Pifithrin ␣ (PFT␣) is a chemical compound isolated for its ability to suppress p53-mediated transactivation. It can protect cells from p53-mediated apoptosis induced by various stimuli and reduce sensitivity of mice to gamma radiation. Identification of molecular targets of PFT␣ is likely to provide new insights into mechanisms of regulation of p53 pathway and is important for predicting potential risks associated with administration of PFT␣-like p53 inhibitors in vivo. We found that PFT␣, in addition to p53, can suppress heat shock and glucocorticoid receptor signaling but has no effect on nuclear factor-B signaling. PFT␣ reduces activation of heat shock transcription factor (HSF1) and increases cell sensitivity to heat. Moreover, it reduces activation of glucocorticoid receptor and rescues mouse thymocytes in vitro and in vivo from apoptotic death after dexamethasone treatment. PFT␣ affected both signaling pathways in a p53-independent manner. These observations suggest that PFT␣ targets some unknown factor that is common for three major signal transduction pathways.Based on the analysis of p53-dependent effects caused by ionizing radiation and chemotherapeutic drugs in mice, p53-mediated apoptosis was defined as a determinant of organism sensitivity to systemic genotoxic stress associated with cancer treatment (1). Temporary reversible pharmacological suppression of p53 was suggested as an approach to reduce cancer treatment side effects. This hypothesis was supported by isolation of a small molecule inhibitor of p53, pifithrin ␣ (PFT␣) 1 that was capable of rescuing mice from lethal genotoxic stress caused by gamma radiation (2). Furthermore, inhibition of p53 was suggested as a therapeutic approach to treatment of other pathological conditions associated with p53 activation (3), some of which have already been experimentally confirmed. Thus, PFT␣ was shown to protect neurons from death induced by DNA-damaging agents, hypoxia and dopamine (4, 5): it had therapeutic effects in animal models of Parkinson disease (6) and acute renal failure (7). In all these works, biological effects of PFT␣ were attributed to its anti-p53 function, although not in all of them has this conclusion been confirmed by genetic approaches. Accurate interpretation of biological effects of PFT␣ requires identification of its molecular target(s) and determination of molecular mechanisms of its activity.PFT␣ was isolated by screening of chemical library in a cell-based readout system for its ability to reduce p53-dependent transactivation (2). This biological effect could be reached by affecting p53 pathway at numerous points and therefore PFT␣ could act by targeting one of numerous factors cooperating with p53 function. Biological effects of PFT␣ on p53 pathway suggested that it acted by interfering with nuclear accumulation of p53 (2). Many transcription factors involved in other signal transduction pathways have the same principles of regulation as p53: after activation in cytoplasm they are translocated to the nucleus, followed by mo...