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Signals generated by cryptochrome (CRY) blue-light photoreceptors are responsible for a variety of developmental and circadian responses in plants. The CRYs are also identified as circadian blue-light photoreceptors in Drosophila and components of the mammalian circadian clock. These flavoproteins all have an N-terminal domain that is similar to photolyase, and most have an additional C-terminal domain of variable length. We present here the crystal structure of the photolyase-like domain of CRY-1 from Arabidopsis thaliana . The structure reveals a fold that is very similar to photolyase, with a single molecule of FAD noncovalently bound to the protein. The surface features of the protein and the dissimilarity of a surface cavity to that of photolyase account for its lack of DNA-repair activity. Previous in vitro experiments established that the photolyase-like domain of CRY-1 can bind Mg·ATP, and we observe a single molecule of an ATP analog bound in the aforementioned surface cavity, near the bound FAD cofactor. The structure has implications for the signaling mechanism of CRY blue-light photoreceptors.

3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the formation of mevalonate, the committed step in the biosynthesis of sterols and isoprenoids. The activity of HMGR is controlled through synthesis, degradation and phosphorylation to maintain the concentration of mevalonate-derived products. In addition to the physiological regulation of HMGR, the human enzyme has been targeted successfully by drugs in the clinical treatment of high serum cholesterol levels. Three crystal structures of the catalytic portion of human HMGR in complexes with HMG-CoA, with HMG and CoA, and with HMG, CoA and NADP(+), provide a detailed view of the enzyme active site. Catalytic portions of human HMGR form tight tetramers. The crystal structure explains the influence of the enzyme's oligomeric state on the activity and suggests a mechanism for cholesterol sensing. The active site architecture of human HMGR is different from that of bacterial HMGR; this may explain why binding of HMGR inhibitors to bacterial HMGRs has not been reported.

Glioblastoma Multiforme (GBM) is the most frequent of malignant tumors of the brain. It is fatal usually within fifteen months after diagnosis. The Cancer Genome Atlas (TCGA) has surveyed the genomes of hundreds of human GBMs for presence of mutations in genes previously associated with cancer. Among those identified, three of the five most frequently mutated genes encode the well-known tumor suppressors: P53, NF1, and Pten (TCGA, 2008). We have generated genetic mouse models of GBM based on mutation in these same three tumor suppressors and they develop GBM with 100% incidence. These mouse GBMs resemble the human counterparts by all commonly used criteria for diagnosis. Extensive study of these mice and tumors has provided novel insights into the natural history of GBM development and moreover pointed at the adult neural stem cell/progenitor compartment as the most likely and frequent source of these tumors (Kwon CH, et al., 2008). We have thus isolated a cancer stem cell and demonstrated a hierarchical growth for these tumors in vivo. (Chen J, et al., 2012) In an effort to identify novel GBM specific molecular pathways that might serve as therapeutic targets, we undertook a 200,000 small compound high throughput screen using primary low passage GBM cells generated by pooling cells from multiple mouse tumors (Mut6 cells). A critical counter screen was designed to minimize compounds that targeted the cell division and DNA replication machinery of cells since many normal cells divide and this property is not unique to cancer cells. We therefore ruled out all compounds that displayed toxicity to primary mouse embryo fibroblasts (MEFs) or to primary astrocytes. At the end of the screen, we selected three lead compounds with amenable physical-chemical properties for further evaluation and development as research discovery reagents and possible translational tools for drug development. We verified the activity of one of these compounds, termed Compound X, on mouse and human GBM primary cells as well as on a subset of established cancer cell lines, but not on MEFs or primary astrocytes. We identified a rapid cellular stress response accompanied by the robust activation of a transcription factor and eventual apoptotic response that can be mimicked by glucose deprivation. Through intensive structure-activity relationship study, analogs were successfully developed for in vivo activity and also for biochemical pull-down assay. Another compound (Compound Y) also induced apoptosis, however displaying response through completely different pathway, which indicates our screen identified multiple vulnerabilities in GBM tumor cells. Citation Format: Sang Kyun Lim, Yufeng Shi, Qiren Liang, Inga Nazarenko, Shuguang Wei, Maya Palnitkar, Yanjiao Li, Woosung Cho, Noelle Williams, Bruce Posner, Jef De Brabander, Luis F. Parada. Identification of small molecules for GBM study. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A63.

Nucleotide excision repair (NER) is the most important DNA-repair mechanism in living organisms. In prokaryotes, three enzymes forming the UvrABC system initiate NER of a variety of structurally different DNA lesions. UvrB, the central component of this system, is responsible for the ultimate DNA damage recognition and participates in the incision of the damaged DNA strand. The crystal structure of Thermus thermophilus UvrB reveals a core that is structurally similar to core regions found in helicases, where they constitute molecular motors. Additional domains implicated in binding to DNA and various components of the NER system are attached to this central core. The architecture and distribution of DNA binding sites suggest a possible model for the DNA damage recognition process.