Electrostatic Impact of Brefeldin A on Thiocyanate Probes Surrounding the Interface of Arf1-BFA-ARNO4M, a Protein-Drug-Protein Complex.

Protein-protein interactions regulate many cellular processes, making them ideal drug candidates. Design of such drugs, however, is hindered by a lack of understanding of the factors that contribute to the interaction specificity. Specific protein-protein complexes possess both structural and electrostatic complementarity, and while structural complementarity of protein complexes has been extensively investigated, fundamental understanding of the complicated networks of electrostatic interactions at these interfaces is lacking, thus hindering the rational design of orthosterically binding small molecules. To better understand the electrostatic interactions at protein interfaces and how a small molecule could contribute to and fit within that environment, we used a model protein-drug-protein system, Arf1-BFA-ARNO4M, to investigate how small molecule brefeldin A (BFA) perturbs the Arf1-ARNO4M interface. By using nitrile probe labeled Arf1 sites and measuring vibrational Stark effects as well as temperature dependent infrared shifts, we measured changes in the electric field and hydrogen bonding at this interface upon BFA binding. At all five probe locations of Arf1, we found that the vibrational shifts resulting from BFA binding corroborate trends found in Poisson-Boltzmann calculations of surface potentials of Arf1-ARNO4M and Arf1-BFA-ARNO4M, where BFA contributes negative electrostatic potential to the protein interface. The data also corroborate previous hypotheses about the mechanism of interfacial binding and confirm that alternating patches of hydrophobic and polar interactions lead to BFA binding specificity. These findings demonstrate the impact of BFA on this protein-protein interface and have implications for the design of other interfacial drug candidates.

Impact of the PrC-210 Radioprotector Molecule on Cancer Deaths in p53-Deficient Mice.

Radiation-induced cancer is an ongoing and significant problem, with sources that include clinics worldwide in which 3.1 billion radiology exams are performed each year, as well as a variety of other scenarios such as space travel and nuclear cleanup. These radiation exposures are typically anticipated, and the exposure is typically well below 1 Gy. When radiation-induced (actually ROS-induced) DNA mutation is prevented, then so too are downstream radiation-induced cancers. Currently, there is no protection available against the effects of such <1 Gy radiation exposures. In this study, we address whether the new PrC-210 ROS-scavenger is effective in protecting p53-deficient (p53-/-) mice against X-ray-induced accelerated tumor mortality; this is the most sensitive radiation tumorigenesis model currently known. Six-day-old p53-/- pups received a single intraperitoneal PrC-210 dose [0.5 maximum tolerated dose (MTD)] or vehicle, and 25 min later, pups received 4.0 Gy X-ray irradiation. At 5 min postirradiation, blood was collected to quantify white blood cell c-H2AX foci. Over the next 250 days, tumor-associated deaths were recorded. Findings revealed that when administered 25 min before 4 Gy X-ray irradiation, PrC-210 reduced DNA damage (c-H2AX foci) by 40%, and in a notable coincidence, caused a 40% shift in tumor latency/incidence, and the 0.5 MTD PrC210 dose had no discernible toxicities in these p53-/- mice. Essentially, the moles of PrC-210 thiol within a single 0.5 MTD PrC-210 dose suppressed the moles of ROS generated by 40% of the 4 Gy X-ray dose administered to p53-/- pups, and in doing so, eliminated the lifetime leukemia/lymphoma risk normally residing "downstream" of that 40% of the 4 Gy dose. In conclusion: 1. PrC-210 is readily tolerated by the 6-day-old p53-/- mice, with no discernible lifetime toxicities; 2. PrC-210 does not cause the nausea, emesis or hypotension that preclude clinical use of earlier aminothiols; and 3. PrC-210 significantly increased survival after 4 Gy irradiation in the p53-/- mouse model.

Reduction of X-ray-induced DNA damage in normal human cells treated with the PrC-210 radioprotector.

The aim of our study was to determine the protective efficacy of the PrC-210 aminothiol radioprotector against X-ray-induced DNA damage in normal human cells and to establish dose- and time-effect models for future PrC-210 use in humans. The PrC-210 structure has a branched structure which enables scavenging of reactive oxygen species (ROS) away from DNA. Normal human blood lymphocytes, fibroblasts and naked genomic DNA were exposed to PrC-210 seconds to hours prior to irradiation. Biological (γ-H2AX foci), chemical (8-oxo-deoxyguanosine) and physical (genomic DNA electrophoretic migration) DNA damage endpoints were scored to determine the ability of PrC-210 to suppress radiation-induced DNA damage. X-ray-induced γ-H2AX foci in blood lymphocytes were reduced by 80% after irradiation with 10, 50 and 100 mGy, and DNA double-strand breaks in fibroblasts were reduced by 60% after irradiation with 20 Gy. Additionally, we observed a reduction of 8-oxo-deoxyguanosine (an ROS-mediated, DNA damage marker) in human genomic DNA to background in a PrC-210 dose-dependent manner. PrC-210 also eliminated radiation-induced cell death in colony formation assays after irradiation with 1 Gy. The protective efficacy of PrC-210 in each of these assay systems supports its development as a radioprotector for humans in multiple radiation exposure settings.

Significant Suppression of CT Radiation-Induced DNA Damage in Normal Human Cells by the PrC-210 Radioprotector.

While computed tomography (CT) is now commonly used and considered to be clinically valuable, significant DNA double-strand breaks (γ-H2AX foci) in white blood cells from adult and pediatric CT patients have been frequently reported. In this study to determine whether γ-H2AX foci and X-ray-induced naked DNA damage are suppressed by administration of the PrC-210 radioprotector, human blood samples were irradiated in a CT scanner at 50-150 mGy with or without PrC-210, and γ-H2AX foci were scored. X-ray-induced naked DNA damage was also studied, and the DNA protective efficacy of PrC-210 was compared against 12 other common "antioxidants." PrC-210 reduced CT radiation-induced γ-H2AX foci in white blood cells to near background ( P < 0.0001) at radiation doses of 50-150 mGy. PrC-210 was most effective among the 13 "antioxidants" in reducing naked DNA X-ray damage, and its addition at 30 s before an •OH pulse reduced to background the •OH insult that otherwise induced >95% DNA damage. A systemic PrC-210 dose known to confer 100% survival in irradiated mice had no discernible effect on micro-CT image signal-to-noise ratio and CT image integrity. PrC-210 suppressed DNA damage to background or near background in each of these assay systems, thus supporting its development as a radioprotector for humans in multiple radiation exposure settings.