Targeting PARP-1 allosteric regulation offers therapeutic potential against cancer.

PARP-1 is a nuclear protein that has important roles in maintenance of genomic integrity. During genotoxic stress, PARP-1 recruits to sites of DNA damage where PARP-1 domain architecture initiates catalytic activation and subsequent poly(ADP-ribose)-dependent DNA repair. PARP-1 inhibition is a promising new way to selectively target cancers harboring DNA repair deficiencies. However, current inhibitors target other PARPs, raising important questions about long-term off-target effects. Here, we propose a new strategy that targets PARP-1 allosteric regulation as a selective way of inhibiting PARP-1. We found that disruption of PARP-1 domain-domain contacts through mutagenesis held no cellular consequences on recruitment to DNA damage or a model system of transcriptional regulation, but prevented DNA-damage-dependent catalytic activation. Furthermore, PARP-1 mutant overexpression in a pancreatic cancer cell line (MIA PaCa-2) increased sensitivity to platinum-based anticancer agents. These results not only highlight the potential of a synergistic drug combination of allosteric PARP inhibitors with DNA-damaging agents in genomically unstable cancer cells (regardless of homologous recombination status), but also signify important applications of selective PARP-1 inhibition. Finally, the development of a high-throughput PARP-1 assay is described as a tool to promote discovery of novel PARP-1 selective inhibitors.

Assay development for histone methyltransferases.

Epigenetic modifications play a crucial role in human diseases. Unlike genetic mutations, however, they do not change the underlying DNA sequences. Epigenetic phenomena have gained increased attention in the field of cancer research, with many studies indicating that they are significantly involved in tumor establishment and progression. Histone methyltransferases (HMTs) are a large group of enzymes that specifically methylate protein lysine and arginine residues, especially in histones, using S-adenosyl-L-methionine (SAM) as the methyl donor. However, in general, HMTs have no widely accepted high-throughput screening (HTS) assay format, and reference inhibitors are not available for many of the enzymes. In this study, we describe the application of a miniaturized, radioisotope-based reaction system: the HotSpot(SM) platform for methyltransferases. Since this platform employs tritiated SAM as a cofactor, it can be applied to the assay of any HMT. The key advantage of this format is that any substrate can be used, including peptides, proteins, or even nucleosomes, without the need for labeling or any other modifications. Using this platform, we have determined substrate specificities, characterized enzyme kinetics, performed compound profiling for both lysine and arginine methyltransferases, and carried out HTS for a small-library LOPAC against DOT1L. After hit confirmation and profiling, we found that suramin inhibited DOT1L, NSD2, and PRMT4 with IC50 values at a low µM range.

Small-molecule inhibitors of acetyltransferase p300 identified by high-throughput screening are potent anticancer agents.

Acetyltransferase p300 (KAT3B) plays key roles in signaling cascades that support cancer cell survival and sustained proliferation. Thus, p300 represents a potential anticancer therapeutic target. To discover novel anticancer agents that target p300, we conducted a high-throughput screening campaign. A library of 622,079 compounds was assayed for cytotoxicity to the triple-negative breast cancer (TNBC) cell line MDA-MB-231 but not to the human mammary epithelial cells. The resulting compounds were tested in a biochemical assay for inhibiting the enzymatic activity of p300. One compound (L002, NSC764414) displayed an IC50 of 1.98 µmol/L against p300 in vitro, inhibited acetylation of histones and p53, and suppressed STAT3 activation in cell-based assays. L002 could be docked to the active site of the p300 catalytic domain. Biochemical tests of a series of related compounds revealed functional groups that may impact inhibitory potency of L002 against p300. Interestingly, these analogs showed inhibitory activities against the cellular paralog of p300 (CBP), p300/CBP-associated factor, and GCN5, but not to other acetyltransferases (KAT5, KAT6B, and KAT7), histone deacetylases, and histone methyltransferases. Among the NCI-60 panel of cancer cell lines, leukemia and lymphoma cell lines were extremely sensitive to L002, whereas it is toxic to only a limited number of cell lines derived from solid tumors. Notably, breast cancer cell lines, especially those derived from TNBC, were highly susceptible to L002. In vivo, it potently suppressed tumor growth and histone acetylation of MDA-MB-468 xenografts. Thus, these new acetyltransferase inhibitors are potential anticancer therapeutics.

Dual roles of PARP-1 promote cancer growth and progression.

UNLABELLED: PARP-1 is an abundant nuclear enzyme that modifies substrates by poly(ADP-ribose)-ylation. PARP-1 has well-described functions in DNA damage repair and also functions as a context-specific regulator of transcription factors. With multiple models, data show that PARP-1 elicits protumorigenic effects in androgen receptor (AR)-positive prostate cancer cells, in both the presence and absence of genotoxic insult. Mechanistically, PARP-1 is recruited to sites of AR function, therein promoting AR occupancy and AR function. It was further confirmed in genetically defined systems that PARP-1 supports AR transcriptional function, and that in models of advanced prostate cancer, PARP-1 enzymatic activity is enhanced, further linking PARP-1 to AR activity and disease progression. In vivo analyses show that PARP-1 activity is required for AR function in xenograft tumors, as well as tumor cell growth in vivo and generation and maintenance of castration resistance. Finally, in a novel explant system of primary human tumors, targeting PARP-1 potently suppresses tumor cell proliferation. Collectively, these studies identify novel functions of PARP-1 in promoting disease progression, and ultimately suggest that the dual functions of PARP-1 can be targeted in human prostate cancer to suppress tumor growth and progression to castration resistance. SIGNIFICANCE: These studies introduce a paradigm shift with regard to PARP-1 function in human malignancy, and suggest that the dual functions of PARP-1 in DNA damage repair and transcription factor regulation can be leveraged to suppress pathways critical for promalignant phenotypes in prostate cancer cells by modulation of the DNA damage response and hormone signaling pathways. The combined studies highlight the importance of dual PARP-1 function in malignancy and provide the basis for therapeutic targeting.

Structural basis for DNA damage-dependent poly(ADP-ribosyl)ation by human PARP-1.

Poly(ADP-ribose) polymerase-1 (PARP-1) (ADP, adenosine diphosphate) has a modular domain architecture that couples DNA damage detection to poly(ADP-ribosyl)ation activity through a poorly understood mechanism. Here, we report the crystal structure of a DNA double-strand break in complex with human PARP-1 domains essential for activation (Zn1, Zn3, WGR-CAT). PARP-1 engages DNA as a monomer, and the interaction with DNA damage organizes PARP-1 domains into a collapsed conformation that can explain the strong preference for automodification. The Zn1, Zn3, and WGR domains collectively bind to DNA, forming a network of interdomain contacts that links the DNA damage interface to the catalytic domain (CAT). The DNA damage-induced conformation of PARP-1 results in structural distortions that destabilize the CAT. Our results suggest that an increase in CAT protein dynamics underlies the DNA-dependent activation mechanism of PARP-1.

Purification of human PARP-1 and PARP-1 domains from Escherichia coli for structural and biochemical analysis.

A general method to express and purify full-length human poly(ADP-ribose) polymerase-1 (PARP-1), individual PARP-1 domains, and groups of PARP-1 domains from Escherichia coli cells is described. The procedure allows for robust production of highly pure PARP-1 that is free of DNA contamination and well-suited for biochemical experiments and for structural and biophysical analysis. Two biochemical assays for monitoring PARP-1 automodification activity are presented that can be used to evaluate purified PARP-1, combinations of PARP-1 domains, or PARP-1 mutants.

Crystal structures of poly(ADP-ribose) polymerase-1 (PARP-1) zinc fingers bound to DNA: structural and functional insights into DNA-dependent PARP-1 activity.

Poly(ADP-ribose) polymerase-1 (PARP-1) has two homologous zinc finger domains, Zn1 and Zn2, that bind to a variety of DNA structures to stimulate poly(ADP-ribose) synthesis activity and to mediate PARP-1 interaction with chromatin. The structural basis for interaction with DNA is unknown, which limits our understanding of PARP-1 regulation and involvement in DNA repair and transcription. Here, we have determined crystal structures for the individual Zn1 and Zn2 domains in complex with a DNA double strand break, providing the first views of PARP-1 zinc fingers bound to DNA. The Zn1-DNA and Zn2-DNA structures establish a novel, bipartite mode of sequence-independent DNA interaction that engages a continuous region of the phosphodiester backbone and the hydrophobic faces of exposed nucleotide bases. Biochemical and cell biological analysis indicate that the Zn1 and Zn2 domains perform distinct functions. The Zn2 domain exhibits high binding affinity to DNA compared with the Zn1 domain. However, the Zn1 domain is essential for DNA-dependent PARP-1 activity in vitro and in vivo, whereas the Zn2 domain is not strictly required. Structural differences between the Zn1-DNA and Zn2-DNA complexes, combined with mutational and structural analysis, indicate that a specialized region of the Zn1 domain is re-configured through the hydrophobic interaction with exposed nucleotide bases to initiate PARP-1 activation.

The Zn3 domain of human poly(ADP-ribose) polymerase-1 (PARP-1) functions in both DNA-dependent poly(ADP-ribose) synthesis activity and chromatin compaction.

PARP-1 is involved in multiple cellular processes, including transcription, DNA repair, and apoptosis. PARP-1 attaches ADP-ribose units to target proteins, including itself as a post-translational modification that can change the biochemical properties of target proteins and mediate recruitment of proteins to sites of poly(ADP-ribose) synthesis. Independent of its catalytic activity, PARP-1 binds to chromatin and promotes compaction affecting RNA polymerase II transcription. PARP-1 has a modular structure composed of six independent domains. Two homologous zinc fingers, Zn1 and Zn2, form the DNA-binding module. Zn1-Zn2 binding to DNA breaks triggers catalytic activity. Recently, we have identified a third zinc binding domain in PARP-1, the Zn3 domain, which is essential for DNA-dependent PARP-1 activity. The crystal structure of the Zn3 domain revealed a novel zinc-ribbon fold and a homodimeric Zn3 structure that formed in the crystal lattice. Structure-guided mutagenesis was used here to investigate the roles of these two features of the Zn3 domain. Our results indicate that the zinc-ribbon fold of the Zn3 domain mediates an interdomain contact crucial to assembly of the DNA-activated conformation of PARP-1. In contrast, residues located at the Zn3 dimer interface are not required for DNA-dependent activation but rather make important contributions to the chromatin compaction activity of PARP-1. Thus, the Zn3 domain has dual roles in regulating the functions of PARP-1.

The carnegie protein trap library: a versatile tool for Drosophila developmental studies.

Metazoan physiology depends on intricate patterns of gene expression that remain poorly known. Using transposon mutagenesis in Drosophila, we constructed a library of 7404 protein trap and enhancer trap lines, the Carnegie collection, to facilitate gene expression mapping at single-cell resolution. By sequencing the genomic insertion sites, determining splicing patterns downstream of the enhanced green fluorescent protein (EGFP) exon, and analyzing expression patterns in the ovary and salivary gland, we found that 600-900 different genes are trapped in our collection. A core set of 244 lines trapped different identifiable protein isoforms, while insertions likely to act as GFP-enhancer traps were found in 256 additional genes. At least 8 novel genes were also identified. Our results demonstrate that the Carnegie collection will be useful as a discovery tool in diverse areas of cell and developmental biology and suggest new strategies for greatly increasing the coverage of the Drosophila proteome with protein trap insertions.