DNA Damage: Drug Discovery

Maintenance of genomic stability and its repair are fundamental processes in safeguarding cellular homeostasis. As outlined before, the human genome is permanently attacked by genotoxic and cytotoxic insults from both, endogenous and exogenous resources leading to aberrant DNA structures, thereby impacting genomic stability and, consequently, health and disease states. In order to assure regular cell metabolism, a complex network of biological processes termed the DNA damage repair (DDR) system has evolved over time that prevents genomic destabilization by accumulating DNA lesions. Herein, the oligomeric MRN (Mre11-Rad50-Nbs1) complex functions as a sensitive detector of DNA lesions in the DSB repair leading  to the recruitment and activation of several down-stream signal transducers, including the serine/threonine kinase ATM (ataxia telangiectasia mutated) and the histone acetyltransferase Tip60 (Tat-interactive protein 60) ^{359, 360, 363, 364, 527}. The tumor suppressor P53-binding protein 1 (53BP1) represents a key regulator in this process as it serves as an interaction platform for numerous DSBR proteins (for a review see Mirza-Aghazadeh-Attari et al., 2019 ³⁴¹; Parnier and Boulton, 2014 ³⁴²). It has been shown previously that the association of the tandem breast cancer C-terminal repeats of 53BP1 and the MRN complex stimulates the activity of ATM and further distal signal transducers such as the check point kinase 2 (CHK2) ⁵²⁸. However, 53BP1 is obviously also involved in SSBR via interaction with the RPA complex, the intrinsic detector of DNA SSB, as evidenced by an increased sensitivity of 53BP1-deficient U2-OS osteosarcoma cells towards camptothecin-induced apoptosis ⁵²⁹.  53BP1 encourages non-homologous end-joining (NHEJ) of DNA DSB and blocks homologous recombination (HR) by neutralizing the function of the BRCA1 protein during HR ⁵³⁰. Moreover, depletion of 53BP1 has been found to abolish the ATM-dependent checkpoint response and G2 cell-cycle arrest induced by DNA breaks in Brca1-deficient mouse embryonic stem cells. 53BP1 depletion also partially restored the HR defect of Brca1-deficient cells and reversed their hypersensitivity towards DNA-damaging agents ⁵³⁰. Irregular expression of 53BP1 has been associated with tumor progression and poor prognosis in breast cancer ⁵³¹. Knockdown of 53BP1 in HCT-116 tumor cells has been shown to block apoptosis, promote cell proliferation and enhance accumulation of cells in S phase, coinciding with a reduced expression of γ-H2AX, CHK2 and p53 ⁵³².

siRNA-mediated silencing of 53BP1 revealed a marked reduction in growth and apoptosis in human hepatocellular carcinoma HepG2 cells together with an enhanced cytotoxic effect of doxorubicin, thereby rendering 53BP1 silencing as being a promising therapeutic tool and adjuvant in chemotherapy ⁵³³. The expression level of 53BP1 has an impact on predicting survival of cancer patients, as patients with non-small cell lung carcinoma subjected to first-line platinum-based chemotherapy and expressing low levels of 53BP1 showed an overall survival being more than twice that of those expressing high 53BP1 levels ⁵³⁴. Transfection of ovarian cancer cells with 53BP1 was reported to decrease proliferation and migration of tumor cells in accordance with an increase in DNA repair and chemoresistance ⁵³⁵. In contrast, depletion of 53BP1 stimulated tumor progression in nude mice and enhanced resistance to 5-fluorouracil (5-FU). It also enhanced proliferation, blocked apoptosis and induced S phase arrest in colorectal cancer cells HCT116 and HT29 after 5-FU treatment in vitro ⁵³⁶. These findings imply that knockdown of 53BP1 might be considered as being a negative factor for chemotherapy efficacy, stimulating cell proliferation and suppressing apoptosis. Phosphorylation of 53BP1 induced by the anti-tumor diketopiperazinedisulfide, Glionitrin A, has been described to decrease the tumor volume in a xenograft model of human prostate cancer in an ATM/ATR/Chk1/2-dependent manner ⁵³⁷. Silencing of the oncogenic BRAF-activated noncoding RNA (BANCR) which directly affects the expression of CSE1L, a regulator of 53BP1 and other DNA repair proteins, was recently identified to inhibit tumor growth and ameliorate adriamycin sensitivity in a mouse model of colorectal cancer (CRC) most likely by triggering the miR-203/CSE1L axis, rendering BANCR as being a putative target for CRC treatment ⁵³⁸. According to Mirza-Aghazadeh-Attari et al. (2019), targeting of 53BP1 aiming at disturbing the balance between error-prone NHEJ and error-free HR might also be a suitable approach in anti-cancer therapies ³⁴¹.

Evidence accumulates indicating a pivotal role of 53BP1 in affecting the function of PARP ⁵³⁹. PARP represents an ADP-ribosylating enzyme which is involved in BER and alternative NHEJ. Blockage of PARP increases the formation of DSB that are commonly fixed by HR ⁵⁴⁰. Studies by the group of Sven Rottenberg revealed that depletion of 53BP1 increased resistance to the PARP inhibitor AZD2461 in mouse mammary tumors that are deficient in BRCA1 ⁵⁴¹. Likewise, several other investigations outline that not only 53BP1 depletion but also depletion of down-stream effectors significantly affect PARP inhibitor resistance ^542-544. In BRCA-mutated cancers, PARP inhibitors such as olaparib, niraparib, and rucaparib have been reported to augment apoptosis owing to synthetic lethality ⁵⁴⁰. It is worth noting that in patients with germline BRCA-mutated and Her2-negative advanced breast cancer, treatment with olaparib or talazoparib demonstrated an improved progression-free survival in comparison to standard chemotherapy in phase III clinical trials ^{545, 546}. Owing to these findings, olaparib and talazoparib have been approved by the FDA for the treatment of advanced germline BRCA-mutated breast cancer while olaparib, rucaparib, and niraparib are currently FDA-approved therapeutics in advanced BRCA-mutated and platinum-sensitive ovarian cancer ^{540, 547, 548}. Clinical trials with PARP inhibitors also revealed significant response rates in the majority of patients with prostate cancer (PCa) bearing mutations in either BRCA1/2 or ATM ⁵⁴⁹. Attempts are being made to optimize the PARP inhibitor olaparib in treating this subset of metastatic PCa patients ⁵⁵⁰. In this regard, the combination of immune check point blockade with PARP inhibition has emerged as a promising tool in the treatment of tumors bearing BRCA1/2 or other DDR gene mutations ⁵⁵¹. A further promising option might be the use of low-dose nitric oxide (NO) donors that have been shown previously to inhibit BRCA1 expression under oxidative stress, accompanied by blockage of error-free HR and stimulation of error-prone NHEJ ^{552, 553}. In combination with PARP inhibitors, NO-donors synergistically stimulate cell death in BRCA1/2-normal cancer cells. Recently, Wilson and colleagues demonstrated that application of NO-donors (SNAP, DETA) in combination with the PARP inhibitor ABT-888 represents an efficient approach for the radio-sensitization of BRCA1/2-competent tumors ⁵⁵⁴.

Sir2-related proteins (collectively known as sirtuins) are well-known players in the DNA repair system, regulation of chromatin structure, and preservation of telomere integrity ^{110, 555}. In this context, the nuclear sirtuin SIRT6, a histone H3 lysine 9 deacetylase, has been shown to prevent telomere dysfunction ^{110, 556} and to stimulate PARP-1, thereby amplifying DSBR by HR and NHEJ, respectively ^{110, 557}.  SIRT6-deficient mice develop hypersensitivity to genotoxic stress and genomic instability, culminating in diverse abnormalities associated with age-related pathology ^{110, 558}. Likewise, Sirt1-deficient mice are subjected to embryonic death based on deterioration of DDR and chromosomal abnormalities ^{110, 559}. It is interesting to note that over-expression of SIRT6 protected transgenic mice from age-related metabolic disorders ^{110, 560}. A crucial role of SIRT6 in DNA DSBR has recently been identified by McCord et al. who described the capacity of SIRT6 to stabilize DNA-PK involved in the NHEJ repair pathway ⁵⁶¹. By using SIRT6-knockout cells, Gao and colleagues recently reported on the function of SIRT6 in the regulation of telomere movement upon oxidative stress thereby underlining its crucial role in telomere maintenance ⁵⁶². It is noteworthy that lamin A could be identified as an endogenous activator of SIRT6 and promoter of SIRT6-mediated DNA DSB repair via mono-ADP ribosylation of PARP-1 ⁵⁶³. Similar to SIRT6, SIRT1 obviously affects the activity of PARP-1 and vice versa since both molecules utilize NAD⁺ for catalysis ⁵⁶⁴. This is supported by the fact that PARP1-deficient mice display up-regulated energy consumption, diminished fat mass and augmented glucose clearance due to increased activity of SIRT1 in skeletal muscle and brown adipose tissue and are consequently protected against metabolic disease ⁵⁶⁵. Furthermore, the pharmacological blockage of PARP in vitro and in vivo up-regulates NAD⁺ levels and SIRT1 activity and augments oxidative metabolism ⁵⁶⁵. From these data it can be concluded that PARP-1 blockage has considerable metabolic influence via triggering SIRT1 functionality and could be effective in the management of metabolic diseases and also cancer.

Various clinical trials are appraising the benefit of a combinatorial treatment of DSBR protein inhibitors and immunotherapy, DNA-damaging agents, topoisomerase inhibitors, or chemotherapy (reviewed by Lama-Sherpa and Shevde, 2020) ⁵⁶⁶. These trials encompass, amongst others, PARP inhibitors in combination with PD-1 inhibitors and topoisomerase inhibitors as well as approaches in targeting ATM, ATR, and CHK together with PARP inhibitors. Combinatorial treatments of ATR inhibitors and PD-1 inhibitors or chemotherapy are also being analyzed. The cyclin-dependent kinase (CDK) inhibitor dinaciclib is known to inhibit the phosphorylation of BRCA1 and Exo1 and has been described to impair HR and sensitize multiple myeloma (MM) cells to the PARP inhibitor, veliparib ⁵⁶⁷. Furthermore, co-treatment of MM cells with dinaciclib and veliparib delayed growth of MM xenografts in SCID mice ⁵⁶⁷. A phase I clinical trial is ongoing to evaluate the safety and tolerability of veliparib (ABT-888) and dinaciclib (SCH727965) in patients with advanced solid tumors. A phase II trial of cediranib, a vascular endothelial growth factor receptor (VEGFR-1,-2,-3) tyrosine kinase inhibitor, in combination with the PARP inhibitor olaparib demonstrated enhanced progression-free survival compared to olaparib alone in women with recurrent platinum-sensitive ovarian cancer ⁵⁶⁸. Cediranib in combination with olaparib is currently being evaluated in a phase II clinical trial in metastatic castration-resistant prostate cancer. Preclinical investigations aiming at analyzing the efficacy of small-molecule inhibitors of ATM, ATR, CHK1, CHK2 and WEE1 also reported encouraging results ⁵⁶⁹. A growing body of evidence indicates a synergistic action of immune check point blockage and PARP inhibition in HR-deficient tumors, and this assumption is currently being evaluated in clinical trials.

 

Ionizing Radiation

Since the discovery of X-rays in 1895 and nuclear radiations in 1896, low-dose ionizing radiation (IR) has emerged as an effective local anti-cancer approach. However, radiotherapy represents a two-edged sword as sub-lethal doses of IR not only induce a nuclear DNA damage response but also facilitate a cellular damage response in tumors via activation of pro-inflammatory pathways predominantly mediated through activation of NF-κB, the central connector between inflammation, carcinogenesis, and radioresistance. IR up-regulates the expression of various immediate early genes ⁵⁷⁰, ICAM-1 ⁵⁷¹, PGHS-2 ⁵⁷², IL-1 ⁵⁷³, IL-6 ^573-576, IL-8 ^{574, 575}, and TNF-α ⁵⁷⁷, and stimulates receptor tyrosine kinase pathways ⁵⁷⁸ as well as mitochondria-associated responses ⁵⁷⁹. Moreover, IR has also been found to activate plasma membrane receptors by inducing ionizing events in the liquid phase of the cytosol that lead to the generation of large amounts of mtROS and mtRNS which in turn suppress protein tyrosine phosphatases (PTPases) ⁵⁸⁰. Suppression of PTPases results in the activation of non-receptor and receptor tyrosine kinases (RTK) such as EGFR and the activation of down-stream signal transduction pathways ^{581, 582}. IR also activates acidic sphingomyelinase and increases the production of ceramide which promotes activation of certain membrane receptors by facilitating the clustering of receptors within lipid rafts ^{583, 584}. Activated RTK in turn induce down-stream pro-survival pathways (e.g., Akt) that might be considered as being potential targets in increasing radiosensitivity of tumors.

Sub-lethal doses of IR are able to eradicate tumor cells and cells of the tumor microenvironment such as endothelial cells and tumor-induced suppressor T cells ⁵⁸⁵. Already in the 1990s, non-lethal IR has been described to promote anti-tumor T cell responses and up-regulate MHC class I/II expression in cancer cells via tumor-specific antigen presentation by dendritic cells, rendering the tumor cells more sensitive to T cell recognition ^{586, 587}. Meanwhile, evidence increased to demonstrate that IR exerts immune stimulatory functions, leading to the recruitment of immune mediators that allow for anti-tumor responses within and outside the radiation field. As summarized by Kang et al., there has been a rapid amplification in the number of clinical trials evaluating the potential of IR to stimulate anti-tumor immunity ⁵⁸⁸. The authors provide an interesting overview of clinical trials combining radiation with various immunotherapies including immune checkpoint blockade, adoptive T cell transfer, cytokine therapy, dendritic cell and peptide vaccines, as well as monoclonal antibody treatment. The combination of immunotherapies with IR has potential to synergistically improve the outcome of IR ⁵⁸⁸.

As discussed above, exposure to IR leads to activation of several transcription factors modulating the expression of numerous factors promoting cancer development. Novel therapeutic approaches thus aim to interfere with the activity or expression of these factors, either in single-agent or combinatorial treatment or as supplements of the existing therapeutic concepts. A broad spectrum of classical or novel drugs such as nutraceuticals have the capacity to interfere with the inflammatory network in cancer and are considered to function as putative radiosensitizers ¹⁰⁴. Consequently, targeting the IR-induced inflammatory signaling pathways offers the opportunity to improve the clinical outcome of radiation therapy by up-regulating radiosensitivity and diminishing putative metabolic effects. Radiosensitizers are promising agents because they enhance injury to tumor tissues by producing free reactive radicals and augmenting DNA damage. Several procedures have been conducted to establish highly efficient and low-toxicity radiosensitizers (for a review see Wang et al., 2018) ⁵⁸⁹.

 

DNA-Damaging Agents in Anti-Cancer Therapy

Most conventional chemo- and radiotherapeutic agents are DNA-damaging agents that kill tumor cells in patients during anti-cancer therapy by forming DNA adducts and breaks, thereby generating replication blocks, and interrupting the ‘untangling’ of DNA during nuclear episodes. DNA-damaging agents in anti-cancer therapies comprise various chemical and physical agents such as cytostatics and ionizing radiation, respectively. The beta-chloroethyl amines or nitrogen mustards were the very first chemotherapeutic drug used as early as 1942 in the treatment of a variety of neoplastic diseases ^{590, 591}. Rapidly replicating cells such as cancer cells are highly susceptible to genotoxic agents in contrast to normal cells, thereby constituting a chemotherapeutic aperture.

 

Anthracycline

Anthracycline and its derivatives (e.g., daunorubicin, doxorubicin, epirubicin, and idarubicin) block DNA and RNA synthesis by intercalating between base pairs ⁵⁹² and also suppress the activity of topoisomerase II (Topo II), crucially involved in transcription and replication of DNA ⁵⁹³. In DNA, anthracyclines induce the formation of DSB followed by the activation of the DNA DSBR which, if left unrepaired, induces apoptosis ⁵⁹⁴. In addition, anthracyclines stimulate the production of mtROS and consequently boost oxidative stress in cells by targeting enzymes involved in redox pathways ⁵⁹⁵. Daunomycin, also termed adriamycin and isolated from Streptomyces peucetius var. caesius, was about the first anthracycline whose anti-tumor activity had been observed in the 1960s ⁵⁹⁶. Other Topo II inhibitors have also been developed, including doxorubicin and its analogs daunorubicin, idarubicin, epirubicin, valrubicin and aclarubicin as well as structurally unrelated drugs such as etoposide. These drugs have been widely used in clinical trials worldwide to explore better combinations. Anthracyclines are frequently utilized as first-line treatment of breast cancer, soft tissue sarcomas, leukemias, and other carcinomas. They may be utilized in monotherapy or combinatorial therapy together with further anti-tumor agents such as bleomycin and vincristine ¹. Since anthracyclines exhibit serious side effects including cardiotoxicity, anthracycline derivatives with lower cardiotoxicity were developed, e.g., mitoxantrone. Mitoxantrone represents an anthracenedione which serves as a Topo II inhibitor and induces the formation of DNA DSB albeit harboring a minor ROS-forming and DNA intercalating capacity ⁵⁹⁷. It has been approved for the treatment of advanced prostate cancer not responding to hormone treatment – used in combination with steroids, acute myelogenous leukemia (AML), beast cancer and Non-Hodgkin’s lymphoma. Since mitoxantrone also possesses potent immunomodulatory activities, it has been approved for the treatment of aggressive forms of multiple sclerosis ^{598, 599}.

 

Alkylating agents

Alkylating agents constitute a heterogenous group of anti-cancer drugs which can be divided into classical and non-classical alkylating agents. While classical alkylating agents directly alkylate DNA bases, the non-classical alkylating agents alkylate DNA bases indirectly after prior metabolization. Consequently, alkylated bases cause conformational rearrangements in DNA that block DNA replication. A major subgroup is represented by nitrogen mustards which gained questionable fame as mustard gas in World War I and II. The mustards have been characterized by the presence of two reactive moieties enabling the interaction with two distinct sites in the DNA. These bifunctional activities culminate in the formation of intra- and interstrand crosslinks on the one hand and, on the other hand, in the generation of DNA-protein crosslinks that inhibit DNA reactivity ^{28, 29}. Some of the most potent and widely used anti-cancer compounds comprise cyclophosphamide ³⁰, dacarbazine ³¹ and cisplatin ³²; all of them working by alkylation of DNA. Cyclophosphamide is frequently used as a first-line treatment of hematological malignancies including leukemia and lymphoma ⁶⁰⁰, and also in chemotherapeutic strategies against solid tumors such as breast cancer ⁶⁰¹. However, cyclophosphamide commonly showes a high toxicity which limits its clinical use. Side effects include hemorrhagic cystitis, bladder cancer, bone marrow suppression, alopecia, and gonadal failure ⁶⁰². In order to decrease the symptoms of haemorrhagic cystitis, the drug is therefore most frequently applied in conjunction with 2-mercaptoethane sulfonate, ¹. Beside its use in chemotherapy, cyclophosphamide is applicated for immunosuppressive therapy against, e.g., SLE ⁶⁰³.

Non-classical alkylating agents comprise, for instance, altretamine, dacarbazine, and procarbazine. These are generally used in combinatorial administration with further anti-cancer drugs. Procarbazine was first synthesized in the late 1950s in a search for a new monoamine oxidase inhibitor, but was promptly established as an anti-cancer agent ^{604, 605}. Procarbazine has been widely utilized in the treatment of a number of cancers including Hodgkin’s lymphomas, brain tumors, multiple myeloma, primary central nervous system lymphoma, malignant melanoma, and lung cancer. Dacarbazine is the single FDA-approved anti-cancer drug, now utilized as a single-agent drug against metastatic melanoma cancer ^{31, 606}.

 

Radiomimetics

Radiomimetics, such as neocarzinostatin, bleomycin and other enediynes, are a group of anti-cancer agents which boost the intracellular production of ROS, thus introducing DSB in DNA ^607-609. Amongst them, anti-tumor enediyne C-1027 has been found to introduce not only DNA strand breaks but also inter-strand crosslinks (ICLs) into cells ⁶¹⁰. Interestingly, an analog of C-1027, 20′-deschloro-C-1027, has been shown to induce inter-strand DNA crosslinks in hypoxic cells and to overcome cytotoxic radioresistance ⁶¹¹. From these findings it can be concluded that rational engineering of the C-1027 family of radiomimetics holds promise toward overcoming the radioresistance associated with hypoxia in solid tumors. The cancer chemotherapeutic drug, bleomycin, is clinically used to treat several neoplasms including testicular and ovarian cancers ^{612, 613}, Hodgkin’s lymphoma and carcinomas of skin, head, and neck ⁶¹⁴. Bleomycin exerts cytotoxicity through its ability to cause DNA SSB and DSB, culminating in extended cell cycle arrest, apoptosis, and cell death ⁶¹⁵. Some inherited disorders such as ataxia telangiectasia and Nijmegen breakage syndrome have been reported as being highly sensitive towards radiomimetics and ionizing radiation ⁶¹⁶. An enhanced susceptibility towards bleomycin has also been associated with a polymorphism in the nucleotide excision repair gene XPC correlating with bleomycin-induced chromosomal aberrations ⁶¹⁷.

 

DNA adduct forming agents

Agents forming bulky DNA adducts comprise platinum-based anti-tumor drugs such as, for instance, cisplatin, carboplatin, oxaliplatin, picoplatin, satraplatin, and triplatin ⁶¹⁸. Platinum compounds were first described in the 1960s by the fundamental work of Rosenberg and collaborators who observed that platinum electrodes were able to block cell division of E. coli ⁶¹⁹ and, later on, that the platinum compound LD50 (cisplatin) diminishes tumor growth in mice ⁶²⁰. Cisplatin represents the first FDA-approved platinum compound for the treatment of cancer in 1978 ⁶²¹. Platinum-based anti-tumor drugs are now among the most widely used and most potent anti-cancer therapeutics, either in single-agent or combination therapy ⁶²². However, their clinical success is restricted due to serious adverse effects and intrinsic or acquired treatment resistance. The most common side effects include nephrotoxicity, neurotoxicity, cardiotoxicity and gastrointestinal disturbances such as nausea and vomiting. Owing to these considerable adverse effects and drug resistance in particular ⁶²³, combinatorial administration of cisplatin with other cancer drugs has been applied as novel therapeutic strategy for many human cancers. Adverse effects are much lower in second-generation platinum compounds such as carboplatin which is more stable than first-generation cisplatin ^{624, 625}. Picoplatin represents a novel platinum-based agent which resolves resistance to cytotoxicity in cancer cells treated with cisplatin and carboplatin, respectively ⁶²⁶. Substantial efforts have been made to develop novel platinum compounds, but only a few of them are undergoing clinical trials or got approval in individual countries such as nedaplatin, lobaplatin and heptaplatin ⁶²⁷. Anti-neoplastic platinum agents are often used as first-line treatment for a broad spectrum of neoplasms including haematological malignancies as well as solid tumors such as ovarian, testicular, lung and bladder cancers ^{32, 622, 627}.

Aziridine derivatives constitute a further group of chemotherapeutics that introduce crosslinks in DNA.  The most prominent members are mitomycin C (MMC) and thiotepa, the latter preferentially being used in pre-transplantation conditioning systems in haematological malignancies ⁶²⁸. Most recently, adjuvant high-dose thiotepa failed to improve overall survival and progression-free survival in patients with relapsed osteosarcomas in a phase II clinical trial ⁶²⁹, thus further restricting its clinical applicability. As single-dose perioperative agents, thiotepa as well as MMC have been found as being associated with similar recurrence-free survival rates in patients with low-grade non-invasive bladder cancer ⁶³⁰. MMC itself has been shown to reduce the recurrence risk significantly in patients with non-muscle invasive bladder cancer (NMIBC) after immediate single intravesical instillation ⁶³¹. A combination of chemohyperthermia and MMC turned out to represent a reliable option for the management of intermediate and high-risk NMIBC, harboring merely low-grade adverse effects ⁶³². It is noteworthy that LiCl increases MMC-induced apoptosis and expression of HMGB1, Bax and Bcl-2 in MDA-MB-231 breast cancer cells which might be of interest as an adjuvant therapy in anti-cancer approaches ⁶³². In a retrospective analysis, intra-arterial infusion of MMC has been reported as being safe and effective in patients with advanced liver metastatic breast cancer ⁶³³. A single-center pilot study currently revealed that sequential treatment of intra-arterial infusion of MMC after selective internal radiation therapy (SIRT) using Yttrium-90 (90Y) resin microspheres was feasible in three quarters of patients with liver metastatic breast cancer ⁶³⁴. MMC is FDA-approved for the treatment of gastric cancer and pancreatic adenocarcinoma alone or in combination with other chemotherapeutics.

 

Nucleotide and nucleoside analogs

Nucleotide analogs comprise modified purine and pyrimidine nucleotides that function as inhibitors of nucleotide biosynthesis. Purine analogs such as methotrexate and pemetrexed block the activity of dihydrofolate reductase (DHFR) which catalyzes the synthesis of the co-enzyme tetrahydrofolate crucially involved in the biosynthesis of purines and thymidine ⁶³⁵. Inhibition of DHFR leads to nucleotide deficiency and ultimately blockage of nucleic acid synthesis. Pemetrexed constitutes a multifunctional enzyme inhibitor as it not only inhibits DHFR but also thymidylate synthase, and the aminoimidazole carboxamide ribonucleotide formyltransferase (AICART) ⁶³⁶. Inhibition of AICART activates the AMP-activated protein kinase followed by an inhibition of the mammalian target of rapamycin (mTOR) and hypophosphorylation of down-stream mTOR targets thereby regulating protein synthesis and cell growth ⁶³⁶. Azathioprine, 6-mercaptopurine, and thioguanine are further representatives of purine analogs, in particular the two latters are substrates for the hypoxanthine-guanine phosphoribosyltransferase and are converted into the ribonucleotides 6-thioguanosine monophosphate (6-thioGMP) and 6-thioinosine monophosphate (T-IMP), respectively. The agglomeration of these nucleotides affects various vital cell functions.

5-FU, capecitabine, cytarabine, gemcitabine are representatives of pyrimidine analogs amongst which gemcitabine acts as an inhibitor of ribonucleotide reductase (RNR). RNR mediates the synthesis of deoxyribonucleotides from ribonucleotides, and its inhibition impedes DNA transcription and repair, resulting in cell apoptosis ^{637, 638}. Gemcitabine is a very active drug against solid tumors while azathioprine and methotrexate are commonly used as immunosuppressants ⁶³⁹. Gemcitabine has been approved by the FDA for the treatment of breast cancer and ovarian cancer as well as non-small cell lung cancer, pancreatic cancer, and bladder cancer.

An exciting approach in epigenetic reprogramming of cancer cells is targeting DNA hypermethylation using nucleoside analogs. Azacitidine, decitabine and FdCyd are cytosine analogs which are intracellularly metabolized and incorporated into DNA ⁶⁴⁰. DNA methyltransferases are able to interact with these modified nucleotides but their modification avoids their methylation. Nucleotide modification additionally inhibits the dissociation of the enzyme thus reducing its activity at distinct sites ⁶⁴⁰. Collectively, nucleoside analogs serve as DNA methyltransferase inhibitors that inhibit proliferation, increase cell differentiation, induce immune recognition, and ultimately cancer cell death ⁶⁴¹. DNA methyltransferase inhibitors have been approved for the treatment of myelodysplastic syndromes, chronic myelomonocytic leukemia, and acute myelogenous leukemia. In order to enhance clinical efficiency and perturb drug resistance, second-generation DNA methyltransferase inhibitors have been developed and are currently evaluated in clinical trials ^{642, 643}.