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.

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.

[Improvement in Aqueous Solubility of Bioactive Molecules by Decreasing Intermolecular Interaction].

Sufficient aqueous solubility is a key requirement for small molecular drug candidates, and improvement of the aqueous solubility of bioactive compounds is often a major issue for medicinal chemists. Decreasing the partition coefficient (Log P) by the introduction of a hydrophilic group is the conventional approach for improving the aqueous solubility of drug candidates, but is not always effective. On the other hand, the solubility of a solid solute in water is also dependent on the crystal packing of the solute suggesting the existence of another principle of solvation. We have developed alternative strategies to improve solubility by means of chemical modification to weaken intermolecular interaction in the solid state, thereby lowering the melting point and increasing the solubility. In this review, we summarize the strategies for improving solubility, that is, modification of molecules in ways that would disrupt molecular planarity by increasing the dihedral angle, that would bend the molecular structure, that would disrupt molecular symmetry, or that introduce a non-flat substituent at the meta position of a benzene substructure. We showed that these strategies can increase the aqueous solubility of molecules even if their hydrophobicity is concomitantly increased. Furthermore, we found that disruption of intermolecular interaction resulted in better aqueous solubility than a decrease of hydrophobicity in some cases.

Conversion of a PROTAC Mutant Huntingtin Degrader into Small-Molecule Hydrophobic Tags Focusing on Drug-like Properties.

The onset of neurodegenerative disorders (NDs), such as Alzheimer's disease, is associated with the accumulation of aggregates of misfolded proteins. We previously showed that chemical knockdown of ND-related aggregation-prone proteins can be achieved by proteolysis targeting chimeras (PROTACs). However, hetero-bifunctional PROTACs generally show poor permeability into the central nervous system, where NDs are located. Here, we document the conversion of one of our PROTACs into hydrophobic tags (HyTs), another class of degraders bearing hydrophobic degrons. This conversion decreases the molecular weight and the number of hydrogen bond donors/acceptors. All the developed HyTs lowered the level of mutant huntingtin, an aggregation-prone protein, with potency comparable to that of the parent PROTAC. Through IAM chromatography analysis and in vivo brain penetration assay of the HyTs, we discovered a brain-permeable HyT. Our results and mechanistic analysis indicate that conversion of protein degraders into HyTs could be a useful approach to improve their drug-like properties.

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.

Improvement in aqueous solubility of achiral symmetric cyclofenil by modification to a chiral asymmetric analog.

Decreasing the partition coefficient (LogP) by the introduction of a hydrophilic group is the conventional approach for improving the aqueous solubility of drug candidates, but is not always effective. Since melting point is related to aqueous solubility, we and other groups have developed alternative strategies to improve solubility by means of chemical modification to weaken intermolecular interaction in the solid state, thereby lowering the melting point and increasing the solubility. Here, we show that converting the symmetrical molecular structure of the clinically used estrogen receptor (ER) antagonist cyclofenil (1) into asymmetrical form by introducing an alkyl group enhances the aqueous solubility. Among the synthesized analogs, the chiral methylated analog (R)-4c shows the highest solubility, being 3.6-fold more soluble than 1 even though its hydrophobicity is increased by the methylation. Furthermore, (R)-4c also showed higher membrane permeability than 1, while retaining a comparable metabolic rate, and equivalent biological activity of the active forms (R)-13a to 2. Further validation of this strategy using lead compounds having symmetric structures is expected.

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.

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.

meta-Non-flat substituents: a novel molecular design to improve aqueous solubility in small molecule drug discovery.

Aqueous solubility is a key requirement for small-molecule drug candidates. Here, we investigated the regioisomer-physicochemical property relationships of disubstituted benzenes. We found that meta-isomers bearing non-flat substituents tend to possess the lowest melting point and the highest thermodynamic aqueous solubility among the regioisomers. The examination of pharmaceutical compounds containing a disubstituted benzene moiety supported the idea that the introduction of a non-flat substituent at the meta position of a benzene substructure would be a promising approach for medicinal chemists aiming to improve the thermodynamic aqueous solubility of drug candidates, even though it might not be universally effective.

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.

Image-based screen capturing misfolding status of Niemann-Pick type C1 identifies potential candidates for chaperone drugs.

Niemann-Pick disease type C is a rare, fatal neurodegenerative disorder characterized by massive intracellular accumulation of cholesterol. In most cases, loss-of-function mutations in the NPC1 gene that encodes lysosomal cholesterol transporter NPC1 are responsible for the disease, and more than half of the mutations are considered to interfere with the biogenesis or folding of the protein. We previously identified a series of oxysterol derivatives and phenanthridine-6-one derivatives as pharmacological chaperones, i.e., small molecules that can rescue folding-defective phenotypes of mutated NPC1, opening up an avenue to develop chaperone therapy for Niemann-Pick disease type C. Here, we present an improved image-based screen for NPC1 chaperones and we describe its application for drug-repurposing screening. We identified some azole antifungals, including itraconazole and posaconazole, and a kinase inhibitor, lapatinib, as probable pharmacological chaperones. A photo-crosslinking study confirmed direct binding of itraconazole to a representative folding-defective mutant protein, NPC1-I1061T. Competitive photo-crosslinking experiments suggested that oxysterol-based chaperones and itraconazole share the same or adjacent binding site(s), and the sensitivity of the crosslinking to P691S mutation in the sterol-sensing domain supports the hypothesis that their binding sites are located near this domain. Although the azoles were less effective in reducing cholesterol accumulation than the oxysterol-derived chaperones or an HDAC inhibitor, LBH-589, our findings should offer new starting points for medicinal chemistry efforts to develop better pharmacological chaperones for NPC1.

Association between work schedules and motivation for lifestyle change in workers with overweight or obesity: a cross-sectional study in Japan.

OBJECTIVE: To investigate the association between work schedules and motivation for behavioural change of lifestyle, based on the transtheoretical model (TTM) in workers with overweight or obesity. DESIGN: A cross-sectional observational study. SETTING: A healthcare examination centre in Japan. PARTICIPANTS: Between April 2014 and March 2016, we recruited 9243 participants who underwent healthcare examination and met the inclusion criteria, namely, age 20-65 years, body mass index (BMI) ≥25 kg/m2 and full-time workers. EXPOSURE: Night and shift (night/shift) workers were compared with daytime workers in terms of motivation for behavioural change. PRIMARY AND SECONDARY OUTCOME MEASURES: The primary outcome was action and maintenance stages of change (SOC) for lifestyle in TTM. In a subgroup analysis, we investigated interactions between characteristics, including age, sex, BMI, current smoking, alcohol habits, hours of sleep and working hours. RESULTS: Overall, 1390 participants (15.0%) were night/shift workers; night/shift workers were younger (median age (IQR): 46 (40-54) vs 43 (37-52) years) and the proportion of men was lesser (75.4 vs 60.9%) compared with daytime workers. The numbers of daytime and night/shift workers in the action and maintenance SOC were 2113 (26.9%) and 309 (22.2%), respectively. Compared with daytime workers, night/shift workers were less likely to demonstrate action and maintenance SOC (adjusted OR (AOR): 0.85, 95% CI: 0.74 to 0.98). In a subgroup analysis that included only those with long working hours (≥10 hours/day), results revealed a strong inverse association between night/shift work and action and maintenance SOC (AOR: 0.65, 95% CI: 0.48 to 0.86). A significant interaction was observed between long working hours and night/shift work (P for interaction=0.04). CONCLUSIONS: In workers with overweight or obesity, a night/shift work schedule was associated with a lower motivation for behavioural change in lifestyle, and the association was strengthened in those with long working hours.

Selective Degradation of Target Proteins by Chimeric Small-Molecular Drugs, PROTACs and SNIPERs.

New therapeutic modalities are needed to address the problem of pathological but undruggable proteins. One possible approach is the induction of protein degradation by chimeric drugs composed of a ubiquitin ligase (E3) ligand coupled to a ligand for the target protein. This article reviews chimeric drugs that decrease the level of specific proteins such as proteolysis targeting chimeric molecules (PROTACs) and specific and nongenetic inhibitor of apoptosis protein (IAP)-dependent protein erasers (SNIPERs), which target proteins for proteasome-mediated degradation. We cover strategies for increasing the degradation activity induced by small molecules, and their scope for application to undruggable proteins.

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.

Efficient Lead Finding, Activity Enhancement and Preliminary Selectivity Control of Nuclear Receptor Ligands Bearing a Phenanthridinone Skeleton.

BACKGROUND: Nuclear receptors (NRs) are considered as potential drug targets because they control diverse biological functions. However, steroidal ligands for NRs have the potential to cross-react with other nuclear receptors, so development of non-steroidal NR ligands is desirable to obtain safer agents for clinical use. We anticipated that efficient lead finding and enhancement of activity toward nuclear receptors recognizing endogenous steroidal ligands might be achieved by exhaustive evaluation of a steroid surrogate library coupled with examination of structure-activity relationships (SAR). METHOD: We evaluated our library of RORs (retinoic acid receptor-related orphan receptors) inverse agonists and/or PR (progesterone receptor) antagonists based on the phenanthridinone skeleton for antagonistic activities toward liver X receptors (LXRs), androgen receptor (AR) and glucocorticoid receptor (GR) and examined their SAR. RESULTS: Potent LXRß, AR, and GR antagonists were identified. SAR studies led to a potent AR antagonist (IC50: 0.059 µM). CONCLUSIONS: Our approach proved effective for efficient lead finding, activity enhancement and preliminary control of selectivity over other receptors. The phenanthridinone skeleton appears to be a promising steroid surrogate.

Novel Nonsteroidal Progesterone Receptor (PR) Antagonists with a Phenanthridinone Skeleton.

The progesterone receptor (PR) plays an important role in various physiological systems, including female reproduction and the central nervous system, and PR antagonists are thought to be effective not only as contraceptive agents and abortifacients but also in the treatment of various diseases, including hormone-dependent cancers and endometriosis. Here, we identified phenanthridin-6(5H)-one derivatives as a new class of PR antagonists and investigated their structure-activity relationships. Among the synthesized compounds, 37, 40, and 46 exhibited very potent PR antagonistic activity with high selectivity for PR over other nuclear receptors. These compounds are structurally distinct from other nonsteroidal PR antagonists, including cyanoaryl derivatives, and should be useful for further studies of the clinical utility of PR antagonists.

Structural development of tetrachlorophthalimides as liver X receptor ß (LXRß)-selective agonists with improved aqueous solubility.

LXRß-selective agonists are promising candidates to improve atherosclerosis without increasing plasma or hepatic TG levels. We have reported a series of tetrachlorophthalimide analogs as an LXRß-selective agonist. However, they exhibited poor aqueous solubility probably due to its high hydrophobicity and highly rigid and plane structure. In this report, we present further structural development of tetrachloro(styrylphenyl)phthalimides as the LXRß-selective agonists with improved aqueous solubility.

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.

Structural basis for specific cleavage of Lys6-linked polyubiquitin chains by USP30.

Parkin ubiquitin (Ub) ligase (also known as PARK2) ubiquitinates damaged mitochondria for their clearance and quality control. USP30 deubiquitinase opposes parkin-mediated Ub-chain formation on mitochondria by preferentially cleaving Lys6-linked Ub chains. Here, we report the crystal structure of zebrafish USP30 in complex with a Lys6-linked diubiquitin (diUb or Ub2) at 1.87-Å resolution. The distal Ub-recognition mechanism of USP30 is similar to those of other USP family members, whereas Phe4 and Thr12 of the proximal Ub are recognized by a USP30-specific surface. Structure-based mutagenesis showed that the interface with the proximal Ub is critical for the specific cleavage of Lys6-linked Ub chains, together with the noncanonical catalytic triad composed of Cys-His-Ser. The structural findings presented here reveal a mechanism for Lys6-linkage-specific deubiquitination.