Hagen Cramer

2-5A ligands - A new concept for the treatment of prostate cancer

Several potent prostate specific membrane antigen (PSMA) inhibitors have been described recently. We generated a PSMA-specific 2-5A ligand called RBI 1033 by linking 2-5A to the N-acetylaspartylglutamate (NAAG)-based inhibitor ZJ-24. We measured the inhibitory activity of RBI 1033 to the folate hydrolase activity of PSMA. Amazingly, we found that compared to ZJ-24 (IC50 = 53.9 nM), RBI 1033 was more than 10 times more potent (IC50 = 4.78 nM) as a folate hydrolase inhibitor, while SMCC 2-5A lacking the ZJ-24 part, did not show much activity (IC50 = 1974 nM). Also, RBI 1033s affinity to PSMA was found to be 10 times higher than ZJ-24 itself.

Nucleotides LXIV[1]: Synthesis, Hybridization and Enzymatic Degradation Studies of 2′-O-Methyl-Oligoribonucleotides and 2′-O-Methyl/Deoxy Gapmers

Abstract 2′-O-Methyloligoribonucleotides, deoxyoligonucleotides and 2′-O-methyl/ deoxy gapmers were synthesized using solid phase phosphoramidite chemistry employing the 2-(4-nitrophenyl)ethyl (npe) protection strategy. Melting temperatures of the synthesized oligonucleotides as well as their stability against degradation by several different nucleases were determined. 2′-O-Methyloligoribonucleotides showed the highest melting temperatures (Tms) whereas 2′-O-methyl/deoxy gapmers revealed either slightly higher or surprizingly no thermal stabilities compared with their deoxy analogs when using selfcomplementary sequences. Gapmers with four 2′-O-methyl nucleotides on both ends showed about the same stability as all 2′-O-methyloligoribonucleotides against micrococal nuclease, nuclease S1, and snake venom phosphodiesterase.

Targeted therapy of respiratory syncytial virus by 2-5A antisense.

Respiratory syncytial virus is a leading cause of respiratory disease in infants, young children, immunocompromized patients, and the elderly. Previous work has shown that RNase L, an antiviral enzyme of the interferon system, can be recruited to cleave RSV genomic RNA by attaching tetrameric 2′-5′-linked oligoadenylates (2 5A) to an antisense oligonucleotide complementary to repetitive intergenic sequences within the RSV genome (2 5A antisense). RBI034, a 2′-O-methyl RNA-modified analogue of the 2 5A anti-RSV compound, was found to have enhanced antiviral activity in cell culture studies while also cleaving RSV genomic RNA in an RNase L· and sequence-specific manner. RBI034′s efficacy in suppressing RSV replication in cell culture is 50 to 100 times better than ribavirin, the only approved drug for RSV infection. Here we show that the activity of 2 5A antisense compound can be further enhanced by a combination treatment with interferon or ribavirin. The anti-RSV activity resulting from combination treatment is more potent than either treatment alone. We also demonstrate that RBI034 is effective against RSV in three different species: mice, cotton rats, and African green monkeys.

The use of triisopropylsilyl-oxymethyl (TOM) in the synthesis of anti-telomerase 2-5A antisense compound RBI 011.

Abstract The 2-5A antisense compound RBI 011 targeting telomerase RNA was synthesized using the triisopropylsilyl-oxymethyl (TOM) group for the 3′-hydroxyl protection of 2′,5′-linked RNA.

Using fluorescence resonance energy transfer (FRET) for measuring 2-5A analogues ability to activate RNase L.

The development of a method for measuring the ability of 2-5A analogues to activate the cleavage of an oligoribonucleotide substrate by RNase L is described. This method is based on fluorescence resonance energy transfer. The method is easily performed with 96-well plates, allowing for quantitative high-throughput analyses of 2-5A analogues under different reaction conditions.

Synthesis of (2′,5′)-Oligoadenylate Antisense Chimeras Targeting Steroid 5α-Reductase

Abstract 2–5A antisense chimeras have been synthesized which target human steroid 5α-reductase mRNA. To enhance the stability of the chimera towards degradative enzymes the terminal phosphodiester bond was isomerized from 3′,5′ to 3′,3′ and the 5′-phosphate group was thiolated.