A small-molecule degron with a phenylpropionic acid scaffold.

Targeted protein degradation (TPD), employing proteolysis-targeting chimeras (PROTACs) composed of ligands for both a target protein and ubiquitin ligase (E3) to redirect the ubiquitin-proteasome system (UPS) to the target protein, has emerged as a promising strategy in drug discovery. However, despite the vast number of E3 ligases, the repertoire of E3 ligands utilized in PROTACs remains limited. Here, we report the discovery of a small-molecule degron with a phenylpropionic acid skeleton, derived from a known ligand of S-phase kinase-interacting protein 2 (Skp2), an E3 ligase. We used this degron to design PROTACs inducing proteasomal degradation of HaloTag-fused proteins, and identified key structural relationships. Surprisingly, our mechanistic studies excluded the involvement of Skp2, suggesting that this degron recruits other protein(s) within the UPS.

Allosteric Hsp70 Modulator YM-1 Induces Degradation of BRD4.

YM-1, an allosteric modulator of heat-shock 70 kDa protein (Hsp70), inhibits cancer cell growth, but the mechanism is not yet fully understood. Here, we show that YM-1 induces the degradation of bromodomain containing 4 (BRD4), which mediates oncogene expression. Overall, our results indicate that YM-1 promotes the binding of HSP70 to BRD4, and this in turn promotes the ubiquitination of BRD4 by C-terminus of Hsc70-interacting protein (CHIP), an E3 ubiquitin ligase working in concert with Hsp70, leading to proteasomal degradation of BRD4. This YM-1-induced decrease of BRD4 would contribute at least in part to the inhibition of cancer cell growth.

Synthesis and Antiviral Activities of Neoechinulin B and Its Derivatives.

We have previously reported that neoechinulin B (1a), a prenylated indole diketopiperazine alkaloid, shows antiviral activities against hepatitis C virus (HCV) via the inactivation of the liver X receptors (LXRs) and the resultant disruption of double-membrane vesicles. In this study, a two-step synthesis of the diketopiperazine scaffold of 1a was achieved by the base-induced coupling of 1,4-diacetyl-3-{[(tert-butyldimethylsilyl)oxy]methyl}piperazine-2,5-dione with aldehydes, followed by the treatment of the resultant coupling products with tetra-n-butylammonium fluoride. Compound 1a and its 16 derivatives 1b-q were prepared using this method. Furthermore, variecolorin H, a related alkaloid, was obtained by the acid treatment of 1a in MeOH. The antiviral evaluation of 1a and its derivatives revealed that 1a, 1c, 1d, 1h, 1j, 1l, and 1o exhibited both anti-HCV and anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) activities. The results of this study indicate that the exomethylene moiety on the diketopiperazine ring is important for the antiviral activities. The antiviral compounds can inhibit the production of HCV and SARS-CoV-2 by inactivating LXRs.

Switching of Photocatalytic Tyrosine/Histidine Labeling and Application to Photocatalytic Proximity Labeling.

Weak and transient protein interactions are involved in dynamic biological responses and are an important research subject; however, methods to elucidate such interactions are lacking. Proximity labeling is a promising technique for labeling transient ligand-binding proteins and protein-protein interaction partners of analytes via an irreversible covalent bond. Expanding chemical tools for proximity labeling is required to analyze the interactome. We developed several photocatalytic proximity-labeling reactions mediated by two different mechanisms. We found that numerous dye molecules can function as catalysts for protein labeling. We also identified catalysts that selectively modify tyrosine and histidine residues and evaluated their mechanisms. Model experiments using HaloTag were performed to demonstrate photocatalytic proximity labeling. We found that both ATTO465, which catalyzes labeling by a single electron transfer, and BODIPY, which catalyzes labeling by singlet oxygen, catalyze proximity labeling in cells.

Proximity Histidine Labeling by Umpolung Strategy Using Singlet Oxygen.

While electrophilic reagents for histidine labeling have been developed, we report an umpolung strategy for histidine functionalization. A nucleophilic small molecule, 1-methyl-4-arylurazole, selectively labeled histidine under singlet oxygen (1O2) generation conditions. Rapid histidine labeling can be applied for instant protein labeling. Utilizing the short diffusion distance of 1O2 and a technique to localize the 1O2 generator, a photocatalyst in close proximity to the ligand-binding site, we demonstrated antibody Fc-selective labeling on magnetic beads functionalized with a ruthenium photocatalyst and Fc ligand, ApA. Three histidine residues located around the ApA binding site were identified as labeling sites by liquid chromatography-mass spectrometry analysis. This result suggests that 1O2-mediated histidine labeling can be applied to a proximity labeling reaction on the nanometer scale.

In vivo synthetic chemistry of proteolysis targeting chimeras (PROTACs).

Chemical knockdown of therapeutic targets using proteolysis targeting chimeras (PROTACs) is a rapidly developing field in drug discovery, but PROTACs are bifunctional molecules that generally show poor bioavailability due to their relatively high molecular weight. Recent developments aimed at the development of next-generation PROTACs include the in vivo synthesis of PROTAC molecules, and the exploitation of PROTACs as chemical tools for in vivo synthesis of ubiquitinated proteins. This short review covers recent advances in these areas and discusses the prospects for in vivo synthetic PROTAC technology.

Epo-C12 inhibits peroxiredoxin 1 peroxidase activity.

Epo-C12 is a synthetic derivative of epolactaene, isolated from Penicillium sp. BM 1689-P. Epo-C12 induces apoptosis in human acute lymphoblastoid leukemia BALL-1 cells. In our previous studies, seven proteins that bind to Epo-C12 were identified by a combination of pull-down experiments using biotinylated Epo-C12 (Bio-Epo-C12) and mass spectrometry. In the present study, the effect of Epo-C12 on peroxiredoxin 1 (Prx 1), one of the proteins that binds to Epo-C12, was investigated. Epo-C12 inhibited Prx 1 peroxidase activity. However, it did not suppress its chaperone activity. Binding experiments between Bio-Epo-C12 and point-mutated Prx 1s suggest that Epo-C12 binds to Cys52 and Cys83 in Prx 1. The present study revealed that Prx 1 is one of the target proteins through which Epo-C12 exerts an apoptotic effect in BALL-1 cells.

Exolytic and endolytic turnover of peptidoglycan by lytic transglycosylase Slt of Pseudomonas aeruginosa.

ß-Lactam antibiotics inhibit cell-wall transpeptidases, preventing the peptidoglycan, the major constituent of the bacterial cell wall, from cross-linking. This causes accumulation of long non-cross-linked strands of peptidoglycan, which leads to bacterial death. Pseudomonas aeruginosa, a nefarious bacterial pathogen, attempts to repair this aberrantly formed peptidoglycan by the function of the lytic transglycosylase Slt. We document in this report that Slt turns over the peptidoglycan by both exolytic and endolytic reactions, which cause glycosidic bond scission from a terminus or in the middle of the peptidoglycan, respectively. These reactions were characterized with complex synthetic peptidoglycan fragments that ranged in size from tetrasaccharides to octasaccharides. The X-ray structure of the wild-type apo Slt revealed it to be a doughnut-shaped protein. In a series of six additional X-ray crystal structures, we provide insights with authentic substrates into how Slt is enabled for catalysis for both the endolytic and exolytic reactions. The substrate for the exolytic reaction binds Slt in a canonical arrangement and reveals how both the glycan chain and the peptide stems are recognized by the Slt. We document that the apo enzyme does not have a fully formed active site for the endolytic reaction. However, binding of the peptidoglycan at the existing subsites within the catalytic domain causes a conformational change in the protein that assembles the surface for binding of a more expansive peptidoglycan between the catalytic domain and an adjacent domain. The complexes of Slt with synthetic peptidoglycan substrates provide an unprecedented snapshot of the endolytic reaction.

Functionalization of Human Serum Albumin by Tyrosine Click.

Human serum albumin (HSA) is a promising drug delivery carrier. Although covalent modification of Cys34 is a well-established method, it is desirable to develop a novel covalent modification method that targets residues other than cysteine to introduce multiple functions into a single HSA molecule. We developed a tyrosine-selective modification of HSA. Three tyrosine selective modification methods, hemin-catalyzed, horseradish peroxidase (HRP)-catalyzed, and laccase-catalyzed reactions were performed, and the modification efficiencies and modification sites of the modified HSAs obtained by these methods were evaluated and compared. We found that the laccase-catalyzed method could efficiently modify the tyrosine residue of HSA under mild reaction conditions without inducing oxidative side reactions. An average of 2.2 molecules of functional groups could be introduced to a single molecule of HSA by the laccase method. Binding site analysis using mass spectrometry suggested Y84, Y138, and Y401 as the main modification sites. Furthermore, we evaluated binding to ibuprofen and found that, unlike the conventional lysine residue modification, the inhibition of drug binding was minimal. These results suggest that tyrosine-residue selective chemical modification is a promising method for covalent drug attachment to HSA.

PROTACs and Other Chemical Protein Degradation Technologies for the Treatment of Neurodegenerative Disorders.

Neurodegenerative disorders (NDs) are a group of diseases that cause neural cell damage, leading to motility and/or cognitive dysfunctions. One of the causative agents is misfolded protein aggregates, which are considered as undruggable in terms of conventional tools, such as inhibitors and agonists/antagonists. Indeed, there is currently no FDA-approved drug for the causal treatment of NDs. However, emerging technologies for chemical protein degradation are opening up the possibility of selective elimination of target proteins through physiological protein degradation machineries, which do not depend on the functions of the target proteins. Here, we review recent efforts towards the treatment of NDs using chemical protein degradation technologies, and we briefly discuss the challenges and prospects.

Application of protein knockdown strategy targeting ß-sheet structure to multiple disease-associated polyglutamine proteins.

Polyglutamine diseases are a class of neurodegenerative diseases associated with the accumulation of aggregated mutant proteins. We previously developed a class of degradation-inducing agents targeting the ß-sheet-rich structure typical of such aggregates, and we showed that these agents dose-, time-, and proteasome-dependently decrease the intracellular level of mutant huntingtin with an extended polyglutamine tract, which correlates well with the severity of Huntington's disease. Here, we demonstrate that the same agents also deplete other polyglutamine disease-related proteins: mutant ataxin-3 and ataxin-7 in cells from spino-cerebellar ataxia patients, and mutant atrophin-1 in cells from dentatorubral-pallidoluysian atrophy patients. Targeting cross-ß-sheet structure could be an effective design strategy to develop therapeutic agents for multiple neurodegenerative diseases.

Isolation, synthesis, and biological activities of a bibenzyl from Empetrum nigrum var. japonicum.

4-(2-Hydroxyphenethyl)-2,6-dimethoxyphenol, a bibenzyl, was isolated from the leaves of Empetrum nigrum var. japonicum, collected from Mount Tateyama. Japanese rock ptarmigans frequently eat the leaves and fruits of this plant. The structure of the bibenzyl was confirmed by NMR spectroscopic analysis and fully characterized. A synthesis of this compound was accomplished by coupling 2-hydroxyphenylacetic acid with syringaldehyde, decarboxylation of the resultant isoaurones, and hydrogenation of the double bond in the corresponding stilbene. This compound displayed cytotoxic activity against human cancer cells (HCT116 and Hela cells) and leukemia cells (HL-60 cells). The present study suggests that this plant serves as a source of biologically active natural products. Also, our findings provide information on the secondary metabolites in the diet of Japanese rock ptarmigans.

Synthesis of nucleotide analogues, EFdA, EdA and EdAP, and the effect of EdAP on hepatitis B virus replication.

4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) and 4'-ethynyl-2'-deoxyadenosine (EdA) are nucleoside analogues which inhibit human immunodeficiency virus type 1 (HIV-1) reverse transcriptase. EdAP, a cyclosaligenyl (cycloSal) phosphate derivative of EdA, inhibits the replication of the influenza A virus. The common structural feature of these compounds is the ethynyl group at the 4'-position. In this study, these nucleoside analogues were prepared by a common synthetic strategy starting from the known 1,2-di-O-acetyl-D-ribofuranose. Biological evaluation of EdAP revealed that this compound reduced hepatitis B virus (HBV) replication dose-dependently without cytotoxicity against host cells tested in this study.

Synthetic and Biological Studies of Juglorubin and Related Naphthoquinones.

Juglorubin, juglorescein, and juglocombins A/B are naturally occurring naphthoquinone dimers isolated from Streptomyces sp. These dimers are proposed to be biogenetically derived from juglomycin C, a monomeric naphthoquinone isolated from the same Streptomyces sp. In this study, the dimerization of a juglomycin C derivative, a key step in the total syntheses of these natural products, was investigated. Juglorubin was synthesized from the minor product of the dimerization via the formation of the juglocombin A/B stereoisomers. A mechanism for the dimerization reaction as well as a plausible biosynthetic pathway to obtain juglorubin from juglomycin C are proposed. Furthermore, the antibacterial and cytotoxic activities of five synthetic compounds were evaluated. Among the compounds tested in this study, 1'-O-methyljuglocombin B dimethyl ester and juglomycin C exhibited antibacterial activity against Bacillus subtilis. 1'-O-Methyljuglocombin B dimethyl ester and juglomycin C showed cytotoxicity against human colon carcinoma HCT116 cells and human leukemia HL-60 cells. 1'-O-Methyljuglocombin B dimethyl ester exhibited cytotoxicity against human normal MRC-5 cells as strong as that against human cancer cells. In contrast, juglomycin C was less toxic against normal MRC-5 cells, indicating a significant selectivity toward cancer cells.

Synthesis, antibacterial and cytotoxic evaluation of flavipucine and its derivatives.

The antibacterial and cytotoxic activity of seven racemic lactams and both enantiomers of flavipucine were evaluated. Of the compounds tested in this study, flavipucine and phenylflavipucine displayed bactericidal activity against Bacillus subtilis. These results indicate that the pyridione epoxide moiety is a pharmacophore for antibacterial activity against B. subtilis. Flavipucine showed cytotoxic activity against several cancer cells. The cytotoxic activity of flavipucine against human leukemia HL-60 cells is as strong as that of SN-38, the active metabolite of irinotecan. In contrast, the cytotoxic activity of flavipucine against nonneoplastic HEK293 cells and human normal MRC-5 cells is weaker than that of SN-38. No significant differences in the biological activity of the racemates or enantiomers of flavipucine were observed.

Degradation of huntingtin mediated by a hybrid molecule composed of IAP antagonist linked to phenyldiazenyl benzothiazole derivative.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by aggregation of mutant huntingtin (mHtt), and removal of mHtt is expected as a potential therapeutic option. We previously reported protein knockdown of Htt by using hybrid small molecules (Htt degraders) consisting of BE04, a ligand of ubiquitin ligase (E3), linked to probes for protein aggregates. Here, in order to examine the effect of changing the ligand, we synthesized a similar Htt degrader utilizing MV1, an antagonist of the inhibitor of apoptosis protein (IAP) family (a subgroup of ubiquitin E3 ligases), which is expected to have a higher affinity and specificity for IAP, as compared with BE04. The MV1-based hybrid successfully induced interaction between Htt aggregates and IAP, and reduced mHtt levels in living cells. Its mode of action was confirmed to be the same as that of the BE04-based hybrid. However, although the affinity of MV1 for IAP is greater than that of BE04, the efficacy of Htt degradation by the MV1-based molecule was lower, suggesting that linker length between the ligand and probe might be an important determinant of efficacy.

Discovery of Small Molecules that Induce the Degradation of Huntingtin.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by the aggregation of mutant huntingtin (mHtt), and removal of toxic mHtt is expected to be an effective therapeutic approach. We designed two small hybrid molecules (1 and 2) by linking a ligand for ubiquitin ligase (cellular inhibitor of apoptosis protein 1; cIAP1) with probes for mHtt aggregates, anticipating that these compounds would recruit cIAP1 to mHtt and induce selective degradation by the ubiquitin-proteasome system. The synthesized compounds reduced mHtt levels in HD patient fibroblasts and appear to be promising candidates for the development of a treatment for HD.

Efficient protein knockdown of HaloTag-fused proteins using hybrid molecules consisting of IAP antagonist and HaloTag ligand.

We previously reported a protein knockdown system for HaloTag-fused proteins using hybrid small molecules consisting of alkyl chloride, which binds covalently to HaloTag, linked to BE04 (2), a bestatin (3) derivative with an affinity for cellular inhibitor of apoptosis protein 1 (cIAP1, a kind of ubiquitin ligase). This system addressed several limitations of prior protein knockdown technology, and was applied to degrade two HaloTag-fused proteins. However, the degradation activity of these hybrid small molecules was not potent. Therefore, we set out to improve this system. We report here the design, synthesis and biological evaluation of novel hybrid compounds 4a and 4b consisting of alkyl chloride linked to IAP antagonist MV1 (5). Compounds 4a and 4b were confirmed to reduce the levels of HaloTag-fused tumor necrosis factor α (HaloTag-TNFα), HaloTag-fused cell division control protein 42 (HaloTag-Cdc42), and unfused HaloTag protein in living cells more potently than did BE04-linked compound 1b. Analysis of the mode of action revealed that the reduction of HaloTag-TNFα is proteasome-dependent, and is also dependent on the linker structure between MV1 (5) and alkyl chloride. These compounds appear to induce ubiquitination at the HaloTag moiety of HaloTag-fused proteins. Our results indicate that these newly synthesized MV1-type hybrid compounds, 4a and 4b, are efficient tools for protein knockdown for HaloTag-fused proteins.

Degradation of HaloTag-fused nuclear proteins using bestatin-HaloTag ligand hybrid molecules.

We have developed a protein knockdown technology using hybrid small molecules designed as conjugates of a ligand for the target protein and a ligand for ubiquitin ligase cellular inhibitor of apoptosis protein 1 (cIAP1). However, this technology has several limitations. Here, we report the development of a novel protein knockdown system to address these limitations. In this system, target proteins are fused with HaloTag to provide a common binding site for a degradation inducer. We designed and synthesized small molecules consisting of alkyl chloride as the HaloTag-binding degradation inducer, which binds to HaloTag, linked to BE04 (2), which binds to cIAP1. Using this system, we successfully knocked down HaloTag-fused cAMP responsive element binding protein 1 (HaloTag-CREB1) and HaloTag-fused c-jun (HaloTag-c-jun), which are ligand-unknown nuclear proteins, in living cells. HaloTag-binding degradation inducers can be synthesized easily, and are expected to be useful as biological tools for pan-degradation of HaloTag-fused proteins.

Structure-activity relationships of bisphenol A analogs at estrogen receptors (ERs): discovery of an ERα-selective antagonist.

Our multi-template approach for drug discovery, focusing on protein targets with similar fold structures, has yielded lead compounds for various targets. We have also shown that a diphenylmethane skeleton can serve as a surrogate for a steroid skeleton. Here, on the basis of those ideas, we hypothesized that the diphenylmethane derivative bisphenol A (BPA) would bind to the ligand-binding domain of estrogen receptors (ERs) in a similar manner to estradiol and act as a steroid surrogate. To test this idea, we synthesized a series of BPA analogs and evaluated their structure-activity relationships, focusing on agonistic/antagonistic activities at ERs and ERα/ERß subtype selectivity. Among the compounds examined, 18 was found to be a potent ERα-antagonist with high selectivity over ERß and androgen receptor under our assay conditions. A computational docking study suggested that 18 would bind to the antagonistic conformation of ERα. ERα-selective antagonists, such as 18, are candidate agents for treatment of breast cancer.