This study has identified novel genomic targets of the small molecule pyridostatin through the use of ChIP-Seq, thereby providing a new, unbiased approach that can be employed for identifying druggable targets of other potential therapeutic agents acting at the DNA level. We have shown that pyridostatin generates DNA damage at specific genomic loci, leading to cell cycle arrest and transcriptional down-regulation of several genes that contain PQS clusters on both of their DNA strands. Our data are consistent with the compound mediating these events by interacting with multiple G-quadruplex motifs in gene bodies during transcription and replication, thereby preventing subsequent gene expression from the affected loci. Whether the drug exerts its effects strictly through producing DNA damage and/or acts as a physical barrier to transcription and replication, however, remains to be determined. Recent physical data have shown that pyridostatin stabilizes G-quadruplexes with mechanical forces able to withstand the load forces generated by RNA and DNA polymerases40
. In light of this and given the requirement for dynamic events such as transcription and replication for pyridostatin to yield DNA damage, it is possible that pyridostatin stalls polymerases during transcription and replication. These processes could in turn result in DNA breakage through physical forces imposed on the DNA substrate. In addition, DNA damage production in response to the small molecule could be mediated by the action of endonucleases, perhaps through mechanisms linked to transcription-coupled-repair poisoning as shown previously41
for the anticancer drug ecteinascidin 743.
Our use of the copper-catalyzed Huisgen reaction to fluorescently tag the drug in cells has allowed us to evaluate the cellular localization of the drug, thereby highlighting how this novel methodology could be used to trace and assess the distribution of any small molecule in cells. In our system, this approach has provided evidence for G-quadruplex structures naturally occurring in unperturbed human cells. The characteristic staining-pattern we observed for hPif1 in the absence of pyridostatin treatment, and the overlap of hPif1 staining with the labelled small molecule further demonstrated the existence of G-quadruplexes in unperturbed cells, and also implicated hPif1 in resolving these secondary structures that are known to be difficult to transcribe and replicate.
A key finding from our work is that, while pyridostatin can target telomeric loci, its most prevalent sites of genomic interaction are non-telomeric at low concentrations. Although we found that genes containing high PQS contents are more likely to be affected by the small molecule, and despite all the genes affected having higher than average PQS frequencies, not all genes with high PQS levels were demonstrably targeted by the compound in our assays. For example, we did not detect any effect on HRAS
expression, even though HRAS
contains one of the highest number of PQS of any human gene. Thus, in addition to there being a requirement for alternative DNA structure formation, additional mechanisms must impact on G-quadruplex folding and/or on the binding of the compound to certain G-quadruplexes. For instance, the local supercoiled nature of DNA at a particular locus is likely to modulate the dynamics of G-quadruplex folding and G-quadruplex interactions with pyridostatin, as previously reported42
. Since we determined that pyridostatin acts during both transcription and replication, it is tempting to speculate that mechanisms regulating these processes might impact G-quadruplex dynamics and small molecule binding. Indeed, as double stranded DNA becomes transiently open during transcription and replication, PQS are prone to form G-quadruplexes during these processes. Consequently, the rate of transcription or replication through a particular DNA locus and/or changes in chromatin structure triggered by such events could have a marked impact on the ability of PQS in the locus to form G-quadruplex structures that can then be targeted. The propensity of PQS to form G-quadruplex structures could also depend on whether they are on the transcribed or non-transcribed strand of a gene. Similarly, whether a PQS is replicated by leading- or lagging-DNA strand synthesis could affect its propensity to form G-quadruplex structures and pyridostatin targeting43
. Our studies have provided a framework upon which future work can generate more accurate predictors of whether or not particular PQS form G-quadruplex structures in vivo
and what determines the druggability of these structures in cells.
It will clearly be of interest to explore whether pyridostatin affects the activities of proteins that operate on G-quadruplex structures and, conversely, whether the actions of such proteins influence the targeting of the small molecule to certain PQS regions. In this regard, we note that DNA helicases have been proposed to regulate G-quadruplex formation and processing because these enzymes are known to catalyze the unwinding of duplex DNA. For example, DNA helicases including hPif1, BLM, WRN and FANCJ can unwind G-quadruplex motifs in vitro28,44-46
, while the ATR-X helicase interacts with PQS clusters and has been linked to transcriptional regulation of genes containing these sequences47
. By establishing a genome-wide map of pyridostatin target sites, our work provides a basis for further defining the molecular mechanisms and consequences of G-quadruplex binding by these and other cellular proteins. Our findings will also facilitate future studies assessing how these enzymes might influence G-quadruplex formation and thereby affect these structures during transcription, replication and potentially DNA damage signalling and repair. Finally, our results highlight the potential druggability of G-quadruplex structures and suggest how pyridostatin, as well as other compounds with similar modes of action, could be exploited as tools for genomic studies and for therapeutic benefit. In particular, the observation that this small molecule can selectively down-regulate the proto-oncogene SRC
and induce DNA damage suggests that pyridostatin and its derivatives could exhibit potential as anticancer agents.