Helquat dyes targeting G-quadruplexes as a new class of anti-HIV-1 inhibitors.

The secondary structure of nucleic acids containing quartets of guanines, termed G-quadruplexes, is known to regulate the transcription of many genes. Several G-quadruplexes can be formed in the HIV-1 long terminal repeat promoter region and their stabilization results in the inhibition of HIV-1 replication. Here, we identified helquat-based compounds as a new class of anti-HIV-1 inhibitors that inhibit HIV-1 replication at the stage of reverse transcription and provirus expression. Using Taq polymerase stop and FRET melting assays, we have demonstrated their ability to stabilize G-quadruplexes in the HIV-1 long-terminal repeat sequence. Moreover, these compounds were not binding to the general G-rich region, but rather to G-quadruplex-forming regions. Finally, docking and molecular dynamics calculations indicate that the structure of the helquat core greatly affects the binding mode to the individual G-quadruplexes. Our findings can provide useful information for the further rational design of inhibitors targeting G-quadruplexes in HIV-1.

Enzymatic Synthesis of 3'-5', 3'-5' Cyclic Dinucleotides, Their Binding Properties to the Stimulator of Interferon Genes Adaptor Protein, and Structure/Activity Correlations.

The 3'-5', 3'-5' cyclic dinucleotides (3'3'CDNs) are bacterial second messengers that can also bind to the stimulator of interferon genes (STING) adaptor protein in vertebrates and activate the host innate immunity. Here, we profiled the substrate specificity of four bacterial dinucleotide synthases from Vibrio cholerae (DncV), Bacillus thuringiensis (btDisA), Escherichia coli (dgcZ), and Thermotoga maritima (tDGC) using a library of 33 nucleoside-5'-triphosphate analogues and then employed these enzymes to synthesize 24 3'3'CDNs. The STING affinity of CDNs was evaluated in cell-based and biochemical assays, and their ability to induce cytokines was determined by employing human peripheral blood mononuclear cells. Interestingly, the prepared heterodimeric 3'3'CDNs bound to the STING much better than their homodimeric counterparts and showed similar or better potency than bacterial 3'3'CDNs. We also rationalized the experimental findings by in-depth STING-CDN structure-activity correlations by dissecting computed interaction free energies into a set of well-defined and intuitive terms. To this aim, we employed state-of-the-art methods of computational chemistry, such as quantum mechanics/molecular mechanics (QM/MM) calculations, and complemented the computed results with the {STING:3'3'c-di-ara-AMP} X-ray crystallographic structure. QM/MM identified three outliers (mostly homodimers) for which we have no clear explanation of their impaired binding with respect to their heterodimeric counterparts, whereas the R2 = 0.7 correlation between the computed ΔG'int_rel and experimental ΔTm's for the remaining ligands has been very encouraging.

Synthesis of phosphonate derivatives of 2'-deoxy-2'-fluorotetradialdose d-nucleosides and tetradialdose d-nucleosides.

Analogs of nucleosides and nucleotides represent a promising pool of potential therapeutics. This work describes a new synthetic route leading to 2'-deoxy-2'-fluorotetradialdose D-nucleoside phosphonates. Moreover, a new universal synthetic route leading to tetradialdose d-nucleosides bearing purine nucleobases is also described. All new compounds were tested as triphosphate analogs for inhibitory potency against a variety of viral polymerases. The fluorinated nucleosides were transformed to phosphoramidate prodrugs and evaluated in cell cultures against various viruses including influenza and SARS-CoV-2.

Kinetic Target-Guided Synthesis of Small-Molecule G-Quadruplex Stabilizers.

The formation of a G-quadruplex motif in the promoter region of the c-MYC protooncogene prevents its expression. Accordingly, G-quadruplex stabilization by a suitable ligand may be a viable approach for anticancer therapy. In our study, we used the 4-(4-methylpiperazin-1-yl)aniline molecule, previously identified as a fragment library screen hit, as a template for the SAR-guided design of a new small library of clickable fragments and subjected them to click reactions, including kinetic target-guided synthesis in the presence of a G-quadruplex forming oligonucleotide Pu24. We tested the clickable fragments and products of click reactions for their G-quadruplex stabilizing activity and determined their mode of binding to the c-MYC G-quadruplex by NMR spectroscopy. The enhanced stabilizing potency of click products in biology assays (FRET, Polymerase extension assay) matched the increased yields of in situ click reactions. In conclusion, we identified the newly synthesized click products of bis-amino derivatives of 4-(4-methylpiperazin-1-yl)aniline as potent stabilizers of c-MYC G-quadruplex, and their further evolution may lead to the development of an efficient tool for cancer treatment.

Switchable highly regioselective synthesis of 3,4-dihydroquinoxalin-2(1H)ones from o-phenylenediamines and aroylpyruvates.

3-Acylmethylidene-3,4-dihydroquinoxalin-2(1H)-ones are compounds which possess a wide range of physical and pharmaceutical applications. These compounds can be easily prepared by cyclocondensation of o-phenylenediamines and aroylpyruvates. Unsymmetrically substituted o-phenylenediamines can be obtained form regioisomeric mixtures of 3,4-dihydroquinoxalin-2(1H)-ones. It is often quite difficult to get a pure regioisomer and determine its structure without controlling the reaction selectivity and exploitation of complex NMR techniques (HSQC, NOESY, HMBC). This article examines the regioselectivity of the cyclocondensation between six monosubstituted o-phenylenediamines (-OMe, -F, -Cl, -COOH, -CN, -NO2) and the derivatives of p-chlorobenzoylpyruvate (ester or acid) which we studied. Six regioisomeric 3,4-dihydroquinoxalin-2(1H)-one pairs were selectively prepared and characterised. Based on our experiences, a simplified methodology for determining the structure of the regioisomers was proposed. We developed two general and highly selective methodologies starting from the same o-phenylenediamines and activated 4-chlorobenzoylpyruvates (ester or acid) enabling switching of 3,4-dihydroquinoxalin-2(1H)-one regioselectivity in a predictable manner. For comparison, all regioselective cyclocondensations were performed with the same standardized conditions (DMF, rt, 3 days), differing only by the additives p-TsOH or HOBt/DIC (hydroxybenzotriazole/N,N'-diisopropylcarbodiimide). Both selected methods are simple, general and highly regioselective (72-97%). A mechanism for the regioselectivity was also proposed and discussed. This study can be used as a guide when choosing the most optimal reaction conditions for the synthesis of the desired 3,4-dihydroquinoxalin-2(1H)-one regioisomers with the best selectivity. The demonstrated methodologies in this article may also be applied to differently substituted 3,4-dihydroquinoxalin-2(1H)-ones in general, which could expand the synthetic impact of our results.

Novel CLK1 inhibitors based on N-aryloxazol-2-amine skeleton - A possible way to dual VEGFR2 TK/CLK ligands.

BACKGROUND: Inhibitors of CLK protein kinases suppress cell growth and induce apoptosis by modulating pre-mRNA splicing in cancer. CLK family kinases are also involved in alternative splicing and RNA processing in Duchenne muscular dystrophy, Alzheimer's disease, HIV-1, and influenza virus. Small inhibitors are valuable tools for better understanding the molecular mechanisms of splicing and may serve as seeds for a novel class of therapeutics. ACHIEVEMENTS: Here we describe a discovery of four novel CLK1 inhibitors possessing N-aryloxazol-2-amine skeleton. Their activity against CLK1 (IC50: 20, 30, 40 and 80 nM) and some other CMGC kinases, predicted CLK binding poses, synthesis and physico-chemical characteristics are also stated. Additionally analysis of all PDB available CLK structures and interactions of their ligands was performed. There are only few powerful dual CLK/VEGFR inhibitors known in the literature. We proposed that our inhibitors have similar binding places and interactions in CLK1, 3 and VEGFR2 TK mostly due to the joint N-aryloxazol-2-amine pharmacophoric fragment. One of our N-aryloxazol-2-amines already proved a good activity against both VEGFR2 and CLK1 enzymes (23/80 nM, resp). We proposed that the presented class of compounds has a potential to be developed in dual VEGFR2/CLK clinical compounds with prospective synergy to treat cancer.

Ynamide Click chemistry in development of triazole VEGFR2 TK modulators.

Structure novelty, chemical stability and synthetic feasibility attracted us to design 1,2,3-triazole compounds as potential inhibitors of VEGFR2 tyrosine kinase. Novel triazoles T1-T7 were proposed by oxazole (AAZ from PDB: 1Y6A)/1,2,3-triazole isosteric replacement, molecular modelling and docking. In order to enable synthesis of T1-T7 we developed a methodology for preparation of ynamide 22. Compound 22 was used for all Click chemistry reactions leading to triazoles T1-T3 and T6-T7. Among the obtained products, T1, T3 and T7 specifically bind VEGFR2 TK and modulate its activity by concentration dependent manner. Moreover predicted binding poses of T1-T7 in VEGFR2 TK were similar to the one known for the oxazole inhibitor AAZ (PDB: 1Y6A). Unfortunately the VEGFR2 inhibition by triazoles e.g. T3 and T7 is lower than that determined for their oxazole bioisosters T3-ox and AAZ, resp. Different electronic properties of 1,2,3-triazole/oxazole heterocyclic rings were proposed to be the main reason for the diminished affinity of T1-T3, T6 and T7 to an oxazole AAZ inhibitor binding site in VEGFR2 TK (PDB: 1Y6A or 1Y6B). Moreover T1-T3 and T6 were screened on cytotoxic activity against two human hepatocellular carcinoma cell lines. Selective cytotoxic activity of T2 against aggressive Mahlavu cells has been discovered indicating possible affinity of T2 to Mahlavu constitutionally active PI3K/Akt pathway.

A development of chimeric VEGFR2 TK inhibitor based on two ligand conformers from PDB: 1Y6A complex--medicinal chemistry consequences of a TKs analysis.

VEGFR2 is an important mediator of angiogenesis and influences fate of some cancer stem cells. Here we analysed all 34 structures of VEGFR2 TK available from PDB database. From them a complex PDB: 1Y6A has an exceptional AAZ ligand bound to TK in form of two conformers (U- and S-shaped). This observation inspired us to develop three chimeric bispyridyl VEGFR2 inhibitors by combining structural features of both AAZ conformers and/or their relative ligand AAX (PDB: 1Y6B). Our most interesting inhibitor 22SYM has an enzymatic VEGFR2 TK activity (IC50: 15.1 nM) comparable or better to the active compounds from clinical drugs Nexavar and Sutent. 22SYM inhibits growth, migration and tube formation of endothelial cells (EC) and selectively induces EC apoptosis. 22SYM also inhibits in vivo angiogenesis in Zebrafish embryo assay. Additionally to the above results, we proved here that tyrosine kinases in an inactive form possessing Type I inhibitors can adopt both a closed or an opened conformation of kinase A-loop independently on their DFG-out arrangement. We proposed here that an activity of certain Type I inhibitors (e.g. 22SYM-like) in complex with DFG-out TK can be negatively influenced by collisions with a dynamically moving TK A-loop.