Design and Synthesis of DDR1 Inhibitors with a Desired Pharmacophore Using Deep Generative Models.

Discoidin domain receptor 1 (DDR1) inhibitors with a desired pharmacophore were designed using deep generative models (DGMs). DDR1 is a receptor tyrosine kinase activated by matrix collagens and implicated in diseases such as cancer, fibrosis and hypoxia. Herein we describe the synthesis and inhibitory activity of compounds generated from DGMs. Three compounds were found to have sub-micromolar inhibitory activity. The most potent of which, compound 3 (N-(4-chloro-3-((pyridin-3-yloxy)methyl)phenyl)-3-(trifluoromethyl)benzamide), had an IC50 value of 92.5 nM. Furthermore, these compounds were predicted to interact with DDR1, which have a desired pharmacophore derived from a known DDR1 inhibitor. The results of synthesis and experiments indicated that our de novo design strategy is practical for hit identification and scaffold hopping.

Elucidation of the Active Conformation of Antiproliferative Sulfonamides, 5N-Arylsulfonyl-1,5-benzodiazepin-2-ones.

The 5N-arylsulfonyl-1,5-benzodiazepin-2-ones with antiproliferative activity were prepared and successfully separated into the (a1R,a2R)- and (a1S,a2S)-atropisomers with extraordinary stability (ΔG⧧ = ∼130 kJ/mol) by freezing the conformation around the sp2-sp2 axis in an Ar-N(SO2) moiety with a C6-methyl group. Also, by introducing a C3-methyl group (central chirality) into the 1,5-benzodiazepine nucleus, the stereochemistry at the axis was biased to take solely one diastereomer with a relative stereochemistry of (a1R*,a2R*,3R*). The (a1S) stereochemistry was crucial for exerting the antiproliferative activity.

In vitro studies on nobiletin isolated from citrus plants and the bioactive metabolites, inhibitory action against gelatinase enzymatic activity and the molecular mechanisms in human retinal Müller cell line.

Diabetic retinopathy (DR) is the most common cause of vision loss in patients with diabetes mellitus. Despite the presence of effective therapy, DR is still a significant health burden. A recent research suggests that matrix metalloproteinases (MMPs) could be promising targets, which exert multiple actions on early- and late-stage pathogenesis of DR. Among the MMP family, gelatinases (MMP-2 and MMP-9) act as potent proinflammatory, proangiogenic, and pro-apoptotic factors. Therefore, the pharmacological inhibitory effect of gelatinases on retinal MMP-producing cells may be useful in the treatment or prevention of DR. Nobiletin isolated from citrus plants is a multi-functional polymethoxylated flavone, which exerts biological effects including inhibitory action against MMP activity in several cancer cells. In the present study, we demonstrated that nobiletin isolated from citrus plants attenuated MMP-9 enzymatic activity through the suppression of transcription for MMP-9 gene expression and augmentation of TIMP-1 production in retinal Müller cells. Nobiletin regulated MMP-9 gene expression and TIMP-1 by inhibiting the PI3K/Akt signaling pathway. In addition, we observed the augmentation of inhibitory action against MMP-9 enzymatic activity by 4'-demethylated nobiletin, which is a major metabolite of nobiletin. We believe that the enhancement of inhibitory action against MMP-9 enzymatic activity by 4'-demethylated nobiletin is through the dual inhibition on Erk1/2 and Akt phosphorylation. The structure-activity relationship analysis revealed that, for the enhancement of inhibitory action against MMP-9 enzymatic activity, demethylation at position 4' in B-ring was a key structural modification in Müller cells, which are an important source of MMPs found in vitreous fluid and retinal tissues in retinal proliferative diseases. These results suggested that nobiletin, derived from a natural source, may serve as a novel MMP inhibitor with minimal side effects, and lead compound for the design of more efficacious drugs.

Development of fluorous lipid-based nanobubbles for efficiently containing perfluoropropane.

Nano-/microbubbles are expected not only to function as ultrasound contrast agents but also as ultrasound-triggered enhancers in gene and drug delivery. Notably, nanobubbles have the ability to pass through tumor vasculature and achieve passive tumor targeting. Thus, nanobubbles would be an attractive tool for use as ultrasound-mediated cancer theranostics. However, the amounts of gas carried by nanobubbles are generally lower than those carried by microbubbles because nanobubbles have inherently smaller volumes. In order to reduce the injection volume and to increase echogenicity, it is important to develop nanobubbles with higher gas content. In this study, we prepared 5 kinds of fluoro-lipids and used these reagents as surfactants to generate "Bubble liposomes", that is, liposomes that encapsulate nanobubbles such that the lipids serve as stabilizers between the fluorous gas and water phases. Bubble liposome containing 1-stearoyl-2-(18,18-difluoro)stearoyl-sn-glycero-3-phosphocholine carried 2-fold higher amounts of C3F8 compared to unmodified Bubble liposome. The modified Bubble liposome also exhibited increased echogenicity by ultrasonography. These results demonstrated that the inclusion of fluoro-lipid is a promising tool for generating nanobubbles with increased efficiency of fluorous gas carrier.

Polymethoxyflavones as agents that prevent formation of cataract: nobiletin congeners show potent growth inhibitory effects in human lens epithelial cells.

Posterior capsular opacification (PCO) is the most frequent complication and the primary reason for visual decrease after extracapsular cataract surgery. The proliferation and migration of leftover lens epithelial cells (LECs) after surgery may contribute to the development of PCO. To prevent PCO, a rational approach would be to inhibit both the proliferation and the migration of LECs using nontoxic xenobiotics. Nobiletin, one of the most abundant polymethoxyflavones (PMFs) in citrus peel, and its synthetic congeners displayed a potent inhibition of LEC proliferation. Structural features which enhance anti-proliferative activity have also been discussed.

B-Ring-modified and/or 5-demethylated nobiletin congeners: inhibitory activity against pro-MMP-9 production.

Three metabolites and 12 analogues of nobiletin (1) were synthesized. Whereas nobiletin derivatives 2-4 inhibited pro-MMP-9 production similarly in both PMA- and TNF-α-stimulated human lens epithelial cells, the 2'-hydroxylated analogue 5a exerted marked inhibitory effects (IC(50): 0.4 µM) on PMA-treated cells, which were 170-fold more potent than those on TNF-α-treated cells. This activity may be closely related to PKC-mediated transcriptional regulation of pro-MMP-9.

Nobiletin metabolites: synthesis and inhibitory activity against matrix metalloproteinase-9 production.

A divergent synthesis of nobiletin metabolites was developed through highly oxygenated acetophenone derivative. We used commercially available methyl 3,4,5-trimethoxybenzoate as a starting material for concise preparation of the key intermediate, 2'-hydroxy-3',4',5',6'-tetramethoxyacetophenone (I). These metabolites showed strong inhibitory activity against matrix metalloproteinase-9 production in human lens epithelial cells.

Dual antitumor mechanisms of Notch signaling inhibitor in a T-cell acute lymphoblastic leukemia xenograft model.

Constitutive activation of Notch signaling is required for the proliferation of a subgroup of human T-cell acute lymphoblastic leukemias (T-ALL). Previous in vitro studies have demonstrated the therapeutic potential of Notch signaling inhibitors for treating T-ALL. To further examine this possibility, we applied a gamma-secretase inhibitor (GSI) to T-ALL xenograft models. Treatment of established subcutaneous tumors with GSI resulted in partial or complete regression of tumors arising from four T-ALL cell lines that were also sensitive to GSI in vitro. To elucidate the mechanism of action, we transduced DND-41 cells with the active form of Notch1 (aN1), which conferred resistance to in vitro GSI treatment. Nevertheless, in vivo treatment with GSI induced a partial but significant regression of subcutaneous tumors that developed from aN1-transduced DND-41 cells, whereas it induced complete regression of tumors that developed from mock-transduced DND-41 cells. These findings indicate that the remarkable efficacy of GSI might be attributable to dual mechanisms, directly via apoptosis of DND-41 cells through the inhibition of cell-autonomous Notch signaling, and indirectly via disturbance of tumor angiogenesis through the inhibition of non-cell-autonomous Notch signaling.

Identification of trimannoside-recognizing peptide sequences from a T7 phage display screen using a QCM device.

Here, we report on the identification of trimannoside-recognizing peptide sequences from a T7 phage display screen using a quartz-crystal microbalance (QCM) device. A trimannoside derivative that can form a self-assembled monolayer (SAM) was synthesized and used for immobilization on the gold electrode surface of a QCM sensor chip. After six sets of one-cycle affinity selection, T7 phage particles displaying PSVGLFTH (8-mer) and SVGLGLGFSTVNCF (14-mer) were found to be enriched at a rate of 17/44, 9/44, respectively, suggesting that these peptides specifically recognize trimannoside. Binding checks using the respective single T7 phage and synthetic peptide also confirmed the specific binding of these sequences to the trimannoside-SAM. Subsequent analysis revealed that these sequences correspond to part of the primary amino acid sequence found in many mannose- or hexose-related proteins. Taken together, these results demonstrate the effectiveness of our T7 phage display environment for affinity selection of binding peptides. We anticipate this screening result will also be extremely useful in the development of inhibitors or drug delivery systems targeting polysaccharides as well as further investigations into the function of carbohydrates in vivo.

Both Notch1 and Notch2 contribute to the regulation of melanocyte homeostasis.

Notch signaling affects a variety of mammalian stem cells, but there has been limited evidence that a specific Notch molecule regulates adult stem cells. Recently, it was reported that the reduced Notch signaling initiated at the embryonic stage results in a gradual hair graying phenotype after birth. Here we demonstrate that the oral administration of a gamma-secretase inhibitor (GSI) to wild-type adult C57/Bl6 mice led to a gradual increase in gray spots, which remained unchanged for at least 20 weeks after discontinuing the GSI. In GSI-treated mice, there was a severe decrease in unpigmented melanocytes in the bulge/subbulge region where melanocyte stem cells are located. While we confirmed that Notch1+/-Notch2+/- double heterozygous mice with a C57/Bl6 background were born with a normal hair color phenotype and gradually turned gray after the second hair cycle, in the c-kit mutant Wv background, Notch1+/- and Notch2+/- mice had larger white spots on the first appearance of hair than did the Wv/+ mice, which did not change throughout life. Notch1+/-Notch2+/-Wv/+ mice had white hair virtually all over the body at the first appearance of hair and the depigmentation continued to progress thereafter. Using a neural crest organ culture system, GSI blocked the generation of pigmented melanocytes when added to the culture during the period of melanoblast proliferation, but not during the period of differentiation. These observations imply roles of Notch signaling in both development of melanocyte during embryogenesis and maintenance of melanocyte stem cells in adulthood, while the degree of requirement is distinct in these settings: the latter is more sensitive than the former to the reduced Notch signaling. Furthermore, Notch1 and Notch2 cooperates with c-kit signaling during embryogenesis, and they cooperate with each other to regulate melanocyte homeostasis after birth.

C-terminal fragment of presenilin is the molecular target of a dipeptidic gamma-secretase-specific inhibitor DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester).

Gamma-secretase is a multimeric membrane protein complex composed of presenilin (PS), nicastrin, Aph-1 and, Pen-2 that is responsible for the intramembrane proteolysis of various type I transmembrane proteins, including amyloid beta-precursor protein and Notch. The direct labeling of PS polypeptides by transition-state analogue gamma-secretase inhibitors suggested that PS represents the catalytic center of gamma-secretase. Here we show that one of the major gamma-secretase inhibitors of dipeptidic type, N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT), targets the C-terminal fragment of PS, especially the transmembrane domain 7 or more C-terminal region, by designing and synthesizing DAP-BpB (N-[N-(3,5-difluorophenacetyl)-l-alanyl]-(S)-phenylglycine-4-(4-(8-biotinamido)octylamino)benzoyl)benzyl)methylamide), a photoactivable DAPT derivative. We also found that DAP-BpB selectively binds to the high molecular weight gamma-secretase complex in an activity-dependent manner. Photolabeling of PS by DAP-BpB is completely blocked by DAPT or its structural relatives (e.g. Compound E) as well as by arylsulfonamides. In contrast, transition-state analogue inhibitor L-685,458 or alpha-helical peptidic inhibitor attenuated the photolabeling of PS1 only at higher concentrations. These data illustrate the DAPT binding site as a novel functional domain within the PS C-terminal fragment that is distinct from the catalytic site or the substrate binding site.

Sulindac sulfide is a noncompetitive gamma-secretase inhibitor that preferentially reduces Abeta 42 generation.

Nonsteroidal anti-inflammatory drugs (NSAIDs) have been known to reduce risk for Alzheimer's disease. In addition to the anti-inflammatory effects of NSAIDs to block cylooxygenase, it has been shown recently that a subset of NSAIDs selectively inhibits the secretion of highly amyloidogenic Abeta42 from cultured cells, although the molecular target(s) of NSAIDs in reducing the activity of gamma-secretase for Abeta42 generation (gamma(42)-secretase) still remain unknown. Here we show that sulindac sulfide (SSide) directly acts on gamma-secretase and preferentially inhibits the gamma(42)-secretase activity derived from the 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate-solubilized membrane fractions of HeLa cells, in an in vitro gamma-secretase assay using recombinant amyloid beta precursor protein C100 as a substrate. SSide also inhibits activities for the generation of Abeta40 as well as for Notch intracellular domain at higher concentrations. Notably, SSide displayed linear noncompetitive inhibition profiles for gamma(42)-secretase in vitro. Our data suggest that SSide is a direct inhibitor of gamma-secretase that preferentially affects the gamma(42)-secretase activity.