Muthian Shanmugasundaram

Locked nucleic acid (LNA)-modified dinucleotide mRNA cap analogue: synthesis, enzymatic incorporation, and utilization.

There has been considerable therapeutic interest in the development of human vaccines against cancers and infectious diseases such as HIV and biowarfare agents by using transfected mRNAs for antigenic proteins of interest. The highest expression levels of these proteins are obtained when the transfected mRNA contains 5-capped ends. In the present study, the locked nucleic acid (LNA)-modified cap analogue 3, m(7(LNA))G[5]ppp[5]G, has been synthesized and its biological properties were examined. The LNA-modified cap analogue was an efficient substrate for T7 RNA polymerase, and the mRNA transcribed, with a poly(A) tail, was efficiently utilized in an in vitro translation process. The RNA with the 5-LNA-modified cap was found to be approximately 1.61- and 1.28-fold more stable than the RNA with the 5-standard 4 and ARCA cap, respectively, and approximately 4.23-fold more stable than the uncapped control RNA. The RNA capped with the m(7(LNA))G[5]ppp[5]G 3 cap analogue was translated the most efficiently, with approximately 3.2-fold more activity than the standard cap, m(7)G[5]ppp[5]G 4. Furthermore, we have developed a nonradioactive analytical HPLC assay to determine that the LNA-modified 3 cap analogue was incorporated solely into the forward orientation. Molecular modeling of the m(7(LNA))G[5]ppp[5]G 3 cap analogue with the cap binding protein elF4E complex indicates that the LNA-modified cap-protein complex is more stable by 47.28 kcal/mol as compared to the standard mCAP-protein complex. These findings suggest that the new antireverse cap analogue m(7(LNA))G[5]ppp[5]G 3 is a potential candidate for RNA-based therapeutic vaccine production as well as studying biochemical processes.

Synthesis and application of a new 2′,3′-isopropylidene guanosine substituted cap analog

The synthesis and biological evaluation of a new cap analog, which is modified at the C2 and C3 positions of N(7)-methylguanosine is reported. The new cap analog, P(1)-2,3-isopropylidene, 7-methylguanosine-5P(3)-guanosine-5triphosphate was assayed with respect to its effects on efficiency of incorporation into RNAs during in vitro transcription, and intracellular stability and translational activity of its 5-capped mRNAs, upon transfection into HeLa cells. The intracellular stability of 5-capped and uncapped full length test mRNAs was measured by using a real-time RT-PCR assay. The RNA with the 5-modified cap was found to be approximately 1.7 times more stable than the RNA with the 5-standard cap and approximately 2.5 times more stable than the uncapped control RNA. The translational efficiency was monitored by measuring the luciferase activity of a variety of in vitro synthesized and capped RNAs coding for a luciferase fusion protein after transfection into HeLa cells. The RNA capped with the 2,3-isopropylidene substituted analog, (m(7,2,3-isopropylidene)G[5]ppp[5]G), was translated the most efficiently, with approximately 2.9-fold more activity than the standard cap (m(7)G[5]ppp[5]G). The observed increase in the level of protein synthesis is likely resulted as a consequence of exclusively forward capped transcripts and increased cellular stability of the 5-modified capped mRNA (Poly A).

Synthesis and biological evaluation of trimethyl-substituted cap analogs

The N(7)-methyl guanosine cap located on the 5-terminus of mRNAs is important for a number of biochemical processes. A new dinucleoside triphosphate cap analog was synthesized with methyl groups on the N(7) of both guanine moieties, as well as the 3-OH of one of the ribose moieties [see text]. The function of this trimethylated cap analog was compared with those of three other, less-methylated cap analogs: one omitting the ribose methylation (m(7)G[5]ppp[5]m(7)G), one omitting the N(7) methylation linked to the unmodified ribose [see text], and the standard cap analog, m(7)G[5]ppp[5]G. These cap modifications were assayed with respect to their effects on capping efficiency, yield of RNAs during in vitro transcription, and the translational activity of these RNAs upon transfection into HeLa cells. The translational activity was monitored by measuring the luciferase activity of a luciferase-fusion protein produced from the in vitro synthesized RNAs. The RNA capped with the trimethylated analog [see text] was translated the most efficiently, with approximately 2.6-fold more activity than the conventional cap (m(7)G[5]ppp[5]G). The other two variants were also more efficient, generating, approximately 2.2 times (for the [see text] analog) and, approximately 1.6 times (for the m(7)G[5]ppp[5]m(7)G analog) more luciferase function than the conventional cap.

Novel serine protease inhibitors

The pursuit of serine protease inhibitors as anticoagulants and anti-hepatitis C virus drugs continues to be an active area of research. Compounds such as P1-P3 macrocyclic peptides and linear peptides have been registered as potent hepatitis C virus protease inhibitors and compounds such as phenylglycinamide derivatives, substituted biaryls, tetrahydroquinoline derivatives, arylpropionamides, arylacrylamides, arylpropynamides, arylmethylurea analogs and peptides as factor XIa inhibitors. Given the recent US FDA approval of telaprevir and boceprevir for the treatment of hepatitis C virus, the development of new serine protease inhibitors is likely to be one of the hottest areas in the pharmaceutical industry. This review covers the patent literature on serine protease inhibitors during the period between 2009 and 2010.