No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Photoregulation of biological activity by photocromic reagents. II. Inhibitors of acetylcholinesterase
Abstract
The enzymic activity of acetylcholinesterase can be photoregulated through the mediation of photochromic inhibitors of the enzyme. N-p-phenylazophenyl-N-phenylcarbamyl fluoride, an irreversible inhibitor of acetylcholinesterase, exists as two geometric isomers which are interconvertible through the action of light. The cis isomer, which predominates after exposure to light of 320 nm, is more active than the trans isomer, which results from exposure to light of 420 nm. It was possible, therefore, to use light energy to regulate the inactivation of the enzyme. Similarly, levels of acetylcholinesterase activity could be photo-regulated in a completely reversible manner by means of the photochromic reversible inhibitor p-phenylazophenyltrimethylammonium chloride. These experiments can serve as models for similar phenomena observed in nature, particularly in photoperiodic rhythms of higher animals.
... Decades later in the 1970s (Bieth et al. 1969(Bieth et al. , 1970Deal et al. 1969), a new development of light as a therapeutic agent was pioneered, which set out to solve a conventional problem with classical medical drugs as outlined in the following. The effect of therapeutic compounds is based on their interaction with protein-based targets such as receptors, ion channels or enzymes (Ritter et al. 2020). ...
... The target proteins must further be specific for the cells that are the cause of the disease, e.g., overproduced proteins in tumor cells or proteins that solely exist in bacteria. While examining the efficacy of the light-responsive drug it became apparent that the difference in activity prior to and after irradiation is more powerful in vivo than in vitro (Bieth et al. 1969;Wainberg and Erlanger 1971). I will use the term light regulation factor (LRF) to describe this difference in activity from now on. ...
Light is essential for various biochemical processes in all domains of life. In its presence certain proteins inside a cell are excited, which either stimulates or inhibits subsequent cellular processes. The artificial photocontrol of specifically proteins is of growing interest for the investigation of scientific questions on the organismal, cellular and molecular level as well as for the development of medicinal drugs or biocatalytic tools. For the targeted design of photocontrol in proteins, three major methods have been developed over the last decades, which employ either chemical engineering of small-molecule photosensitive effectors (photopharmacology), incorporation of photoactive non-canonical amino acids by genetic code expansion (photoxenoprotein engineering), or fusion with photoreactive biological modules (hybrid protein optogenetics). This review compares the different methods as well as their strategies and current applications for the light-regulation of proteins and provides background information useful for the implementation of each technique.
... One central motivation for this work was found in the recognition that light can be precisely controlled in space and time and offers non-invasive 'remote' control in transparent matrices. Inspired by classic work dating back as far as to the 1960s Bieth et al. 1969;Deal et al. 1969), photochromes were recently re-introduced in the ion channel field to contribute these experimental advantages to our current research. Researchers began to exploit photochromes with the help of molecular, chemical and genetic engineering in the fields of photopharmacology and optochemical genetics. ...
... The first PCLs were designed and synthesized in the 1960s and 1970s in the form of AB-substituted acetylcholines and carbachols. These molecules were mono-or bi-functional with a quaternary ammonium (QA) and were applied to control nicotinic acetylcholine receptors (nAChRs) and acetylcholine esterase to study kinetics of ion channel activation and membrane potential shifts in excitable tissue Bieth et al. 1969;Deal et al. 1969;Chabala et al. 1985;Nargeot et al. 1982). Despite these early successful examples for the optical control of ion channel function, further design and application of PCLs was not revisited until nearly four decades later. ...
In this chapter we discuss the strengths, caveats and technical considerations of three approaches for reprogramming the chemical composition of selected amino acids within a membrane protein. In vivo nonsense suppression in the Xenopus laevis oocyte, evolved orthogonal tRNA and aminoacyl-tRNA synthetase pairs and protein ligation for biochemical production of semisynthetic proteins have been used successfully for ion channel and receptor studies. The level of difficulty for the application of each approach ranges from trivial to technically demanding, yet all have untapped potential in their application to membrane proteins.
... One central motivation for this work was found in the recognition that light can be precisely controlled in space and time and offers non-invasive 'remote' control in transparent matrices. Inspired by classic work dating back as far as to the 1960s Bieth et al. 1969;Deal et al. 1969), photochromes were recently re-introduced in the ion channel field to contribute these experimental advantages to our current research. Researchers began to exploit photochromes with the help of molecular, chemical and genetic engineering in the fields of photopharmacology and optochemical genetics. ...
... The first PCLs were designed and synthesized in the 1960s and 1970s in the form of AB-substituted acetylcholines and carbachols. These molecules were mono-or bi-functional with a quaternary ammonium (QA) and were applied to control nicotinic acetylcholine receptors (nAChRs) and acetylcholine esterase to study kinetics of ion channel activation and membrane potential shifts in excitable tissue Bieth et al. 1969;Deal et al. 1969;Chabala et al. 1985;Nargeot et al. 1982). Despite these early successful examples for the optical control of ion channel function, further design and application of PCLs was not revisited until nearly four decades later. ...
Ion channels are membrane-spanning proteins that control the flow of ions across biological membranes through an aqueous pathway. The opening or closing of this pore can be controlled by a myriad of physiological inputs (voltage, ligands, temperature, metabolites, pH), which in turn allow for the controlled flux of ions across membranes, resulting in the generation of minute electrical signals. The functional implications of ion channel function on physiological processes are vast. Electrical impulses, in the form of action potentials or diverse chemo-electrical signals, coordinate the syncytium of the heart beat, support a myriad of neuronal communication pathways, insulin secretion, and are central to the immune response, with more roles being discovered virtually everyday. Thus, ion channel function is a biophysical process that is central to biological life at many levels. And with over 500 channel-forming subunits known today in humans, this large class of proteins is also increasingly recognised as important drug targets, as inherited or acquired ion channel dysfunction are known causes of disease.
... 18 The technology also provides new opportunities to study biological processes 18,19 and to develop novel therapeutic strategies. 20 Photoregulation of acetylcholinesterase 21,22 activity was first investigated in the 1960s, and then, photoswitches for ion channels, 23−28 transporters, pumps, 29,30 G protein-coupled receptors, 31−34 enzymes, 35−39 cytoskeletons, 40,41 and other applications 42−44 in biological systems were subsequently reported. Photochromic ligands (PCLs) exhibit different affinities and/or efficacies toward their biological targets and have varied pharmacodynamics in two (or more) different isomeric forms upon irradiation with light. ...
... Photopharmacology originated as an effort to provide more reliable tools to optogenetics and in the last few years has grown noticeably due to its applicability in living systems and its role in complementing the conventional optogenetic techniques. The first breakthrough in this area dated as early as the 1960s, when Erlanger and Nachmansohn investigated azobenzene-based inhibitors of acetylcholinesterase (33,34). However, it was only back in 2012 that Trauner and Kramer matured the idea of developing drugs containing synthetic light-switching molecules. ...
Pain afflicts billions of people worldwide, who suffer especially from long-term chronic pain. This gruelling condition affects the nervous system at all levels: from the brain to the spinal cord, the Dorsal Root Ganglia (DRG) and the peripheral fibres innervating the skin. The nature of the different molecular and cellular components of the somatosensory modalities, as well as the complexity of the peripheral and central circuitry are yet poorly understood. Light-based techniques such as optogenetics, in concert with the recent advances in single-cell genetic profiling, can help to elucidate the role of diverse neuronal sub-populations in the encoding of different sensory and painful stimuli by switching these neurons on and off via optically active proteins, namely opsins. Recently, photopharmacology has emerged from the efforts made to advance optogenetics. The introduction of azo-benzene-based light-sensitive molecular switches has been applied to a wide variety of molecular targets, from ion channels and receptors to transporters, enzymes and many more, some of which are paramount for pain research and therapy.
In this Review, we summarise the recent advances in the fields of optogenetics and photopharmacology and we discuss the use of light-based techniques for the study of acute and chronic pain physiology, as well as their potential for future therapeutic use to improve pain treatment.
... Beyondt heir relevance as pharmacological tools in basic research,t he use of photoswitchable enzyme modulators as tunable (pro-)drugsi nt herapeutic applications is the vision of photopharmacology.P otential advantages of photopharmacology,r elative to classical pharmacotherapy,i nclude highly focused treatment, decreased side effects, preventing environmental contamination, and lowering emergence of resistance. [5,6] Photoswitchable enzyme modulators reported so far are often azo-functionalized compounds and comprise, among others, inhibitors of acetylcholinesterase, [7][8][9][10] antibacterial agents, [11,12] microtubule targeting agents, [13,14] modulators of GABA A receptors, [15,16] and agonists for m-opioid receptors. [17] Regarding the field of protein kinases,o nly af ew photosensitive small-molecule compounds have been reported. ...
Axitinib is an approved drug targeting tyrosine kinases including the vascular endothelial growth factor receptor 2 (VEGFR2) and is licensed for second‐line therapy of renal cell carcinoma. Interestingly, axitinib contains a stilbene‐like double bond allowing for E/Z isomerization. In this study, we investigated the photoinduced E/Z isomerization of axitinib to explore if its inhibitory effect can be turned "on" and "off", triggered by light. Compared to the E‐isomer, the absorption of the Z‐isomer is red‐shifted indicating reversible photoswitching properties. When both isolated isomers were measured in a commercial available profiling under ambient light, no differences of biological activities could be determined. However, under controlled light conditions we could demonstrate that (Z) axitinib is 43 times less active compared to the (E) isomer in an VEGFR2 assays. Furthermore, we proved that kinase activity in HUVEC assays is reduced by (E)‐axitinib, but only weakly affected by (Z) axitinib. By irradiating (Z) axitinib in vitro with UV light (385 nm) it is possible to switch it almost quantitatively to the (E)‐isomer and to completely restore the biological activity of (E) axitinib. However, vice versa switching the biological activity "off" from (Z)‐ to (E)‐axitinib was not possible in aqueous solution due to a competing irreversible [2+2]‐photocycloaddition yielding a biologically inactive axitinib‐dimer.
... [125] AChE inhibition has also been associated with myasthenia gravis and glaucoma. [125] Erlanger and co-workers pioneered the concept of photopharmacology around 1969, [126][127][128][129] after reporting the control of AChE activity with light by using azobenzene-based inhibitors. Inspired by the known AChE inhibitor phenyltrimethylammonium, [130] compounds 13 and 14 were developed. ...
Das Feld der Photopharmakologie nutzt durch Licht schaltbare Moleküle, um Kontrolle über die Wirkung biologisch aktiver Verbindungen zu erlangen. Das Ziel ist es, die systemische Toxizität von Wirkstoffen wie auch die Herausbildung von Resistenzen zu vermindern und gleichzeitig eine extrem hohe Präzision in der Behandlung zu erreichen. Durch die Verwendung niedermolekularer Verbindungen stellt die Photopharmakologie eine valide Alternative zur Optogenetik dar. Wir präsentieren hier einen Überblick über die pharmakologischen Zielstrukturen in verschiedenen Organen sowie über Organsysteme im menschlichen Körper, die auf nicht-invasive Weise angesteuert werden können. Wir diskutieren die Perspektiven einer selektiven Lichtzuführung zu diesen Organen sowie die spezifischen Anforderungen lichtaktivierbarer Wirkstoffe. Über anwendungsorientierte Aspekte hinaus sind wir bestrebt, die Wirkstofftauglichkeit (“druggability”) pharmazeutischer Zielstrukturen anhand aktueller Ergebnisse zu illustrieren. Ferner wollen wir aufzeigen, an welchen Stellen neue Ansätze erforscht werden müssen, damit die Umsetzung von “intelligentem” Moleküldesign in der Photopharmakologie zur klinischen Anwendung gelingt.
... [125] AChE inhibition has also been associated with myasthenia gravis and glaucoma. [125] Erlanger and co-workers pioneered the concept of photopharmacology around 1969, [126][127][128][129] after reporting the control of AChE activity with light by using azobenzene-based inhibitors. Inspired by the known AChE inhibitor phenyltrimethylammonium, [130] compounds 13 and 14 were developed. ...
The field of photopharmacology uses molecular photoswitches to establish control over the action of bioactive molecules. It aims to reduce systemic drug toxicity and the emergence of resistance, while achieving unprecedented precision in treatment. By using small molecules, photopharmacology provides a viable alternative to optogenetics. We present here a critical overview of the different pharmacological targets in various organs and a survey of organ systems in the human body that can be addressed in a non-invasive manner. We discuss the prospects for the selective delivery of light to these organs and the specific requirements for light-activatable drugs. We also aim to illustrate the druggability of medicinal targets with recent findings and emphasize where conceptually new approaches have to be explored to provide photopharmacology with future opportunities to bring "smart" molecular design ultimately to the realm of clinical use.
... This strategy has been used to prepare photoswitchable inhibitors of e.g. acetylcholinesterase 18,19 and proteases 20 . Previously we reported the design, synthesis and biological evaluation of a small library of 3-substituted pyrazolopyrimidines that inhibit the RET kinase in low nanomolar concentrations (down to 8 nM) in vitro 21 . ...
REarranged during Transfection (RET) is a transmembrane receptor tyrosine kinase required for normal development and maintenance of neurons of the central and peripheral nervous systems. Deregulation of RET and hyperactivity of the RET kinase is intimately connected to several types of human cancers, most notably thyroid cancers, making it an attractive therapeutic target for small-molecule kinase inhibitors. Novel approaches, allowing external control of the activity of RET, would be key additions to the signal transduction toolbox. In this work, photoswitchable RET kinase inhibitors based on azo-functionalized pyrazolopyrimidines were developed, enabling photonic control of RET activity. The most promising compound displays excellent switching properties and stability with good inhibitory effect towards RET in cell-free as well as live-cell assays and a significant difference in inhibitory activity between its two photoisomeric forms. As the first reported photoswitchable small-molecule kinase inhibi
Photoisomerization of azobenzenes from their stable E isomer to the metastable Z state is the basis of numerous applications of these molecules. However, this reaction typically requires ultraviolet light, which limits applicability. In this study, we introduce disequilibration by sensitization under confinement (DESC), a supramolecular approach to induce the E -to- Z isomerization by using light of a desired color, including red. DESC relies on a combination of a macrocyclic host and a photosensitizer, which act together to selectively bind and sensitize E -azobenzenes for isomerization. The Z isomer lacks strong affinity for and is expelled from the host, which can then convert additional E- azobenzenes to the Z state. In this way, the host–photosensitizer complex converts photon energy into chemical energy in the form of out-of-equilibrium photostationary states, including ones that cannot be accessed through direct photoexcitation.
The process of vision begins with the absorption of light by retinal, which triggers isomerization around a double bond and, consequently, a large conformational change in the surrounding protein opsin. However, certain organisms evolved different visual systems; for example, deep-sea fishes employ chlorophyll-like antennas capable of capturing red light and sensitizing the nearby retinal molecule via an energy-transfer process. Similar to retinal, most synthetic photochromic molecules, such as azobenzenes and spiropyrans, switch by double-bond isomerization. However, this reaction typically requires shortwavelength (ultraviolet) light, which severely limits the applicability of these molecules. Here, we introduce DisEquilibration by Sensitization under Confinement (DESC) – a supramolecular approach to switch various azoarenes from the E isomer to the metastable Z isomer using visible light of desired color, including red. DESC relies on a combination of a coordination cage and a photosensitizer (PS), which act together to bind and selectively sensitize E-azoarenes. After switching to the Z isomer, the azoarene loses its affinity to—and is expelled from—the cage, which can convert additional copies of E into Z. In this way, the cage⋅PS complex acts as a light-driven supramolecular machine, converting photon energy into chemical energy in the form of out-of-equilibrium photostationary states, including ones that cannot be accessed via direct photoexcitation.
Photoswitchable ligands as biological tools provide an opportunity to explore the kinetics and dynamics of the clinically relevant μ‐opioid receptor. These ligands can potentially activate or deactivate the receptor when desired by using light. Spatial and temporal control of biological activity allows for application in a diverse range of biological investigations. Photoswitchable ligands have been developed in this work, modelled on the known agonist fentanyl, with the aim of expanding the current “toolbox” of fentanyl photoswitchable ligands. In doing so, ligands have been developed that change geometry (isomerize) upon exposure to light, with varying photophysical and biochemical properties. This variation in properties could be valuable in further studying the functional significance of the μ‐opioid receptor.
Photoswitchable cytotoxins (PSCs) have high value as spatiotemporally precise research reagents to modulate critical biological processes. Compared to other photopharmaceuticals, they face intriguing challenges in design and validation, due to the ubiquitous importance of their targets: but their rewards may be correspondingly greater. The utility of PSCs has been confirmed by research applications across a range of biological settings, involving targets from cytoskeleton to signaling, and structural to genomic integrity. The stage is set for further development and broader applications of PSCs throughout the physical and life sciences, particularly harnessing the unique assay precision that they allow.
Diarylethene (DAE) photoswitch is a new and promising family of photochromic molecules and has shown superior performance as a smart trigger in stimulus-responsive materials. During the past few decades, the DAE family has achieved a leap from simple molecules to functional molecules and developed toward validity as a universal switching building block. In recent years, the introduction of DAE into an assembly system is an attractive strategy that enables the photochromic behavior of the building blocks to be manifested at the level of the entire system, beyond the DAE unit itself. This assembly-based strategy will bring many unexpected results that promoting the design and manufacture of a new generation of advanced materials. This review summarizes recent advances in the design and fabrication of diarylethene as a trigger in materials science, chemistry, and biomedicine.
This article is protected by copyright. All rights reserved
Azobenzenes are archetypal molecules that have a central role in fundamental and applied research. Over the course of almost two centuries, the area of azobenzenes has witnessed great achievements; azobenzenes have evolved from simple dyes to ‘little engines’ and have become ubiquitous in many aspects of our lives, ranging from textiles, cosmetics, food and medicine to energy and photonics. Despite their long history, azobenzenes continue to arouse academic interest, while being intensively produced for industrial purposes, owing to their rich chemistry, versatile and straightforward design, robust photoswitching process and biodegradability. The development of azobenzenes has stimulated the production of new coloured and light-responsive materials with various applications, and their use continues to expand towards new high-tech applications. In this Review, we highlight the latest achievements in the synthesis of red-light-responsive azobenzenes and the emerging application areas of photopharmacology, photoswitchable adhesives and biodegradable materials for drug delivery. We show how the synthetic versatility and adaptive properties of azobenzenes continue to inspire new research directions, with limits imposed only by one’s imagination.
The process of two-photon-induced isomerization occurring in various organic molecules, among which azobenzene derivatives hold a prominent position, offers a wide range of functionalities, which can be used in both material and life sciences. This review provides a comprehensive description of nonlinear optical (NLO) properties of azobenzene (AB) derivatives whose geometries can be switched through two-photon absorption (TPA). Employing the nonlinear excitation process allows for deeper penetration of light into the tissues and provides opportunities to regulate biological systems in a non-invasive manner. At the same time, the tight focus of the beam needed to induce nonlinear absorption helps to improve the spatial resolution of the photoinduced structures. Since near-infrared (NIR) wavelengths are employed, the lower photon energies compared to usual one-photon excitation (typically, the azobenzene geometry change from trans to cis form requires the use of UV photons) cause less damage to the biological samples. Herein, we present an overview of the strategies for optimizing azobenzene-based photoswitches for efficient two-photon excitation (TPE) and the potential applications of two-photon-induced isomerization of azobenzenes in biological systems: control of ion flow in ion channels or control of drug release, as well as in materials science, to fabricate data storage media, optical filters, diffraction elements etc., based on phenomena like photoinduced anisotropy, mass transport and phase transition. The extant challenges in the field of two-photon switchable azomolecules are discussed.
Analogs of the known inhibitor (peptide pDI) of the p53/MDM2 protein–protein interaction are reported, which are stapled by linkers bearing a photoisomerizable diarylethene moiety. The corresponding photoisomers possess significantly different affinities to the p53-interacting domain of the human MDM2. Apparent dissociation constants are in the picomolar-to-low nanomolar range for those isomers with diarylethene in the “open” configuration, but up to eight times larger for the corresponding “closed” isomers. Spectroscopic, structural, and computational studies showed that the stapling linkers of the peptides contribute to their binding. Calorimetry revealed that the binding of the “closed” isomers is mostly enthalpy-driven, whereas the “open” photoforms bind to the protein stronger due to their increased binding entropy. The results suggest that conformational dynamics of the protein-peptide complexes may explain the differences in the thermodynamic profiles of the binding.
Synthetic photoswitches have been known for many years, but their usefulness in biology, pharmacology, and medicine has only recently been systematically explored. Over the past decade photopharmacology has grown into a vibrant field. As the photophysical, pharmacodynamic, and pharmacokinetic properties of photoswitches, such as azobenzenes, have become established, they have been applied to a wide range of biological targets. These include transmembrane proteins (ion channels, transporters, G protein-coupled receptors, receptor-linked enzymes), soluble proteins (kinases, proteases, factors involved in epigenetic regulation), lipid membranes, and nucleic acids. In this review, we provide an overview of photopharmacology using synthetic switches that have been applied in vivo, i.e., in living cells and organisms. We discuss the scope and limitations of this approach to study biological function and the challenges it faces in translational medicine. The relationships between synthetic photoswitches, natural chromophores used in optogenetics, and caged ligands are addressed.
Diarylethene derivatives, whose biological activity can be reversibly changed by irradiation with light of different wavelengths, have shown promise as scientific tools and as candidates for photocontrollable drugs. However, examples demonstrating efficient photocontrol of their biological activity are still relatively rare. This concept article discusses the possible reasons for this situation and presents a critical analysis of existing data and hypotheses in this field, in order to extract the design principles enabling the construction of efficient photocontrollable diarylethene‐based molecules. Papers addressing biologically relevant interactions between diarylethenes and biomolecules are analyzed; however, in most published cases, the efficiency of photocontrol in living systems remains to be demonstrated. We hope that this article will encourage further discussion of design principles, primarily among pharmacologists and synthetic and medicinal chemists.
The control of ligand-gated receptors with light using photochromic compounds has evolved from the first handcrafted examples to accurate, engineered receptors, whose development is supported by rational design, high-resolution protein structures, comparative pharmacology and molecular biology manipulations. Photoswitchable regulators have been designed and characterized for a large number of ligand-gated receptors in the mammalian nervous system, including nicotinic acetylcholine, glutamate and GABA receptors. They provide a well-equipped toolbox to investigate synaptic and neuronal circuits in all-optical experiments. This focused review discusses the design and properties of these photoswitches, their applications and shortcomings and future perspectives in the field.
Linked articles:
This article is part of a themed section on Nicotinic Acetylcholine Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.11/issuetoc.
The unique photochemistry of diarylethene dyes can be tuned to design photoactivatable drugs. However, their biological mechanism of action has not yet been determined but tentatively ascribed to photoinduced effects on DNA. Herein, we performed theoretical simulations to provide comprehensive insights into the cell death that occurs after the irradiation of diarylethenes-dosed tissues. It is shown that diarylethenes intercalate between DNA base pairs only if the central ring is closed, while two possible pathways might be envisioned for such DNA-photochrome adducts: (i) the use of visible light to open the core ring and release the diarylethene without damaging DNA; or (ii) to produce a degradation of the DNA upon 300–400 nm light radiation. The latter photodamage is characterized by a large charge transfer in the DNA diarylethene direction. The described computational protocol might be used to open up new synthetic routes for diarylethenes with clinical applications, e.g., by screening electroacceptor substituent groups to optimize the rates of radiation-induced damage to DNA.
Membrane receptors and ion channels respond to various stimuli and relay that information across the plasma membrane by triggering specific and timed processes. These include activation of second messengers, allowing ion permeation, and changing cellular excitability, to name a few. Gaining control over equivalent processes is essential to understand neuronal physiology and pathophysiology. Recently, new optical techniques have emerged proffering new remote means to control various functions of defined neuronal populations by light, dubbed optogenetics. Still, optogenetic tools do not typically address the activity of receptors and channels native to neurons (or of neuronal origin), nor gain access to their signaling mechanisms. A related method-synthetic optogenetics-bridges this gap by endowing light sensitivity to endogenous neuronal receptors and channels by the appending of synthetic, light-receptive molecules, or photoswitches. This provides the means to photoregulate neuronal receptors and channels and tap into their native signaling mechanisms in select regions of the neurons, such as the synapse. This review discusses the development of synthetic optogenetics as a means to study neuronal receptors and channels remotely, in their natural environment, with unprecedented spatial and temporal precision, and provides an overview of tool design, mode of action, potential clinical applications and insights and achievements gained.
Many small molecules, natural ones and synthetic ones, act on biological membranes by controlling ionic channels in the membrane. Our research has focused on the channel associated with the acetylcholine receptor in the postsynaptic membrane of the nicotinic synapse. For several decades this junction has provided advances in our understanding of synaptic transmission.
The flow of energy from the Sun out into the universe, absorbed by day and radiated out at night, has brought about on Earth the molecular ordering from which life originated [1]. The same flow has since supplied the necessary energy for the continuation of life. The way in which living organisms utilize solar energy can be roughly divided in two classes. It is either trapped and subsequently stored in chemical bonds, to be used as source of energy, or it triggers a physiological response for information purposes. It is this second class of phenomena that provided the inspiration for the investigations reported in this article. To place this research in its proper context we will briefly discuss three systems of the type we are interested in that have been reported in the literature. These are the process of vision, the photoregulation of enzyme inhibition and the photoregulation of the viscosity of a polymer solution. Of these three the first one is purely biological, the last one completely artificial, while the second system is more or less in between.
This chapter discusses the photocontrol of biologically relevant systems using synthetically derived photochromic molecules. Then, it presents a general overview of recent progress made in the field of light-controlled biomolecules in a variety of contexts. Today, there is a wide range of tools capable of modifying various biochemical functions. Those relevant to artificial photochromic biosystems are discussed in the chapter. The design of artificial light-controlled biological systems using photochromic molecules relies on harnessing the light-induced structural changes that accompany the chromophore's photoisomerization process and exploiting these changes to influence biomolecule function. There are a number of factors to consider when designing a light-controlled biomolecule that has an incorporated photochromic tether. The chapter focuses on photocontrol of proteins and peptides, the drawbacks that accompany the use of ultraviolet light to control channel function in living systems have prompted researchers to design photoswitches that can be isomerized using longer wavelengths of light.
Physiological processes that are influenced by light abound in nature. Organisms as complex as the human being and as “simple” as bacteria show evidence of having developed photoresponsive systems. Examples are the phototactic behavior of bacteria, plants and animal cells, diurnal variations in social and sexual behavior of animals (including man), and season-related migration patterns in birds. The process of vision is a highly sophisticated photoresponsive process as are the processes in plants that are controlled by the phytochrome systems. (Two reviews that can prove useful are Er langer, 1976 and Briggs and Rice, 1972).
All photographic processes are based on the ability of a material to change its properties, especially transparency and color, under the action of light. These changes which arise from photochemical conversions of light sensitive compounds, result in the formation of a photographic image. There are very many light sensitive compounds; but only a few have found application in photography. Examples are 1) silver salts that are reduced to silver metal by light, 2) complex salts of iron which are converted to divalent iron by light and in turn can reduce salts of other metals, 3) bichromates of alkaline metals that can tan a gelatin carrier under the action of light, 4) diazo compounds that undergo degradation under the action of light; 5) dyes that change their color under the action of light, and 6) monomers or low molecular weight soluble polymers that under the action of light are changed into insoluble high molecular weight compounds (1–3). As things are today, light signals can be recorded and converted into visible images in any region of the spectrum.
Although photochromic azobenzene molecules with the ability to modulate biological activity are not found in nature, they can be used to probe naturally occurring systems and to enable us to learn something about the mechanisms by which these systems function. Studies on their ability to mimic or to interfere with biochemical activity can provide information that can compliment data obtained by other methods, including physical (X-ray crystallography, NMR, circular dichroism), chemical, physiological or immunological procedures. Although no single methodology provides a complete, integrated picture of a biochemical process, each can contribute data that serve to clarify the overall system. Even such a powerful technique as X-ray crystallography shows the structure and configuration as it is in a crystal only; this structure can be different from the configuration that directly participate in the biological process.
This chapter discusses the role of acetylcholine (AcCh) in excitable membranes and nerve activities. AcCh acts as a signal within excitable membranes; it triggers a series of molecular changes that result in increased permeability. AcCh is intrinsically associated with excitability; its action is an integral part of the processes generating and propagating electrical currents. The role of AcCh is fundamentally similar in the membranes of nerve and muscle fibers and in the pre- and postsynaptic membranes of junctions. AcCh is present in the membrane in bound form, probably associated with a protein or a lipoprotein. It is released by excitation and acts as a signal recognized by a receptor protein located at a distance of a few angstroms within the membrane. The reaction induces a conformational change of the protein. The change may possibly release by allosteric action calcium (Ca2+) ions bound to carboxyl groups of the proteins. Calcium ions may induce further conformational changes of phospholipids and other polyelectrolytes. The end result of the sequence of chemical reactions is the change of permeability to ions permitting the movements across the membrane of many thousands of ions per molecule of AcCh released. Among the components of a cell, only proteins have the ability to recognize a specific ligand such as AcCh and thus respond to a signal. The series of reactions described in the chapter act as typical amplifiers of the signal released by excitation.
The discovery, in the latter part of the 18th century, that the powerful shock of certain fish is an electric discharge immediately raised the question as to the mechanism by which living cells produce electricity. The importance of this problem for biology in general became apparent when it was firmly established during the 19th century that nerve impulses are propagated by electric currents. Thus, the understanding of one of the most vital functions of the organism became linked to the knowledge of the mechanism of bioelectricity. At the turn of this century, two notions were widely accepted. First, in a fluid system, such as the living cell, ions must be the carriers of electric currents. Since it was known that Na+ ions are highly concentrated in the outer environment of cells whereas K+ ion concentrations are high in the interior, Overton (1902) proposed, on the basis of simple experiments, that during activity Na+ ions move into the cell interior and an equivalent number of K+ ions flow to the outside. Overton’s assumption was borne out when the availability of radioactive ions after World War II made it possible to measure the ion movements during rest and during activity. The second notion was concerned with the control of these ion movements; it was postulated that the cell membranes surrounding nerve and muscle fibers must be able to change their permeability to ions during activity.
Photoresponses have been described for bacteria, fungi, protozoa, algae, plants, invertebrates, and higher animals. These responses include phototaxis, phototropism, photomorphogenesis, photoperiodism, vision, and photocontrol of biological rhythms. The molecular details for the translation of a light stimulus to the observed biological response remain largely unknown for most responses to light. One possibility for the primary process in the stimulation of biological responses is the enhancement by light of an enzyme reaction. This can involve direct absorption of photons by an immediate component of the enzyme system (e.g., substrate), or indirect effects of light on enzymes such as the enhancement of protein synthesis, and enzyme activation which requires an additional protein acting as a light activation factor. The light activation of enzymes represents a topic that is developing rapidly, with particular emphasis in the areas of vision, photochromic enzyme inhibitors, photoreactivation, and enzyme activation in photosynthetic organisms. A few years ago there were not many reports on the activation of enzymes by light, whereas the photomactivation of enzymes has received widespread attention for years. Light activates specific enzymes, whereas inactivation by exposure to far-UV* radiation or by photodynamic action is not enzyme specific. Some inactivations by visible or near-UV light are selective, but are usually not reversible.
Nature has incorporated small photochromic molecules, colloquially termed 'photoswitches', in photoreceptor proteins to sense optical cues in phototaxis and vision. While Nature's ability to employ light-responsive functionalities has long been recognized, it was not until recently that scientists designed, synthesized and applied synthetic photochromes to manipulate many of which open rapidly and locally in their native cell types, biological processes with the temporal and spatial resolution of light. Ion channels in particular have come to the forefront of proteins that can be put under the designer control of synthetic photochromes. Photochromic ion channel controllers are comprised of three classes, photochromic soluble ligands (PCLs), photochromic tethered ligands (PTLs) and photochromic crosslinkers (PXs), and in each class ion channel functionality is controlled through reversible changes in photochrome structure. By acting as light-dependent ion channel agonists, antagonist or modulators, photochromic controllers effectively converted a wide range of ion channels, including voltage-gated ion channels, 'leak channels', tri-, tetra- and pentameric ligand-gated ion channels, and temperature-sensitive ion channels, into man-made photoreceptors. Control by photochromes can be reversible, unlike in the case of 'caged' compounds, and non-invasive with high spatial precision, unlike pharmacology and electrical manipulation. Here, we introduce design principles of emerging photochromic molecules that act on ion channels and discuss the impact that these molecules are beginning to have on ion channel biophysics and neuronal physiology.
Here the synthesis and characterization of a new class of spiropyran-based protease inhibitor is reported that can be reversibly photoswitched between an active spiropyran (SP) isomer and a less active merocyanine (MC) isomer upon irradiation with UV and visible light, respectively, both in solution and on a surface of a microstructured optical fiber (MOF). The most potent inhibitor in the series (SP-3 b) has a C-terminal phenylalanyl-based α-ketoester group and inhibits α-chymotrypsin with a Ki of 115 nM. An analogue containing a C-terminal Weinreb amide (SP-2 d) demonstrated excellent stability and photoswitching in solution and was attached to the surface of a MOF. The SP isomer of Weinreb amide 2 d is a competitive reversible inhibitor in solution and also on fiber, while the corresponding MC isomer was significantly less active in both media. The ability of this new class of spiropyran-based protease inhibitor to modulate enzyme activity on a MOF paves the way for sensing applications.
© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Photochrome Liganden lassen sich dazu verwenden, um diverse biologische Funktionen, insbesondere in neuronalen Systemen, zu steuern. In letzter Zeit wurde die Photokontrolle von Ionenkanälen und G-Protein-gekoppelten Rezeptoren, die in der Synapse zu finden sind, intensiv beforscht. Nun beschreiben wir die Erweiterung unseres photopharmakologischen Ansatzes zur Lichtsteuerung eines Enzyms. Unsere Untersuchungen zu photochromen Inhibitoren eines der wichtigsten Enzyme in der synaptischen Übertragung, der Acetylcholinesterase (AChE), führten zum strukturbasierten Design mehrerer Azobenzol-Analoga des bekannten AChE-Hemmers Tacrin (THA). Eine unserer Verbindungen, AzoTHA, ist ein reversibler photochromer Blocker, der AChE in vitro und ex vivo mit hoher Affinität und schneller Kinetik hemmt. Daher kann AzoTHA über den lichtabhängigen Abbau eines Neurotransmitters dazu verwendet werden, die synaptische Übertragung an der neuromuskulären Endplatte zu steuern.
Photochromic ligands have been used to control a variety of biological functions, especially in neural systems. Recently, much effort has been invested in the photocontrol of ion channels and G-protein coupled receptors found in the synapse. Herein, we describe the expansion of our photopharmacological approach toward the remote control of an enzyme. Building on hallmark studies dating from the late 1960s, we evaluated photochromic inhibitors of one of the most important enzymes in synaptic transmission, acetylcholinesterase (AChE). Using structure-based design, we synthesized several azobenzene analogues of the well-known AChE inhibitor tacrine (THA) and determined their effects on enzymatic activity. One of our compounds, AzoTHA, is a reversible photochromic blocker of AChE in vitro and ex vivo with high affinity and fast kinetics. As such, AzoTHA can be used to control synaptic transmission on the neuromuscular endplate based on the light-dependent clearance of a neurotransmitter.
Pharmacotherapy is often severely hindered by issues related to poor drug selectivity, including side-effects, environmental toxicity and the emergence of resistance. Lack of selectivity is caused by the inability to control drug activity in time and space. Photopharmacology aims at solving these issues by incorporating photoswitchable groups into the molecular structure of bioactive compounds. These switching units allow for the use of light as an external control element for pharmacological activity, which can be delivered with very high spatiotemporal precision. This perspective presents the reader with the current state and outlook on photopharmacology. In particular, the principles behind photoregulation of bioactivity, the challenges of molecular design and the possible therapeutic scenarios are discussed.
Der Sehvorgang sowie andere durch Licht ausgelöste biochemische Umsetzungen in Pflanzen und Lebewesen sind ausgeklügelte biologische Vorgänge, in denen optische Signale registriert und in (physiko)chemische umgesetzt werden. Photoschaltbare Biomakromoleküle sind eine neue Klasse von Substanzen, in denen optische Signale zwei diskrete Ein-und Aus-Zustände biologischer Funktionen erzeugen und die damit den Schaltelementen in Computern gleichen, die bei Änderung des elektrischen Stroms zwischen den Zuständen 0 und 1 wechseln. Die (Photo-)Chemie photochromer Stoffe wurde in den letzten vier Jahrzehnten sehr stark weiterentwickelt. Diese Substanzen isomerisieren bei Lichtabsorption, und die photoisomeren Zustände haben unterschiedliche spektroskopische und chemische Eigenschaften. Der Einbau photoisomerisierbarer (oder photochromer) Einheiten in Biomakromoleküle ermöglicht es, deren sekundäre Funktionen wie Biokatalyse, Bindung und Elektronentransfer nach Wunsch ein- und ausschaltbar zu machen. Dies gelingt durch chemische Modifizierung des Biomakromoleküls durch photoisomerisierbare Einheiten oder durch Einbettung des Biomakromoleküls in photoisomerisierbare Mikroumgebungen wie Monoschichten oder Polymere. Die Photoschaltbarkeit ist im ersten Fall der lichtinduzierten Bildung und Störung des aktiven Zentrums über die photoisomeren Zustände zuzuschreiben und im zweiten durch Licht beeinflußbaren physikalischen oder chemischen Merkmalen der photoisomerisierbaren Systeme aus Polymeren, Monoschichten oder Membranen. Die Aktivierung katalytisch wirkender Biomakromoleküle durch Licht eröffnet eine Möglichkeit, das aufgenommene optische Signal durch biochemische Umwandlungen zu verstärken, und photostimulierte biochemische Redoxschalter ermöglichen die elektrochemische Weiterleitung und Verstärkung der registrierten optischen Signale. Über Photoschalter auf der Basis von Biomakromolekülen wurde in den letzten Jahren als Teil der Suche nach molekularen Schalteinrichtungen und Mikromaschinen intensiv gearbeitet. Das umfangreiche Wissen über die Modifizierung von Biomakromolekülen, ihre gentechnische Erzeugung und die Herstellung von durch biologisch aktive Verbindungen modifizierten Oberflächen macht Biomakromoleküle mit verbesserten optischen Schalteigenschaften zugänglich. Ihr Einsatz in optoelektronischen/bioelektronischen Bauelementen ist nicht mehr nur eine Idee, sondern Realität. So wurde ihre Verwendung zur Informationsspeicherung und -verarbeitung (Biocomputer), in Sensoren, reversiblen Immunsensoren und biologischen Verstärkern optischer Signale bereits gezeigt; für die Zukunft bleiben aber noch bedeutende Herausforderungen.
Model systems help us understand naturally-occuring photoregulated processes and how they might have evolved.
A photochromic α-amylase was prepared by chemical modification with a spiropyran compound and isolated with DEAE-Sephadex chromatography. The modified α-amylase showed the reverse photochromism in water. UV- or VS-light irradiation caused a decrease in enzyme activity of the modified enzyme. The initial activity could be recovered when the modified enzyme irradiated was kept in the dark.
Plasticized poly(vinyl chloride)(PVC) membranes entrapping photoresponsive bis-(15-crown-5) with an azo-linkage (2) and its monomeric analogue (1) were prepared for the purpose of regulating their membrane potential by u.v. or visible light irradiation. The azobenzene moieties of (1) and (2) were found to undergo photochemical trans–cis isomerization reversibly in the membrane matrix. Alternating U.V. and visible light irradiation induced a significant reversibly potential change across the PVC–(2) membrane but did not for PVC–(1) membrane. Large potential shifts were induced by photoirradiation in the presence of KCI or RbCl, while the potential shifts, if observed, were small (–2.5 mV or less) in the presence of LiCl or NaCl. The photoinduced potential changes across the membranes were explained in terms of the change in binding ability of (2), resulting from trans–cis photoisomerization, for K+ and Rb+ at the membrane–solution interface. In other words the surface potentials at the two membrane–solution interfaces were considered to be perturbed by photoirradiation.
Vision and other light-triggered biochemical transformations in plants and living organisms represent a sophisticated biological processes in which optical signals are recorded and transduced as (physico)chemical events. Photoswitchable biomaterials are a new class of substances in which optical signals generate discrete “On” and “Off” states of biological functions, resembling logic gates that flip between 0 and 1 states in response to the changes in electric currents in computers. The (photo)chemistry of photochromic materials has been extensively developed in the past four decades. These materials isomerize reversibly upon light absorption, and the discrete photoisomeric states exhibit distinct spectral and chemical features. Integration of photoisomerizable (or photochromic) units into biomaterials allow their secondary functions such as biocatalysis, binding, and electron transfer to be tailored so that they can be switched on or off. This can be accomplished by chemical modification of the biomaterial by photoisomerizable units and by integration of biomaterials in photoisomerizable microenvironments such as monolayers or polymers. The photoswitchable properties of chemically modified biomaterials originate from the light-induced generation or perturbation of the biologically active site, whereas in photoisomerizable matrices they depend upon the regulation of the physical or chemical features of the photoisomerizable assemblies of polymers, monolayers, or membranes. Light-triggered activation of catalytic biomaterials provides a means of amplifying the recorded optical signal by biochemical transformations, and photostimulated biochemical redox switches allow its electrochemical transduction and amplification. The field of photoswitches based on biomaterials has developed extensively in the past few years within the general context of molecular switching devices and micromachinery. The extensive knowledge on the manipulation of biomaterials through genetic engineering and the fabrication of surfaces modified by biologically active materials enables us to prepare biomaterials with improved optical-switching features. Their application in optoelectronic or bioelectronic devices has been transformed from fantasy to reality. The use of photoswitchable biomaterials in information storage and processing devices (biocomputers), sensors, reversible immunosensors, and biological amplifiers of optical signals has already been demonstrated, but still leaves important future challenges.
Controlling the conformation and activity of biomolecules in a reversible manner is a fascinating challenge that has an outstanding potential for the study of and interference with complex processes in living cells. Spatial and temporal control of cellular processes could provide unparalleled opportunities for studying organism development or disease progression. A variety of synthetic photoswitches, that can undergo a reversible change in their structure upon irradiation with light, have been designed. They are commonly characterized by the absorption maxima of their isomeric firms, as well as by the photostationary state (PSS), defined as the equilibrium composition during irradiation. Azobenzenes remain the most widely used photoswitches in biological applications. The reasons behind this include their easy synthesis, relatively high photostationary states and quantum yields, fast photoisomerization, and low rate of photobleaching.
Abstract The central role played by acetylcholine in the conduction of the nerve impulse is now generally accepted after decades of controversy. It is assumed that acetylcholine acts as a trigger, inducing changes in the cation permeability of electrically excitable membranes through attachment to a receptor biopolymer. While an enormous amount of scientific work has been centered on the receptor proteins to which acetylcholine is attached and on the enzyme (acetylcholinesterase) responsible for the hydrolysis of acetylcholine, much less effort has been centered on studying the synthesis and the regulation of the synthesis of this molecule.
The enzyme alpha-chymotrypsin was immobilized in acrylamide copolymers which contain photoisomerizable components. The resulting enzyme-copolymer assemblies reveal photoswitchable ''on-off'' biocatalytic activities. Three kinds of acrylamide copolymers cross-linked with 4-(methacryloylamino)azobenzene (polymer 1) 1-[beta-(methacryloxy)-ethyl]-3,3-dimethyl-6-nitrospiro[indoline-2,2'-[2H-1]benzopyran](polymer2), and bis[4-(dimethylamino)phenyl](4-vinylphenyl)methyl leucohydroxide (polymer 3) were used to immobilize the enzyme. The enzyme reveals bioactivity (position ''on'') in the copolymer isomer states 1b, 2b, and 3b, respectively, while its activity is blocked (position ''off'') in copolymers 1a, 2a, and 3a, respectively. The activity of the enzyme is assayed toward the hydrolysis of N-(3-carboxypropionyl)-L-phenylalanine p-nitroanilide (7). The photostimulated activities of the enzyme entrapped in the different copolymers correlate with the permeability properties of the substrate 7 across the photoisomer forms of the copolymers. While the copolymer isomer forms 1a, 2a, and 3a exhibit poor permeability toward the substrate 7, the copolymers 1b, 2b, and 3b are permeable toward the substrate 7.
Macromolecules exhibiting photoswitchable physical or chemical properties are extensively examined as information storage and signal amplification materials. Photoregulated "on-of" biomaterials provide a novel means to design targeted therapeutic agents activated and deactivated by external light signals. Various means to photoregulate biotransformations by light-switchable enzymes have been described and include the modification of the enzyme active site and protein backbone by photochromic components and immobilization of enzymes in photochromic copolymers. Here we wish to report on the photoregulation of the binding properties of a protein by its chemical modification with photochromic units. We describe the photoswitchable binding of saccharides to concanavalin A modified by thiophenefulgide dye.
Nine populations of Juniperus virginiana were sampled at approximately 150-mile intervals along a 1500-mile transect from northeastern Texas to Washington, D.C. Individual plants were examined for terpenoids by gas liquid chromatography and the resulting data analyzed by numerical classification methods using characters weighted according to their estimated variance in the natural populations.
The results of the analysis show that these populations of J. virginiana cluster clinally from northeast to southwest, the more homogeneous populations occurring in the Appalachian region of North America; the more divergent populations found in progressively more distant regions, as measured along the transect from the northeast toward the southwest. No biochemical evidence could be found to support the hypothesis that hybridization with J. ashei might be causing this variability, as had been widely supposed previously.
THE activity of the melatonin-forming enzyme in the rat pineal gland, hydroxyindole-O-methyl-transferase (HIOMT), is high in animals kept in constant darkness and low when they are kept in constant light1. The response to light depends on the integrity of one component of the central retinal projections, the accessory optic system. Rats subjected to bilateral section of the inferior accessory optic tracts do not show a pineal HIOMT response to light although their visual behaviour is otherwise normal2,3. In contrast, animals with bilateral transection of the primary optic tracts behave as though they were blind but exhibit a normal pineal response to light2,3. There seem to be two separate functions carried out by the central retinal projections in the rat; one projection, the primary optic tracts, maintains visually guided behaviour, and the other, the accessory optic system, shares in the control of a pineal response to light.
A specific inactivator of chymotrypsin, p-azophenyldiphenylcarbamyl chloride, exists as two geometric isomers, cis and trans, which are interconvertible by means of light. The cis-isomer is five times more reactive than the more stable trans-isomer, and is obtained by exposure of the latter to light of 320 nanometer wavelength. The trans-isomer can be regained by exposure of the cis-isomer to light of 420 nanometer wavelength. This interconversion can be made to occur in aqueous solution in the presence of the enzyme under conditions in which the trans-isomer reacts relatively slowly with chymotrypsin. Thus, it is possible to regulate the rate of inactivation of chymotrypsin by using light of the appropriate wavelength. This system is presented as a model for some of the light-sensitive metabolic systems present in living organisms.
Diphenylcarbamyl chloride (DPCC) was found to inactivate chymotrypsin and trypsin by means of a 1:1 stoichiometric reaction. The reaction was relatively specific for chymotrypsin, which was inactivated 80 times faster than trypsin. Km for the reaction with chymotrypsin was found to be 0.6 ± 0.2 × 10-4 M at pH 8.0. The inactivation of chymotrypsin could be competitively inhibited by indole. DPCC was unreactive toward chymotrypsinogen, diethylphosphorylchymotrypsin, and pepsin. The pH-rate profile of the reaction of DPCC with chymotrypsin was bell shaped, with an optimum at 7.8. Its proton release curve was complex; only in the region near pH 5.0-5.5 was 1 equiv of H+ released. The inactive product, DPC-chymotrypsin, could be reactivated by a number of nucleophilic agents including hydroxylamine, isonitrosoacetone, and acyl hydroxamic acids. The experimental data are interpreted with respect to their contribution toward an understanding of the enzymic mechanism of chymotrypsin.
1.1. A new preparation has been developed in which an isolated single electroplax of Electrophorus electricus may be used for the study of the properties of the cell.2.2. The electroplax is kept between a nylon sheet having a window adjusted to the dimensions of the cell and of a grid consisting of nylon threads. It is placed between two chambers in a way that the cell separates two pools of fluid.3.3. The innervated membrane of the electroplax is bathed by the fluid of one chamber, the non-innervated one is bathed by the fluid of the other chamber.4.4. This preparation permits one to test the effect of chemical and physical factors separately on the innervated one non-innervated faces of the cell and is suitable for studying ion flux across the cell.5.5. The actions of acetylcholine and of related tertiary and quaternary compounds have been tested with this single preparation.6.6. Previous observations have been confirmed, viz. that tertiary nitrogen compounds block electrical activity but do not depolarize, they are receptor inhibitors. Quaternary nitrogen derivatives block and depolarize simultaneously, they are receptor activators.7.7. Whereas, however, in previous observations the effects were irreversible, tested with this preparation the following compounds were found to act reversibly: acetylcholine, carbamylcholine, eserine, prostigmine, DFP, d-tubocurarine and procain. The following compounds were found to act irreversibly: tabun, decamethonium and stilbamidine.8.8. The action of non-depolarizing agents is completely reversible, that of depolarizing compounds can be only partially reversed and then only temporarily.9.9. The compounds act whether they are applied in the solution bathing the innervated or non-innervated membranes.10.10. The depolarization of the cell produced by the quarternary derivatives is due to an action upon the synaptic region.11.11. Blocking and depolarizing action by quarternary compounds cannot be dissociated. Experiments with curare show that they act exclusively upon the synaptic junction.12.12. New evidence is offered that the tertiary and quarternary compounds tested compete for the same receptor.
Diphenylcarbamyl chloride, diphenylcarbamyl fluoride, methylphenylcarbamyl chloride, and methylphenylcarbamyl fluoride were studied as acid-transferring inhibitors of chymotrypsin, trypsin, acetylcholinesterase, and serum cholinesterase. The fluorides were the better inhibitors in all cases. They are quite potent inhibitors and are the best of the known inhibitors of chymotrypsin and trypsin. The greater activity of the fluorides is evidence that there is an electrophilic component in the enzymic mechanism.
- H Kaufman
- S M Vratsanos
- B F Erlanger
Kaufman, H., S. M. Vratsanos, and B. F. Erlanger, Science, 162, 1487 (1968).
) is a Fulbright Scholar and was supported in part by a generous grant from the Irene Heinz Given and John LaPorte Given Foundation, Inc. The authors wish to thank Dr
- France
France) is a Fulbright Scholar and was supported in part by a generous grant from the Irene
Heinz Given and John LaPorte Given Foundation, Inc. The authors wish to thank Dr. R.
Kitz for helpful discussions during this work.
This formula is based on a turnover number of 7.4 X 10-5 min
X 10-. This formula is based on a turnover number of 7.4 X 10-5 min' given by
Wilson, I. B., and M. A. Harrison, J. Biol. Chem., 236, 2292 (1961).