**4. Conclusions**

Morpholino nucleosides are readily available derivatives of natural ribonucleosides that found a broad application in the synthesis of morpholino oligonucleotides as nucleic acid mimetics. These are important and widely used molecular tools in molecular biology and are now even in clinic for an antisense-based treatment. However, a high potential of morpholino nucleoside monomers as terminators in gene sequencing, polymerase inhibitors (in phosphorylated form), or as a part of other low molecular weight biological regulators is not investigated in its entirety. In this regard, it is essential that the morpholine ring is a well-known and important pharmacophore widely used in numerous medicines [93]. The present study allowed for a broader involvement of morpholino nucleosides into a pool of potentially biologically active compounds. We proposed a novel class of PARP-1 and PARP-2 inhibitors consisting of various types of morpholino nucleosides and ADP [45].

The highest inhibition activity against PARP-1 was found for ADP conjugates containing 5-iodouracil morpholino nucleosides. These compounds were predicted to target the NA binding site by a common mechanism to known for potential PARP-1 inhibitors.

Surprisingly, in relation to PARP-2 the most active compound with IC50~50 µM was dinucleotide-containing adenine morpholino nucleoside. The activity of 20 -aminomethylmorpholino nucleoside of adenine could be achieved by targeting to the acceptor substrate binding site by mimicking interactions of the PAR substrate. In particular, it has been shown that the amine group of the morpholine ring can mimic an interaction of the 20–OH group of adenine riboside with Glu558. The replacement of P–O to P–N bond leads to the enhanced stabilization of pyrophosphate conformation due to the formation of an intramolecular hydrogen bond, and followed to a subsequently higher inhibition activity. Stabilizing interactions with variable regions lead to enhanced selectivity in relation to PARP-2. Strikingly, these predictions are validated by a fluorescence anisotropy assay that suggests the mixed mode of inhibitory activity by targeting the active and allosteric centers simultaneously. This is in contrast to 5-iodouracil morpholino nucleosides as well as the **3-AB** compound, a well known PARP inhibitor targeting nicotinamide binding site.

Lack of activity of **10A** against PARP-3 that has a different amino acid composition of the acceptor binding site as well as substrates selectivity further confirms the predicted mechanism of action. It has to be noted that only 5-iodouracil morpholino nucleosides **10IU** demonstrated low activity against PARP-3 at mM concentration. These data are in accordance with generally observed up to two orders lower activity of known PARP-1/2 inhibitors against PARP-3, targeting the donor nicotinamide binding pocket [94].

Interestingly, it has been shown that occupancy of the NAD+-binding site with benzamide adenine dinucleotide (BAD) locks PARP-1 on a DNA break [38]. It makes a design of new NAD+ analogs a promising strategy to create a wide spectrum of the compounds for future clinical usage under PARP inhibitor therapy. Conducted analysis suggests that compounds based on 20 -aminomethylmorpholino nucleosides target PARP-1/2 by a novel molecular mechanism distinct from a known class of potent inhibitors directed on the NA donor subsite, but not on the acceptor substrate binding site. It opens new horizons for the development of additional classes of PARP-1/2 selective inhibitors.

Taking into account our enzyme assay and molecular modeling calculations, a new strategy for conformation stabilization and enhancement of the activity and selectivity of a novel class of NAD+ analogs may be proposed.

**Supplementary Materials:** Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/1/214/s1.

**Author Contributions:** T.V.A, O.I.L. and V.N.S. have conceptualized the research. Y.V.S and T.V.A performed the synthesis. N.V.I. and V.A.I. carried out molecular modeling study. R.Y.P. and I.V.E. performed NMR analysis. A.L.Z., M.V.S., M.M.K., T.A.K. and E.A.B. carried out an enzyme purification and enzyme inhibition assays. T.V.A., A.L.Z., N.V.I., Y.V.S. and E.A.B. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** The work was partially supported by the Russian Foundation for Basic Research (RFBR, Grant No. 18-04-00352: the design of the study, the collection, analyses and interpretation of data, the writing of the manuscript, the decision to publish the results, compound synthesis and molecular modeling; and Grant No. 17-00-00097: the collection, analyses and interpretation of data, PARP-1 and PARP-2 assay); the Russian Science Foundation (RSF, Grant No. 17-74-20075: the writing of the manuscript, the decision to publish the results PARP-3 purification and assay); and by Russian State funded budget projects of ICBFM SB RAS No. AAAA-A17-117020210022-4: the collection, analyses and interpretation of data, the writing of the manuscript, the decision to publish the results.

**Conflicts of Interest:** The authors declare no conflict of interest.
