Development of a fluorescent-labeled trapping reagent to evaluate the risk posed by acyl-CoA conjugates.

Although acyl-CoA conjugates are known to have higher reactivity than acyl glucuronides, few studies have been conducted to evaluate the risk of the conjugates. In the present study, we aimed to develop a trapping assay for acyl-CoA conjugates using trapping reagents we have developed previously. It was revealed that Cys-Dan, which has both a thiol and an amino group, was the most effective in forming stable adducts containing an amide bond after intramolecular acyl migration. Additionally, we also developed a hepatocyte-based trapping assay in the present study to overcome the shortcomings of liver microsomes. Although liver microsomes are commonly used as enzyme sources in trapping assays, they lack some of the enzymes required for drug metabolism and detoxification systems. In human hepatocytes, our three trapping reagents, CysGlu-Dan, Dap-Dan and Cys-Dan, captured CYP-dependent reactive metabolites, reactive acyl glucuronides, and reactive acyl-CoA conjugates, respectively. The work suggests that the trapping assay with the reagents in hepatocytes is useful to evaluate the risk of reactive metabolites in drug discovery.

Fullerene derivatives as inhibitors of the SARS-CoV-2 main protease.

COVID-19 is an ongoing worldwide pandemic. Even today, there is a need for the development of effective therapeutic agents. SARS-CoV-2 is known as the causative virus of COVID-19, and its main protease is one of the enzymes essential for its growth and is considered a drug discovery target. In this study, we evaluated the inhibitory activities of a variety of fullerene derivatives, including newly synthesized derivatives, against the main protease of SARS-CoV-2. As a result, the malonic acid-type fullerene derivatives showed the strongest inhibitory activity.

Formation of reactive metabolites of benzbromarone in humanized-liver mice.

Benzbromarone, a uricosuric drug, has the potential to cause serious hepatotoxicity. Several studies have shown the formation of reactive metabolites of benzbromarone and their association with hepatotoxicity in mice. However, it is unknown whether those reactive metabolites are generated in humans in vivo. In the present study, we firstly investigated the pharmacokinetic profiles of benzbromarone in chimeric TK-NOG mice transplanted with human hepatocytes (humanized-liver mice) and then investigated whether reactive metabolites could be generated. The area under the plasma concentration-time curve ratio of benzbromarone and its major metabolites (benzbromarone: 1'-hydroxy benzbromarone: 6-hydroxy benzbromarone) in humanized-liver mice was 1: 1.2: 0.7, which was similar to that reported in humans. In addition, glutathione conjugates and their further metabolites derived from the epoxidation of the benzofuran ring and 1',6-dihydroxylation of benzbromarone were detected in the livers, urine and plasma. Furthermore, their peak intensities in mass spectrometry showed markedly higher levels compared with those of TK-NOG mice. These results suggested that the metabolic profiles of benzbromarone in humanized-liver mice were similar to those in humans and that the reactive metabolites detected in humanized-liver mice could be generated and are associated with the benzbromarone-induced hepatotoxicity in humans.

Synthesis and evaluation of tofacitinib analogs designed to mitigate metabolic activation.

Tofacitinib (TFT), a JAK inhibitor used for the treatment of rheumatoid arthritis and other diseases, is associated with severe liver injury that is believed to be caused by its reactive aldehyde or epoxide metabolites. In this study, we synthesized six tofacitinib analogs designed to avoid the formation of reactive metabolites and evaluated their JAK3 inhibitory activity, metabolic stability, CYP3A time-dependent inhibition, and cytotoxicity. Our data indicated that purine analog 3, which showed little inhibition of CYP3A and cytotoxicity and inhibited JAK3 in the nanomolar range, could be a safer drug candidate than TFT. In addition, the results of the bioactivation study using TFT and its analogs suggest that the epoxide metabolite might contribute to TFT-induced CYP3A4 mechanism-based inhibition and hepatic toxicity.

Novel Mechanism by a Bis-Pyridinium Fullerene Derivative to Induce Apoptosis by Enhancing the MEK-ERK Pathway in a Reactive Oxygen Species-Independent Manner in BCR-ABL-Positive Chronic Myeloid Leukemia-Derived K562 Cells.

In the treatment of breakpoint cluster region-Abelson (BCR-ABL)-positive chronic myeloid leukemia (CML) using BCR-ABL inhibitors, the appearance of a gatekeeper mutation (T315I) in BCR-ABL is a serious issue. Therefore, the development of novel drugs that overcome acquired resistance to BCR-ABL inhibitors by CML cells is required. We previously demonstrated that a bis-pyridinium fullerene derivative (BPF) induced apoptosis in human chronic myeloid leukemia (CML)-derived K562 cells partially through the generation of reactive oxygen species (ROS). We herein show that BPF enhanced the activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase-extracellular signal-regulated kinase (MEK-ERK) pathway in a ROS-independent manner. BPF-induced apoptosis was attenuated by trametinib, suggesting the functional involvement of the MEK-ERK pathway in apoptosis in K562 cells. In addition, the constitutive activation of the MEK-ERK pathway by the enforced expression of the BRAFV600E mutant significantly increased the sensitivity of K562 cells to BPF. These results confirmed for the first time that BPF induces apoptosis in K562 cells through dual pathways-ROS production and the activation of the MEK-ERK pathway. Furthermore, BPF induced cell death in transformed Ba/F3 cells expressing not only BCR-ABL but also T315I mutant through the activation of the MEK-ERK pathway. These results indicate that BPF is as an effective CML drug that overcomes resistance to BCR-ABL inhibitors.

A pyridinium­type fullerene derivative suppresses primary effusion lymphoma cell viability via the downregulation of the Wnt signaling pathway through the destabilization of ß­catenin.

Primary effusion lymphoma (PEL) is defined as a rare subtype of non­Hodgkin's B cell lymphoma, which is caused by Kaposi's sarcoma­associated herpesvirus (KSHV) in immunosuppressed patients. PEL is an aggressive type of lymphoma and is frequently resistant to conventional chemotherapeutics. Therefore, the discovery of novel drug candidates for the treatment of PEL is of utmost importance. In order to discover potential novel anti­tumor compounds against PEL, the authors previously developed a pyrrolidinium­type fullerene derivative, 1,1,1',1'­tetramethyl [60]fullerenodipyrrolidinium diiodide (derivative #1), which induced the apoptosis of PEL cells via caspase­9 activation. In the present study, the growth inhibitory effects of pyrrolidinium­type (derivatives #1 and #2), pyridinium­type (derivatives #3 and #5 to #9) and anilinium­type fullerene derivatives (derivative #4) against PEL cells were evaluated. This analysis revealed a pyridinium­type derivative (derivative #5; 3­â€‹5'­(etho xycarbonyl)­1',5'­dihydro­2'H­[5,6]fullereno­C60­Ih­[1,9­c]pyrrol­2'­yl]­1­methylpyridinium iodide), which exhibited antitumor activity against PEL cells via the downregulation of Wnt/ß­catenin signaling. Derivative #5 suppressed the viability of KSHV­infected PEL cells compared with KSHV­uninfected B­lymphoma cells. Furthermore, derivative #5 induced the destabilization of ß­catenin and suppressed ß­catenin­TCF4 transcriptional activity in PEL cells. It is known that the constitutive activation of Wnt/ß­catenin signaling is essential for the growth of KSHV­infected cells. The Wnt/ß­catenin activation in KSHV­infected cells is mediated by KSHV latency­associated nuclear antigen (LANA). The data demonstrated that derivative #5 increased ß­catenin phosphorylation, which resulted in ß­catenin polyubiquitination and subsequent degradation. Thus, derivative #5 overcame LANA­mediated ß­catenin stabilization. Furthermore, the administration of derivative #5 suppressed the development of PEL cells in the ascites of SCID mice with tumor xenografts derived from PEL cells. On the whole, these findings provide evidence that the pyridinium­type fullerene derivative #5 exhibits antitumor activity against PEL cells in vitro and in vivo. Thus, derivative #5 may be utilized as a novel therapeutic agent for the treatment of PEL.

A bis-pyridinium fullerene derivative induces apoptosis through the generation of ROS in BCR-ABL-positive leukemia cells.

A fusion protein, Breakpoint cluster region-Abelson (BCR-ABL) is responsible for the development of chronic myeloid leukemia (CML) and acute lymphocytic leukemia (ALL). Inhibitors against BCR-ABL are effective for the treatment of leukemia; however, a gatekeeper mutation (T315I) in BCR-ABL results in resistance to these inhibitors, which markedly impedes their efficacy. We herein demonstrated that a bis-pyridinium fullerene derivative (BPF) significantly induced apoptosis in human CML-derived K562 cells and ALL-derived SUP-B15 cells via the generation of reactive oxygen species (ROS). BPF reduced the expression of Bcr-Abl mRNA by inhibiting expression of c-Myc through ROS production. BPF also accelerated protein degradation of BCR-ABL through ROS production. Furthermore, BPF down-regulated the expression of not only BCR-ABL but also T315I-mutated BCR-ABL in ROS-dependent manner. As a result, BPF effectively induced apoptosis in transformed Ba/F3 cells expressing both BCR-ABL and T315I-mutated BCR-ABL. Collectively, these results indicate the potential of BPF as an effective leukemia drug that overcomes resistance to BCR-ABL inhibitors.

Development of a Fluorescent-Labeled Trapping Reagent to Detect Reactive Acyl Glucuronides.

Acyl glucuronides are common metabolites of carboxylic acid-containing compounds. Since acyl glucuronides sometimes show high reactivity, they are considered to be involved in drug toxicity. Therefore, it is important to evaluate the risk posed by acyl glucuronides in the development of safe drugs; however, there are no suitable evaluation methods for the early stages of drug discovery. We aimed to develop a trapping reagent that detects reactive acyl glucuronides to assess their risk. We designed a diamine-structured trapping reagent, Dap-Dan, and compared its trapping ability with the reported one that has an amino group, and results showed that Dap-Dan showed higher accuracy. In the trapping assay with 17 medicines containing a carboxylic acid, Dap-Dan trapped acyl glucuronides that had a higher risk of toxicity. In conclusion, Dap-Dan can be useful for evaluating the risk of reactive acyl glucuronides.

Novel pyridinium-type fullerene derivatives as multitargeting inhibitors of HIV-1 reverse transcriptase, HIV-1 protease, and HCV NS5B polymerase.

In the present study, we newly synthesized four types of novel fullerene derivatives: pyridinium/ethyl ester-type derivatives 3b-3l, pyridinium/carboxylic acid-type derivatives 4a, 4e, 4f, pyridinium/amide-type derivative 5a, and pyridinium/2-morpholinone-type derivative 6a. Among the assessed compounds, cis-3c, cis-3d, trans-3e, trans-3h, cis-3l, cis-4e, cis-4f, trans-4f, and cis-5a were found to inhibit HIV-1 reverse transcriptase (HIV-RT), HIV-1 protease (HIV-PR), and HCV NS5B polymerase (HCV NS5B), with IC50 values observed in the micromolar range. Cellular uptake of pyridinium/ethyl ester-type derivatives was higher than that of corresponding pyridinium/carboxylic acid-type derivatives and pyridinium/amide-type derivatives. This result might indicate that pyridinium/ethyl ester-type derivatives are expected to be lead compounds for multitargeting drugs to treat HIV/HCV coinfection.

Development of fluorescent-labeled trapping reagents based on cysteine to detect soft and hard electrophilic reactive metabolites.

Trapping assays are conducted at lead optimization stages to detect reactive metabolites (RMs) that can contribute to drug toxicity. The commonly used dansyl glutathione (dGSH) provides a sensitive analysis owing to the fluorescent label, however, it captures only soft electrophilic RMs. TRs for hard electrophilic RMs, few of which are labeled fluorescently, can detect hard electrophilic aldehydes only by forming unstable imine derivatives. In this study, we aimed to develop novel fluorescently labeled TRs that detect both soft and hard electrophilic RMs and form stable ring structures with aldehydes. We designed four dansylated TRs based on cysteine, which has both soft and hard nucleophilic groups. To evaluate the reactivity of the TRs, we incubated them with several substrates and found that one of the TRs (CysGlu-Dan) detected all the soft and hard electrophilic RMs. We also examined the inhibition potential of each TR for seven major CYPs involved in drug metabolism and found that CysGlu-Dan showed an inhibitory profile similar to that of dGSH. In conclusion, CysGlu-Dan can be used to evaluate the risk of RMs in drug discovery.

p62/SQSTM1-droplet serves as a platform for autophagosome formation and anti-oxidative stress response.

Autophagy contributes to the selective degradation of liquid droplets, including the P-Granule, Ape1-complex and p62/SQSTM1-body, although the molecular mechanisms and physiological relevance of selective degradation remain unclear. In this report, we describe the properties of endogenous p62-bodies, the effect of autophagosome biogenesis on these bodies, and the in vivo significance of their turnover. p62-bodies are low-liquidity gels containing ubiquitin and core autophagy-related proteins. Multiple autophagosomes form on the p62-gels, and the interaction of autophagosome-localizing Atg8-proteins with p62 directs autophagosome formation toward the p62-gel. Keap1 also reversibly translocates to the p62-gels in a p62-binding dependent fashion to activate the transcription factor Nrf2. Mice deficient for Atg8-interaction-dependent selective autophagy show that impaired turnover of p62-gels leads to Nrf2 hyperactivation in vivo. These results indicate that p62-gels are not simple substrates for autophagy but serve as platforms for both autophagosome formation and anti-oxidative stress.

Fullerene derivatives as dual inhibitors of HIV-1 reverse transcriptase and protease.

In the present study, we newly synthesized three types of novel fullerene derivatives: pyridinium-type derivatives trans-3a and 4a-5b, piperidinium-type derivative 9, and proline-type derivatives 10a-12. Among the assessed compounds, 5a, 10e, 10f, 10i, 11a-d, and 12 were found to inhibit both HIV reverse transcriptase and HIV protease (HIV-PR), with IC50 values in the low micromolar range being observed. Regarding HIV-PR inhibition activity, proline-type derivatives 11a-11d and 12, bearing an alkyl chain between the hydroxylmethylcarbonyl (HMC) moiety and pyrrolidine ring, were more potent than other derivatives. This result might indicate that connecting HMC moieties with proline-type fullerene derivatives through properly sized alkyl chain leads to improved HIV-PR inhibitory activity.

Development of Novel Diclofenac Analogs Designed to Avoid Metabolic Activation and Hepatocyte Toxicity.

Diclofenac (DCF) is widely used as a nonsteroidal anti-inflammatory drug; however, it is associated with severe liver injury. This adverse reaction is thought to be related to the reactive quinone imine (QI) and acyl glucuronide (AG) metabolites of DCF, but it remains controversial which reactive metabolites mainly contribute to DCF-induced toxicity. In this study, we synthesized five types of DCF analogs that were designed to mitigate the formation of reactive QI and/or AG metabolites and evaluated their metabolic stability, cyclooxygenase (COX) inhibitory activity, and toxicity to cryopreserved human hepatocytes. Compounds with fluorine at the 5- and 4'-positions of aromatic rings exhibited modest and high metabolic stability to oxidation by cytochrome P450, respectively, but induced cytotoxicity comparable to DCF. Replacing the carboxylic group of DCF with its bioisosteres was effective in terms of stability to oxidative metabolism and glucuronidation; however, sulfonic acid and sulfonamide groups were not preferable for COX inhibition, and tetrazole-containing analogs induced strong cytotoxicity. On the other hand, compounds that have fluorine at the benzylic position were resistant to glucuronidation and showed little toxicity to hepatocytes. In addition, among these compounds, those with hydrogen at the 4'-position (2a and 2c) selectively inhibited the COX-2 enzyme. Throughout these data, it was suggested that compounds 2a and 2c might be novel safer and more efficacious drug candidates instead of DCF.

Synthesis and evaluation of nevirapine analogs to study the metabolic activation of nevirapine.

Nevirapine (NVP) is widely used as a non-nucleoside reverse transcriptase inhibitor of HIV-1, however, it is associated with severe skin and liver injury. The mechanisms of these adverse reactions are not yet clear, but the metabolic activation of NVP is thought to be related to the injury process. Until now, several metabolic activation pathways of NVP have been reported. In this study, in order to identify the reactive metabolite of NVP mainly responsible for CYP inhibition and liver injury, we synthesized five NVP analogs designed to avoid the proposed bioactivation pathway and evaluated their metabolic stabilities, CYP3A4 time-dependent inhibitory activities, and cytotoxicity. As a result, only a pyrimidine analog of NVP, which could avoid the formation of a reactive epoxide intermediate, did not inhibit CYP3A4. Outside of this compound, the other synthesized compounds, which could avoid the generation of a reactive quinone-methide intermediate, inhibited CYP3A4 equal to or stronger than NVP. The pyrimidine analog of NVP did not induce cytotoxicity in HepG2 and transchromosomic HepG2 cells, expressing major four CYP enzymes and CYP oxidoreductase. These results indicated that the epoxide intermediate of NVP might play an important role in NVP-induced liver injury.

Inhibitors of the protein-protein interaction between phosphorylated p62 and Keap1 attenuate chemoresistance in a human hepatocellular carcinoma cell line.

Resistance to anticancer agents has been an obstacle to developing therapeutics and reducing medical costs. Whereas sorafenib is used for the treatment of human hepatocellular carcinoma (HCC), resistance limits its efficacy. p62, a multifunctional protein, is overexpressed in several HCC cell lines, such as Huh-1 cells. Phosphorylated p62 (p-p62) inhibits the protein-protein interaction (PPI) between Keap1 and Nrf2, resulting in the Nrf2 overactivation that causes drug resistance. We have found a unique Nrf2 inactivator, named K67, that inhibited the PPI between Keap1 and p-p62 and attenuated sorafenib resistance in Huh-1 cells. Herein, we designed and synthesised novel K67 derivatives by modification of the substituent at the 4-position of the two benzenesulfonyl groups of K67. Although these new derivatives inhibited the Keap1-p-p62 PPI to a level comparable to or weaker than that of K67, the isopropoxy derivative enhanced the sensitivity of Huh-1 cells to sorafenib to a greater extent than K67 without any influence on the viability of Huh-7 cells, which is a non-resistant HCC cell line. The isopropoxy derivative also increased the sensitivity of Huh-1 cells to regorafenib, which suggests that this derivative has the potential to be used as an agent to overcome chemoresistance based on Nrf2 inactivation.

N-Acetyl cysteine prevents activities of STAT3 inhibitors, Stattic and BP-1-102 independently of its antioxidant properties.

BACKGROUND: Inhibitors for signal transducer and activator of transcription 3 (STAT3), Stattic, BP-1-102, and LLL12 significantly induce apoptosis in transformed Ba/F3 cells expressing an oncogenic fusion protein, nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) that induces the activation of STAT3. We found that the antioxidant reagent, N-acetyl cysteine (NAC) prevented the abilities of Stattic and BP-1-102, but not LLL12 to induce apoptosis in transformed cells expressing NPM-ALK, providing a novel problem in use of STAT3 inhibitors. We herein investigated the mechanisms how NAC prevented the effects of Sttatic and BP-1-102. METHODS: Ba/F3 cells expressing NPM-ALK and SUDHL-1 cells were treated with antioxidants such as NAC, Trolox or edaravone in combination with STAT3 inhibitors. Phosphorylation of STAT3, cell proliferation rate, cell viability, cell cycle, internucleosomal DNA fragmentation and the intracellular accumulation of reactive oxygen species (ROS) was investigated. The binding of STAT3 inhibitors and NAC was analyzed by LC-MS. RESULTS: NAC but not Trolox and edaravone diminished the abilities of Stattic and BP-1-102 to induce apoptosis in cells expressing NPM-ALK. The ROS levels in cells expressing NPM-ALK were not markedly affected by the treatments with Stattic and BP-1-102 in combination with NAC, suggesting that NAC inhibited the activity of Stattic and BP-1-102 independent of its antioxidant activity. LC-MS analysis revealed that NAC directly bound to Stattic and BP-1-102. Furthermore, these NAC adducts exhibited no cytotoxicity, and failed to affect the activity of STAT3. CONCLUSIONS: NAC antagonizes the activities of Stattic and BP-1-102, which inhibit STAT3 activation by interacting with cysteine residues in STAT3.

Synthesis and antitumor activity of novel pyridinium fullerene derivatives.

PURPOSE: We have previously reported that some cationic fullerene derivatives exhibited anticancer activity, and they are expected to be a potential lead compound for an anti-drug resistant cancer agent. However, they are bis-adducts and a mixture of multiple regioisomers, which cannot be readily separated due to the variability of substituent positions on the fullerene cage. To overcome this issue, we evaluated the antiproliferative activities of a set of mono-adduct derivatives and examined their structure-activity relationship. In addition, the in vivo antitumor activity of selected derivatives was also examined. METHODS: Nineteen pyridinium fullerene derivatives were newly designed and synthesized in this study. Their antiproliferative activities were evaluated using several cancer cell lines including drug-resistant cells. Furthermore, in vivo antitumor activity of several derivatives was investigated in mouse xenograft model of human lung cancer. RESULTS: The derivatives inhibited the proliferation of cancer cell lines, including cisplatin-resistant cells and doxorubicin-resistant cells. It was also shown that compound 10 (10 µM), 13 (10 µM) and cis-14 (10 µM) induced the intracellular oxidative stress. In addition, compound 13 (20 mg/kg) and cis-14 (15 mg/kg) significantly exhibited antitumor activity in mouse xenograft model of human lung cancer. CONCLUSION: We synthesized a novel set of mono-adduct fullerene derivatives functionalized with pyridinium groups and found that most of them show potent antiproliferative activities against cancer cell lines and some of them show significant antitumor activities in vivo. We propose that these fullerene derivatives serve as the lead compounds for a novel type of antitumor agents.

Synthesis of novel benzbromarone derivatives designed to avoid metabolic activation.

We synthesized six novel BBR derivatives that were designed to avoid metabolic activation via ipso-substitution and evaluated for their degree of toxicity and hURAT1 inhibition. It was found that all of the derivatives demonstrate lower cytotoxicity in mouse hepatocytes and lower levels of metabolic activation than BBR, while maintaining their inhibitory activity toward the uric acid transporter. We propose that these derivatives could serve as effective uricosuric agents that have much better safety profiles than BBR.

[Hit-to-Lead in Academia: Discovery of a Protein-Protein Interaction Inhibitor of Keap1-Nrf2].

In the process of recent hit-to-lead studies, not only in industry but also in academia, early evaluation of metabolic properties has been one of the key aspects supporting a higher probability of success in drug discovery. In this review, we introduce the development of chemical seeds targeting the Kelch-like ECH-associated protein-1 (Keap1) as an example of an academic hit-to-lead study considering metabolic stability. Keap1 regulates the function of nuclear factor erythroid 2-related factor 2 (Nrf2), which induces various antioxidative or detoxification proteins. An inhibitor of protein-protein interaction (PPI) between Keap1 and Nrf2 to activate Nrf2 is expected to be a novel target for drug discovery. However, Nrf2 is also activated in several cancers, such as human hepatocellular carcinoma, and causes chemoresistance, which is mediated by phosphorylated p62/Sqstm1 (p-p62), an autophagy-related protein that also undergoes a PPI with Keap1. In this case, an Nrf2 suppressor could be used to attenuate drug resistance. We discovered inhibitors against the Nrf2-Keap1 PPI and p-p62-Keap1 PPI using high-throughput screening and established the synthetic routes for the hit compounds and their derivatives. Furthermore, we assessed the metabolic stability of both of the PPI inhibitors in human liver microsomes and identified the metabolic sites.

Discovery of benzo[g]indoles as a novel class of non-covalent Keap1-Nrf2 protein-protein interaction inhibitor.

The Keap1-Nrf2 system is an attractive target for drug discovery regarding various unmet medical needs. Only covalent inhibitors for protein-protein interaction (PPI) between Keap1 and Nrf2 to activate Nrf2 have been approved or are under clinical trials, but such electrophilic compounds lack selectivity. Therefore, specific non-covalent Keap1-Nrf2 PPI inhibitors are expected to be safer Nrf2 activators. We found a novel class of non-covalent Keap1-Nrf2 PPI inhibitor that has a benzo[g]indole skeleton and an indole-3-hydroxamic acid moiety and that exhibits significant PPI inhibitory activity. Additionally, the benzo[g]indole-3-carbohydrazide derivatives were newly prepared. The benzo[g]indole derivatives showed a stronger Keap1-Nrf2 PPI inhibitory activity than Cpd16, a previously reported non-covalent PPI inhibitor. Moreover, most of the PPI inhibitors showed a high metabolic stability in a human microsome system with a low cytotoxicity against HepG2 cell lines, which suggests that novel benzo[g]indole-type Keap1-Nrf2 PPI inhibitors are expected to be biological tools or lead compounds for Nrf2 activators.