There are currently four radiation medical countermeasures that have been approved by the United States Food and Drug Administration to mitigate hematopoietic acute radiation syndrome, all of which are repurposed radiomitigators. The evaluation of additional candidate drugs that may also be helpful for use during a radiological/nuclear emergency is ongoing. A chlorobenzyl sulfone derivative (organosulfur compound) known as Ex-Rad, or ON01210, is one such candidate medical countermeasure, being a novel, small-molecule kinase inhibitor that has demonstrated efficacy in the murine model. In this study, nonhuman primates exposed to ionizing radiation were subsequently administered Ex-Rad as two treatment schedules (Ex-Rad I administered 24 and 36 h post-irradiation, and Ex-Rad II administered 48 and 60 h post-irradiation) and the proteomic profiles of serum using a global molecular profiling approach were assessed. We observed that administration of Ex-Rad post-irradiation is capable of mitigating radiation-induced perturbations in protein abundance, particularly in restoring protein homeostasis and immune response and mitigating hematopoietic damage, at least in part after acute exposure. Taken together, restoration of functionally significant pathway perturbations may serve to protect damage to vital organs and provide long-term survival benefits to the afflicted population.

Data Acquisition

Nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) was used to analyze the peptides from human serum digests, performed on a ThermoFisher Ultimate 3000 HPLC System (ThermoFisher Scientific, Bremen, Germany) connected to a quadrupole-Orbitrap mass spectrometer (Q-Exactive, ThermoFisher Scientific, Bremen, Germany) equipped with an online nano-electrospray flexi ion source.

Firstly, the peptides were redissolved in 40 μl solvent (100% water with 0.1% formic acid) and then 1 μg peptide sample was loaded on the analytical column (Acclaim PepMap C18, 75 mm and 25 cm). Subsequently, the peptides were separated and identified under a 65 min method, which was composed of 2 min in 25% B, 40 min of 95% B, 48 min of 95% B, 50 min of 1% B, 50.1 min of 95% B, 55 min of 95%, and 57 min of 1% B. The column was then reequilibrated at initial conditions for 8 min with a constant flow rate at 300 nl/min. The column temperature kept at ambient temperature and the electrospray voltage of 2.2 kV versus the inlet of the mass spectrometer was applied. For the library development on the fractions, the Q-Exactive mass spectrometer was operated in data-dependent mode and each scan cycle consisted of one full-MS scan at the m/z ranging from m/z 375 to 2,000 with a mass resolution of 70 K. Then the selected parent ions were fragmented in ten sequential high-energy collisional dissociations (HCD) MS/MS scans with a resolution of 17.5 K. In addition, for MS/MS, the precursor ions were activated with 30% normalized collision energy and 2 m/z of the isolation window. In all cases, single charge state was rejected and a micro-scan was recorded using dynamic exclusion of 30 s.

Samples were acquired in the Data Independent Acquisition (DIA) mode after a full precursor ion scan (range between 375 – 2,000 m/z). The resolution of the instrument was set to 35,000 and the isolation window was 20 Da. For both DDA and DIA, the normalized collision energy (NCE) was 30 ev.

Database retrieval and statistical tests

Tandem mass spectra were searched by Pulsar search engine (Biognosys) and the database searched was the Human UniProt obtained on 11/23/2021. The data retrieval parameters for the Q-Exactive instrument were as follows: precursor mass tolerance: 5 ppm; fragment mass tolerance: 50 mmu; enzyme: trypsin; missed cleavages: 2; fixed modification: Carbamidomethyl on cysteine (C); variable modifications: Oxidation on methionine (M). The percolator algorithm was used to control the false discovery rate (FDR) of peptides below 1%.

The test of radiation effect and radiomitigative effects of Ex-Rad were calculated either by Mann–Whitney U test or two-tailed unpaired Student t-test, whereas p-values of less than 0.05 exclusively were considered significant. Furthermore, p-values were corrected for multiple testing using Benjamini–Hochberg procedure which limits the FDRs. All statistical analyses were performed using R (version 4.2.0).