Takahiro Oyama

Structure-activity relationships of the thujaplicins for inhibition of human tyrosinase.

Tyrosinase inhibitors have become increasingly critical agents in cosmetic, agricultural, and medicinal products. Although a large number of tyrosinase inhibitors have been reported, almost all the inhibitors were unfortunately evaluated by using commercial available mushroom tyrosinase. Here, we examined the inhibitory effects of three isomers of thujaplicin (α, β, and γ) on human tyrosinase and analyzed their binding modes using homology model and docking studies. As the results, γ-thujaplicin was found to strongly inhibit human tyrosinase with the IC50 of 1.15 μM, extremely superior to a well-known tyrosinase inhibitor kojic acid (IC50 = 571.17 μM). MM-GB/SA binding free energy decomposition analyses suggested that the potent inhibitory activity of γ-thujaplicin may be due to the interactions with His367, Ile368, and Val377 (hot spot amino acid residues) in human tyrosinase. Furthermore, the binding mode of α-thujaplicin indicated that Val377 and Ser380 may cause van der Waals clashes with the isopropyl group of α-thujaplicin. These results provide a novel structural insight into the hot spot of human tyrosinase for the specific binding of γ-thujaplicin and a way to optimize not only thujaplicins but also other lead compounds as specific inhibitors for human tyrosinase in a rational manner.

Characterization of 5’-Flanking Regions of Various Human Telomere Maintenance Factor-Encoding Genes

Telomeres are the unique nucleoprotein complex structures located at the end of linear eukaryotic chromosomes (Blackburn, 2000; de Lange, 2006). They are composed of TTAGGG repeats that are typically 10 kb at birth and gradually shorten with cell divisions (de Lange, 2006). Telomerase is composed of the protein subunit TERT and the RNA subunit TERC (TR). It elongates the telomere by adding telomeric repeats (Greider & Blackburn, 1987). The 50 to 300 nucleotides from the terminal end of the telomeres are single stranded 3’-protluded Goverhang structures which make the t-loop configuration (de Lange, 2006; Griffith et al., 1999). Mammalian telomeres are included in heterochoromatin and attached to the nuclear matrix (Oberdoerffer & Sinclair, 2007; Gonzalez-Suarez & Gonzalo, 2008). Telomere shortening causes instability of the ends of chromosomes to lead to replicative senescence (O’Sullivan & Karlseder, 2010; Lundblad & Szostak, 1989). Therefore, the ends of telomeres should be protected from damaging or cellular activities. The t-loop structures are regulated by shelterin protein factors, TRF1, TRF2, Rap1, TIN2, TPP1, POT1 (Gilson & Geli, 2007; O’Sullivan & Karlseder, 2010), and Rec Q DNA helicases, WRN and BLM (Chu & Hickson, 2009). TRF1 and TRF2, which bind to duplex telomeric DNA and retain shelterin on the telomere repeats, were shown to interact with various functional proteins (Giannone et al., 2010). Molecular structural analysis of Rap1 revealed that its mechanism of action involves interaction with TRF2 and Taz1 proteins (Chen et al., 2011). A recent study showed that depletion of TPP1 and its partner TIN2 causes a loss of telomerase recruitment to telomeres (Abreu et al., 2010). POT1 is an important regulator of telomerase length, in stimulating the RecQ helicases WRN and BLM (Opresko et al., 2005). Tankyrase-1 (TANK1), which is classified as a poly(ADP-ribose) polymerase family protein, is also known to regulate telomere homeostasis by modifying TRF1 (Smith et al., 1998; Schreiber et al., 2006). Dyskerin, which is encoded by the DKC1 gene, is a key auxiliary protein that is contained in a Cajal body with TERT (Cohen et al., 2007). Defects in the shelterin components and telomerase are thought to down-regulate telomere structure

New Insights into the Roles of NAD+-Poly(ADP-ribose) Metabolism and Poly(ADP-ribose) Glycohydrolase.

Accumulating evidence has suggested the fundamental functions of NAD+-poly(ADP-ribose) metabolism in cellular and physiological processes, including energy homeostasis, signal transduction, DNA transaction, genomic stability and cell death or survival. The NAD+ biosynthesis and poly(ADP-ribose) [(ADP-R)n] turnover are tightly controlled by several key enzymes, such as nicotinamide phosphoribosyltransferase (NmPRT), nicotinamide mononucleotide adenylyltransferases (NMNATs), poly(ADP-ribose) polymerase (PARP), poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribose pyrophosphorylase (ADPRPPL). Many researches investigating the roles of these enzymes in cells have revealed the physiological and pathological importance, and thereby the therapeutical values. Among these enzymes, the polymer degrading enzyme PARG has not yet been intensively studied, because of the low cellular content, lack of cell-available PARG chemical inhibitors and PARG genetic models. So, the biological roles of (ADP-R)n catabolism by PARG are still being elucidated as compared to those of synthesis by PARP. However, recent studies delineate that PARG-dependent (ADP-R)n degradation is critical for many pathological conditions, and thus PARG is an important target for chemical therapeutics for several diseases. This review will present the recent progresses about the roles of NAD+-(ADP-R)n metabolism and the structures and functions of PARG, with a focus on its role in DNA repair and cell death by apoptosis in relation to central regulatory network, and the therapeutic potentials of PARG inhibitors in cancer chemotherapy.

Discovery of a new type of scaffold for the creation of novel tyrosinase inhibitors

Tyrosinase is known as the key enzyme for melanin biosynthesis, which is effective in preventing skin injury by ultra violet (UV). In past decades, tyrosinase has been well studied in the field of cosmetics, medicine, agriculture and environmental sciences, and a lot of tyrosinase inhibitors have been developed for their needs. Here, we searched for new types of tyrosinase inhibitors and found phenylbenzoic acid (PBA) as a unique scaffold. Among three isomers of PBA, 3-phenylbenzoic acid (3-PBA) was revealed to be the most potent inhibitor against mushroom tyrosinase (IC50=6.97μM, monophenolase activity; IC50=36.3μM, diphenolase activity). The kinetic studies suggested that the apparent inhibition modes for the monophenolase and diphenolase activities were noncompetitive and mixed type inhibition, respectively. Analyses by in silico docking studies using the crystallographic structure of mushroom tyrosinase indicated that the carboxylic acid group of the 3-PBA could adequately bind to two cupric ions in the tyrosinase. To prove this hypothesis, we examined the effect of modification of the carboxylic acid group of the 3-PBA on its inhibitory activity. As expected, the esterification abrogated the inhibitory activity. These observations suggest that 3-PBA is a useful lead compound for the generation of novel tyrosinase inhibitors and provides a new insight into the molecular basis of tyrosinase catalytic mechanisms.

A New Protocol to Discover Novel Anti-Aging Compounds

Several natural and chemical compounds have been suggested to have effects as anti-aging drugs. For example, caloric restriction (CR) mimetics trans-resveratrol (Rsv) and 2-deoxy-D-glucose (2DG) are candidate compounds that can elongate life span of organisms, and they might also have roles in the regulation of telomere maintenance and mitochondrial functions. Recently, pharmaceutical medicaments rapamycin, an immunosuppressant and metformin, a medicine for diabetes, have been shown to act on the insulin/IGF1 signaling pathway. Therefore, they are also expected to have anti-aging effects. Here we propose a new protocol to discover novel compounds that can be used as a remedy to slow senescence and control aging in the aspect of promoter activities of telomere and energy metabolism-regulating factor encoding genes.