APPROACH
One of the ways to create new antiviral drugs that are effective in
treating diseases caused by RNA viruses is the selection of
RNA-dependent RNA polymerase (RdRp) inhibitors using molecular modeling
methods [16–18]. Docking led to the development of molnupiravir, the
only drug effective in mitigating the effects of SARS-CoV-2 [19,20].
Although the drug alleviates symptoms, it does not significantly reduce
patient mortality from complications [21]. In addition, it is effective
only in the initial stages of infection. It was previously discovered
that cobolamines in high doses can inhibit the replication of several
viruses in cell cultures [6,22]. The mechanism of action of cobalamin
was determined to be inhibition of RdRp through simultaneous
interaction with specific aptamer fragments of viral RNA and the
peptide fragment of RdRp. At the same time, the dose of cyanocobalamin
was quite high and exceeded what was permissible for use in vivo. We
hypothesized that it is possible to increase the affinity of cobalamin
for the binding sites of RNA aptamers and RdRp by carboxylation of
ribose residues. This may increase the affinity of such a product for
other binding sites and reduce the effective dose of the drug [23].
Figure 1.
An example of the combinatorial synthesis of one of the MoLviRx variants (modifiers succinic and acetic
anhydrides)
As can be seen from Figure 1, the carboxylation reaction results in a
mixture of derivatives substituted at the ribose residue with different
combinations and substitutions. A total of 8 different cyanocobalamin
derivatives are formed. Each component of this mixture synergistically
enhances the effects of the others because it has different tropism to
different points of interaction with the RNA-aptamer (Fig. 2-3).
Preliminary results
In silico study
Based on the results of molecular modeling (Yasara, Autodock Vina,
aptamer 1et4, cyanocobalamin alone (Fig. 2 and dynamic structure
MoLviRx from 8 derivatives Fig. 3), we have shown that the degree of
affinity of B12 to RNA aptamer fragments of viral RNA and the protein
component of the SARS-CoV-2 RdRp can be increased by several times if
the ribose fragment of cyanocobalamin is combinatorial carboxylated
with several modifiers simultaneously. Such structures containing a
mixture of combinatorial derivatives with the same core but different
dicarboxylic acid residues in the ribose fragment are called dynamic.
They can form supramolecular structures with the revert RdRp and viral
RNA complex and integrate into various regions of this structure,
different from those unmodified B12 binds.
Figure 2. Yasara, Autodock Vina, multiligand docking,
aptamer 1et4, cyanocobalamin alone. 3 binding points.
As can be seen from Figure 1, cyanocobalamin has 3 binding points to
the aptamer on its surface, while the dynamic structure MoLviRx (Figure
2) binds to the aptamer at 7 points. Thus, MoLviRx's multiple targeting
makes it possible to reduce its effective dose and reduce the
likelihood of the virus adapting to the drug.
We have successfully synthesized a novel structure - dynamic
carboxylated cyanocobalamin, which includes a mixture of 8 derivatives
(MoLviRx ). Combinatorial synthesis involved using non-equivalent
amounts of two modifiers simultaneously - maleic and succinic
anhydride. This innovative approach allows all 8 derivatives to
function as a combination chemotherapy, targeting different regions of
viral RNA aptamers and blocking various RNA replication mechanisms.
This unique strategy eliminates the risk of drug resistance
development. Initial in vitro studies have revealed a broad spectrum of
action of MoLviRx on viruses such as influenza (H1N1-09), New Castle
disease virus (live vaccinia virus Merck), and coronavirus (str.D52) at
doses 100 times lower (8-10 mcM/mL) than the original Cyanocobalamin
(900-1200 mcM/mL). A culture virus titer decrease was observed from 8
lg to 2 lg. The MoLviRx product we developed is protected
by two valid patents US11160878B1 and US11191806B2. In addition to its
higher antiviral activity, MoLviRx has shown significant
nephroprotective properties and a higher affinity for renal megalin
than native B12.
Next, we provide an example of studies of the developed product's
antiviral activity on the TGP pig gastroenteritis coronavirus model.
Figure 3. Yasara, Autodock Vina, multi-ligand docking,
aptamer 1et4, MoLviRx 7 binding points
Antiviral activity in vitro to coronavirus str.D52
The purpose of the study is to determine the virucidal activity of the
MoLviRx substance in a model of coronavirus transmissible
gastroenteritis of pigs (TGP).
Materials and methods
Virus: TGEV - the etiological agent of transmissible gastroenteritis of
pigs (TGP) - a highly contagious intestinal disease of pigs
(coronavirus strain str.D52) [24]. D52-5 (BRE79) - is a highly
pathogenic virus for pigs of all ages at 5 passages in transplanted
monolayer culture of testicular cells of piglets ST. The tropism of the
virus in the gastrointestinal tract and respiratory tract is shown. Dr.
Hubert Laude provided the strain from the Laboratory of Molecular
Virology and Immunology of the INRA Center for Biotechnology in
Jouy-en-Josas (France). Cell culture: CHEB - is a transplanted culture
of kidney cells of a pig embryo [25]; substance: MOLVIRX -
dynamic derivatives of cyanocobalamin 1% solution.
Determination of antiviral action. A str.D52 with an infectious titer
8,5 lg ID50 was used. These studies aim to identify the antiviral
effect of the MoLviRx substance. The viral suspension, at a dose of
TCD50/ml, was incubated with a cell culture CHEB for 4 hours. After
that, 0.001% and 0.0001% (up to the final concentration in the medium)
MoLviRx solution was added to the culture, and the cells were allowed
to continue incubating. The infectious titer (viral RNA copies in terms
of titer) of the virus was then determined the next day by RT-PCR. A
decrease in the infectious titer of the virus by 1.5-2.0 lgTCD50
compared to the control indicates the severity of the antiviral effect.
Detection of RNA of transmissible gastroenteritis virus of pigs str.D52
by reverse polymerase chain reaction (RT-PCR). RNA
isolation was performed using a set of "Fish-sorb" following the
manufacturer's instructions (AmpliSens). The reverse transcription
reaction was performed using a kit called "RevertAidTM H Minus First
Strand DNA Synthesis Kit" according to the manufacturer's instructions
(ThermoScientific) [26]. For PCR were used gene-specific nucleoprotein
oligonucleotide primers of the following sequence: direct Uni_1
(5'-TGCACTGATCAATGTGCTAG-3) and reversed Uni 2 (5'
TGAAAACACTGTGGCACCCТТ-3") [27]. A fragment amplified by size 309 P.M.
M-marker "100 bpPlus DNA Ladder" ("ThermoFisherScientific") [28]. TGP
virus from different cultures was propagated in CHEB culture, and the
amplification products were by distributing DNA fragments in 1.5%
aerosol gel.
Statistical processing of research results. The digital material
presented in the work is processed statistically. Statistical
evaluation of the significance levels of differences in the obtained
figures was performed using Student's t-test using Microsoft Excel and
Microcal Origin. Differences at p <0.05 were considered authentic.
Research results
The results showed that the MoLviRx substance inhibited the
reproduction of extracellular coronavirus TGP by 5.0 lg at dose 0,0001%
min exposure and 6.0 lg at dose 0,001%.
Conclusions
MoLviRx's antiviral activity is dose-dependent. Research has determined
that it is an effective antiviral substance and can be used to develop
new anticoronaviral drugs.
Team Expertise: Our team uniquely integrates the Theory of Inventive
Problem Solving (TRIZ) into pharmaceutical development and includes
Ph.D. scientists with diverse educational and practical backgrounds. We
are composed of experts in pharmacology, microbiology, and drug design
and have recently secured 10 new patents in drug design, including
three in antivirals, bringing our total to over 220 granted patents,
with several industry-implemented innovations. Our principal
investigator and consultant are the authors of the proposal’s granted
patents and peer-reviewed published articles. Key personnel include a
clinician experienced in anticancer trials and multicenter studies. Our
diverse expertise and systems approach position us to tackle complex
challenges effectively.