Design of Novel Aminoglycoside Derivatives with Enhanced Suppression of Diseases-Causing Nonsense Mutations

Article abstract of DOI:10.1021/acsmedchemlett.6b00006

New pseudotrisaccharide derivatives of aminoglycosides that exploit additional interaction on the shallow groove face of the decoding-site rRNA of eukaryotic ribosome were designed, synthesized and biologically evaluated. Novel lead structures (6 and 7 with an additional 7′-OH), exhibiting enhanced specificity to eukaryotic cytoplasmic ribosome, and superior nonsense mutation suppression activity than those of gentamicin, were discovered. The comparative benefit of new leads was demonstrated in four different nonsense DNA-constructs underling the genetic diseases cystic fibrosis, Usher syndrome, and Hurler syndrome.

Full text of DOI:10.1021/acsmedchemlett.6b00006

Letter
pubs.acs.org/acsmedchemlett
Design of Novel Aminoglycoside Derivatives with Enhanced
Suppression of Diseases-Causing Nonsense Mutations
Narayana Murthy Sabbavarapu, Michal Shavit, Yarden Degani, Boris Smolkin, Valery Belakhov,
and Timor Baasov*
The Edith and Joseph Fischer Enzyme Inhibitors Laboratory, Schulich Faculty of Chemistry, Technion−Israel Institute of
Technology, Haifa 32000, Israel
*
S Supporting Information
ABSTRACT: New pseudotrisaccharide derivatives of aminoglycosides that exploit
additional interaction on the shallow groove face of the decoding-site rRNA of
eukaryotic ribosome were designed, synthesized and biologically evaluated. Novel
lead structures (6 and 7 with an additional 7′-OH), exhibiting enhanced specificity to
eukaryotic cytoplasmic ribosome, and superior nonsense mutation suppression
activity than those of gentamicin, were discovered. The comparative benefit of new
leads was demonstrated in four different nonsense DNA-constructs underling the
genetic diseases cystic fibrosis, Usher syndrome, and Hurler syndrome.
KEYWORDS: Aminoglycosides, genetic diseases, nonsense mutations, translational readthrough, cystic fibrosis
onsense mutations are caused by single nucleotide base
changes within a gene that result in an in-frame
conventional AGs. For example, by initially identifying the
N
pseudodisaccharide moiety of G418 (1) as an important
fragment that induces significant read-through, we then used 17
as a common scaffold and developed the lead compounds 2
premature termination codon (PTC) and lead to the formation
1
,2
of truncated, nonfunctional proteins. Nonsense mutations
account for about 11% of all described gene lesions that cause
inherited human diseases including cystic fibrosis (CF),
Duchenne muscular dystrophy (DMD), Usher syndrome
5
and 3 (also named NB74 and NB124, respectively, Figure 1),
exhibiting improved read-through activity and reduced toxicity.
Thus, while this strategy until now has provided encouraging
results, the “rational design” of synthetic AGs for suppression
therapy is still far from being well established. Recently
reported three-dimensional structure of 80S yeast ribosome in
(
USH), mucopolysaccharidosis type I−H (MPS I−H), and
3
numerous types of cancer. For many of those diseases there is
presently no effective treatment. In the last several years, it was
shown that some aminoglycoside (AG) antibiotics (including
gentamicin and G418, Figure 1) have the ability to allow the
mammalian ribosome to selectively read past a false-stop signal,
but not a normal termination signal, and generate full-length
8
9
complex with G418, along with the structures of G418 and
1
0
paromomycin bound to their putative binding site of
Leishmania ribosomes, have provided important structural
insights that paved the way for structure-based rational design
of novel read-through inducers. Based on these structural data,
herein we report our pilot study on the design, synthesis, and
evaluation of new lead structures 6 and 7 (also named NB156
and NB157, respectively, Figure 1) that differ from the previous
leads (2 and 3) by the presence of an additional hydroxyl group
on the side-chain of the glucosamine ring (ring I) and show
substantially higher readthrough activity in comparison to those
of the parent structures.
3
functional proteins. This unique ability of AGs, also called
“
PTC suppression” or “translational readthrough”, has been
demonstrated in a series of in vitro and in vivo studies including
clinical trials, which highlighted them as promising therapeutics
for treatment of PTC underlined genetic disorders. Despite this
promise, high human toxicity of conventional AGs and reduced
PTC suppression efficacy at subtoxic doses have limited their
3
,4
clinical advantage for suppression therapy.
High-resolution X-ray crystal structure of 80S ribosome from
To address these issues we systematically developed a series
8
5
Saccharomyces cerevisiae in complex with G418, along with our
of lead compounds of NB series, which was found to improve
6
own modeling studies, suggested that extension of the side-
chain methyl group at the glucosamine moiety (ring I) of G418
with a hydroxyl group to generate 6′,7′-diol might allow for
readthrough activity and reduce toxicity as demonstrated in
various in vitro, ex vivo, and in vivo models of a variety of
4
different diseases underlined by PTC mutations. Note that,
due to the lack of detailed structural data on the interaction of
AGs within their eukaryotic target site, the design and
development of all those lead structures were based mainly
on the earlier reported biochemical and toxicity data on
Received: January 7, 2016
Accepted: February 8, 2016
©
XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.6b00006
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a
Scheme 1
a
Reagents and conditions: (i) (a) TIPSCl, DMF, 4-DMAP, 0−25 °C,
3%; (b) PMBCl, NaH, DMF, 0−25 °C, 84%; (ii) (a) TBAF, THF,
−25 °C, 88%; (b) IBX, EtOAc, 80 °C, 85%; (c) CH P(Ph) I, n-BuLi,
Figure 1. Chemical structures of natural and synthetic AGs that were
investigated in this study.
8
0
3
3
THF, 0−25 °C, 56%; (iii) K OsO ·2H O, NMO, acetone/H O/t-
BuOH, 93% (3:1 ratio); (iv) (a) DDQ, CH Cl /H O; (b) Ac O, Py,
0
NaOH (0.1 M), 73% [6′-(R)-4]; 78% [6′-(S)-5].
2
4
2
2
additional H-bond interaction between the added 7′-OH and
2
2
2
2
the O2 atom of cytosine 1409 (Figure 2). Initially, the validity
−25 °C, 91% for 2 steps; (c) NaOMe, MeOH, 60%; (v) PMe , THF,
3
afford the terminal alkene 10. The alkene 10 was dihydroxy-
13
lated to provide the diol 11 as an inseparable mixture of 6′-
diastereomers. Treatment of 11 with DDQ was followed by
acetylation (Ac O) and deacetylation (NaOMe) steps to afford
2
the mixture of 6′-diastereomers (∼3:1 ratio), which was
successfully separated by column chromatography to give the
major diastereomer 12 and the minor diastereomer 13. The
1
absolute configuration at 6′-position was assigned by using H
NMR magnetic anisotropy (for the details see Supporting
Information), which established 6′-(R)- and 6′-(S)-configu-
ration for the major and minor diastereomers, respectively. The
two diastereomers 12 and 13 were separately subjected to
Staudinger reaction to produce the target pseudodisaccharides
Figure 2. Three-dimensional structure of 80S ribosome from S.
cerevisiae in complex with G418 (PDB code 4U4O), highlighting the
interactions of G418 ring I (green) into the decoding-site rRNA
8
(
magenta, E. coli numbering). The ring I oxygen atoms (red) are
4
and 5.
specified by numbers, including the modeled O7′-oxygen (PyMol
software). H-bonds (dashed black lines) and C−H···π stacking (dotted
red lines) are shown. The inset shows chemical structure of ring I of
G418 with the modeled additional 7′-OH.
Preliminary comparative PTC suppression activity tests in
Figure 3 show that installation of C7′-hydroxyl group
compound 6′-(R)-4) on compound 1 dramatically increases
its in vitro readthrough activity, whereas this effect in compound
′-(S)-5 is comparatively small. These data, while providing
(
6
of this design strategy was tested on pseudodisaccharides; we
synthesized compounds 1, 4, and 5 (Figure 1) and tested their
comparative in vitro readthrough efficacy.
proof-of-concept of our design strategy also highlights the
pivotal role of stereochemistry at 6′ position in RNA target
recognition; compound 4 that retains the same 6′-(R)
configuration as in G418 (and in compound 1) exhibits higher
activity than that of compound 5 with an opposite, 6′-(S)
configuration. The observed somewhat higher activity of 5 to
that of 1 suggests that the additional 7′-hydroxyl in compound
5 can overcome the configurational penalty at 6′ position.
Nevertheless, the observed significantly higher PTC suppres-
sion activity of 4 compared to that of 1 suggested that the
7
Compound 1 was synthesized as previously reported by us.
The syntheses of 4 and 5 are illustrated in Scheme 1. The
11
known perazido derivative 8 was selectively protected by
TIPSCl, and the remaining hydroxyls were protected by p-
methoxybenzyl (PMB) groups to afford 9. Selective depro-
tection of silyl group with TBAF was followed by oxidation
12
with 2-iodoxy benzoic acid (IBX) and Wittig reaction to
B
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Figure 3. In vitro stop codon suppression levels induced by
compounds 1 (-■-), (R)-4 (-▲-), and (S)-5 (-△-) in R3X nonsense
mutation construct representing USH1 genetic disease.
compound 4 can serve as an improved pseudodisaccharide
scaffold for the generation of new lead compounds.
Encouraged, we choose 6′-(R)-4 to assemble the correspond-
ing pseudotrisaccharides 6 and 7 (Figure 1). The syntheses of 6
and 7 were accomplished from the intermediate 12 by using
essentially the same chemical transformations as illustrated in
Scheme 2.
a
Scheme 2
Figure 4. Comparative in vitro stop codon suppression levels induced
by compounds 2 (-△-), 6 (-▲-), and gentamicin (--■--) (left) and by
3
(-Δ-), 7 (-▲-), and gentamicin (--■--) (right) in a series of nonsense
constructs representing various genetic diseases (shown in paren-
theses): (A) R3X (USH1), (B) R245X (USH1), (C) Q70X (HS), and
a
Reagents and conditions: (i) Ac O, Py., −20 °C, 53%; (ii) BF −OEt ,
2
3
2
(
D) G542X (CF). The assays were performed as previously described
CH Cl , −30 °C, 85% of 17, 93% of 18; (iii) (a) MeNH , r.t., 80% (R
=
6
17
2
2
2
by us. The results are averages of at least three independent
experiments.
H), 98% (R = CH ); (b) PMe , THF, NaOH (0.1 M), 60% of 6,
3 3
4% of 7.
Regioselective acetylation of 12 with Ac O at low temper-
the PCDH15, CFTR, and IDUA genes that underline USH1,
CF, and MPS I−H, respectively. The nonsense reporters we
choose were R3X and R245X for USH1, G542X for CF, and
Q70X for MPS I−H. The observed data in Figure 4 clearly
show that the positive impact of the C7′-hydroxyl group in
scaffold 4 is largely retained in the subsequent pseudotrisac-
charides 6 and 7. In all mutations tested, the in vitro
readthrough activity of 6 is substantially better than that of
its parent 2, and the activity of 7 is better than its parent 3. In
addition, in all mutations tested, the activities of both 6 and 7
were significantly better than that of the clinical drug
gentamicin.
2
ature gave the corresponding C5 acceptor 14. For the
glycosylation of 14 we used the trichloroacemidate donors
14
15
1
5
and 16, which furnished the corresponding pseudo-
trisaccharides 17 and 18 in 85% and 93% isolated yields,
respectively, exclusively as β-anomers. Treatment with methyl-
amine was followed by Staudinger reaction to afford
compounds 6 and 7.
The impact of the additional 7′-hydroxyl in compounds 6
and 7 was separately evaluated against their parent structures 2
and 3, respectively, and the observed data are illustrated in
Figure 4. Since the read-through efficiency induced by AGs is
highly dependent on the identity of a stop codon and the
Previous studies have demonstrated that the increased
specificity toward eukaryotic cytoplasmic ribosome correlates
1
6
sequence contest around the stop codon, for broader
evaluation of the structure−activity relationship, we used a
collection of dual-luciferase reporter plasmids previously
5
with the increased PTC suppression activity. To test the
validity of this trend, we examined comparative protein
translation inhibition of pseudotrisaccharides 2, 3 and 6, 7 in
eukaryotic system, using coupled transcription/translation
1
7
developed by us. These reporter plasmids contain different
sequence contests around premature stop codons derived from
C
DOI: 10.1021/acsmedchemlett.6b00006
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ACS Medicinal Chemistry Letters
Letter
17
assays as previously described (Table 1). The observed data
indicate that the efficacy with which compound 7 (half-maximal
Table 1. Comparative Antibacterial Activity and Inhibition
a
of Translation in Eukaryotic and Prokaryotic Systems
b
c
translation inhibition
antibacterial activity MIC (μM)
E. coli R
477/100
B. subtilis
ATCC6633
Euk
Pro
compd
IC5 (μM)
IC (μM)
0
50
Genta.
G418
1
62 ± 9
0.03 ± 0.00
0.01 ± 0.00
6.0 ± 1.0
11.0 ± 1.2
91.9 ± 8.4
1.0 ± 0.1
0.7 ± 0.1
1.1 ± 0.2
1.2 ± 0.1
6
9
<0.75
<1.25
2.0 ± 0.3
347.1 ± 34.3
120.5 ± 14.5
515.8 ± 15
13.9 ± 1.2
7.5 ± 0.5
1.5 ± 0.1
1.2 ± 0.1
(
(
R)-4
S)-5
>311
>375
680
311
>375
42
2
6
3
7
>273
1267
>257
34
156
64
a
In all biological tests, all AGs were in their sulfate salt forms, and the
b
concentrations refer to the free amine form of each AG. The
eukaryotic and prokaryotic half-maximal-inhibition values (IC50 and
IC5 ) were quantified in coupled transcription/translation assays by
Euk
Pro
0
5
using active luciferase detection as previously described by us.
c
Minimal inhibitory concentration (MIC) values were determined by
using the double-microdilution method.
inhibitory concentration value IC 5E 0u k = 1.2 μM) inhibits
eukaryotic translation is greater than that of compound 6
IC5 = 13.9 μM) and gentamicin (IC E5 0u k= 62 μM), a similar
Euk
(
0
trend observed for PTC suppression activity: 7 > 6 >
Euk
gentamicin (Figure 5). In addition, the comparison of IC50
values reveals that 6 and 7 are 1.85-fold and 1.25-fold more
specific to the eukaryotic ribosome than their parent structures
2
and 3, respectively. These data indicate that the elevated PTC
suppression activities of 6 and 7 are associated with their
increased specificity to the eukaryotic ribosome; these data
further support the validity of our design strategy.
Pro
The measured IC50 and MIC values in Table 1 show that the
efficacy with which 6 and 7 inhibit the prokaryotic ribosome
and their subsequent antibacterial activity are very similar to
5
those of the parent compounds 2 and 3. We have recently
demonstrated that the observed significantly decreased
6
18
prokaryotic ribosome specificity of 2 and 3 is also linked
to their reduced mitochondrial translation inhibition and
subsequently to their reduced ototoxic effects than gentamicin
and G418. The observed similar impact on bacterial ribosome
by the current (6 and 7) and previous leads (2 and 3) therefore
suggest that 6 and 7 are perhaps less ototoxic than gentamicin
and G418. Further studies on the impact of 6 and 7 on
mitochondrial translation inhibition, along with their ototox-
icity tests, are currently underway.
Figure 5. (A) Secondary structures of rRNA A-sites of E. coli (left) and
of eukaryotes (right). The conserved residues, A1492 and A1493 (E.
coli numbering), are highlighted in blue and red, respectively. (B)
Superimposition of G418 bound to the E. coli A-site (green, PDB code
2
0
8
1
MWL) and the S. serevisiae A-site (magenta, PDB code 4U4O),
using the PyMol software align algorithm (r.m.s.d. = 1.5 Å). (C)
Expansion of the panel B highlighting different interactions of G418
ring I in the E. coli (green) and the S. serevisiae (magenta). The
relevant H-bond interactions are shown by dashed lines with the bond
lengths in the same color.
Finally, it is of note that a very similar rational design
strategy, but for the bacterial ribosome, has been reported
19
previously by Hermann and co-workers. Using 3D-structure
of paromomycin bound to the bacterial decoding-site rRNA
and modeling study, this group synthesized a series of C6′-
modified pseudodisaccharides, including 4 but as an unassigned
mixture of C6′ diastereomers, and tested them as inhibitors of
bacterial in vitro translation in comparison to paromamine
the C6′-diasteromeric mixture of 4/5 (∼3:1 ratio) was a 6-fold
Pro
0
poorer inhibitor than the parent paromamine (IC5 values of
24 and 3.9 μM, respectively), concluding that the 6′-position
19
(
rings I and II of paromomomycin, containing a primary 6′-
plays a pivotal role in bacterial rRNA recognition. Our results
in regards to the bacterial ribosome are in agreement with these
previously reported data. Thus, even though instead of 6′-
diastereomeric mixture reported in the latter study, we used the
hydroxyl at ring I). The results obtained were somewhat
discouraging: all the tested 6′-modifications resulted in about
1−2-orders of magnitude decrease in inhibition potency, and
D
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ACS Medicinal Chemistry Letters
Letter
pure 6′-diastereomers 1, 4, and 5, the data in Table 1 shows
that 4 and 5 are 1.8-fold and 15.3-fold poorer inhibitors of
prokaryotic translation than the parent compound 1 (IC50
In summary, the recently solved X-ray crystal structure of
eukaryotic ribosome in complex with the aminoglycoside
G418 provided, for the first time, a clear picture of differences
Pro
8
values of 6.0, 11.0, and 91.9 μM for 1, 4, and 5, respectively).
The scenario in eukaryotic translation is, however, different:
between the prokaryotic and eukaryotic sites, which was
exploited here for the rational design of new compounds that
selectively target the eukaryotic cytoplasmic rRNA A site, a key
target for PTC suppression activity. Using this tool, we
discovered a new pharmacophore, 7′-hydroxyl group, as a
valuable structural element of the glucosamine ring (ring I) that
significantly affects eukaryotic versus prokaryotic selectivity and
the subsequent PTC suppression activity. In addition, the
observed data support the feasibility of using the rational design
strategy employed here for the construction of new AG
derivatives that may act as drug for the treatment of PTC
underlined genetic disorders.
compound 4 is 2.9-fold more potent inhibitor than its parent 1
Euk
0
(
IC5 values of 120.5 and 347.1 μM, respectively), which is
further supported by the observed elevated PTC suppression
activity of 4 in comparison to that of 1 (Figure 3).
The observed differential selectivity of 4 can be explained by
the differences between its putative binding sites in eukaryotic
versus prokaryotic ribosome. A brief comparison between the
secondary structures of A sites in Figure 5A clearly shows that
while the bacterial A site is largely closed between the two C
G Watson−Crick base pairs, the eukaryotic A site is more open
due to unpaired C1409 and A1491 (E. coli numbering),
allowing higher flexibility of the eukaryotic decoding center at
this region. The two major bases that differ between bacteria
and eukaryotes are at positions 1408 and 1491: A1408 and
G1491 in bacteria; and G1408 and A1491 in eukaryotes (Figure
ASSOCIATED CONTENT
Supporting Information
■
*
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.6b00006.
5
A). These differences make AGs potent antibiotics being
highly selective toward the bacterial versus the eukaryotic
ribosomes. For example, the data in Table 1 show that G418 is
Biochemical assays and synthetic procedures, assignment
of absolute configuration at 6′-position in 4 and 5, and
copies of NMR spectra (PDF)
225-fold more selective toward the prokaryotic versus the
eukaryotic ribosome. The interactions of AGs ring I with the
rRNA target sites have been suggested to contribute
2
0
AUTHOR INFORMATION
Corresponding Author
*Tel: +972-4-829-2590. Fax: +972-4-829-5703. E-mail:
chtimor@tx.technion.ac.il.
significantly to the observed selectivity of AGs. Super-
■
8
imposition of the G418−Yeast structure with the crystal
20
structure of G418 bound to the bacterial A site (G418−Bact)
indicates that the overall conformation of the binding site upon
G418 binding in both structures is relatively similar (r.m.s.d.
values of 1.5 Å, Figure 5B), yet there are significant differences
in ligand−host interactions. Due to the lack of a strong
C1409G1491 Watson−Crick pairing that is present in
G418−Bact structure, the A1491 in G418−Yeast structure is
significantly shifted down toward the deep/major groove and
moves all the ligand molecule to the same direction. This makes
G418 relatively poorly bound in eukaryotic versus prokaryotic
target site as it is highlighted only for its ring I in Figure 5C.
Funding
This work was supported by Eloxx Pharmaceutical LTD
Research Fund (Grant No. 2019230).
Notes
The authors declare the following competing financial
interest(s): T.B. declares that the compounds 2, 3, 6, and 7
discussed in this publication are subject to license agreement
granted to a commercial third party.
20
In the bacterial A-site (Figure 5C, green) ring I forms a
pseudo base pair with A1408, by forming two hydrogen bonds
involving the ring oxygen and the 6′-OH group. These
interactions position the pyranose ring hydrogens in C−H···π
stacking with G1491, whereas two other hydroxyls on the
opposite side of the ring I, 3′-OH and 4′-OH, make H-bonds
with the phosphate oxygen atoms. The chiral (R)-6′-Me points
in the middle of the G1491C1409 pair, thus providing some
additional hydrophobic stabilization of ring-I. Because of such
compact interactions of ring I in the bacterial A-site, the
modifications at 6′-position could affect negatively to the
binding and to the subsequent biological activity as it is
observed for the compounds 4 and 5 (Table 1). In the G418−
ACKNOWLEDGMENTS
■
N.M.S. thanks the Fine Postdoctoral Fellowship at the
Technion. V.B. acknowledges the Ministry of Science and
Technology, Israel (Kamea Program).
ABBREVIATIONS
■
AG, aminoglycoside; PTC, premature termination codon; CF,
cystic fibrosis; DMD, Duchenne muscular dystrophy; USH,
Usher syndrome; MPS I−H, mucopolysaccharidosis type I−H
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DOI: 10.1021/acsmedchemlett.6b00006
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