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
(a)
https://t.me/med1917
Ligand
8.1 Introduction 205
Drug
Drug
5c 5c
Natural ligand binds riboswitch aptamer
Gene Gene
Drug mimics binding of natural ligand
(b)
OFFOFF
Protein of
interest
Ligand
Drug
Protein of
interest
Drug
Ligand
Ligand
Ligand
5c
Riboswitch-based sensors identify compounds
that disrupt homeostasis of the target ligand
Ligand
Reporter
(c)
Drug
Drug
5c
Transgene
OFF
5c
“Designer riboswitches” turn ON transgene
expression when drug is administered
Transgene
ON
Figure 8.3 Riboswitches as targets and tools in drug development. (a) Riboswitch
aptamers are targeted by small molecule drugs that, for example, can mimic the binding
of the native ligand. (b) Riboswitches used as sensors for their native ligand can be
employed to screen for small molecule inhibitors of a protein of interest that processes
the ligand. (c) “Designer riboswitches” that sense drug or drug-like compounds can be
used to turn on expression of a therapeutic transgene in response to the drug.
provide information about how small-molecule compounds in the screen may
cause a buildup or depletion of the ligand, presumably by disrupting the function
of a metabolic enzyme or a protein involved in a key aspect of bacterial physiology
[22–24] (Figure 8.3b). The goal of this approach is to produce novel antibiotic
compounds that disrupt new targets and pathways. A third application entails
engineering riboswitches that regulate gene expression in response to drug-like
compounds [25] (Figure 8.3c). Such a device could be employed in human gene
therapy applications to precisely regulate the expression of a therapeutic transgene.
8.1.3 The Need for Novel Antibiotics
The development of antibiotics was one of the dening medical breakthroughs of
the twentieth century [26]. The common use (and misuse) of antibiotics combined
with the ability of bacterial pathogens to rapidly evolve has led to the development and spread of antimicrobial resistance [27]. Following the current trend,
206 8 Prospects for Riboswitches in Drug Development
https://t.me/med1917
Key
Present
Absent
Cobalamin
FMN
Glycine
Guanidine-I
Guanine-I
Lysine
2+
Mg
2+
Mg
Mn
Moco
+
Na
+
Na
PreQ
1-I
PreQ
1-II
SAH
Acinetobacter baumannii
Pseudomonas aeruginosa
Enterobacteriaceae
-
F
-I
-II
2+
-I
-II
Enterococcus faecium
Staphylococcus aureus
Helicobacter pylori
Campylobacter
Salmonella
Neisseria gonorrhoeae
Streptococcus pneumoniae
Haemophilus influenzae
Shigella
SAM-I, -I/IV
SAM-III
THF
TPP
ZTP
Priority: 1 – Critical 2 – High
本书版权归John Wiley & Sons Inc.所有
3 – Medium
8.2 Riboswitches as Drug Targets 207
https://t.me/med1917
antimicrobial resistance is predicted to lead to 10million deaths per year by 2050
[26]. To address this urgent threat, new classes of chemical compounds that target
novel targets or pathways are needed [23, 28]. Two of the three drug development
modalities introduced above are related to the development of novel antibiotic
compounds.
8.2 Riboswitches as Drug Targets
8.2.1 Why Target Riboswitches?
RNA is widely recognized as a clinically useful target in bacterial pathogens and
humans [29, 30]. RNA molecules, like proteins, possess dynamic structures that
undergo conformation changes, which can be inuenced by small molecules [20].
For example, most antibiotics in clinical use target bacterial ribosomes, and nearly
all of these compounds have been shown to bind ribosomal RNAs rather than ribosomal proteins [31].
Among the diverse types of RNAs present in cells, riboswitches are especially
attractive as drug targets [32]. Whereas the interaction between other RNA targets
and small molecules is mostly fortuitous, riboswitch aptamers often form structurally sophisticated binding pockets that specically bind small-molecule ligands
even in the presence of highly similar compounds that exist within cells [19].
Thus, compounds that target RNA ligand-binding pockets might be identied in
a manner analogously to that for pockets made by proteins. Bacterial pathogens
might also have greater diculty developing resistance to a drug that targets a
riboswitch aptamer because a mutation to a conserved nucleotide in the aptamer
that avoids drug binding might also confer loss of binding to the native ligand.
Because riboswitches often control the expression of genes important to bacterial
survival, it is likely that disrupting riboswitch function would aect bacterial
viability or virulence [20].
Additionally, various riboswitch classes are predicted to be present in bacterial
pathogens identied by the World Health Organization’s global priority pathogens
list [19, 21] (Figure 8.4), which includes 12 species of bacteria that exhibit medium,
Figure 8.4 Riboswitch classes predicted to be present in the 12 members of the World
Health Organization global priority pathogens list. These riboswitch classes include
Cobalamin [33], Fluoride (F−) [34], FMN (flavin mononucleotide) [35], Glycine [36],
Guanidine-I [37], Guanine-I [38], Lysine [39], Mg2+-I [40], Mg2+-II [41], Mn2+[42],
molybdenum cofactor (Moco) [43], Na+-I [44], Na+-II [45], PreQ1-I (pre-queuosine1) [46],
PreQ1-II [47], SAH (S-adenosylhomocysteine) [48], SAM-I (S-adenosylmethionine) [49],
SAM-I/IV [50], SAM-III [51], THF (tetrahydrofolate) [52], TPP (thiamin pyrophosphate) [53],
and ZTP (5-aminoimidazole-4-carboxamide riboside 5′-triphosphate) [54]. Though many
classes are represented, not all are likely to make useful targets (see Section 8.2.2).
Riboswitches for the same ligand that employ different binding pockets are separated into
different rows, whereas those that are known to employ highly similar binding pockets
(e.g. SAM-I and SAM-I/IV [55]) are grouped. The data summarized in this graphic was
collected from Rfam 14.9 [56].
208 8 Prospects for Riboswitches in Drug Development
https://t.me/med1917
high, or critical antimicrobial resistance [57]. None of these riboswitches have
been discovered to occur naturally in humans. It is thus possible that drugs targeted
to one of these riboswitches may have a low chance of interfering with human
physiology.
8.2.2 Features of a Druggable Riboswitch
Riboswitches that control essential genes and function as OFF switches are perhaps
more pharmacologically relevant [21]. A drug compound that binds such a
riboswitch with anity comparable to or greater than that of the native ligand
would force the riboswitch to be in the OFF state regardless of the concentration
of the native ligand inside the cell. Thus, the riboswitch will be unable to regulate
essential downstream genes in response to uctuating concentrations of the native
ligand. Specically, the riboswitch is tricked into acting as if there is an abundance
of an essential ligand when in fact the cell is being starved of the molecule. However,
some riboswitches might not fully suppress gene expression [58] and permit a level
of gene expression that is sucient for bacterial survival even when there is a high
concentration of a ligand analog.
Features of the aptamer-binding pocket can also aect the suitability of a
riboswitch as a drug target. Many existing drug compounds have complex,
hydrophobic chemical structures. Thus, aptamer-binding pockets that are small,
polar, or solvent-exposed might be unable to bind molecules with drug-like properties [59, 60]. The challenge for medicinal chemists is to identify compounds with
the appropriate mix of drug-like properties and the characteristics needed to be
recognized by natural aptamers.
Finally, riboswitch classes that are widespread over many genera would make
ideal target candidates for development of broad-spectrum antibiotics. The common riboswitch classes that sense FMN, TPP, SAM, or AdoCbl appear to be the
best-suited targets to achieve broad-spectrum ecacy. Other riboswitch classes that
are more narrowly distributed could be targets for developing narrow-spectrum
antibiotics. Historically, broad-spectrum antibiotic agents have been preferred,
but narrow-spectrum compounds could help to avoid damaging the human
microbiome and present a lower risk for developing antimicrobial resistance in the
environment [61].
8.2.3 Riboswitch-Targeted Drugs
8.2.3.1 Small Molecules Targeting FMN Riboswitches
FMN (avin mononucleotide, Figure 8.5) riboswitches exhibit many of the druggable features discussed above [35, 65, 66]. FMN riboswitches from Bacillus subtilis
turn o expression of essential genes in response to increased FMN levels in
the cell [35]. Subsequently, it was discovered that the natural antibacterial agent
roseoavin targets FMN riboswitches, mimicking the binding of the native ligand
[58]. The synthetic compound 5FDQD (Figure 8.5), developed as an FMN mimic
using medicinal chemistry principles, also targets FMN riboswitches [62]. 5FDQD
8.2 Riboswitches as Drug Targets 209
https://t.me/med1917
O
N
N
O
P
OH
O
OH
FMN
HO
Figure 8.5 Chemical structures of flavin mononucleotide (FMN) (the native ligand of
FMN riboswitches [35]) and FMN riboswitch-targeting compounds 5FDQD [62], ribocil-C
[63], and ribocil C-PA [64].
N
OH
OH
NH
O
N
N
5FDQD
O
NH
N
O
N
F
ribocil-C: R = H
ribocil C-PA:
R =
R
N
N
N
N OH
S
N
H
N
+
NH
3
was found to be nearly as eective as broad-spectrum antibacterial compounds
vancomycin and metronidazole at curing mice infected with Clostridioides dicile
[62]. Notably, 5FDQD showed markedly less alteration in culturable cecal ora
relative to vancomycin and metronidazole [62]. Compounds that bind FMN
riboswitches with even greater anity have also been reported [67], suggesting that
medicinal chemistry approaches can be used to make additional improvements to
this class of compounds.
Another example of a synthetic compound that targets FMN riboswitches is
ribocil, which was discovered through a phenotypic screen [63]. Ribocil is a racemic
mixture of ribocil-A (R enantiomer) and ribocil-B (S enantiomer), the latter of
which was determined to be the active compound [63]. Ribocil-B was further
improved by medicinal chemistry approaches, which led to the development of
ribocil-C [63]. Moreover, the chemical scaold of ribocil diers substantially from
that of FMN and other compounds that target FMN riboswitches (Figure 8.5).
Whereas FMN binds the E. coli FMN riboswitch with a KDof <10 nM [35], ribocil
binds the same riboswitch with a KDof 13 nM [68] and ribocil-C binds with a KDof
<1 nM [63]. Ribocil-C was found to be eective at treating Gram-positive bacteria
in infectious settings, but less so with Gram-negative bacteria, which are known
to exclude or eject many compounds [21, 69]. Recently, an improved ribocil C-PA
was developed that was 16-fold more potent against Gram-negative bacteria E. coli
and K. pneumoniae [64]. Using rational design principles, a quaternary amine was
installed at a solvent-exposed site, which had the desired eects of increased entry
and accumulation in Gram-negative bacteria [64].
8.2.3.2 Other Riboswitches Targeted in Proof-of-Principle Demonstrations
There are numerous proof-of-principle reports describing novel ligand development
for various riboswitches [70–81]. Much work has been performed in developing
synthetic ligands that target glmS ribozymes, riboswitches that use glucosamine-6phosphate
as a cofactor for self-cleavage [70–72]. Several reports describe synthetic
210 8 Prospects for Riboswitches in Drug Development
https://t.me/med1917
ligand mimics of guanine that bind Guanine-I riboswitches with high anity
[73–75]. Synthetic ligands and probes have been developed using a combination
of HTS and structure–activity relationship (SAR) approaches that target PreQ
riboswitches [76, 77]. ZTP riboswitches were targeted with synthetic ZMP analogs
in which the sugar-phosphate moiety is replaced with various functional groups,
including simple heterocycles [78]. Fragment-based approaches have led to the
development of compounds that bind TPP riboswitches [79–81]. The antibacterial
and antifungal compound pyrithiamine is converted to pyrithiamine pyrophosphate
inside cells, which also binds and disrupts TPP riboswitches [82, 83]. Moreover, its
discovery predates that of TPP riboswitches by more than half a century [53, 65].
Though many of the compounds described in these reports are not as advanced
as the FMN-targeting compounds, the diversity of riboswitches that have been
targeted is indicative of the potential of this technology.
8.2.4 Barriers and Future Developments
A potential pitfall is that ligands developed in vitro, either through rational design
or by screening compound libraries, that tightly bind a riboswitch aptamer target in
a test tube may fail to cause antibacterial eects in vivo. High-throughput screening
approaches sometimes use in vivo assays, which can circumvent this problem, but
access to suciently diverse chemical libraries remains a substantial barrier that
must be addressed [21]. Another related possibility is that antibacterial compounds
that are intended to disrupt riboswitch function might instead target other aspects
of bacterial physiology. For example, synthetic lysine analogs that bind Lysine
riboswitches in vitro also display antibacterial activity, but the mechanism of
antibacterial activity is likely via lysyl-tRNA synthetases [84, 85].
In addition to continuing to develop ligands that bind known riboswitches,
additional riboswitch targets are likely to emerge as orphan riboswitches (ligand
unknown) [86] and yet-undiscovered riboswitches are likely to continue to be
experimentally validated. It is hypothesized that most – if not all – of the widely
distributed riboswitch classes have already been discovered [12]. Undiscovered
riboswitch classes are therefore likely to be rare and narrowly distributed. Some of
these latter classes likely will present intriguing candidates for narrow-spectrum
antibiotic development.
1
8.3 Riboswitches as Tools for Antibiotic Drug
Development
8.3.1 Riboswitches as Biosensors
Riboswitches can be used as tools to identify compounds that disrupt key aspects
of bacterial metabolism or physiology. Riboswitches or riboswitch aptamers can be
exploited in the construction of biosensors that report to researchers the relative
concentrations of their target ligands [87, 88]. This is achieved by installing a
8.3 Riboswitches as Tools for Antibiotic Drug Development 211
https://t.me/med1917
riboswitch upstream of a reporter gene such as lacZ (β-galactosidase) [22–24], or
by producing an allosteric device in which the riboswitch regulates a uorogenic
aptamer [89–91]. Bacterial cells that express this biosensor are then subjected to a
high-throughput screen to identify small molecules that alter the intracellular concentration of the target ligand. A plausible cause for changes in ligand concentration
could be that the small molecule disrupts the activity of a protein that is important for
ligand homeostasis. This can subsequently be conrmed by performing the requisite
assays with the protein of interest. Hit compounds identied through this approach
could be further improved through medicinal chemistry approaches to enhance
their disruptive eects, leading to the development of new antibacterial compounds.
8.3.2 A Riboswitch-Based Fluoride Sensor Illuminates Agonists
of Fluoride Toxicity
In a prior report [22], the uoride riboswitch was exploited using this approach to
identify agonists of uoride toxicity. Fluoride possesses substantial antibacterial
eects [92, 93] and is generally toxic to organisms in all three domains of life [22].
Thus, compounds that specically increase uoride toxicity in bacteria could exploit
the antibacterial action of this anion, and these could be valuable to combating
antimicrobial resistance, particularly for topical or surface treatments. Researchers
constructed a uoride sensor by inserting a Fluoride riboswitch upstream of a
lacZ reporter gene. The model gram-positive bacterial organism Escherichia coli
expressing this reporter was subjected to a high-throughput screen in the presence
of uoride. Several hit compounds associated with increased reporter signals were
identied. The most active hit compound was further improved by structure–
activity relationship analysis to yield a series of diaryl urea and diaryl thiourea
compounds that were conrmed experimentally as potent agonists of uoride
toxicity (Figure 8.6a). Various types of uoride agonists presumably could be made
to shuttle uoride into cells, block its export, or act by inhibiting cell wall or lipid
biosynthesis, thus increasing uoride transport into the cell [22, 94–96]. Although
the reported compounds do not appear potent enough to gain widespread use
as antibiotics in combination with uoride, the results showcased the utility of
riboswitches as tools for developing drugs for other bacterial targets.
8.3.3 A Riboswitch-Based ZTP Sensor Identifies Inhibitors of Folate
Biosynthesis
In a subsequent report [23], researchers employed another riboswitch to search for
inhibitors of folate biosynthesis in E. coli. The folate metabolic pathway is essential
in all species and therefore is an attractive target for antibiotic drug development
[97]. Dihydropteroate synthase is a folate biosynthesis enzyme that is not found
in humans, and therefore can be targeted without concern for disrupting folate
metabolism in patients [98]. A class of antibacterial agents known as sulfonamides,
which contain a characteristic sulfonamide linkage, was developed long ago that
specically target this enzyme [99].
212 8 Prospects for Riboswitches in Drug Development
https://t.me/med1917
(a) (b) (c)
EWG
Figure 8.6 Chemical scaffolds of compounds identified in HTS campaigns that employed a
riboswitch-based sensor. (a) Agonists of fluoride toxicity in E. coli and S. mutans discovered
through a HTS effort that employed an F−riboswitch reporter. The best-performing
compounds employed a diaryl urea or diaryl thiourea scaffold in which one aryl moiety
contained electron withdrawing groups at the meta and/or para positions. (b) Inhibitors
of folate biosynthesis in E. coli discovered through a HTS effort that employed a ZTP
riboswitch reporter. The best-performing compounds employed a sulfanilamide scaffold.
(c) Inhibitors of SAH nucleosidase in E. coli discovered through a HTS effort that employed a
SAH riboswitch-based sensor. A confirmed active compound identified is carmofur
(1-hexylcarbamoyl-5-fluorouracil).
X = O, S
X
NHN
Diaryl (thio)ureas Sulfanilamides
F
F
H2N
H
F
R = variable groups
O
H
S R
N
F
OO
O
N
N
O
H
Carmofur
N
H
To search for inhibitors of folate biosynthesis with dierent chemical structures
or novel biochemical mechanisms of action, researchers exploited a riboswitch
that senses ZTP (5-aminoimidazole-4-carboxamide riboside 5′-triphosphate)
[23, 54]. ZTP is proposed to function as an alarmone that produces a stress
response when the cell is starved of essential folate derivatives, particularly 10-formyl-tetrahydrofolate [100]. Grafting a ZTP riboswitch upstream
of the open reading frame (ORF) of a lacZ mRNA allowed for monitoring
of intracellular ZTP levels. Numerous hit compounds were identied in a
high-throughput screen that produced an increase in ZTP concentration. Most
of the hits carry a sulfanilamide scaold containing the characteristic sulfonamide linkage (Figure 8.6b). Based on this chemical similarity, these compounds
presumably employ a similar mechanism of action to previously reported sulfonamide antimicrobials. It is likely that this screening campaign mostly yielded
sulfonamide-containing compounds and few other types of folate inhibitor classes
at least in part due to the relative low chemical diversity of compounds in the
library.
8.3.4 A Riboswitch-Based SAH Sensor Reveals an Inhibitor of SAH
Nucleosidase
S-adenosylhomocysteine (SAH) is a by-product of cellular transmethylation
biochemistry involving S-adenosylmethionine (SAM) [101]. SAH buildup can
cause inhibition of cellular transmethylation reactions, rendering its detoxication essential [101]. SAH nucleosidase is the enzyme responsible for recycling
SAH in bacteria [102], identifying it as a critical target for antibiotic drug development [103]. Immucillin-based compounds have been developed to target
SAH nucleosidase, but unfortunately there is reported cross-reactivity with
human methylthioadenosine phosphorylase as well as poor cellular permeability
[103–105].
8.4 Application of Riboswitches in Gene Therapy 213
https://t.me/med1917
To develop screening methods that address these issues, researchers recently
exploited a SAH riboswitch as an in vivo sensor for this by-product of SAMdependent
to search for new compounds that disrupt SAH nucleosidase activity in E. coli [24].
An active compound, carmofur (1-hexylcarbamoyl-5-uorouracil), was identied
(Figure 8.6c) and validated using genetic (riboswitch mutant) and biochemical
(enzyme kinetics) assays [24]. Carmofur is approved for use as an antineoplastic
drug for cancer treatment [106], making its immediate implementation as an
antibacterial agent unlikely. However, it presents a novel chemical scaold that
could presumably be further improved by medicinal chemistry techniques.
methylation reactions. A riboswitch-reporter fusion construct was used
8.3.5 Barriers and Future Developments
A major barrier to any high-throughput screening eort is access to a large and
diverse chemical library. Additionally, because the reporters often rely on uorescence signal to detect changes in ligand concentration, many autouorescent
compounds are typically discounted as false positives. In principle, active
compounds could also be autouorescent, but they are not suitable for the assay
in its current format. For future screening eorts, multiple reporters with distinct
emission wavelengthscould be used to accommodate uorescent compounds whose
emissions overlap with that of one of the reporters. Also, bactericidal compounds
may be so potent that they cause bacterial cell death at the concentrations used in the
screening assay.In this case, the reporteractivity would be low,despite the possibility
that the compound has the desired eect. While it is dicult to avoid this problem,
one method might be to screen certain compounds at reduced concentrations.
Future eorts will likely target pathways that are unique to bacteria, to avoid
o-target eects in humans. Pathways widespread in pathogenic bacteria, but
less common in bacteria that compose the human gut microbiome, are of particular interest. Prime candidates for future endeavors include TPP riboswitches,
which represent the most widespread class and thus might be well suited for the
development of broad-spectrum antibiotics [14, 21].
8.4 Application of Riboswitches in Gene Therapy
8.4.1 Considerations for Designer Riboswitches
Genetically encodable synthetic devices that allow for conditional regulation of
gene expression are proving to be increasingly useful in a host of applications
including synthetic biology, functional genomics, biosensing, and therapeutics
[107, 108]. Such genetic switches would be ideally suited for regulating the
expression of a transgene in human gene therapy applications, in which transgene
expression is chemically induced by a drug. Genetic switches could be employed
as important components of gene therapy systems to treat a wide array of human
health issues including pathological diseases, genetic disorders, and cancer.
214 8 Prospects for Riboswitches in Drug Development
https://t.me/med1917
Protein-based genetic switches, such as the tetracycline-responsive Tet-ON/
Tet-OFF systems, have achieved success in preclinical gene therapy applications
[109–111]. However, these switches are immunogenic and prohibitively large
(>1 kb), producing a substantial genetic footprint and hindering delivery [112, 113].
Riboswitches oer tremendous potential for overcoming these challenges. However,
natural examples respond to fundamental metabolites such as nucleic acid derivatives, ions, or amino acids [14]. The concentrations of these ligands cannot be safely
manipulated in humans, precluding the use of most known natural riboswitches
for human gene therapy applications. Recently, riboswitches with alternative
aptamer and expression platform domains have been engineered to regulate gene
expression in response to other ligands [114]. Relative to protein switches, these
“designer riboswitches” are notably smaller (<200 bases), nonimmunogenic [112],
and modular in design [115]. The relatively compact size makes it easier to include
RNA-based switches in a delivery vector alongside a therapeutic protein of interest.
Additionally, while it is a considerable challenge to modify a protein switch to
respond to a dierent drug, it is comparatively straightforward to evolve aptamers
for new compounds.
The rst consideration in developing a designer riboswitch is the target ligand.
In principle, an RNA aptamer can be generated as a receptor for almost any target
using in vitro selection [116, 117]. Drug-like compounds that exhibit excellent
bioavailability, safety, pharmacodynamics, and pharmacokinetics are ideal candidates for this eort. Recently, researchers have exploited natural riboswitch
scaolds to generate aptamers for new compounds that maintain the natural
scaold [118–120]. This strategy appears to increase the likelihood that RNA
aptamers will fold reliably (and function) inside cells. Aptamers can then be grafted
onto expression platforms to produce engineered riboswitches [120]. In some
reports, aptamer and expression platform domains are evolved together [121], or a
communication module that enables allostery is developed [115, 122–124].
An important consideration is that designer riboswitches should be implemented
as ON switches, so that the drug is only administered while treatment is required.
Ideally, there would be zero gene expression in the absence of the drug. Administering the drug at carefully measured doses would enable exploring the dynamic
range of the switch, a valuable feature of this type of personalized medicine. If the
aptamer and drug exhibit 1 : 1 binding, it should theoretically be possible to explore
intermediate gene expression levels between fully OFF and ON states over two
orders of magnitude of drug concentrations. Furthermore, developing an aptamer
with high anity for the drug is imperative, as low concentrations of the drug will
minimize o-target eects.
8.4.2 Eukaryotic Expression Platforms
With most bacterial riboswitches, the aptamer domain regulates a transcription
terminator stem or an anti-Shine-Dalgarno sequence [8, 9]. These expression
platforms do not readily conform to the mechanisms used for gene expression and
regulation in humans. Thus, dierent expression platforms must be developed to
Соседние файлы в папке Библиотека им академика М.И. Перельмана