In Silico Prediction of N-Nitrosamine Formation Pathways of Pharmaceutical Products.

The recent discovery of N-nitrosodimethylamine (NDMA), a mutagenic N-nitrosamine, in pharmaceuticals has adversely impacted the global supply of relevant pharmaceutical products. Contamination by N-nitrosamines diverts resources and time from research and development or pharmaceutical production, representing a bottleneck in drug development. Therefore, predicting the risk of N-nitrosamine contamination is an important step in preventing pharmaceutical contamination by DNA-reactive impurities for the production of high-quality pharmaceuticals. In this study, we first predicted the degradation pathways and impurities of model pharmaceuticals, namely gliclazide and indapamide, in silico using an expert-knowledge software. Second, we verified the prediction results with a demonstration test, which confirmed that N-nitrosamines formed from the degradation of gliclazide and indapamide in the presence of hydrogen peroxide, especially under alkaline conditions. Furthermore, the pathways by which degradation products formed were determined using ranitidine, a compound previously demonstrated to generate NDMA. The prediction indicated that a ranitidine-related compound served as a potential source of nitroso groups for NDMA formation. In silico software is expected to be useful for developing methods to assess the risk of N-nitrosamine formation from pharmaceuticals.

Structural analysis of an lysergic acid diethylamide (LSD) analogue N-methyl-N-isopropyllysergamide (MiPLA): Insights from Rotamers in NMR spectra.

Lysergic acid diethylamide (LSD) is a hallucinogenic compound that binds to and activates the serotonin 2A receptor and is classified as a controlled narcotic in Japan. Recently, MiPLA, an N-methyl-N-isopropyl derivative of LSD, has been detected in paper-sheet products in several countries. This study focuses on the synthesis of MiPLA and includes a comprehensive analysis involving structural and liquid chromatography-mass spectrometry (LC-MS). Particularly, MiPLA was synthesized in three-steps starting from ergometrine maleate, which resulted in the formation of (8S)-isomer, iso-MiPLA, as a by-product. The LC-MS results showed that LSD, MiPLA, and iso-MiPLA exhibited different retention times. Their chemical structures were determined using nuclear magnetic resonance spectroscopy, which revealed the presence of rotamers involving the N-methyl-N-isopropyl groups of tertiary amides in MiPLA and iso-MiPLA.

Synthesis of Norgestomet and its 17ß-isomer and evaluation of their agonistic activities against progesterone receptor.

Norgestomet is a synthetic progesterone derivative applied in veterinary medicine to control estrus and ovulation in cattle. Norgestomet has been widely used in the livestock industry to promote the synchronization of estrus in cattle and increase pregnancy rates. However, highly reproducible synthetic methods for Norgestomet have been rarely reported. Here, we described a method for the synthesis of Norgestomet and performed quantitative NMR analysis to determine the purity of the products. Moreover, the agonistic activity of the synthesized compounds against progesterone receptors (PRs) was evaluated using an alkaline phosphatase assay. We synthesized Norgestomet with 97.9% purity that exhibited agonistic activity against PR with EC50 values of 4.5 nM. We also synthesized the 17ß-isomer of Norgestomet with 92.7% purity that did not exhibit any PR agonistic activity. The proposed synthetic route of Norgestomet can facilitate the assessment of residual Norgestomet in foods.

Discovery of non-proteinogenic amino acids inhibiting biofilm formation by S. aureus and methicillin-resistant S. aureus.

Bacterial biofilms often cause medical complications and there has been a great deal of interest in the discovery of small-molecule agents that can inhibit the formation of biofilms. Among these agents, it has been reported that several d-amino acids, such as d-Leu, d-Trp, d-Tyr, and d-Met, exhibit weak inhibitory activity toward bacterial biofilm formation. In this study, we have screened a library of 332 non-proteinogenic amino acids for new biofilm inhibitory agents and discovered several compounds exhibiting biofilm-inhibitory activity against Gram-positive bacteria. In particular, H-DL-ß-(3,4-dihydroxyphenyl)-dl-Ser-OH (253) showed potent activity against S. aureus, including methicillin-resistant S. aureus.

TRIP12 promotes small-molecule-induced degradation through K29/K48-branched ubiquitin chains.

Targeted protein degradation is an emerging therapeutic paradigm. Small-molecule degraders such as proteolysis-targeting chimeras (PROTACs) induce the degradation of neo-substrates by hijacking E3 ubiquitin ligases. Although ubiquitylation of endogenous substrates has been extensively studied, the mechanism underlying forced degradation of neo-substrates is less well understood. We found that the ubiquitin ligase TRIP12 promotes PROTAC-induced and CRL2VHL-mediated degradation of BRD4 but is dispensable for the degradation of the endogenous CRL2VHL substrate HIF-1α. TRIP12 associates with BRD4 via CRL2VHL and specifically assembles K29-linked ubiquitin chains, facilitating the formation of K29/K48-branched ubiquitin chains and accelerating the assembly of K48 linkage by CRL2VHL. Consequently, TRIP12 promotes the PROTAC-induced apoptotic response. TRIP12 also supports the efficiency of other degraders that target CRABP2 or TRIM24 or recruit CRBN. These observations define TRIP12 and K29/K48-branched ubiquitin chains as accelerators of PROTAC-directed targeted protein degradation, revealing a cooperative mechanism of branched ubiquitin chain assembly unique to the degradation of neo-substrates.

Approach to Establishment of Control Strategy for Oral Solid Dosage Forms Using Continuous Manufacturing.

As a result of the research activities of the Japan Agency for Medical Research and Development (AMED), this document aims to show an approach to establishing control strategy for continuous manufacturing of oral solid dosage forms. The methods of drug development, technology transfer, process control, and quality control used in the current commercial batch manufacturing would be effective also in continuous manufacturing, while there are differences in the process development using continuous manufacturing and batch manufacturing. This document introduces an example of the way of thinking for establishing a control strategy for continuous manufacturing processes.

Critical role of mitochondrial ubiquitination and the OPTN-ATG9A axis in mitophagy.

Damaged mitochondria are selectively eliminated in a process called mitophagy. Parkin and PINK1, proteins mutated in Parkinson's disease, amplify ubiquitin signals on damaged mitochondria with the subsequent activation of autophagic machinery. Autophagy adaptors are thought to link ubiquitinated mitochondria and autophagy through ATG8 protein binding. Here, we establish methods for inducing mitophagy by mitochondria-targeted ubiquitin chains and chemical-induced mitochondrial ubiquitination. Using these tools, we reveal that the ubiquitin signal is sufficient for mitophagy and that PINK1 and Parkin are unnecessary for autophagy activation per se. Furthermore, using phase-separated fluorescent foci, we show that the critical autophagy adaptor OPTN forms a complex with ATG9A vesicles. Disruption of OPTN-ATG9A interactions does not induce mitophagy. Therefore, in addition to binding ATG8 proteins, the critical autophagy adaptors also bind the autophagy core units that contribute to the formation of multivalent interactions in the de novo synthesis of autophagosomal membranes near ubiquitinated mitochondria.

Targeted Protein Degradation by Chimeric Compounds using Hydrophobic E3 Ligands and Adamantane Moiety.

Targeted protein degradation using small chimeric molecules, such as proteolysis-targeting chimeras (PROTACs) and specific and nongenetic inhibitors of apoptosis protein [IAP]-dependent protein erasers (SNIPERs), is a promising technology in drug discovery. We recently developed a novel class of chimeric compounds that recruit the aryl hydrocarbon receptor (AhR) E3 ligase complex and induce the AhR-dependent degradation of target proteins. However, these chimeras contain a hydrophobic AhR E3 ligand, and thus, degrade target proteins even in cells that do not express AhR. In this study, we synthesized new compounds in which the AhR ligands were replaced with a hydrophobic adamantane moiety to investigate the mechanisms of AhR-independent degradation. Our results showed that the compounds, 2, 3, and 16 induced significant degradation of some target proteins in cells that do not express AhR, similar to the chimeras containing AhR ligands. However, in cells expressing AhR, 2, 3, and 16 did not induce the degradation of other target proteins, in contrast with their response to chimeras containing AhR ligands. Overall, it was suggested that target proteins susceptible to the hydrophobic tagging system are degraded by chimeras containing hydrophobic AhR ligands even without AhR.

Development of Small Molecule Chimeras That Recruit AhR E3 Ligase to Target Proteins.

Targeted protein degradation using chimeric small molecules such as proteolysis-targeting chimeras (PROTACs) and specific and nongenetic inhibitors of apoptosis protein [IAP]-dependent protein erasers (SNIPERs) is an emerging modality in drug discovery. Here, we expand the repertoire of E3 ligases capable of ubiquitylating target proteins using this system. By incorporating ß-naphthoflavone (ß-NF) as a ligand, we developed a novel class of chimeric molecules that recruit the arylhydrocarbon receptor (AhR) E3 ligase complex. ß-NF-ATRA, a chimeric degrader directed against cellular retinoic acid binding proteins (CRABPs), induced the AhR-dependent degradation of CRABP-1 and CRABP-2 via the ubiquitin-proteasome pathway. A similar compound ITE-ATRA, in which an alternative AhR ligand was used, also degraded CRABP proteins. Finally, we developed a chimeric compound ß-NF-JQ1 that is directed against bromodomain-containing (BRD) proteins using ß-NF as an AhR ligand. ß-NF-JQ1 induced the interaction of AhR and BRD proteins and displayed effective anticancer activity that correlated with protein knockdown activity. These results demonstrate a novel class of chimeric degrader molecules based on the ability to bring a target protein and an AhR E3 ligase into close proximity.

Design and synthesis of cell-permeable fluorescent nitrilotriacetic acid derivatives.

Fluorescence labeling of the target molecules using a small molecule-based probe is superior than a method using genetically expressed green fluorescence protein (GFP) in terms of convenience in its preparation and functionalization. Fluorophore-nitrilotriacetic acid (NTA) conjugates with several ester protecting groups were synthesized and evaluated for their cell membrane permeability by fluorescence microscopy analysis. One of the derivatives, acetoxymethyl (AM)-protected NTA conjugate is hydrolyzed, resulting in intracellular accumulation, thus providing localized fluorescence intensity in cells. This modification is expected as an effective method for converting a non-cell membrane permeable NTA-BODIPY conjugates to a cell membrane permeable derivatives.

Development of a Small Hybrid Molecule That Mediates Degradation of His-Tag Fused Proteins.

In recent years, the induction of target-protein degradation via the ubiquitin-proteasome system (UPS) mediated by small molecules has attracted attention, and this approach has applications in pharmaceutical development. However, this technique requires a ligand for the target protein that can be incorporated into tailor-made molecules, and there are many proteins for which such ligands have not been found. In this study, we developed a protein-knockdown method that recognizes a His-tag fused to a protein of interest. This strategy theoretically allows comprehensive targeting of proteins of interest by a particular molecule recognizing the tag. As expected, our hybrid molecule 10 [SNIPER(CH6)] efficiently degraded His-tagged CRABP-II and Smad2 in cells. This system provides an easy method to determine the susceptibility of proteins of interest to UPS-mediated degradation. Furthermore, we hope that this method will become an efficient tool to analyze the function of the UPS.

Development of an ON/OFF switchable fluorescent probe targeting His tag fused proteins in living cells.

The fluorescent labeling of target proteins is useful for analyzing their functions and localization in cells, and several fluorescent probes have been developed. However, the fusion of tags such as green fluorescent protein (GFP) to target proteins occasionally affects their functions and/or localization in living cells. Therefore, an imaging method that uses short peptide tags such as hexa-histidine (the His tag) has been attracting increasing attention. Few studies have investigated ON/OFF switchable fluorescent probes for intracellular His-tagged proteins. We herein developed a novel ON/OFF switchable probe for imaging targeted intracellular proteins fused with a CH6 tag, which is composed of one cysteine residue and six histidine residues.

Design and synthesis of novel selective estrogen receptor degradation inducers based on the diphenylheptane skeleton.

Estrogen receptors (ERs) are a family of nuclear receptors (NRs) that regulate physiological effects such as reproduction and bone homeostasis. It has been reported that approximately 70% of human breast cancers are hormone-dependent and ERα-positive. Recently, novel anti-breast cancer drugs based on different mechanisms of action have received significant attention. In this article, we have designed and synthesized a selective ER degradation inducer based on the diphenylheptane skeleton. Western blotting analysis revealed that PBP-NC10 degraded ERα through the ubiquitin-proteasome system. We also performed computational docking analysis to predict the binding mode of PBP-NC10 to ERα.

Targeted Degradation of Proteins Localized in Subcellular Compartments by Hybrid Small Molecules.

Development of novel small molecules that selectively degrade pathogenic proteins would provide an important advance in targeted therapy. Recently, we have devised a series of hybrid small molecules named SNIPER (specific and nongenetic IAP-dependent protein ERaser) that induces the degradation of target proteins via the ubiquitin-proteasome system. To understand the localization of proteins that can be targeted by this protein knockdown technology, we examined whether SNIPER molecules are able to induce degradation of cellular retinoic acid binding protein II (CRABP-II) proteins localized in subcellular compartments of cells. CRABP-II is genetically fused with subcellular localization signals, and they are expressed in the cells. SNIPER(CRABP) with different IAP-ligands, SNIPER(CRABP)-4 with bestatin and SNIPER(CRABP)-11 with MV1 compound, induce the proteasomal degradation of wild-type (WT), cytosolic, nuclear, and membrane-localized CRABP-II proteins, whereas only SNIPER(CRABP)-11 displayed degradation activity toward the mitochondrial CRABP-II protein. The small interfering RNA-mediated silencing of cIAP1 expression attenuated the knockdown activity of SNIPER(CRABP) against WT and cytosolic CRABP-II proteins, indicating that cIAP1 is the E3 ligase responsible for degradation of these proteins. Against membrane-localized CRABP-II protein, cIAP1 is also a primary E3 ligase in the cells, but another E3 ligase distinct from cIAP2 and X-linked inhibitor of apoptosis protein (XIAP) could also be involved in the SNIPER(CRABP)-11-induced degradation. However, for the degradation of nuclear and mitochondrial CRABP-II proteins, E3 ligases other than cIAP1, cIAP2, and XIAP play a role in the SNIPER-mediated protein knockdown. These results indicate that SNIPER can target cytosolic, nuclear, membrane-localized, and mitochondrial proteins for degradation, but the responsible E3 ligase is different, depending on the localization of the target protein.

Tamoxifen and Fulvestrant Hybrids Showed Potency as Selective Estrogen Receptor Down-Regulators.

BACKGROUND: Estrogen receptors (ERs) are an important target for the management of breast cancers. Selective estrogen receptor down-regulators (SERDs) block ER activity, as well as reduce ERα protein levels in cells, and therefore are promising therapeutic agents for the treatment of breast cancers. OBJECTIVE: In order to develop potent SERDs, we prepared tamoxifen and fulvestrant hybrids and evaluated their binding activity and down-regulation of ERα. METHODS: We designed and synthesized tamoxifen derivatives, which had a 4,4,5,5,5- pentafluoropentyl group on the terminal alkyl chain. The oxidation state of the sulfur atom and alkyl length between the sulfur and nitrogen atoms were varied. Western blotting was performed to determine the ability to down-regulate ERα. Binding affinities of synthesized compounds were evaluated by a fluorescence polarization-based competitive binding assay. RESULTS: We successfully prepared nine compounds. Treatment with 11, 14, and 17 effectively reduced ERα protein levels in MCF-7 cells in a concentration-dependent manner. This reduction was inhibited by a proteasome inhibitor. The ability of 14 to down-regulate the ERα protein level was equal to fulvestrant. All compounds showed a largely equal affinity for ERα. CONCLUSION: As indicated by Western blots, the ERα degradation activity was observed only in the series of butyl linker derivatives, namely, 11, 14, and 17. These findings suggest that the specific length of the alkyl chain is an important factor in controlling the down-regulation of ER. These results provide useful information for designing promising SERD candidates.

Development of BCR-ABL degradation inducers via the conjugation of an imatinib derivative and a cIAP1 ligand.

The manipulation of protein stability with small molecules has great potential as a technique for aiding the development of clinical therapies, including treatments for cancer. In this study, BCR-ABL protein degradation inducers called SNIPER(ABL) (Specific and Non-genetic inhibitors of apoptosis protein [IAP]-dependent Protein Erasers) were developed. The designed molecules contained two biologically active scaffolds: one was an imatinib derivative that binds to BCL-ABL and the other was a methyl bestatin that binds to cellular IAP 1 (cIAP1). The hybrid molecules, SNIPER(ABL), were expected to recruit BCR-ABL to cIAP1 for removal by proteasomes. In fact, SNIPER(ABL) induced the degradation of BCR-ABL protein and a subsequent reduction in cell growth. Thus, the degradation of BCR-ABL by SNIPER(ABL) is one potential strategy for treating BCR-ABL driven chronic myelogenous leukemia.

Synthesis and evaluation of raloxifene derivatives as a selective estrogen receptor down-regulator.

Estrogen receptors (ERs) play a major role in the growth of human breast cancer cells. A selective estrogen receptor down-regulator (SERD) that acts as not only an inhibitor of ligand binding, but also induces the down-regulation of ER, would be useful for the treatment for ER-positive breast cancer. We previously reported that tamoxifen derivatives, which have a long alkyl chain, had the ability to down-regulate ERα. With the aim of expanding range of the currently available SERDs, we designed and synthesized raloxifene derivatives, which had various lengths of the long alkyl chains, and evaluated their SERD activities. All compounds were able to bind ERα, and RC10, which has a decyl group on the amine moiety of raloxifene, was shown to be the most potent compound. Our findings suggest that the ligand core was replaceable, and that the alkyl length was important for controlling SERD activity. Moreover, RC10 showed antagonistic activity and its potency was superior to that of 4,4'-(heptane-4,4-diyl)bis(2-methylphenol) (18), a competitive antagonist of ER without SERD activity. These results provide information that will be useful for the development of promising SERDs candidates.

An Activity-Based Probe for Studying Crosslinking in Live Bacteria.

Penicillin-binding proteins (PBPs) catalyze the crosslinking of peptidoglycan (PG), an essential process for bacterial growth and survival, and a common antibiotic target. Yet, despite its importance, little is known about the spatiotemporal aspects of crosslinking­largely because of a lack of experimental tools for studying the reaction in live bacteria. Here we introduce such a tool: an activity-based probe that enables visualization and relative quantitation of crosslinking in vivo. In Staphylococcus aureus, we show that fluorescent mimics of the natural substrate of PBPs (PG stem peptide) are covalently incorporated into the cell wall, installing fluorophores in place of natural crosslinks. These fluorescent stem peptide mimics (FSPMs) are selectively recognized by a single PBP in S. aureus: PBP4. Thus, we were able to use FSPM pulse-labeling to localize PBP4 activity in live cells, showing that it is recruited to the septum in a manner dependent on wall teichoic acid.

Synthesis and evaluation of tamoxifen derivatives with a long alkyl side chain as selective estrogen receptor down-regulators.

Estrogen receptors (ERs) play a major role in the growth of human breast cancer cells. An antagonist that acts as not only an inhibitor of ligand binding but also an inducer of the down-regulation of ER would be useful for the treatment for ER-positive breast cancer. We previously reported the design and synthesis of a selective estrogen receptor down-regulator (SERD), (E/Z)-4-(1-{4-[2-(dodecylamino)ethoxy]phenyl}-2-phenylbut-1-en-1-yl)phenol (C12), which is a tamoxifen derivative having a long alkyl chain on the amine moiety. This compound induced degradation of ERα via a proteasome-dependent pathway and showed an antagonistic effect in MCF-7 cells. With the aim of increasing the potency of SERDs, we designed and synthesized various tamoxifen derivatives that have various lengths and terminal groups of the long alkyl side chain. During the course of our investigation, C10F having a 10-fluorodecyl group on the amine moiety of 4-OHT was shown to be the most potent compound among the tamoxifen derivatives. Moreover, computational docking analysis suggested that the long alkyl chain interacted with the hydrophobic region on the surface of the ER, which is a binding site of helix 12 and coactivator. These results provide useful information to develop promising candidates as SERDs.

Illuminating HIV gp120-Ligand Recognition through Computationally-Driven Optimization of Antibody-Recruiting Molecules.

Here we report on the structure-based optimization of antibody-recruiting molecules targeting HIV gp120 (ARM-H). These studies have leveraged a combination of medicinal chemistry, biochemical and cellular assay analysis, and computation. Our findings have afforded an optimized analog of ARM-H, which is ~1000 fold more potent in gp120-binding and MT-2 antiviral assays than our previously reported derivative. Furthermore, computational analysis, taken together with experimental data, provides evidence that azaindole- and indole-based attachment inhibitors bind gp120 at an accessory hydrophobic pocket beneath the CD4-binding site and can also adopt multiple unique binding modes in interacting with gp120. These results are likely to prove highly enabling in the development of novel HIV attachment inhibitors, and more broadly, they suggest novel applications for ARMs as probes of conformationally flexible systems.