In this work, we designed, synthesized and explored a new class of mechanism-based mitochondria-targeted inhibitors that act as strong ligands of iron in complexes of cyt c
with CL. The compounds “lock” the catalytic site of the enzymatic complex, inhibiting its ability to facilitate the development of pro-apoptotic oxidative events, and suppress release of cyt c
from mitochondria into the cytosol thus inhibiting apoptotic cell death. We demonstrated that imidazole fatty acids specifically interact with partially unfolded cyt c
and not with intact cyt c
. Indeed, low-temperature (He) EPR experiments indicate that liganding of heme-iron in cyt c
by IOA was dependent on the presence of CL. While it is possible that imidazole fatty acids may interact with other hemoproteins – e.g., cytochromes P45016
their effects on the oxygenase/peroxidase activity of the hemoproteins might depend on several parameters such as redox potential, heme-coordination state, spin states. Notably, many hemoproteins with peroxidase function – cytochromes P450, myeloperoxidase, cyclooxygenase - do not require anionic (phospho)lipids for their activation.
Importantly, TPP-ISA and TPP-IOA exerted strong radoprotective/radiomitigative effects in vivo
against lethal doses of irradiation of mice (from 1 hr before through 24 hrs after the irradiation). One can envision two possible applications of our findings in clinical practice. One of them may be associated with the employment of the newly discovered radiomitigators for the treatment of victims of terroristic attacks with nuclear devices as well as individuals inadvertently exposed to irradiation resulting from catastrophic nuclear accidents. Intraperitoneal injections of TPP-IOA and TPP-ISA employed in this study is not the preferred route of drug administration in clinical practice although it has been used for selected drugs in several disease states29–31
. It is likely that pharmacologists would have to derive new formulations that could be given orally or as a skin patch. For radiation counterterrorism, a topical or trans-dermal delivery system would be preferred.
The second kind of applications may be relevant to organ specific delivery of TPP-IOA and TPP-ISA to protect normal tissues from ionizing irradiation effects in clinical radiotherapy. Several highly effective chemotherapy drugs are known to interact with ionizing irradiation to promote tumor cell killing, but unfortunately also exacerbate normal tissue toxicity. Local, tissue specific delivery of TPP-IOA and TPP-ISA may be also important in amelioration of the toxicity of this combined modality cancer therapy. The design of a pharmacological formulation by which to facilitate delivery of TPP-IOA and TPP-ISA to the oral cavity or oropharynx during radiotherapy of head and neck cancer; to the esophagus during radiotherapy of non-small cell lung cancer; to the bladder during brachytherapy or fractionated pelvic radiotherapy of endometrial or cervix cancer; and to the rectum during fractionated radiotherapy of prostate cancer, could provide both normal tissue protection and decreased morbidity for conventional treatment protocols and potentially allow radiation dose escalation to improve local control.
γ-Irradiation is a potent carcinogen by virtue of its ability to cause single- and double-strand DNA breaks32
- the effect that is also associated with a massive p53-dependent cell death in radiosensitive targets (primary lymphoid organs and intestinal epithelium) leading to acute injury33,34
. While it is known that p53 acts as a potent and important tumor suppressor35
, it is believed that this tumor suppressor activity is not directly related to p53’s acute pathological response to irradiation induced systemic genotoxicity34
. Indeed there is substantial pharmacologic36
evidence supporting the notion that temporary and reversible suppression of p53, resulting in massive rescue of cells in radiosensitive tissues, is not associated with an increase in carcinogenicity. Rather, the tumor suppressor activity of p53 is related to activities in cells several days after the irradiation induced systemic genotoxicity. Thus, p53 inhibitors are expected to represent tissue protective drugs to be used under pathological conditions associated with massive apoptotic cell death37
. Although p53 inhibitors hold therapeutic promise, their potential applications are limited by the tissue specificity of p53-dependent radiosensitivity. For example, despite the pronounced p53-dependent apoptosis that occurs among epithelial cells of the small intestine after irradiation, clinical gastrointestinal acute radiation syndrome develops independently of p53. Thus radioprotection of the gastrointestinal tract should be based on p53-independent strategies38
, possibly other types of apoptosis inhibitors. The current work capitalizes on this principle by utilizing mitochondria-targeted inhibitors of cyt c
/CL peroxidase activity – TPP-IOA and TPP-ISA – that demonstrated effectiveness in protecting against radiation induced apoptosis as well as radioprotective and radiomitigative effects in vivo
. The mechanism of radiation mitigation proposed herein will prevent acute radiation sickness while having no impact on p53-mediated tumor suppression. When treatment with the mitochondrial-targeted inhibitors of cyt c
is withdrawn, those few cells that have acquired a radiation-induced oncogenic chromosome aberration will be eliminated by p53-mediated apoptosis. This acquired oncogenic stress will be present in these cells for many weeks prior to tumor onset allowing ample time for p53 to execute its function as a tumor suppressor.
This study was aimed at the discovery, rather than the development, of novel mitochondria-targeted radiomitigators and radioprotectors. However, further improvements of structural features and pharmacological characteristics of the proposed inhibitors may lead to an optimized series of radioprotectors and radiomitigators with a broad spectrum of biomedical applications in the biodefense area as well as in radiotherapy of cancer by achieving increased resistance of normal tissues surrounding the tumor.