Diketopiperazine-Based, Flexible Tadalafil Analogues: Synthesis, Crystal Structures and Biological Activity Profile

Phosphodiesterase 5 (PDE5) is one of the most extensively studied phosphodiesterases that is highly specific for cyclic-GMP hydrolysis. PDE5 became a target for drug development based on its efficacy for treatment of erectile dysfunction. In the present study, we synthesized four novel analogues of the phosphodiesterase type 5 (PDE5) inhibitor—tadalafil, which differs in (i) ligand flexibility (rigid structure of tadalafil vs. conformational flexibility of newly synthesized compounds), (ii) stereochemistry associated with applied amino acid building blocks, and (iii) substitution with bromine atom in the piperonyl moiety. For both the intermediate and final compounds as well as for the parent molecule, we have established the crystal structures and performed a detailed analysis of their structural features. The initial screening of the cytotoxic effect on 16 different human cancer and non-cancer derived cell lines revealed that in most cases, the parent compound exhibited a stronger cytotoxic effect than new derivatives, except for two cell lines: HEK 293T (derived from a normal embryonic kidney, that expresses a mutant version of SV40 large T antigen) and MCF7 (breast adenocarcinoma). Two independent studies on the inhibition of PDE5 activity, based on both pure enzyme assay and modulation of the release of nitric oxide from platelets under the influence of tadalafil and its analogues revealed that, unlike a reference compound that showed strong PDE5 inhibitory activity, the newly obtained compounds did not have a noticeable effect on PDE5 activity in the range of concentrations tested. Finally, we performed an investigation of the toxicological effect of synthesized compounds on Caenorhabditis elegans in the highest applied concentration of 6a,b and 7a,b (160 μM) and did not find any effect that would suggest disturbance to the life cycle of Caenorhabditis elegans. The lack of toxicity observed in Caenorhabditis elegans and enhanced, strengthened selectivity and activity toward the MCF7 cell line made 7a,b good leading structures for further structure activity optimization and makes 7a,b a reasonable starting point for the search of new, selective cytotoxic agents

Phosphorylation of Thymidylate Synthase and Dihydrofolate Reductase in Cancer Cells and the Effect of CK2α Silencing

Our previous research suggests an important regulatory role of CK2-mediated phosphorylation of enzymes involved in the thymidylate biosynthesis cycle, i.e., thymidylate synthase (TS), dihydrofolate reductase (DHFR), and serine hydroxymethyltransferase (SHMT). The aim of this study was to show whether silencing of the CK2α gene affects TS and DHFR expression in A-549 cells. Additionally, we attempted to identify the endogenous kinases that phosphorylate TS and DHFR in CCRF-CEM and A-549 cells. We used immunodetection, immunofluorescence/confocal analyses, reverse transcription–quantitative polymerase chain reaction (RT-qPCR), in-gel kinase assay, and mass spectrometry analysis. Our results demonstrate that silencing of the CK2α gene in lung adenocarcinoma cells significantly increases both TS and DHFR expression and affects their cellular distribution. Additionally, we show for the first time that both TS and DHFR are very likely phosphorylated by endogenous CK2 in two types of cancer cells, i.e., acute lymphoblastic leukaemia and lung adenocarcinoma. Moreover, our studies indicate that DHFR is phosphorylated intracellularly by CK2 to a greater extent in leukaemia cells than in lung adenocarcinoma cells. Interestingly, in-gel kinase assay results indicate that the CK2α’ isoform was more active than the CK2α subunit. Our results confirm the previous studies concerning the physiological relevance of CK2-mediated phosphorylation of TS and DHFR

Synthesis and Physico-Chemical Properties in Aqueous Medium of All Possible Isomeric Bromo Analogues of Benzo-1H-Triazole, Potential Inhibitors of Protein Kinases.

In ongoing studies on the role of the individual bromine atoms of 4,5,6,7-tetrabromobenzotriazole (TBBt) in its relatively selective inhibition of protein kinase CK2α, we have prepared all the possible two mono-, four di-, and two tri- bromobenzotriazoles, and determined their physico-chemical properties in aqueous medium. They exhibited a general trend of a decrease in solubility with an increase in the number of bromines on the benzene ring, significantly modulated by the pattern of substitution. For a given number of attached bromines, this was directly related to the electronic effects resulting from different sites of substitution, leading to marked variations of pKa values for dissociation of the triazole proton. Experimental data (pKa, solubility) and ab initio calculations demonstrated that hydration of halogenated benzotriazoles is driven by a subtle balance of hydrophobic and polar interactions. The combination of QM-derived free energies for solvation and proton dissociations was found to be a reasonably good predictor of inhibitory activity of halogenated benzotriazoles vs CK2α. Since the pattern of halogenation of the benzene ring of benzotriazole has also been shown to be one of the determinants of inhibitory potency vs some viruses and viral enzymes, the present comprehensive description of their physico-chemical properties should prove helpful in efforts to elucidate reaction mechanisms, including possible halogen bonding, and the search for more selective and potent inhibitors

Cellular aspects of folate and antifolate membrane transport.

Folates - one carbon carriers - take part in the metabolism of purine, thymidylate and some amino acids. Internalization of these compounds employs several mechanisms of transport systems. Reduced folate carriers and folate receptors play the most important role in this process. The physiological role of these molecules in normal and neoplastic cells is described regarding changes in transport activity and connection of transport systems with resistance to antifolates and cancer development

Isomeric Mono-, Di-, and Tri-Bromobenzo-1H-Triazoles as Inhibitors of Human Protein Kinase CK2α.

To further clarify the role of the individual bromine atoms of 4,5,6,7-tetrabromotriazole (TBBt), a relatively selective inhibitor of protein kinase CK2, we have examined the inhibition (IC(50)) of human CK2α by the two mono-, the four di-, and the two tri- bromobenzotriazoles relative to that of TBBt. Halogenation of the central vicinal C(5)/C(6) atoms proved to be a key factor in enhancing inhibitory activity, in that 5,6-di-Br(2)Bt and 4,5,6-Br(3)Bt were almost as effective inhibitors as TBBt, notwithstanding their marked differences in pK(a) for dissociation of the triazole proton. The decrease in pK(a) on halogenation of the peripheral C(4)/C(7) atoms virtually nullifies the gain due to hydrophobic interactions, and does not lead to a decrease in IC(50). Molecular modeling of structures of complexes of the ligands with the enzyme, as well as QSAR analysis, pointed to a balance of hydrophobic and electrostatic interactions as a discriminator of inhibitory activity. The role of halogen bonding remains debatable, as originally noted for the crystal structure of TBBt with CK2α (pdb1j91). Finally we direct attention to the promising applicability of our series of well-defined halogenated benzotriazoles to studies on inhibition of kinases other than CK2

Structures of all possible halogenated derivatives of benzotriazole.

<p>Structures of all possible halogenated derivatives of benzotriazole.</p

Inhibitory activities (IC₅₀) of brominated Bt derivatives predicted on the basis of: (A) V_mol and experimental pK_a; (B) <i>ab initio</i> derived ΔG_solv(anion) and ΔG_diss free energies; and (C) autodock-derived free energy of binding (ΔG_bind).

<p>All relations point to predominance of hydrophobic interactions (V_mol or ΔG_solv(anion)), accompanied by protonation of the anionic form of the ligand upon binding to CK2α (pK_a or ΔG_diss) (see text for details).</p

Distribution of short halogen-acceptor (O, N, S, π system) contacts identified in 21 accessible structures of complexes of CK2α with halogenated ligands.

<p>The Gaussian cumulative distribution was fitted to the crystallographic data for halogen to donor distance (solid line in panel A) and, according to the Anderson-Darling test, experimental data up to 3.7 Å agrees with a normal distribution (panel B). However, pairs separated by more than 3.7 Å are overrepresented, clearly limiting the maximal distance for eventual halogen-bonding interactions to the sum of donor and acceptor VdW radii. Note that an isolated water molecule (red triangles in panels A, C) is an equally favorable acceptor to the protein (O, N, S, π-electrons). The cumulative distribution of the experimental data is visibly better represented by a bi-normal distribution (panel C), in which the contribution of an additional narrow peak represents putative halogen-bonding (panel D). This is additionally supported by the distribution of angles X…Acc-C and C-X…Acc, which, for short halogen-acceptor distances, are substantially restricted to the regions favoring halogen bond formation (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048898#pone.0048898.s002" target="_blank">Figure S2</a>).</p

Schematic representation of the effects of stepwise bromination of the benzene ring of benzotriazole (Bt), including interdependence of the molecular volume (A) and pK_a (B) of the products with their IC₅₀ for inhibition of protein kinase CK2α: (a) Green arrows follow sequential bromination of the central vicinal C(5)/C(6) atoms, leading to a moderate decrease of pK_a and a large decrease in IC₅₀; (b) Red arrows illustrate the lesser effects of bromination of the peripheral C(4)/C(7) atoms, resulting in a significant decrease of pK_a, with virtually no gain in inhibitory activity; (c) Black lines link products with the same number of bromine atoms, but substantially differing in pK_a and IC₅₀, culminating in the di- and tri-brominated derivatives with inhibitory activities comparable to that of TBBt.

<p>Note that the parent Bt (pKa 8.56), which is not an inhibitor (IC₅₀>2 mM), is located outside the diagrams.</p

Location of all nine halogenated Bt derivatives in complex with CK2α.

<p>For each ligand the average location determined from the 3 ns trace of Molecular Dynamics performed in the presence of explicit aqueous solvent is presented in relation to X-ray structure of CK2α (pdbj91). All ligands were found to bind in the same orientation (see panel A), in the position almost identical to that found for TBBt in the crystal structure of the complex with CK2α (see panel B, TBBt, from PDB, in magenta and putative location of Bt in green). The lowest-energy structures identified in 3 ns MD traces are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048898#pone.0048898.s003" target="_blank">Figure S3</a>.</p