Добавил:
Sekretar
kiopkiopkiop18@yandex.ru
t.me/Prokururor I Вовсе не секретарь, но почту проверяю
Опубликованный материал нарушает ваши авторские права? Сообщите нам.
Вуз:
Предмет:
Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5644_Библиотеки_им_академика_М_И_Перельмана
.pdf
10.4 Targeting Frameshifting in SARS-CoV-1 and SARS-CoV-2 265
https://t.me/med1917
L1
S1
5ʹ-GGGUUU
S3
3ʹ
3ʹ
S2
3ʹ
L1
S2
S1
5ʹ
(b)
S3
S1
L1
L3
L3
5ʹ
L2
3ʹ
L3
S1
S3
S2
5ʹ
40
S3
L2
(a) (c)
L2
Figure 10.11 (a) SARS-CoV-2 FSE. Slippery sequence (red), stem 1 (blue), stem 2 (yellow),
stem 3 (purple), loops (gray). (b) 5′end of the FSE threaded (left) and unthreaded (right)
through the middle of the structure, modeled with molecular dynamics simulations. Source:
[49, 50]/PLOS, CC BY 4.0; /Springer Nature/CC-BY 4.0. (c) Cryo-EM structure from Bhatt et al.
Source: Adapted from Bhatt et al. [18].
structure harbors the stop codon for orf1a within stem 1 (Figure 10.11, blue), and
was generated based on molecular dynamics simulations by the Woodside lab and
cryo-EM data from the Ban lab. Interestingly, the predicted structures included a
conformer with the 5′end (Figure 10.11, red) threaded through loop 3 and also
a conformer with the 5′end remaining on the exterior of the construct. It was
also posited that it was likely that this element may be likely to fold into multiple
conformations as the RNA undergoes multiple cycles of unfolding and refolding
[49, 50].
266 10 Approaches to the Identification of Molecules Altering Programmed Ribosomal Frameshifting
https://t.me/med1917
AS
13445
13445
13435
(a)
13445
13426
AS
HSS
13475
13435
13465
SL1
13485
No Data
13455
HSS
13495
13505
Reactivity
0.0 0.4 0.85
13465
13485
SL1
13515
13515
13495
13551
13535
13545
13535
13525
13545
13551
SL3
(b)
Figure 10.12 SHAPE (selective 2′hydroxyl acylation analyzed by primer extension) data
from Vero E6 cells infected with SARS-CoV-2 identified two possible conformations, (a) and
(b). (a) had a higher probability of formation than (b). Source: Huston et al. [45]/with
permission of Elsevier.
Later structural analyses by the Pyle lab indicated that the canonical pseudoknot
could, in fact, form multiple conformations in a cellular environment [45]. While
SHAPE-MaP data from multiple reads encompassing the FSE and surrounding
RNA indicated that the canonical three stem-three loop H-type pseudoknot could
form (Figure 10.12b), a second conformer was also likely to form that, rather than
13426
13475
13525
SL2
13505
10.4 Targeting Frameshifting in SARS-CoV-1 and SARS-CoV-2 267
https://t.me/med1917
13,460
Attenuator Hairpin
Slippery Site
13,440
Attenuator Hairpin
13,440
Alternative Stem 1
13,419
153 nt
13,265
13,256
Pseudoknot Stem 3
13,500
13,460
Slippery Site
13,480
13,534
14,695
13,541
14,680
Alternative Stem 1
13,419
Pseudoknot stem 2
13,500
Normalized
DMS reactivity
1.00
0.67
0.33
0.00
no data
Long-distance interaction
13,559
13,559
13,480
13,568
13,541
Pseudoknot Stem 3
13,520
14,652
1082 nt
13,568
Figure 10.13 DMS reactivities of the SARS-CoV-2 FSE analyzed by full-genome RT-PCR in
Huh7 cells. Abundance of each cluster: 45% (left), 55% (right). The FSE is shown with
extensive interactions with upstream and downstream nucleotides. Source: Lan et al.
[46]/Springer Nature/CC BY 4.0.
a tightly structured pseudoknot, formed two distinct stem loops connected by an
unstructured region that was capable of interacting with other regions of RNA
(Figure 10.12a). In fact, when they expanded the length of RNA studied to a 749
nucleotide surrounding the FSE, they observed that the FSE formed interactions
with RNA located 260 or 470 nucleotides upstream of the FSE [45].
This nding that the SARS-CoV-2 orf1ab FSE is capable of extended interactions in a cellular environment was corroborated by the Rouskin Lab, who used
DMS-MaPSeq with the DREEM clustering algorithm to show that the canonical
13,520
268 10 Approaches to the Identification of Molecules Altering Programmed Ribosomal Frameshifting
https://t.me/med1917
pseudoknot, while capable for forming the 3 stem/3 loop structure in isolation, had
extensive upstream and downstream interactions with other regions of the viral
RNA in infected Vero and Huh7 cells (Figure 10.13) [46].
Early research on targeting the SARS-CoV-2 FSE relied on previous research
on the SARS-CoV-1 FSE. The two RNA elements have nearly identical sequences,
diering by only two nucleotides in the loop 3 region. In 2011, the small-molecule
2-{[4-(2-methylthiazol-4-ylmethyl)-[1,4]diazepane-1-carbonyl]amino}benzoic acid
ethyl ester (MTDB (11), Figure 10.14) was reported as a frameshifting modulator
for the SARS-CoV-1 FSE after it was identied as binding to the FSE via a virtual
docking screen. Though its binding anity was quite low (Kd= 210± 20 μM),
cell-free and in cellulo studies conrmed that it was eective at inhibiting frameshift
eciency for SARS-CoV-1 (Figure 10.14) [53]. With the emergence of SARS-CoV-2,
MTDB showed similar activity with the SARS-CoV-2 FSE (Figure 10.14)3;this
nding along with the sequence similarity between the two viruses [44] allowed
researchers to gain valuable early insights into the novel virus’ frameshifting
behavior and its impact on viral replication.
O O
H
N
O
N
N
N
S
MTDB (11)
70
60
50
40
30
–1 RF (%)
20
10
0
DMSO 0.08 0.8 8 (uM)
/ml
6
50
TCID
5
10
4
3
SARS-CoV-2 log
0
5
MTDB concentration in μMCompound 43
102050
100
150
200
50
40
30
20
–1 PRF (%)
10
0
MTDB:
+–
Figure 10.14 MTDB (11, also labeled compound 43, top). (Left) Concentration-dependent
inhibition of frameshift efficiency in HEK293T cells containing the SARS-CoV-1 FSE
sandwiched between an upstream renilla luciferase and a downstream firefly-renilla
luciferase fusion protein in the −1 reading frame, treated with MTDB. Source: Adapted from
Park et al. [51]. Though MTDB inhibits frameshifting at sub-micromolar concentrations (left)
and viral infectivity at micromolar concentrations (middle). Source: [18, 52], it is unlikely
that it does so by binding to the FSE, to which it binds only weakly [53]. MTDB (5 μM) also
reduces frameshifting of the SARS-CoV-2 ORF1ab FSE in a dual luciferase assay in rabbit
reticulocyte lysate (right). Source: Kelly et al. [44]/with permission of Elsevier.
3 Reprinted (adapted) with permission from Park, S.J., Kim, Y.G., Park, H.J. (2011). Identication
of RNA pseudoknot-binding ligand that inhibits the −1 ribosomal frameshifting of SARScoronavirus
by structure-based virtual screening. J. Am. Chem. Soc. 133 (26): 10094–10100. https://
doi.org/10.1021/ja1098325. Copyright 2011 American Chemical Society.
10.4 Targeting Frameshifting in SARS-CoV-1 and SARS-CoV-2 269
https://t.me/med1917
Meraoxacin (12) was identied as an inhibitor of SARS-CoV-2 −1 PRF via a
dual reporter assay combined with a high-throughput screen of 4434 compounds
from collections of FDA-approved drugs, the Pharmakon 1600 collection, and the
Tested-In-Human collection [19]. Meraoxacin is a member of the uoroquinolone
class of antibacterial agents, and the only uoroquinolone of 40 tested in the screen
to have a signicant impact on frameshifting [19].
HN
F
N
F
N
OH
O O
Merafloxacin (12)
18
Merafloxacin (12). Source: Borthwick [40]/with permission of American Society for
Microbiology.
While the mechanism of inhibition has yet to be elucidated, shortening the distal sidechain meraoxacin and adding a terminal alcohol group partially restored
frameshifting activity and viral titer, and frameshifting activity and viral titer were
completely restored by replacing the pyrrolidine moiety with a piperidine moiety
(Figure 10.15) [19]. These results suggest that frameshifting activity of meraoxacin
is tied to the specic structure of the compound rather than the underlying uoroquinolone backbone.
Meraoxacin does appear to specically aect the frameshifting of betacoronaviruses, with some impact on frameshifting of alphacoronaviruses, as observed
in HEK293T cells transfected with a dual reporter plasmid containing the FSE of
various viruses and subsequently treated with meraoxacin [19].
Rather than target the FSE itself, the Disney Lab turned its focus to the upstream
attenuator hairpin, shown to assist in regulation of frameshifting (Figure 10.16)
[54]. Using an RNA-focused compound library, they identied a small molecule (C5
(15), Figure 10.17) capable of binding to the 1x1 UU internal loop in the attenuator
N
HO
F
13
N NH2N
O
F
14
Figure 10.15 Analogs of merafloxacin partially (13,20μM) and mostly (14,20μM) restore
frameshift efficiency (middle) and viral titer (right) in Vero E6 cells infected with
SARS-CoV-2. Source: Sun et al. [19]/National Academy of Sciences/CC BY 4.0.
F
F
N
OO
O O
1
OH
PRF inhibition
OH
0.01 0.1 1
DMSO
Merafloxacin
**
***
0 0.5
14
100 15050
Cell viability
Merafloxacin
Relative viral titer (%)
0
0 2513
Relative PRF efficiency (%)
14
13
50 75 100
270 10 Approaches to the Identification of Molecules Altering Programmed Ribosomal Frameshifting
https://t.me/med1917
C
Attenuator
Hairpin (AH)
Slippery
site
Pseudoknot
FSE
Small-molecule
binding site
3ʹ5ʹ
CCCA CAAACG
A
G
U
C
G
AAU
C
G
U
U U
C
G
–
G
C
–
U
A
SARS-CoV-2 AH
Figure 10.16 (left) Diagram of the location of the attenuator hairpin in relation to the
slippery site and FSE in SARS-CoV-1 and SARS-CoV-2. (right) The SARS-CoV-2 attenuator
hairpin contains a small-molecule-binding site in a 1 × 1 U-U internal loop that is not
present in the SARS-CoV-1 attenuator hairpin. Differences between the SARS-CoV-1 and -2
attenuator hairpins are shown by the nucleotides in red. Source: Haniff et al. [48]/with
permission of American Chemical Society.
1.25
1.00
0.75
Frameshift efficiency
0.50
0 0.2 2 20
[Covidcil-19], μm
**
***
HO
O
NH
N
N
N
H
15
Figure 10.17 Compound C5 (Covidcil-19 (15), right) reduced frameshift efficiency in a
concentration-dependent manner when a dual luciferase reporter plasmid containing the
slippery sequence and FSE between in-frame Renilla luciferase and firefly luciferase in the
−1 frame is transfected in HEK293T cells (top). At 2 μM, frameshift efficiency was reduced
to roughly 50% in the presence of C5 versus vehicle alone. Source: Haniff et al. [48]/with
permission of American Chemical Society.
hairpin [48], which is absent in the attenuator hairpin of SARS-CoV-1 [48, 54].
They then used Chem-CLIP (chemical cross-linking and isolation by pull-down)
to selectively target and isolate the SARS-CoV-2 RNA in a cellular environment,
then built onto that design with an RNA-degrading chimera—linking a RNase
L-recruiter molecule to C5 to selectively target and cleave the attenuator hairpin
(Figure 10.18). The eect on frameshifting was measured with a dual luciferase
reporter system in HEK293T cells. Not only did C5 inhibit frameshifting by binding
to the attenuator hairpin, the C5-Chem-CLIP construct allowed the SARS-CoV-2
RNA to be isolated without pulling down cellular RNA, and the C5-RIBOTAC
construct recruited RNase L for the selective degradation of the entire SARS-CoV-2
RNA while having no eect on SARS-CoV-1 RNA counts [48].
10.4 Targeting Frameshifting in SARS-CoV-1 and SARS-CoV-2 271
https://t.me/med1917
SARS-CoV-2 SARS-CoV
0
**
0.02
****
0.2
248
[C5-RIBOTAC], μM
125
100
75
50
% Firefly luminescence
25
0
0.02
0.2
***
248
125
100
% Renilla luminescence
SARS-CoV-2 SARS-CoV
***
*
**
75
50
25
0
248
0.2
0.02
[C5-RIBOTAC], μM
0
0.02
0.2
248
Figure 10.18 The C5-RIBOTAC (ribonuclease targeting chimera) recruits Rnase L for
degradation of SARS-CoV-2 RNA in HEK293T cells. A dual luciferase reporter system shows
a decrease in both Renilla (upstream of attenuator hairpin and FSE) and firefly (downstream
of FSE) luciferases, as the ribonuclease does not differentiate between different sections of
the reporter system when it is recruited by C5-RIBOTAC binding to the attenuator hairpin.
Source: Haniff et al. [48]/with permission of American Chemical Society.
16
Structure
O
+
N
N
N
O
–
–
O
N
NH
+
F
EC50 (µM)
13.0
HO
H
N
HO
O
HO
H
HO
O
HO
OH
Geneticin (19)
O
NH
2
NH
O
NH
2
18
17
N
N
O
25.2
O
NH
N
N
O
–
N
N
O
+
N
+
N
N
O
N
–
O
12.0
Figure 10.19 Analogs (16–18) of geneticin (19) have improved antiviral activity and
inhibition of frameshift efficiency over geneticin, an aminoglycoside capable of reducing
frameshift efficiency. Source: Varricchio et al. [55]/with permission of Elsevier. Frameshift
efficiency was measured with a dual luciferase assay in Vero E6 cells.
2
272 10 Approaches to the Identification of Molecules Altering Programmed Ribosomal Frameshifting
https://t.me/med1917
NH
N
NH
2
2
H2N
O
O
NH
Nafamostat (20)
S
O
CH
3
MERS-CoV
SARS-CoV-2
F
N N
F
MERS-CoV
SARS-CoV-2
H2C
OH
H3C
CH
H3C
O
H3C
H3C
3
H3C
10
0
–10
–20
–30
–40
–50
–60
HIV
PEMV1
KY770854
KF294282
CH
3
H2N
OH
10
O
O
0
–10
–20
–30
–40
–50
NafamostatMerafloxacinPalbociclib
–60
HIV
PEMV1
KY770854
KF294282
(f)
CH
O
10
0
–10
–20
–30
–40
–50
MTDB Valnemulin Abemaciclib
–60
3
O
NH
O
S
N
CH
N
3
N
O O
CH
3
N
H3C
H
H3C
NH
2
10
0
–10
–20
–30
–40
–50
–60
HIV
N
N
PEMV1
NH
HIV
PEMV1
10
–10
–20
–30
–40
–50
–60
(e)
KY770854
KF294282
KU182958
H3C
0
KY770854
KF294282
KU182958
LC469308
H
N
LC469308
KY770854
KF294282
LC469308
KU182958
(a) (b) (c)
CH
CH
3
O
ONN
MERS-CoV
SARS-CoV-2
3
N
N
H
10
0
–10
–20
–30
–40
–50
–60
Change in −1 PRF from 20 μM ligand (%) Change in −1 PRF from 20 μM ligand (%)
KY770854
KF294282
LC469308
KU182958
MERS-CoV
(d)
SARS-CoV-2
F
N
N
CH
F
3
LC469308
KU182958
NH
958
LC469308
KU182
H
NNN
N
MERS-CoV
SARS-CoV-2
O
O
MERS-CoV
SARS-CoV-2
N
HIV
N
H
HIV
CH
N
PEMV1
NH
NH
PEMV1
3
2
Figure 10.20 Results of an in vitro dual luciferase assay in rabbit reticulocyte lysate
observing the effects of six compounds, including MTDB (a), merafloxacin (e), and
nafamostat (f) on frameshift efficiency for FSEs from different coronaviruses, as well as HIV
and PEMV1 as controls. Source: Munshi et al. [57]/MDPI/CC BY 4.0. Valnemulin (b) is an
antibiotic approved for veterinary use, Abemaciclib (c) and Palbociclib (d) are CDK4/6 kinase
inhibitors approved for breast cancer treatment. Viruses KY770854 and KF294282 are alpha
coronaviruses isolated from bats, and KU182958 and LC469308 are beta coronaviruses also
isolated from bats. PEMV1 is the pea enation mosaic virus, which has a 2 stem H-type
pseudoknot FSE.
10
https://t.me/med1917
10
10
10.4 Targeting Frameshifting in SARS-CoV-1 and SARS-CoV-2 273
Vero 76 Calu-3
6
5
4
10
10
10
6
5
4
3
10
2
Titer [PFU/ml]
10
1
10
0
10
DMEM
100 μ
M
10 μM
μM
μM
0.1
μM
0.01
1
3
10
2
Titer [PFU/ml]
10
1
10
0
10
DMEM
*** *** *** *** ***
m
1 μ
100 μm
10 μm
0.1 μm
μm
0.01
Figure 10.21 Nafamostat does not reduce viral titer at 100 μM in Vero E6 cells but does
reduce viral titer at all concentrations in Calu-3 cells. Cells were treated with nafamostat for
one hour, then infected with SARS-CoV-2 before being washed and treated with nafamostat
again for another 24 hours. Source: Jäger et al. [58]/MDPI/CC BY 4.0.
As mentioned above, aminoglycosides are a class of compounds known to bind
to RNA secondary structures. One aminoglycoside, geneticin (19), shows antiviral
activity against multiple viruses. Geneticin is also capable of reducing frameshift
eciency for SARS-CoV-2 at high concentration (600 μM) in VeroE6 cells, as shown
with a dual reporter assay [55]. Further analysis with in silico docking experiments
with geneticin and the SARS-CoV-2 FSE suggested 3 potential docking sites on
the FSE, one of which was conrmed in vitro by mutating the residues involved
and observing a corresponding decrease in frameshift inhibition in the presence
of geneticin and meraoxacin (as a control) [55]. These docking experiments were
followed up by virtually screening the RNA against a library of RNA binders;
the most potent of these compounds had EC50= 12 μM and reduced frameshift
eciency in a dual luciferase assay to ∼65% versus untreated Vero E6 cells (500 μM
compound) (Figure 10.19) [55]. The authors of this study hypothesized that the
compounds shown in the table have a similar binding mode to geneticin based on
the docking studies, but further experiments are needed to support this hypothesis.
Nafamostat (20) was in clinical trials as a protease inhibitor for SARS-CoV-2 during the COVID-19 pandemic [56]. However, it also showed inhibition of frameshifting for a spectrum of betacoronaviruses, including SARS-CoV-2, while only slightly
modulating frameshifting for two other frameshifting viruses not belonging to the
coronavirus family [57]. Nafamostat was identied by Munshi et al. as an inhibitor
of frameshifting through a screen of 1814 FDA-approved drugs combined with a
cell-free dual reporter assay,along with valnemulin (antibiotic), palbociclib,and abemaciclib (kinase inhibitors). Out of those four, nafamostat, despite its broader eect,
consistently strongly inhibited frameshifting (<40% inhibition) across most of the
coronavirus strains tested (Figure 10.20) [57].
274 10 Approaches to the Identification of Molecules Altering Programmed Ribosomal Frameshifting
https://t.me/med1917
Nafamostat reduces viral titer in Calu-3 cells, which express the protease
TMPRSS2, but not Vero 76 cells, which do not express TMPRSS2 (Figure 10.21)
[58]. It can be inferred, therefore, that while nafamostat is a frameshifting inhibitor,
its eects on viral infection in cells are likely to be a result of its protease inhibition
activity rather than its frameshifting inhibition [58].
10.5 Conclusions
The RNAs used by viruses to regulate essential frameshift processes are intriguing
drug targets, as they circumvent many of the potential pitfalls that are encountered
in more traditional drug targets such as proteins and DNA. Eorts to date targeting
these RNAs also highlight the need for greater chemical diversity in RNA-targeted
compounds, especially for small-molecule binders with lower weight and higher
specicity. Of the viruses discussed herein, the Miller group at Rochester has
identied compounds that bind to the HIV-1 FSE RNA, building from initial identication via a Resin-Bound Dynamic Combinatorial Library (RBDCL) to aord
compounds with exceptionally high anity and selectivity. These compounds have
been demonstrated to alter frameshift eciency, and interfere with replication
of HIV-1, including a multidrug-resistant patient isolate, in human cells [16].
Frameshifting of the SARS-CoV-2 orf1ab FSE has been targeted by a variety of
small molecules [19, 48, 51, 55, 57]. Many of these are capable of binding to the
SARS-CoV-2 orf1ab FSE and decreasing frameshift eciency, which is disruptive to
viral replication, though more work is needed in this area to develop a more robust
and non-toxic small-molecule binder.
As it stands, there is a tremendous amount of potential in targeting RNA that
extends beyond the work presented here for HIV-1 and SARS-CoV-2 RNA. 90% of
the human genome consists of non-coding RNAs, which have important and varied roles in most aspects of gene expression, ranging from transcription, translation
and serving as scaolds for membrane-less organelles [59]. With advances in structural biology, these RNAs and others that were historically dicult to characterize will benet from adaptation and further development of technologies that have
been previously used for protein targets, including mass spectrometry screening and
DNA-encoded libraries [59]. These advancements are welcome, as commonly used
methods for targeting RNA rely on the use of single-stranded antisense oligonucleotides (ASO) and double-stranded small interfering RNAs (siRNA) [60]. While
exciting research tools, both ASOs and siRNAs have proven challenging in clinical
application. For example,siRNAs often cause allergic reactions,display poor cell permeability, and have limited ability to cross the brain–blood barrier [60]. Currently,
there are precious few therapeutically relevant compounds that target RNA [60],
underscoring the unmet need for a greater understanding of the chemical space of
RNA binders.
References
1 Mei, H.-Y., Mack, D.P., Galan, A.A. et al. (1997). Discovery of selective,
small-molecule inhibitors of RNA complexes—1. The tat protein/TAR RNA
Соседние файлы в папке Библиотека им академика М.И. Перельмана