In a recent study published in Nature Communications, researchers investigated the ability of kinetin (N6-furfurylaminopurine, MB-905) to inhibit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proliferation in vitro.
The continual emergence of SARS-CoV-2 variants has threatened the efficacy of coronavirus disease 2019 (COVID-19) vaccines and therapeutic agents such as monoclonal antibodies, warranting the development of novel antiviral drugs. Severe SARS-CoV-2 infections are characterized by virus-induced inflammation and immunological dysfunction. Therefore, drugs with anti-inflammatory and antiviral properties may widen the therapeutic landscape of COVID-19.
About the study
In the present study, researchers evaluated the therapeutic potential of kinetin (MB-905) against SARS-CoV-2 infections in vitro.
To identify molecules with anti-SARS-CoV-2 activity, a molecular library comprising nitrogenous nucleosides, nucleotide analogs, and bases was tested using SARS-CoV-2-infected huh-7 (human hepatoma cell line) cells, Calu 3 (human lung epithelial cell line) cells and primary monocytes. Next, the team investigated whether kinetin and its associated compounds and molnupiravir could decrease SARS-CoV-2 ribonucleic acid (RNA) levels and SARS-CoV-2-induced inflammatory marker expression in human primary monocytes. Cytotoxicity assays and yield-reduction assays were performed.
Since kinetin was the most likely pro-drug of its corresponding nucleotide triphosphate, the team investigated whether inhibition of SARS-CoV-2 proliferation was due to a direct action on SARS-CoV-2 RNA polymerase. To test whether kinetin would be incorporated into the SARS-CoV-2 genome from kinetin-treated and SARS-CoV-2-infected Calu-3 human epithelial cells, anti-kinetin immunoglobulin G (IgG) was utilized for immunoprecipitating (IP) SARS-CoV-2 RNA, followed by RNA quantification using quantitative reverse transcription-polymerase chain reaction (RT-qPCR) analysis.
The full-length SARS-CoV-2 genome from MB-905- and nil-treated SARS-CoV-2-infected cells were sequenced, and the double-stranded RNA structure was modeled. Further, the team investigated whether the co-inhibition of SARS-CoV-2 exonuclease increased the antiviral activity of the MB molecules in vitro. The team combined MB-905 with suboptimal dosages of control RNA polymerase inhibitors, DTG (dolutegravir), RTG (raltegravir), PIB (pibrentasvir), OMB (ombitasvir), or daclatasvir, to evaluate potential synergistic effects on SARS-CoV-2 replication using SARS-CoV-2 ribonucleic acid polymerase inhibition assays.
Additional preclinical studies for kinetin were performed, including pharmacokinetics analysis in rats and mice, maximum tolerable dosage, 28.0-day repeated oral dosage, toxicokinetics analysis, in vitro and in vivo potential cardiotoxicity, and serum albumin binding.
Kinetin inhibited SARS-CoV-2 replication in a dosage-based fashion in calu-3 and huh-7 cells. On infected monocytes, 10.0 µM of kinetin suppressed SARS-CoV-2 replication, interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) levels. However, N9 position blockade or the addition of an unphysiological adenine excess impaired the antiviral activity of MB-905, indicating that kinetin might need to be activated by host cells via the APRT (adenine phosphoribosyl transferase) pathway. The riboside 5′-triphosphate of kinetin inhibited SARS-CoV-2 ribonucleic acid polymerase with a half-maximum inhibitory concentration (IC50) three-fold greater than that of remdesivir controls and induced error-prone SARS-CoV-2 replication.
The concomitant inhibition of SARS-CoV-2 exonuclease, the proofreading enzyme for correcting erroneous nucleotide insertion during SARS-CoV-2 RNA replication, enhanced SARS-CoV-2 inhibition by kinetin. Kinetin showed good oral absorption, with metabolite stability, achieving durable serological and pulmonary concentrations. Additionally, the drug showed no cardiotoxic or mutagenic effects. SARS-CoV-2-positive K18-human angiotensin-converting enzyme 2 (hACE2) mice and 140 mg/kg (in one or two daily doses) of kinetin-treated hamsters showed reduced SARS-CoV-2 proliferation, pulmonary necrosis, inflammation, and hemorrhage. MB-711, MB-801, and Kinetin showed comparable SARS-CoV-2 proliferation inhibition in calu-3 human epithelial cells.
Even though adenine-associated MBs were less potent than RDV, MB-711, MB-801, and MB-905 showed 6.0-fold to 40.0-fold greater selective indices compared to molnupiravir and one order of magnitude lesser cytotoxicity compared to molnupiravir. Further, MB-711, MB-801, and MB-905 lowered cell-related SARS-CoV-2 RNA levels in a concentration-dependent fashion, and molnupiravir demonstrated high cytotoxicity in myeloid cells. Remarkably, SARS-CoV-2 RNA extracted from the anti-kinetin IP of kinetin-treated and Calu-3 human epithelial cells infected by SARS-CoV-2 was enhanced by >10.0-fold SARS-CoV-2 RNA levels in comparison to nil-treated and control cells.
Thus, kinetin appeared to be inserted into the SARS-CoV-2 genome upon kinetin treatment. Sequencing analysis showed greater alterations at the level of the initial base, particularly cytosine (C) → adenine (A) and thymine (T)uracil (U) → adenine. The combined effects of 4.0 SARS-CoV-2 passages among calu-3 human epithelial cells in molnupiravir and kinetin presence were similar. The N6-furfuryl group of kinetin appeared to impact the structure and pairing of bases between the two RNA strands.
Combining RNA polymerase and repurposed SARS-CoV-2 non-structural protein 14 inhibitors enhanced kinetin potency. Effective inhibition was achieved at the EC99 level after combining kinetin with human immunodeficiency virus (HIV) integrase inhibitors. MBs inhibited SARS-CoV-2 proliferation by 1.0-log10 at 10.0 µM, which doubled on combining with 5.0 µM DTG or RTG. Kinetin showed >50.0% bioavailability when administered orally in mice in vivo. Upon oral administration, kinetin was converted to its active metabolite by phosphorylation in the respiratory tract for potent RNA polymerase inhibition and, therefore, SARS-CoV-2 replication inhibition.
Overall, the study findings highlighted the opportunity for the rapid clinical development of a novel therapeutic option for COVID-19, given kinetin has been clinically investigated for familial dysautonomia, a rare genetic illness, at daily concentrations of ≥11.0 mg/kg, beyond the estimated concentrations of anti-inflammatory and antiviral actions-mediated SARS-CoV-2 inhibition.