Guanine-rich DNA and RNA sequences can fold into unique structures known as G-quadruplexes. The structures of G-quadruplexes can be divided into several classes, depending on the parallel or antiparallel nature of the strands and the number of G-rich tracts present in an oligonucleotide. Oligonucleotides with single tracts of guanines form intermolecular parallel tetrameric G-quadruplexes. Oligonucleotides with two tracts of guanosines separated by two or more bases can form both intermolecular antiparallel fold-back dimeric and parallel tetrameric G-quadruplexes, and those with four tracts of guanosines can form both intramolecular parallel and antiparallel structures. Intramolecular G-qaudruplexes can fold into several folding topologies including antiparallel crossover basket, antiparallel chair, and parallel propeller. The ability to control the folding of G-quadruplexes would allow the physical, biochemical, and biological properties of these various folding topologies to be studied. Previously, the known methods to control the folding topology of G-quadruplexes included changing the buffer by varying the mono- and divalent cations that are present, and by changing the DNA sequence. Because the glycosidic bonds in the G-quartets of G-quadruplexes with parallel strands are in the anti conformation, we reasoned that incorporation of nucleoside analogues that prefer the anti conformation of the glycosidic bond into G-rich sequences would increase the preference for parallel G-quadruplex formation. As predicted, by positioning the conformationally constrained nucleotide analogue 2'-O-4'-C-methylene-linked ribonucleotide into specific positions of a DNA G-quadruplex we were able to shift the thermodynamically favored structure of a G-quadruplex from an antiparallel to a parallel structure.

Ribose cysteine (RibCys) is a cysteine prodrug that increases both hepatic and renal glutathione with documented antagonism of acetaminophen (APAP)-induced hepatotoxicity. To determine if RibCys could also protect against APAP-induced kidney damage, mice were injected with APAP (600 mg/kg) or APAP and RibCys (1.0 g/kg) (APAP/RIB) followed by additional RibCys injections 1 and 2 hours later. Mice were euthanatized 10-12 hours after APAP administration, and liver and kidney toxicity were assessed by plasma sorbitol dehydrogenase (SDH) activity and blood urea nitrogen (BUN), respectively, and by histopathology. APAP treatment resulted in elevation of SDH activity and BUN to 2,490 U/ml and 47 mg/dl, respectively. By contrast, SDH and BUN values for APAP/RIB-treated mice were not different from controls, 0 U/ml and 31 mg/dl, respectively. Histopathologic examination revealed moderate to severe hepatic centrilobular necrosis in 9/11 and renal proximal tubular necrosis in 10/11 APAP-treated mice. However, no evidence of hepatic or renal toxicity was noted in any of the 12 APAP/RIB-treated mice. Utilizing the same treatment regimen, APAP covalent binding to hepatic and renal cytosolic proteins was assessed 4 hours after APAP challenge. RibCys cotreatment decreased covalent binding to the 58-kDa acetaminophen-binding protein in both liver and kidney. RibCys decreased both toxicity and covalent binding after APAP administration, and in addition to protecting the liver, this cysteine prodrug can also effectively protect the kidney from APAP-induced injury.

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Ribose cysteine (2(R,S)-D-ribo-(1ø,2ø,3ø,4ø-tetrahydroxybutyl)thiazolidine-4(R)-carboxylic acid) protects against acetaminophen-induced hepatic and renal toxicity. The mechanism for this protection is not known, but may involve inactivation of the toxic electrophile via enhancement of glutathione (GSH) biosynthesis. Therefore, the goal of this study was to determine if GSH biosynthesis was required for the ribose cysteine protection. Male CD-1 mice were injected with either acetaminophen or acetaminophen and ribose cysteine. The ribose cysteine cotreatment antagonized the acetaminophen-induced depletion of non-protein sulfhydryls in liver as well as GSH in kidney. Moreover, ribose cysteine cotreatment significantly increased the concentration of acetaminophen-cysteine, hepatic acetaminophen-mercapturate in liver and renal acetaminophen-GSH metabolites in kidney 4 hr after acetaminophen. To determine whether protection against acetaminophen-induced liver and kidney damage involved ribose cysteine dependent GSH biosynthesis, buthionine sulfoximine was used to selectively block g-glutamylcysteine synthetase (g-GCS). Plasma sorbitol dehydrogenase (SDH) activity and blood urea nitrogen from mice pretreated with buthionine sulfoximine and challenged with acetaminophen indicated that both liver and kidney injury had occurred. While co-treatment with ribose cysteine had previously protected against acetaminophen-induced liver and kidney injury, it did not diminish the acetaminophen-induced damage to either organ in the buthionine sulfoximine-treated mice. In conclusion, ribose cysteine serves as a cysteine prodrug that facilitates GSH biosynthesis and protects against acetaminophen-induced target organ toxicity.

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Ribose cysteine (2(R,S)-D-ribo-(1ø,2ø,3ø,4ø-tetrahydroxybutyl)thiazolidine-4(R)-carboxylic acid) protects against acetaminophen-induced hepatic and renal toxicity. The mechanism for this protection is not known, but may involve inactivation of the toxic electrophile via enhancement of glutathione (GSH) biosynthesis. Therefore, the goal of this study was to determine if GSH biosynthesis was required for the ribose cysteine protection. Male CD-1 mice were injected with either acetaminophen or acetaminophen and ribose cysteine. The ribose cysteine cotreatment antagonized the acetaminophen-induced depletion of non-protein sulfhydryls in liver as well as GSH in kidney. Moreover, ribose cysteine cotreatment significantly increased the concentration of acetaminophen-cysteine, hepatic acetaminophen-mercapturate in liver and renal acetaminophen-GSH metabolites in kidney 4 hr after acetaminophen. To determine whether protection against acetaminophen-induced liver and kidney damage involved ribose cysteine dependent GSH biosynthesis, buthionine sulfoximine was used to selectively block g-glutamylcysteine synthetase (g-GCS). Plasma sorbitol dehydrogenase (SDH) activity and blood urea nitrogen from mice pretreated with buthionine sulfoximine and challenged with acetaminophen indicated that both liver and kidney injury had occurred. While co-treatment with ribose cysteine had previously protected against acetaminophen-induced liver and kidney injury, it did not diminish the acetaminophen-induced damage to either organ in the buthionine sulfoximine-treated mice. In conclusion, ribose cysteine serves as a cysteine prodrug that facilitates GSH biosynthesis and protects against acetaminophen-induced target organ toxicity.

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