MTr1 (2'-O-ribose methyltransferase 1)

In step three, researchers evaluated which of the identified compounds had an inhibitory effect at the lowest concentration and finally identified Trifluoromethyl-tubercidin (TFMT) as the most effective compound (Fig. 2AOpens in image viewer and fig. S8, A and B). We confirmed the binding of TFMT to the SAM binding pocket of MTr1 and recombinant MTr1 with in silico docking and in vitro thermal shift assay (Fig. 2BOpens in image viewer and fig. S8C), respectively. Furthermore, we confirmed TFMT inhibition of the MTase activity of the recombinant MTr1 protein (fig. S8D). We also confirmed TFMT was active against IAV and IBV but not HAZV or STBV (Fig. 2COpens in image viewer), exactly matching the phenotypes of MTr1 deficiency shown in Fig. 1Opens in image viewer and fig. S6. The median inhibitory concentration (IC50) for TFMT against IAV infection was 0.30 μM, and no notable in vitro toxicity was observed in the range of effective concentrations, as measured with water-soluble tetrazolium (WST)–8 cell viability assay (Fig. 2DOpens in image viewer). TFMT treatment also greatly inhibited IAV replication when administered 3 to 4 hours after infection; however, the effect was reduced or not visible if the drug was administered later (fig. S8E) [1].

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Anti-influenza efficacy of Trifluoromethyl-tubercidin/TFMT [1]
Next, researchers examined the anti-IAV activity of Trifluoromethyl-tubercidin/TFMT in normal human bronchial epithelial (NHBE) cells. The RNA and protein levels of IAV (H1N1, PR8) were significantly reduced by TFMT treatment in a dose-dependent manner (Fig. 2, E and FOpens in image viewer). Histological analysis also revealed a profound reduction of the IAV NP levels in TFMT-treated NHBE cells without cytotoxicity (Fig. 2GOpens in image viewer). TFMT treatment did not inhibit HAZV replication (Fig. 2HOpens in image viewer), indicating that specific efficacy against certain viruses by this compound was retained even in human primary cells. Because this compound was effective in human NHBE cells, this prompted us to evaluate TFMT in human lung explants as an ex vivo setting (fig. S9A). We infected lung tissues with IAV (H1N1, seasonal isolate in 2019), and the viral titer in the supernatant was determined with plaque assay at 1, 24, 48, and 72 hours after infection. As shown in Fig. 3AOpens in image viewer, the titer in the nontreated samples increased >105 plaque-forming units (PFU)/ml at 48 or 72 hours after infection, whereas the titer from the TFMT-treated lung explants remained <103 PFU/ml, indicating 100- to 1000-fold suppression by the treatment. The sum of the titers of all six independent donors revealed differences between the control and TFMT treatment–reduced IAV titers in culture supernatants (Fig. 3BOpens in image viewer and fig. S9B). TFMT treatment at 12 hours IAV after infection significantly impaired virus growth in human lung explants (fig. S9C). Consistent with virus titers observed, no IAV NP–positive cells or morphological changes were observed in IAV-infected lung tissues treated with TFMT. These results indicate that TFMT inhibits replication of the seasonal IAV isolate ex vivo and shows potential for clinical translation.

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TFMT/Trifluoromethyl-tubercidin inhibits IAV cap snatching [1]
As shown in Fig. 4AOpens in image viewer, the effect of TFMT treatment on IAV replication was independent of IFIT1-dependent sequestration of RNA or RIG-I or MDA5 signaling. We found no replication of IAV in RIG-I–MTr1 double KO cells or IFIT1-MTr1 double KO cells, similarly to MTr1 KO cells (fig. S11, A and B). In addition, IFN signaling blockade by JAK inhibitor tofacitinib was not accompanied by IAV replication in MTr1 KO cells (fig. S12, C and D). Influenza A virus with a deletion of nonstructural protein 1 (IAVΔNS1) is known to induce high IFN responses, and its replication was prevented in MTr1-deficient cells without IFN and ISG (interferon-stimulated gene) induction but was rescued by MTr1 overexpression (fig. S2, D and E). TFMT treatment did not induce IFN-β or antiviral ISGs in A549 cells nor in PBMC (Fig. 4BOpens in image viewer and fig. S12). These results confirm that the observed antiviral effect does not depend on activation of antiviral IFN responses. Replication of IFN-sensitive non–cap-snatching RNA viruses such as Sendai virus (SeV), vesicular stomatitis virus (VSV), and encephalomyocarditis virus (EMCV) were not altered by TFMT treatment (Fig. 4COpens in image viewer)—likewise, in MTr1 KO cells (fig. S13). Expression of IAV mRNA (segment 1) snatched specifically from U2 spliceosomal snRNA was impaired by TFMT treatment, similar to the effect of MTr1 deficiency. Hence, we conclude that TFMT treatment inhibits IAV replication by directly affecting the cap-snatching activity of IAV and not through immune modulation.

Researchers tested the in vivo efficacy of this compound in mice. They first confirmed that TFMT/Trifluoromethyl-tubercidin showed inhibitory activity in the IAV-infected mouse cell line LA-4, albeit with lower potency (IC50 = 7.7 μM) than in human cells (fig. S10A). Next, we assessed in vivo toxicity in mice with intranasal inoculation once a day for 2 days. Although treatment with the parental compound tubercidin caused substantial weight loss of mice, we did not observe any weight loss or cytotoxicity in lungs with the selected derivative TFMT (Fig. 3DOpens in image viewer and fig. S10B). Last, we examined the effect of TFMT treatment at 2 days after infection with IAV. At this point, NP and PB2 mRNA levels were significantly reduced by TFMT treatment in mouse lungs, indicating that the trifluoromethyl substitution of tubercidin reduces in vivo toxicity to levels we could not detect, but retains anti-IAV efficacy (Fig. 3EOpens in image viewer). We also confirmed the antiviral efficacy of baloxavir marboxil (BXM) in vivo in this setting (fig. S10C). Taken together, TFMT shows potential to inhibit IAV replication in all tested systems, including a human cell line and NHBE cells in vitro, human lung explants ex vivo, and mice in vivo [1].

[1]. Inhibition of cellular RNA methyltransferase abrogates influenza virus capping and replication. Science. 2023 Feb 10;379(6632):586-591.

Orthomyxoviruses and Bunyaviruses steal the 5' cap structure of the host RNA to initiate their own transcription, a process known as "cap stealing". We found that RNA modification of the cap structure by the host 2'-O-ribose methyltransferase 1 (MTr1) is crucial for the initiation of influenza A and B virus replication, but not for other cap stealing viruses. We identified a streptococcal natural product derivative, trifluoromethyltuberculin (TFMT), by interacting with the S-adenosine-L-methionine binding pocket of MTr1 to inhibit MTr1 and thus limit influenza virus replication. Mechanistically, TFMT weakens the binding of the host cap RNA to the basic protein 2 subunit of the viral polymerase, a phenomenon observed in human lung tissue explants and in vivo mice. TFMT has a synergistic effect with approved anti-influenza drugs. [1]

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There are currently approved drugs targeting influenza virus proteins; however, all drugs have been found to have drug-resistant viral mutants. Host-targeted antiviral drugs have a low likelihood of inducing resistance. The host mitogen-activated protein kinase inhibitor ATR-002 has been shown to have broad efficacy against various RNA viruses, including influenza and SARS-CoV-2, through its mechanism of action by inhibiting viral replication and modulating the inflammatory response. In this study, we demonstrated the anti-influenza efficacy of the cellular RNA methyltransferase inhibitor trifluoromethyltuberculin/TFMT. Considering the potential toxicity of long-term host-targeting, dose reduction and combination with other approved antiviral drugs (such as BXM and oseltamivir) are feasible. We found that TFMT is a highly specific and non-toxic MTr1 inhibitor that specifically inhibits the replication of cap-dependent viruses (such as IAV and IBV) (Figure S19). Overall, our data indicate that TFMT's inhibition of MTr1 cap-dependent replication specifically inhibits the replication of various IAV and IBV strains, including seasonal H1N1 isolates and a highly pathogenic avian influenza virus resistant to BXM. Mechanistically, TFMT leads to MTr1 dysfunction and cap0 RNA accumulation, thereby impairing the binding of the viral polymerase subunit PB2 to the host cap RNA, thus hindering IAV polymerase-mediated cap capture and viral RNA synthesis. TFMT and BXM have a synergistic effect because the two drugs target different polymerase subunits, PB2 and PA, respectively. TFMT-dependent IAV inhibition is independent of RIG-I and IFIT1-mediated innate immune responses, and TFMT has no effect on the replication of interferon-sensitive viruses (such as VSV and EMCV). Comparison of the PB2 subunits of influenza virus and THOV shows that the primary structure of the cap RNA binding region (e.g., N1-2′-O-Me interacting amino acids) of IAV PB2 is conserved in IBV PB2, but not in ICV, IDV, or THOV PB2 (Figure S14B). Furthermore, IAV requires 10 to 13 nucleotides to capture the cap structure, while THOV is reported to capture the 5′-terminal m7G cap residue. The difference in the mechanism of cap-capping by viral polymerases may explain the specificity of TFMT restriction to IAV and IBV. [1]

C12H13F3N4O4

334.251232862473

334.088

1854086-05-7

118636125

White to off-white solid powder

-0.5

4

10

2

23

443

4

C1=C(C2=C(N=CN=C2N1[C@H]3[C@@H]([C@@H]([C@H](O3)CO)O)O)N)C(F)(F)F

RSOXZOFDCJMRMK-IOSLPCCCSA-N

InChI=1S/C12H13F3N4O4/c13-12(14,15)4-1-19(10-6(4)9(16)17-3-18-10)11-8(22)7(21)5(2-20)23-11/h1,3,5,7-8,11,20-22H,2H2,(H2,16,17,18)/t5-,7-,8-,11-/m1/s1

(2R,3R,4S,5R)-2-[4-amino-5-(trifluoromethyl)pyrrolo[2,3-d]pyrimidin-7-yl]-5-(hydroxymethyl)oxolane-3,4-diol

Trifluoromethyl-tubercidin; 1854086-05-7; (2R,3R,4S,5R)-2-[4-amino-5-(trifluoromethyl)pyrrolo[2,3-d]pyrimidin-7-yl]-5-(hydroxymethyl)oxolane-3,4-diol; TFMT?; SCHEMBL17406905; RSOXZOFDCJMRMK-IOSLPCCCSA-N; NSC793694; NSC-793694;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)