Connecting dots between nucleotide biosynthesis and DNA lesion repair/bypass in cancer.

Purine and pyrimidine nucleotides are crucial building blocks for the survival of cells, and there are layers of pathways to make sure a stable supply of them including de novo nucleotide biosynthesis. Fast-growing cells including cancer cells have high demand for nucleotide, and they highly utilize the nucleotide biosynthesis pathways. Due to the nature of the fast-growing cells, they tend to make more errors in replication compared with the normal cells. Naturally, DNA repair and DNA lesion bypass are heavily employed in cancer cells to ensure fidelity and completion of the replication without stalling. There have been a lot of drugs targeting cancer that mimic the chemical structures of the nucleobase, nucleoside, and nucleotides, and the resistance toward those drugs is a serious problem. Herein, we have reviewed some of the representative nucleotide analog anticancer agents such as 5-fluorouracil, specifically their mechanism of action and resistance is discussed. Also, we have chosen several enzymes in nucleotide biosynthesis, DNA repair, and DNA lesion bypass, and we have discussed the known and potential roles of these enzymes in maintaining genomic fidelity and cancer chemotherapy.

Novel insights into the role of translesion synthesis polymerase in DNA incorporation and bypass of 5-fluorouracil in colorectal cancer.

5-Fluorouracil (5-FU) is the first-line chemotherapeutic agent in colorectal cancer, and resistance to 5-FU easily emerges. One of the mechanisms of drug action and resistance of 5-FU is through DNA incorporation. Our quantitative reverse-transcription PCR data showed that one of the translesion synthesis (TLS) DNA polymerases, DNA polymerase η (polη), was upregulated within 72 h upon 5-FU administration at 1 and 10 µM, indicating that polη is one of the first responding polymerases, and the only TLS polymerase, upon the 5-FU treatment to incorporate 5-FU into DNA. Our kinetic studies revealed that 5-fluoro-2'-deoxyuridine triphosphate (5FdUTP) was incorporated across dA 41 and 28 times more efficiently than across dG and across inosine, respectively, by polη indicating that the mutagenicity of 5-FU incorporation is higher in the presence of inosine and that DNA lesions could lead to more mutagenic incorporation of 5-FU. Our polη crystal structures complexed with DNA and 5FdUTP revealed that dA:5FdUTP base pair is like dA:dTTP in the active site of polη, while 5FdUTP adopted 4-enol tautomer in the base pairs with dG and HX increasing the insertion efficiency compared to dG:dTTP for the incorrect insertions. These studies confirm that polη engages in the DNA incorporation and bypass of 5-FU.

Effects of N7-Alkylguanine Conformation and Metal Cofactors on the Translesion Synthesis by Human DNA Polymerase η.

Non-enzymatic alkylation on DNA often generates N7-alkyl-2'-deoxyguanosine (N7alkylG) adducts as major lesions. N7alkylG adducts significantly block replicative DNA polymerases and can be bypassed by translesion synthesis (TLS) polymerases such as polymerase η (polη). To gain insights into the bypass of N7alkylG by TLS polymerases, we conducted kinetic and structural studies of polη catalyzing across N7BnG, a genotoxic lesion generated by the carcinogenic N-nitrosobenzylmethylamine. The presence of templating N7BnG in the polη catalytic site decreased the replication fidelity by ∼9-fold, highlighting the promutagenicity of N7BnG. The catalytic efficiency for dCTP incorporation opposite N7BnG decreased ∼22-fold and ∼7-fold compared to the incorporation opposite undamaged guanine in the presence of Mg2+ and Mn2+, respectively. A crystal structure of the complexes grown with polη, templating N7BnG, incoming dCTP, and Mg2+ ions showed the lack of the incoming nucleotide and metal cofactors in the polη catalytic site. Interestingly, the templating N7BnG adopted a syn conformation, which has not been observed in the published N7alkylG structures. The preferential formation of syn-N7BnG conformation at the templating site may deter the binding of an incoming dCTP, causing the inefficient bypass by polη. In contrast, the use of Mn2+ in place of Mg2+ in co-crystallization yielded a ternary complex displaying an anti-N7BnG:dCTP base pair and catalytic metal ions, which would be a close mimic of a catalytically competent state. We conclude that certain bulky N7-alkylG lesions can slow TLS polymerase-mediated bypass by adopting a catalytically unfavorable syn conformation in the replicating base pair site.

Translesion synthesis of the major nitrogen mustard-induced DNA lesion by human DNA polymerase η.

Nitrogen mustards are among the first modern anticancer chemotherapeutics that are still widely used as non-specific anticancer alkylating agents. While the mechanism of action of mustard drugs involves the generation of DNA interstrand cross-links, the predominant lesions produced by these drugs are nitrogen half-mustard-N7-dG (NHMG) adducts. The bulky major groove lesion NHMG, if left unrepaired, can be bypassed by translesion synthesis (TLS) DNA polymerases. However, studies of the TLS past NHMG have not been reported so far. Here, we present the first synthesis of an oligonucleotide containing a site-specific NHMG. We also report kinetic and structural characterization of human DNA polymerase η (polη) bypassing NHMG. The templating NHMG slows dCTP incorporation ∼130-fold, while it increases the misincorporation frequency ∼10-30-fold, highlighting the promutagenic nature of NHMG. A crystal structure of polη incorporating dCTP opposite NHMG shows a Watson-Crick NHMG:dCTP base pair with a large propeller twist angle. The nitrogen half-mustard moiety fits snugly into an open cleft created by the Arg61-Trp64 loop of polη, suggesting a role of the Arg61-Trp64 loop in accommodating bulky major groove adducts during lesion bypass. Overall, our results presented here to provide first insights into the TLS of the major DNA adduct formed by nitrogen mustard drugs.

The structure of importin α and the nuclear localization peptide of ChREBP, and small compound inhibitors of ChREBP-importin α interactions.

The carbohydrate response element binding protein (ChREBP) is a glucose-responsive transcription factor that plays a critical role in glucose-mediated induction of genes involved in hepatic glycolysis and lipogenesis. In response to fluctuating blood glucose levels ChREBP activity is regulated mainly by nucleocytoplasmic shuttling of ChREBP. Under high glucose ChREBP binds to importin α and importin ß and translocates into the nucleus to initiate transcription. We have previously shown that the nuclear localization signal site (NLS) for ChREBP is bipartite with the NLS extending from Arg158 to Lys190. Here, we report the 2.5 Šcrystal structure of the ChREBP-NLS peptide bound to importin α. The structure revealed that the NLS binding is monopartite, with the amino acid residues K171RRI174 from the ChREBP-NLS interacting with ARM2-ARM5 on importin α. We discovered that importin α also binds to the primary binding site of the 14-3-3 proteins with high affinity, which suggests that both importin α and 14-3-3 are each competing with the other for this broad-binding region (residues 117-196) on ChREBP. We screened a small compound library and identified two novel compounds that inhibit the ChREBP-NLS/importin α interaction, nuclear localization, and transcription activities of ChREBP. These candidate molecules support developing inhibitors of ChREBP that may be useful in treatment of obesity and the associated diseases.

Copper metallothioneins.

Metallothioneins (MTs) are a class of low molecular weight and cysteine-rich metal binding proteins present in all the branches of the tree of life. MTs efficiently bind with high affinity several essential and toxic divalent and monovalent transition metals by forming characteristic polynuclear metal-thiolate clusters within their structure. MTs fulfil multiple biological functions related to their metal binding properties, with essential roles in both Zn(II) and Cu(I) homeostasis as well as metal detoxification. Depending on the organism considered, the primary sequence, and the specific physiological and metabolic status, Cu(I)-bound MT isoforms have been isolated, and their chemistry and biology characterized. Besides the recognized role in the biochemistry of divalent metals, it is becoming evident that unique biological functions in selectively controlling copper levels, its reactivity as well as copper-mediated biochemical processes have evolved in some members of the MT superfamily. Selected examples are reviewed to highlight the peculiar chemical properties and biological functions of copper MTs. © 2016 IUBMB Life, 69(4):236-245, 2017.

Metabolite Regulation of Nuclear Localization of Carbohydrate-response Element-binding Protein (ChREBP): ROLE OF AMP AS AN ALLOSTERIC INHIBITOR.

The carbohydrate-response element-binding protein (ChREBP) is a glucose-responsive transcription factor that plays an essential role in converting excess carbohydrate to fat storage in the liver. In response to glucose levels, ChREBP is regulated by nuclear/cytosol trafficking via interaction with 14-3-3 proteins, CRM-1 (exportin-1 or XPO-1), or importins. Nuclear localization of ChREBP was rapidly inhibited when incubated in branched-chain α-ketoacids, saturated and unsaturated fatty acids, or 5-aminoimidazole-4-carboxamide ribonucleotide. Here, we discovered that protein-free extracts of high fat-fed livers contained, in addition to ketone bodies, a new metabolite, identified as AMP, which specifically activates the interaction between ChREBP and 14-3-3. The crystal structure showed that AMP binds directly to the N terminus of ChREBP-α2 helix. Our results suggest that AMP inhibits the nuclear localization of ChREBP through an allosteric activation of ChREBP/14-3-3 interactions and not by activation of AMPK. AMP and ketone bodies together can therefore inhibit lipogenesis by restricting localization of ChREBP to the cytoplasm during periods of ketosis.

The identification of novel Mycobacterium tuberculosis DHFR inhibitors and the investigation of their binding preferences by using molecular modelling.

It is an urgent need to develop new drugs for Mycobacterium tuberculosis (Mtb), and the enzyme, dihydrofolate reductase (DHFR) is a recognised drug target. The crystal structures of methotrexate binding to mt- and h-DHFR separately indicate that the glycerol (GOL) binding site is likely to be critical for the function of mt-DHFR selective inhibitors. We have used in silico methods to screen NCI small molecule database and a group of related compounds were obtained that inhibit mt-DHFR activity and showed bactericidal effects against a test Mtb strain. The binding poses were then analysed and the influence of GOL binding site was studied by using molecular modelling. By comparing the chemical structures, 4 compounds that might be able to occupy the GOL binding site were identified. However, these compounds contain large hydrophobic side chains. As the GOL binding site is more hydrophilic, molecular modelling indicated that these compounds were failed to occupy the GOL site. The most potent inhibitor (compound 6) demonstrated limited selectivity for mt-DHFR, but did contain a novel central core (7H-pyrrolo[3,2-f]quinazoline-1,3-diamine), which may significantly expand the chemical space of novel mt-DHFR inhibitors. Collectively, these observations will inform future medicinal chemistry efforts to improve the selectivity of compounds against mt-DHFR.

Folate pathway disruption leads to critical disruption of methionine derivatives in Mycobacterium tuberculosis.

In this study, we identified antifolates with potent, targeted activity against whole-cell Mycobacterium tuberculosis (MTB). Liquid chromatography-mass spectrometry analysis of antifolate-treated cultures revealed metabolic disruption, including decreased pools of methionine and S-adenosylmethionine. Transcriptomic analysis highlighted altered regulation of genes involved in the biosynthesis and utilization of these two compounds. Supplementation with amino acids or S-adenosylmethionine was sufficient to rescue cultures from antifolate treatment. Instead of the "thymineless death" that characterizes folate pathway inhibition in a wide variety of organisms, these data suggest that MTB is vulnerable to a critical disruption of the reactions centered around S-adenosylmethionione, the activated methyl cycle.