Libmonster ID: KZ-1576
Author(s) of the publication: Alexander SHPAKOV

by Alexander SHPAKOV, Dr. Sc. (Biol.), laboratory head, Molecular Endocrinology Laboratory, Sechenov Institute of Evolutionary Physiology and Biochemistry, St. Petersburg, Russia

Cells live on only if they respond adequately to external signals sent by hormones and growth factors involved mostly in a specific interaction with serpentine receptors in the plasma membrane. Such receptors make use of GTP-binding proteins to get the signal into the cell. The complex natural mechanism of this action calls for great intellectual effort. Therefore any headway in this area is valued high by the world scientific community: four Nobel prizes have been awarded in these last twenty years for progress made. Such breakthrough explorations expand the boundaries of knowledge. And not only that: receptors coupled to GTP-binding proteins are targets for as much as 40 percent medical drugs used today, and so they are naturally in the focus of attention. These last few years have seen new revolutionary technologies for high-selectivity regulators of like receptors ushering in dramatic changes in treating many diseases and on the pharmaceutical market as well.

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Cycle of hormonal activation of serpentine receptors and coupled G protein. 1--hormone gets to receptor associated with the G protein; 2-hormone associates with the receptor binding site; 3--receptor and G protein change their conformation, thus lowering the affinity of G protein forguanosine diphosphate (GDP); 4--guanosine triphosphate (GTP) in the G protein a-subunit is substituted for GDP; 5--á-subunit dissociates from the βγ-complex and, acting severally, they activate inferior signaling cascades; 6-α-subunit reduces GTP to GDP, which results in its association with the βγ-complex.


None of the biochemical and physiological processes in man and animals can be out of control. Highly specialized signaling molecules act as their remote control regulators, and these are the hormones and growth factors: noted for high specificity, they bind to protein receptors on cell surface. Such receptors may be described as an "informational gate" since they are implicated in transcription factors regulation, either suppressing or, vice versa, intensifying gene expression. Thereby they control all vital cell processes like metabolism, growth, differentiation and motion.

In between a receptor opening the signaling pathway and transcription factors at its end and in immediate contact with genes are many specialized proteins modifying the signal and amplifying it. Any dysfunctions in the signaling cascade distort cell response to external controlling signals and lead to diseases of the endocrine, nervous, cardiovascular and other systems, and cause malignancies and autoimmune pathologies. Small wonder that hormonal systems and the search for new strategies to their regulation and correction are a live issue in biology and medicine.

Today we know of many kinds of hormonal receptors, with serpentine receptors certainly in the lead-man has over 1,300 receptors like that. Their curves, coils and twists make them indeed look serpentine, that is winding about like a snake's body. Remarkable for seven hydrophobic sites across the plasma membrane, they form an internal channel, with a receptor's "holy of holies" inside this channel--the site of a hormone's specific binding. This framework is known to molecular biologists as "key to lock", with only one hormone, the "key", fitting the "keyhole", the receptor. Recognizing a "friendly" signal, the receptor sends it into the cell. So just one particular hormone can get in. Yet another characteristic feature: the receptor interacts with GTP*-binding protein (or G proteins for short) located on the inner side of the plasma membrane and composed of three subunits, α, β and γ.

G proteins discovered by two American scientists, Martin Rodbell and Alfred Gilman (Nobel prize in physiology and medicine, 1994), are the main connective link between receptors and signaling cascades. When inactive, the a subunit of the G protein contains the nucleotide guanosine-5'-diphosphate and forms a tight bond with the βγ-complex. When hormone-activated, the G protein changes its structure, with guanosine-5'-diphosphate of the a subunit substituted

* GTP, guanosine-5'-triphosphate: a nucleotide which is a substrate tor RNA synthesis in the transcription process; energy source in protein biosynthesis.--Ed.

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GPCR spatial structure. Extracell loops, seven transmembrane sites and intracell receptor loops shown.

by guanosine-5'-triphosphate and disassociated from the βγ-complex. "Free" at last, the a subunit and the βγ-complex send a signal to intercell effector proteins evoking a functional cell response to the hormonal effect. Such "freedom" is but momentary: the a subunit destroys GTP, and reassociates with the βγ-complex to bring the signal reception system back to the inactive state.

The tight bond between serpentine receptors and G proteins detected back in the 1980s is now known as G protein-coupled receptors, or GPCR. Since serpentine receptors were discovered but recently (such receptors transmitting a hormonal signal with no participation of G proteins), the very name, GPCR, appears to be not quite correct.


Each hormone fits its receptor really like a key does its keyhole. It is highly specific in its interaction with the receptor's ligand*-binding site in its transmembrane canal. Such specificity of binding, however, in no way ensures a high specificity of response. Why?

* Ligand, here an atom or a molecule bound to some center (acceptor). In biochemistry this term denotes agents combining with biological acceptors, receptors in particular.--Ed.

First, because one hormone has a set of several types of receptors to fit in, each regulating different signaling cascades that may be opposite in the end cell response. Thus serotonin, an all-important neurohormone, binds selectively to as many as 18 subtypes of serotin receptors located in different organs and tissues and regulating a good many biochemical and physiological processes. Secondly, even if one hormone is adjusted to one receptor only, the hormone-G protein interaction may involve a complex ramification (branching) pattern and lead to separation of signaling cascades, for GPCR usually interact with several types of G proteins, each coupled by its receptor to a definite signaling channel.

So, research scientists have to wrestle with hard and often imponderable problems bearing on selective GPCR regulators. This search is very important since G protein-coupled receptors are targets, as we have said, for 40 percent of drugs in use today. Lately impressive results have been achieved in identifying high-selectivity GPCR ligands and also in mapping out new strategies of their synthesis. Robert J. Lefkowitz and Brian K. Kobilka of the United States merited a Nobel prize in chemistry in 2012 for their breakthrough explorations in GPCR. In 2007 they discovered a three-dimensional structure of the β adrenergic receptor (which belongs to the GPCR family) in its natural environment within the plasma membrane. First this receptor was crystallized and then studied in X-rays. The configuration of its ligand-binding site was determined with high accuracy, which made it possible to predict the chemical structure of compounds implicated in specific interaction with it. Between 2008 and 2014 a three-dimensional structure was identified for something like fifteen G protein-coupled receptors. Criteria were devised for obtaining GPCR ligands of pre-assigned activity. These include full agonists (GPCR activators), inversional agonists (bringing down GPCR activity and inhibiting the effects of full agonists) and neutral antagonists (preventing GPCR binding to agonists).

Whereas hundreds and thousands of compounds had to be sorted out before in search of a desired ligand, (even using molecular modeling, and this search often carried out at random to cover hundreds of thousands of substances, the situation changed dramatically with the deciphering of the GPCR spatial structure: the effectiv-ity of this work has soared manifold. More than that,

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Search for new GPCR agonists and antagonists via drug screening. The computer model shows a selected chemical building into the receptor ligand-binding pocket, with the receptor's spatial structure identified. Effect of the tested compound/receptor interaction evaluated. Prospective substance synthesized and tested biologically.

while formerly the guiding principle was--first find an active substance and then study its analogs changing but slightly their structure--now the use of a new strategy (structure-based drug screening) allowed to design GPCR ligands of an absolutely new structure. The results thus obtained were just fantastic!

Of 25 ligands designed for the B2 adrenergetic receptor six exhibited high affinity in binding to the receptor, with two ligands having an absolutely new chemical structure. In the case of the A2A adenosine receptor, 30 substances from among the 76 synthesized ones proved active, that is 39 percent; for the D3 dophamine receptor, six in 26 were active, or 23 percent; and for the H1 histamine receptor, 19 out of 26 were found active, or 76 percent.

The high efficiency of the search for GPCR ligands means a cardinal change in the drug-making strategy. A research team that is the first to decipher the spatial structure of yet another GPCR will be capable of designing--and doing it fast--actually the entire set of functionally active ligands for this receptor to rule the pharmaceutical market. Pharmaceutical companies for dozens of years specializing in the total screening of chemical compounds will be put out of business.

The discovery of GPCR multiple active conformations (spatial structures) made by Robert Lefkowitz, Brian Kobilka, and Kurt Wiitrich of Switzerland (Nobel prize, 2002) in 2011 and 2012 is of exceptional importance both in theoretical and in practical terms. These research scientists found that a receptor has several active conformations; not one, and that its transition into one of them depends on a ligand's chemical characteristics. And thus already at the ligand's binding stage it will be possible to adopt a strategy for sending in a signal and an adequate cell response. Work is now in progress in designing GPCR ligands highly selective both to receptors and to the signaling cascades.


For all the strides made in search of classical GPCR ligands, they cannot be used for effective regulation of all G protein-coupled receptors, not counting in certain side effects. So the search is on for other regulators capable of interacting with receptor sites which, though not within the ligand-binding site, affect its active conformation and GPCR contacts with G proteins. GPCR peptides structurally corresponding to functionally significant cytoplasmic receptor sites, are of the utmost interest in this respect. These peptides, albeit detected late in the 1980s and early 1990s, have been in for intensive studies only in these last few years with the knowledge of the molecular mechanism of their action on cells, and thus their high-activity analogs could be designed.

The first steps in search of high-activity GPCR peptides were made in 2002 by the research team of Atan Kouliopulos of the Institute of Molecular Cardiology in Boston, USA. A hydrophobic radical (part of a molecule in effective interaction with nonpolar substances like lipids or the lipid phase of membranes), if bound to GPCR peptides, increases manifold their biological activity in biochemical experiments in vitro. Used as hydrophobic radicals are fatty acid residues if large enough. Peptides thus modified are called pepducins. They were found active in vivo, too, by regulating effectively biochemical and physiological processes in iso-

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lated organs and tissues in experimental animals. The high activity of these compounds is due to their effective transmembrane transport by a hydrophobic radical that gets them "anchored" on the membrane internal surface and makes them interact efficiently with target proteins. The main pepducin targets are the complementary sites of a homologous receptor; in cells and tissues with no homologous receptor present, the pepducin effect is not observed. Pepducins cannot interact with "aliens" lacking structural affinity and with complementary sites. All that underlies the pepducin receptor and tissue specificity and in many ways determines the value of pepducins as prototypes of drugs.

Pepducins are active as intercell agonists and antagonists capable as they are, even in the absence of a hormone, of transferring the receptor into an active confirmation and of triggering a signaling cascade. It is important that the pepducin interaction with a homologous receptor renders it into a definite confirmation providing for high selectivity in activating the intracell signaling cascades and their targeted action upon biochemical processes in the cell. Here are a few examples.

In 2006 Atan Kouliopulos and coworkers synthesized a pepducin corresponding to the cytoplasmic site of the protease-activated receptor (PAR) of type 4 which, if administered to test animals, prevented arterial throm-

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bosis causing myocardial infarction and ishemic insult. In 2014 Arnold Speck and coworkers (USA) demonstrated that pepducin Plpal-12 active as a PAR antagonist of type 1 suppresses lung fibrosis in mice and can be used in treating idiopathic* pneumosclerosis not amenable to medication. Even more unique characteristics are proper to the PZ-128 preparation synthesized by Ping Ziang and colleagues from Tufts University in the United States from a peptoducin derived from PAC of type 1. It suppresses arterial thrombosis in guinea pigs and monkeys, restores the functions of blood platelets after coronal surgery, while not causing any side effects; PZ-128 also inhibits mammary, ovarian and lung malignancies and secondary cancers, and is not second to antitumor drugs now in use.

Working in the Molecular Endocrinology Laboratory (Sechenov Institute of Evolutionary Physiology and Biochemistry), in 2005 we got down to pepducin synthesis, with selective regulators of the central nervous and endocrine systems in the focus. We designed pepdu-cins corresponding in their structure to the cytoplasmic sites of serotonin, relaxin, luteinizing and thyrotropic hormone receptors; we found them to be highly specific and effective in vitro. Given to rats, pepducin 612-627 (Pal), a thyrotropic hormone derivative, increased the level of thyroidal hormones; so it has a stimulating ef-

* Idiopathic: of a disease the cause of which is unknown.--Tr.

feet on thyroid gland functions. We published our results in 2012. This preparation, 612-627 (Pal), also boosts the efficacy of thyroid and goiter tumor treatment. Treated by pepducin 562-572 (Pal), a luteinizing hormone receptor, introduced into their reproductive organs, male rats showed a higher level of testosterone, the male hormone; this compound has good prospects for androgenic insufficiency (male impotence).

Even minor structural modifications of GPCR peptides as well as ramified networks created on their basis can boost the selectivity and efficacy of this compound and even work cardinal changes in its biological activity spectrum. All that opens up really boundless possibilities for targeted action drugs based on GPCR peptides.


Dysfunctions in the activity of receptors are one of the causes of many diseases since they are touched off by anomalies in the information exchange between the cell and the environment. G protein-coupled receptors are a key link in this regard and, consequently, compensation and normalization of their functions are in the mainstream of prevention and therapy of such pathologies.

The cause-and-effect dependencies between GPCR activity changes and pathologies may be very complex. One of the causative factors of many maladies is due to

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Pepducin action mechanisms (Shpakov, 2013). Bright green, pepducin with an associated hydrophobic radical (black wavy line). Receptor homologous sites color-coded in light green, complementary sites, in violet. I--immediate interaction of pepducin with the C-terminus of the G protein α-subunit realized at low specificity is of no interest for design of selective GPCR regulators; II--pepducin interacting with homologous receptor complementary sites controls receptor activity and conjugated signaling cascade. Therapeutic effect is based on such interaction; lll-pepducin interacting with a dimeric complex of two receptor molecules. This interaction affects the stability of the complex and accounts for pepducin specific biological activity.

GPCR mutations lowering GPCR sensitivity to hormones and other functions; one such symptom relates to antibodies to receptor extracell sites. For instance, many thyroid gland diseases spring from malfunctions of the thyrotropic hormone receptor. The activating mutations render it utterly uncontrollable by causing hyperactivation of signaling cascades dependent on it, and ultimately lead to congenital hyperthyroidism, toxic goiter and thyroid cancer. The stimulating antibodies to this hormone receptor activate thyroid gland growth and cause thyrotoxicosis. Intensive human genome studies are now underway so as to identify and systematize GPCR mutations and evaluate their role in hereditary somatic morbidity. Especially in the focus are combined mutations of several receptors affecting signaling proteins--this provokes combined diseases and composite symptoms. For example, inactivating mutations in the melanocortin receptor 4 may lead to adiposity and metabolic syndromes; say, if such anomalies of functionally related proteins are on simultaneously, the probability and gravity of disease go up considerably.

Autoimmune diseases caused by GPCR antibodies are likewise of major interest. While formerly only the

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role of tyrotropic hormone receptor antibodies in thyroid pathologies was considered by and large, today we have conclusive evidence on GPCR antibodies being the primary cause of such diseases as cardyomyopathy, pervasive pain syndrome as well as certain cognitive disorders, with this list expanding all along. In idiopathic cardyomyopathy patients, antibodies were detected on extracellular sites of the β-adrenergetic receptor; blocking it, these antibodies are the cause of the pathology. Ordinary medication is of little effect in such cases since the main causative agent, the antibodies, are not eliminated. But immunosorbents, as shown by practical application, allow to do so, and thus they have a curative effect. Working with much success in this area is the Russian Cardiological R&D Complex in Moscow in search of treating cardiovascular diseases provoked by β-adrenergetic receptor antibodies.

All that speaks of the significance of experimental studies in further identification of such pathologies. For instance, test animals are immunized by peptides containing antigen receptor determinants to see what pathologies this could involve. Working within the framework of the Russian Fundamental Research Foundation, we are creating models of autoimmune diseases in test animals immunized by GPCR peptide conjugates. In our experiments on rats we used peptides which included antigen determinants of melanocortin receptor 4; that caused obesity, lower sensitivity to insulin and other dysfunctions characteristic of diabetes mellitus (sugar diabetes) II, lower cognitive functions and lower inquisitiveness. We shall note in this context that a composite research program for a wide-scale screening of autoimmune diseases caused by GPCR antibodies holds out good prospects.

Yet another important sidelight. Since host cell GPCR are needed to let in certain viruses, antibodies to these receptors will knock them out and prevent contagion. Viruses making use of GPCR for infection include the human immunodeficiency virus (HIV) I which needs the GPR1 receptor to infiltrate lymphocytes. It has been established recently that monoclonal antibodies to the GPR1 receptor keep HIV I from penetrating the cell and block its infection.

We and other researchers have shown that the initial stage of many diseases sees changes in the number and functional activity of GPCR, and these changes are compensatory. They are reversible for a time. But the lasting effect of pathogenic factors induces irreversible changes in receptor functions--the disease makes strong progress, and so do its complications. Consequently, the GPCR functional status may be used for differential diagnosis of a pathology, its gravity and complications. Higher GPCR compensatory capabilities are needed for preventing progressive maladies, and here farmacological approaches can be used in the first place. We have watched the dynamics of GPCR activity changes in animals in sugar diabetes I and II models. Anomalies of these receptors and related cascades surface long before complications in the nervous, cardiovascular, reproductive and other systems. Therefore timely diagnostics and compensation of such disorders allows, for one, to mitigate the gravity of sugar diabetes, and prevent--partly or in full--the ensuing complications.

Our scientists have made good progress in studying the structural-and-functional organization of GPCR, and the role of GPCR in morbidity, and in creating selective regulators and modulators of these receptors. Working successfully in this field are the research schools by Yuri Ovchinnikov, Vsevolod Tkachuk, Mikhail Kirpichnikov and other premier researchers. Yet the latest economic crisis has resulted in a bad lag in this most promising and go-ahead area of our biology, farmacology and biotechnology. So there is a felt need of making GPCR research into a separate priority research trend. If we fail to do that, we shall land on the sidelines watching a triumphant march of GPCR technologies abroad, and have to shell out big money--much more than research programs of our own could take.


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