Vinylphosphonate-based cyclic dinucleotides enhance STING-mediated cancer immunotherapy.

Cyclic dinucleotides (CDNs) trigger the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway, which plays a key role in cytosolic DNA sensing and thus in immunomodulation against infections, cell damage and cancer. However, cancer immunotherapy trials with CDNs have shown immune activation, but not complete tumor regression. Nevertheless, we designed a novel class of CDNs containing vinylphosphonate based on a STING-affinity screening assay. In vitro, acyloxymethyl phosphate/phosphonate prodrugs of these vinylphosphonate CDNs were up to 1000-fold more potent than the clinical candidate ADU-S100. In vivo, the lead prodrug induced tumor-specific T cell priming and facilitated tumor regression in the 4T1 syngeneic mouse model of breast cancer. Moreover, we solved the crystal structure of this ligand bound to the STING protein. Therefore, our findings not only validate the therapeutic potential of vinylphosphonate CDNs but also open up opportunities for drug development in cancer immunotherapy bridging innate and adaptive immunity.

Design, Synthesis, and Biochemical and Biological Evaluation of Novel 7-Deazapurine Cyclic Dinucleotide Analogues as STING Receptor Agonists.

Cyclic dinucleotides (CDNs) are second messengers that activate stimulator of interferon genes (STING). The cGAS-STING pathway plays a promising role in cancer immunotherapy. Here, we describe the synthesis of CDNs containing 7-substituted 7-deazapurine moiety. We used mouse cyclic GMP-AMP synthase and bacterial dinucleotide synthases for the enzymatic synthesis of CDNs. Alternatively, 7-(het)aryl 7-deazapurine CDNs were prepared by Suzuki-Miyaura cross-couplings. New CDNs were tested in biochemical and cell-based assays for their affinity to human STING. Eight CDNs showed better activity than 2'3'-cGAMP, the natural ligand of STING. The effect on cytokine and chemokine induction was also evaluated. The best activities were observed for CDNs bearing large aromatic substituents that point above the CDN molecule. We solved four X-ray structures of complexes of new CDNs with human STING. We observed π-π stacking interactions between the aromatic substituents and Tyr240 that are involved in the stabilization of CDN-STING complexes.

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.

Enzymatic Preparation of 2'-5',3'-5'-Cyclic Dinucleotides, Their Binding Properties to Stimulator of Interferon Genes Adaptor Protein, and Structure/Activity Correlations.

Cyclic dinucleotides are second messengers in the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, which plays an important role in recognizing tumor cells and viral or bacterial infections. They bind to the STING adaptor protein and trigger expression of cytokines via TANK binding kinase 1 (TBK1)/interferon regulatory factor 3 (IRF3) and inhibitor of nuclear factor-κB (IκB) kinase (IKK)/nuclear factor-κB (NFκB) signaling cascades. In this work, we describe an enzymatic preparation of 2'-5',3'-5'-cyclic dinucleotides (2'3'CDNs) with use of cyclic GMP-AMP synthases (cGAS) from human, mouse, and chicken. We profile substrate specificity of these enzymes by employing a small library of nucleotide-5'-triphosphate (NTP) analogues and use them to prepare 33 2'3'CDNs. We also determine affinity of these CDNs to five different STING haplotypes in cell-based and biochemical assays and describe properties needed for their optimal activity toward all STING haplotypes. Next, we study their effect on cytokine and chemokine induction by human peripheral blood mononuclear cells (PBMCs) and evaluate their cytotoxic effect on monocytes. Additionally, we report X-ray crystal structures of two new CDNs bound to STING protein and discuss structure-activity relationship by using quantum and molecular mechanical (QM/MM) computational modeling.

Solid-Phase Synthesis of Phosphorothioate/Phosphonothioate and Phosphoramidate/Phosphonamidate Oligonucleotides.

We have developed a robust solid-phase protocol which allowed the synthesis of chimeric oligonucleotides modified with phosphodiester and O-methylphosphonate linkages as well as their P-S and P-N variants. The novel O-methylphosphonate-derived modifications were obtained by oxidation, sulfurization, and amidation of the O-methyl-(H)-phosphinate internucleotide linkage introduced into the oligonucleotide chain by H-phosphonate chemistry using nucleoside-O-methyl-(H)-phosphinates as monomers. The H-phosphonate coupling followed by oxidation after each cycle enabled us to successfully combine H-phosphonate and phosphoramidite chemistries to synthesize diversely modified oligonucleotide strands.

Tuning the hybridization properties of modified oligonucleotides: from flexible to conformationally constrained phosphonate internucleotide linkages.

The concept of conformational restriction leading to the preorganization of modified strands has proven to be successful and has afforded nucleic acid analogues with many interesting properties suitable for various biochemical applications. We utilized this concept to prepare a set of constrained oligonucleotides derived from 1,4-dioxane and 1,3-dioxolane-locked nucleoside phosphonates and evaluated their hybridization affinities towards their complementary RNA strands. With an increase of ΔTm per modification up to +5.2 °C, the hybridization experiments revealed the (S)-2',3'-O-phosphonomethylidene internucleotide linkage as one of the most Tm-increasing modifications reported to date. Moreover, we introduced a novel prediction tool for the pre-selection of potentially interesting chemical modifications of oligonucleotides.

4-Toluenesulfonyloxymethyl-(H)-phosphinate: A Reagent for the Introduction of O- and S-Methyl-(H)-phosphinate Moieties.

The straightforward synthesis of sodium 4-toluenesulfonyloxymethyl-(H)-phosphinate and (H)-phosphinomethylisothiouronium tosylate as new reagents for the preparation of O- and S-methyl-(H)-phosphinic acid derivatives, respectively, is described. The reactivity of both reagents was demonstrated by the preparation of protected 2'-deoxyribonucleoside-O-methyl-(H)-phosphinates in the 5'- and 3'-series and 2',5'-dideoxyribonucleoside-5'-S-methyl-(H)-phosphinates. These compounds represent a new class of monomers compatible with the solid phase synthesis of oligonucleotides by H-phosphonate chemistry, as it was proved with the synthesis of a fully phosphonate heptamer.

Dr Jekyll and Mr Hyde: a strange case of 5-ethynyl-2'-deoxyuridine and 5-ethynyl-2'-deoxycytidine.

5-Ethynyl-2'-deoxyuridine (EdU) and 5-ethynyl-2'-deoxycytidine (EdC) are mainly used as markers of cellular replicational activity. Although EdU is employed as a replicational marker more frequently than EdC, its cytotoxicity is commonly much higher than the toxicity of EdC. To reveal the reason of the lower cytotoxicity of EdC, we performed a DNA analysis of five EdC-treated human cell lines. Surprisingly, not a single one of the tested cell lines contained a detectable amount of EdC in their DNA. Instead, the DNA of all the cell lines contained EdU. The content of incorporated EdU differed in particular cells and EdC-related cytotoxicity was directly proportional to the content of EdU. The results of experiments with the targeted inhibition of the cytidine deaminase (CDD) and dCMP deaminase activities indicated that the dominant role in the conversion pathway of EdC to EdUTP is played by CDD in HeLa cells. Our results also showed that the deamination itself was not able to effectively prevent the conversion of EdC to EdCTP, the conversion of EdC to EdCTP occurs with much lesser effectivity than the conversion of EdU to EdUTP and the EdCTP is not effectively recognized by the replication complex as a substrate for the synthesis of nuclear DNA.

Straightforward Synthesis of Purine 4'-Alkoxy-2'-deoxynucleosides: First Report of Mixed Purine-Pyrimidine 4'-Alkoxyoligodeoxynucleotides as New RNA Mimics.

Purine and pyrimidine 4'-alkoxy-2'-deoxynucleosides were efficiently prepared from nucleoside 4'-5'-enol acetates in three steps by N-iodosuccinimide promoted alkoxylation, hydrolysis, and reduction followed by conversion to phosphoramidite monomers for the solid-phase synthesis of the oligonucleotides. Fully modified 4'-alkoxyoligodeoxynucleotides, which are characterized by a prevalent N-type (RNA-like) conformation, exhibited superior chemical and nuclease resistance as well as excellent hybridization properties with a strong tendency for RNA-selective hybridization, suggesting a potential application of 4'-alkoxy-oligodeoxynucleotides in antisense technologies.

The Fingerprint of Anti-Bromodeoxyuridine Antibodies and Its Use for the Assessment of Their Affinity to 5-Bromo-2'-Deoxyuridine in Cellular DNA under Various Conditions.

We have developed a simple system for the analysis of the affinity of anti-bromodeoxyuridine antibodies. The system is based on the anchored oligonucleotides containing 5-bromo-2'-deoxyuridine (BrdU) at three different positions. It allows a reliable estimation of the reactivity of particular clones of monoclonal anti-bromodeoxyuridine antibodies with BrdU in fixed and permeabilized cells. Using oligonucleotide probes and four different protocols for the detection of BrdU incorporated in cellular DNA, we identified two antibody clones that evinced sufficient reactivity to BrdU in all the tested protocols. One of these clones exhibited higher reactivity to 5-iodo-2'-deoxyuridine (IdU) than to BrdU. It allowed us to increase the sensitivity of the used protocols without a negative effect on the cell physiology as the cytotoxicity of IdU was comparable with BrdU and negligible when compared to 5-ethynyl-2'-deoxyuridine. The combination of IdU and the improved protocol for oxidative degradation of DNA provided a sensitive and reliable approach for the situations when the low degradation of DNA and high BrdU signal is a priority.

5'-O-Methylphosphonate nucleic acids--new modified DNAs that increase the Escherichia coli RNase H cleavage rate of hybrid duplexes.

Several oligothymidylates containing various ratios of phosphodiester and isopolar 5'-hydroxyphosphonate, 5'-O-methylphosphonate and 3'-O-methylphosphonate internucleotide linkages were examined with respect to their hybridization properties with oligoriboadenylates and their ability to induce RNA cleavage by ribonuclease H (RNase H). The results demonstrated that the increasing number of 5'-hydroxyphosphonate or 5'-O-methylphosphonate units in antisense oligonucleotides (AOs) significantly stabilizes the heteroduplexes, whereas 3'-O-methylphosphonate AOs cause strong destabilization of the heteroduplexes. Only the heteroduplexes with 5'-O-methylphosphonate units in the antisense strand exhibited a significant increase in Escherichia coli RNase H cleavage activity by up to 3-fold (depending on the ratio of phosphodiester and phosphonate linkages) in comparison with the natural heteroduplex. A similar increase in RNase H cleavage activity was also observed for heteroduplexes composed of miRNA191 and complementary AOs containing 5'-O-methylphosphonate units. We propose for this type of AOs, working via the RNase H mechanism, the abbreviation MEPNA (MEthylPhosphonate Nucleic Acid).

Inhibition of human thymidine phosphorylase by conformationally constrained pyrimidine nucleoside phosphonic acids and their "open-structure" isosteres.

A series of conformationally constrained uridine-based nucleoside phosphonic acids containing annealed 1,3-dioxolane and 1,4-dioxane rings and their "open-structure" isosteres were synthesized and evaluated as potential multisubstrate-like inhibitors of the human recombinant thymidine phosphorylase (TP, EC 2.4.2.4) and TP obtained from peripheral blood mononuclear cells (PBMC). From a large set of tested nucleoside phosphonic acids, several potent compounds were identified that exhibited Ki values in the range of 0.048-1 µM. The inhibition potency of the studied compounds strongly depended on the degree of conformational flexibility of the phosphonate moiety, the stereochemical arrangement of the sugar-phosphonate component, and the substituent at position 5 of the pyrimidine nucleobase.

Most anti-BrdU antibodies react with 2'-deoxy-5-ethynyluridine -- the method for the effective suppression of this cross-reactivity.

5-Bromo-2'-deoxyuridine (BrdU) and 2'-deoxy-5-ethynyluridine (EdU) are widely used as markers of replicated DNA. While BrdU is detected using antibodies, the click reaction typically with fluorescent azido-dyes is used for EdU localisation. We have performed an analysis of ten samples of antibodies against BrdU with respect to their reactivity with EdU. Except for one sample all the others evinced reactivity with EdU. A high level of EdU persists in nuclear DNA even after the reaction of EdU with fluorescent azido-dyes if the common concentration of dye is used. Although a ten-time increase of azido-dye concentration resulted in a decrease of the signal provided by anti-BrdU antibodies, it also resulted in a substantial increase of the non-specific signal. We have shown that this unwanted reactivity is effectively suppressed by non-fluorescent azido molecules. In this respect, we have tested two protocols of the simultaneous localisation of incorporated BrdU and EdU. They differ in the mechanism of the revelation of incorporated BrdU for the reaction with antibodies. The first one was based on the use of hydrochloric acid, the second one on the incubation of samples with copper(I) ions. The use of hydrochloric acid resulted in a significant increase of the non-specific signal. In the case of the second method, no such effect was observed.

Atomic scissors: a new method of tracking the 5-bromo-2'-deoxyuridine-labeled DNA in situ.

A new method of the light microscopy detection of BrdU-labeled DNA in situ is described. It is based on the oxidative attack at the deoxyribose moiety by copper(I) in the presence of oxygen, which leads to the abstraction of hydrogen atom from deoxyribose culminating in the elimination of the nucleobase, scission of the nucleic-acid strand and formation of frequent gaps. The gaps allow the reaction of the antibodies with the commonly used markers of replication (e.g. 5-bromo-2'-deoxyuridine), which are otherwise masked. The method developed makes it possible to detect nuclear and mitochondrial DNA replication efficiently. In most cases, it does not inhibit effective protein detections and in addition enables simultaneous localization of newly-synthesized RNA. The alternative presently-used methods result in protein denaturation and/or extensive DNA cleavage followed by the DNA-bound proteins peeling off.

4'-Alkoxy oligodeoxynucleotides: a novel class of RNA mimics.

4'-Alkoxy-oligothymidylates were prepared as model compounds to study the influence of a C4'-alkoxy group on hybridisation. The phosphodiester homooligomers (15 units long) containing either a 4'-methoxy or 4'-(2-methoxyethoxy) group were found to display increased hybridisation with both dA(15) and rA(15) complementary counterparts compared to the natural oligothymidylate. In addition, we found their hybridisation behaviour to be similar to that of the regioisomeric 2'-O-methyl-oligothymidylate. The formed complexes (duplexes and triplexes) were studied using UV spectroscopy and polyacrylamide gel electrophoresis (PAGE). Structural background of the hybridization behaviour was examined using NMR and MDS. The favourable hybridisation properties of the 4'-alkoxyoligothymidylates indicated that 4'-alkoxy modified nucleotides are promising compounds for the assembly of chimeric oligonucleotides with tunable properties.

Unusual activity pattern of leucine aminopeptidase inhibitors based on phosphorus containing derivatives of methionine and norleucine.

Ligands containing bulky aliphatic P1 residues exhibit a high affinity towards cytosolic leucine aminopeptidase, a bizinc protease of biomedical significance. According to this specificity, a series of phosphonic and phosphinic compounds have been put forward as novel putative inhibitors of the enzyme. These phosphonic and phosphinic compounds were derivatives of methionine and norleucine as both single amino acids and dipeptides. The designed inhibitors were synthesised and tested towards the peptidase isolated from porcine kidneys using an improved separation procedure affording superior homogeneity. Unexpectedly, organophosphorus derivatives of methionine and norleucine exhibited moderate activity with K(i) values in the micromolar range.

Structural diversity of nucleoside phosphonic acids as a key factor in the discovery of potent inhibitors of rat T-cell lymphoma thymidine phosphorylase.

Structurally diverse, sugar-modified, thymine-containing nucleoside phosphonic acids were evaluated for their ability to inhibit thymidine phosphorylase (TP, EC 2.4.2.4) purified from spontaneous T-cell lymphomas of an inbred Sprague-Dawley rat strain. From a large set of tested compounds, among them a number of pyrrolidine-based derivatives, 10 nucleotide analogues with IC(50) values below 1 microM were selected. Out of them, four compounds strongly inhibited the enzyme with IC(50) values lying in a range of 11-45 nM. These most potent compounds might be bi-substrate analogues.

Chiral phosphonate internucleotide linkage: a promising modification for chimeric oligonucleotides?

We have synthesized two different groups of oligonucleotides containing chiral isopolar nonisosteric phosphonate internucleotide linkages, and studied their properties in combination with natural phosphodiester ones. The improved synthetic procedures for the monomers preparation are also reported.

Prolinol-based nucleoside phosphonic acids: synthesis and properties.

Commercially available trans-4-hydroxy-L-proline has been used as a starting material for the synthesis of prolinol-based nucleotide analogues with N-phosphonomethyl moiety attached to the nitrogen atom of prolinol ring. The synthetic methodology based on the inversion of configuration at both 1- and 4- positions led, in result, to all diastereoisomeric O-protected 4-mesyloxyprolinol-N-methylphosphonates. Alkylation of nucleobases using the synthons afforded the nucleotide analogues corresponding to alpha- and beta-nucleotides in both L- and D-series. The NMR-based conformational study of alpha- and beta-nucleotides in aqueous solution performed at two different pH values securing either N-fully protonated or deprotonated forms revealed in both cases occurrence of the same mostly populated conformer. All final prolinol-based nucleoside phosphonic acids were tested for cytotoxic and antiviral properties, but no significant activity was found.

Nucleoside phosphonic acids in thymidine phosphorylase inhibition: structure-activity relationship.

A number of structurally diverse nucleoside phosphonic acids have been tested against human recombinant thymidine phosphorylase and human platelets supernatant using 2'-deoxy-5-nitrouridine as the substrate. We have selected several inhibitors working at micromolar level as lead structures for further evaluation.