Evaluating a Retro-Inverso Type Inhibitor of HTLV-1 Protease as a Competitive Inhibitor.

The inhibition mode of a retro-inverso (RI) inhibitor containing a hydroxyethylamine dipeptide isostere against the human T-cell leukemia virus type-1 (HTLV-1) protease was examined. Enzymatic evaluation of the RI-modified inhibitor containing a D-allo-Ile residue revealed that HTLV-1 was competitively inhibited. IC50 values of the RI-modified inhibitor and pepstatin A, a standard inhibitor of aspartic proteases, were nearly equivalent.

7,8-Dihydroxy-3-(4'-hydroxyphenyl)coumarin inhibits invasion and migration of osteosarcoma cells.

Advances in pharmacy and medicine have led to the development of many anti-cancer and molecular targeted agents; however, there are few agents capable of suppressing metastasis. To prevent cancer recurrence, it is essential to develop novel agents for inhibiting metastasis. Coumarin-based compounds have multiple pharmacological activities including anti-cancer effects. We screened a compound library constructed at Kyoto Pharmaceutical University and showed that 7,8-dihydroxy-3-(4'-hydroxyphenyl)coumarin (DHC) inhibited invasion and migration of LM8 mouse osteosarcoma cells and 143B human osteosarcoma cells in a concentration-dependent manner. DHC decreased intracellular actin filament formation by downregulating Rho small GTP-binding proteins such as RHOA, RAC1, and CDC42, which regulate actin reorganization. However, DHC did not downregulate the corresponding mRNA transcripts, whereas it downregulated Rho small GTP-binding proteins in the presence of cycloheximide, suggesting that DHC enhances the degradation of these proteins. DHC treatment inhibited metastasis and prolonged overall survival in a spontaneous metastasis mouse model. These results indicate that DHC has the potential to suppress metastasis of osteosarcoma cells by downregulating Rho small GTP-binding proteins.

Development of an activity-based chemiluminogenic probe for γ-glutamylcyclotransferase.

While γ-glutamylcyclotransferase (GGCT) has been implicated in cancer-cell proliferation, the role of GGCT enzymatic activity in the regulation of cancer-cell growth remains unclear. Toward further understanding of GGCT in vivo, here we report a novel cell-permeable chemiluminogenic probe "MAM-LISA-103" that detects intracellular GGCT activity and apply it to in vivo imaging. We first developed a chemiluminogenic probe LISA-103, which simply and sensitively detects the enzymatic activity of recombinant GGCT through chemiluminescence. We then designed the cell-permeable GGCT probe MAM-LISA-103 and applied it to several biological experiments. MAM-LISA-103 successfully detected the intracellular GGCT activity in GGCT-overexpressing NIH-3T3 cells. Moreover, MAM-LISA-103 demonstrated tumor-imaging ability when administered to a xenograft model using immunocompromised mice inoculated with MCF7 cells.

Copper-mediated radioiodination and radiobromination via aryl boronic precursor and its application to 125I/77Br-labeled prostate-specific membrane antigen imaging probes.

Prostate-specific membrane antigen (PSMA), expressed in prostate cancer cells, is being investigated extensively worldwide as a target for imaging and therapy of prostate cancer. Various radioiodinated PSMA imaging probes have been developed, and their structure has a peptidomimetic urea-based skeleton as a pharmacophore. For direct radioiodination of molecules containing these peptidomimetic structures, prior studies performed radioiododestannylation or electrophilic radioiodination of tyrosine residues. However, although these radiolabeling methods are frequently used, there are some issues with precursor toxicity and by-product production. Therefore, it is required to investigate a radiolabeling method that can be used for the radiosynthesis of radioiodinated PSMA imaging probes with urea-based peptidomimetic structures. We recently reported that copper-mediated radioiodination via a boronic precursor is an effective method for directly labeling a peptide. This radiohalogenation method was expected to be an effective method for radiosynthesis of PSMA imaging probes with a peptidomimetic structure. In this study, to confirm that this labeling method applies to the synthesis of the PSMA imaging probe, we synthesized PSMA imaging probes labeled with 125I and 77Br ([125I]mIB-PS and [77Br]mBrB-PS) using a copper-mediated radiohalogenation via common boronic precursors and investigated optimal boronic precursor and labeling conditions. As a result, the radiochemical yields of [125I]mIB-PS and [77Br]mBrB-PS were improved to > 93% at room temperature by optimizing the structure of the boronic precursor. We demonstrate that copper-mediated nucleophilic radiochemistry using a boronic precursor is a promising radiosynthetic method of PSMA imaging probes. Although we focused on the synthesis of PSMA imaging probes, the results in this study will also be useful for the synthesis of various radioiodine or radiobromine-labeled bioactive molecules.

The Effects of Side-Chain Configurations of a Retro-Inverso-Type Inhibitor on the Human T-Cell Leukemia Virus (HTLV)-1 Protease.

In this study, the effects of side-chain configurations of D-Ile residues of a retro-inverso (RI)-type inhibitor on the human T-cell leukemia virus type 1 (HTLV-1) protease containing a hydroxyethylamine dipeptide isostere were clarified. Prior to evaluation using the RI-type inhibitor, the effects of side-chain configurations of Ile residues of the substrate peptide on the HTLV-1 protease were examined to estimate the influence of side-chain configurations on enzyme activity. Based on the estimation of the influence of side-chain configurations on protease affinity, the RI-type inhibitors containing a D-allo-Ile residue in the corresponding substrate sequence, instead of a D-Ile residue, were synthesized via 9-fluorenylmethoxycarbonyl-based solid-phase peptide synthesis. Refolded recombinant HTLV-1 protease (1-116, L40I) was used for the simple and short evaluation of the inhibitory activities of the synthesized RI-type inhibitors. The results clearly indicated that mimicking the whole topology, comprising both the main- and side-chain structures of the parent inhibitor, is effective for the design of potent RI-modified protease inhibitors.

A novel dipeptide type inhibitor of the Wnt/ß-catenin pathway suppresses proliferation of acute myelogenous leukemia cells.

The Wnt/ß-catenin pathway is an attractive target for the treatment of acute myelogenous leukemia (AML), since aberrant activation of the Wnt/ß-catenin pathway contributes to carcinogenesis in various types of cancers including AML. Screening of an in-house compound library, constructed at Kyoto Pharmaceutical University, identified a novel compound designated "31" that was found to be an inhibitor of the Wnt/ß-catenin pathway. The compound inhibited T-cell factor (TCF) activity in a TCF firefly luciferase-reporter assay and suppressed the proliferation of several human AML cell lines in a dose-dependent manner. Compound 31 arrested the cell cycle of AML cells at the G1 stage and induced apoptosis. Decrease in protein and mRNA expression level of Wnt pathway-related molecules was confirmed by the analyses of western blotting and quantitative reverse transcription-polymerase chain reaction. In addition, compound 31 combined with idarubicin synergistically inhibited the proliferation of AML cells. In conclusion, these results strongly suggest that compound 31 has potential as a novel anti-AML agent targeting the Wnt/ß-catenin signaling pathway.

Synthesis of (+)-Muconin via Diastereoselective Oxypalladation.

Synthesis of (+)-muconin isolated from Rollinia mucosa (Annonaceae) was achieved. Stereoselective construction of a tetrahydrofuran-terahydropyran (THF-THP) ring moiety was performed using diastereoselective oxypalladation in the presence of CuCl2. The cross-coupling reaction of the THF-THP moiety with the γ-lactone portion followed by reduction of the enyne and removal of the protecting groups afforded (+)-muconin.

Macrocyclic BACE1 inhibitors with hydrophobic cross-linked structures: Optimization of ring size and ring structure.

Based on the X-ray crystallography of recombinant BACE1 and a hydroxyethylamine-type peptidic inhibitor, we introduced a cross-linked structure between the P1 and P3 side chains of the inhibitor to enhance its inhibitory activity. The P1 and P3 fragments bearing terminal alkenes were synthesized, and a ring-closing metathesis of these alkenes was used to construct the cross-linked structure. Evaluation of ring size using P1 and P3 fragments with various side chain lengths revealed that 13-membered rings were optimal, although their activity was reduced compared to that of the parent compound. Furthermore, the optimal ring structure was found to be a macrocycle with a dimethyl branched substituent at the P3 ß-position, which was approximately 100-fold more active than the non-substituted macrocycle. In addition, the introduction of a 4-carboxymethylphenyl group at the P1' position further improved the activity.

Structure-activity relationship study of hydroxyethylamine isostere and P1' site structure of peptide mimetic BACE1 inhibitors.

An aromatic substituent has been introduced into a known hydroxyethylamine (HEA)-type BACE1 inhibitor containing the superior substrate sequence to enhance inhibitory activity. The HEA-type isosteres bearing different hydroxyl group and methyl group configurations were prepared through a branched synthesis approach using intra- and inter-molecular epoxide opening reactions. The effect of their configuration was evaluated, showing that an R-configuration improved the inhibitory activity, while introduction of a methyl group on the isostere decreased the activity. Based on the non-substituted isostere with an R-configuration, 21 derivatives containing various substituents at the P1' site were synthesized. Our evaluation of the derivatives showed that the structure of the P1' site had a clear effect on activity, and highly potent inhibitor 40g, which showed sub-micromolar activity against recombinant BACE1 (rBACE1), was identified. The docking simulation of 40g with rBACE1 suggested that a carboxymethyl group at the para-position of the P1' benzene ring interacted with Lys285 in the S1' pocket.

Synthesis of (+)-solenopsin via Pd-catalyzed N-alkylation and cyclization.

Synthesis of (+)-solenopsin, a 2,6-disubstituted piperidine alkaloid, isolated from fire ants (Solenopsis), was achieved. Stereoselective construction of trans-2,6-piperidine ring moiety was performed using palladium-catalyzed cyclization. Chain elongation using Grubbs 2nd catalyst followed by the reduction of double bond and the deprotection of the Cbz group afforded (+)-solenopsin.

Copper-mediated radioiodination reaction through aryl boronic acid or ester precursor and its application to direct radiolabeling of a cyclic peptide.

A copper-mediated radioiodination using aryl boronic precursors is attracting attention as a solution to oxidative iododestannylation and nickel-mediated radioiodination drawbacks. The copper-mediated radiolabeling method allows radioiodination at room temperature with stable aryl boronic precursors without preparing complex starting materials or reagents and can be performed in a reaction vessel exposed to air. This method has good potential in radiochemistry; however, studies on the scope of copper-mediated radioiodination through boronic precursors are insufficient. In particular, few reports have demonstrated the effect of protecting groups on radiolabeling efficiency. Therefore, the effect of the protecting group of aryl boronic acids on the copper-mediated radioiodination was investigated. In addition, this method, which does not require heating, is expected to be useful for direct radiolabeling of peptides. Thus, we attempted direct radioiodination of c(RGDyk) as an example. The resulting radioiodination method was well tolerated in various substrates and was unaffected by the pinacol ester-type protecting group. Also, c(RGDyk) was labeled with 125 I via copper-mediated radioiodination using an aryl boronic acid precursor. The reaction time and yield were improved, compared with the indirect method. Furthermore, the large difference in polarity between the boronic acid precursor and the radiolabeled compound facilitated purification.

Evaluation of an octahydroisochromene scaffold used as a novel SARS 3CL protease inhibitor.

An octahydroisochromene scaffold has been introduced into a known SARS 3CL protease inhibitor as a novel hydrophobic core to interact with the S2 pocket of the protease. An alkyl or aryl substituent was also introduced at the 1-position of the octahydroisochromene scaffold and expected to introduce additional interactions with the protease. Sharpless-Katsuki asymmetric epoxidation and Sharpless asymmetric dihydroxylation were employed to construct the octahydroisochromene scaffold. The introductions of the P1 site His-al and the substituent at 1-position was achieved using successive reductive amination reactions. Our initial evaluations of the diastereo-isomeric mixtures (16a-d) revealed that the octahydroisochromene moiety functions as a core hydrophobic scaffold for the S2 pocket of the protease and the substituent at the 1-position may form additional interactions with the protease. The inhibitory activities of the diastereoisomerically-pure inhibitors (3a-d) strongly suggest that a specific stereo-isomer of the octahydroisochromene scaffold, (1S, 3S) 3b, directs the P1 site imidazole, the warhead aldehyde, and substituent at the 1-position of the fused ring to their appropriate pockets in the protease.

Evaluation of a non-prime site substituent and warheads combined with a decahydroisoquinolin scaffold as a SARS 3CL protease inhibitor.

A non-prime site substituent and warheads combined with a decahydroisoquinolin scaffold was evaluated as a novel inhibitor for severe acute respiratory syndrome (SARS) chymotrypsin-like protease (3CLpro). The decahydroisoquinolin scaffold has been demonstrated to be an effective hydrophobic center to interact with S2 site of SARS 3CLpro, but the lack of interactions at S3 to S4 site is thought to be a major reason for the moderate inhibitory activity. In this study, the effects of an additional non-prime site substituent on the scaffold as well as effects of several warheads are evaluated. For the introduction of a desired non-prime site substituent, amino functionality was introduced on the decahydroisoquinolin scaffold, and the scaffold was constructed by Pd(II) catalyzed diastereoselective ring formation. The synthesized decahydroisoquinolin inhibitors showed about 2.4 times potent inhibitory activities for SARS 3CLpro when combined with a non-prime site substituent. The present results indicated not only the expected additional interactions with the SARS 3CLpro but also the possibility of new inhibitors containing a fused-ring system as a hydrophobic scaffold and a new warhead such as thioacetal.

Convergent Synthesis of trans-2,6-Disubstituted Piperidine Alkaloid, (-)-iso-6-Spectaline by Palladium-Catalyzed Cyclization.

The plant alkaloids, iso-6-spectaline and spectaline, isolated from the Cassia or Senna genera contain a characteristic 2,6-disubstituted piperidin-3-ol scaffold. Although both natural products are reported to exhibit a variety of interesting biological activities, few stereo-selective schemes for the construction of the 2,6-disubstituted scaffold have been reported. Following our previous studies regarding the synthesis of (+)-spectaline, herein we report the first convergent synthesis of (-)-iso-6-spectaline using a cross-metathesis under thermal conditions where the cis-2,6-disubstituted piperidin-3-ol scaffold is condensed with a long alkyl chain containing a terminal olefin. The cis-2,6-disubstituted piperidin-3-ol used in the synthesis was prepared simply via Pd(II)-catalyzed diastereoselective cyclization. It was confirmed that (+)-spectaline, an epimer of (-)-iso-6-spectaline, was selectively synthesized by the cross-metathesis reaction under less intense thermal conditions starting from the same cis-2,6-disubstituted piperidin-3-ol derivative.

Effects of replacement and addition of an amino acid contained in a cyclic peptide corresponding to a ß-hairpin loop sequence of human EGF receptor.

Effects of replacement and addition of an amino acid in a cyclic decapeptide 1 (cyclic-CYNPTTYQMC) for inhibitory activity to dimerization of human epidermal growth factor receptor (EGFR) were examined. By alanine scanning of 1 corresponding to the arm structure (residues 246-254) of a ß-hairpin loop sequence (residues 242-259) of EGFR, it was confirmed that replacement of any amino acid in the loop structure lowered the dimerization inhibitory activity of 1. Among the residues examined, Tyr at position 246 and Thr at 250 were found to be crucial for dimer formation. Addition of an amino acid to the N-terminus of 1 also affected the dimerization inhibitory activity. Addition of an amino acid containing a moderately hydrophilic side-chain increased the inhibitory activity. In contrast, an intramolecular hydrogen bond of 1 is not thought to be crucial for holding the dimer structure on the basis of the dimerization inhibitory activities of N-methylated analogues of 1. These results will be useful for the design and evaluation of a potent dimerization inhibitor as an anti-proliferation agent. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Structural basis for the development of SARS 3CL protease inhibitors from a peptide mimic to an aza-decaline scaffold.

Design of inhibitors against severe acute respiratory syndrome (SARS) chymotrypsin-like protease (3CL(pro) ) is a potentially important approach to fight against SARS. We have developed several synthetic inhibitors by structure-based drug design. In this report, we reveal two crystal structures of SARS 3CL(pro) complexed with two new inhibitors based on our previous work. These structures combined with six crystal structures complexed with a series of related ligands reported by us are collectively analyzed. To these eight complexes, the structural basis for inhibitor binding was analyzed by the COMBINE method, which is a chemometrical analysis optimized for the protein-ligand complex. The analysis revealed that the first two latent variables gave a cumulative contribution ratio of r(2) = 0.971. Interestingly, scores using the second latent variables for each complex were strongly correlated with root mean square deviations (RMSDs) of side-chain heavy atoms of Met(49) from those of the intact crystal structure of SARS-3CL(pro) (r = 0.77) enlarging the S2 pocket. The substantial contribution of this side chain (∼10%) for the explanation of pIC50 s was dependent on stereochemistry and the chemical structure of the ligand adapted to the S2 pocket of the protease. Thus, starting from a substrate mimic inhibitor, a design for a central scaffold for a low molecular weight inhibitor was evaluated to develop a further potent inhibitor. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 391-403, 2016.

Effective internalization of U251-MG-secreted exosomes into cancer cells and characterization of their lipid components.

Exosomes, the natural vehicles of various biological molecules, have been examined in several research fields including drug delivery. Although understanding of the biological functions of exosomes has increased, how exosomes are transported between cells remains unclear. We hypothesized that cell tropism is important for effective exosomal intercellular communication and that parental cells regulate exosome movement by modulating constituent exosomal molecules. Herein, we demonstrated the strong translocation of glioblastoma-derived exosomes (U251exo) into their parental (U251) cells, breast cancer (MDA-MB-231) cells, and fibrosarcoma (HT-1080). Furthermore, disruption of proteins of U251exo by enzymatic treatment did not affect their uptake. Therefore, we focused on lipid molecules of U251exo with the expectation that they are crucial for effective incorporation of U251exo by cancer cells. Phosphatidylethanolamine was identified as a unique lipid component of U251-MG cell-derived extracellular vesicles. From these results, valuable insight is provided into the targeting of U251exo to cancer cells, which will help to develop a cancer-targeted drug delivery system.

Fused-ring structure of decahydroisoquinolin as a novel scaffold for SARS 3CL protease inhibitors.

The design and evaluation of a novel decahydroisoquinolin scaffold as an inhibitor for severe acute respiratory syndrome (SARS) chymotrypsin-like protease (3CL(pro)) are described. Focusing on hydrophobic interactions at the S2 site, the decahydroisoquinolin scaffold was designed by connecting the P2 site cyclohexyl group of the substrate-based inhibitor to the main-chain at the α-nitrogen atom of the P2 position via a methylene linker. Starting from a cyclohexene enantiomer obtained by salt resolution, trans-decahydroisoquinolin derivatives were synthesized. All decahydroisoquinolin inhibitors synthesized showed moderate but clear inhibitory activities for SARS 3CL(pro), which confirmed the fused ring structure of the decahydroisoquinolin functions as a novel scaffold for SARS 3CL(pro) inhibitor. X-ray crystallographic analyses of the SARS 3CL(pro) in a complex with the decahydroisoquinolin inhibitor revealed the expected interactions at the S1 and S2 sites, as well as additional interactions at the N-substituent of the inhibitor.

Evaluation of transition-state mimics in a superior BACE1 cleavage sequence as peptide-mimetic BACE1 inhibitors.

A superior substrate sequence for BACE1 containing transition-state mimics at the scissile site was evaluated as a protease inhibitor. Hydroxymethylcarbonyl (HMC) and hydroxyethylamine (HEA) isosteres were selected as the transition state mimics, and incorporated into the scissile site of the superior sequence covering the P4 to P1' sites (Glu-Ile-Thi-Thi(*)Nva; (*)denotes the cleavage site). Isosteres having different absolute configurations of the hydroxyl group were synthesized separately, and the effect of the configuration was evaluated. Configuration of the hydroxyl group of each isostere showed a marked effect on the inhibitory activity; anti-configuration to the scissile site substituent had potent inhibitory activity in an HMC-type inhibitor, whereas anti-configuration of HEA-type inhibitors showed no inhibitory activity. Structural evaluations based on X-ray crystallographic analyses of recombinant BACE1 in complex with each inhibitor provided insights into the protein-ligand interactions, especially at the prime sites.