVPro. Compound 16, an analog of 1
containing a pyrrolidine-amide in the P1 position, was found to
be the most potent inhibitor of NVPro with a Ki value of 75 nM.
The slightly enhanced activity should be due to the additional
van der Waals interactions of the 5-membered pyrrolidine ring
with Lys162 and Ser163 in the S1 pocket.
Scheme 1 Isomerization of glutaminal.
well as larger/bipolar His only results in ꢀ6-fold activity
decrease (0.31 / 0.59 / 1.9 mM). Similarly, #10ꢁ potency
variation is found when altering the P3 Leu in compound 1 to
the Asn in 7, or the P3 Val in 6 to the Leu in 8. This observation
could be attributed to the less organized S3 pocket of NVPro,
which is actually half exposed to the solvent.
Inhibition of Houston virus protease (HOVPro)
Houston virus (HOV) belongs to the genotype II noroviruses.5 As
shown in Fig. 2a, these two noroviral proteases exhibit a high
sequence homology of 66% identity and 85% similarity.
Because of its high prevalence in noroviral infections in recent
years, we also tested 4 selected inhibitors, 1–3 and 5, against the
protease of HOV (HOVPro) and the results are shown in Table 2.
As compared with NVPro, these compounds exhibited reduced
inhibitory activities against HOVPro with Ki values of 4.3–14 mM.
Compound 1 with P2-P1 of Phe-Gln(diMe) shows ꢀ60-fold
activity reduction, while compounds 2 and 5 with a Leu at the P2
have ꢀ10-fold decreases, being the best HOVPro inhibitors (Ki ¼
4.6 and 4.3 mM, respectively). Although having the same P3-P2
Acetyl containing compound 9 was synthesized to nd if the Z
(benzyloxycarbonyl) protecting group is important for the inhibi-
tion. As compared to compound 1 with the same P3–P1 sequence,
9 (Ki ¼ 39 mM) exhibits a large activity loss (>300-fold) for NVPro,
showing Z contributes considerably to the tight binding of 1.
We next investigated the SARs with respect to the P1 residue.
Since the aldehyde functionality is highly reactive and may be
deactivated by cellular or plasma thiols such as glutathione,17 we
synthesized compounds 10 and 11 with a methylketone and a, b-
unsaturated ethyl ester as a nucleophile, respectively. These two
functional groups have been successfully used in inhibitor
discovery targeting other viral proteases. The P3–P1 sequence of
10 and 11 is the same as that of compound 1. However, ketone 10
only exhibits a very weak inhibitory activity with a Ki value of
150 mM, being 1200-fold less active than 1 (Table 1). a, b-Unsat-
urated ester 11 was found to be essentially inactive. Because the
a, b-unsaturated ester functionality is particularly attractive as it
is chemically stable to non-specic nucleophilic attacks by
cellular thiols,18,19 compounds 12 (with an unmasked Gln P1
residue) and 13 (with a b-acetyl diaminopropionic acid as the P1
residue) were also synthesized to test if a different P1 side chain
affects the activity. Neither 12 nor 13 exhibits inhibitory activity
against NVPro. These results show a highly reactive electrophile
such as aldehyde is required to potently inhibit the protease.
Compounds 3, 14–16 were synthesized to optimize the P1 side
chain. As compared with compound 1 with the same P3–P2
sequence of Leu-Phe, compound 3 with a reversed amide P1 side
chain is ꢀ12ꢁ less active (Ki ¼ 1.5 mM). Compound 14 was
designed to nd if both N-Me of 1 is important for the activity. 14
was, however, found to possess a largely reduced inhibitory
activity with a Ki value of 5.1 mM, being 42-fold less active than 1.
Although the crystal structure of NVPro:1 indicates the N-Me
trans to the carbonyl O atom in 1 has hydrophobic interactions
with Ala160, it seems unlikely that the reduced van der Waals
contacts for 14 (without the corresponding N-Me) is mainly
responsible for such a large activity loss. More possibly,
compound 14 exists as equilibrium between an aldehyde form
Fig. 2 (A) Alignment of NVPro and HOVPro protein sequence. The conserved
residues that compose the S2 pockets of the two proteins are labeled with a
and a cyclic hemiglutaminal (Scheme 1), with the latter species bracket and a major difference is highlighted red letters; (B) superposition of the
being inactive against NVPro. Indeed, the 1H NMR of compound
14 shows the aldehyde form accounts for <10%. Similarly,
crystal structure of NVPro (PDB: 2FYQ) shown in cyan and a homology model of
HOVPro structure in gold. The S2 pocket loop (bII–cII) shows a different confor-
mation in the HOV protease, which appears to be due to the Gly115 residue. In
compound 15 (Ki ¼ 1.4 mM) with an N-cyclopropyl-Gln side chain
the NV protease the residue at the 115 position is His; (C) tables depict the
is also considerably less active than compound 1. As compared to
conserved residues that compose the S1, S2 and S4 pockets, in NV and HOV
14, the ꢀ3ꢁ improved inhibition for 15 might be attributed to a proteases.
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Table 2 Ki (mM) against NVPro and HOVPro
Compound
NVPro
HOVPro
1
2
3
5
0.12
0.59
1.5
7.5
4.6
14
0.31
4.3
residues as 1, compound 3 with a b-acetyl diaminopropionic
acid as the P1 residue also show ꢀ10ꢁ activity loss.
To nd out possible reasons for the activity changes, a
homology modeling of HOVPro was performed using the
program Phyre20 and the result is shown in Fig. 2b, superimposed
together with the crystal structure of NVPro. There is only one
major conformational difference between the two structures. The
S2 loop of HOVPro (104–115) moves more closely towards the
protein active site, with the b-sheet kinked at Gly115. However, in
NVPro, the side chain of the corresponding residue His115 forms
a H-bond with Glu75, which stabilizes the NVPro S2 loop and
prevents it from turning inwards (Fig. 2b). The consequence for
the closer S2 loop of HOVPro is a smaller S2 pocket, which
favours a smaller P2 residue of an inhibitor. This homology
model seems to be reasonable, since compounds 2 and 5 with a
smaller Leu as a P2 show better inhibitory activities than
compound 1 with a bulkier Phe at this position (Table 2). The
generally reduced activities of these inhibitors could also be due
to the same reason. Gly115 renders the S2 loop of HOVPro more
exible (with a higher overall entropy), which could reduce the
binding affinity of an inhibitor. In addition, as shown in Fig. 2c,
the S1 pocket of HOVPro contains more polar residues (i.e.,
Thr135 and 158 vs. Ile135 and A158 in NVPro). Our experimental
results (ꢀ10ꢁ activity reduction for 3 vs. >60ꢁ for 1) suggest
b-acetyl diaminopropionate at the P1 position could be more
favourable for HOVPro.
Scheme 2 General synthetic methods. Reagents and conditions: (i) X1X2NH,
EDC, HOBT, triethylamine; (ii) H2, 5% Pd/C; (iii) Ac2O, AcOH; (iv) LiBH4; (v) Dess–
Martin periodinane; (vi) triethyl phosphonoacetate, NaH; (vii) MeMgBr.
followed by Dess–Martin periodinane oxidation. For making
acetyl containing compound 9, Ac-Leu-OH was used to react with
compound 20 to obtain an acetyl protected tripeptide 21, which
was reduced and oxidized to give compound 9.
Conclusion
In conclusion, a series of 16 tripeptide based compounds were
designed and synthesized targeting the protease of noroviruses,
which is a potential drug target for treating noroviral infections.
Compounds 1 and 16 with a general structure of Z-Leu-Phe-
Gln(N,N-X1,X2)-CHO were found to be the most potent inhibi-
tors of NVPro with Ki values of 120 and 75 nM, respectively.
From the activities of this small set of compounds, SAR analysis
shows that (1) a potent NVPro inhibitor requires to have an
aldehyde group to be the P1 electrophile; (2) N,N-disubstituted
Gln is the most favorable P1 side chain; (3) Phe or Leu is
favorable for the P2, while Pro at this position is bad for the
activity; and (4) the P3 residue seems to play a minor role in
tight binding. Selected NVPro inhibitors were tested against the
protease of the genotype II Houston virus. Despite a high
homology of these two proteases, NVPro inhibitors exhibited a
considerably reduced activity against HOVPro. A homology
model of HOVPro was generated, which can rationalize the
observed activity changes.
Compound synthesis
The general methods for synthesis of compounds 1–16 are shown
in Scheme 2 and detailed synthesis as well as compound char-
acterization are described in ESI.† EDC (N-(3-dimethylamino-
propyl)-N0-ethylcarbodiimide
hydrochloride)
and
HOBT
(1-hydroxybenzotriazole) were used as a general peptide/amide
coupling method. Amino acid methyl ester 19, representing the
P1 component of the nal products, was synthesized from Z-Glu-
OMe (a-Z-protected glutamic acid methyl ester) or a-Z-protected
(S)-2,3-diaminopropionic acid methyl ester. Using an EDC/HOBT
coupling reaction followed by hydrogenation to remove the Z
protection group, tripeptide 21 was prepared from 19, via a
dipeptide intermediate 20. The methyl ester of 21 was selectively
reduced to a primary alcohol followed by Dess–Martin period-
inane oxidation, to give compounds 1–8 and 14–16 having an
aldehyde functionality. For synthesizing compounds 11–13, the
corresponding aldehyde was treated with the sodium salt of
triethyl phosphonoacetate in tetrahydrofuran to convert the
Acknowledgements
aldehyde to a, b-unsaturated ethyl ester. Compound 10 was This work was supported by grants from the NIH (PO1 AI057788
prepared by treating compound 1 with MeMgBr at ꢂ78 ꢃC to M.K.E., T.P. and B.V.V.P., and P30DK56338 to M.K.E.), the
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