Tuning PARP-1 retention and release on DNA breaks

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Breaks in the structure of DNA are a persistent stress on the integrity of the genome, and they pose a substantial risk of chromosomal rearrangement and genetic mutation that can challenge the well-being of an organism and promote the development of cancer. There are several cellular mechanisms that monitor the state of the genome and rapidly initiate repair mechanisms in response to DNA damage so that a healthy genome is passed on to the next generation. Poly(ADP-ribose) Polymerase-1, or PARP-1, is a primary responder to breaks in the structure of DNA. PARP-1 has a unique catalytic activity that synthesizes polymers of ADP- ribose as a posttranslational modification on target proteins, primarily on PARP-1 itself (automodification). Upon binding to DNA breaks, PARP-1 activity is “turned on” to modulate DNA damage repair pathways and thereby promote cell survival. In contrast, excessive DNA damage leads to an elevated level of PARP-1 activity that results in cell death. Regulation of PARP-1 activity is therefore a critical factor in determining the fate of a cell. Importantly, inhibitors of PARP-1 (PARPi) have recently emerged as promising therapeutic agents for the treatment of cancer and inflammation. Despite a growing interest in PARPi and the discovery of expanded roles for PARP-1 activity in DNA repair, transcriptional regulation, and apoptotic signaling, there are still limited insights into the mechanism of PARP-1 catalytic activity and regulation. The objective of this research program is to fill major gaps in our knowledge of how PARP-1 is activated, modulated by a critical accessory protein (HPF1), and subsequently silenced in the process of detecting DNA damage in healthy cells and how it can be best inhibited by small molecules in current efforts to target PARP-1 in cancer and inflammation. Hydrogen-deuterium exchange coupled with mass spectrometry (HXMS) and crystallography are the major structural tools that we will apply to understand the impact of PARPi on PARP-1 dynamics and how PARP-1 in the DNA damage response is initially activated and then subsequently silenced through automodification. The structural and protein dynamics experiments will be combined with biochemical analysis of PARP-1 catalysis and DNA binding, and cell-based analysis of PARP-1 function based on our structural and biochemical work. In addition, medicinal chemistry will be employed to engineer allosteric PARP-1 “trapping” into PARPi compounds in order to increase the efficacy of targeting this enzyme in the cancer clinic. Moreover, an emerging PARPi-based imaging approach using established tumor assays with breast cancer patient-derived xenografts will determine the degree to which the PARPi compounds that we generate engage/kill cancer cells and impact survival of mice carrying tumor xenografts. The proposed studies of PARP-1 activity and regulation will advance current models of PARP-1 biological functions and generate new small molecule tools that will advance the understanding of PARP-1 biology and potentially represent new medicines for the cancer clinic.