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Ultraviolet (UV) radiation is among the most prevalent environmental factors that influence human health and disease. Even one hour of UV irradiation extensively damages the genome. To cope with resulting deleterious DNA lesions, cells activate a multitude of DNA damage response pathways, including DNA repair. Strikingly, UV-induced DNA damage formation and repair are affected by chromatin state. When cells enter S phase with these lesions, a distinct mutation signature is created via error-prone translesion synthesis. Chronic UV exposure leads to high mutation burden in skin and consequently the development of skin cancer, the most common cancer in the United States. Intriguingly, UV-induced oxidative stress has opposing effects on carcinogenesis. Elucidating the molecular mechanisms of UV-induced DNA damage responses will be useful for preventing and treating skin cancer with greater precision. Excitingly, recent studies have uncovered substantial depth of novel findings regarding the molecular and cellular consequences of UV irradiation. In this review, we will discuss updated mechanisms of UV-induced DNA damage responses including the ATR pathway, which maintains genome integrity following UV irradiation. We will also present current strategies for preventing and treating nonmelanoma skin cancer, including ATR pathway inhibition for prevention and photodynamic therapy for treatment.

Unique chemical methodology enables the synthesis of innovative and diverse scaffolds and chemotypes and allows access to previously unexplored “chemical space.” Compound collections based on such new synthetic methods can provide small-molecule probes of proteins and/or pathways whose functions are not fully understood. We describe the identification, characterization, and evolution of two such probes. In one example, a pathway-based screen for DNA damage checkpoint inhibitors identified a compound, MARPIN (AT M and A T R p athway in hibitor) that sensitizes p53-deficient cells to DNA-damaging agents. Modification of the small molecule and generation of an immobilized probe were used to selectively bind putative protein target(s) responsible for the observed activity. The second example describes a focused library approach that relied on tandem multicomponent reaction methodologies to afford a series of modulators of the heat shock protein 70 (Hsp70) molecular chaperone. The synthesis of libraries based on the structure of MAL3-101 generated a collection of chemotypes, each modulating Hsp70 function, but exhibiting divergent pharmacological activities. For example, probes that compromise the replication of a disease-associated polyomavirus were identified. These projects highlight the importance of chemical methodology development as a source of small-molecule probes and as a drug discovery starting point.

The 24-residue peptide Humanin (HN), containing two Ser residues at positions 7 and 14, protects neuronal cells from insults of various Alzheimer's disease (AD) genes and Aβ. It was not known why the rescue function of (S14G)HN is more potent than HN by two to three orders of magnitude. Investigating the possibility that the post-translational modification of Ser14 might play a role, we found that HN with d-Ser at position 14 exerts neuroprotection more potently than HN by two to three orders of magnitude, whereas d-Ser7 substitution does not affect the rescue function of HN. On the other hand, S7A substitution nullified the HN function. Multiple series of experiments indicated that Ser7 is necessary for self-dimerization of HN, which is essential for neuroprotection by this factor. These findings indicate that the rescue function of HN is quantitatively modulated by d-isomerization of Ser14 and Ser7-relevant dimerization, allowing for the construction of a very potent HN derivative that was fully neuroprotective at 10 pm against 25 µm Aβ1–43. This study provides important clues to the understanding of the neuroprotective mechanism of HN, as well as to the development of novel AD therapeutics.

Amyloid precursor protein (APP), the precursor of Abeta, has been shown to function as a cell surface receptor that mediates neuronal cell death by anti-APP antibody. The c-Jun N-terminal kinase (JNK) can mediate various neurotoxic signals, including Abeta neurotoxicity. However, the relationship of APP-mediated neurotoxicity to JNK is not clear, partly because APP cytotoxicity is Abeta independent. Here we examined whether JNK is involved in APP-mediated neuronal cell death and found that: (i) neuronal cell death by antibody-bound APP was inhibited by dominant-negative JNK, JIP-1b and SP600125, the specific inhibitor of JNK, but not by SB203580 or PD98059; (ii) constitutively active (ca) JNK caused neuronal cell death and (iii) the pharmacological profile of caJNK-mediated cell death closely coincided with that of APP-mediated cell death. Pertussis toxin (PTX) suppressed APP-mediated cell death but not caJNK-induced cell death, which was suppressed by Humanin, a newly identified neuroprotective factor which inhibits APP-mediated cytotoxicity. In the presence of PTX, the PTX-resistant mutant of Galphao, but not that of Galphai, recovered the cytotoxic action of APP. These findings demonstrate that JNK is involved in APP-mediated neuronal cell death as a downstream signal transducer of Go.

Antibodies against APP, a precursor of Abeta deposited in Alzheimer's disease brain, have been shown to cause neuronal death. Therefore, it is important to determine whether Abeta mediates antibody-induced neurotoxicity. When primary neurons were treated with anti-APP antibodies, Abeta40 and Abeta42 in the cultured media were undetectable by an assay capable of detecting 100 nM Abeta peptides. However, exogenously treated Abeta1-42 or Abeta1-43 required >3 microM to exert neurotoxicity, and 25 microM Abeta1-40 was not neurotoxic. Glutathione-ethyl-ester inhibited neuronal death by anti-APP antibody, but not death by Abeta1-42, whereas serum attenuated toxicity by Abeta1-42, but not by anti-APP antibody. Using immortalized neuronal cells, we specified the domain responsible for toxicity to be cytoplasmic His(657)-Lys(676), but not the Abeta1-42 region, of APP. This indicates that neuronal cell death by anti-APP antibody is not mediated by secreted Abeta.

In the yeast Saccharomyces cerevisiae, Sic1, an inhibitor of Clb-Cdc28 kinases, must be phosphorylated and degraded in G1for cells to initiate DNA replication, and Cln-Cdc28 kinase appears to be primarily responsible for phosphorylation of Sic1. The Pho85 kinase is a yeast cyclin-dependent kinase (Cdk), which is not essential for cell growth unless both CLN1 andCLN2 are absent. We demonstrate that Pho85, when complexed with Pcl1, a G1cyclin homologue, can phosphorylate Sic1 in vitro, and that Sic1 appears to be more stable inpho85Δ cells. Three consensus Cdk phosphorylation sites present in Sic1 are phosphorylated in vivo, and two of them are required for prompt degradation of the inhibitor. Pho85 and other G1Cdks appear to phosphorylate Sic1 at different sites in vivo. Thus at least two distinct Cdks can participate in phosphorylation of Sic1 and may therefore regulate progression through G1.

New approaches to prevent and reverse UV damage are needed to combat rising sunlight-induced skin cancer rates. Mouse studies have shown that oral or topical caffeine promotes elimination of UV-damaged keratinocytes through apoptosis and markedly inhibits subsequent skin cancer development. This potentially important therapeutic effect has not been studied in human skin cells. Here, we use primary human keratinocytes to examine which of several caffeine effects mediates this process. In these cells, caffeine more than doubled apoptosis after 75 mJ cm(-2) of ultraviolet light B (UVB). Selectively targeting two of caffeine's known effects did not alter UVB-induced apoptosis: inhibition of ataxia-telangiectasia mutated and augmentation of cyclic AMP levels. In contrast, siRNA against ataxia-telangiectasia and Rad3-related (ATR) doubled apoptosis after UV through a p53-independent mechanism. Caffeine did not further augment apoptosis after UVB in cells in which ATR had been specifically depleted, suggesting that a key target of caffeine in this effect is ATR. Inhibition of a central ATR target, checkpoint kinase 1 (Chk1), through siRNA or a new and highly specific inhibitor (PF610666) also augmented UVB-induced apoptosis. These data suggest that a relevant target of caffeine is the ATR-Chk1 pathway and that inhibiting ATR or Chk1 might have promise in preventing or reversing UV damage.

Chemical genetics employs diverse small-molecule compounds to elucidate biological processes in a manner analogous to the mutagenesis strategies at the core of classical genetics. Screening small-molecule libraries for compounds that induce a phenotype of interest represents the forward chemical genetic approach, whereas the reverse approach involves small molecules targeting a single protein. Here, we review key differences between the goals for small-molecule screening in industry versus academia, recent developments in high-throughput screening, and publicly available resources of compound collections, screening facilities, and databases. A particularly exciting outcome of a chemical genetic screen is the discovery of a previously unknown role for a protein in a pathway together with compounds that affect the function of that protein. In illustrative cases, such discoveries have led to progress toward therapeutic development and more commonly have increased the size of the small molecule “toolbox” available to the research community for the study of biological processes.

Using a yeast two-hybrid method, we searched for amyloid precursor protein (APP)-interacting molecules by screening mouse and human brain libraries. In addition to known interacting proteins containing a phosphotyrosine-interaction-domain (PID)-Fe65, Fe65L, Fe65L2, X11, and mDab1, we identified, as a novel APP-interacting molecule, a PID-containing isoform of mouse JNK-interacting protein-1 (JIP-1b) and its human homolog IB1, the established scaffold proteins for JNK. The APP amino acids Tyr(682), Asn(684), and Tyr(687) in the G(681)YENPTY(687) region were all essential for APP/JIP-1b interaction, but neither Tyr(653) nor Thr(668) was necessary. APP-interacting ability was specific for this additional isoform containing PID and was shared by both human and mouse homologs. JIP-1b expressed by mammalian cells was efficiently precipitated by the cytoplasmic domain of APP in the extreme Gly(681)-Asn(695) domain-dependent manner. Reciprocally, both full-length wild-type and familial Alzheimer's disease mutant APPs were precipitated by PID-containing JIP constructs. Antibodies raised against the N and C termini of JIP-1b coprecipitated JIP-1b and wild-type or mutant APP in non-neuronal and neuronal cells. Moreover, human JNK1beta1 formed a complex with APP in a JIP-1b-dependent manner. Confocal microscopic examination demonstrated that APP and JIP-1b share similar subcellular localization in transfected cells. These data indicate that JIP-1b/IB1 scaffolds APP with JNK, providing a novel insight into the role of the JNK scaffold protein as an interface of APP with intracellular functional molecules.

An important goal in cancer therapy has been to selectively sensitize cancers to existing drugs that typically work by inducing DNA damage. In particular, many cancers are p53-defective and chemoresistant, thus novel approaches to target p53-defective cancers are needed. One promising strategy to sensitize p53-deficient cells has been to inhibit DNA damage response kinases such as ATM/ATR via ATP-competitive kinase inhibitors. To better understand ATM/ATR activation mechanisms and discover novel probes for the ATM/ATR pathway, we performed a phenotype-based screen of 9,195 small-molecule compounds for inhibitors of hydroxyurea-induced phosphorylation of Chk1, a key ATR substrate. After subsequent biological screens, we selected 4 compounds that inhibited ATR and ATM pathways; 3 were known bioactive agents and 1 was a diversity-oriented-synthetic product. These compounds sensitized p53-deficient cells to diverse DNA-damaging agents. Xenograft experiments were performed on one compound, and it showed synergistic suppression of p53-deficient tumor growth with cisplatin. Importantly, these compounds did not suppress ATR kinase catalytic activity in vitro, unlike typical ATM/ATR kinase inhibitors that are ATP-competitive. To identify molecular targets of one compound, “MARPIN” (ATM and ATR pathway inhibitor), we defined its active site through structure-activity relationship analysis, resulting in synthesis of inactive derivatives of MARPIN. Identification of proteins that specifically bind MARPIN, but not its inactive derivatives, is underway. This phenotype-based chemical genetic screen identified novel ATM/ATR pathway inhibitors that are mechanistically distinct from kinase catalytic inhibitors, and these compounds could serve as probes to identify previously unrecognized druggable targets in DNA damage response pathways. Citation Format: Masaoki Kawasumi, James E. Bradner, Nicola Tolliday, Renee Thibodeau, Heather Sloan, Kay M. Brummond, Paul Nghiem. A chemical genetic screen identifies novel ATM/ATR pathway inhibitors that sensitize p53-deficient cells to DNA-damaging agents without affecting ATR kinase catalytic activity. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr B04.

Background The replication (G2/M) checkpoint is principally mediated by the serine/threonine protein kinase ATR (ataxia telangiectasia mutated and Rad3-related). ATR is a large (350 kD) member of the phosphatidylinositol kinase related kinase family. After exposure to genotoxic or replication stress, ATR halts cell cycle progression, allowing DNA repair complexes time enough to restore the fidelity of the genome prior to cell division. Previous experiments have demonstrated that cancer cells with p53 mutation are critically dependent on ATR-mediated arrest of the cell cycle. Industrial approaches to identify ATR inhibitors have failed likely as a result of protein insolubility. Methods We have undertaken a novel chemical genetic approach employing small molecule microarrays (SMMs) to identify molecules with high binding specificity for ATR. Three diversity-oriented combinatorial chemical libraries of more than 15,000 entities were generated by split-pool synthesis in solid phase on polystyrene macrobead supports. Compounds were robotically printed in microarray format on glass slides. Four analogs of FK506 were printed as positive controls. Extracts were prepared from mammalian cells transfected with over-expression constructs of FLAG-tagged ATR, FKBP12 and GFP. A protocol was developed and optimized for screening employing a primary anti-FLAG mouse monoclonal antibody and Cy5-fluorophore labeled anti-mouse antibody. Data analysis for small molecule binders was performed with GenePix software on an Axon Scanner. Biological activity of these molecules was analyzed in the context of mitotic spread and chromosomal fragility assays. Results Protein expression and antibody fidelity was verified by Western blot. The lysate-based SMM screening approach was optimized and validated by recognition of an interaction between over-expressed, epitope-tagged FKBP12 and analogs of FK506. Six small molecule hits suggesting ATR binding were identified and verified by triplicate microarray assays. Positive compounds were structurally similar members of a dihydropyrancarboxamide library suggesting recognition of a common target. Mitotic spread analysis of cells treated with two of these molecules and hydroxyurea demonstrated the premature chromatin condensation phenotype characteristic of replication checkpoint inhibition. Chromosomal fragility was notably augmented by these molecules as well. Chemosensitivity following replication stress was witnessed in p53-negative cells relative to an otherwise identical wild-type cell line. Conclusions Classical approaches to drug discovery are often limited by challenges in protein biochemistry such as protein size, solubility, activity and yield. We present compelling data that the small molecule microarray format can effectively be tailored for use with cellular lysates over-expressing a protein target of biological interest. Furthermore, we have used an optimized protocol to identify two novel, active small molecule inhibitors of the replication checkpoint (SMIRC-1 and SMIRC-2). The enhanced chemosensitivity in p53-negative cell lines supports a plausible role for ATR inhibitors as potentially useful chemotherapeutic agents.