Molecular mechanism underlying the TLR4 antagonistic and antiseptic activities of papiliocin, an insect innate immune response molecule.

SignificanceSimilar to mammalian TLR4/MD-2, the Toll9/MD-2-like protein complex in the silkworm, Bombyx mori, acts as an innate pattern-recognition receptor that recognizes lipopolysaccharide (LPS) and induces LPS-stimulated expression of antimicrobial peptides such as cecropins. Here, we report that papiliocin, a cecropin-like insect antimicrobial peptide from the swallowtail butterfly, competitively inhibits the LPS-TLR4/MD-2 interaction by directly binding to human TLR4/MD-2. Structural elements in papiliocin, which are important in inhibiting TLR4 signaling via direct binding, are highly conserved among insect cecropins, indicating that its TLR4-antagonistic activity may be related to insect Toll9-mediated immune response against microbial infection. This study highlights the potential of papiliocin as a potent TLR4 antagonist and safe peptide antibiotic for treating gram-negative sepsis.

Separation of native and C106-oxidized DJ-1 proteins by using column chromatography.

Mutations in PARK7, the gene encoding the DJ-1 protein, are associated with early onset of Parkinson's disease. The C106 residue of DJ-1 is highly susceptible to oxidation, and its oxidation status is essential for various in vivo neuroprotective roles. Since C106 is readily oxidized to sulfinic acid that is not reduced by dithiothreitol, no method to separate native DJ-1 protein from the oxidized one creates challenges in the in vitro study of the biological relevance of C106-oxidation state. Here, we report an efficient column chromatography method to purify native, C106-sulfinic, and mixed (combination of the priors) forms of DJ-1. This method will be useful for systematic in vitro studies of DJ-1 functions by providing specific native and C106-sulfinic DJ-1 proteins.

New Inhibitors of Laccase and Tyrosinase by Examination of Cross-Inhibition between Copper-Containing Enzymes.

Coppers play crucial roles in the maintenance homeostasis in living species. Approximately 20 enzyme families of eukaryotes and prokaryotes are known to utilize copper atoms for catalytic activities. However, small-molecule inhibitors directly targeting catalytic centers are rare, except for those that act against tyrosinase and dopamine-ß-hydroxylase (DBH). This study tested whether known tyrosinase inhibitors can inhibit the copper-containing enzymes, ceruloplasmin, DBH, and laccase. While most small molecules minimally reduced the activities of ceruloplasmin and DBH, aside from known inhibitors, 5 of 28 tested molecules significantly inhibited the function of laccase, with the Ki values in the range of 15 to 48 µM. Enzyme inhibitory kinetics classified the molecules as competitive inhibitors, whereas differential scanning fluorimetry and fluorescence quenching supported direct bindings. To the best of our knowledge, this is the first report on organic small-molecule inhibitors for laccase. Comparison of tyrosinase and DBH inhibitors using cheminformatics predicted that the presence of thione moiety would suffice to inhibit tyrosinase. Enzyme assays confirmed this prediction, leading to the discovery of two new dual tyrosinase and DBH inhibitors.

Antiseptic 9-Meric Peptide with Potency against Carbapenem-Resistant Acinetobacter baumannii Infection.

Carbapenem-resistant A. baumannii (CRAB) infection can cause acute host reactions that lead to high-fatality sepsis, making it important to develop new therapeutic options. Previously, we developed a short 9-meric peptide, Pro9-3D, with significant antibacterial and cytotoxic effects. In this study, we attempted to produce safer peptide antibiotics against CRAB by reversing the parent sequence to generate R-Pro9-3 and R-Pro9-3D. Among the tested peptides, R-Pro9-3D had the most rapid and effective antibacterial activity against Gram-negative bacteria, particularly clinical CRAB isolates. Analyses of antimicrobial mechanisms based on lipopolysaccharide (LPS)-neutralization, LPS binding, and membrane depolarization, as well as SEM ultrastructural investigations, revealed that R-Pro9-3D binds strongly to LPS and impairs the membrane integrity of CRAB by effectively permeabilizing its outer membrane. R-Pro9-3D was also less cytotoxic and had better proteolytic stability than Pro9-3D and killed biofilm forming CRAB. As an LPS-neutralizing peptide, R-Pro9-3D effectively reduced LPS-induced pro-inflammatory cytokine levels in RAW 264.7 cells. The antiseptic abilities of R-Pro9-3D were also investigated using a mouse model of CRAB-induced sepsis, which revealed that R-Pro9-3D reduced multiple organ damage and attenuated systemic infection by acting as an antibacterial and immunosuppressive agent. Thus, R-Pro9-3D displays potential as a novel antiseptic peptide for treating Gram-negative CRAB infections.

NMR mapping of the highly flexible regions of 13C/15N-labeled antibody TTAC-0001-Fab.

Monoclonal antibody (mAb) drugs are clinically important for the treatment of various diseases. TTAC-0001 is under development as a new anti-cancer antibody drug targeting VEGFR-2. As the less severe toxicity of TTAC-0001 compared to Bevacizumab, likely due to the decreased in vivo half-life, seems to be related to its structural flexibility, it is important to map the exact flexible regions. Although the 13C/15N-labeled protein is required for NMR analyses, it is difficult to obtain antibody fragments (Fab and scFv) containing disulfide bonds through general cytosolic expression in Escherichia coli (E. coli). Here, we notably increased the periplasmic expression of the 13C/15N-labeled TTAC-0001-Fab (13C/15N-TTAC-Fab) through simple isopropyl ß-D-1-thiogalactopyranoside (IPTG)-induction at an increased optical density (1.5 OD600nm). Through NMR triple resonance experiments, two loop insertions (LI-1 between the VH and CH1; LI-2 between the VL and CL) were confirmed to be highly flexible. The additional LIs could be another way to engineer the antibody by changing the pharmacokinetic properties.

A Novel Peptide Antibiotic, Pro10-1D, Designed from Insect Defensin Shows Antibacterial and Anti-Inflammatory Activities in Sepsis Models.

Owing to the challenges faced by conventional therapeutics, novel peptide antibiotics against multidrug-resistant (MDR) gram-negative bacteria need to be urgently developed. We had previously designed Pro9-3 and Pro9-3D from the defensin of beetle Protaetia brevitarsis; they showed high antimicrobial activity with cytotoxicity. Here, we aimed to develop peptide antibiotics with bacterial cell selectivity and potent antibacterial activity against gram-negative bacteria. We designed 10-meric peptides with increased cationicity by adding Arg to the N-terminus of Pro9-3 (Pro10-1) and its D-enantiomeric alteration (Pro10-1D). Among all tested peptides, the newly designed Pro10-1D showed the strongest antibacterial activity against Escherichia coli, Acinetobacter baumannii, and MDR strains with resistance against protease digestion. Pro10-1D can act as a novel potent peptide antibiotic owing to its outstanding inhibitory activities against bacterial film formation with high bacterial cell selectivity. Dye leakage and scanning electron microscopy revealed that Pro10-1D targets the bacterial membrane. Pro10-1D inhibited inflammation via Toll Like Receptor 4 (TLR4)/Nuclear factor-κB (NF-κB) signaling pathways in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Furthermore, Pro10-1D ameliorated multiple-organ damage and attenuated systemic infection-associated inflammation in an E. coli K1-induced sepsis mouse model. Overall, our results suggest that Pro10-1D can potentially serve as a novel peptide antibiotic for the treatment of gram-negative sepsis.

Thiopurine Drugs Repositioned as Tyrosinase Inhibitors.

Drug repositioning is the application of the existing drugs to new uses and has the potential to reduce the time and cost required for the typical drug discovery process. In this study, we repositioned thiopurine drugs used for the treatment of acute leukaemia as new tyrosinase inhibitors. Tyrosinase catalyses two successive oxidations in melanin biosynthesis: the conversions of tyrosine to dihydroxyphenylalanine (DOPA) and DOPA to dopaquinone. Continuous efforts are underway to discover small molecule inhibitors of tyrosinase for therapeutic and cosmetic purposes. Structure-based virtual screening predicted inhibitor candidates from the US Food and Drug Administration (FDA)-approved drugs. Enzyme assays confirmed the thiopurine leukaemia drug, thioguanine, as a tyrosinase inhibitor with the inhibitory constant of 52 µM. Two other thiopurine drugs, mercaptopurine and azathioprine, were also evaluated for their tyrosinase inhibition; mercaptopurine caused stronger inhibition than thioguanine did, whereas azathioprine was a poor inhibitor. The inhibitory constant of mercaptopurine (16 µM) was comparable to that of the well-known inhibitor kojic acid (13 µM). The cell-based assay using B16F10 melanoma cells confirmed that the compounds inhibit mammalian tyrosinase. Particularly, 50 µM thioguanine reduced the melanin content by 57%, without apparent cytotoxicity. Cheminformatics showed that the thiopurine drugs shared little chemical similarity with the known tyrosinase inhibitors.

Ensemble-Based Virtual Screening Led to the Discovery of New Classes of Potent Tyrosinase Inhibitors.

In this study, we report new classes of potent tyrosinase inhibitors identified by enhanced structure-based virtual screening prediction; the enzyme and melanin content assays were also confirmed. Tyrosinase, a type-3 copper protein, participates in two distinct reactions, hydroxylation of tyrosine to DOPA and conversion of DOPA to dopaquinone, in melanin biosynthesis. Although numerous inhibitors of this reaction have been reported, there is a lag in the discovery of the new functional moieties. In order to improve the performance of virtual screening, we first produced an ensemble of 10,000 structures using molecular dynamics simulation. Quantum mechanical calculation was used to determine the partial charges of catalytic copper ions based on the met and deoxy states. Second, we selected a structure showing an optimal receiver operating characteristic (ROC) curve with known direct binders and their physicochemically matched decoys. The structure revealed more than 10-fold higher enrichment at 1% of the ROC curve than those observed in X-ray structures. Third, high-throughput virtual screening with DOCK 3.6 was performed using a library consisting of approximately 400,000 small molecules derived from the ZINC database. Fourth, we obtained the top 60 molecules and tested their inhibition of mushroom tyrosinase. The extended assays included 21 analogs of the 21 initial hits to test their inhibition properties. Here, the moieties of tetrazole and triazole were identified as new binding cores interacting with the dicopper catalytic center. All 42 inhibitors showed inhibitory constant, Ki, values ranging from 11.1 nM and 33.4 µM, with a tetrazole compound exhibiting the strongest activity. Among the 42 molecules, five displayed more than 30% reduction in melanin production when treated in B16F10 melanoma cells; cell viability was >90% at 20 µM. Particularly, a thiosemicarbazone-containing compound reduced melanin content by 55%.

Repositioning of Thiourea-Containing Drugs as Tyrosinase Inhibitors.

Tyrosinase catalyzes two distinct sequential reactions in melanin biosynthesis: The hydroxylation of tyrosine to dihydroxyphenylalanine (DOPA) and the oxidation of DOPA to dopaquinone. Developing functional modulators of tyrosinase is important for therapeutic and cosmetic purposes. Given the abundance of thiourea moiety in known tyrosinase inhibitors, we studied other thiourea-containing drugs as potential tyrosinase inhibitors. The thiourea-containing drugs in clinical use were retrieved and tested for their ability to inhibit tyrosinase. We observed that methimazole, thiouracil, methylthiouracil, propylthiouracil, ambazone, and thioacetazone inhibited mushroom tyrosinase. Except for methimazole, there was limited information regarding the activity of other drugs against tyrosinase. Both thioacetazone and ambazone significantly inhibited tyrosinase, with IC50 of 14 and 15 µM, respectively. Ambazone decreased melanin content without causing cellular toxicity at 20 µM in B16F10 cells. The activity of ambazone was stronger than that of kojic acid both in enzyme and melanin content assays. Kinetics of enzyme inhibition assigned the thiourea-containg drugs as non-competitive inhibitors. The complex models by docking simulation suggested that the intermolecular hydrogen bond via the nitrogen of thiourea and the contacts via thione were equally important for interacting with tyrosinase. These data were consistent with the results of enzyme assays with the analogues of thiourea.

Degradation of Polo-like Kinase 1 by the Novel Poly-Arginine N-Degron Pathway PROTAC Regulates Tumor Growth in Nonsmall Cell Lung Cancer.

Polo-like kinase 1 (PLK1), which is crucial in cell cycle regulation, is considered a promising anticancer drug target. Herein, we present the N-degron pathway-based proteolysis targeting chimera (PROTAC) for PLK1 degradation, targeting the Polo-box domain (PBD). We identified DD-2 as the most potent PROTAC that selectively induces PLK1 degradation in cancer cells, including HeLa and nonsmall cell lung cancer (NSCLC), through the N-degron pathway. DD-2 exhibited significant in vitro anticancer effects, inducing G2/M arrest and apoptosis in HeLa and NSCLC cell lines. DD-2 showed significant tumor growth inhibition in a xenograft mouse model using HeLa and NSCLC cell lines, highlighting its potential in cancer treatment. Furthermore, the combination of DD-2 with tyrosine kinase inhibitor (TKI), osimertinib, effectively suppressed tumor growth in double-mutated H1975 cell lines, emphasizing DD-2's potential in combination cancer therapies. Collectively, this study demonstrates the potential of the N-degron pathway, especially using DD-2, for targeted cancer therapies.

Kinetic evidence in favor of glyoxalase III and against deglycase activity of DJ-1.

DJ-1, a protein encoded by PARK7 plays a protective role against neurodegeneration. Since its glyoxalase III activity catalyzing methylglyoxal (MG) to lactate was discovered, DJ-1 has been re-established as a deglycase decomposing the MG-intermediates with amino acids and nucleotides (hemithioacetal and hemiaminal) rather than MG itself, but it is still debatable. Here, we have clarified that human DJ-1 directly recognizes MG, and not MG-intermediates, by monitoring the detailed catalytic processes and enantiomeric lactate products. The hemithioacetal intermediate between C106 of 15 N-labeled DJ-1 (15N DJ-1) and MG was also monitored by NMR. TRIS molecule formed stable diastereotopic complexes with MG (Kd , 1.57 ± 0.27 mM) by utilizing its three OH groups, which likely disturbed the assay of deglycase activity. The low kcat of DJ-1 for MG and its MG-induced structural perturbation may suggest that DJ-1 has a regulatory function as an in vivo sensor of reactive carbonyl stress.

Hydroxamic Acid as a Potent Metal-Binding Group for Inhibiting Tyrosinase.

Tyrosinase, a metalloenzyme containing a dicopper cofactor, plays a central role in synthesizing melanin from tyrosine. Many studies have aimed to identify small-molecule inhibitors of tyrosinase for pharmaceutical, cosmetic, and agricultural purposes. In this study, we report that hydroxamic acid is a potent metal-binding group for interacting with dicopper atoms, thereby inhibiting tyrosinase. Hydroxamate-containing molecules, including anticancer drugs targeting histone deacetylase, vorinostat and panobinostat, significantly inhibited mushroom tyrosinase, with inhibitory constants in the submicromolar range. Of the tested molecules, benzohydroxamic acid was the most potent. Its inhibitory constant of 7 nM indicates that benzohydroxamic acid is one of the most potent tyrosinase inhibitors. Results from differential scanning fluorimetry revealed that direct binding mediates inhibition. The enzyme kinetics were studied to assess the inhibitory mechanism of the hydroxamate-containing molecules. Experiments with B16F10 cell lysates confirmed that the new inhibitors are inhibitory against mammalian tyrosinase. Docking simulation data revealed intermolecular contacts between hydroxamate-containing molecules and tyrosinase.

Repositioning Food and Drug Administration-Approved Drugs for Inhibiting Biliverdin IXß Reductase B as a Novel Thrombocytopenia Therapeutic Target.

Biliverdin IXß reductase B (BLVRB) has recently been proposed as a novel therapeutic target for thrombocytopenia through its reactive oxygen species (ROS)-associated mechanism. Thus, we aim at repurposing drugs as new inhibitors of BLVRB. Based on IC50 (<5 µM), we have identified 20 compounds out of 1496 compounds from the Food and Drug Administration (FDA)-approved library and have clearly mapped their binding sites to the active site. Furthermore, we show the detailed BLVRB-binding modes and thermodynamic properties (ΔH, ΔS, and KD) with nuclear magnetic resonance (NMR) and isothermal titration calorimetry together with complex structures of eight water-soluble compounds. We anticipate that the results will serve as a novel platform for further in-depth studies on BLVRB effects for related functions such as ROS accumulation and megakaryocyte differentiation, and ultimately treatments of platelet disorders.

Regioselectivity of an arachidonate 9S-lipoxygenase from Sphingopyxis macrogoltabida that biosynthesizes 9S,15S- and 11S,17S-dihydroxy fatty acids from C20 and C22 polyunsaturated fatty acids.

Lipoxygenases (LOXs) biosynthesize lipid mediators (LMs) as human signaling molecules. Among LMs, specialized pro-resolving mediators (SPMs) are involved in the resolution of inflammation and infection in humans. Here, the putative LOX from the bacterium Sphingopyxis macrogoltabida was identified as arachidonate 9S-LOX. The enzyme catalyzed oxygenation at the n-12 position of C20 and C22 polyunsaturated fatty acids (PUFAs) to form 9S- and 11S-hydroperoxy fatty acids, which were reduced to 9S- and 11S-hydroxy fatty acids (HFAs) by cysteine, respectively, and it catalyzed again oxygenation at the n-6 position of HFAs to form 9S,15S- and 11S,17S-DiHFAs, respectively. The regioselective residues of 9S-LOX were determined as lle395 and Val569 based on the amino acid alignment and homology models. The regioselectivity of the I395F variant was changed from the n-12 position on C20 PUFA to the n-6 position to form 15S-HFAs. This may be due to the reduction of the substrate-binding pocket by replacing the smaller Ile with a larger Phe. The V569W variant had a significantly lower second­oxygenating activity compared to wild-type 9S-LOX because the insertion of the hydroxyl group of the first­oxygenating products at the active site was seemed to be hindered by substituting a larger Trp for a smaller Val. The compounds, 11S-hydroxydocosapentaenoic acid, 9S,15S-dihydroxyeicosatetraenoic acid, 9S,15S-dihydroxyeicosapentaenoic acid, 11S,17S-hydroxydocosapentaenoic acid, and 11S,17S-dihydroxydocosahexaenoic acid, were newly identified by polarimeter, LC-MS/MS, and NMR. 11S,17S-DiHFAs as SPM isomers biosynthesized from C22 PUFAs showed anti-inflammatory activities in mouse and human cells. Our study contributes may stimulate physiological studies by providing new LMs.

MUL1-RING recruits the substrate, p53-TAD as a complex with UBE2D2-UB conjugate.

The RING domain of MUL1 (RINGMUL1 ) alone mediates ubiquitylation of the p53-transactivation domain (TADp53 ). To elucidate the mechanism underlying the simultaneous recruitment of UBE2D2 and the substrate TADp53 by RINGMUL1 , we determined the complex structure of RINGMUL1 :UBE2D2 and studied the interaction between RINGMUL1 and TADp53 in the presence of UBE2D2-UB thioester (UBE2D2~UB) mimetics. The RINGMUL1 -binding induced the closed conformation of UBE2D2S22R/C85S -UBK48R oxyester (UBE2D2RS -UBR OE ), and strongly accelerated its hydrolysis, which was suppressed by the additional N77A-mutation of UBE2D2. Interestingly, UBE2D2S22R/N77A/C85S -UBK48R oxyester (UBE2D2RAS -UBR OE ) already formed a closed conformation in the absence of RINGMUL1 . Although TADp53 exhibited weak binding for RINGMUL1 or UBE2D2 alone, its binding affinity was enhanced and even further for RINGMUL1 :UBE2D2 and RINGMUL1 :UBE2D2RAS -UBR OE , respectively. The recognition of TADp53 by RINGMUL1 as a complex with UBE2D2~UB is related to the multivalency of the binding events and underlies the ability of RINGMUL1 to ubiquitylate the intrinsically disordered protein, TADp53 .

Anti-Endotoxin 9-Meric Peptide with Therapeutic Potential for the Treatment of Endotoxemia.

Inflammatory reactions activated by lipopolysaccharide (LPS) of gram-negative bacteria can lead to severe septic shock. With the recent emergence of multidrug-resistant gram-negative bacteria and a lack of efficient ways to treat resulting infections, there is a need to develop novel anti-endotoxin agents. Antimicrobial peptides have been noticed as potential therapeutic molecules for bacterial infection and as candidates for new antibiotic drugs. We previously designed the 9-meric antimicrobial peptide Pro9-3 and it showed high antimicrobial activity against gram-negative bacteria. Here, to further examine its potency as an anti-endotoxin agent, we examined the antiendotoxin activities of Pro9-3 and elucidated its mechanism of action. We performed a dye-leakage experiment and BODIPY-TR cadaverine and limulus amebocyte lysate assays for Pro9-3 as well as its lysine-substituted analogue and their enantiomers. The results confirmed that Pro9-3 targets the bacterial membrane and the arginine residues play key roles in its antimicrobial activity. Pro9-3 showed excellent LPS-neutralizing activity and LPS-binding properties, which were superior to those of other peptides. Saturation transfer difference-nuclear magnetic resonance experiments to explore the interaction between LPS and Pro9-3 revealed that Trp3 and Tlr7 in Pro9-3 are critical for attracting Pro9-3 to the LPS in the gram-negative bacterial membrane. Moreover, the anti-septic effect of Pro9-3 in vivo was investigated using an LPS-induced endotoxemia mouse model, demonstrating its dual activities: antibacterial activity against gram-negative bacteria and immunosuppressive effect preventing LPS-induced endotoxemia. Collectively, these results confirmed the therapeutic potential of Pro9-3 against infection of gram-negative bacteria.

Development of Novel Peptides for the Antimicrobial Combination Therapy against Carbapenem-Resistant Acinetobacter baumannii Infection.

Carbapenem-resistant Acinetobacter baumannii (CRAB) infection has a high mortality rate, making the development of novel effective antibiotic therapeutic strategies highly critical. Antimicrobial peptides can outperform conventional antibiotics regarding drug resistance and broad-spectrum activity. PapMA, an 18-residue hybrid peptide, containing N-terminal residues of papiliocin and magainin 2, has previously demonstrated potent antibacterial activity. In this study, PapMA analogs were designed by substituting Ala15 or Phe18 with Ala, Phe, and Trp. PapMA-3 with Trp18 showed the highest bacterial selectivity against CRAB, alongside low cytotoxicity. Biophysical studies revealed that PapMA-3 permeabilizes CRAB membrane via strong binding to LPS. To reduce toxicity via reduced antibiotic doses, while preventing the emergence of multi-drug resistant bacteria, the efficacy of PapMA-3 in combination with six selected antibiotics was evaluated against clinical CRAB isolates (C1-C5). At 25% of the minimum inhibition concentration, PapMA-3 partially depolarized the CRAB membrane and caused sufficient morphological changes, facilitating the entry of antibiotics into the bacterial cell. Combining PapMA-3 with rifampin significantly and synergistically inhibited CRAB C4 (FICI = 0.13). Meanwhile, combining PapMA-3 with vancomycin or erythromycin, both potent against Gram-positive bacteria, demonstrated remarkable synergistic antibiofilm activity against Gram-negative CRAB. This study could aid in the development of combination therapeutic approaches against CRAB.

Development of a Polo-like Kinase-1 Polo-Box Domain Inhibitor as a Tumor Growth Suppressor in Mice Models.

Polo-like kinase-1 (Plk1) plays a key role in mitosis and has been identified as an attractive anticancer drug target. Plk1 consists of two drug-targeting sites, namely, N-terminal kinase domain (KD) and C-terminal polo-box domain (PBD). As KD-targeting inhibitors are associated with severe side effects, here we report on the pyrazole-based Plk1 PBD inhibitor, KBJK557, which showed a remarkable in vitro anticancer effect by inducing Plk1 delocalization, mitotic arrest, and apoptosis in HeLa cells. Further, in vivo optical imaging analysis and antitumorigenic activities in mouse xenograft models demonstrate that KBJK557 preferentially accumulates in cancer cells and selectively inhibits cancer cell proliferation. Pharmacokinetic profiles and partition coefficients suggest that KBJK557 was exposed in the blood and circulated through the organs with an intermediate level of clearance (t1/2, 7.73 h). The present investigation offers a strategy for specifically targeting cancer using a newly identified small-molecule inhibitor that targets the Plk1 PBD.

Structural ensemble-based docking simulation and biophysical studies discovered new inhibitors of Hsp90 N-terminal domain.

Heat shock protein 90 (Hsp90) is one of the most abundant cellular proteins and plays a substantial role in the folding of client proteins. The inhibition of Hsp90 has been regarded as an attractive therapeutic strategy for treating cancer because many oncogenic kinases are Hsp90 client proteins. In this study, we report new inhibitors that directly bind to N-terminal ATP-binding pocket of Hsp90. Optimized structure-based virtual screening predicted candidate molecules, which was followed by confirmation using biophysical and cell-based assays. Among the reported crystal structures, we chose the two structures that show the most favourable early enrichments of true-positives in the receiver operating characteristic curve. Four molecules showed significant changes in the signals of 2D [1H, 15N] correlation NMR spectroscopy. Differential scanning calorimetry analysis supported the results indicating direct binding. Quantified dissociation constant values of the molecules, determined by a series of 2D NMR experiments, lie in the range of 0.1-33 µM. Growth inhibition assay with breast and lung cancer cells confirmed the cellular activities of the molecules. Cheminformatics revealed that the molecules share limited chemical similarities with known inhibitors. Molecular dynamics simulations detailed the putative binding modes of the inhibitors.

Antithrombotic properties of JJ1, a potent and novel thrombin inhibitor.

The development of new anticoagulants is an important goal for the improvement of thrombosis treatment. Recent studies have suggested the importance of thrombin inhibitors in the modulation of thromboembolic disorders. The aim of this study was to discover a new small-molecule thrombin inhibitor. In this study, the compound JJ1, which has a novel scaffold, was selected by structure-based docking simulation to determine its potential inhibitory activity against thrombin. JJ1 was shown to inhibit the catalytic activity of human α-thrombin with a K i of 0.019 µM by direct binding to the active site and with at least 10,000-fold selectivity relative to that reported for the inhibition of other biologically important serine proteases. JJ1 prolonged clotting times (activated partial thromboplastin time and prothrombin time) and inhibited the activity and production of thrombin. Furthermore, it inhibited thrombin-catalyzed fibrin polymerization and platelet aggregation. Similar to its in vitro antithrombotic activities, JJ1 showed enhanced antithrombotic effects in an in vivo pulmonary embolism and arterial thrombosis model. It also exhibited anticoagulant effects in mice. Collectively, these results demonstrated that JJ1 was a potent, direct, and selective thrombin inhibitor that may be useful in the management of various thrombotic disorders.