Fluorescence probes are now widely used as indispensable tools in many cell functional analyses. At present, the design of fluorescent probes largely depends on the limited numbers of established sensing mechanisms such as photo-induced electron transfer (PET), photo-induced charge transfer (PCT), and fluorescence resonance energy transfer (FRET). Although these mechanisms are versatile in metal ion sensing, introduction of a new sensing mechanism is highly desirable not only to design a more sophisticated probe with high selectivity and sensitivity but also to expand the diversity of the sensing targets to unveil biological phenomena. In this article, we report the design of dual emission fluorescent probes for metal ions based on a unique fluorescence-sensing mechanism. The fluorescent probes incorporating this sensing mechanism displayed a large emission red-shift on complexation with metal ions such as CdII and AgI. X-ray crystallography and theoretical computational calculations of the CdII and AgI complexes revealed that the emission shift was induced by non-coordination contact between the aromatic ring of fluorophore and the metal ion (arene-metal ion contact; AM-contact), which modulates the energy levels of molecular orbitals. The fluorescent probe was successfully applied to in cell ratiometric bioimaging of bioactive hydrogen sulfide (H2S). These successful applications highlight the usefulness of this sensing mechanism in biological fluorescence analysis.
Synthetic small-molecule ligands that control the intracellular location of proteins would be powerful tools for regulating cellular systems. However, the creation of such molecules has long remained unexplored because of the lack of a design methodology. Here, we introduce a new type of synthetic ligands, self-localizing ligands (SLLs), which spontaneously localize to specific subcellular regions in mammalian cells. We show that SLLs bind their target (exogenously expressed and endogenous) proteins and relocate them rapidly from the cytoplasm to their targeting sites. SLL-induced protein translocation is applicable to manipulate diverse synthetic/endogenous signaling pathways. These results validate the utility of SLLs in the spatial control of intracellular protein localization and signaling processes, opening a new direction in the design of small-molecule-based chemical tools or drugs for cell regulation.
X-rays are widely used in imaging applications such as diffraction imaging of crystals and medical imaging. In particular, X-ray computed tomography (CT) is a critical tool for clinical and disease diagnostics. The principle of conventional CT is based on X-ray attenuation caused by photoelectric absorption and scattering. In addition to conventional CT, a number of novel methodologies are presently under development, including state-of-the-art instrument technologies and chemical probes to fulfill diagnosis criteria. Among these novel methodologies, we have utilized hard X-ray-excited optical luminescence (hXEOL) as a new methodology to enhance the contrast of the image. Herein, we explored the possibility of hXEOL via iridium-doped polymer nanoparticles and biomolecule-directed metal clusters and propose it as a potential platform for new X-ray imaging.
There are several approaches to understanding the physiological roles of biomolecules: (1) by observing the localization or activities of biomolecules (based on microscopic imaging experiments with fluorescent proteins or fluorescent probes) and (2) by investigating the cellular response via activation or suppression of functions of the target molecule (by using inhibitors, antagonists, siRNAs, etc.). In this context, protein-labeling technology serves as a powerful tool that can be used in various experiments, such as for fluorescence imaging of target proteins. Recently, we developed a protein-labeling technology that uses a mutant β-lactamase (a bacterial hydrolase) as the tag protein. In this protein-labeling technology, also referred to as the BL-tag technology, various β-lactam compounds were used as specific ligands that were covalently labeled to the tag. One major advantage of this labeling technology is that various functions can be carried out by suitably designing both the functional moieties such as the fluorophore and the β-lactam ligand structure. In this review, we briefly introduce the BL-tag technology and describe our future outlook for this technology, such as in fluorescence imaging of biomolecules and functional regulation of cellular proteins in living cells.
Nitric oxide (NO) is historically well known as a toxic gas but now recognized as a physiological cellular mediator acting at very low concentrations. It is biosynthesized within the body, and modulates many signal transduction processes. For investigation of the functions of this gaseous mediator, it is necessary to use chemical donors that release NO specifically, and it is highly advantageous if the release can be made with precise spatiotemporal control. For this purpose, we have developed caged NO (photocontrollable NO-releasing compounds) with unique releasing mechanisms. One employs the photoinduced rearrangement of an arylnitro group and subsequent release of NO, and another uses photoinduced electron transfer to release NO. One of our caged NO was confirmed to induce a NO-dependent cellular response in vivo under photocontrol. Photocontrollable NO releasers are expected to become indispensable tools for physiological experiments, and are also potential therapeutic agents for photodynamic therapy.
Recently, human serum albumin (HSA) has emerged as a versatile carrier for therapeutic agents against diabetes, cancer, and infectious diseases. Market-approved products include fatty acid derivatives of human insulin for diabetes and the paclitaxel-HSA nanoparticle for various cancers such as metastatic breast cancer and advanced pancreatic cancer. In this review, we focus on the next-generation approach including HSA-binding bioactive gas such as nitric oxide (NO) for treating ischemic/reperfusion injury, cancer, and bacterial infection. To date, pharmacologically active compounds that release NO within the body, such as organic nitrates, have been used as therapeutic agents, but their efficacy is significantly limited by unwanted side effects. Therefore, novel NO donors with better pharmacological and pharmacokinetic properties are highly desirable. The S-nitrosothiol fraction in plasma is largely composed of endogenous S-nitrosated HSA (SNO-HSA), which is why we are investigating whether this albumin form can be therapeutically useful. Recently, we have developed SNO-HSA analogues such as SNO-HSA with many conjugated SNO groups (poly-SNO-HSA) prepared using chemical modification. Unexpectedly, we found striking inverse effects between poly-SNO-HSA and SNO-HSA. Despite the fact that SNO-HSA inhibits apoptosis, poly-SNO-HSA possesses very strong pro-apoptotic effects against tumor cells. Furthermore, poly-SNO-HSA can reduce or even completely eliminate the multidrug resistance often developed by cancer cells. In this review, we put forward the possibility that poly-SNO-HSA can be used as a safe, effective multifunctional antitumor agent.
Exosomes are secretory membrane vesicles containing lipids, proteins, and nucleic acids. They act as intercellular transporters by delivering their components to exosome recipient cells. Based on their endogenous delivery system properties, exosomes are expected to become drug delivery systems (DDS) for various molecules such as nucleic acid-based drugs. Important factors such as drug loading to exosomes, production, and pharmacokinetics of exosomes need to be considered for the development of exosome-based DDS. Of these, the pharmacokinetics of exosomes have rarely been studied, probably because of the lack of quantitative evaluation methods of in vivo exosomal pharmacokinetics. We selected lactadherin as an exosome tropic protein and developed it as a fusion protein with Gaussia luciferase to label exosomes for in vivo imaging. In addition, a fusion protein of lactadherin and streptavidin was developed, and the tissue distribution of exosomes was quantitatively evaluated by radiolabeling the exosomes using 125I-labeled biotin. Using labeled exosomes, we found that intravenously injected exosomes were rapidly cleared from the systemic circulation by macrophages. In addition, the exosomes were mainly distributed to the liver, lung, and spleen. We also examined the effect of exosome isolation methods on their physicochemical and pharmacokinetic properties. We found that exosomes collected by the ultracentrifugation-based density-gradient method were more dispersed than exosomes collected by other methods, including the ultracentrifugation-based pelleting method. The gradient method is more time-consuming than others; therefore the development of a more efficient method for exosome isolation will advance the development of exosome-based DDS.
A variety of human diseases, including neurodegenerative disorders, ischemic heart disease, diabetes, and cancer have been reported to be associated with mitochondrial dysfunction. Because of this, mitochondrial therapy is expected to be useful and productive in the treatment of such diseases. We previously reported on the development of a MITO-Porter, a liposome-based nanocarrier that permits macromolecular cargos to be delivered into mitochondria via membrane fusion. Intracellular observations using the green fluorescence protein as a model macromolecule provided confirmation that a macromolecule could be delivered to mitochondria in living cells by the MITO-Porter. Here, we present our current findings on the development of mitochondrial medicine and mitochondrial gene therapy based on our mitochondrial drug delivery system (DDS). In this review, we propose “mitochondrial DDS” as a theme for “DDS research for innovative drug development” and discuss the contribution of mitochondrial DDS to innovative drug development.
Cases of people experiencing disturbed consciousness or dyspnea, causing traffic accidents, or requiring ambulance transport to hospital due to abuse of law-evading chemical substances have become a serious social problem in Japan. Most law-evading herbal products are marketed as incense or herbs and consist of finely chopped, dry vegetative matter mixed with chemical substances (drugs). Analysis of the chemical substances in these herbal products has demonstrated that they contain synthetic cannabinoids. Because there are many cannabinoid compounds, even if a particular drug is regulated, similar compounds that differ only slightly in structure may be added in their place. Therefore a cat-and-mouse game exists between regulations on chemical substances and their propagation. This paper summarizes the pharmacological actions and dangers of chemical substances contained in law-evading herbal products by focusing on synthetic cannabinoids, as a group of chemical substances contained in these products. Furthermore, comprehensive designations of synthetic cannabinoids have been introduced as a new method of regulation that emphasizes the similarity of chemical structures; this paper also outlines the comprehensive designations. We established a psychic-dependence liability and cytotoxicity screening system for synthetic cannabinoids using animals (behavioral analysis in vivo) and cell cultures (cytotoxicity analysis in vitro). With our drug-screening system, we were able rapidly to evaluate and quantify psychic-dependence liabilities and cytotoxicity of synthetic cannabinoids contained in law-evading herbal products. These scientific data using our screening system contributed to the establishment of legislation for comprehensive designations of synthetic cannabinoids.
Benzodiazepines (BZs) work by agonising gamma-aminobutyric acid (GABA)-BZ-receptor complex and thereby produce sedation and anti-anxiety effects. BZs are commonly used in several clinical areas as hypnotics or anti-anxiety drugs. However, these drugs once supplied by medical institutions often lead to abuse and dependence. Thus it is important for institutions to supply and manage BZs properly. At Tokyo Women's Medical University Hospital educational activities about proper use of BZs are performed by not only medical doctors but also pharmacists. We coordinate distribution of leaflets and run an educational workshop. As a result of these activities, the number of patients receiving BZ prescriptions was reduced. Performing these activities, pharmacists were required to work for patients, doctors, and nurses; they acquired knowledge about BZs such as action mechanisms, efficacy, adverse effects, problems about co-prescription, and methods of discontinuing BZs, as well as information on coping techniques other than medication. The most important point to attend the patients is to answer their anxieties.
The non-medical use or abuse of prescription drugs, including benzodiazepines, is a growing health problem in Japan. An association between prescription drug overdose and suicide risk has also been reported. The Japanese Ministry of Health, Labour and Welfare has expected pharmacists to act as “gatekeepers”, facilitating early identification of individuals at high risk of prescription drug abuse including overdose, supplying medication counseling to patients, and helping to introduce these patients to appropriate medical care. Prescription drugs such as benzodiazepines are widely used in psychiatry. However, these drugs are prescribed not only by psychiatrists but also by other healthcare professionals including primary care physicians. Moreover, in recent years, the dispensing of prescriptions has moved rapidly from inside to outside hospitals, with prescription drugs being dispensed mainly at community pharmacies. Although all healthcare professionals including hospital pharmacists can play a role in preventing prescription drug abuse, the role of the community pharmacist is vital in addressing this problem. Formerly, community pharmacists were recognized as “community scientists”, low-threshold accessible healthcare advisors. Now, community pharmacists should return to the role of community scientists to prevent prescription drug abuse. This article begins by reviewing the current situation of prescription drug abuse and dependence in Japan. The role of pharmacists as gatekeepers in preventing prescription drug abuse is then examined. Finally, this article discusses the effect of intervention in the form of gatekeeper training for community pharmacists.
The increasing number of law-evading drug users in Japan is becoming a serious social problem. Previous studies have shown that law-evading drug users are younger, more educated, and less antisocial than methamphetamine users. They also tend to have some type of psychiatric disorder before starting drug use; therefore one of the reasons that they start using drugs may be to alleviate certain psychiatric symptoms. Furthermore, if drug users are successful in avoiding arrest, they often lack the motivation to stop, which makes treatment difficult. Therapists are required to be non-confrontational, to keep pace with their patients, and to take their patients' other existing disorders into account. Recently, the Matrix Model has shown promise as a new treatment strategy for drug abusers in Japan. The Matrix Model, which was originally developed in response to the 1980s cocaine epidemic in the USA, is an intensive outpatient treatment approach for drug abuse and dependence. The Matrix Model integrates cognitive-behavioral therapy, contingency management, motivational interviewing, 12-step facilitation, family involvement, and so on, with a directive, non-confrontational approach, and this style of therapy seems to fit with law-evading drug users. A Matrix Model-based treatment program was first established in Japan in 2006. The aim of this report is to introduce and assess the benefits of the TAMA Mental Health and Welfare Center Relapse Prevention Program, a Matrix Model-based treatment program established at the Tama Mental Health and Welfare Center in 2007.
Intermolecular interactions are key features in the stabilization or destabilization of complexes. In particular, interactions involving aromatic rings have been extensively studied both theoretically and experimentally. Studies have shown that aromatic-aromatic interactions can be categorized by ring-ring orientation into a variety of different types, such as stacking interactions and T-shaped interactions. Because these different orientations affect stabilization, analyses of such interactions, for example ab initio molecular orbital calculations, are applied to pairs of aromatic rings, both in model systems and real systems. An important series of aromatic-aromatic interactions include those between pairs of aromatic residues in proteins. These residues have been studied computationally using both a theoretical chemistry approach and a knowledge-based analys. Protein 3D structural information is essential for knowledge-based studies of aromatic-aromatic interactions in protein-ligand complexes. Some databases filter entries from the Protein Data Bank (PDB) using criteria that make them suitable for computational approaches involving specific research targets. Lanzarotti et al. have shown that aromatic clusters in which three or more aromatic residues are in close proximity to each other are found in many protein structures, expanding pairwise aromatic-aromatic interactions. Moreover, these clusters are thought to be important in terms of protein function, structural stability and ligand recognition. Here, we show that aromatic clusters, as well as individual proteins, are found in a variety of protein-ligand complexes. As such, we anticipate that these clusters might have a significant role in ligand binding and could help in efficient ligand design.
De novo molecular design aims to propose molecules exhibiting desired properties and/or activities, as constructed from scratch. Although this approach opposes the widely used virtual screening (VS), the same criteria should be applied, such as ones based on substructure filters, and quantitative structure-property relationship (QSPR) and quantitative structure-activity relationship (QSAR) regression models. QSPR/QSAR, which enables us to predict properties/activities by making use of experimental data, are widely used in academia as well as in industry. Herewith, we present a novel chemical structure generation system by combining fragments whose final chemical structures satisfy the aforementioned criteria. Using inverse analysis, QSPR/QSAR models determine a specific region in chemical space corresponding to a set of desired values by a designer. Chemical structures are generated by combining ring systems, as well as atom fragments, in every possible way until violating the upper bounds of that region. We also show the results of inverse-QSAR analysis for the human Alpha-2A adrenergic receptor. This suggests that our system has features preferable to VS-like methods in terms of the number of generated structures.
Computational drug design plays an important role in the discovery of new drugs. Recently, we proposed an algorithm for designing new drug-like molecules utilizing the structure of a known active molecule. To design molecules, three types of fragments (ring, linker, and side-chain fragments) were defined as building blocks, and a fragment library was prepared from molecules listed in G protein-coupled receptor (GPCR)-SARfari database. An evolutionary algorithm which executes evolutionary operations, such as crossover, mutation, and selection, was implemented to evolve the molecules. As a case study, some GPCRs were selected for computational experiments in which we tried to design ligands from simple seed fragments using the Tanimoto coefficient as a fitness function. The results showed that the algorithm could be used successfully to design new molecules with structural similarity, scaffold variety, and chemical validity. In addition, a docking study revealed that these designed molecules also exhibited shape complementarity with the binding site of the target protein. Therefore, this is expected to become a powerful tool for designing new drug-like molecules in drug discovery projects.
Molecular simulations have been widely used in biomolecular systems such as proteins, DNA, etc. The search for stable conformations of proteins by molecular simulations is important to understand the function and stability of proteins. However, finding the stable state by conformational search is difficult, because the energy landscape of the system is characterized by many local minima separated by high energy barriers. In order to overcome this difficulty, various sampling and optimization methods for the conformation of proteins have been proposed. In this study, we propose a new conformational search method for proteins based on a genetic algorithm. We applied this method to an α-helical protein. We found that the conformations obtained from our simulations are in good agreement with the experimental results.
Recently, ab initio quantum mechanical calculations have been applied to large molecules, including biomolecular systems. The fragment molecular orbital (FMO) method is one of the most efficient approaches for the quantum mechanical investigation of such molecules. In the FMO method, dividing a target molecule into small fragments reduces computational effort. The clear definition of inter-fragment interaction energy (IFIE) as an expression of total energy is another valuable feature of the FMO method because it provides the ability to analyze interactions in biomolecules. Thus, the FMO method is expected to be useful for drug discovery. This study demonstrates applications of the FMO method related to drug discovery. First, IFIE, according to FMO calculations, was used in the optimization of drug candidates for the development of anti-prion compounds. The second example involved interaction analysis of the human immunodeficiency virus type 1 (HIV-1) protease and a drug compound that used a novel analytical method for dispersion interaction, i.e., fragment interaction analysis based on LMP2 (FILM).
“Pharmacist-scientists” are needed in the clinical setting. However, research competency, including logical thinking, differs among pharmacists. This difference stems from the varying experience of research during university and graduate school. Thus, to ascertain the research experience within different educational systems, we evaluated pharmacists in Kobe City Medical Center General Hospital. In most instances, there was a direct correlation between the duration of research (in the laboratory at university or graduate school), and research experience gained, such as independent thinking and presentations at seminars or academic conferences. Respondents who graduated from the recently introduced 6-year pharmaceutical science course had less research experience than those who graduated with a combination 4-year degree and subsequent master's course. Conversely, the number of presentations at academic conferences and the number of published papers postgraduation were independent of research experience during university and graduate school. These results indicate that there is a considerable difference in the research experience during university and graduate school among pharmacists, and this is likely to impact their pharmaceutical skills.
Despite being damaged by the 2011 Tohoku earthquake, tsunami, and nuclear disaster, Soma General Hospital, located approximately 40 km north of Fukushima Daiichi nuclear power plant, was able to fulfill its role as a key regional hospital in northeast Fukushima. To elucidate the pharmaceutical service in response to the disaster, we investigated the hospital's operations in 2011 according to the medical records and prescriptions. One of the difficulties that the department of pharmaceutical service faced at that time was the increase in emergency healthcare requests by evacuated patients from other hospitals and clinics. Herein, we propose the following countermeasures to be considered in future disaster preparations: (1) establishing a medical and pharmaceutical service coordinator for disaster relief; (2) sharing all local patients' medical information in emergencies (at least contraindicated drugs or allergy history); and (3) reviewing disaster stockpiles, especially pharmaceuticals (both at the hospital and in nearby locations).