br Concluding Remarks br Financial Disclosures br Author

Concluding Remarks
Financial Disclosures
Author Contributions
Role of the Funding Source Partial funding for this study was provided by a service directed grant from the U.S. Department of Veterans Affairs and the University of Minnesota (Brain and Genomics Fund and the American Legion Brain Sciences Chair). The sponsors had no role in the current study design, analysis or interpretation, or in the writing of this paper. The contents do not represent the views of the U.S. Department of Veterans Affairs or the United States Government.
Acknowledgments
Introduction Poly(ADP-ribose) polymerase 1 (PARP-1) is an abundant and ubiquitous nuclear enzyme. When active, it captures NAD+ to assemble long and branching polymers of Poly(ADP-ribose) (pADPr), modifying itself, as well as surrounding proteins (D\'Amours et al., 1999). Although DNA repair is commonly accepted as its main function, recent findings indicate that PARP-1 also participates in numerous nuclear processes, including regulation of chromatin and gene expression, ribosome biogenesis, nuclear traffic, and epigenetic bookmarking (Krishnakumar et al., 2008; Thomas & Tulin, 2013). PARP-1 tends to control the expression of genes involved in cell adhesion and cell-to-cell signaling (Krishnakumar et al., 2008). Given the multitude of PARP-1 functions, inhibitors of PARP-1 hold promise for several branches of medicine. Preclinical data have shown that PARP-1 inhibitors may mitigate inflammation, circulatory shock, stroke, and myocardial infarction (Curtin & Szabo, 2013). The most extensive research on PARP-1 inhibitors has been carried out in oncology (Alberts, 2009; Curtin, 2005). PARP-1 inhibitors have been shown to selectively eliminate several types of tumorigenic cells, namely BRCA1/2-deficient breast and ovarian purchase CP-673451 (Bryant et al., 2005). Indeed, one PARP-1 inhibitor (Olaparib/Lynparza™, AstraZeneca) has been approved by both the European Medicines Agency and FDA to treat advanced ovarian cancer with BRCA1/2 mutations (Brown et al., 2016) Several PARP-1 inhibitors are currently undergoing phase II/III clinical trials in cancers with or without genetic predisposition, as a monotherapy or in combination with other drugs (Brown et al., 2016). Therefore, PARP-1 inhibitors have already been shown to have the potential required to treat a variety of cancer types, including prostate, colorectal and pancreatic tumors, beyond BRCA1/2 ovarian cancer (Brown et al., 2016; Deshmukh & Qiu, 2015; Lupo & Trusolino, 1846). Unfortunately, a number of clinical studies reported setbacks in research on PARP-1-based anticancer therapies (Guha, 2011). One of the most disappointing studies thus far was the phase III trial of iniparib with gemcitabine and carboplatin in metastatic triple-negative breast cancer (O\'Shaughnessy et al., 2014). The majority of currently available PARP-1 inhibitors (Supplementary Table 1) were designed as NAD competitors and generally represent various memes of nicotinamide pharmacophore (Fig. 1A,B) (Jayle & Curtin, 2011). Ubiquity of NAD in a cell makes it difficult to completely eliminate PARP-1 activity by using NAD competitors. Specificity of these inhibitors to PARP-1 has been tested and confirmed within the PARP superfamily (Wahlberg et al., 2012). However, little is known about their effect on other NAD-dependent pathways. Since classical PARP-1 inhibitors display strong structural similarities to nucleotides, they tend to obstruct functions of other enzymes that utilize nucleotides as cofactors, such as kinases (Chuang et al., 2012; Antolín et al., 2012; Antolin & Mestres, 2014; Passeri et al., 2015). To overcome the limitation of NAD-like PARP-1 inhibitors, we set out to identify molecules that inhibit PARP-1, but maintain structural independence from NAD (Tulin, 2011). To accomplish this, we employed a blind screen of a random small- molecule collection containing 50,000 compounds. As noted above, PARP-1 can be regulated by competing for binding with NAD (D\'Amours et al., 1999), as well as by two additional routes: obstruction of PARP-1 binding with DNA (Kirsanov et al., 2014) and disruption of PARP-1 interaction with histone H4 (Pinnola et al., 2007). The latter rout of activation is highly specific to PARP-1. Instead of targeting NAD-binding to PARP-1, we searched for molecules that could disrupt PARP-1 activation by the core histone H4 (Fig. 1C). H4-dependent PARP-1 activation is stronger and better sustained than DNA-dependent activation (Fig. 1D). Thus, we designed a new automated approach to screen a large collection of small molecules, using the H4-dependent route. Besides identifying NAD competitors, our screen has identified molecules that show no similarity to NAD, other nucleotides, or to any known PARP-1 inhibitor. Further testing of a subset of these compounds demonstrated their efficacy toward PARP-1 inhibition in cancer cells, as well as their ability to suppress tumorigenesis in prostate and kidney cancer with greater efficacy when compared to current clinically approved drugs and the NAD-competitor Olaparib.