So far, many inhibitors of tyrosinase have been discovered for cosmetic and clinical agents. However, the molecular mechanisms underlying the inhibition in the active site of tyrosinase have not been well understood. To explore this problem, we examined here the inhibitory effects of 4′-hydroxylation and methoxylation of phenylbenzoic acid (PBA) isomers, which have a unique scaffold to inhibit mushroom tyrosinase. The inhibitory effect of 3-PBA, which has the most potent inhibitory activity among the isomers, was slightly decreased by 4′-hydroxylation and further decreased by 4′-methoxylation against mushroom tyrosinase. Surprisingly, 4′-hydroxylation but not methoxylation of 2-PBA appeared inhibitory activity. On the other hand, both 4′-hydroxylation and methoxylation of 4-PBA increased the inhibitory activity against mushroom tyrosinase. In silico docking analyses using the crystallographic structure of mushroom tyrosinase indicated that the carboxylic acid or 4′-hydroxyl group of PBA derivatives could chelate with cupric ions in the active site of mushroom tyrosinase, and that the interactions of Asn260 and Phe264 in the active site with the adequate-angled biphenyl group are involved in the inhibitory activities of the modified PBAs, by parallel and T-shaped π-π interactions, respectively. Furthermore, Arg268 could fix the angle of the aromatic ring of Phe264, and Val248 is supposed to interact with the inhibitors as a hydrophobic manner. These results may enhance the structural insight into mushroom tyrosinase for the creation of novel tyrosinase inhibitors.Download high-res image (125KB)Download full-size image

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 = 6.97 μM, monophenolase activity; IC50 = 36.3 μM, 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.

High mobility group box1 (HMGB1) is a non-histone chromatin chromosomal protein playing an important role in chromatin architecture and transcriptional regulation. Recently, HMGB1 has been shown to be secreted into extracellular milieu in necrosis and apoptosis, and involved in inflammatory responses. However, the mechanism by which apoptotic cells release HMGB1 is unclear. In this study, to investigate the mechanism of HMGB1 release, we searched inhibitors of HMGB1 release from apoptotic cells. As a result, three compounds, 4-(4,6-dichloro-[1,3,5]-triazin-2-ylamino)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-benzoic acid (DR396), Pontacyl Violet 6R (PV6R), and Fmoc-D-Cha-OH (FDCO) in our in-house chemical library were found to inhibit HMGB1 release from staurosporine (STS)-induced apoptotic HeLa S3 cells. Interestingly, these three compounds have been previously categorized into apoptotic DNase γ inhibitors. Therefore, we examined whether apoptotic nucleosomal DNA fragmentation is involved in the release of HMGB1 during apoptosis. Expectedly, DR396, which is the most potent and specific inhibitor of DNase γ, was found to almost completely inhibit both HMGB1 release and internucleosomal DNA cleavage in HeLa S3 cells transfected with DNase γ expression vector and stably expressing DNase γ (HeLa S3/γ cells). These results clearly suggest that nucleosomal DNA fragmentation catalyzed by DNase γ is critical in the release of HMGB1 from apoptotic cells.

The human glyoxalase I (hGLO I), which is a rate-limiting enzyme in the pathway for detoxification of apoptosis-inducible methylglyoxal (MG), has been expected as an attractive target for the development of new anti-cancer drugs. We have previously identified a natural compound myricetin as a substrate transition-state (Zn2+-bound MG-glutathione (GSH) hemithioacetal) mimetic inhibitor of hGLO I. Here, we constructed a hGLO I/inhibitor 4-point pharmacophore based on the binding mode of myricetin to hGLO I. Using this pharmacophore, in silico screening of chemical library was performed by docking study. Consequently, a new type of compound, which has a unique benzothiazole ring with a carboxyl group, named TLSC702, was found to inhibit hGLO I more effectively than S-p-bromobenzylglutathione (BBG), a well-known GSH analog inhibitor. The computational simulation of the binding mode indicates the contribution of Zn2+-chelating carboxyl group of TLSC702 to the hGLO I inhibitory activity. This implies an important scaffold-hopping of myricetin to TLSC702. Thus, TLSC702 may be a valuable seed compound for the generation of a new lead of anti-cancer pharmaceuticals targeting hGLO I.A new type inhibitor of human glyoxalase I, named TLSC702, was discovered by myricetin-based 4-point pharmacophore.

Glyoxalase I (GLO I) is the rate-limiting enzyme for detoxification of methylglyoxal (MG), a side-product of glycolysis, which is able to induce apoptosis. Since GLO I is known to be highly expressed in the most tumor cells and little in normal cells, inhibitors of this enzyme has been expected to be new anticancer drugs. Here, we examined the inhibitory abilities to the human GLO I of anthocyanidins, such as delphinidin, cyanidin and pelargonidin. Among them, delphinidin was found to have the most potent inhibitory effect on human GLO I. Also, only delphinidin-induced apoptosis in HL-60 cells in a dose- and time-dependent manner. Furthermore, we determined a pharmacophore for delphinidin binding to the human GLO I by computational simulation analyses of the binding modes of delphinidin, cyanidin and pelargonidin to the enzyme hot spot. These results suggest that delphinidin could be a useful lead compound for the development of novel GLO I inhibitory anticancer drugs.The structure of delphinidin and its predicted binding mode on the human GLO I.

Tyrosinase inhibitors are important agents for cosmetic products. We examined here the inhibitory effects of three isomers of thujaplicins (α, β and γ) on mushroom tyrosinase and analyzed their binding modes using a homology model from the crystal structure of Streptomyces castaneoglobisporus tyrosinase (PDB ID: 1wx2). All the thujaplicins were found to be competitive inhibitors and γ-thujaplicin has the most potent inhibitory activity (IC50 = 0.07 μM). It is noted that there are good correlations between their observed IC50 values and their binding free energies calculated by MM-GB/SA. The binding modes of thujaplicins were predicted to be similar to that of Tyr98 of caddie protein (ORF378), which was co-crystallized with S. castaneoglobisporus tyrosinase. Furthermore, free energy decomposition analysis indicated that the potent inhibitory activity of γ-thujaplicin is due to the interactions with His242, Val243 and Pro257 (hot spot amino acid residues) at the active site of tyrosinase. These results provide a novel structural insight into the hot spot of mushroom tyrosinase for the specific binding of γ-thujaplicin.

Nuclear factor-κB (NF-κB) has been considered as a good target for the treatment of many diseases. Although a lot of NF-κB inhibitors have already been reported, many of them have several common problems. Thus, we attempted to identify novel NF-κB inhibitors to be unique lead compounds for creating new pharmaceuticals. In the present study, we screened our chemical library for compounds that directly inhibit the DNA binding of NF-κB by using fluorescence correlation spectroscopy (FCS). Consequently, we identified a promising compound, 4,6-dichloro-N-phenyl-1,3,5-triazin-2-amine, referred to as NI241. It mediated a dose-dependent inhibition of the DNA binding of NF-κB p50. Its analogues also showed dose-dependent inhibition and their inhibitory effects were altered by the substituents on the N-phenyl group. Furthermore, we predicted the binding mode of NI241 with p50 in silico. In this model, NI241 forms three hydrogen bonds with Tyr60, His144, and Asp242 on p50, which are important amino acid residues for the interaction with DNA. These results suggest that NI241 with structural novelty may serve as a useful scaffold for the creation of new NF-κB inhibitors by rational optimization.

Glyoxalase I (GLO I) is the rate-limiting enzyme for detoxification of methylglyoxal (MG), a side product of glycolysis, which is able to induce apoptosis. Since GLO I is known to be highly expressed in the most tumor cells and little in normal cells, specific inhibitors of this enzyme have been expected as effective anticancer drugs. The purpose of this study is a good construction of the human GLO I/inhibitor pharmacophore to obtain unique human GLO I inhibitory seed compounds for the development of useful anticancer drugs. Here, we selected natural flavonoid compounds that possess a plane configuration of cis C-4 ketone and C-5 hydroxy groups as the substrate (MG) transition-state mimetic structure. These compounds were examined the inhibitory abilities to human GLO I activity and analyzed their structure–activity relationships to determine an important pharmacophore of flavonoids for the human GLO I binding. Our results point to the contribution of hydroxy groups at the B ring of flavonoids to the effective inhibition of the human GLO I. Based on the binding mode of flavonoids, we constructed the human GLO I/inhibitor pharmacophore. This work delivers the first three-dimensional (3D) structural data and explains certain flavonoids interact specifically with the human GLO I.

Ac-DNLD-CHO is a novel caspase-3 specific peptide inhibitor that was rationally designed by our computational strategy. The specificity was shown to be due to the specific interaction of NLD moiety with the active site of caspase-3 on the basis of docking mode and site-directed mutagenesis analyses. Here, we computationally screened non-peptidic small molecular inhibitors of caspase-3 from our chemical library using a reliable pharmacophore derived from the specific binding mode of NLD. Through in vitro enzyme assay of the screened candidate compounds, we discovered a novel caspase-3 specific small molecular inhibitor, CS4566, which has a unique scaffold structure. The binding mode of CS4566 to caspase-3 mimics that of NLD, especially LD moiety. This represents a promising lead compound for creating non-peptidic pharmaceuticals for caspase-mediated diseases, such as neurodegenerative disorders.

Here, we describe the non-redundant roles of caspase-activated DNase (CAD) and DNaseγ during apoptosis in the immature B-cell line WEHI-231. These cells induce DNA-ladder formation and nuclear fragmentation by activating CAD during cytotoxic drug-induced apoptosis. Moreover, these apoptotic manifestations are accompanied by inhibitor of CAD (ICAD) cleavage and are abrogated by the constitutive expression of a caspase-resistant ICAD mutant. No such nuclear changes occur during oxidative stress-induced necrosis, indicating that neither CAD nor DNaseγ functions under necrotic conditions. Interestingly, the DNA-ladder formation and nuclear fragmentation induced by B-cell receptor ligation occur in the absence of ICAD cleavage and are not significantly affected by the ICAD mutant. Both types of nuclear changes are preceded by the upregulation of DNaseγ expression and are strongly suppressed by 4-(4,6-dichloro-[1, 3, 5]-triazin-2-ylamino)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-benzoic acid (DR396), which is a specific inhibitor of DNaseγ. Our results suggest that DNaseγ provides an alternative mechanism for inducing nuclear changes when the working apoptotic cascade is unsuitable for CAD activation.

The rational design of peptide-based specific inhibitors of the caspase family members using their X-ray crystallographies is an important strategy for chemical knockdown to define the critical role of each enzyme in apoptosis and inflammation. Recently, we designed a novel potent peptide inhibitor, Ac-DNLD-CHO, for caspase-3 using a new computational screening system named the Amino acid Positional Fitness (APF) method (BMC Pharmacol. 2004, 4:7). Here, we report the specificity of the DNLD sequence against caspase-3 over other major caspase family members that participate in apoptosis by computational docking and site-directed mutagenesis studies.Ac-DNLD-CHO inhibits caspases-3, -7, -8, and -9 activities with Kiapp values of 0.68, 55.7, >200, and >200 nM, respectively. In contrast, a well-known caspase-3 inhibitor, Ac-DEVD-CHO, inhibits all these caspases with similar Kiapp values. The selective recognition of a DNLD sequence by caspase-3 was confirmed by substrate preference studies using fluorometric methylcoumarin-amide (MCA)-fused peptide substrates. The bases for its selectivity and potency were assessed on a notable interaction between the substrate Asn (N) and the caspase-3 residue Ser209 in the S3 subsite and the tight interaction between the substrate Leu (L) and the caspase-3 hydrophobic S2 subsite, respectively, in computational docking studies. Expectedly, the substitution of Ser209 with alanine resulted in loss of the cleavage activity on Ac-DNLD-MCA and had virtually no effect on cleaving Ac-DEVD-MCA. These findings suggest that N and L residues in Ac-DNLD-CHO are the determinants for the selective and potent inhibitory activity against caspase-3.On the basis of our results, we conclude that Ac-DNLD-CHO is a reliable, potent and selective inhibitor of caspase-3. The specific inhibitory effect on caspase-3 suggests that this inhibitor could become an important tool for investigations of the biological function of caspase-3. Furthermore, Ac-DNLD-CHO may be an attractive lead compound to generate novel effective non-peptidic pharmaceuticals for caspase-mediated apoptosis diseases, such as neurodegenerative disorders and viral infection diseases.

DNase γ, a member of the DNase I family, has been suggested to cause DNA fragmentation during apoptosis. We recently identified 4-(4,6-dichloro-[1,3,5]-triazine-2-ylamino)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-benzoic acid (DR396) as a novel specific inhibitor for human DNase γ [Sunaga, S.; Kobayashi, T.; Yoshimori, A.; Shiokawa, D.; Tanuma, S. Biochem. Biophys. Res. Commun.2004, 325, 1292]. However, the binding mode (coordinate) of DR396 to DNase γ has not yet been defined. Here, we examined the molecular basis for the inhibitory activity of DR396 to DNase γ by structure-based computational docking studies. In the blind-docking study using a human DNase γ homology model, a unique binding site of DR396 was predicted, which is tentatively named the ‘DNA trapping site’ because of the binding domain of the unhydrolyzed DNA strand, but not the active site. Targeting the DNA trapping site as a hot spot, new human DNase γ inhibitors were obtained from our diverse chemical library in silico. These inhibitors showed high correlations between their predicted binding-free energies (ΔGs) and observed IC50 values in the DNA trapping site but not the active site. The IC50 of a regioisomer of DR396, 5-(4,6-dichloro-[1,3,5]-triazine-2-ylamino)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-benzoic acid (DF365), was 73 μM (ΔG = −9.75 kcal/mol), a 20-fold weaker inhibitory ability than that of DR396 (IC50 = 3.2 μM, ΔG = −11.22 kcal/mol). Fluorescein and triazine derivatives, partial structures of DR396, had little inhibitory activity for DNase γ. Docking analyses of the interaction between DR396 and DNase γ revealed that DR396 binds tightly to three subsites (S1, S2, and S3) in the trapping site of DNase γ by forming six hydrogen bonds, whereas DF365 and the partial structures are unable to form hydrogen bonds at all three subsites. These findings suggest that the specificity and potency of the inhibitory activity of DR396 for DNase γ is due to the specific interaction of DR396 with three subsites in the DNA trapping site of DNase γ.

In this study, we investigate the roles of two apoptotic endonucleases, CAD and DNase , in neuronal apoptosis. High expression of CAD, but not DNase , is detected in proliferating N1E-115 neuroblastoma cells, and apoptotic DNA fragmentation induced by staurosporine under proliferating conditions is abolished by the expression of a caspase-resistant form of ICAD. After the induction of neuronal differentiation, CAD disappearance and the induction of DNase occur simultaneously in N1E-115 cells. Apoptotic DNA fragmentation that occurs under differentiating conditions is suppressed by the downregulation of DNase caused by its antisense RNA. The induction of DNase is also observed during neuronal differentiation of PC12 cells, and apoptotic DNA fragmentation induced by NGF deprivation is inhibited by the antisense-mediated downregulation of DNase . These observations suggest that DNA fragmentation in neuronal apoptosis is catalyzed by either CAD or DNase depending on the differentiation state. Furthermore, DNase is suggested to be involved in naturally occurring apoptosis in developing nervous systems.

Poly(ADP-ribosyl)ation, which is mainly involved in DNA repair and replication, is catalyzed mainly by poly(ADP-ribose) polymerase-1 (PARP-1) and poly(ADP-ribose) glycohydrolase (PARG). Although recombinant human PARP-1 (hPARP-1) is commercially available, there are no reports on the preparation of recombinant human PARG (hPARG). Here, we report the efficient expression and purification of a recombinant hPARG-catalytic domain (hPARG-CD) from Escherichia coli (E. coli). hPARG-CD was expressed as a fusion protein with a glutathione S-transferase (GST) tag at the N-terminus and a hexahistidine (6His) tag at the C-terminus. Both high cell density and low temperature culture conditions were important for the maximum production of soluble recombinant hPARG-CD. After sequential affinity chromatography using immobilized metal affinity resin and glutathione-Sepharose (GSH-Sephasrose), more than 95% pure recombinant hPARG-CD was obtained with a yield of approximately 2 mg per 1 L of E. coli culture medium. The km and Vmax values of purified recombinant hPARG-CD were 9.0 μM and 35.6 μmol/min/mg protein, respectively. These kinetic values were similar to those of purified endogenous hPARG reported previously. Furthermore, the recombinant hPARG-CD was inhibited by known PARG inhibitors such as adenosine diphosphate (hydroxymethyl) pyrrolidinediol (ADP-HPD), eosin Y, and phloxine B. These results show that the recombinant hPARG-CD is useful to search for specific inhibitors and to elucidate the regulatory mechanisms of hPARG.