Exploring inhibitor structural features required to engage the 216-loop of human parainfluenza virus type-3 hemagglutinin-neuraminidase

Article abstract of DOI:10.1039/c6md00519e

Human parainfluenza virus type-3 is a leading cause of acute respiratory infection in infants and children. There is currently neither vaccine nor clinically effective treatment for parainfluenza virus infection. Hemagglutinin-neuraminidase glycoprotein is a key protein in viral infection, and its inhibition has been a target for inhibitor development. In this study, we explore the structural features required for Neu2en derivatives to efficiently lock-open the 216-loop of the human parainfluenza virus type-3 hemagglutinin-neuraminidase protein.

Full text of DOI:10.1039/c6md00519e

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RESEARCH ARTICLE
Exploring inhibitor structural features required to
engage the 216-loop of human parainfluenza virus
type-3 hemagglutinin-neuraminidase†‡
Cite this: DOI: 10.1039/c6md00519e
*
*
*
Ibrahim M. El-Deeb,§ Patrice Guillon, Larissa Dirr and Mark von Itzstein
Human parainfluenza virus type-3 is a leading cause of acute respiratory infection in infants and children.
There is currently neither vaccine nor clinically effective treatment for parainfluenza virus infection.
Hemagglutinin-neuraminidase glycoprotein is a key protein in viral infection, and its inhibition has been a
target for inhibitor development. In this study, we explore the structural features required for Neu2en deriv-
atives to efficiently lock-open the 216-loop of the human parainfluenza virus type-3 hemagglutinin-neur-
aminidase protein.
Received 12th September 2016,
Accepted 4th October 2016
DOI: 10.1039/c6md00519e
www.rsc.org/medchemcomm
three functions of the HN protein have made it a major target
for anti-hPIV inhibitor design.
Introduction
Human parainfluenza viruses (hPIVs) are a group of respira-
tory viruses that belong to the Paramyxoviridae family and
cause a range of human respiratory diseases in infants, chil-
dren, immunocompromised patients and elderly people.1 Hu-
man parainfluenza virus type-3 (hPIV-3) is an enveloped, sin-
gle-stranded, negative-sense RNA virus that affects children
worldwide causing croup, bronchiolitis, and pneumonia.2 It
comes second only to respiratory syncytial virus (RSV) as the
leading cause of acute respiratory infections that require hos-
pitalization of infants and children.3 It is also an important
cause of morbidity and occasional mortality in immunocom-
promised patients.4,5 Despite continuing efforts, there are cur-
rently no effective vaccines or specific therapies to control or
treat parainfluenza virus infection.
The envelope of hPIV-3 contains two viral glycoproteins,
the hemagglutinin-neuraminidase (HN) and the fusion pro-
tein (F).2 The HN protein recognizes sialic acid-containing re-
ceptors on respiratory epithelial cell surfaces and uses these
sialic acid residues to anchor the virus to the host cell. A sec-
ond function for the HN protein is promoting F-mediated fu-
sion to allow penetration of the virus genome into the host
cell. In addition to its receptor attachment and fusion promo-
tion functions, HN possesses neuraminidase activity that pro-
motes the release of newly formed virions from the cell sur-
face and prevents them from self-agglutination.2,6 These
Over the past decades, numerous inhibitors that target
sialidase proteins of other related viruses, such as influenza
A virus (IAV), have been developed. Representative examples
are the naturally occurring inhibitor 2-deoxy-2,3-didehydro-N-
acetylneuraminic acid (Neu5Ac2en, 1, Fig. 1)7 and the potent
anti-IAV 4-deoxy-4-guanidino-Neu5Ac2en inhibitor (zanamivir,
2, Fig. 1).8,9 However, as a result of the structural differences
between hPIV-HN and other sialidases, inhibitory potencies
for these unsaturated sialic acid-based inhibitors were very
weak against hPIV-HN.10,11 The first potent Neu2en-based in-
hibitors to be rationally designed to specifically target hPIV-
HN were BCX 2798 (3) and BCX 2855 (4).12–14 In spite of their
in vitro potency against hPIV, these two inhibitions failed to
continue in development as a result of their poor in vivo
efficiency.12–14 We have recently reported11 the development
of potent Neu2en-based inhibitors (5 and 6) that carry bulky
C-4 substituents. These bulky C-4 substituents target a
Institute for Glycomics, Griffith University, Gold Coast Campus, Queensland,
4222, Australia. E-mail: m.vonitzstein@griffith.edu.au
† The authors declare no competing financial interest.
‡ Electronic supplementary information (ESI) available: Complete experimental
procedures, characterization data, and 1H and 13C NMR spectra of new interme-
diates and final compounds. See DOI: 10.1039/c6md00519e
§ Current address: RCSI Bahrain, Adilya, Bahrain.
Fig. 1 Structures of the anti-influenza sialidase inhibitors (1 and 2), the
hPIV inhibitors (3 and 4), and the potent hPIV-3 designer inhibitors (5
and 6).
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unique feature in the hPIV-3 HN protein, the flexible 216-
loop, locking open the loop and efficiently occupying the cre-
ated 216-cavity.11 This efficient binding to the HN protein re-
sults in potent inhibition of both HN functions, as well as
significant blockade of virus propagation.
The ability of these new designer inhibitors (5 and 6) to
force the 216-loop open and reveal the 216-cavity is attributed
to their bulky C-4 functionality (the 4-phenyl-1,2,3-triazole
group). This outcome has proved the notion that the binding
pocket of the hPIV-3 HN protein can efficiently accommodate
Neu2en derivatives carrying bulky, hydrophobic C-4 substitu-
ents.15 Such bulky inhibitors are not only able to fit into the
binding pocket but also benefit from the extra hydrophobic
binding interactions exerted by their C-4 bulky substituents,
resulting finally in more potent inhibition of HN functions.
The STD NMR experiments carried out for these triazole
inhibitors in complex with either the intact virus or the re-
combinant HN protein have shown that the most significant
interactions with the protein were exerted by the phenyl moi-
ety carried by the 1,2,3-triazole ring.11 Accordingly, in this
study, we verify the importance of the triazole ring as a
spacer carrying the hydrophobic phenyl group at the C-4 posi-
tion of 4-deoxy-Neu2en, by replacing it with other acyclic
spacers (urea,16 amide and sulfonamide functionalities), in
order to explore the structural features of the C-4 substituent
and define the essential inhibitor characteristics required to
force the 216-loop open to reveal and efficiently occupy the
216-cavity.
Fig. 2 Distance between the nitrogen atom at C-4 of the 4-deoxy-
Neu5Ac2en skeleton and C-1 of the phenyl ring in triazoles (a), ureas
(b), amides (c) and sulfonamides (d).
shown in Scheme 1, the fully protected 4-amino-4-deoxy-
Neu5Ac2en intermediate (7) was reacted with a number of
substituted phenyl and benzyl isocyanate reagents in
dichloromethane, in the presence of a catalytic amount of
DMAP, to yield the protected urea derivatives 8a–g in excel-
lent yields. The deprotection of compounds 8a–g by treat-
ment with NaOH in MeOH : H2O (1 : 1), provided the
deprotected target 4-deoxy-4-arylureido-Neu5Ac2en derivatives
9a–g.
The same intermediate 7 was utilized (Scheme 2) for the
synthesis of amides 11a–c by reaction with three different
benzoyl chlorides in the presence of triethylamine to yield
the fully protected amides 10a–c, which upon stirring in a 1 :
1 mixture of methanol : water at pH 13–14 (adjusted by
NaOH) afforded the final deprotected amides 11a–c.
In Scheme 3, amine 7 was converted to the protected sul-
fonamides 12a–d by reaction with the appropriate sulfonyl
chloride in the presence of triethylamine. The resultant sul-
fonamides 12a–d were then deprotected by treatment with
NaOH in 50% MeOH to afford the target sulfonamides
13a–d.
Results and discussion
Computational chemistry17
In the lead inhibitors (5 and 6), the distance between the
triazole N-1 and C-1 of the phenyl ring in the energy mini-
mized structures was found to be 3.58 Å. Accordingly, the
triazole ring was replaced by a number of acyclic spacers that
hold the phenyl ring at comparable distances from the
4-deoxy-NeuAc2en skeleton. The selected spacers, which can
be readily synthesized using common 4-deoxy-Neu5Ac2en in-
termediates, are urea-, amide- and sulfonamide-based. These
three linkers were found to generate distances of 3.65 Å, 2.44
Å and 2.87 Å, respectively, between the 4-amino-4-deoxy-
Neu5Ac2en C-4 nitrogen and C-1 of the phenyl ring (Fig. 2).
As predicted from these preliminary calculations and by
aligning the structures of these 4-deoxy-Neu5Ac2en derivatives
that contain these three acyclic linkers, the urea-based linker
creates the most comparable distance between the 4-deoxy-
Neu5Ac2en skeleton and the phenyl ring in the triazole inhib-
itor 5 (Fig. 3).
In order to evaluate the effect of a C-5 modification on the
inhibitory potency of these derivatives, the reported 4-amino-
4-deoxy-5-isobutyramido-Neu2en intermediate (14) (ref. 10)
was utilized to synthesize amide 15 as shown in Scheme 4.
Chemistry
Based on the initial computational chemistry calculations, a
series of 4-deoxy-4-phenylureido-Neu5Ac2en-based inhibitors
that contain variable substituents at the phenyl group (9a–g,
Scheme 1) was synthesized. The synthesis of these urea deriv-
atives was achieved by using the known intermediate 7.10 As
Fig.
3 Alignment of 4-deoxy-4-phenylureido-Neu5Ac2en (a),
4-deoxy-4-phenylamido-Neu5Ac2en (b) and 4-deoxy-4-phenylsulfo-
namido-Neu5Ac2en (c) to the 4-deoxy-4-(4-phenyl-1,2,3-triazol-1-yl)-
Neu5Ac2en inhibitor (5).
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Thus, stirring amine 14 in the presence of triethylamine with
4-chlorobenzoyl chloride afforded amide 15 in 85% yield.
Deprotection of the resultant amide by exposure to NaOH in
aq. MeOH yielded the final deprotected inhibitor 16 in 94%
yield.
Biological screening
Evaluation of the synthesized urea-, amide- and sulfonamide-
based 4-deoxy-Neu2en derivatives for their capacity to block
the neuraminidase function of hPIV-3 HN, as well as a com-
parison of their potencies to those of the lead triazole inhibi-
tors 5 and 6, was carried out by using a standard neuramini-
dase inhibition (NI) assay (Fig. 4).11
Scheme 1 Synthesis of compounds 9a–g. Reagents and conditions: (a)
R-NCO, DMAP, DCM, rt, o/n, (8a, 87%; 8b, 96%; 8c, 90%; 8d, 84%; 8e,
89%; 8f, 91%; 8g, 82%); (b) NaOH, MeOH/H2O (1 : 1), rt, o/n (9a, 80%;
9b, 91%; 9c, 78%; 9d, 87%; 9e, 77%; 9f, 88%; 9g, 85%).
Surprisingly, and in spite of the close spacer length mea-
surements between the triazole ring and the urea functionali-
ties, all of the synthesized C-4 ureido-Neu5Ac2en derivatives
failed to show NI IC50 values below the maximal-threshold
concentration used in this assay (150 μM, data not shown).
On the other hand, the neuraminidase inhibition activities
exerted by amide and sulfonamide derivatives were relatively
more potent and comparable to each other. Within each of
these two series (amides and sulfonamides), the most potent
neuraminidase inhibition activity was exerted by the
4-chlorophenyl derivatives (11b and 13b), with the amide 11b
showing an IC50 value of 45.1 μM and the sulfonamide 13b
showing an IC50 value of 48.7 μM. By comparing the three
amides, the next potent neuraminidase inhibitor within this
group was found to be the phenylamido derivative 11a with
an IC50 value of 103.5 μM, while derivative 11c with a
4-methoxyphenylamido group failed to show an IC50 value be-
low the detection threshold. Within the sulfonamide series,
the 4-toluenesulfonamido derivative 13d was a poorer inhibi-
tor compared to the 4-chlorophenylsulfonamide 13b with an
IC50 value of 64.8 μM. The 4-methoxyphenylsulfonamide 13c
was found to be slightly less potent with an IC50 of 71.0 μM,
while the weakest neuraminidase inhibitor within the
Scheme 2 Synthesis of compounds 11a–c. Reagents and conditions:
(a) R-COCl, Et3N, DCM, argon, rt, o/n (10a, 80%; 10b, 77%; 10c, 89%);
(b) NaOH, MeOH : H2O (1 : 1), rt, o/n (11a, 89%; 11b, 85%; 11c, 91%).
Scheme 3 Synthesis of compounds 13a–d. Reagents and conditions:
(a) R-SO2Cl, Et3N, DCM, argon, rt, o/n (12a, 83%; 12b, 89%; 12c, 82%;
12d, 74%); (b) NaOH, MeOH : H2O (1 : 1), rt, o/n (13a, 85%; 13b, 92%;
13c, 89%; 13d, 88%).
Fig. 4 NI IC50 values of inhibitors 11a–c, 13a–d and 16. Plain bars
represent 4-deoxy-Neu2en derivatives with a C-5 acetamido group,
while bars with stripes represent 4-deoxy-Neu2en derivatives with a
C-5 isobutyramido group. The IC50 values are the result of 3 indepen-
dent experiments performed in duplicate. Error bars represent the SEM
of the NI IC50 values of the compounds.
Scheme 4 Synthesis of compound 16. Reagents and conditions: (a)
4-Cl-Ph-COCl, Et3N, DCM, argon, rt, o/n, 85%; (b) NaOH, MeOH : H2O
(1 : 1), rt, o/n, 94%.
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sulfonamides was the phenylsulfonamido derivative 13a with
an IC50 of 99.6 μM.
inhibition of hPIV-3 HN functions. This was illustrated by the
NI IC50 values of the urea, amide and sulfonamide derivatives
evaluated in this study, where the weakest inhibition was ob-
served for the most flexible derivatives (the urea series), while
intermediate inhibition was found for the derivatives that
have linkers with moderate rigidity (the amides and sulfon-
amides). These results set the basis for the design of novel
Neu2en derivatives carrying bulky C-4 substituents targeting
hPIV-3 HN with the aim of reorienting the 216-loop and occu-
pying the 216-cavity.
The unexpected weak inhibition observed for all of the
urea derivatives can be rationalized on the basis of the signif-
icant flexibility of the phenyl moiety-bearing urea linker in
these derivatives, in contrast to the rigid triazole ring in the
potent inhibitors 5 and 6. It is reasonable to conclude from
these results that the linker that carries the bulky phenyl sub-
stituent at the C-4 position of Neu2en needs to be rigid
enough to keep the phenyl ring maximally oriented towards
the 216-loop and occupy the open loop 216-cavity. In both
the amide and the sulfonamide derivatives, the linker holds
the phenyl group at a shorter distance relative to that in the
C-4 urea derivatives. Moreover, these linkers are less flexible
and direct the C-4 phenyl moiety towards the open loop 216-
cavity to provide the observed improved potency within these
two series compared with the C-4 urea derivatives. Conversely,
in the inhibitor series 11 and 13 the combination of C-4
phenyl moieties positioned at a shorter distance from the 216
Author contributions
The manuscript was written through contributions of all au-
thors. All authors have given approval to the final version of
the manuscript. I. M. E.-D. and P. G. contributed equally and
are joint senior authors.
loop with the use of more flexible linkers, compared to the Acknowledgements
rigid triazole linker in inhibitors 5 and 6, resulted in a
The Australian Research Council (DP1094549) is gratefully ac-
decreased potency. This combination presumably limits the
inhibitors' capability to lock open the 216-loop and efficiently
occupy the 216-cavity.
knowledged for its financial support (MvI) and for the award
of an Australian Postdoctoral Award (PG). The National
Health and Medical Research Council (1047824 and 1071659)
is thanked for its financial support (MvI). Griffith University
is gratefully acknowledged for the award of a Griffith Univer-
sity Postdoctoral Award (IMED).
In order to evaluate the effect of C-5 substitution in these
new inhibitors on their NI potencies, the C-5 acetamido
group of the derivative that showed the most potent inhibi-
tion (the 4-chlorophenylamide 11b) was replaced by the iso-
butyramido group that has been reported in previous studies
to further improve the inhibition of Neu2en derivatives
against hPIV HN.10,11,18 Accordingly, inhibitor 16 that con-
tains a C-4 4-chlorophenylamido substituent and a C-5 iso-
butyramido group was synthesized and tested for its capacity
to inhibit hPIV-3 HN neuraminidase activity. Indeed, the new
C-5 isobutyramido-based inhibitor proved to be slightly more
potent with a NI IC50 value of 38.9 μM (vs. 45.1 μM for the
parent C-5 acetamido derivative 11b), suggesting a similar
binding mode to that of the triazole derivatives.
Notes and references
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Conclusions
In conclusion, a group of C-4 phenylureido, phenylamido
and phenylsulfonamido 4-deoxy-Neu2en derivatives, in which
the triazole linker carrying the phenyl moiety in inhibitors 5
and 6 was replaced by more flexible linkers, have been syn-
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HN. The results have shown that the triazole ring in inhibi-
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it suitably orients the phenyl ring towards the 216-loop. This
optimal linker can induce 216-loop movement to reveal the
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