We have examined the changes in physical properties of aqueous solutions of the plasmid pUC18 that take place on the addition of the cationic oligopeptide penta-arginine. An increase in sedimentation rate and static light scattering, and changes in the nucleic acid CD spectrum all suggest that this ligand acts to condense the plasmid. Dynamic light scattering suggests the hydrodynamic radii of the condensate particles are a few micrometers, ca. 50-fold larger than that of the monomeric plasmid. Condensation of the plasmid also produces a ca. 100-fold decrease in the strand break yield produced by gamma irradiation. This extensive protection against reactive intermediates in the bulk of the solution implies that condensed plasmid DNA may offer a model system with which to study the direct effect of ionizing radiation (ionization of the DNA itself). The use of peptide ligands as condensing agents in this application is attractive because the derivatives of several amino acids (particularly tryptophan and tyrosine) have been shown to modify the radiation chemistry of DNA extensively.

Coumarin derivatives have found application as probes for the hydroxyl radical because one of the products of the reaction between them is a highly fluorescent umbelliferone. We have examined the interaction in aqueous solution between a cationic coumarin-labeled hexa-arginine peptide ligand and plasmid DNA, and compared after gamma irradiation the yields of products derived from both of them. At low ionic strengths, the ligand binds very tightly to the plasmid. Compared with the structurally similar 4-methylumbelliferone (phenolic pK(a) = 7.8), the fluorescent product derived from gamma irradiation of the coumarin labeled cationic peptide is significantly more acidic (pK(a) = 6.1), making it a very convenient probe for solutions of pH in the physiological range. The yield of this product is generally in excellent agreement over a wide range of conditions with that of the single strand break product produced by the reaction of the hydroxyl radical with the plasmid. Thus coumarin-labeled peptide ligands offer promise as hydroxyl radical probes for locations in close proximity to DNA.

In the presence of cationic ligands, DNA molecules can become aggregated into larger particles in a process known as condensation. DNA condensates are of interest as models for the dense packing found in naturally occurring structures such as phage heads and chromatin. They have found extensive application in DNA transfection and also provide convenient models with which to study DNA damage by the direct effect of ionizing radiation. Further, conjugates of cationic peptides with fatty acids may represent a class of attractive ligands for these areas because of their simple synthesis. When plasmid pUC18 is used as the DNA target and N-caproyl-penta-arginine amide (Cap-R(5)-NH(2)) is used as the ligand, the physical properties of the resulting mixtures were characterized using static and dynamic light scattering, sedimentation, dye exclusion, circular dichroism, nanoparticle tracking, and atomic force microscopy. Their chemical properties were assayed using solvent extraction and protection against hydroxyl radical attack and nuclease digestion. Titration of the plasmid with the Cap-R(5)-NH(2) ligand produced sharply defined changes in both chemical and physical properties, which was associated with the formation of condensed DNA particles in the 100-2000 nm size range. The caproyl group at the ligand's N-terminus produced a large increase in the partitioning of the resulting condensate from water into chloroform and in its binding to the neutral detergent Pluronic F-127. Both the physical and chemical data were all consistent with condensation of the plasmid by the ligand where the presence in the ligand of the caproyl group conferred an extensive lipophilic character upon the condensate.

The four members of the Janus family of nonreceptor tyrosine kinases play a significant role in immune function. The JAK family kinase inhibitor, tofacitinib 1, has been approved in the United States for use in rheumatoid arthritis (RA) patients. A number of JAK inhibitors with a variety of JAK family selectivity profiles are currently in clinical trials. Our goal was to identify inhibitors that were functionally selective for JAK1 and JAK3. Compound 22 was prepared with the desired functional selectivity profile, but it suffered from poor absorption related to physical properties. Use of the phosphate prodrug 32 enabled progression to a murine collagen induced arthritis (CIA) model. The demonstration of a robust efficacy in the CIA model suggests that use of phosphate prodrugs may resolve issues with progressing this chemotype for the treatment of autoimmune diseases such as RA.

Guanine bases are the most easily oxidized sites in DNA and therefore electron deficient guanine radical species are major intermediates in the direct effect of ionizing radiation (ionization of the DNA itself) on DNA as a consequence of hole migration to guanine. As a model for this process we have used gamma-irradiation in the presence of thiocyanate ions to generate single electron oxidized guanine radicals in a plasmid target in aqueous solution. The stable species formed from these radicals can be detected and quantified by the formation of strand breaks in the plasmid after a post-irradiation incubation using a suitable enzyme. If a tyrosine derivative is also present during irradiation, the production of guanine oxidation products is decreased by electron transfer from tyrosine to the intermediate guanyl radical species. By using cationic tyrosine containing ligands we are able to observe this process when the tyrosine is electrostatically bound to the plasmid. The driving force dependence of this reaction was determined by comparing the reactivity of tyrosine with its 3-nitro analog. The results imply that the electron transfer reaction is coupled to a proton transfer. The experimental conditions used in this model system provide a reasonable approximation to those involved in the radioprotection of DNA by tightly bound proteins in chromatin.