: March 20, 2008, Evolution in the shadow of dinosaurs, volume 19, no. 1
In the cells of our body - complexly arranged "chemical factories" - a countless variety of biochemical reactions are constantly taking place. Moreover, the processes of destruction, destruction of unnecessary or foreign substances for the cell are no less significant for it than the processes of synthesis. They are carried out, as a rule, with the help of enzymes - natural catalysts of a protein nature...
Ribonucleic acids, which serve as templates for protein synthesis, are an essential component of cellular biochemical “production”. However, in some cases, during infections or other pathological conditions of the body, it is necessary to stop the processes of synthesis of a particular protein. And one way to do this is to destroy the corresponding RNA using special enzymes.
It is not surprising that scientists began to think about the possibility of creating fundamentally new drugs based on them, capable of selectively destroying, for example, viral RNA, without affecting the vital ribonucleic acids of the body’s cells.
Indications for use
The drug is recommended for use in the following diseases and pathological conditions:
- Exudative type pleurisy
- Fistulas and lung abscess
- Respiratory ailments in which there is poor sputum discharge
- Otitis
- Osteomyelitis
- Viral meningitis
- Bronchiectasis
- Tick-borne encephalitis
- Sinusitis
- Dental diseases (periodontal disease, gingivitis)
- Trophic ulcers
- Thrombophlebitis
- Lung atelactasis.
Universal "soldiers"
Where can artificial ribonucleases be used? First of all, where natural enzymes are now used. For example, in modern biotechnology - when isolating genomic DNA and preparing it for PCR analysis, which is widely used in a variety of fields, from medical diagnostics to forensics.
Artificial ribonucleases are completely inert towards other biomolecules, in particular proteins. This property turned out to be indispensable in the creation of vaccines. After all, in order to obtain safe antiviral vaccines, viral particles must be “disarmed,” that is, their genetic material must be destroyed. Formaldehyde, which combines high activity with low cost, is currently used most often for this purpose. However, formaldehyde also partially destroys viral proteins, namely: the body develops an immune response to them during vaccination. As a result, the effectiveness of vaccination decreases. Artificial ribonucleases do not have this drawback, which was confirmed by preliminary experiments conducted on laboratory mice infected with the influenza virus. The survival rate of animals receiving such a vaccine was significantly higher compared to the control group vaccinated in a standard manner.
Finally, artificial enzymes can successfully replace natural ones in studies of various RNA-protein complexes. For example, when deciding the question of what determined the specificity of protoenzymes at the dawn of life - their RNA-binding certain bonds in the RNA chain (as is observed in modern ribonucleases) or was this function originally performed by their catalytically active centers? Such work is directly related to one of the most important fundamental evolutionary problems - the problem of the formation of protein life.
Medicinal properties
Ribonuclease is classified as an enzymatic preparation; its main component is obtained by processing the secretion of the pancreas of cattle.
The mechanism of action is based on the process of depolymerization of ribonucleic acids to oligopeptides or monopeptides.
The main function of ribonuclease is to liquefy excessively viscous sputum, pus, and mucus. In addition, this substance can relieve the severity of the inflammatory process.
This tool inhibits the spread of certain types of viruses containing RNA. This effect is achieved by activating the process of destruction of nucleic acids.
Ribonuclease activity can be determined biologically by taking into account the total number of acid-soluble components that are released due to RNA hydrolysis. A unit of activity is equal to 1 mg of ribonuclease.
Everything ingenious is simple
Ribonucleases
- enzymes of the class of hydrolases that cleave RNA - are widely distributed in the cells of all organisms. The most famous of them is pancreatic ribonuclease (or RNase A), isolated from the bovine pancreas.
When talking about this enzyme, we constantly have to add the word “for the first time.” RNase A is the first enzyme protein for which in the early 60s. last century, the sequence of its constituent amino acids was completely determined. And when the first chemical synthesis of the enzyme was carried out in 1969, it turned out to be the same pancreatic ribonuclease. Moreover, it also became the first enzyme to enter medical practice (it is used as an antiviral agent). As an aside, we note that the enzyme onconase is an analogue of RNase A, isolated from the oocytes of the frog Rana pipiens
, today is a completely new anticancer drug, the clear success of clinical trials of which suggests the emergence of a new class of anticancer drugs - a serious alternative to classical chemotherapy.
Natural ribonucleases - enzymes that destroy unnecessary or foreign (viral) RNA - are present in the cells of all living organisms. The
next milestone in the transformation of ribonucleases into drugs can be considered the 1980s, when the American biochemist R. Breslow discovered that small molecules of the heterocyclic substance imidazole capable of cleaving RNA like natural pancreatic ribonuclease. He came up with the idea that it was possible to create artificial analogues of enzymes - low-molecular compounds with the functions of natural catalysts. This idea was brilliant in its simplicity: to achieve the result, it is enough to take catalytically active groups found in the active centers of natural enzymes, fix them in a certain way in space - and the synthetic analogue of the enzyme is ready!
Ribonucleases were the first enzymes for which artificial analogues were created - low molecular weight compounds that act like catalyst proteins
A few years later, R. Breslow managed to put his idea into practice. And although the “artificial” ribonucleases he obtained were capable of cleaving phosphodiester bonds only in a substrate specially synthesized for these purposes, and not in natural RNA, a start had been made.
The appearance of the first works on artificial ribonucleases coincided with the peak of research on another very interesting topic - the so-called antisense oligonucleotides
, work on which was carried out in the Department of Biochemistry of the Institute of General Chemistry SB RAS (Novosibirsk), headed by Academician D.G.
Knorre. It should be clarified here that shorter nucleic acid sequences are unique in their structure. Therefore, they can be targeted by complementary
(antisense) oligonucleotides, which can specifically “recognize” and attach only to certain sections of a particular nucleic acid.
The combination of these two technologies—antisense and low-molecular-weight artificial ribonucleases—promised a breakthrough in the production of synthetic enzymes for medical purposes. And since good ideas often come to many people at the same time, it is not surprising that work in this direction in the 90s. last century began almost simultaneously all over the world - in Japan, Europe, the United States and Russia.
Ribonuclease: instructions for use
In case of local use, the wound or ulcer should be sprinkled with powder from a bottle in a volume of 25-50 mg. On top you need to place a gauze pad, which has been previously moistened with saline solution.
To carry out the inhalation procedure, you will need to use a fine aerosol: for 1 procedure, take 25 mg of lyophilisate, which is diluted with 4 ml of saline solution.
For endobronchial administration, you will need to take a dose of the drug of 25-50 mg, the procedure is carried out using a catheter or a laryngeal syringe.
For intrapleural administration, take ribonuclease in a volume of 25-50 mg, as well as 5-10 ml of saline solution.
In the case of sinusitis, a dosage of the drug of 5-10 m is indicated, which is diluted with 5 ml of saline solution. The drug is injected directly into the nasal sinus after the rinsing procedure. For otitis media, injection of 1 ml of 0.1% solution into the ear canal is indicated.
Intramuscular injection procedure: 1 ml of saline solution is taken for 5-10 mg of the drug. Treatment involves several injections - from 2 to 10 injections (1 or 2 throughout the day).
The highest single dosage for intramuscular administration is 10 mg; for local administration, the dose of the drug is 50 mg.
When treating tick-borne encephalitis, the drug is administered intramuscularly up to 6 times throughout the day with a dosage of 25-30 mg. A single dosage for children aged 1 to 3 years is 5-8 mg; 4-6 years – drug dosage 10-14 mg; the age group of 7-11 years is recommended to use 15-18 mg of the drug; 12-15 years old - use 20 mg of ribonuclease.
Before the injection procedure, the required volume of the drug should be diluted with 2 ml of 0.5% procaine solution.
Immediately before the course of treatment, skin tests should be performed to show sensitivity to the drug.
No worse than natural ones
Unfortunately, during the implementation of antisense technology in its original version, a number of problems were discovered, as a result of which the creation of real drugs based on such compounds was postponed indefinitely.
These problems primarily concerned the penetration of synthetic oligonucleotide derivatives through cell membranes, as well as their stability in living systems. In addition, the curtailment of work in this direction was influenced by a “non-technological” reason. At the initial stage of its development, antisense technology seemed so easy to implement that commercial companies began to appear like mushrooms all over the world, promising to create a “panacea” in the coming years. Huge amounts of money have been invested in this area; when the promised quick returns did not materialize, companies quickly began to go bankrupt, and there was a significant reduction in funding for basic research. For Russian scientists, this period coincided with the general collapse of the Russian economy and a significant reduction in funding for science in general.
Analogues of natural enzymes - a convenient tool for molecular biology and genetic engineering
When Novosibirsk scientists from the Institute of Chemical Biology and Fundamental Medicine SB RAS began their work in the field of constructing artificial ribonucleases based on oligonucleotide conjugates, they knew about all the difficulties, although, like many at that time, they considered them temporary. Unfortunately, ideal carriers for our ribonucleases - synthetic analogues of nucleic acids that easily penetrate into cells and are not destroyed by natural enzymes - have not yet been created.
However, despite all the difficulties, scientists did not stop working on creating artificial ribonucleases, starting with the modeling of that same RNase A. Dozens of potential artificial enzymes were synthesized in the laboratory of organic synthesis. All of them, to one degree or another, cleaved phosphodiester bonds in various natural RNA molecules, without affecting DNA at all. Each molecule of these synthetic enzymes was capable of catalyzing the destruction of tens and hundreds of bonds in RNA.
Artificial ribonucleases can become highly selective, low-toxic drugs for the treatment of viral, oncological and hereditary diseases
Scientists were able to finally say that structural and functional analogs of RNase A had been created after they had studied in detail the features of RNA destruction in the presence of both natural and synthetic enzymes. The fact is that in addition to high efficiency, natural enzymes are also characterized by high selectivity of action. For example, RNase A most effectively cleaves bonds in the RNA chain between the nucleotides adenine (A) and cytosine (C) and somewhat less efficiently between adenine and uridine (U). So, it turned out that artificial enzymes worked almost the same as natural ones.
Of course, even the most active of the created ribonucleases are hundreds and even thousands of times inferior in activity to the natural enzyme. However, if you consider that RNase A accelerates the destruction of RNA by 1014 times, then the results obtained look impressive. At the same time, artificial ribonucleases have a number of advantages: first of all, they are very cheap compared to natural enzymes. In addition, these compounds are very stable and can operate under a wide range of conditions.
Journal "Child's Health" 5 (32) 2011
Introduction
Representatives of the group of endonucleases, ribonucleases A (RNase-A), represent a large family of divergent proteins, the molecules of which contain specific elements of homologous amino acid sequences with a unique disulfide tertiary structure. RNase-A endonucleases are among the most studied mammalian proteins. The first representative of this family was isolated by René Dubos in 1938 from the bovine pancreas and identified as an enzyme involved in the digestion process [4, 8, 36, 37]. Subsequently, it was shown that RNase-A is involved not only in the processes of food RNA degradation in the intestinal lumen, but also performs RNA depolymerization in body tissues, regulating the processes of transcription and translation. Currently, 13 members of the RNase-A superfamily have been identified in humans. The genes of all members of the human RNase-A superfamily are located on chromosome 14 [31]. Numerous scientific findings have significantly changed the understanding of the physiological significance of this protein superfamily. In particular, it was found that representatives of RNase-A have cytotoxic and antibacterial properties [39], which allow them to participate in a variety of physiological processes, including the regulation of angiogenesis, cell apoptosis, and nonspecific protection of the respiratory tract [2, 8, 15].
Brief characteristics of ribonucleases A
Human ribonucleases A are a superfamily consisting of 13 enzymes (12–16 kDa) (Table 1) [10, 28, 32, 52].
Depending on the presence of ribonuclease activity, active and inactive human RNase-A are distinguished.
Eight representatives of the RNase-A superfamily are active, the molecules of which have three specific catalytic amino acid residues: one lysine within the conserved sequence CKXXNTF and two histidines in the N- and C-terminal regions, forming the catalytic cleft and determining ribonuclease activity. The molecules of the seven mature members of the RNase-A superfamily contain 8 cysteine residues, which form 4 disulfide bridges. The ANG molecule contains 6 cysteine residues. The shape of RNase-A molecules resembles a kidney (Fig. 1, using RNASE1 as an example) [3, 4, 10, 44, 52].
Analysis of the phylogenetic relationships of members of the RNase-A superfamily showed that human RNASEs are divided into two groups—the active (A) subfamily and the inactive (HA) ribonuclease subfamily (Fig. 2).
In turn, active RNase-A represents two subgroups, the first of which includes RNASE1, which is involved in digestion, RNASE4, RNASE5, which stimulates the formation of blood vessels and has a bactericidal effect; the second is two eosinophilic cationic proteins (RNASE2/eosinophilic neurotoxin, RNASE3/eosinophilic cationic protein) and RNASE6, RNASE7, RNASE8 [5, 21, 24, 27, 39, 40, 42, 47].
Five representatives of the human RNase-A superfamily are inactive: RNASE9, RNASE10, RNASE11, RNASE12, RNASE13, which have lost their catalytic elements during evolution [10, 28, 32].
The genes encoding RNase-A are located on the long arm of chromosome 14 [10, 28, 32, 55].
It has been shown that representatives of the RNase-A superfamily, such as RNASE2, RNASE3, RNASE7, participate in protection against infectious agents tropic to the respiratory tract.
The main producers of RNASE2, RNASE3 (Fig. 3, 4) are eosinophils. These two members of RNase-A are part of the group of four major eosinophil proteins, which also includes eosinophil peroxidase (EPO) and major basic protein (MBP) [7, 50]. Helene F. Rosenberg [39] believes that the RNASE2, RNASE3 genes arose about 50 million years ago as a result of the duplication process. In addition to eosinophils, RNASE2 is also produced by hepatocytes and neutrophils [41].
The RNASE2 molecule (molecular weight 15.5 kDa) consists of 134 amino acid residues. RNASE2 was identified in 1981 as an eosinophilic protein that has a toxic effect on cells and causes damage to the cerebellum (Gordon phenomenon), and therefore received the name “eosinophil-derived neurotoxin” (EDN) [14]. RNASE3 (15.5 kDa) is an arginine-rich (19 of 133 amino acids) cationic protein [33]. Both proteins are similar in biochemical characteristics, but RNASE3 exhibits more pronounced anthelmintic activity [39]. In the inactive state of eosinophils, ribonucleases RNASE2 and RNASE3 accumulate in large specific cytoplasmic granules [41].
The RNASE7 protein (14.5 kDa) was identified by Jürgen Harder and Jens-Michael Schröder in 2002 (Fig. 5) [23]. The RNASE7 molecule is enriched in lysine residues (18 out of 128 amino acids) [53]. It has been shown that RNASE7 is produced by both skin keratinocytes and epithelial cells of the mucous membrane of the nasal cavity, trachea and bronchi. RNASE7 is characterized by constitutive and inducible production. The main triggers of the RNASE7 synthesis mechanisms are pathogen-associated molecular structures, IL-1b, IFN-g [11, 23].
Mechanism of action of ribonucleases A
The main function of RNase-A is the depolymerization of ribonucleic acids by hydrolysis of the 3',5'-phosphodiester bonds of single-stranded RNA (ssRNA) with the participation of two histidine residues (His12 and His119) and one lysine residue (Lis41), forming the catalytic cleft [3, 12, 37 , 43].
Thanks to their catalytic activity, representatives of the RNase-A superfamily are involved both in the regulation of protein synthesis, predetermining the rate of hydrolysis of messenger RNA, protection of the macroorganism from RNA-containing viruses [39], and in a variety of physiological and pathological processes (Table 2) [8, 15, 39].
Anti-infective effect of RNASE2, RNASE3 and RNASE7
Almost from the moment of identification of RNASE3 and RNASE7, it was found that they have bactericidal activity against gram-negative and gram-positive bacteria [1, 8, 53]. RNASE3 has been shown to suppress the growth of Staphylococcus aureus and Escherichia coli colonies [23, 40]. RNASE7 is characterized by both very powerful antibacterial activity, superior to defensins, against various pathogenic microorganisms, including Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Enterococcus faecium, Propionibacterium acnes, and antifungal activity, in particular against Candida albicans [23, 53] . Jürgen Harder and Jens-Michael Schröder showed [23] that RNASE7 exhibits very high bactericidal activity against vancomycin-resistant Enterococcus faecium (LD90 < 0.03 microM), even more pronounced than human b-defensin-3 (LD90 ~ 0.5 microM ). In all likelihood, RNASE3 plays a significant role in the process of sanogenesis of acute infectious diseases of the respiratory tract caused by bacterial intracellular infections. Thus, Chun-Jen Chen et al. [47] showed that in the blood serum of children with mycoplasma pneumonia, the concentration of RNASE3 significantly increases, reaching a level of 28.88 μg/ml compared with the norm of 4.48 μg/ml.
The molecular mechanisms of the bactericidal action of RNASE3 and RNASE7 remain unclear. Currently, there are several hypotheses. It is assumed that the RNASE3 protein, by destabilizing the lipid layer of membranes, organizes pores in the membranes of bacterial cells [51, 53] and activates bacterial autolysis [22]. Yu-Chie Huang et al. [46] believe that the bactericidal effect of RNASE7 is associated with the lytic activity of four specifically grouped lysine residues (Lys1, Lys3, Lys111, Lys112). The RNASE7 molecule can bind with cationic amino acid residues to negatively charged components of the bacterial membrane and lyse the bacterial membrane. However, the authors do not exclude the possibility of interaction of RNASE7 with RNASE7-binding proteins of the bacterial membrane, which are subunits of bacterial membrane pores. This interaction leads to the opening of the pores and the fatal release of the intracellular contents of microorganisms.
RNASE2 and RNASE3 have antiviral activity against respiratory syncytial virus [13, 17, 41, 54]. Simon Phipps et al. [20] showed that respiratory syncytial virus ssRNA, being a TLR7 ligand, induces the functional activity of eosinophils, in particular, specific degranulation, release of ribonucleases, and expression of the phagocytic receptor CD11b. The participation of eosinophils in the response to infection is accompanied by faster elimination of the virus from the body. It has also been demonstrated that recombinant RNASE2 reduces the ability of respiratory syncytial virus to enter epithelial cells [38].
RNASE2 also has antiviral activity against the human immunodeficiency virus [30]. In all likelihood, the antiviral effect of RNASE is based not on the direct proteolytic effect of ribonuclease on extracellular forms of the virion, but on the interaction of RNASE with both the target cell and viral surface components [55]. RNASE7 has no established antiviral effect [53].
Immunomodulatory effect of RNASE2
Currently, RNASE2 has been shown to have a direct effect on the immune system. Thus, it has been established that RNASE2 is an exceptional chemoattractant with a highly specific effect on dendritic cells (DC). RNASE2, directly or indirectly interacting with an unidentified pertussis-sensitive G-protein receptor, causes activation of p44/42 MAPK, causing DC chemotaxis [18]. The strength of chemotactic activity of RNASE2 is comparable to the effect of stromal-derived growth factor 1a/SDF-1a. RNASE2 also determines the maturation of DC (CD34), which are responsible for the production of physiologically significant amounts of IL-6, regulator of activation of normal T-cell expression and secretion (RANTES, CCL5), TNF-a, macrophage chemoattractant protein 2 (MCP-2), macrophage inflammatory protein 1a (MIP-1a, CCL3) and interferon-g-inducible protein 10 kDa (IP-10, CXCL10) [39]. It has been demonstrated that RNASE2 is an endogenous ligand of TLR2, interaction with which leads to activation of the adapter molecule MyD88 and excitation of intracellular signaling pathways. RNASE2 promotes the induction of a Th2 response [19, 39]. Some researchers, not without reason, attribute RNASE2 to a special molecular group, whose representatives are called death-associated molecular patterns (DAMPs). Representatives of the DAMP group are involved in the functioning of the early warning signaling system of the immune system. When released from the intracellular space, DAMPs exhibit high pro-inflammatory or even damaging activity [6, 34].
Involvement of RNASE3 in lung tissue remodeling
RNASE3 stimulates the production of TGF-b1 by fibroblasts of lung tissue, predetermining the development of fibrosis [16]. Currently, the question remains unexplored whether the cationic protein RNASE2, like RNASE3, EPO and MBP [35], is involved in the remodeling of lung tissue.
Conclusion
The phylogenetically ancient RNase-A superfamily, which in humans is organized into 13 representatives of endonucleases, is involved in many physiological processes of the body, including the regulation of transcription and translation, angiogenesis, cell apoptosis, nonspecific protection of the respiratory tract and remodeling of lung tissue. RNASE3 and RNASE7 have pronounced bactericidal activity against gram-negative and gram-positive bacteria. RNASE3 plays an essential role in the process of sanogenesis of acute infectious diseases of the respiratory tract caused by bacterial intracellular infections. RNASE2 and RNASE3 have antiviral activity against respiratory syncytial virus. RNASE2 has direct effects on the immune system, promoting DC maturation and chemotaxis, as well as the induction of Th2 responses. The development of new drugs based on Rnase-A, which for the most part are characterized by high activity and minimal immunogenicity, will help solve many anti-infective and oncological problems [45]. In particular, the drug Ranpirnase (Onconase), derived from frog RNase (Rana pipiens) (ONC, Alfacell Inc, New Jersey, USA) is currently undergoing clinical trials for the treatment of malignant mesothelioma and lung cancer [46].
