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Background: Patients with glioblastoma (GBM) have a poor prognosis and limited treatment options. The mRNA decapping enzyme scavenger (DCPS) is a cap-hydrolyzing enzyme. The DCPS inhibitor RG3039 exhibited excellent central nervous system bioavailability in vivo and was safe and well tolerated in healthy volunteers in a phase 1 clinical trial. In this study, we investigated the expression of DCPS in GBM and the anti-tumor activity of RG3039 in various preclinical models of GBM. Methods: DCPS expression was examined in human GBM and paired peritumoral tissues. Its prognostic role was evaluated together with clinicopathological characteristics of patients. The anti-GBM effect of RG3039 was determined using GBM cell lines, patient-derived organoids, and orthotopic mouse models. The therapeutic mechanisms of DCPS inhibition were explored. Results: DCPS is overexpressed in GBM and is associated with poor survival of patients with GBM. The DCPS inhibitor RG3039 exhibited robust anti-GBM activities in GBM cell lines, patient-derived organoids and orthotopic mouse models, with drug exposure achievable in humans. Mechanistically, RG3039 downregulated STAT5B expression, thereby suppressing proliferation, survival and colony formation of GBM cells. Conclusions: DCPS is a promising target for GBM. Inhibition of DCPS with RG3039 at doses achievable in humans downregulates STAT5B expression and reduces proliferation, survival and colony formation of GBM cells. Given the excellent anti-cancer activity and central nervous system bioavailability in vivo and good tolerance in humans, RG3039 warrants further study as a potential GBM therapy. [ABSTRACT FROM AUTHOR]

Nuclear magnetic resonance (NMR) spectroscopy is uniquely suited to study the dynamics of biomolecules in solution. Most NMR studies exploit the spins of proton, carbon and nitrogen isotopes, as these atoms are highly abundant in proteins and nucleic acids. As an alternative and complementary approach, fluorine atoms can be introduced into biomolecules at specific sites of interest. These labels can then be used as sensitive probes for biomolecular structure, dynamics or interactions. Here, we address if the replacement of tryptophan with 5-fluorotryptophan residues has an effect on the overall dynamics of proteins and if the introduced fluorine probe is able to accurately report on global exchange processes. For the four different model proteins (KIX, Dcp1, Dcp2 and DcpS) that we examined, we established that 15N CPMG relaxation dispersion or EXSY profiles are not affected by the 5-fluorotryptophan, indicating that this replacement of a proton with a fluorine has no effect on the protein motions. However, we found that the motions that the 5-fluorotryptophan reports on can be significantly faster than the backbone motions. This implies that care needs to be taken when interpreting fluorine relaxation data in terms of global protein motions. In summary, our results underscore the great potential of fluorine NMR methods, but also highlight potential pitfalls that need to be considered. [ABSTRACT FROM AUTHOR]

DCPS gene encodes for a protein involved in gene expression regulation through promoting cap degradation during mRNA decapping processes. Mutations altering the DCPS function have been associated to a distinct disorder, Al‐Raqad syndrome, so far described only in two families. We report on a patient harboring a novel homozygous missense mutation in DCPS, presenting with growth retardation, craniofacial anomalies, skin dyschromia, and neuromuscular defects. This case study explains the molecular spectrum of DCPS mutations and might contribute to the phenotypic delineation of this rare condition. [ABSTRACT FROM AUTHOR]

DcpS (scavenger decapping enzyme) from nematode C. elegans readily hydrolyzes both monomethyl- and trimethylguanosine cap analogues. The reaction was followed fluorimetrically. The marked increase of fluorescence intensity after the cleavage of pyrophosphate bond in dinucleotides was used to determine Km and Vmaxvalues. Kinetic parameters were similar for both classes of substrates and only slightly dependent on pH. The hydrolysis was strongly inhibited by methylene cap analogues (m7Gp(CH2)ppG and m7Gpp(CH2)pG) and less potently by ARCA (m7,3' OGpppG). [ABSTRACT FROM AUTHOR]

A series of new dinucleotide cap analogs with methylene groups replacing oxygens within the pyrophosphate moieties have been synthesized. All the compounds were resistant to the human scavenger decapping hydrolase, DcpS. Binding constants of the modified caps to eIF4E are comparable to those obtained for m 7 GpppG. This suggests these methylene modifications in the pyrophosphate chain do not significantly affect cap-binding at least for eIF4E. These cap analogs are also good inhibitors of in vitro translation. mRNAs capped with novel analogs were translated similarly to the mRNA capped with the parent m 7 GpppG. [ABSTRACT FROM AUTHOR]

Nucleosides and oligonucleotides with an oxygen replaced by sulfur atom are an interesting class of compounds because of their improved stability toward enzymatic cleavage by nucleases. We have synthesized several dinucleotide mRNA cap analogs containing a phosphorothioate moiety in the α, β, or γ position of 5′,5′-triphosphate chain [m 7 Gp(s)ppG, m 7 Gpp(s)pG, and m 7 Gppp(s)G]. These are the first examples of the biologically important 5′mRNA cap analogs containing a phosphorothioate moiety, and these compounds may be useful in a variety of biochemical and biotechnological applications. Incorporation of a sulfur atom in the α or γ position within the dinucleotide cap analog was achieved using PSCl 3 in a nucleoside phosphorylation reaction followed by coupling the phosphorothioate of nucleoside with a second nucleotide. Synthesis of cap analogs with the phosphorothioate moiety in β position was performed using an organic phosphorothioate salt in a coupling reaction with an activated nucleotide. The structures of newly synthesized compounds was confirmed using MS and 1 H and 31 P NMR spectroscopy. We present here the results of preliminary studies on their interaction with translation initiation factor eIF4E and enzymatic hydrolysis with human and nematode DcpS scavengers. [ABSTRACT FROM AUTHOR]

Dinucleotide analogs of the messenger RNA cap (m7GpppN) are useful research tools and have potential applications as translational inhibitors or reagents for modification of in vitro transcribed mRNAs. It has been previously reported that replacing the methyl group at the N7-position with benzyl (Bn) produces a dinucleotide cap with superior properties. Here, we followed up on this finding by synthesizing 17 novel Bn7GpppG analogs and determining their structure–activity relationship regarding translation and translational inhibition. The compounds were prepared in two steps, including selective N7-alkylation of guanosine 5′-monophosphate by arylmethyl bromide followed by coupling with imidazole-activated GDP, with total yields varying from 22% to 62%. The compounds were then evaluated by determining their affinity for eukaryotic translation initiation factor 4E (eIF4E), testing their susceptibility to decapping pyrophosphatase, DcpS—which is most likely the major cellular enzyme targeting this type of compound—and determining their translation inhibitory properties in vitro. We also synthesized mRNAs capped with the evaluated compounds and tested their translational properties in A549 cells. Our studies identified N7-(4-halogenbenzyl) substituents as promising modifications in the contexts of either mRNA translation or translational inhibition. Finally, to gain more insight into the consequences at the molecular level of N7-benzylation of the mRNA cap, we determined the crystal structures of three compounds with eIF4E. [ABSTRACT FROM AUTHOR]

The mRNA 5′ cap consists of N7-methylguanosine bound by a 5′,5′-triphosphate bridge to the first nucleotide of the transcript. The cap interacts with various specific proteins and participates in all key mRNA-related processes, which may be of therapeutic relevance. There is a growing demand for new biophysical and biochemical methods to study cap–protein interactions and identify the factors which inhibit them. The development of such methods can be aided by the use of properly designed fluorescent molecular probes. Herein, we synthesized a new class of m7Gp3G cap derivatives modified with an alkyne handle at the N1-position of guanosine and, using alkyne-azide cycloaddition, we functionalized them with fluorescent tags to obtain potential probes. The cap derivatives and probes were evaluated in the context of two cap-binding proteins, eukaryotic translation initiation factor (eIF4E) and decapping scavenger (DcpS). Biochemical and biophysical studies revealed that N1-propargyl moiety did not significantly disturb cap–protein interaction. The fluorescent properties of the probes turned out to be in line with microscale thermophoresis (MST)-based binding assays. [ABSTRACT FROM AUTHOR]