Designing the Ribonucleases: The Scissor Enzymes

Ribonucleic acids, which serve as templates for protein synthesis, make an important part of the cellular “biochemical industry”. However, in some situations, such as infections or other cell disorders, it is necessary to shut down the synthesis of a particular protein. Destruction of mRNA template encoding for that protein by means of specific enzymes is one of the opportunities to that end.

It comes as no surprise that the researchers started considering the possibility of drug design on their basis. Those fundamentally novel drugs would be able to selectively destroy viral RNAs, while leaving all critically important cellular RNAs intact.

Genius in simplicity

Ribonucleases are the enzymes belonging to a wider class of enzymes, hydrolases, known to break up RNA. RNAses are widely represented in the cells of all living organisms. The best known RNAse is the pancreatic enzyme, RNAse A, isolated from calf pancreas.

When one mentions this enzyme, there is no way to avoid using “first”. RNAse A was the first sequenced enzyme —its complete amino acid sequence was determined in the early 1960s. When in 1969 the first chemically synthesized enzyme was obtained, it turned out to be pancreatic RNAse as well. Moreover, it was the first enzyme introduced into clinical practices as an antiviral drug. Despite the digression from the main subject, it is worth to note Onconase, an enzyme similar to RNAse A but isolated from the oocytes of the frog, Rana pipiens. An apparently successful completion of clinical trials with this novel antitumor drug offers a chance to speculate upon the generation of a new class of anticancer drugs, a serious alternative to conventional chemotherapy.

Natural endonucleases are the enzymes able to destroy superfluous or foreign (viral) RNAs, which are present in the cells of every living organism

The next milestone on the way of conversion of RNAses into therapeutic drugs was set up in the 1980s by an American biochemist, R. Breslow, who found out that the small molecules of heterocyclic compound, imidazole, were able to split RNA in a similar way as the natural pancreatic RNAse does. He realized that the man-made enzymes can be produced on the basis of low-molecular compounds possessing the functions of natural catalysts. The idea was as simple as it was brilliant: it seemed sufficient to arrange the catalytically active groups derived from active centers of natural enzymes in a particular spatial order, and a synthetic analog of the enzyme would be there.

Ribonucleases werethe first enzymes for which low-molecular synthetic mimics were designed

Several years later R.Breslow successfully implemented his idea in experiment. Despite the fact that his “man-made” ribonucleases were able to cleave phosphodiester bonds only in a specific substrate particularly synthesized for the purpose of this experiment, and not in naturally occurring RNAs, the commencement was set.

The first publications on artificial ribonucleases concurred with the research boom in another interesting field, the so-called antisense oligonucleotides. The research on the latter was carried out, in particular, at the Division of Biochemistry of the Institute of General Chemistry of the Siberian Branch of Russian Academy of Sciences, headed by academician D. G. Knorre*. It is worth to stress here that more or less extensive nucleic acid sequences are unique in their structure. This feature makes feasible the directed intervention by complementary (antisense) oligonucleotides able to specifically recognize and bind the particular regions of a specific nucleic acid.

Linking together the two technologies—the antisense approach and that of low-molecular artificial ribonucleases—promised a breakthrough in the manufacture of synthetic enzymes for medical purposes. As many other bright ideas, this one has dawned on many researchers simultaneously, so no wonder that experiments in this field were commenced in the 1990s in labs all over the world—in Japan, Europe, the United States, and Russia.

None the worse than natural

Unfortunately, the implementation of antisense technology in its original version involved a number of problems, due to which the development of real antisense-based drugs has been indefinitely delayed. In the first place, these problems were associated with the penetration of synthetic oligonucleotides across the plasma membrane as well as with the oligonucleotide stability in living organisms.

Analogs of natural enzymes are a convenient tool for molecular biology and genetic engineering

Research in this field was further curtailed by a reason that was not technological at all. The implementation of antisense technology seemed so feasible at its initial stage that multiple companies promising to design a cure-all drug in the nearest future started to crop out. The field has been massively funded, however, when the expected financial return did not occur, the companies rapidly went bankrupt, and the situation was further deteriorated by a significant curtail of fundamental research funding in the field. In Russia the decline was further aggravated by the overall collapse of national economy followed by a heavy cut-back of financial support of the research.

The obstacles were evident although seemed to be only temporary when Novosibirsk researchers from the Institute of Chemical Biology and Fundamental Medicine SB RAS set about their project on the design of artificial endonucleases on the basis of oligonucleotide conjugates. Unfortunately, the perfect vehicles for our ribonucleases – the synthetic analogs of nucleic acids capable of effortlessly crossing the cell membrane and resistant to the naturally occurring enzymes—have not been designed yet.

Despite all obstacles, the artificial ribonucleases project was not stopped and initially focused on artificial reconstruction of RNAse A. The lab of organic synthesis produced dozens of candidate artificial enzymes. All of them were, to a variable degree, capable of splitting phosphodiester bonds in different naturally occurring RNA molecules, while leaving the DNA intact. Every molecule of those man-made enzymes was able to catalyze the break-up of tens and hundreds of bonds in an RNA molecule.

Artificial endonucleasescan become highly selectiveand low toxic antiviral, antitumor,or genetic disease treatment drugs

Only upon a detailed analysis of the RNA splitting by natural and man-made enzymes, the researchers were able to definitely testify to the making of the structural and functional RNAse A analog. The point is that besides their high efficiency, the natural enzymes are intrinsically capable of hydrolizing the bonds between adenine and cytosine better than those between adenine and uracil. The artificial enzymes proved to simulate the work of the natural ones in every detail.

Apparently, the most efficient synthetic analogs were 2-3 orders of magnitude less active than the natural enzyme. However, considering the fact that RNAse A catalysis of RNA degradation yields a 1014 acceleration of the rate of degradation, the achievement looked impressive. In addition, artificial ribonucleases have a number of advantages: first, they are way cheaper than the natural enzymes. Further, they demonstrate an extraordinary stability and tolerate a wide range of reaction conditions.

Universal soldiers

Where might those artificial ribonucleases be of use? Primarily, they can replace the natural ones in the applications where the latter are currently used. An example would be the genomic DNA isolation and sample preparation for PCR tests widely used in different fields, from medical testing to forensic investigation.

Artificial ribonucleases are completely inert to the other biomolecules, particularly, proteins. This feature makes them indispensible for vaccines manufacture. In order to produce a safe vaccine, virus particles are supposed to be “disarmed” in a sense that their genetic information should be destroyed. Currently, formaldehyde combining high efficiency and low cost is being used for this purpose. Unfortunately, formaldehyde partially destroys virus proteins to which the immune response is elicited in vaccinated persons. This naturally results in lower vaccine efficiency. Man-made ribonucleases are lacking this disadvantage, as one can judge from the preliminary experiments with influenza virus infection of lab mice. The mortality rate in the group of animals vaccinated with rybozyme-treated vaccine was significantly lower than in the control animal vaccinated with a conventional preparation.

After all, man-made enzymes may successfully replace the natural ones in RNA-protein complex studies. For example, they can offer an answer to the problem of the source of protoferment specificity at the origin of planetary life. Possible answers could be their RNA-binding center, which recognizes specific bonds in the RNA molecule chain (as it is true for the contemporary ribonucleases); otherwise, the catalytic centers of enzymes could have initially possessed that function. This research avenue is directly linked with one of the central evolutionary problems, the origin of the protein form of life.

A bull’s-eye shot

An important discovery resulted from the studies of long RNAs (for example, influenza virus genome fragments) splitting by synthetic ribonucleases, which turned out to hydrolyze not all theoretically accessible phosphodiester bonds but only those localized in specific spatial RNA segments. This offered an opportunity to reconsider the idea of selective antiviral drug design but based on the use of low-molecular compounds, which allowed us to avoid the antisense technology. Those enzymes do not require helper compounds to recognize and destroy the targeted RNA.

Many doubted the success of that venture, particularly, taking into account the costs of comprehensive antiviral activity trials. A helpful occasion was a meeting of Novosibirsk researchers with virologists from Odessa Antiplague Institute, who have agreed to perform such trials for 15 drugs. This happened to be a bull’s-eye shot: ten out of fifteen compounds under test demonstrated clear-cut anti-influenza activity. Moreover, the activity of top four drugs was comparable or even 50—100-fold higher than that of known anti-flu medications, such as remantadine or deiteforin. Provided the low toxicity of tested ribonucleases, this was a clear success, which drew the attention not only of Ukrainian but also of Belorussian and Italian colleagues, as well as of some pharmaceutical companies. The interest was also expressed by regional authorities. However, the most valuable piece of appreciation came from the founding-father of artificial enzymes, R. Breslow, who was quoted to express his “admiration for the chemists from Novosibirsk who have managed to achieve such a success”!

References
M. Zenkov, V. Silnikov, and V. Vlasov. Chemical ribonucleases // Molecular Biology—1998—V. 32.—№ 1. Pp. 62— 70.
I. Garipova and V. Silnikov. Synthetic site-specific ribonucleases developed on the basis of oligonucleotide conjugates with metal-independent organic catalyzers of phosphodiester bond hydrolis // Bulletin of the RussianAcademy of Sciences. Chemistry series. —2002—№ 7. Pp. 1025—1030
V. Silnikov and V. Vlasov. Design of reagents for the directed cleavage of ribonucleic acids // Advances in Chemistry.—2001. —v.70—№ 6. Pp. 562—580.
Kuznetsova I. L., Silnikov V. N. Small ribonuclease mimics. Ed. Marina A. Zenkova «Artificial Nucleases» in Nucleic Acids and Molecular Biology—2004.—V. 13. Pp. 111—128, Springer Verlag Berlin.

* For more details, see SCIENCE First Hand, # 2 (14), 2007