Guanine Quadruplexes and Disease: Insight Into Mechanisms and Therapeutic Potential

Guanine quadruplexes and disease: Insight into mechanisms and therapeutic potential

Katie Manas and Celena Hoeve

Abstract Understanding Guanine Quadruplexes (G4s) provides valuable insight into a wide array of diseases at a genetic level and their potential therapy. These unusual architectures can form in DNA and RNA, composed of planar, stacked tetrads of four hydrogen bonded guanines. G4s are widely implicated in a range of disease categories, including parasites, viral infections, cancer, and neurodegenerative diseases, with varied roles within each. Of current relevance, a connection to the coronavirus family may be an intriguing avenue of investigation for recent outbreaks. The planar surface of the G4 tetrad also provides an attractive target for small molecule binders that can provide therapeutic value. Overall, this review aims to address the exciting relevance of G4s in these felds, a few major mechanisms of action, and their potential as therapeutic targets.

1. Introduction arise from the orientation of the four guanine segments with Though most are familiar with DNA as a double stranded respect to the 3’ and 5’ ends. Parallel G4s have all strands helical structure composed of four nucleobases (ACTG), it oriented in the same direction, while antiparallel G4s have also has the ability to form other higher-order architectures, the four strands in alternating directions. Hybrid G4s have such as the well-known guanine quadruplex (G4). G4s were three strands oriented in one direction and the fourth strand frst proposed in 1962 (Gellert et al. 1962), as three-dimen- in the other direction. G4s can also be described based on sional architectures that form in guanine-rich environments. the loops, which create a unique environment surrounding Four guanine nucleobases self-assemble via hydrogen bond- the stacked guanine tetrads of the G4. The structural features ing into a planar guanine tetrad; these tetrads then stack on of G4s have previously been reviewed (Keniry 2001). These one another to form the three-dimensional G4 architecture structures are often determined by techniques such as solu- in the presence of stabilizing monovalent cations (Figure 1) tion-state NMR, which has been used with the c-Myc G4, (Pinnavaia et al. 1978). found in the promoter region preceding the gene (Figure 2) (Ambrus et al. 2005).

A B Figure 1. A) Chemical structure of a guanine tetrad, showing the hydrogen bonding interactions of four guanine nucleobases and stabilized by a central cation (M+). Figure 2. Solution-state NMR structure of the c-Myc pro- B) G4 formed by stacked tetrads (stabilizing cations are omit- moter G4 with stabilizing cations (PDB ID: 1XAV) (Ambrus ted). et al. 2005).

G4s can adopt parallel, anti-parallel, and hybrid topologies, G4s can assemble when guanine-rich regions of nucleic acid which can be useful for classifcation purposes. The topologies are in single-stranded forms. This can occur during activi-

40 40 ties such as replication and transcription, where the typically helicase, which is suggested to have the ability to unwind double stranded DNA is temporarily separated into single G4s. Therefore, P. falciparum can use the RecQ helicase to turn strands to allow for processing. It can also occur in RNA on transcription by unwinding the G4 structures upstream of molecules, which are intrinsically more diverse in shape, and the var genes (Smargiasso et al. 2009). often exist as single strands (Ji et al. 2011). G4 structures play important roles in nucleic acid processing in both humans and other organisms, which has made G4s an active area of research in recent years. We aim to provide a review that demonstrates the relevance of G4s by describing their im- portance in parasitic infection, viral infection, cancer, and neurodegeneration. We will cover G4 mechanisms of action in these areas, as well as the potential of G4s as targets for therapeutic treatments.

2. Parasites 2.1. History of G4s in parasites Although G4s are now better known for their roles in cancer and viral activity, they were originally characterized in protozoa such as Oxytricha and Tetrahymena (Williamson et al. 1989). Early crystal structures were also determined in Oxytri- cha (Kang et al. 1992). Despite this early interest in protozoan G4s, research in this area decreased when the focus shifted Figure 3. Trypanosoma parasites to G4s in cancer and infectious disease. In recent years, new A) Transcription-on state in mediated by the formation of research into the roles of G4s in parasites has started to re- RNA G4s. B) Replication-on state mediated by the formation surface. The new research has provided further insight into of DNA/RNA hybrid G4s. the action mechanisms of G4s in protozoa, and the potential to exploit these structures in treatments of parasitic protozoa. There is also evidence suggesting that G4s play a role in recombination events in P. falciparum. A strong relationship 2.2. G4 mechanisms of action in parasites has been established between breakpoints and PQSs. A possi- One interesting aspect of protozoan genomes is that some ble mechanism could be that breaks occur when the replica- of them contain a high proportion of A and T nucleotides tion fork is stalled by a G4, and recombination occurs when compared to C and G nucleotides. For example, Plasmodium repair is carried out (Stanton et al. 2016). Parasites of the falciparum, the most severe of the malaria-causing parasites, Trypanosoma genus, implicated in conditions such as sleeping has a genome with an A+T composition of 80.6% (Gardner et sickness (T. brucei) and Chagas disease (T. cruzi), also use G4s al. 2002). However, despite such AT-rich genomes, G4-form- in their genome. Here, G4s are used as a switch to mediate ing sequences have been preserved in P. falciparum as well as transcription and replication. As RNA polymerase synthesiz- several other species (Smargiasso et al. 2009), suggesting that es pre-mRNA, there are two options for G4 formation. The they play an important role in the genome. frst is G4 formation using exclusively the pre-mRNA. This In P. falciparum, 16 putative G-quadruplex sequences (PQSs) leads to RNA editing and continued transcription. The sec- have been identifed upstream of the group B var genes ond is the formation of a DNA/RNA hybrid G4. This blocks (Smargiasso et al. 2009). The var genes encode P. falciparum transcription, allowing replication to occur instead. Making erythrocyte membrane protein 1 (PfEMP1), which is a sur- transcription and replication mutually exclusive allows these face antigen. At any given time, only a single var gene is two processes to be carried out on the circular DNA without being expressed, and switching this expression is a method interfering with each other (Figure 3) (Leeder et al. 2016). used by P. falciparum to evade immunity (Kyes et al. 2007). The Another species in which G4 activity has been investigated presence of PQSs upstream of the group B var genes suggests is Leishmania tarentolae, a parasite that causes leishmaniasis. In that these structures may play a role in regulating the var L. tarentolae, as well as in several other kinetoplastid protists, genes by blocking transcription. P. falciparum encodes a RecQ some T nucleobases in the genome are altered through glu- cosylation and hydroxylation to give base J. The role of base

41 41 J is as a transcription terminator; it has been suggested that CoV) (Kusov et al. 2015). The link with SARS-CoV could be of the absence of base J would cause transcription to continue particular interest because of the recent outbreak of corona- beyond the points where it should be stopped, killing L. taren- virus disease (COVID-19), which is caused by viral infection tolae (van Luenen et al. 2012). The presence of G4s has been with SARS-CoV-2. Considering that both diseases are of the correlated with J insertion, suggesting that G4s may serve as coronavirus family, it may be worth investigating potential a landmark for this process (Genest et al. 2015). Therefore, G4 mechanisms in SARS-CoV-2. targeting G4s in L. tarentolae could be used to kill the parasite by preventing proper transcriptional termination. 3.1. G4s and antigenic variation G4s in viruses have been shown to play a role in antigenic 2.3. G4s as therapeutic targets in parasites variation (Cahoon and Seifert 2009; Kuryavyi et al. 2012), a It can be clearly seen that G4s are implicated in a variety process by which pathogens switch out the antigen-encoding of important processes for several parasitic species, which genes that are being expressed to alter recognizable charac- makes them a potential target for therapeutic purposes. Of- teristics and thus avoid being detected by the immune sys- ten, G4s are targeted with binders composed of a fat, aromat- tem (Domingo et al. 2016). G4-mediated antigenic variation ic core which can stack on top of the terminal guanine tetrad is well modelled by Neisseria gonorrhoeae, where the antigenic in the G4 to provide a stabilizing efect. Several known G4 variation of pilin proteins, which are found on the surface of ligands were tested to determine their stabilization of P. falci- the bacteria, has been linked to a G4 structure (Cahoon and parum G4s. Compounds such as Phen-DC3, Phen-DC6, and Seifert 2009). A G4 motif near the pilE gene forms a parallel telomestatin were shown to have strong stabilization of G4s G4, the structure of which seems to be crucial, as replace- in P. falciparum, which is promising for the use of these com- ment with other G4s resulted in the loss of pilin antigenic pounds as therapeutic tools. However, the G4 binders did not variation (Cahoon and Seifert 2009). This G4 recruits the show strong selectivity for P. falciparum G4s over human G4s. RecA recombinase protein which mediates non- reciprocal This area would need to be further explored to determine if recombination between a silent pilS locus and the pilE locus, selectivity is necessary to achieve efective malaria treatment thus altering the pilin proteins being expressed (Figure 4) without major side efects (De Cian et al. 2008). Drug resis- (Kuryavyi et al. 2012). Understanding the process of antigenic tance is a concern with malaria, so the development of new variation is important because it is a strategy that allows vi- treatment methods would be benefcial to combat this issue. ruses to evade the immune system and therefore continue to More recently, in vitro trials have shown that quarfoxin has proliferate. activity in P. falciparum by suppressing the transcription of a G4-containing gene (Harris et al. 2018). This provides another 3.2. G4s and antigenic diversifcation possible option for G4 targeting in the fght against malaria. Like antigenic variation, antigenic diversifcation is another Using in vitro trials, it has also been found that naphthalene strategy used by viruses to evade the immune system. An- diimide G4 ligands have an antiparasitic efect against the tigenic diversifcation uses processes such as recombination trypanosome T. brucei, as well as showing potential for low and mutation to generate new versions of genes that can then toxicity in humans since the ligands demonstrate selectivity be used in antigenic variation. Using Saccharomyces cerevisiae as for T. brucei G4s over human G4s (Belmonte-Reche et al. 2018). a model, a mechanism has been proposed for how G4s could This shows promise that the challenge of achieving selectivity promote antigenic diversifcation: unresolved G4s can lead for parasite G4s over human G4s can be overcome. to breaks in the genome by causing replication fork stalling and recombination can occur during repair of these breaks 3. Viral infections (Ribeyre et al. 2009). Recombination can create new versions In addition to their role in parasites, G4s have been linked of genes to contribute to the pool of genes used for antigenic to a number of viruses. G4s are present in DNA viruses such variation. Overall, this provides an efective and sustainable as human papillomavirus (HPV) (Tlučková et al. 2013), hu- tactic for avoiding detection by the immune system. man herpesviruses (Biswas et al. 2016), and hepatitis B virus (HBV) (Biswas et al. 2017). They are also present in certain RNA viruses, including hepatitis C virus (HCV) (Wang et al. 2016), Zika (Fleming et al. 2016), Ebola (Krafčíková et al. 2017), and severe acute respiratory syndrome coronavirus (SARS-

42 42 BRACO-19 is a G4 binder that has been studied with several viruses. With HIV-1 it is active against both the replicating and latent life stages of the virus. The activity against latent HIV-1 is especially promising as it could provide an opportunity to fully eliminate the virus in patients. This would prevent the possibility of the virus reactivating, an achievement that is not possible with treatments that only target the active virus (Piekna-Przybylska et al. 2017). Beyond HIV-1, BRA- CO-19 has shown antiviral activity against herpes simplex virus 1 (HSV-1) (Artusi et al. 2015). However, despite the promise shown by BRACO-19, there are barriers that prevent it from being viable for clinical use. It is soluble in water but does not interact easily with lipids, causing difculties with permeability through the phospho- lipid membranes of cells (Taetz et al. 2006). Another more general issue with the use of G4 ligands to target viruses is selectivity for viral G4s over cellular G4s. Many G4 binders, including BRACO-19 (Zhou et al. 2016) and TMPyP4 (Grand et al. 2002), also have potential for anti-cancer applications in humans, which means that they are capable of binding Figure 4. Transcription by RNA polymerase opens dou- to cellular G4s. One route that could be further explored to ble-stranded DNA to allow for G4 formation in a single achieve selectivity for specifc G4s is attaching side arms to strand. This leads to G4 assistance in RecA-catalyzed N. gonor- the aromatic core. Since loops on diferent G4s are distinct rhoeae antigenic variation. from each other, these side arms could be tuned to interact selectively with a single G4 based on its loops. There has 3.3. G4s and viral latency been some encouraging news with respect to achieving partial A third viral strategy that can involve a G4 mechanism is viral selectivity in G4 binders for antiviral activity. The G4 ligand latency. A G-rich sequence in the human immunodefcien- c-exNDI, initially developed to target HIV-1, has been tested cy virus type 1 (HIV-1) can adopt a variety of G4 structures for activity against HSV-1 (Callegaro et al. 2017). It showed se- by forming interactions between diferent G-rich sequences. lectivity for G4s in HSV- 1 over telomeric G4s, which are the It is possible that diferent conformations act as a switch- most abundant cellular G4s. However, it was not selective for ing mechanism to precisely control transcriptional output HSV-1 G4s over the c-Myc and c-kit2 G4s, which are cellular (Piekna-Przybylska et al. 2014). This G4-forming sequence in G4s found in promoter regions. Despite the lack of complete HIV-1 contains three binding sites where interaction with the selectivity, it showed strong antiviral activity and low toxicity. Sp1 transcription factor can occur. Once bound to the G4, This is likely due to a combination of the partial selectivity Sp1 then binds c-Myc, and c-Myc binds histone deacetylase 1 for viral G4s over cellular G4s and the large excess of viral (HDAC1). This forms a DNA- protein complex with the Sp1, G4s compared to cellular G4s during the HSV-1 viral life cy- c-Myc, and HDAC1 proteins to contribute to the maintenance cle (Callegaro et al. 2017). This shows promise that progress is of a state of viral latency (Jiang et al. 2007). G4 activity in the being made in addressing the current barriers preventing the latent stage is of note because it may provide an opportunity clinical use of G4 binders as antiviral therapies. to target latent viruses. 4. Cancer 3.4. G4s as antiviral targets Cancer-related G4s are perhaps the best studied occurrences The relevance of G4 structures for several virulence mecha- of these higher order structures. Generally, G4s are enhanced nisms makes them a desirable target for treatment options. in the promoter and telomeric regions of DNA which are Several known G4 binders have been shown to have promis- both critical regions of our genome that are involved in the ing antiviral activity. For example, TMPyP4 and PDP have development of diferent cancers (Wright et al. 1997; Kham- both been shown to inhibit HCV replication and protein bata-Ford et al. 2003). More recently, the important roles of translation (Wang et al. 2016).

43 43 RNA G4s have emerged, providing another target of study for al. 1997; Blackburn 2001). In normal somatic cells, telomeres therapeutic potential (Agarwala et al. 2015). shorten during progressive cell division acting as a ‘biological clock’ that can trigger cellular senescence (prevention 4.1. G4s in DNA promoter regions of cell division) when they reach a critical length. Cancer G4s are enriched in the promoter regions of DNA, which are cells often develop the ability to bypass this normal process regulatory sequences involved in transcription initiation (Fig- of senescence and thereby become ‘immortal’ with unlimit- ure 5A) (Khambata-Ford et al. 2003). Several bioinformatic ed potential for proliferation. Telomeres frequently contain a studies have been conducted to map out putative quadruplex single stranded G-rich sequence on the 3’ terminus extending sequences (PQSs) that can form G4s (Huppert 2005). Hup- beyond the duplex DNA, known as the G-overhang (Wright pert and Balasubramanian (2007) found that >40% of ‘known’ et al. 1997; Jafri et al. 2016). The G-overhang can loop back to human genes contain one or more PQS in their promoter interact with duplex telomere DNA forming a protective cap region, with a high probability of existing in close proxim- conformation, assisted by various proteins such as telomere ity to the transcription start site, indicating a mechanistic repeat factors (Blackburn 2001). When unfolded and unpro- connection with transcription. Even more relevant is the ele- tected, the G-rich and single-stranded nature of the G-over- vated frequency of PQS in promoters of pre-cancerous genes hang allows G4 structures to form (Figure 5B). Stabilization known as proto-oncogenes, with 69% having at least one of G4s, mediated by small molecule ligands, can interfere PQS, especially those involved in transcription factor activity, with the protective cap conformation and afect the length development, neurogenesis, and kinase activity (Huppert and of the G-overhang to disrupt the function of telomeres and Balasubramanian 2007). Therefore, G4s are heavily involved activate a DNA damage response (Wright et al. 1997; Gomez as regulatory elements in transcription regulation, with spe- et al. 2004; Gomez, O’Donohue, et al. 2006; Gomez, Wenner, cial relevance in cancerous oncogene promoters. et al. 2006). This can lead to the onset of senescence (Gomez, The c-Myc oncogene is well known for its overexpression in Wenner, et al. 2006) or apoptosis (Douarre et al. 2005). a variety of malignancies, including lymphomas, myeloid leukaemias, breast and colon cancers, cervical carcinomas, and osteosarcomas (Facchini and Penn 1998; Simonsson et al. 1998). c-Myc expression is involved in cell proliferation by acting as a transcriptional activator of proliferation-related genes, repressor of growth arrest genes, and also has a role in cell death (Facchini and Penn 1998; Siddiqui-Jain et al. 2002). Thus, dysregulation of c-Myc and resulting oncogenic activity are associated with the progression of cancers. A study by Siddiqui-Jain et al. (2002) indicated that G4s are involved in c-Myc transcriptional repression. They determined that a biologically relevant G4 structure in the c-Myc promoter can be destabilized through point mutations resulting in a three-fold increase in transcriptional activity (Siddiqui-Jain et al. 2002). This suggests that the absence of G4 regulatory elements and Figure 5. A) G4 in promoter regions of DNA. B) The forma- their repressive function can lead to the proliferation of can- tion of G4s in the G-overhang of telomere DNA. cer cells. Thus, G4s in the c-Myc promoter have become an immense topic of study for cancer therapy, with a focus on G4 4.3. G4s in RNA stabilization using small molecules to downregulate c-Myc In addition to the well-studied DNA G4s, these higher or- overexpression. Current research into ligand-mediated G4 der structures have also been characterized in RNA. In fact, stabilization is highlighted in 4.4. RNA G4s are more likely to exist due to the presence of the 2’ hydroxyl group that enhances its stability due to increased 4.2. G4s in DNA telomeres: Telomere Targeting intramolecular interactions (Sacca et al. 2005; Bif et al. 2014; Another signifcant area for G4 related cancer research is Agarwala et al. 2015). They can only form a parallel topol- telomere targeting. Telomeres are repetitive G-rich DNA with ogy, thus reducing the diversity of possible RNA G4 con- associated proteins that cap the ends of eukaryotic chromo- formations, so designing selective binding of ligands is less somes to ofer protection during DNA replication (Wright et challenging. RNA G4s have been studied for their potential

44 44 functions as regulatory elements in translation, mRNA processing, and alternative splicing (Agarwala et al. 2015). For example, Kumari et al. (2008) discovered a highly conserved G4 in the 5’ untranslated region (UTR) in the RNA transcript of the human neuroblastoma RAS (NRAS) pro- to-oncogene, whose oncogenic expression activates signalling pathways responsible for cell proliferation and diferentiation. They also demonstrated that the NRAS RNA G4 modulated translational repression (Kumari et al. 2007). G4-forming motifs exist in the 5’ UTRs of approximately 3000 known human genes (Kumari et al. 2007), so the NRAS RNA G4 Figure 6. Solution-state NMR structure showing top view of may act as a model for other oncogenes, thus providing an Phen-DC3 (blue) bound to the c-Myc promoter G4 (PDB ID: additional approach for potential cancer therapy. Since RNA 2MGN) (Chung et al. 2014). can be found outside of the nucleus, they may also provide an easier target for stabilizing ligands. 5. Neurodegenerative diseases

4.4. G4s in cancer therapy 5.1. G4-related mechanisms of toxicity in C9FTD/ALS Cancer therapy using the G4 targeting approach has mainly Guanine quadruplexes have recently been discovered to play focused on the development of small molecule ligands that an interesting role in neurological diseases through the ex- can stabilize the G4 structures to afect the mechanisms at pansion of G-rich sequences of DNA and RNA. Amyotrophic work in promoters, telomeres, and RNA. Many of these li- lateral sclerosis (ALS) and frontotemporal dementia (FTD) gands have a planar, aromatic core to take advantage of π/π provide the most well-studied illustration of this role. ALS is interactions to stack with terminal G-tetrads. Telomestatin a rapidly progressive neurodegenerative disease that is spo- is an example of a highly potent macrocyclic G4 ligand with radic in 90% of cases and familial in around 5-10% causing selectivity for tumour cells with minimal efect on normal the loss of motor neurons and muscle atrophy (Kiernan et cells (Monchaud et al. 2010). It was originally isolated from al. 2011). There is a large overlap of patients with ALS and the Streptomyces anulatus bacteria (Shin- ya et al. 2001), with FTD, which is the second most common cause of dementia the goal of targeting telomeres to interfere with capping (Go- in the presenile age group (<65 years old), characterised by mez, O’Donohue, et al. 2006; Gomez, Wenner, et al. 2006), but progressive deterioration in behaviour, personality, and lan- was also discovered to act on the c-Myc promoter G4 as well guage, with relative preservation of memory (Rademakers et (Lemarteleur et al. 2004). However, synthesizing telomestatin al. 2012). at an industrial scale provided a challenge, as well as low wa- In 2011, an expansion of the hexanucleotide GGGGCC ter solubility, preventing it from becoming a mainstream can- (G4C2) repeat in the non-coding region of the C9orf72 gene cer treatment (Monchaud et al. 2010). Analogues with similar was found to be the most common genetic cause of FTD/ALS macrocyclic structures have been developed with varying de- and has been considered pathogenic for neurodegeneration grees of success to attempt to mimic telomestatin’s excellent (DeJesus-Hernandez et al. 2011; Renton et al. 2011; Majounie et quadruplex recognition abilities (Ma et al. 2019). Biosynthesis al. 2012). Healthy individuals have less than 25 G4C2 repeats methods are also being researched to improve and simplify in the C9orf72 gene, whereas FTD/ALS patients are estimat- telomestatin production (Amagai et al. 2017). Other ligands ed to have 700-1600 repeat units (DeJesus-Hernandez et al. such as Phen-DC3, a bis-quinolinium derivative, also provide 2011). This G-rich expanded sequence has the ability to form very potent binding capabilities due to extensive π overlap stable G4s in DNA and RNA (DeJesus-Hernandez et al. 2011; with the planar G-tetrad as can be seen in Figure 6 (Piazza Fratta et al. 2012), as well as hairpin structures (Su et al. 2014) et al. 2010). This compound can be used as a ‘benchmark’ for illustrated in Figure 7. G4s have been implicated in two major comparison of novel G4 ligands (Miron and Petitjean 2018). mechanisms for C9FTD/ALS related neurodegeneration: i) RNA toxicity due to the formation of foci that sequester RNA binding proteins, and ii) repeat-associated non-ATG (RAN) translation of dipeptide repeat (DPR) proteins. Interestingly, RNA foci and DPR are rarely observed in the same cells so

45 45 these mechanisms represent two distinct ways the formation of G4s from the G4C2 repeat expansion can generate toxicity (Gendron et al. 2013). Both mechanisms are described herein: i) Stable G4s formed in the expanded C9orf72 DNA can im- pair RNA polymerase processing within this region to stall transcription (Haeusler et al. 2014). During transcription, G4s promote the unwinding of duplex DNA and enhance the formation of DNA:RNA template hybrids, known as R-loops, in the transcription bubble that can result in aborted transcription (Figure 7) (Haeusler et al. 2014; Wang et al. 2015). The accumulation of incomplete RNA transcripts containing the G4C2 repeats can agglomerate into toxic RNA foci that sequester RNA binding proteins and interfere with their function (Donnelly et al. 2013). For example, Haeusler et al. (2014) found that the protein nucleolin is sequestered in the RNA foci through preferential recognition of the RNA G4 motif thus causing nucleolar stress and associated functional defects in C9FTD/ALS patients. Other RNA binding proteins can also be sequestered, thus perturbing their involvement in roles such as transcription regulation, splicing, and RNA transport (Vatovec et al. 2014). G4s play a critical role in the formation of abortive RNA transcripts and the sequestering of specifc RNA binding proteins to produce RNA toxicity in Figure 7. G4s and R-loops formed in the C9orf72 G4C2 re- C9FTD/ALS patients. peat region of DNA leads to the formation of abortive RNA ii) RNA transcripts containing the G4C2 repeat can also es- transcripts which can be involved in the RNA foci and RAN cape the nucleus to be translated through RAN translation translation mechanisms of toxicity in C9ALS/FTD patients. mechanisms (Haeusler et al. 2016). RAN translation does not use the canonical ATG/AUG start codon to initiate transla- 5.2 G4s in C9FTD/ALS therapy tion (Cleary et al. 2018). G4s may play a role in the uncon- To address these C9FTD/ALS mechanisms of toxicity, there ventional translation mechanisms as regulatory elements that are two major treatment strategies being explored: i) antisense repress or promote translation, but not much else is defn- oligonucleotides (ASOs), and ii) small molecule-based ap- itively known about underlying mechanisms (Bugaut and proaches. The antisense mediated intervention approach uses Balasubramanian 2012). RAN translation of the G4C2 sense small chemically modifed single stranded DNA sequences and C4G2 antisense RNA produces DPR proteins. The accu- that are complementary to the target RNA. Binding of the mulation of DPR proteins is a hallmark feature of C9FTD/ ASO to target RNA can trigger mechanisms of RNase-medi- ALS that have been found in neurons and glia throughout ated degradation or mRNA modifcation (Schoch and Mill- the central nervous system (Cleary et al. 2018). They can dis- er 2017). Jiang et al. (2016) have established the potency of rupt the cell through interactions with RNA binding pro- multiple ASOs in mitigating the accumulation of sense RNA teins, nucleoporin proteins, ribosomal proteins, translation foci and certain DPR proteins. Although efective, ASO ther- initiation and elongation factors (Cleary and Ranum 2017). apeutics also have a high cost and require invasive injections Furthermore, Mori et al. (2013) detected insoluble aggregates (Jiang et al. 2016; Schoch and Miller 2017), so other approach- of DPR proteins accumulating in clinically relevant regions es are under investigation. of the brain, including the hippocampus and frontotemporal More specifc to G4 targeting is the use of small molecule neocortex, that can be pathogenic for neurodegeneration. The binders, in a similar manner to proposed cancer treatments. RAN translation of C9orf72 transcribed RNA directly leads Targeting and stabilizing RNA G4s can address both the to DPR pathology, thus preventing this mechanism upstream inhibition of RAN translation and RNA foci formation in at the RNA level providing a potential therapeutic target of C9FTD/ALS. There is also the possibility of specifc recogni- interest. The role of G4s in RAN translation regulation may tion of certain G4 structures with a ligand. In 2018, Simone be an interesting strategy to investigate further. et al. identifed three promising small molecules with simi-

46 46 lar structures containing connected aromatic rings that tar- lenges encountered with achieving specifcity, the poor lipid get and specifcally stabilize the G4C2 repeat G4 RNA. They solubility and thus poor cellular membrane permeability of frst determined preferential stabilization of G4s in the RNA certain G4 binders provides additional complications. These sequence over the DNA sequence, then studied their efect in remaining obstacles must be overcome prior to the clinical vitro using cortical and spinal motor neurons containing the use of G4-targeting drugs, which likely means that these expanded repeat. The three binders reduced the amount of types of drugs won’t be hitting the market in the near future. RNA foci by ~50% in cortical neurons and only two reduced However, with their broad range of potential applications, the burden in motor neurons. There was also a signifcant G4-targeting drugs show great promise and could eventually reduction in the DPR protein, poly(GP), in motor neurons. play an important role in therapeutic treatments. Excitingly, the small molecules were tested in vivo in G4C2 repeat-expressing Drosophila, demonstrating a decrease in References poly(GP) levels and improved survival, with no observable Agarwala, P., Pandey, S. and Maiti, S. (2015) ‘The tale of RNA toxicity in the control group (Simone et al. 2018). This is a G-quadruplex’, Organic & Biomolecular Chemistry, 13(20), pp. promising preliminary step to validate small molecule G4 5570–5585. doi: 10.1039/C4OB02681K. targeting as a viable therapeutic strategy for neurodegen- Amagai, K. et al. (2017) ‘Identifcation of a gene cluster for telomestatin biosynthesis and heterologous expression using a specifc pro- erative diseases. 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