Cysteinylprolyl ester-mediated drug release from a lipid-drug conjugate

For small-molecule drugs, lipidation via a cleavable linkage can extend half-life in circulation through interaction with albumin. Here we modified the cysteinylprolyl ester (CPE) system used in peptide thioester synthesis, which normally requires basic conditions, for use as an self-immolative linker and release device for a lipid-gemcitabine conjugate. To improve release under physiological conditions for medical application, a methyl group at the α-position of cysteine on the CPE unit was incorporated in anticipation of the Thorpe-Ingold effect. As a result, Ac-Gly-(α-Me)Cys(SH)-Pro-gemcitabine 11 drastically promoted the release of gemcitabine in comparison with Ac-Gly-Cys(SH)-Pro-gemcitabine 10. Furthermore, in the presence of bovine serum albumin and/or 2-mercaptoethanesulfonic acid, the gentle and continuous release of gemcitabine from the lipid-gemcitabine conjugate 16 was achieved. In addition to gemcitabine, this method could allow high clearance drugs, including nucleic acid and prostacyclin derivatives, to maintain their biological activity long enough to become effective.

Bioorganic & Medicinal Chemistry Letters


Sulfoxide-Mediated Cys-Trp-Selective Bioconjugation that Enables Protein Labeling and Peptide Heterodimerization


Late-stage formation of a sactionine linkage enabled by Lossen rearrangement of glycyl hydroxamic acid

Late-stage formation of a sactionine thioether bond connecting a Gly α-carbon and Cys thiol was achieved by Lossen rearrangement of a glycyl hydroxamic acid (GlyHA) residue in a peptide. Lossen rearrangement allowed conversion of GlyHA within a peptide to an -acyl iminium equivalent, which subsequently reacted with -acetamidomethyl Cys (Cys(Acm)) in TFA in the presence of guanidine hydrochloride (Gn·HCl) to yield the desired thioether linkage in the final stage.


Synthesis of N-Glyoxylyl Peptides Enabled by a Lossen Rearrangement-Induced Intramolecular Redox Reaction of N-Terminal Glycyl Hydroxamic Acid

An oxidant-free approach to the synthesis of -glyoxylyl peptides has been developed that utilizes the Lossen rearrangement of the N-terminal glycyl hydroxamic acid residue. The synthesis proceeds via an intramolecular redox mechanism to yield the glyoxylyl peptides, which are then subjected to various peptide cyclization procedures. The reaction scheme is suitable for oxidation-sensitive moieties including amino acids.


Advanced Insulin Synthesis by One-pot/Stepwise Disulfide Bond Formation Enabled by S-Protected Cysteine Sulfoxide

An advanced insulin synthesis is presented that utilizes one-pot/stepwise disulfide bond formation enabled by acid-activated S-protected cysteine sulfoxides in the presence of chloride anion. S-chlorocysteine generated from cysteine sulfoxides reacts with an S-protected cysteine to afford S-sulfenylsulfonium cation, which then furnishes the disulfide or reversely returns to the starting materials depending on the S-protection employed and the reaction conditions. Use of S-acetamidomethyl cysteine (Cys(Acm)) and its sulfoxide (Cys(Acm)(O)) selectively give the disulfide under weak acid conditions in the presence of MgCl2 even if S-p-methoxybenzyl cysteine (Cys(MBzl)) and its sulfoxide (Cys(MBzl)(O)) are also present. In contrast, the S-MBzl pair yields the disulfide under more acidic conditions in the presence of a chloride anion source. These reaction conditions allowed a one-pot insulin synthesis. Additionally, lipidated insulin was prepared by a one-pot disulfide-bonding/lipidation sequence.


Identification of Low-Density Lipoprotein Receptor-Related Protein 1 as a CXCL14 Receptor Using Chemically Synthesized Tetrafunctional Probes

CXCL14 is a primordial CXC-type chemokine that transports CpG oligodeoxynucleotides (ODN) into endosomes and lysosomes in dendritic cells, thereby leading to the activation of the Toll-like receptor 9 (TLR9)-mediated innate immune system. However, the underlying molecular mechanism by which the CXCL14-CpG ODN complex enters cells remains elusive. Herein, we describe the chemical synthesis of CXCL14-derived photoaffinity probes and their application to the identification of target receptors for CXCL14 using quantitative proteomics. By utilizing native chemical ligation and maleimide-thiol coupling chemistry, we synthesized site-specifically modified CXCL14-based photoaffinity probes that contain photoreactive 2-aryl-5-carboxytetrazole (ACT) and a hydrazine-labile cleavable linker. CXCL14-based probes were found to be capable of binding CpG ODN to immune cells, whose bioactivities were comparable to native CXCL14. Application of CXCL14-derived probes to quantitative proteomic experiments enabled the identification of dozens of target receptor candidates for CXCL14 in mouse macrophage-derived RAW264.7 cells, and we discovered that low-density lipoprotein receptor-related protein 1 (LRP1) is a novel receptor for CXCL14 by competitive proteome profiling. We further showed that disruption of LRP1 affected the incorporation of the CXCL14-CpG ODN complex in the cells. Overall, this report highlights the power of synthetic CXCL14-derived photoaffinity probes combined with chemical proteomics to discover previously unidentified receptors for CXCL14, which could promote an understanding of the molecular functions of CXCL14 and the elaborate machinery of innate immune systems.


CA9 and PRELID2; hypoxia-responsive potential therapeutic targets for pancreatic ductal adenocarcinoma as per bioinformatics analyses.

A strong hypoxic environment has been observed in pancreatic ductal adenocarcinoma (PDAC) cells, which contributes to drug resistance, tumor progression, and metastasis. Therefore, we performed bioinformatics analyses to investigate potential targets for the treatment of PDAC. To identify potential genes as effective PDAC treatment targets, we selected all genes whose expression level was related to worse overall survival (OS) in The Cancer Genome Atlas (TCGA) database and selected only the genes that matched with the genes upregulated due to hypoxia in pancreatic cancer cells in the dataset obtained from the Gene Expression Omnibus (GEO) database. Although the extracted 107 hypoxia-responsive genes included the genes that were slightly enriched in angiogenic factors, TCGA data analysis revealed that the expression level of endothelial cell (EC) markers did not affect OS. Finally, we selected CA9 and PRELID2 as potential targets for PDAC treatment and elucidated that a CA9 inhibitor, U-104, suppressed pancreatic cancer cell growth more effectively than 5-fluorouracil (5-FU) and PRELID2 siRNA treatment suppressed the cell growth stronger than CA9 siRNA treatment. Thus, we elucidated that specific inhibition of PRELID2 as well as CA9, extracted via exhaustive bioinformatic analyses of clinical datasets, could be a more effective strategy for PDAC treatment.


Enhanced tumor specific drug release by hypoxia sensitive dual-prodrugs based on 2-nitroimidazole

Hypoxia in cancer is important in the development of cancer-selective medicines. Here, a novel hypoxia-responsible dual-prodrug is described. We designed and synthesized 2-nitroimidazole derivatives which spontaneously release both a PYG inhibitor and gemcitabine under hypoxic conditions. One such derivative, a prodrug 9 was found to be stable against chemical and enzymatic hydrolysis, and upon chemical reduction of the nitro group on imidazole, successfully releases both drugs. In an in vitro proliferation assay using human pancreatic cells, compound 9 exhibited significant anti-proliferative effects in hypoxia but fewer effects in normoxia. Consequently, prodrug 9 should be useful for cancer treatment due to its improved cancer selectivity and potential to overcome drug resistance.


Structural characterization of the optical isomers esomeprazole and omeprazole using the JADER and FAERS databases

It is unclear whether the s (-) form of esomeprazole (EPZ) has an improved safety profile when compared with its racemic form omeprazole (OPZ). We assessed the potential complications of these optical isomers when combined with cilostazol, clopidogrel, and prasugrel, which are frequently used concomitant medications. Using two adverse event spontaneous reporting databases, Japanese Adverse Drug Event Report (JADER) and FDA Adverse Event Reporting System (FAERS), adverse event names for hemorrhage, venous/arterial embolization, and thrombus were obtained from the Medical Dictionary for Regulatory Activities. Reported odds ratios were calculated using a 2 × 2 contingency table, and a signal was considered present if the lower limit of the 95% confidence interval was >1. In combination with cilostazol, a hemorrhagic signal for OPZ in JADER and arterial emboli and thrombus signals for EPZ were detected in both databases. In combination with clopidogrel, OPZ showed arterial emboli and thrombus signals in JADER and venous/arterial emboli and thrombus signals in FAERS, while EPZ displayed arterial emboli and thrombus signals in FAERS. In contrast, when in combination with prasugrel, there were no adverse event signals in either database. This study has confirmed using big data, that EPZ, the optical isomer and racemic form of omeprazole, has the beneficial characteristics of being less sensitive to CYP, as was intended by its design.


Residue-selective CH sulfenylation enabled by acid-activated S-acetamidomethyl cysteine sulfoxide with application to one-pot stapling and lipidation sequence

A tyrosine (Tyr)- or tryptophan (Trp)-selective metal-free C-H sulfenylation reaction using an acid-activated S-acetamidomethyl cysteine (Cys) sulfoxide, Cys(Acm)(O), has been achieved. The dually protonated intermediate produced from Cys(Acm)(O) under acidic conditions allows the sulfenylation of Tyr. Significantly, the reaction in the presence of trimethylsilyl trifluoromethanesulfonate (TMSOTf) mainly affords a Cys-Tyr-linked peptide even in the presence of Trp residues. In contrast, a Cys-Trp-linked peptide was selectively obtained from the reaction in the presence of guanidine hydrochloride (Gn ⋅ HCl) under acidic conditions. Established Tyr- and Trp-selective sulfenylation methods were used in the Cys-Tyr stapling and Trp lipidation of glucagon-like peptides 1 in a one-pot/stepwise manner. Investigation of the mechanism showed that orbital- and charge-controlled reactions are responsible for the Trp and Tyr selectivity, respectively.


S-Protected cysteine sulfoxide-enabled tryptophan-selective modification with application to peptide lipidation


Identification of an endoplasmic reticulum proteostasis modulator that enhances insulin production in pancreatic β cells.

Perturbation of endoplasmic reticulum (ER) proteostasis is associated with impairment of cellular function in diverse diseases, especially the function of pancreatic β cells in type 2 diabetes. Restoration of ER proteostasis by small molecules shows therapeutic promise for type 2 diabetes. Here, using cell-based screening, we report identification of a chemical chaperone-like small molecule, KM04794, that alleviates ER stress. KM04794 prevented protein aggregation and cell death caused by ER stressors and a mutant insulin protein. We also found that this compound increased intracellular and secreted insulin levels in pancreatic β cells. Chemical biology and biochemical approaches revealed that the compound accumulated in the ER and interacted directly with the ER molecular chaperone BiP. Our data show that this corrector of ER proteostasis can enhance insulin storage and pancreatic β cell function.


Late-stage macrolactonisation enabled by tandem acyl transfers followed by desulphurisation

Intramolecular -acylation of a thiol-installed threonine with a thioester unit, followed by S-O acyl transfer and subsequent desulphurisation, allows the synthesis of lactone peptides. A protocol has been developed enabling the cyclisation of a linear peptide, a reaction which has not been achieved by conventional methods.


Potential Application of Measuring Serum Infliximab Levels in Rheumatoid Arthritis Management: A Retrospective Study based on KURAMA Cohort Data

Infliximab (IFX) therapy has considerably improved the treatment of rheumatoid arthritis (RA). However, some patients still do not respond adequately to IFX therapy, or the efficacy of the treatment diminishes over time. Although previous studies have reported a relationship between serum IFX levels and therapeutic efficacy, the potential applications of IFX therapeutic drug monitoring (TDM) in clinical practice remain unclear. The purpose of this study was to investigate the potential applications of IFX TDM by analyzing a Japanese cohort database. Data were collected retrospectively from the Kyoto University Rheumatoid Arthritis Management Alliance cohort between January 1, 2011, and December 31, 2018. Serum IFX levels were measured using a liquid chromatography-tandem mass spectrometer. Out of the 311 RA patients that used IFX, 41 were eligible for the analysis. Serum IFX levels were significantly higher in responders than in non-responders. An optimal cut-off value was determined to be 0.32 μg/mL based on a receiver operating characteristic curve. At the IFX measurement point, a better therapeutic response was observed in the high IFX group (n = 32) than in the low IFX group (n = 9). Conversely, at the maximum effect point, when DAS28-ESR was the lowest between IFX introduction and measurement points, there were no differences in responder proportions between the low and high IFX groups. IFX primary ineffectiveness could be avoided with appropriate dose escalation without blood concentration measurement in clinical practice. In conclusion, IFX TDM could facilitate the identification of secondary non-responders and in turn, proper IFX use.


Copper (II)-mediated C-H sulphenylation or selenylation of tryptophan enabling macrocyclization of peptides


Peptide Cyclization Mediated by Metal-Free S-Arylation: S-Protected Cysteine Sulfoxide as an Umpolung of Cysteine Nucleophile


Sequence-independent traceless method for preparation of peptide/protein thioesters using CPaseY-mediated hydrazinolysis

Proteins incorporating artificial moieties such as fluorophores and drugs have enjoyed increasing use in chemical biology and drug development research. Preparation of such artificial protein derivatives has relied mainly on native chemical ligation in which peptide/protein thioesters chemoselectively react with N-terminal cysteine peptides to afford protein molecules. The protein thioesters derived from expressed proteins represent thioesters that are very useful for the preparation of artificial proteins by native chemical ligation with synthetic peptides with N-terminal Cys. We recently have developed a traceless thioester-producing protocol using carboxypeptidase Y (CPaseY) which is compatible with an expressed protein. The traceless character is ensured by CPaseY-mediated hydrazinolysis of C-terminal Xaa (X)-Cys-Pro-Leu-OH sequence followed by an auto-processing of the Cys-Pro (CP) dipeptide unit, affording the corresponding X-thioester (X-SR). However, hydrazinolysis of the amide bond in the prolyl leucine junction depends significantly on the nature of X. In the case of hydrophobic X residues, the hydrazinolysis overreacts to give several hydrazides while the reaction of hydrophilic X residues proceeds slowly. In this research, we attempted to develop an X-independent CPaseY-mediated protocol and found that the incorporation of a triple CP sequence into the C-terminal end (X-(CP)-Leu-OH) allows for efficient X-SR formation in a manner that is independent of X.


Deprotection of S-Acetamidomethyl Cysteine with Copper (II) and 1,2-Aminothiols under Aerobic Conditions


Sulfanylmethyldimethylaminopyridine as a Useful Thiol Additive for Ligation Chemistry in Peptide/Protein Synthesis

Sulfanylmethyl-installed dimethylaminopyridine, 2-sulfanylmethyl-4-dimethylaminopyridine (), has an acidic thiol group comparable to that in aryl thiols due to the formation of a zwitterion consisting of a thiolate anion and a pyridinium cation. It can be used as an additive for native chemical ligation. The alkyl thiol in allows it to be used for the one-pot native chemical ligation-desulfurization protocol in peptide synthesis. The utility of in the synthesis of cyclic peptides is demonstrated.


Cisplatin, rather than oxaliplatin, increases paracellular permeability of LLC-PK1 cells via activating protein kinase C.

The clinical use of cisplatin is limited by its adverse events, particularly serious nephrotoxicity. It was clarified that cisplatin is transported by a kidney-specific organic cation transporter (OCT2). OCT2 also mediates the uptake of oxaliplatin into renal proximal tubular cells; however, this agent does not lead nephrotoxicity. In the present study, we carried out comparative experiments with cisplatin and oxaliplatin using porcine kidney LLC-PK1 cell monolayers. In the fluorescein-labeled isothiocyanate-dextran flux assay, the basolateral application of cisplatin, but not oxaliplatin, resulted in an increase in the paracellular permeability of cell monolayers. Even though the cellular accumulation of platinum at 50 μM oxaliplatin could reach the same level at 30 μM cisplatin, oxaliplatin did not induce hyper-permeability in cell monolayers. Cisplatin, but not oxaliplatin, significantly activated PKC. In addition, the combination of PKC inhibitors recovered the increase in paracellular permeability. In conclusion, pharmacodynamic mechanisms via PKC could explain the difference in nephrotoxicity between cisplatin and oxaliplatin.


Concentration and Glycoform of Rituximab in Plasma of Patients with B Cell Non-Hodgkin's Lymphoma.

Inter-individual variations in plasma concentrations of rituximab were found in some B-NHL patients. We could analyze a change in glycoforms of rituximab in the patients, and this finding may affect the pharmacokinetics of rituximab.

Of recruited twenty, 3 patients exhibited rapid rituximab clearance. Nine types of carbohydrate chains were detected in rituximab isolated from the blood. The composition ratios in some glycoforms were significantly different between at 1 h after administration and at the trough, although consisted amino acids remained unchanged. The patients with high clearance showed extensive alterations of glycoform composition ratios. However, pharmacodynamics makers were not different.


Advanced Insulin Synthesis by One-pot/stepwise Disulfide Bond Formation Enabled by Acid-activated S-Protected Cysteine Sulfoxide in the Presence of Chloride Anion


Sulfoxide-mediated Cys-Trp-selective bioconjugation that enables protein labeling and peptide heterodimerization


Residue-Specific Modification Reaction Using S-Acetamidomethyl Cysteine Sulfoxide, Cys(Acm)(O)


Development of copper and iron-mediated Cys-Trp-linking reaction


A New Thiol Additive for One-pot Sequential Peptide Ligation-Desulfurization Chemistry


Deprotection of S-Acetamidomethyl Cysteine Mediated by Copper Salts