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(a) Regular (0 frame)
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translation
(b) –1 Frameshift
translation
AAAAA
U
UUU UUU UGGG G
A A A A
90 − 95%
10.1 Introduction 255
3′
Gag
HIV-1
Structural
proteins
AAAAAA
A A A AUUU UUU UGGG G
3′
GagPol
HIV-1
structural proteins
5 − 10%
and enzymes
Figure 10.1 During translation of an mRNA containing a frameshift-stimulating element
(FSE), the ribosome translates the transcript, reaches the FSE, overcomes its hindrance to
translation and unwinds it, and terminates translation when it reaches the stop codon
within the FSE (a, top). However, some of the time the ribosome is unable to easily
overcome the hindrance to translation presented by the FSE, resulting in a shifting of the
ribosome into the −1 open reading frame (b, bottom). Source: Anokhina and Miller
[12]/with permission of Springer Nature. For HIV, a −1 frameshifting event is required for
the production of viral enzymes (Gag and GagPol) essential for viral replication.
GTP
3
H
3ʹ
5ʹ
E P A EPA
FSS-mediated inhibition
of A-site tRNA binding
5ʹ
FSS-mediated inhibition
of tRNA/mRNA translocation
GTP
EF-Tu
EF-G
3ʹ
Cy3-labeled L9
Cy5-labeled S6
(a) (b)
Figure 10.2 Mechanisms of frameshifting, as described by Bao et al. [20] (a, left,
Mechanism 1) Frameshift-stimulating stem loops can dock in the A-site of the ribosome,
preventing the incoming tRNA from docking, resulting in the slippage of the tRNA in the
P-site. (b, right, Mechanism 2) Alternatively, frameshift-stimulating elements can interact
with the exterior of the ribosome, including the mRNA entry channel, preventing
translocation of the ribosome and inducing frameshifting of the tRNAs in the P- and A-sites.
In both cases, the ribosome must unfold the hindering RNA structure to continue
translation. Source: Bao et al. [20]/Springer Nature/CC BY 4.0.
256 10 Approaches to the Identification of Molecules Altering Programmed Ribosomal Frameshifting
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Instead, it has been posited that a frameshift signal inhibits tRNA binding by
interacting with the A-site. This was demonstrated using a construct that expanded
the length of a hairpin frameshift-stimulating element (FSE) by six base pairs; this
failed to inhibit tRNA binding to the A-site, pointing to the rst mechanism [20].
The human immunodeciency virus type 1 (HIV-1) is one of the more well-known
examples of retroviruses employing frameshifting as a mechanism to produce and
regulate the relative quantities of viral enzymes and structural proteins. The gag-pol
HIV-1 mRNA codes for translation of two polyproteins: Gag (which is proteolytically
processed to yield HIV matrix, capsid, and nucleocapsid) and Pol, which only forms
as a Gag-Pol fusion polyprotein. Pol is proteolytically processed to yield protease,
reverse transcriptase, and integrase enzymes. Gag-Pol forms when -1 PRF causes
the ribosome to move in to a new reading frame, skipping the stop codon on the gag
gene during translation [21, 23]. The frameshift eciency, resulting in the formation of the Gag-Pol polyprotein, is approximately 5–10%, and this stoichiometry has
been shown to be necessary for proper packaging of virus particles [21]. For example,
mutants that result in Gag-Pol being produced 100% of the time were unable to make
viral particles [23]. As discussed above in a general context, a frameshift stimulatory
element (FSE, Figure 10.3) regulates the frequency of −1 PRF and hence the ratio
of Gag and Gag-Pol. Therefore, we and others hypothesized several years ago that
a compound that could bind to the FSE mRNA might modulate frameshifting, and
consequently inhibit viral assembly and replication [23, 25].
10.2 Mechanisms of Frameshifting
To begin to understand how to target the FSE RNA, it is useful to understand what
is necessary to facilitate a frameshifting event. Frameshifting can only occur when
two elements are present: a highly conserved UUUUUUA sequence, known as the
slippery sequence (Figure 10.3, boxed in blue), and a downstream frameshift stimulatory stem–loop (Figure 10.3, upper stem) [23, 24]. The slippery sequence is where
the shift of one nucleotide causes a change in reading frame during translation,
while the stimulatory stem has been posited to pause the ribosome [24]. Studies
elucidating the structure of the HIV-1 frameshift element mRNA determined that
the slippery sequence is proceeded by the frameshift stimulatory signal, which is an
irregular helix made up of an upper and lower stem [24, 25]. Of note, shortening
or removing the stem–loop, along variations of the HIV FSE sequence do not aect
tRNA binding to the A-site [20]. It has been posited that the interaction of the FSE
stem–loop is not sequence-specic, most likely involving recognition of the RNA’s
sugar-phosphate backbone [20].
A common method for investigating frameshifting that can also be employed
to test the inuence of compounds is through use of a dual reporter assay. In
the assay, the slippery sequence and FSE are sandwiched between two dierent
reporters, typically Renilla luciferase (Rluc) and rey luciferase (Fluc) or other
reporter genes. Rluc and Fluc yield enzymes acting on substrates to produce luminescence at dierent wavelengths (colors) [26]. In cellular systems (or sometimes
1620
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10.3 Targeting Frameshifting in HIV 257
1660
C
AAA
C G
G
C
C
G
UUA
C
C
1650
G
G
U
C
2
C
U
G
A
C
G
A
G
A
G
CU
G
A
AA
U
UUUUU
UU
A
G
U
1
G
A
GG
AA A A
GG
CC
A
U
AA
1600
1580
1560
U
A
G
A
A
A
C
A
G
G
A
3
A
G
A
A
A
G
G
U
G
U
A
A
A
G
U
U
5 6
G
U
C
G
G
G
Upper stem
A
G
G
G
C
1670
A
G
G
G
A
A
U
U
Alternate lower
U
U
stem
C
U
U
CG
A
G
A
G
C
A
G
A
C
C
1690
A
G
A
G
CG
C
C
G
C
C
C C CAAAAGAU AAG A A AG G GA AC
A
A
A
Anchoring helix
1710
4
SHAPE-directed model
Figure 10.3 The SHAPE-determined model of the HIV gag-pol frameshift sequence RNA.
Source: Low et al. [24]/with permission of American Chemical Society.
in a cellular lysate such as rabbit reticulocyte lysate), the downstream reporter is
only expressed when frameshifting occurs while the upstream reporter is always
expressed, allowing for an assay capable of measuring frameshifting eciency for
a given FSE [26].
10.3 Targeting Frameshifting in HIV
Dual-reporter assays were employed extensively in analyzing one of the more
prominent classes of compounds that bind the FSE mRNA: aminoglycosides. Guanidinoneomycin B, an exhaustively guanidine-derivatized analog of neomycin B, is
an example of this, and was studied extensively by Butcher and Tor [27]. However,
aminoglycosides are generally regarded as binding to RNA targets in a non-selective
258 10 Approaches to the Identification of Molecules Altering Programmed Ribosomal Frameshifting
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manner [28]. The nature of aminoglycosides to bind indiscriminately to RNA has
been attributed to their conformational exibility, which allows aminoglycosides to
conform to varied RNA targets [28, 29]. Recently, nucleic acid dye tags were used
to study the frameshifting event for HIV-1 [30]. Peptide-based and peptidomimetic
compounds have been used with considerable success for targeting the HIV-1 FSE.
These compounds were studied over a period of several years by the Miller group.
Initial hit compounds were identied using a resin-bound variant of a library
generation method called dynamic combinatorial chemistry. This uses reversible
reactions to create dynamic combinatorial libraries, which can undergo evolution
based on the selection pressure of library members binding preferentially to a
nucleic acid (or other) target [31]. While many exchange chemistries are possible,
here the reversible exchange was facilitated by the presence of cysteine residues
present in each library member, allowing for the formation of resin-bound disuldes
and subsequent reversible disulde exchange between resin-bound materials and
solution-phase compounds [31]. In the screen targeting the HIV-1 FSE RNA,
an 11,325 member Resin Bound Dynamic Combinatorial Library (RBDCL) was
prepared. The equilibrium of the reversible exchange between the solid-phase and
solution-phase monomers was altered when uorescently tagged RNA was introduced, allowing for the identication of an initial hit compound (Figure 10.4, 1)2via
C A
AA
C-G
C-G
C-G
U-A
U-A
C-G
C-G
G-G
G-C
U-A
C-G
G
A
U-A
A-U
G-U
A-U
A-U
G-U
5ʹ-UUUUUUAG U-3ʹ
Slippery
sequence
G-U
stem-loop
Purine bulge
G
Upper
H2N
NH
3
O
HN
O
EtNN
O
O
NH
N
S
S
HN
O
O
N
Et
NH
O
HN
O
NH
2
3
1
Figure 10.4 The initial lead compound (right) determined via the RBDCL [32] for the HIV
gag-pol FSE RNA (left) [33]. Source: (a) Palde et al. [33]/with permission of American
Chemical Society; (b) McNaughton et al. [32]/with permission of American Chemical Society.
2 Reprinted (adapted) with permission from Palde, P.B., Ofori, L.O., Gareiss, P.C. et al. (2010).
Strategies for recognition of stem-loop RNA structures by synthetic ligands: application to the
HIV-1 frameshift stimulatory sequence. J. Med. Chem. 53 (16): 6018–6027. https://doi.org/10.1021/
jm100231t. Copyright 2010 American Chemical Society.
10.3 Targeting Frameshifting in HIV 259
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uorescence microscopy, followed by mass spectrometry [32]. The library members
incorporated heterocyclic moieties to facilitate binding to the HIV-1 FSE RNA; the
primary compound identied from the screen was symmetrical, with quinoline
groups as the heterocycle [32, 33]. Post identication validation involved testing
for anity and specicity via surface plasmon resonance (SPR). This yielded a K
of 4.1± 2.4 μM and showed that the compound displayed no apparent anity to
related RNA sequences [32].
Based on this initial hit, several structural hypotheses were tested to yield
compounds with higher anity and selectivity for the FSE mRNA. One set of initial
experiments focused on structure activity relationships of the 2-ethylquinoline
3-carboxamide moiety. Modifying the 2-ethyl to methyl or proton yielded insignicant
decreases in anity of the compound for the FSE RNA [33]. However, reducing
the π-surface by replacing 2-ethylquinoline with 2-methyl-3-carboxypyridine
resulted in ablation of binding to the FSE RNA [33]. Another point of modication
on the lead compound was to test a suitable bioisostere for the disulde bridge
of the compound, due to susceptibility of disuldes to reduction or exchange in
a cellular environment [16, 33]. While disuldes have served a structural role
in bioactive peptides, thioesters and carbon linkers have been demonstrated to
enhance biostability without aecting function of the compound [33]. To that
end, the Miller lab synthesized and tested the activity of olen and hydrocarbon
analogs of the lead compound (Figure 10.5). It was found that the Z-isomer of
the olen displayed greater anity for the FSE RNA over the E-isomer [33].
The Z-isomer olen analog displayed over fourfold increase of anity relative
to the hydrocarbon analog; it was reasoned that this was due to the increased
exibility and hydrophobicity of the fully saturated analog [33]. Further experiments demonstrated that analogs were selective for binding the stem–loop RNA.
Interestingly, the monomeric compound, while able to bind to the FSE RNA,
had binding ablated in the presence of yeast tRNA, showing that its binding to
the FSE RNA was nonspecic [33]. Similar binding behavior was observed with
2-ethylquinoline 3-carboxylic acid, which binds to RNA non-specically, suggesting
that the entire peptide and quinoline is necessary for selective binding to the
FSE RNA [33].
Increasing the pi-surface area available for binding by replacing the 2-ethylquinoline with a benzo[g]quinoline enhanced anity (Figure 10.5, 2–8). These
analogs were tested to determine binding kinetics, as the equilibrium dissociation
constant K
on its own does not provide the full picture of the binding event. The
D
dissociation rate, kd, is regarded as an important determinant of selectivity for
protein binders, and this represented an opportunity to test the idea in the context
of RNA [34]. Slower dissociation rates indicate longer residence times and greater
target selectivity [35]. With that in mind, binding experiments using SPR were
carried out with immobilized biotinylated FSE RNA on the surface. The determined
o-rates for compound 1, along with the analogs, were 10−2to 10−3s−1, which is a
stark contrast to the fast on- and o-rate of a tested aminoglycoside, Neomycin [33].
With promising results, these analogs were tested in cells via a MTT assay, serving
D
260 10 Approaches to the Identification of Molecules Altering Programmed Ribosomal Frameshifting
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H2N
H2N
H2N
N
HN
O
N
O
OOHN
HN
O
NH
N
O
NH
O
NH
O
H2N
NH
2
N
HN
O
2
N
O
HN
O
N
HN
HN
O
O
O
N
N
N
O
NH
O
N
NH
NH
O
N
H2N
N
HN
NH
2
O
N
O
N
N
O
O
O
N
N
O
NH
O
N
O
N
NH
2
3
N
N
O
N
O
O
N
N
O
O
NH
NH
O
N
O
N
NH
N
NH
2
54
N
O
N
O
N
HN
HN
O
O
O
N
N
N
O
NH
O
N
NH
N
O
N
NH
H2N
N
HN
2
O
O
6
N
O
HN
O
N
O
N
N
N
O
N
NH
NH
O
O
N
N
NH
2
7
Figure 10.5 The N-methylation and triazole analogs of the olefin FSE RNA-binding
compound. Source: Hilimire et al. [16]/with permission of American Chemical Society.
H2N
N
N
O
N
N
HN
O
O
O
N
N
O
O
NH
N
O
N
O
N
N
N
NH
2
8
10.3 Targeting Frameshifting in HIV 261
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as a reporter for mitochondrial activity, and found no statistically signicant cell
toxicity with cells treated with compounds up to 1 mM [33].
Two further modications introduced were N-methylation and 1,4-triazole as
an alternative disulde bioisotere to the olen. N-methylation was used to further
enhance binding selectivity of the olen analog (Figure 10.5, 3), as this modication
often improves anity, selectivity, and activity of peptides [36, 37]. Triazole was
chosen as a bioisotere (Figure 10.5, 4–8) due to the robust metal-catalyzed Huisgen
cycloaddition chemistry that was readily available, and that despite the precedent
for triazole to be used in other targets, including RNA, they have not been used as a
bioisostere for disuldes [16]. The structural similarities between disuldes, olens,
and triazoles were clear, based on density functional theory (DFT) calculations,
though the disulde torsional bond rotation is distinct from that of olens and
triazoles [16]. What was found from these experiments is that inclusion of a triazole
in the structure had no eect on anity, while N-methylation had a modest eect
[16]. It was observed that N -methyl analog 3 had a dissociation constant (KD) for the
FSE RNA of 13.0 ± 5.0 nM, a ve-fold increase in anity over the olen-containing
compound [16]. Cell permeability and toxicity experiments revealed that compound
6 was non-toxic up to 110 μM, and compound 8 was the most cell permeable as
determined via ow cytometry [16]. These N-methyl analogs were also studied
for frameshift modulating and anti-HIV activity using pseudotyped HIV, and were
found to enhance frameshifting at levels consistent with their relative potencies,
and likewise to interfere with the infectivity of pseudotyped HIV. Finally, compounds were found to be active against both laboratory HIV-1 strains (HIV
IIIB
) and
a multi-drug-resistant patient isolate of HIV-1 [16, 36].
Success with replacing portions of the peptide with non-peptidic bioisosteres
led to the question: could one incorporate other bioisosteric peptide mimics to
further evolve these HIV-1 FSE binders to something entirely non-peptidic? To
address that question, we hypothesized that the 2,5-diketopiperazine (DKP) could
be integrated into FSE-binding molecules, and perhaps serve more broadly as
desirable scaold for RNA-binding compounds. DKP is a considered a “privileged
structure,” dened as a substructure exhibiting good drug-like properties and often
molecules able to specically bind a broad range of biological targets [39]. DKPs
are known to oer bioavailability and biostability advantages relative to simple
peptides, due to improved stability, protease resistance, and conformational rigidity
[40, 41]. The DKP has at least four points of functionality, making it a particularly
diversiable scaold that can cover a great deal of chemical space. However, prior
to our work, it had yet to be used as part of an RNA-binding compound. Thus a
DKP-containing compound (10) was designed, synthesized, and tested alongside
a non-DKP-containing compound (9) to determine the eects this new scaold
has on binding to the FSE RNA (Figure 10.6). Initial DFT simulations suggested
that the DKP and non-DKP analogs adopted similar orientations and distances,
having only a 1 Å RMS dierence between the two [38]. Anity and selectivity
experiments for both analogs determined that the incorporation of the DKP scaold
did not impair binding, and competition with ve-fold excess yeast tRNA did not
yield notable changes in binding constants [38]. In contrast, a DKP containing
262 10 Approaches to the Identification of Molecules Altering Programmed Ribosomal Frameshifting
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NH
2
HN
O
NH
N
O
N
H2N
HN
O
O
NH
N
HN
O
N
O
NH
HN
O
O
H2N
O
N
N
N
N
O NH
O
HNH2N
NN
NN
NH
NN
O
OO
NN
H2N
O
O
NH
HN
O
O
N
HN
O
N
HN
N
N
109
Figure 10.6 The DKP (10) and non-DKP (9) containing analogs. They both adopt similar
structural and 3D space. Source: Arévalo et al. [38]/with permission of Royal Society of
Chemistry.
half-compound displayed no binding to the RNA, demonstrating the necessity of
the entire compound [38].
To determine where the DKP-containing compound was binding onto the FSE
RNA, a series of surface plasmon resonance (SPR) competition experiments were
performed by co-injecting the DKP compound with ve-fold excess of stem and loop
swapped FSE RNA mutants, along with FSE RNA (Figure 10.7). The competition
experiment revealed that WT HIV-1 FSE RNA had ablated approximately 40% of
total binding based on R
value, suggesting that the anity of the DKP-containing
max
compound is impacted by whether the RNA is immobilized or in solution [38]. Of
NH
2
O
A
C
A A
C
C
C
U
U
C
C
G
G
HIV-1 FSS
5 µM 10 + 25 µM “stem-swapped” RNA
5 µM 10 + 25 µM “loop mutant”
Figure 10.7 DKP containing compound co-inject competition experiments with WT HIV-1
FSE RNA, “stem-swapped” and “loop” mutant RNAs via SPR. Source: Arévalo et al. [38]/with
permission of Royal Society of Chemistry.
G
G
G
A
A
G
G
C
C
“stem-swapped” RNA “loop mutant” RNA
Conditions
5 µM 10
5 µM 10 + 25 µM HIV − 1 FSS
C
A A
G
G
G
A
A
G
G
C
C
A
C
C
C
U
U
C
C
G
G
K
D
5.04×e
6.5×e
6.4×e
6.3×e
R(max)
–7
15.9 0
–7
9.6
–7
–7
15.2
Δ[R(max)], %
13
C C
C
C
U
U
C
C
G
G
40
18
4
C
GAC
G
G
A
A
G
G
C
C
10.4 Targeting Frameshifting in SARS-CoV-1 and SARS-CoV-2 263
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p = 0.046
7
3×10
7
2×10
7
1×10
Fluorescence intensity
0
0
25
μM
100 150
10
9
P24, pg/mL
800,000
600,000
400,000
200,000
Mock
0
0
Media μM
p = 0.067
p = 0.007
15 25 100
p = 0.022
10
9
8
150
(b)(a)
Figure 10.8 The toxicity and anti-HIV activity assays, from left to right. Both analogs are
non-toxic up to 150 μM in MT-2 cells and display modest anti-HIV activity. Source: Arévalo
et al. [38]/with permission of Royal Society of Chemistry.
the two mutant RNA that were co-injected, the stem-swapped RNA sequence caused
an 18% decrease in R
, while the loop-swapped mutant only competed o 4%
max
(Figure 10.7). This nding suggests that most of the interactions of DKP analog to
the FSE RNA is at the loop region [38]. Toxicity assays performed with MT-2 cells
treated with both analogs have shown that they both do not show any notable toxicity up to 150 μM (Figure 10.8). The anti-HIV activity was not impacted with the
inclusion of the DKP scaold, though there was a loss of activity compared to the
control [38].
10.4 Targeting Frameshifting in SARS-CoV-1
and SARS-CoV-2
Much like HIV-1, frameshifting in coronaviruses such as SARS-Cov-1 and -Cov-2
acts as a regulatory mechanism that controls the ratio of the production of
non-structural proteins and enzymes. Specically, genes orf1a and orf1b have an
overlap in reading frames in the genome, requiring a frameshifting event during
translation to produce the out-of-frame downstream polyprotein pp1ab in addition
to the in-frame upstream polyprotein pp1a [42]. These polyproteins are further
processed into nonstructural viral proteins such as proteases for viral protein
processing (part of pp1a) and replication enzymes, including the RNA-dependent
RNA polymerase (in pp1ab) [42].
While disrupting frameshifting of the HIV gag-pol polyprotein can abolish viral
reproduction, changing the frameshift eciency of beta coronaviruses such as
SARS-CoV-1 and 2 impacts early viral growth kinetics, viral infectivity, and viral
replication (Figure 10.9) [13, 14].
As previously stated, frameshifting requires an upstream slippery sequence and
downstream frameshift-stimulating structure. In the case of beta coronaviruses, the
canonical FSE is a three-stem, three-loop H-type pseudoknot [43, 44] capable of
forming upstream and downstream interactions with neighboring RNA [45, 46] and
dimerizing with another FSE [47]. This structure is conserved in coronaviruses, and
264 10 Approaches to the Identification of Molecules Altering Programmed Ribosomal Frameshifting
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Slippery site sequence
U UUA AAC (WT)
U UUU UUU
A AAA AAU
U UUG AAC
% –1 PRF Standard deviation Plaque size
14.4
4.88
2.33
0.15
2.35 4.53+ + +
1.31
0.55
0.03
+ +
+
–
TCID
<1
<1
ND
50
Figure 10.9 Mutating the SARS-CoV-1 slippery sequence reduces frameshift efficiency,
which negatively impacts viral plaque size and infectivity. Source: Plant et al. [13]/with
permission of American Society for Microbiology. Frameshift efficiency was measured in
Vero E6 cells by incorporating either a wild-type or mutant slippery sequence and the FSE
into a dual luciferase reporter plasmid. Clones of the SARS-CoV-1 genome were transfected
into Vero E6 cells to measure infectivity of transcripts.
Loop 1
Slippery site
Stem 2
Stem 1
Stem 3
Loop 2
Loop 3
pseudoknot
mRNA
Pseudoknot
tRNA
LSU
Stem 3
Head
h16
Stem 2
Stem 1
Shoulder
Beak
Body
Attenuator
loop
Figure 10.10 SARS-CoV-2 orf1ab FSE (green, purple, and blue) and ribosome (large
subunit, light blue; small subunit, light green) captured together prior to a frameshifting
event. The viral RNA is in the 0 frame, with a stop codon incorporated in the upstream
slippery sequence. The presence of a mutant eRF1 prevents the release of the nascent
polypeptide. The experiment was performed in rabbit reticulocyte lysate. Cryo-EM
resolution ranged from 2.2 to 7. 4 Å. Source: Bhatt et al. [18]/American Association for the
Advancement of Science/CC BY 4.0.
changing the structure results in a corresponding change in frameshift eciency
[13]. An upstream hairpin stem loop, known as the attenuator stem loop, adds additional regulation to frameshift eciency [44, 48].
The mechanism of frameshifting in coronaviruses is still under investigation, but
cryo-EM and crystallography data from the Ban Lab shows the FSE wedged into the
mRNA entry channel during translation (Figure 10.10) [18], providing evidence of
a mechanism for inhibiting ribosomal translocation and causing ribosomal stalling
required for a frameshifting event to occur.
One of the challenges of targeting frameshifting in SARS-CoV-2 is the complexity of the frameshift-stimulating element (FSE). Originally believed to be
a three-stemmed, three-looped H-type pseudoknot, research by the Woodside
group and others have shown that while this conformation occurs in isolation
(Figure 10.11), the RNA element is context-dependent and can assume multiple
conformations and associations with upstream and downstream RNA sequences,
especially in a cellular environment (Figures 10.12 and 10.13) [18, 45, 49].
Initial structural data was obtained based on the canonical pseudoknot structure
located at the 3′end of orf1a. Composed of three stems and three loops, this
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