Substrate-Based Design of Cytosolic Nucleotidase IIIB Inhibitors and Structural Insights into Inhibition Mechanism.

Cytosolic nucleotidases (cNs) catalyze dephosphorylation of nucleoside 5'-monophosphates and thereby contribute to the regulation of nucleotide levels in cells. cNs have also been shown to dephosphorylate several therapeutically relevant nucleotide analogues. cN-IIIB has shown in vitro a distinctive activity towards 7-mehtylguanosine monophosphate (m7GMP), which is one key metabolites of mRNA cap. Consequently, it has been proposed that cN-IIIB participates in mRNA cap turnover and prevents undesired accumulation and salvage of m7GMP. Here, we sought to develop molecular tools enabling more advanced studies on the cellular role of cN-IIIB. To that end, we performed substrate and inhibitor property profiling using a library of 41 substrate analogs. The most potent hit compounds (identified among m7GMP analogs) were used as a starting point for structure-activity relationship studies. As a result, we identified several 7-benzylguanosine 5'-monophosphate (Bn7GMP) derivatives as potent, unhydrolyzable cN-IIIB inhibitors. The mechanism of inhibition was elucidated using X-ray crystallography and molecular docking. Finally, we showed that compounds that potently inhibit recombinant cN-IIIB have the ability to inhibit m7GMP decay in cell lysates.

Phosphodiester modifications in mRNA poly(A) tail prevent deadenylation without compromising protein expression.

Chemical modifications enable preparation of mRNAs with augmented stability and translational activity. In this study, we explored how chemical modifications of 5',3'-phosphodiester bonds in the mRNA body and poly(A) tail influence the biological properties of eukaryotic mRNA. To obtain modified and unmodified in vitro transcribed mRNAs, we used ATP and ATP analogs modified at the α-phosphate (containing either O-to-S or O-to-BH3 substitutions) and three different RNA polymerases-SP6, T7, and poly(A) polymerase. To verify the efficiency of incorporation of ATP analogs in the presence of ATP, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantitative assessment of modification frequency based on exhaustive degradation of the transcripts to 5'-mononucleotides. The method also estimated the average poly(A) tail lengths, thereby providing a versatile tool for establishing a structure-biological property relationship for mRNA. We found that mRNAs containing phosphorothioate groups within the poly(A) tail were substantially less susceptible to degradation by 3'-deadenylase than unmodified mRNA and were efficiently expressed in cultured cells, which makes them useful research tools and potential candidates for future development of mRNA-based therapeutics.

Exploring tryptamine conjugates as pronucleotides of phosphate-modified 7-methylguanine nucleotides targeting cap-dependent translation.

Eukaryotic translation initiation factor 4E (eIF4E) is overexpressed in many cancers deregulating translational control of the cell cycle. mRNA 5' cap analogs targeting eIF4E are small molecules with the potential to counteract elevated levels of eIF4E in cancer cells. However, the practical utility of typical cap analogs is limited because of their reduced cell membrane permeability. Transforming the active analogs into their pronucleotide derivatives is a promising approach to overcome this obstacle. 7-Benzylguanosine monophosphate (bn7GMP) is a cap analog that has been successfully transformed into a cell-penetrating pronucleotide by conjugation of the phosphate moiety with tryptamine. In this work, we explored whether a similar strategy is applicable to other cap analogs, particularly phosphate-modified 7-methylguanine nucleotides. We report the synthesis of six new tryptamine conjugates containing N7-methylguanosine mono- and diphosphate and their analogs modified with thiophosphate moiety. These new potential pronucleotides and the expected products of their activation were characterized by biophysical and biochemical methods to determine their affinity towards eIF4E, their ability to inhibit translation in vitro, their susceptibility to enzymatic degradation and their turnover in cell extract. The results suggest that compounds containing the thiophosphate moiety may act as pronucleotides that release low but sustainable concentrations of 7-methylguanosine 5'-phosphorothioate (m7GMPS), which is a translation inhibitor with in vitro potency higher than bn7GMP.

ExciTides: NTP-derived probes for monitoring pyrophosphatase activity based on excimer-to-monomer transitions.

We describe a new type of nucleotide-derived fluorescent probe designed for monitoring pyrophosphatase activity based on excimer-to-monomer transitions, called ExciTide. The nucleotides were designed with two self-interacting dye moieties and synthesised using copper-catalysed azide-alkyne cycloaddition click chemistry. We applied these probes for enzyme activity monitoring and inhibitor evaluation. Some of the probes permeated into living cells, yielding interesting prospects for future applications.

Analysis of mononucleotides by tandem mass spectrometry: investigation of fragmentation pathways for phosphate- and ribose-modified nucleotide analogues.

Synthetic nucleotide and nucleic acid analogues are useful research tools and modern therapeutics. Hence, methods for the rapid and unambiguous identification of mononucleotides derived from organic syntheses or biological materials are of broad interest. Here, we analysed over 150 mononucleotides (mostly nucleoside 5'-mono-, 5'-di-, and 5'-triphosphates) and their structurally related nucleobase-, phosphate-, and ribose-modified analogues by electrospray tandem mass spectrometry (ESI/MS/MS), identifying characteristic fragmentation ions that may be helpful in structure determination. While positive-ion mode yielded fragments derived mainly from nucleobases, negative-ion mode provided insight into the structures of phosphoryl and phosphoribosyl moieties, enabling the determination of structural features such as the number of phosphate groups and the presence of ribose or phosphate substitutions. Based on these data, we proposed fragmentation pathways that were confirmed by experiments with [18O]-isotopologues. We demonstrated the utility of ESI(-)/MS/MS in the analysis of structurally related compounds by analysing isomeric and isobaric nucleotides and applying ESI(-)/MS/MS to rapid identification of nucleotide synthesis products. We formulated general rules regarding nucleotide structure-fragmentation pattern relationships and indicating characteristic fragmentation ions for the interpretation of ESI(-)/MS/MS spectra of nucleotides and their analogues. The ESI(-)/MS/MS spectra of all nucleotides are available in an on-line database, msTide, at www.msTide-db.com.

Selective and Cooperative Ligand Binding to Antiparallel Human Telomeric DNA G-Quadruplexes.

The quest for ligands that specifically bind to particular G-quadruplex nucleic acid structures is particularly important to conceive molecules with specific effects on gene expression or telomere maintenance, or conceive structure-specific molecular probes. Using electrospray mass spectrometry in native conditions, we reveal a highly cooperative and selective 2:1 binding of Cu(II) -tolylterpyridine complexes to human telomeric G-quadruplexes. Circular dichroism and comparisons of affinities for different sequences reveal a marked preference for antiparallel structures with diagonal loops and/or wide-medium-narrow-medium groove-width order. The cooperativity is attributed to conformational changes in the polymorphic telomeric G-quadruplex sequences, which convert preferably into an antiparallel three-quartet topology upon binding of two ligands.

Evaluation of dimethyl sulfoxide (DMSO) as a mobile phase additive during top 3 label-free quantitative proteomics.

Dimethyl sulfoxide (DMSO) has been advocated as a beneficial additive to electrospray solvents for peptide analysis due to the improved ionisation efficiency conferred. Previous reports have shown that the resultant improvements in peptide ion signal intensities are non-uniform. As a result, it was hypothesised that inclusion of DMSO in electrospray solvents could be detrimental to the outcome of intensity-based label-free absolute quantification approaches, specifically the top 3 method. The effect of DMSO as a mobile phase additive in top 3 label-free quantification was therefore evaluated. We show that inclusion of DMSO enhances data quality, improving the precision and number of proteins quantified, with no significant change to the quantification values observed in its absence.