Effect of Two-Photon Excitation to 8-Azacoumarin Derivatives as Photolabile Protecting Groups.

An improvement of the two-photon excitation was achieved using 8-azacoumarin-type caged compounds, which showed large values of the two-photon uncaging action cross-section (δu >0.1 Goeppert-Mayer (GM)). In particular, the 7-hydroxy-6-iodo-8-azacoumarin (8-aza-Ihc)-caged compound showed an excellent uncaging action cross-section value (δu = 1.28 GM). Therefore, 8-azacoumarin-type photolabile protecting groups (PPGs) can be used as two-photon excitation sources.

Exploratory studies on soluble small molecule CD4 mimics as HIV entry inhibitors.

Several small molecule CD4 mimics, which inhibit the interaction of gp120 with CD4, have been developed. Original CD4 mimics such as NBD-556, which has an aromatic ring, an oxalamide linker and a piperidine moiety, possess significant anti-HIV activity but with their hydrophobic aromatic ring-containing structures are poorly soluble in water. We have developed derivatives with a halopyridinyl group in place of the phenyl group, such as KKN-134, and found them to have excellent aqueous solubility. Other leads that were examined are YIR-821, a compound with a cyclohexane group in a spiro attachment to a piperidine ring and a guanidino group on the piperidine nitrogen atom, and its PEGylated derivative, TKB-002. YIR-821 and TKB-002 retain potent anti-HIV activity. Here, new CD4 mimics, in which the phenyl group was replaced by a halopyridinyl group with the halogen atoms in different positions, their derivatives without a cyclohexane group on the piperidine ring and their hybrid molecules with PEG units were designed and synthesized. Some of these compounds show significantly higher aqueous solubility with maintenance of certain levels of anti-HIV activity. The present data should be useful in the future design of CD4 mimic molecules.

Endocannabinoid hydrolysis inhibition unmasks that unsaturated fatty acids induce a robust biosynthesis of 2-arachidonoyl-glycerol and its congeners in human myeloid leukocytes.

The endocannabinoid (eCB) 2-arachidonoyl-gycerol (2-AG) modulates immune responses by activating cannabinoid receptors or through its multiple metabolites, notably eicosanoids. Thus, 2-AG hydrolysis inhibition might represent an interesting anti-inflammatory strategy that would simultaneously increase the levels of 2-AG and decrease those of eicosanoids. Accordingly, 2-AG hydrolysis inhibition increased 2-AG half-life in neutrophils. Under such setting, neutrophils, eosinophils, and monocytes synthesized large amounts of 2-AG and other monoacylglycerols (MAGs) in response to arachidonic acid (AA) and other unsaturated fatty acids (UFAs). Arachidonic acid and UFAs were ~1000-fold more potent than G protein-coupled receptor (GPCR) agonists. Triascin C and thimerosal, which, respectively, inhibit fatty acyl-CoA synthases and acyl-CoA transferases, prevented the UFA-induced MAG biosynthesis, implying glycerolipid remodeling. 2-AG and other MAG biosynthesis was preceded by that of the corresponding lysophosphatidic acid (LPA). However, we could not directly implicate LPA dephosphorylation in MAG biosynthesis. While GPCR agonists poorly induced 2-AG biosynthesis, they inhibited that induced by AA by 25%-50%, suggesting that 2-AG biosynthesis is decreased when leukocytes are surrounded by a pro-inflammatory entourage. Our data strongly indicate that human leukocytes use AA and UFAs to biosynthesize biologically significant concentrations of 2-AG and other MAGs and that hijacking the immune system with 2-AG hydrolysis inhibitors might diminish inflammation in humans.

Fluorescence resonance energy transfer-based screening for protein kinase C ligands using 6-methoxynaphthalene-labeled 1,2-diacylglycerol-lactones.

Protein kinase C (PKC) is associated with a central cellular signal transduction pathway and disorders such as cancer and Alzheimer-type dementia and is therefore a target for the treatment of these diseases. The development of simple methods suitable for high-throughput screening to find potent PKC ligands is desirable. We have developed an assay based on fluorescence-quenching screening with a solvatochromic fluorophore attached to a competitive probe and its alternative method based on Förster/fluorescence resonance energy transfer (FRET) phenomena. Here, an improved FRET-based PKC binding assay using a diacylglycerol (DAG) lactone labeled with a donor fluorescent dye, 6-methoxynaphthalene (6MN), was developed. The 6MN-labeled DAG-lactone has a higher binding affinity for the PKCδ C1b domain and the fluorescent PKCδ C1b domain labeled by fluorescein as an acceptor fluorescent dye (Fl-δC1b) than the diethylaminocoumarin (DEAC)-labeled DAG-lactone. The combination of the 6MN-labeled DAG-lactone and Fl-δC1b showed a change in fluorescence response larger than that of the DEAC-labeled DAG-lactone and Fl-δC1b. The IC50 values of known PKC ligands calculated by the present FRET-based method using 6MN-labeled DAG-lactone agree well with the Ki values obtained by the conventional radioisotope-based assays. Some false positive compounds, identified by the previous solvatochromic fluorophore-based method, were found to be negative by this method. The present FRET-based PKC binding assay is more sensitive and could be more useful.

Construction of 7-Diethylaminocoumarins Promoted by an Electron-Withdrawing Group.

The coumarin skeleton has been a focus of attention for many years, and its fluorescence properties vary depending on the substituents. Fluorescent coumarin derivatives are useful tools for many strategies have been developed for their synthesis. Although 7-diethylaminocoumarin has excellent fluorescence properties, it is unstable. We have developed a facile strategy for the synthesis of 7-diethylaminocoumarin derivatives by increasing the electrophilicity of the ynone moiety to promote nucleophilic addition reactions and cyclization. The reaction tolerates a variety of substitutions at the 4-position.

Synthesis of a Coumarin-Based PPARγ Fluorescence Probe for Competitive Binding Assay.

Peroxisome proliferator-activated receptor γ (PPARγ) is a molecular target of metabolic syndrome and inflammatory disease. PPARγ is an important nuclear receptor and numerous PPARγ ligands were developed to date; thus, efficient assay methods are important. Here, we investigated the incorporation of 7-diethylamino coumarin into the PPARγ agonist rosiglitazone and used the compound in a binding assay for PPARγ. PPARγ-ligand-incorporated 7-methoxycoumarin, 1, showed weak fluorescence intensity in a previous report. We synthesized PPARγ-ligand-incorporating coumarin, 2, in this report, and it enhanced the fluorescence intensity. The PPARγ ligand 2 maintained the rosiglitazone activity. The obtained partial agonist 6 appeared to act through a novel mechanism. The fluorescence intensity of 2 and 6 increased by binding to the ligand binding domain (LBD) of PPARγ and the affinity of reported PPARγ ligands were evaluated using the probe.

Synthesis of hydrophilic caged DAG-lactones for chemical biology applications.

The 6-bromo-7-hydroxy-coumarin-4-ylmethyl (Bhc) group has been used widely in cage chemistry because of its high molar absorptivity and photolytic efficiency. One of the drawbacks of coumarins however is their low aqueous solubility. Aqueous solubility is important in the behavior of caged compounds because hydrophobic caged compounds might be aggregated in physiological conditions and consequently the photocleavage would be impaired. The 8-azacoumarin-4-ylmethyl derivatives with bromine (8-aza-Bhc) or iodine (8-aza-Ihc), which were previously developed in this laboratory, have aqueous solubilities that are higher than those of related coumarins. Here, to improve the hydrophilicity and management of caged diacylglycerol lactones (DAG-lactones), 8-aza-Bhc and 8-aza-Ihc were introduced into the DAG-lactone structure. The synthesized caged compounds showed high hydrophilicity compared with the parent Bhc-caged DAG-lactone, and the 8-aza-Ihc-caged DAG-lactone (2) showed excellent photolytic efficiency, which allows rapid release of the DAG-lactone (1) by brief photoirradiation. The 8-aza-7-hydroxy-6-iodo-coumarin-4-ylmethyl group might be useful for caging of bioactive compounds, especially hydrophobic compounds.

Bivalent HIV-1 fusion inhibitors based on peptidomimetics.

Membrane fusion is a valid target for inhibition of HIV-1 replication. A 34-mer fragment peptide (C34), which is contained in the HIV-1 envelope protein gp41, has significant anti-HIV activity. Previously, a dimeric derivative of C34 linked by a disulfide bridge at its C-terminus was found to have more potent anti-HIV activity than the C34 peptide monomer. To date, several peptidomimetic small inhibitors have been reported, but most have lower potency than peptide derivatives related to C34. In the present study we applied this dimerization concept to these peptidomimetic small inhibitors and designed several bivalent peptidomimetic HIV-1 fusion inhibitors. The importance of the length of linkers crosslinking two peptidomimetic compounds was demonstrated and several potent bivalent inhibitors containing tethered peptidomimetics were produced.

Soluble-type small-molecule CD4 mimics as HIV entry inhibitors.

Several small molecule CD4 mimics have been reported previously as HIV-1 entry inhibitors, which block the interaction between the Phe43 cavity of HIV-1 gp120 and the host CD4. Known CD4 mimics such as NBD-556 possess significant anti-HIV activity but are less soluble in water, perhaps due to their hydrophobic aromatic ring-containing structures. Compounds with a pyridinyl group in place of the phenyl group in these molecules have been designed and synthesized in an attempt to increase the hydrophilicity. Some of these new CD4 mimics, containing a tetramethylpiperidine ring show significantly higher water solubility than NBD-556 and have high anti-HIV activity and synergistic anti-HIV activity with a neutralizing antibody. The CD4 mimic that has a cyclohexylpiperidine ring and a 6-fluoropyridin-3-yl ring has high anti-HIV activity and no significant cytotoxicity. The present results will be useful in the future design and development of novel soluble-type molecule CD4 mimics.

Flexibility of small molecular CD4 mimics as HIV entry inhibitors.

CD4 mimics such as YIR-821 and its derivatives are small molecules which inhibit the interaction between the Phe43 cavity of HIV-1 gp120 with host CD4, an interaction that is involved in the entry of HIV to cells. Known CD4 mimics generally possess three structural features, an aromatic ring, an oxalamide linker and a piperidine moiety. We have shown previously that introduction of a cyclohexyl group and a guanidine group into the piperidine moiety and a fluorine atom at the meta-position of the aromatic ring leads to a significant increase in the anti-HIV activity. In the current study, the effects of conformational flexibility were investigated by introduction of an indole-type group in the junction between the oxalamide linker and the aromatic moiety or by replacement of the oxalamide linker with a glycine linker. This led to the development of compounds with high anti-HIV activity, showing the importance of the junction region for the expression of high anti-HIV activity. The present data are expected to be useful in the future design of novel CD4 mimic molecules.

Synthesis and Evaluation of Dimeric Derivatives of Diacylglycerol-Lactones as Protein Kinase C Ligands.

Protein kinase C (PKC) mediates a central cellular signal transduction pathway involved in disorders such as cancer and Alzheimer's disease. PKC is regulated by binding of the second messenger sn-1,2-diacylglycerol (DAG) to its tandem C1 domains, designated C1a and C1b, leading both to PKC activation and to its translocation to the plasma membrane and to internal organelles. Depending on the isoform, there may be differences in the ligand selectivity of the C1a and C1b domains, and there is different spacing between the C1 domains of the conventional and novel PKCs. Bivalent ligands have the potential to exploit these differences between isoforms, yielding isoform selectivity. In the present study, we describe the synthesis of a series of dimeric derivatives of conformationally constrained diacylglycerol (DAG) analogs (DAG-lactones). We characterize the derivatives in vitro for their binding affinities, both to a single C1 domain (the C1b domain of PKCδ) as well as to the conventional PKCα isoform and the novel PKCδ isoform, and we measure their abilities to cause translocation of PKCδ and PKCε in intact cells. The dimeric compound with the 10-carbon linker was modestly more effective for the isolated PKCδ C1b domain than was the monomeric compound. For the intact PKCα and PKCδ, the shortest DAG-lactone dimer had similar affinity to the monomer and affinity decreased progressively up to the 16-carbon linker. The dimeric derivatives did not cause the Golgi accumulation of PKCδ. The present results provide important insights into the development of new chemical tools for biological studies on PKC.

Development of anti-HIV peptides based on a viral capsid protein.

Peptide inhibitors with cell permeability targeting an HIV-1 capsid (CA) protein might make therapeutic by regulating HIV-1 replication. Overlapping fragment peptide libraries covering the whole sequence of an HIV-1 CA protein have been synthesized with the addition of an octa-arginyl moiety to increase their cell permeability. Amongst these peptides, several compounds which inhibit the HIV-1 replication cycle have been found. Conjugation of cell-penetrating functions such as an octa-arginyl group to individual peptides in combination with the addition of chloroquine in cell-based anti-HIV assays was previously proven to be a useful assay method with which to search for active peptides. Anti-HIV assays have been performed in the presence or absence of chloroquine and found that most of compounds have higher anti-HIV activity in the presence, rather than in the absence of chloroquine. Some potent seeds as anti-HIV agents might naturally lie hidden in CA proteins, and could become useful leads to HIV inhibitors.

A minimally cytotoxic CD4 mimic as an HIV entry inhibitor.

Several CD4 mimics have been reported as HIV-1 entry inhibitors which can block the interaction between the viral envelope glycoprotein gp120 and the cell surface protein CD4. We previously found a lead compound 2 (YYA-021) with high anti-HIV activity and low cytotoxicity. Pharmacokinetic analysis however showed compound 2 to have wide tissue distribution and relatively high distribution volumes in rats and rhesus macaques. In the present study we searched for more hydrophilic CD4 mimics with a view to reducing tissue distribution. A new compound (5) with a 1,3-benzodioxolyl moiety was found to have relatively high anti-HIV activity and no significant cytotoxicity. Compound 5 is more hydrophilic than compound 2 and the pharmacokinetics of the intravenous administration of compound 5 in a rhesus macaque showed that compound 5 has lower tissue distribution than compound 2, suggesting that compound 5 possesses a better profile.

An in-cell fluorogenic Tag-probe system for protein dynamics imaging enabled by cell-penetrating peptides.

Fluorogenic probes are useful as molecular tools in chemical biology because they can overcome noise associated with background emission. Previously, using a leucine zipper assembly, we developed a fluorogenically active ZIP tag-probe pair. A probe peptide was designed as an α-helical peptide containing 4-nitrobenzo-2-oxa-1,3-diazole, a solvatochromic fluorescent dye. Tag peptides were designed as antiparallel 2 α-helical peptides, and the tag and probe together form the 3 α-helical bundle structure of the leucine zipper. The use of the system was limited to membrane proteins or targets on the cellular surface because the probe peptide was not compatible with cell penetration. In this study, a challenge for the fluorescent imaging of proteins inside the cells was conducted by development of the ZIP tag-probe system as the second generation. To enable the cell penetration of the probe peptide, the addition of a cell penetrating peptide sequence was tested and a probe peptide with a C-terminal octa-arginine was shown to have high affinity for the tag peptide. In addition to attachment of a CPP structure, pretreatment of cells by 1-pyrenebutyrate enhanced distribution of the probe peptide into the cytosol. Observed colocalization of fluorescence of monomer Kusabira Orange and 4-nitrobenzo-2-oxa-1,3-diazole indicates our fluorogenic tag-probe system can be utilized with tagged proteins. Following stimulation by phorbol ester, the translocation of protein kinase C was tracked by the fluorescence of 4-nitrobenzo-2-oxa-1,3-diazole, suggesting the formation of the noncovalently assembled tag-probe pairing is maintained during the translocation, even when the concentration of the probe peptide is reduced to 0.1 µM. The results indicated that the dynamic change of the protein localization by chemical stimulations can be revealed by the ZIP tag-probe system. Above all, the system is simple to handle and highly compatible with virtually any protein inside the cells.

Trivalent ligands for CXCR4 bearing polyproline linkers show specific recognition for cells with increased CXCR4 expression.

The assembly status of G protein-coupled receptors (GPCR) on the cell surface is of interest because the multimerization of GPCR could play pivotal roles in cellular functions. A bivalent ligand with polyproline linkers for CXCR4 has been shown to serve as a "molecular ruler" as a result of the rigid structure of polyproline helices. To expand the utility of the ligands with rigid linkers and explore the possible multimeric forms of GPCR, trivalent ligands with polyproline helices were newly designed and synthesized. The binding affinities of the trivalent ligands for CXCR4 suggested that the ligands recognize the dimeric form of CXCR4 on the cell surface. The fluorescence imaging and analysis by flow cytometry revealed that the ligand with 9 proline linkers binds to CXCR4 with remarkable specificity. The results of the present study suggest that the ligand design with rigid linkers is useful in the multimeric form, but the design of trivalent ligands requires different strategic approaches.

Anti-HIV screening for cell-penetrating peptides using chloroquine and identification of anti-HIV peptides derived from matrix proteins.

Previously, compounds which inhibit the HIV-1 replication cycle were found in overlapping peptide libraries covering the whole sequence of an HIV-1 matrix (MA) protein constructed with the addition of an octa-arginyl group. The two top lead compounds are sequential fragments MA-8L and MA-9L. In the present study, the addition of chloroquine in cell-based anti-HIV assays was proven to be an efficient method with which to find anti-HIV compounds among several peptides conjugated by cell-penetrating signals such as an octa-arginyl group: the conjugation of an octa-arginyl group to individual peptides contained in whole proteins in combination with the addition of chloroquine in cells is a useful assay method to search active peptides. To find more potent fragment peptides, individual peptides between MA-8L and MA-9L, having the same peptide chain length but with sequences shifted by one amino acid residue, were synthesized in this paper and their anti-HIV activity was evaluated with an anti-HIV assay using chloroquine. As a result, the peptides in the C-terminal side of the series, which are relatively close to MA-9L, showed more potent inhibitory activity against both X4-HIV-1 and R5-HIV-1 than the peptides in the N-terminal side.

Screening for protein kinase C ligands using fluorescence resonance energy transfer.

Protein kinase C (PKC) is correlated with cell signaling pathways and also receives attention as a therapeutic target for cancer and Alzheimer-type dementia. The application of Förster/fluorescence resonance energy transfer (FRET) phenomena to detect binding between proteins and small molecules, for example, PKC and its ligands, underlies a fluorescence-based assay method suitable for high-throughput screening. To accelerate studies on PKC functions in processing signals using small molecules and the development of drugs that target PKC, novel methods for the assessment of the PKC binding affinity of compounds are necessary. We previously developed solvatochromic fluorophore-based methods for that assessment. In this study, a novel method for a FRET-based PKC binding assay was developed and is expected to overcome the limitations of solvatochromic fluorophores.

Correction: 2-cyanopyridine derivatives enable N-terminal cysteine bioconjugation and peptide bond cleavage of glutathione under aqueous and mild conditions.

[This corrects the article DOI: 10.1039/D4RA00437J.].

2-cyanopyridine derivatives enable N-terminal cysteine bioconjugation and peptide bond cleavage of glutathione under aqueous and mild conditions.

Inspired by the chemical reactivity of apalutamide, we have developed an efficient method for N-terminal cysteine bioconjugation with 2-cyanopyridine derivatives. Systematic investigations of various 2-cyanopyridines revealed that 2-cyanopyridines with electron-withdrawing groups react efficiently with cysteine under aqueous and mild conditions. Moreover, the highly reactive 2-cyanopyridines enable the peptide bond cleavage of glutathione. The utility of our method is demonstrated by its application to the cysteine-selective chemical modification of bioactive peptides.

Discovery of Potent DAG-Lactone Derivatives as HIV Latency Reversing Agents.

Toward human immunodeficiency virus type-1 (HIV-1) cure, cells latently infected with HIV-1 must be eliminated from people living with HIV-1. We previously developed a protein kinase C (PKC) activator, diacylglycerol (DAG)-lactone derivative 3, with high HIV-1 latency-reversing activity, based on YSE028 (2) as a lead compound and found that the activity was correlated with binding affinity for PKC and stability against esterase-mediated hydrolysis. Here, we synthesized new DAG-lactone derivatives not only containing a tertiary ester group or an isoxazole surrogate but also several symmetric alkylidene moieties to improve HIV-1 latency reversing activity. Compound 9a, with a dimethyl group at the α-position of the ester group, exerted twice higher HIV-1 latency reversing activity than compound 3, and compound 26, with the isoxazole moiety, was significantly active. In addition, DAG-lactone derivatives with moderate hydrophobicity and potent biostability showed high biological activity.