DNA-dependent DNA and RNA polymerases are important modulators of biological functions such as replication, transcription, recombination, or repair. In this work performed in cell-free media, we studied the ability of selected DNA polymerases to overcome a monofunctional adduct of the cytotoxic/antitumor platinum-acridinylthiourea conjugate [PtCl(en)(L)](NO3)2 (en = ethane-1,2-diamine, L = 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea) (ACR) in its favored 5'-CG sequence. We focused on how a single site-specific ACR adduct with intercalation potency affects the processivity and fidelity of DNA-dependent DNA polymerases involved in translesion synthesis (TLS) and repair. The ability of the G(N7) hybrid ACR adduct formed in the 5'-TCGT sequence of a 24-mer DNA template to inhibit the synthesis of a complementary DNA strand by the exonuclease-deficient Klenow fragment of DNA polymerase I (KFexo-) and human polymerases eta, kappa, and iota was supplemented by thermodynamic analysis of the polymerization process. Thermodynamic parameters of a simulated translesion synthesis across the ACR adduct were obtained by using microscale thermophoresis (MST). Our results show a strong inhibitory effect of an ACR adduct on enzymatic TLS: there was only small synthesis of a full-length product (less than 10%) except polymerase eta (~20%). Polymerase eta was able to most efficiently bypass the ACR hybrid adduct. Incorporation of a correct dCMP opposite the modified G residue is preferred by all the four polymerases tested. On the other hand, the frequency of misinsertions increased. The relative efficiency of misinsertions is higher than that of matched cytidine monophosphate but still lower than for the nonmodified control duplex. Thermodynamic inspection of the simulated TLS revealed a significant stabilization of successively extended primer/template duplexes containing an ACR adduct. Moreover, no significant decrease of dissociation enthalpy change behind the position of the modification can contribute to the enzymatic TLS observed with the DNA-dependent, repair-involved polymerases. This TLS could lead to a higher tolerance of cancer cells to the ACR conjugate compared to its enhanced analog, where thiourea is replaced by an amidine group: [PtCl(en)(L)](NO3)2 (complex AMD, en = ethane-1,2-diamine, L = N-[2-(acridin-9-ylamino)ethyl]-N-methylpropionamidine).

• Keywords
• DNA polymerases, antitumor, cytotoxic, drug resistance, lesion bypass, metal–intercalator, microscale thermophoresis, platinum–acridine, thermodynamic, translesion synthesis,
• MeSH
• DNA Adducts chemistry   MeSH
• DNA-Directed DNA Polymerase metabolism   MeSH
• Intercalating Agents chemistry   MeSH
• Humans MeSH
• Urea analogs & derivatives   chemistry   MeSH
• DNA Repair * MeSH
• Organoplatinum Compounds chemistry   MeSH
• DNA Damage * MeSH
• DNA Replication MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• 1-(2-(acridin-9-ylamino)ethyl)-1,3-dimethylthiourea MeSH Browser
• DNA Adducts MeSH
• DNA-Directed DNA Polymerase MeSH
• Intercalating Agents MeSH
• Urea MeSH
• Organoplatinum Compounds MeSH

The search for more effective platinum anticancer drugs has led to the design, synthesis, and preclinical testing of hundreds of new platinum complexes. This search resulted in the recognition and subsequent FDA approval of the third-generation Pt(II) anticancer drug, [Pt(1,2-diaminocyclohexane)(oxalate)], oxaliplatin, as an effective agent in treating colorectal and gastrointestinal cancers. Another promising example of the class of anticancer platinum(II) complexes incorporating the Pt(1,n-diaminocycloalkane) moiety is kiteplatin ([Pt(cis-1,4-DACH)Cl2], DACH = diaminocyclohexane). We report here our progress in evaluating the role of the cycloalkyl moiety in these complexes focusing on the synthesis, characterization, evaluation of the antiproliferative activity in tumor cells and studies of the mechanism of action of new [Pt(cis-1,3-diaminocycloalkane)Cl2] complexes wherein the cis-1,3-diaminocycloalkane group contains the cyclobutyl, cyclopentyl, and cyclohexyl moieties. We demonstrate that [Pt(cis-1,3-DACH)Cl2] destroys cancer cells with greater efficacy than the other two investigated 1,3-diamminocycloalkane derivatives, or cisplatin. Moreover, the investigated [Pt(cis-1,3-diaminocycloalkane)Cl2] complexes show selectivity toward tumor cells relative to non-tumorigenic normal cells. We also performed several mechanistic studies in cell-free media focused on understanding some early steps in the mechanism of antitumor activity of bifunctional platinum(II) complexes. Our data indicate that reactivities of the investigated [Pt(cis-1,3-diaminocycloalkane)Cl2] complexes and cisplatin with glutathione and DNA binding do not correlate with antiproliferative activity of these platinum(II) complexes in cancer cells. In contrast, we show that the higher antiproliferative activity in cancer cells of [Pt(cis-1,3-DACH)Cl2] originates from its highest hydrophobicity and most efficient cellular uptake.

• Keywords
• Antitumor, Cellular uptake, DNA, Glutathione, Oxaliplatin, Pt(1,n-diaminocycloalkane) moiety,
• MeSH
• Apoptosis drug effects   MeSH
• Cisplatin pharmacology   standards   MeSH
• Hydrocarbons, Cyclic chemical synthesis   MeSH
• DNA chemistry   MeSH
• Glutathione chemistry   MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Organometallic Compounds chemical synthesis   pharmacology   MeSH
• Cell Membrane Permeability MeSH
• Platinum chemistry   MeSH
• Cell Proliferation drug effects   MeSH
• Antineoplastic Agents chemical synthesis   pharmacology   MeSH
• Drug Screening Assays, Antitumor MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cisplatin MeSH
• Hydrocarbons, Cyclic MeSH
• DNA MeSH
• Glutathione MeSH
• Organometallic Compounds MeSH
• Platinum MeSH
• Antineoplastic Agents MeSH

Oxidative stress in cells can lead to the accumulation of reactive oxygen species and oxidation of DNA precursors. Oxidized nucleotides such as 2'-deoxyribo-5-hydroxyuridin (HdU) and 2'-deoxyribo-5-hydroxymethyluridin (HMdU) can be inserted into DNA during replication and repair. HdU and HMdU have attracted particular interest because they have different effects on damaged-DNA processing enzymes that control the downstream effects of the lesions. Herein, we studied the chemically simulated translesion DNA synthesis (TLS) across the lesions formed by HdU or HMdU using microscale thermophoresis (MST). The thermodynamic changes associated with replication across HdU or HMdU show that the HdU paired with the mismatched deoxyribonucleoside triphosphates disturbs DNA duplexes considerably less than thymidine (dT) or HMdU. Moreover, we also demonstrate that TLS by DNA polymerases across the lesion derived from HdU was markedly less extensive and potentially more mutagenic than that across the lesion formed by HMdU. Thus, DNA polymerization by DNA polymerase η (polη), the exonuclease-deficient Klenow fragment of DNA polymerase I (KF-), and reverse transcriptase from human immunodeficiency virus type 1 (HIV-1 RT) across these pyrimidine lesions correlated with the different stabilization effects of the HdU and HMdU in DNA duplexes revealed by MST. The equilibrium thermodynamic data obtained by MST can explain the influence of the thermodynamic alterations on the ability of DNA polymerases to bypass lesions induced by oxidative products of pyrimidines. The results also highlighted the usefulness of MST in evaluating the impact of oxidative products of pyrimidines on the processing of these lesions by damaged DNA processing enzymes.

• Keywords
• 2’-deoxyribo-5-hydroxymethyl- uridin, 2’-deoxyribo-5-hydroxyuridin, DNA polymerases, microscale thermophoresis, oxidized nucleotides, translesion DNA synthesis,
• MeSH
• DNA-Directed DNA Polymerase metabolism   MeSH
• DNA biosynthesis   drug effects   MeSH
• HIV-1 MeSH
• Humans MeSH
• Mutagens chemistry   metabolism   pharmacology   MeSH
• DNA Repair MeSH
• Oxidation-Reduction MeSH
• Oxidative Stress * MeSH
• Pentoxyl analogs & derivatives   chemistry   metabolism   pharmacology   MeSH
• DNA Damage MeSH
• Pyrimidines chemistry   metabolism   pharmacology   MeSH
• DNA Replication drug effects   MeSH
• Thermodynamics MeSH
• Uracil analogs & derivatives   chemistry   metabolism   pharmacology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• 5-hydroxymethyluracil MeSH Browser
• 5-hydroxyuracil MeSH Browser
• DNA-Directed DNA Polymerase MeSH
• DNA MeSH
• Mutagens MeSH
• Pentoxyl MeSH
• Pyrimidines MeSH
• Rad30 protein MeSH Browser
• Uracil MeSH