In this study we carried out a genome-wide screen of yeast single deletion strains to better understand the mechanisms of action of TMPyP4, hypothesising that deletion of telomerase-, telomere-, or DNA damage response-associated genes would result in a change in sensitivity to TMPyP4 compared to wild type strains. However, we found no evidence of an over-representation of telomere associated genes amongst the strains found to be most sensitive to TMPyP4, instead observing that genes associated with the pentose phosphate pathway (PPP), the oxidative stress response and tubulin folding demonstrated highest TMPyP4-sensitivity upon deletion.
The PPP plays an important role in both nucleotide production and NADPH generation. However, the pathway is also significant in cancer cell metabolism, through the Warburg effect and the overexpression of a mutant form of the human transketolase (TKTL1) in various cancer cell lines 
. Interestingly there may also exist a link between the oxidative phase of the PPP and the DNA damage response (DDR), through modulation of glucose-6-phosphate dehydrogenase activity by the DDR effector ATM 
. The TMPyP4-sensitivity displayed by ppp
Δ strains in all likelihood stems from a reduction in NADPH-generation. NADPH is a cofactor key for antioxidant function and therefore links the PPP to the oxidative stress response. Consequently, null mutants of PPP genes, including tal1
Δ strains, are sensitive to a wide range of oxidative agents 
. However, there may also be an NADPH-independent role for the PPP in the oxidative stress response, which is proposed to exert its effects through transcriptional alterations 
. In addition to the ppp
Δ strains, we also found several oxidative stress response-linked strains to be sensitive to TMPyP4, including null mutants for CCS1
, YAP1, SOD1
. We also found that a number of TMPyP4-sensitive strains were also sensitive to hydrogen peroxide (H2
). We hypothesise therefore that the sensitivity of the ppp
Δ and sod
Δ strains to TMPyP4 is linked to a deficiency in the oxidative stress response.
It was previously noted through transcriptional studies that oxidative stress-linked genes were upregulated in response to TMPyP4 treatment in human cell lines, which suggested that ROS production is occurring due to the presence of TMPyP4 
. TMPyP4 is a member of the porphyrin family, a group of compounds historically used in photodynamic therapy, wherein reactive oxygen species are produced upon stimulation by light 
. Interestingly, TMPyP4 has also been utilised in the photocleavage of DNA, which may also link to a potential reaction of the DDR 
. The photoreactive property of TMPyP4 therefore provides a potential explanation for our observation that defects in the oxidative stress response cause TMPyP4-sensitivity. Indeed, we found that exposure to light dramatically increased the toxicity of TMPyP4. Our data is supported by a recent study investigating the photodynamic killing of human pathogens using TMPyP4 and exposure to visible light 
. Therefore, we conclude that treatment of S. cerevisiae
with TMPyP4 and exposure to light causes the production of ROS and, interestingly, the PPP is instrumental in protection against the phototoxic effects of the ligand.
Strains deficient in tubulin folding and microtubule formation (cin1
Δ and tub3
Δ) were also found to be TMPyP4-sensitive. Microtubules are targeted by certain anti-cancer drugs, which either inhibit tubulin polymerisation or cause stabilisation of microtubules 
. TMPyP4 does not, as far as we are aware target microtubules; however, it has been demonstrated that TMPyP4, along with other G-quadruplex binding ligands, induces elongated chromosomes incapable of separating in anaphase 
. Difficulties in chromosome segregation may therefore be exacerbated by deletion of key microtubule formation genes, resulting in increased sensitivity to TMPyP4. For that reason, the response of tubulin processing mechanisms to TMPyP4 could be an important area of study with regards to anti-cancer use of TMPyP4.
A study by Hershman et al. (2008) investigated the function of N
-methyl mesoporphyrin (NMM), which selectively binds G-quadruplexes in vitro
at a higher affinity than TMPyP4 
. Similar to the work described here, the authors screened for yeast mutants that enhance or suppress growth inhibition by NMM, finding that deletion of genes related to chromatin remodelling or modification, transcriptional regulation and those impacting upon telomere function led to increased sensitivity to the agent. This contrasts with our findings, dominated by genes related to the oxidative stress response, and suggests that the increased affinity for G-quadruplexes of NMM may make it a more reliable agent to use in the study of G-quadruplexes, at least in yeast.
There may be additional targets for TMPyP4 or effects of TMPyP4 treatment which remain to be identified. For example, Morris et al. recently demonstrated that TMPyP4 also has the ability to unfold G-quadruplexes in RNA and potentially affect translation in eukaryotes 
. Our high-throughput data provides a resource to help identify other intracellular targets of TMPyP4, HU, RHPS4 and H2