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.

Development of versatile solid-phase methods for syntheses of PROTACs with diverse E3 ligands.

Developing highly active proteolysis-targeting chimeras (PROTACs) requires investigating a variety of ubiquitin ligase (E3 ligase) ligands and linker structures as well as their lengths. In this study, we developed a solid-phase synthesis method that affords PROTAC design diversity. We expanded the E3 ligand range to include Von Hippel-Lindau (VHL) and inhibitor of apoptosis protein (IAP) ligands because only the cereblon (CRBN) ligand thalidomide and its derivatives have been investigated for solid-phase synthesis of PROTACs. Moreover, we examined the suitability of a polyethylene glycol (PEG) rather than an alkyl linker used in our previous study for synthesizing PROTACs. Facile and rapid solid-phase synthesis methods using the above E3 ligands for developing PROTACs targeting bromodomain-containing protein 4 (BRD4) were accomplished. Western blotting analysis revealed that minor differences in the E3 ligand and linker type significantly affected the activity of the synthesized PROTACs. Our solid-phase PROTAC synthesis methods enable rapid synthesis of multiple PROTACs with various combinations of ligands for the protein-of-interest and E3 ligands and linkers that connect these ligands.

CRBN ligand expansion for hematopoietic prostaglandin D2 synthase (H-PGDS) targeting PROTAC design and their in vitro ADME profiles.

An increasing number of research reports are describing modifications of the E3 ligand, in particular, cereblon (CRBN) ligands, to improve the chemical and metabolic stabilities as well as the physical properties of PROTACs. In this study, phenyl-glutarimide (PG) and 6-fluoropomalidomide (6-F-POM), recently used as CRBN ligands for PROTAC design, were applied to hematopoietic prostaglandin D2 synthase (H-PGDS)-targeted PROTACs. Both PROTAC-5 containing PG and PROTAC-6 containing 6-F-POM were found to have potent activities to induce H-PGDS degradation. Furthermore, we obtained in vitro ADME data on the newly designed PROTACS as well as our previously reported PROTACs(H-PGDS) series. Although all PROTACs(H-PGDS) are relatively stable toward metabolism, they had poor PAMPA values. Nevertheless, PROTAC-5 showed Papp values similar to TAS-205, which is in Phase 3 clinical trials, and is expected to be the key to improving the pharmacokinetics of PROTACs.

In silico optimization of peptides that inhibit Wnt/ß-catenin signaling.

The Wnt/ß-catenin signaling pathway causes transcriptional activation through the interaction between ß-catenin and T cell-specific transcription factor (TCF) and regulates a wide variety of cellular responses, including proliferation, differentiation and cell motility. Excessive transcriptional activation of the Wnt/ß-catenin pathway is implicated in developing or exacerbating various cancers. We have recently reported that liver receptor homolog-1 (LRH-1)-derived peptides inhibit the ß-catenin/TCF interaction. In addition, we developed a cell-penetrating peptide (CPP)-conjugated LRH-1-derived peptide that inhibits the growth of colon cancer cells and specifically inhibits the Wnt/ß-catenin pathway. Nonetheless, the inhibitory activity of the CPP-conjugated LRH-1-derived peptide was unsatisfactory (ca. 20 µM), and improving the bioactivity of peptide inhibitors is required for their in vivo applications. In this study, we optimized the LRH-1-derived peptide using in silico design to enhance its activity further. The newly designed peptides showed binding affinity toward ß-catenin comparable to the parent peptide. In addition, the CPP-conjugated stapled peptide, Penetratin-st6, showed excellent inhibition (ca. 5 µM). Thus, the combination of in silico design by MOE and MD calculations has revealed that logical molecular design of PPI inhibitory peptides targeting ß-catenin is possible. This method can be also applied to the rational design of peptide-based inhibitors targeting other proteins.

Origin of the kinetic HDAC2-selectivity of an HDAC inhibitor.

A newly synthesized small molecule, KTT-1, exhibits kinetically selective inhibition of histone deacetylase 2, HDAC2, over its homologous enzyme, HDAC1. KTT-1 is hard to be released from the HDAC2/KTT-1 complex, compared to the HDAC1/KTT-1 complex and the residence time of KTT-1 in HDAC2 is longer than that in HDAC1. To explore the physical origin of this kinetic selectivity, we performed replica-exchange umbrella sampling molecular dynamics simulations for formation of both complexes. The calculated potentials of mean force suggest that KTT-1 is stably bound to HDAC2 and that it is easily disassociated from HDAC1. In the direct vicinity of the KTT-1 binding site in both enzymes, there exists a conserved loop consisting of four consecutive glycine residues (Gly304-307 for HDAC2; Gly299-302 for HDA1). The difference between the two enzymes comes from a single un-conserved residue behind this loop, namely, Ala268 in HDAC2 and Ser263 in HDAC1. The Ala268 contributes to the tight binding of KTT-1 to HDAC2 by the linear orientation of Ala268, Gly306, and one carbon atom in KTT-1. On the other hand, Ser263 cannot stabilize the binding of KTT-1 to HDAC1, because it is relatively further away from the glycine loop and because the directions of the two forces are not in line.

Structure-activity relationship study of PROTACs against hematopoietic prostaglandin D2 synthase.

Degradation of hematopoietic prostaglandin D2 synthase (H-PGDS) by proteolysis-targeting chimeras (PROTACs) is expected to be important in the treatment of allergic diseases and Duchenne's muscular dystrophy. We recently reported that PROTAC(H-PGDS)-7 (PROTAC1), which is composed of H-PGDS inhibitor (TFC-007) and cereblon (CRBN) E3 ligase ligand (pomalidomide), showed potent H-PGDS degradation activity. Here, we investigated the structure-activity relationships of PROTAC1, focusing on the C4- or C5-conjugation of pomalidomide, in addition, the H-PGDS ligand exchanging from TFC-007 with the biaryl ether to TAS-205 with the pyrrole. Three new PROTACs were evaluated for H-PGDS affinity, H-PGDS degrading activity, and inhibition of prostaglandin D2 production. All compounds showed high H-PGDS degrading activities, but PROTAC(H-PGDS)-4-TAS-205 (PROTAC3) was slightly less active than the other compounds. Molecular dynamics simulations suggested that the decrease in activity of PROTAC3 may be due to the lower stability of the CRBN-PROTAC-H-PGDS ternary complex.

Oligoarginine-Conjugated Peptide Foldamers Inhibiting Vitamin D Receptor-Mediated Transcription.

The vitamin D receptor (VDR) is a nuclear receptor, which is involved in several physiological processes, including differentiation and bone homeostasis. The VDR is a promising target for the development of drugs against cancer and bone-related diseases. To date, several VDR antagonists, which bind to the ligand binding domain of the VDR and compete with the endogenous agonist 1α,25(OH)D3, have been reported. However, these ligands contain a secosteroidal skeleton, which is chemically unstable and complicated to synthesize. A few VDR antagonists with a nonsecosteroidal skeleton have been reported. Alternative inhibitors against VDR transactivation that act via different mechanisms are desirable. Here, we developed peptide-based VDR inhibitors capable of disrupting the VDR-coactivator interaction. It was reported that helical SRC2-3 peptides strongly bound to the VDR and competed with the coactivator in vitro. Therefore, we designed and synthesized a series of SRC2-3 derivatives by the introduction of nonproteinogenic amino acids, such as ß-amino acids, and by side-chain stapling to stabilize helical structures and provide resistance against digestive enzymes. In addition, conjugation with a cell-penetrating peptide increased the cell membrane permeability and was a promising strategy for intracellular VDR inhibition. The nona-arginine-conjugated peptides 24 with side-chain stapling and 25 with cyclic ß-amino acids showed strong intracellular VDR inhibitory activity, resulting in suppression of the target gene expression and inhibition of the cell differentiation of HL-60 cells. Herein, the peptide design, structure-activity relationship (SAR) study, and biological evaluation of the peptides are described.

Cancer-Cell-Selective Targeting by Arylcyclopropylamine-Vorinostat Conjugates.

Anticancer drug delivery by small molecules offers a number of advantages over conventional macromolecular drug delivery systems. We previously developed phenylcyclopropylamine (PCPA)-drug conjugates (PDCs) as small-molecule-based drug delivery vehicles for targeting lysine-specific demethylase 1 (LSD1)-overexpressing cancers. In this study, we applied this PDC strategy to the HDAC-inhibitory anticancer agent vorinostat. Among three synthesized PCPA or arylcyclopropylamine (ACPA)-vorinostat conjugates 1, 9, and 32, conjugate 32 with a 4-oxybenzyl linker showed sufficient stability in buffer solutions, potent LSD1 inhibition, efficient LSD1-dependent vorinostat release, and potent and selective antiproliferative activity toward LSD1-expressing human breast cancer and small-cell lung cancer cell lines. These results indicate that the conjugate selectively releases vorinostat in cancer cells. A similar strategy may be applicable to other anticancer drugs.

Development of a penetratin-conjugated stapled peptide that inhibits Wnt/ß-catenin signaling.

Wnt/ß-catenin pathway triggers the formation of a complex between ß-catenin and T cell-specific transcription factor (TCF), which induces transcriptional activation. Excessive transcriptional activation of this pathway is associated with the development, cause, and deterioration of various cancers. Therefore, the Wnt/ß-catenin pathway is an attractive drug target for cancer therapeutics and small molecule- and peptide-based protein-protein interaction (PPI) inhibitors have been developed. However, peptide-based PPI inhibitors generally have low cell-membrane permeability because of their large molecular size. To improve cell-membrane permeability, conjugating cell-penetrating peptides (CPPs) to PPI-inhibiting peptides is a useful method for developing intracellularly targeted PPI inhibitors. In this study, we focused on the interaction between ß-catenin and liver receptor homologue-1 (LRH-1) and designed and synthesized a series of LRH-1-derived peptides to develop inhibitors against Wnt/ß-catenin signaling. The results showed that a penetratin-conjugated LRH-1-derived peptide (Penetratin-st7) predominantly inhibited DLD-1 cell growth at 20 µM treatment via inhibition of the Wnt signaling pathway. This result suggests that Penetratin-st7 is one of promising PPI inhibitors between TCF and ß-catenin.

Development of Rapid and Facile Solid-Phase Synthesis of PROTACs via a Variety of Binding Styles.

Optimizing linker design is important for ensuring efficient degradation activity of proteolysis-targeting chimeras (PROTACs). Therefore, developing a straightforward synthetic approach that combines the protein-of-interest ligand (POI ligand) and the ligand for E3 ubiquitin ligase (E3 ligand) in various binding styles through a linker is essential for rapid PROTAC syntheses. Herein, a solid-phase approach for convenient PROTAC synthesis is presented. We designed azide intermediates with different linker lengths to which the E3 ligand, pomalidomide, is attached and performed facile PROTACs synthesis by forming triazole, amide, and urea bonds from the intermediates.

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.

Peptide Stapling Improves the Sustainability of a Peptide-Based Chimeric Molecule That Induces Targeted Protein Degradation.

Peptide-based target protein degradation inducers called PROTACs/SNIPERs have low cell penetrability and poor intracellular stability as drawbacks. These shortcomings can be overcome by easily modifying these peptides by conjugation with cell penetrating peptides and side-chain stapling. In this study, we succeeded in developing the stapled peptide stPERML-R7, which is based on the estrogen receptor alpha (ERα)-binding peptide PERML and composed of natural amino acids. stPERML-R7, which includes a hepta-arginine motif and a hydrocarbon stapling moiety, showed increased α-helicity and similar binding affinity toward ERα when compared with those of the parent peptide PERML. Furthermore, we used stPERML-R7 to develop a peptide-based degrader LCL-stPERML-R7 targeting ERα by conjugating stPERML-R7 with a small molecule LCL161 (LCL) that recruits the E3 ligase IAPs to induce proteasomal degradation via ubiquitylation. The chimeric peptide LCL-stPERML-R7 induced sustained degradation of ERα and potently inhibited ERα-mediated transcription more effectively than the unstapled chimera LCL-PERML-R7. These results suggest that a stapled structure is effective in maintaining the intracellular activity of peptide-based degraders.

Identification of Novel Histone Deacetylase 6-Selective Inhibitors Bearing 3,3,3-Trifluorolactic Amide (TFLAM) Motif as a Zinc Binding Group.

Pharmacological inhibition of histone deacetylase 6 (HDAC6) is an effective therapeutic strategy for cancer and immunological diseases. Most of the previously reported HDAC6 inhibitors have a hydroxamate group as a zinc binding group (ZBG), which coordinates to the catalytic zinc ion of HDAC6. The hydroxamate group is liable to metabolically generate mutagenetic hydroxylamine; therefore, non-hydroxamate HDAC6 inhibitors would be advantageous. In this study, to identify novel non-hydroxamate HDAC6-selective inhibitors, screening of a chemical library and the subsequent structural optimization were performed, which led to the identification of HDAC6-selective inhibitors with 3,3,3-trifluorolactic amide (TFLAM) as a novel ZBG. The identified inhibitor showed potent and selective HDAC6-inhibitory activity in cells and induced regulatory T (Treg) cell differentiation.

Discovery of novel tetrahydrobenzo[b]thiophene-3-carbonitriles as histone deacetylase inhibitors.

The discovery and development of isoform-selective histone deacetylase (HDAC) inhibitor is a challenging task because of the sequence homology among HDAC enzymes. In the present work, novel tetrahydro benzo[b]thiophene-3-carbonitrile based benzamides were designed, synthesized, and evaluated as HDAC inhibitors. Pharmacophore modeling was our main design strategy, and two novel series of tetrahydro benzo[b]thiophene-3-carbonitrile derivatives with piperidine linker (series 1) and piperazine linker (series 2) were identified as HDAC inhibitors. Among all the synthesised compounds, 9h with 4-(aminomethyl) piperidine linker and 14n with piperazine linker demonstrated good activity against human HDAC1 and HDAC6, respectively. Both the compounds also exhibited good antiproliferative activity against several human cancer cell lines. Both these compounds (9h and 14n) also induced cell cycle arrest and apoptosis in U937 and MDA-MB-231 cancer cells. Overall, for the first time, this research discovered potent isoform-selective HDAC inhibitors using cyclic linker instead of the aliphatic chain and aromatic ring system, which were reported in known HDAC inhibitors.

Design, Synthesis, and Biological Evaluation of Lysine Demethylase 5†C Degraders.

Lysine demethylase 5 C (KDM5C) controls epigenetic gene expression and is attracting great interest in the field of chemical epigenetics. KDM5C has emerged as a therapeutic target for anti-prostate cancer agents, and recently we identified triazole 1 as an inhibitor of KDM5C. Compound 1 exhibited highly potent KDM5C-inhibitory activity in in vitro enzyme assays, but did not show strong anticancer effects. Therefore, a different approach is needed for the development of anticancer agents targeting KDM5C. Here, we attempted to identify KDM5C degraders by focusing on a protein-knockdown strategy. Compound 3 b, which was designed based on compound 1, degraded KDM5C and inhibited the growth of prostate cancer PC-3 cells more strongly than compound 1. These findings suggest that KDM5C degraders are more effective as anticancer agents than compounds that only inhibit the catalytic activity of KDM5C.

Identification of Diketopiperazine-Containing 2-Anilinobenzamides as Potent Sirtuin 2 (SIRT2)-Selective Inhibitors Targeting the "Selectivity Pocket", Substrate-Binding Site, and NAD+-Binding Site.

The NAD+-dependent deacetylase SIRT2 represents an attractive target for drug development. Here, we designed and synthesized drug-like SIRT2-selective inhibitors based on an analysis of the putative binding modes of recently reported SIRT2-selective inhibitors and evaluated their SIRT2-inhibitory activity. This led us to develop a more drug-like diketopiperazine structure as a "hydrogen bond (H-bond) hunter" to target the substrate-binding site of SIRT2. Thioamide 53, a conjugate of diketopiperazine and 2-anilinobenzamide which is expected to occupy the "selectivity pocket" of SIRT2, exhibited potent SIRT2-selective inhibition. Inhibition of SIRT2 by 53 was mediated by the formation of a 53-ADP-ribose conjugate, suggesting that 53 is a mechanism-based inhibitor targeting the "selectivity pocket", substrate-binding site, and NAD+-binding site. Furthermore, 53 showed potent antiproliferative activity toward breast cancer cells and promoted neurite outgrowth of Neuro-2a cells. These findings should pave the way for the discovery of novel therapeutic agents for cancer and neurological disorders.

Identification of ortho-hydroxy anilide as a novel scaffold for lysine demethylase 5 inhibitors.

Fe(II)/α-ketoglutarate-dependent lysine demethylases (KDMs) are attractive drug targets for several diseases including cancer. In this study, we designed and screened ortho-substituted anilides that are expected to function as Fe(II) chelators, and identified ortho-hydroxy anilide as a novel scaffold for KDM5A inhibitors. Treatment of human lung cancer A549 cells with a prodrug form of 4-carboxy-2-hydroxy-formanilide (9c) increased trimethylated lysine 4 on histone H3 level, suggesting KDM5 inhibition in the cells.

N+-C-H···O Hydrogen bonds in protein-ligand complexes.

In the context of drug design, C-H···O hydrogen bonds have received little attention so far, mostly because they are considered weak relative to other noncovalent interactions such as O-H···O hydrogen bonds, π/π interactions, and van der Waals interactions. Herein, we demonstrate the significance of hydrogen bonds between C-H groups adjacent to an ammonium cation and an oxygen atom (N+-C-H···O hydrogen bonds) in protein-ligand complexes. Quantum chemical calculations revealed details on the strength and geometrical requirements of these N+-C-H···O hydrogen bonds, and a subsequent survey of the Protein Data Bank (PDB) based on these criteria suggested that numerous protein-ligand complexes contain such N+-C-H···O hydrogen bonds. An ensuing experimental investigation into the G9a-like protein (GLP)-inhibitor complex demonstrated that N+-C-H···O hydrogen bonds affect the activity of the inhibitors against the target enzyme. These results should provide the basis for the use of N+-C-H···O hydrogen bonds in drug discovery.

Synthesis of ß-allylbutenolides via one-pot copper-catalyzed hydroallylation/cyclization of γ-hydroxybutynoate derivatives.

One-pot copper-catalyzed hydroallylation/lactone cyclization of γ-hydroxybutynoate derivatives was developed to afford ß-allylbutenolides.