Primer-Like Inhibitors for DNA Repair Enzymes of the AML-HL60 and WERI-1A/Y79 Malignant Cells

A conventionally synthesized thio- and cyano-modified single-stranded poly(dNTP) sequences of different molecular sizes (20n - 200n) and the same lengths routine poly(dNTP) and poly(NTP) species were obtained through the good services provided by the Russian Federal Bioorganic Products Group and by the ThermoFischer, Inc., and then tested for their impact on catalytic activities of β-like DNA polymerases from chromatin of HL-60, WERI-1A and Y-79 cells as well as for the affinity patterns in DNApolβ-poly(dNTP)/ (NTP) pairs, respectively. An essential link between the lengths of ultrashort (50n - 100n) single-stranded poly(dNTP) sequences of different structures and their inhibitory effects towards the cancer-specific DNA polymerases β has been found. A possible significance of this phenomenon for both DNA repair suppression in tumors and a consequent anti-cancer activity of the DNA repair related short poly(dNTP) fragments has been for the first time emphasized with a respect to their pharmacophore revealing potential. Thus, this work presents an experimental attempt to upgrade a contemporary atti-tude towards the DNA derived products applied for anti-cancer agenda, par-ticularly, for acute myeloid leukemia and retinoblastoma cell DNA repair machinery breakdown. In this study, tumor specific DNA polymerases β were found of being the targets for attack promoted with the primer-like sin-gle-stranded DNA fragments followed by consequent cytostatic phenomena. A novel concept of the DNA related anti-cancer medicines is under discussion.


Introduction
A background-lying platform of this work derives from experimental data on magnetic isotope effects (MIE) towards both DNApolβ catalytic activity [1] [2] [3] and the viability of cancer cells [1] [2]. Thus, a sharp decrease of the survival ability patterns of 25 Mg 2+ -treated AML/HL-60 cells as compared to abundant spinless, non-magnetic, magnesium ions impact has been found [1]. Furthermore, a similar result was then obtained on the same leukemia cells treated with magnetic, nuclear spin possessing, 43 Ca 2+ and 67 Zn 2+ ions [1] [3] as well as on human retinoblastoma cells subjected to these metal isotopes [2] [3].
It was also shown that a processivity of beta-like DNA polymerases isolated from the all above mentioned malignant cells depends on MIE, so the resulted DNA fragment sizes becomes shorter within a 40n -250n range following the increase of magnetic isotope content in a total metal pool [2] [3]. For instance, an up to 58% elevation of 43 Ca 2+ content leads to a monotonic decrease of sizes of polynucleotides processed from average 230n -250n to an abnormal (DNA repair invalid) 36n -40n as directed by beta-like DNA polymerases from Y-79 and WERI-1A retinoblastoma cells [1].
This nuclear-magnetic control over the DNA synthesis, therefore, allows coming up with a firm statement that a replication mechanism involves some ion-radical steps consisting of the ion-radical pairs formation [1]. A key element of this mechanism deals with an electron transfer from the nascent DNA deoxyribose anion to a bivalent ion coordinated inside the DNA polymerase catalytic site [1]  "compartment" for this reaction (10 -15 nm or less), all physical parameters describing a molecular machinery in normal and cancer cells at proliferation rates observed are identical or pretty close to each other [5]- [10].
Obviously, the DNA synthesis reaction [1] [2] [3] is unlikely a source for apoptosis of tumor cells as relates to dependence of their viability on MIE [1].
Nonetheless, this MIE-viability dependence might have a link to DNA repair activity expressed by beta-like DNA polymerases in neoplasma [2] [3] [11].
According to our assumption, these enzymes are to produce the ultrashort PDRN sequences (≈40n) playing a role of DNApolβ inhibitors. Acting as the DNA repair limiting/damaging factors, these endogenous inhibitors may promote an essential anti-cancer effect. In case of 25

Enzyme Activity Estimation
Beta

DNA and Protein Measurements
The DNA ultramirco amounts measurements were performed in diluted water solutions according to [12] [13]. The protein ultramicro amounts were estimated by method [14] modified in [12].

Ligand-Enzyme Binding Measurements
To quantify a ligand-enzyme binding patterns, a conventional criteria such as K dis (K d ) and A c were taken into account. In this approach, a ligand-receptor interaction R L RL α β  → + ←  , where R is the receptor, L is the ligand, RL is the ligand-receptor complex, α is the probability of formation of a complex molecule, and β is the probability of its dissociation. If the random number of ligand-receptor complex molecules is x, and the initial number of receptors is 0 R , the number of free receptors makes 0 R x − . Assume that the process unfolds under the condition of large ligand surplus, so that the number of ligand molecules stays equal to its initial value 0 L .
The formation of ligand-receptor complexes is described by the function ( ) Bailey's equation system is: Following this algorithm, an experimental data on the ligand-enzyme complexes stability in water solutions obtained by techniques described in [7] [15] and modified in [16], and affected by temperature/ultrasound/ionic strength were processed using a LQ170 SigmaLab software in HP9000 analytical system (Hewlett Packard, USA).
In all enzyme-ligand coupling tests, the enzyme required optimal catalytic parameters [2] [11] were held for 40 min, +37˚C, while the following ligand con-

Statistics
A Dunnett's non-parametric (n ≤ 6) technique was used to elucidate reproductability of the data along with a significance of differences in control-experiment comparisons [19].

Results
A variable set of polynucleotide ligands (see Methods) was tested for both inhibitory effects and the ligand-enzyme binding properties using beta-like DNA polymerase species from acute myeloid leukemia (HL-60) and retinoblastoma (WERI-1A, Y-79) cells.
As seen from the data presented in Figure 2 and  The data presented in Figure 2 and Figure 3 leaves no doubt about a maximal inhibitory efficiency provided by ultrashort, 40n -50n in length, cyano-polydeoxyribothymidyl derivatives. These agents are to show as high as nearly 6.5-fold extent of enzyme suppression. That does not mean, however, that   this particular type of "pseudoprimers" is the only one capable to play a role of a powerful DNApolβ inhibitor. A truly crucial parameter to reveal a potential pharmacophore signs have nothing to do with a structure of DNA fragments being entirely related to its molecular size (Figure 2 and Figure 3).
So the results observed looks worthy of not indicating to any particular and perfect pharmacophore found but, instead, of a detailed analysis as a new platform to upgrade a whole approach towards the DNA derivatives in a current  anti-cancer agenda. This a Discussion of our work is all about.

Discussion
A wide diversity of investigated synthetic homopolynucleotides and their monotonic thio-and cyano-derivatives is itself a prove for universal, indiscriminate, mode of DNApolβ inhibition effect we described. This is in a favor with our assumption stated that the key role in this phenomenon belongs exclusively to molecular sizes of small DNA fragments regardless on their primary structure peculiarities.
The ligands diversity mentioned makes no way to explain a formation of the ligand-enzyme complexes just by a routine docking ("complimentary") paradigm. This DNA-protein binding act may involve some random appearing hydrogen bonds, Coulomb hyperfine coupling along with dispersion interaction and, last not least, the hydrophobic connections, might also be a case in this scenario. A key point of this model derives from the homopolynucleotides related data and reveals that the mean energy of these "weak" interaction depends on the length of a ligand but not of its structure. A similar event occurs in the DNA repair process as long as a randomly appeared local DNA damage comes first. In this case, a clearly indiscriminate mode of the ligand-enzyme coupling is a consequence of unpredictable variability in primary structures of DNA fragments suitable for the repair purposes. Therefore, a polynucleotide length alone is an impact-making factor towards the DNApolβ catalytic function. This factor might determine a formation of the above listed "weak" non-covalent interactions and, hence, to control the mean amount of hydrogen bonds per one nucleotide of DNA-primer (ligand) inside the DNA polymerase catalytic site. That is indeed critical for the enzyme suppression in situ.
All the affinity isotherms presented and a variable ligand-enzyme binding data (Figures 4-6) reveals a maximal strength of this peculiar intermolecular interaction within the short interval of ligand sizes, 50n -100n. This is to confirm that the enzyme inhibition we observed (Figures 2(B)-(E), Figure 3) is indeed a consequence of the stable ligand-enzyme pair formation which has nothing to do with either the solution properties changes or the enzyme surrounding disperse phase modulations.
A comparison between the ligand amounts bound with (a) DNApolβ and (b) HSA show that the ligand-enzyme complex includes two molecules of ligand per one molecule of enzyme, while the ligand-HSA (control) pair contains just one ligand per HSA molecule (Figure 4, Figure 7, Figure 8). This is in a favor to a mere fact of existance of two separate Mg 2+ -coordinating catalytic sites in βand β-like DNA polymerases [8] [10] [20] [21] [22].
Let's evaluate the activation energy value for a ligand-enzyme complex dissociation following the data listed in Figure 4 and Figure 8. Obviously, a   dependence of K d on temperature has a clear Ahrrenius mode: where R-a universal gas constant and T-enzyme functioning temperature.
Taking into account a low level of the temperature interval tested as compared to an initial T 0 = 298K, we'll use further a linear decay as T in (1). To estimate E, a linear reguliarity must be treated as: where T ∆ is a temperature change in experiment (Figure 7 and Figure 8).
The Equation (2) [24]. This certainly takes into account the superficial hydrophobic bounds as well which are nothing but the result of a cooperative (cumulative) act of all the above specified forces [23] [24].
So the key role in formation of Poly(dNTP)/DNApolβ complexes belongs to Van der Waals bounds which energy corresponds directly to either 1) the efficient surface of ligand-enzyme pair or 2) a length of an enzyme-bound ligand.
Furthermore, since the mean value of the U d dispersion patterns is hardly dependent on the element composition, and as long as the amounts of charged groups in U q -determining Poly(dNTP) ligands tested are about the same, a very similar parameters of their inhibitory activity should be expected then. This explains well a lack of dependence of inhibitory capabilities of these ligands on their nucleotide composition (Figures 2(B)-(E), Figure 3).
Once ~RT ε , we might suppose that the short ligands (50n -100n, or shorter) are to get bound with the enzyme molecule throughout a whole linear chain, i.e. through nearly every nucleotide of Poly(dNTP) sequence.
Using the Ahrrenius equation, we may evaluate a ratio between the ligand- τ does not allow n 20 -ligand to promote a sharp suppression of the enzyme activity. A shorter time for the ligand "neighboring" around the enzyme catalytic site, the lesser inhibitory effect to be expressed. On other hand, the longest ligands tested were found to be a rather poor inhibitors as well (Figure 2(E), Figure 4) due to their compactization-caused small square of the enzyme-ligand efficient interaction [23] [25] as also seen from Equation (3).
As per a zero inhibition affect shown by RNA-like ligands tested (Figure 3(A) and Figure 3(B) The inhibitory power of Poly(dNTP) species we found has nothing to do with their structures and composition being entirely dependent on molecular size (Figures 2(B)-(E), Figure 3(A), Figure 3(C), Figure 3(D), Figure 3(E)). This allows to "turn the page" in a long record of polynucleotide medicinal applica- Knowing that the abundance of 25 Mg is relatively high (≈10%) and considering a remarkable role of magnesium in enzymatic phosphorylation processes [1] [24] [34], it would be logical to suppose the existence of so called "hidden" effects of Magnesium-25 on numerous metabolic pathways. In this case, a high content of endogenous Fe 2+ in cells and tissues of a living organism becomes a natural limiting factor to prohibit any expression of magnetic isotope effects in vivo. This is in accordance with the data on correlations between the 25 Mg 2+ -affected ATP synthesis levels in mitochondria and the Fe 2+ contents in these mitochondria isolated from different rat tissues [34] as well as with the results stated an increase of Fe/Mg ratios in several types of cancer (ovarian cancer, renal adeno- single-stranded DNA fragmets in a blood plasma of oncology patients [26] [35] may now also be treated under a point of view of the probable DNA repair related origin of these tumor-released Poly(dNTP) sequences.
Last not least, the infamous quorum sensing phenomena [36] could be engaged with an effort to uncover a biological meaning of what we've found testing the capabilities of different Poly(dNTP) species to affect some tumor-specific DNA repair key enzymes.
Manifesting a positive correlation between these inhibitory capabilities and the strength of affinity in enzyme-ligand pairs, such poly(dNTP) species promote a sharp suppression of enzymatic catalysis regardless on their primary structures. Only molecular size matters.
The data presented show a clear regulatory potential of some short ssDNA entities once they are about to interact with the key DNA repair enzymes.
This may provide a promising platform for further research aiming to shut down the DNA repair machinery in malignancies.