Natural product from Streptomyces diastatochromogenes; XBP1 mRNA splicing; CDK9/cyclinT1 (IC50 = 79 nM)

Inhibiting both IRE1α-XBP1 buffer activation caused below the endoplasmic reticulum and XBP1 mRNA splicing elicited above it, toyocamycin (0-0.3 μM; 4 h) blocks both processes concurrently [1]. In MM cell lines, constitutive activation of XBP1 is inhibited by toyocamycin (0-0.3 μM; 24 h) and toyocamycin (250 nM; 48 h) [1]. Toyocamycin (50 μM–0.05 nM; 48 and 72 hours). Toyocamycin (0-100 nM; 24 or 48 hours) did not immediately cause cytotoxicity against YB5 and HCT116 with cell survival above 50%; nevertheless, when treated with 10 nM for 24 hours, cancer cells are eradicated two weeks later [2]. By blocking p38 on ERK MAPK, toyocamycin (60 nM; 0-48 h) inhibits ROS-mediated cell fluorescence and enhances p38/ERK MAPK activation [3].

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Here, we screened small-molecule inhibitors of ER stress-induced XBP1 activation, and identified Toyocamycin from a culture broth of an Actinomycete strain. Toyocamycin was shown to suppress thapsigargin-, tunicamycin- and 2-deoxyglucose-induced XBP1 mRNA splicing in HeLa cells without affecting activating transcription factor 6 (ATF6) and PKR-like ER kinase (PERK) activation. Furthermore, although toyocamycin was unable to inhibit IRE1α phosphorylation, it prevented IRE1α-induced XBP1 mRNA cleavage in vitro. Thus, toyocamycin is an inhibitor of IRE1α-induced XBP1 mRNA cleavage. Toyocamycin inhibited not only ER stress-induced but also constitutive activation of XBP1 expression in MM lines as well as primary samples from patients. It showed synergistic effects with bortezomib, and induced apoptosis of MM cells including bortezomib-resistant cells at nanomolar levels in a dose-dependent manner. [1]

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After screening a natural product drug library, we identified that Toyocamycin, an adenosine-analog, induces potent GFP reactivation and loss of clonogenicity in human colon cancer cells. Connectivity-mapping analysis revealed that toyocamycin produces a pharmacological signature mimicking cyclin-dependent kinase (CDK) inhibitors. RNA-sequencing revealed that the toyocamycin transcriptomic signature resembles that of a specific CDK9 inhibitor (HH1). Specific inhibition of RNA Pol II phosphorylation level and kinase assays confirmed that toyocamycin specifically inhibits CDK9 (IC50 = 79 nM) with a greater efficacy than other CDKs (IC50 values between 0.67 and 15 µM). Molecular docking showed that toyocamycin efficiently binds the CDK9 catalytic site in a conformation that differs from other CDKs, explained by the binding contribution of specific amino acids within the catalytic pocket and protein backbone. Altogether, we demonstrated that toyocamycin exhibits specific CDK9 inhibition in cancer cells, highlighting its potential for cancer chemotherapy. [2]

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We investigated the apoptotic effect of Toyocamycin and the underlying molecular mechanism in prostate cancer PC-3 cells. Toyocamycin treatment resulted in reduced cell viability of PC-3 cells, but not of non-malignant RWPE-1 cells. Toyocamycin enhanced apoptosis, mitochondrial dysfunction, and ROS production in PC-3 cells. In addition, MAPK proteins were activated upon Toyocamycin treatment. The p38 and extracellular signal-regulated kinases (ERK) activities were regulated by ROS-mediated signaling pathway underlying the Toyocamycin-induced apoptosis. Pretreatment with N-acetyl-l-cysteine (NAC) recovered the Toyocamycin-induced mitochondrial dysfunction, ROS, and apoptosis. Additionally, p38 stimulated ROS production and inhibitory effects on ERK activation, while ERK inhibited the ROS production and had no effect on p38 activation. Conclusion: ROS-mediated activation of p38/ERK partially contributes to Toyocamycin-induced apoptosis, and p38/ERK MAPKs regulate the ROS production in PC-3 cells[3].

Toyocamycin (0.5 mg/kg, 1.0 mg/kg; intraperitoneally; twice weekly; 2 weeks) exhibits anticancer activity in a human multiple myeloma (MM) xenograft model.

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In vivo anti-tumor activity of Toyocamycin alone and in combination with BTZ in a human MM xenograft model [1]
To evaluate in vivo efficacy of toyocamycin on MM cells, SCID mice subcutaneously inoculated with RPMI8226 were treated with twice- or once-weekly intraperitoneal toyocamycin at either 0.5 or 1.0 mg/kg. In addition, the combination treatment of toyocamycin with BTZ was tested. Toyocamycin alone showed robust anti-tumor activity resulting in smaller tumor volumes compared with controls on day 15. This was similar to the effect of BTZ (Figures 6a and b). No obvious difference in tumor inhibitory effect was seen on twice- or once-weekly injection of toyocamycin.
The combination treatment of BTZ with Toyocamycin, either at 0.5 mg/kg or 1.0 mg/kg, showed a trend toward enhancing anti-tumor activity represented as smaller tumor volumes when compared with BTZ or toyocamycin alone (Figures 6a and b).

Cyclin-Dependent Kinase Inhibition Assays [2]
Enzymatic activities of recombinant human CDK9/Cyclin T1, CDK2/Cyclin 2A, CDK4/Cyclin D3, CDK6/Cyclin D3, and CDK7/Cyclin H/MAT1 were tested against Toyocamycin and known CDK inhibitors (staurosporine, dinaciclib, palbociclib). The in vitro enzymatic assays were conducted at BPS Bioscience. CDK9/Cyclin T1, CDK2/Cyclin 2A, CDK4/Cyclin D3, and CDK6/Cyclin D3 enzymatic activities were tested with an ATP Kinase-Glo Plus Luminescence kinase assay kit, while the CDK7/Cyclin H/MAT1 activities were tested using ADP-Glo Kinase. These assays measure kinase activity by quantifying the amount of ATP or ADP remaining in solution following a kinase reaction. The luminescent signal from the assay is correlated to the amount of ATP or ADP present and is inversely correlated to the kinase activity. The compounds were diluted to 10% DMSO and were added to the reaction so that the final concentration of DMSO was 1% in all reactions. All enzymatic reactions were conducted at 30 °C for 60 min. The reaction mixture contained 40 mM Tris, pH 7.4, 10 mM MgCl2, 0.1 mg/mL BSA, 1 mM DTT, 10 µM ATP, kinase substrate, and the enzyme. After the enzymatic reaction, the luminescence signal was measured using a BioTek Synergy 2 microplate reader. Kinase activity assays were performed in duplicate at each concentration and the percent of kinase activity was calculated as a ratio of luminescence intensities in the absence or in presence of the compounds. The values of percent activity versus a series of compound concentrations were plotted using non-linear regression analysis of the sigmoidal dose-response curve. Data were repeated three times for CDK9 and two times for the rest of the CDKs. IC50 values were determined by the concentration causing a half-maximal percent activity .

Western Blot analysis [1]
Cell Types: HeLa, HEK293
Tested Concentrations: 0, 0.03, 0.1, 0.3 μM
Incubation Duration: 4 hrs (hours)
Experimental Results: Neither inhibited tunicamycin-induced ATF6 nor PERK activation. Inhibited IRE1α-induced XBP1 mRNA cleavage without affecting phosphorylation on IRE1α Ser724.

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Western Blot Analysis[3]
Cell Types: Human prostate cancer PC-3 cells
Tested Concentrations: 60 nM
Incubation Duration: 12, 24, 36, 48 hrs (hours)
Experimental Results: Inhibited the phosphorylation level of AKA, while reducing the phosphorylation level of ERK and p38.

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Cell viability assay[3]
Cell Types: PC-3 and RWPE-1 Cell
Tested Concentrations: 0, 20, 40, 60, 80, 100 nM
Incubation Duration: 24 or 48 hrs (hours)
Experimental Results: Inhibited cell viability and induced apoptosis by 62%.

Animal/Disease Models: SCID (severe combined immunodeficient) mouse are injected with human multiple myeloma (MM) cells [1].
Doses: 0.5 mg/kg, 1.0 mg/kg.
Route of Administration: intraperitoneal (ip) injection; tumor activity [1]. Twice a week; 2-week
Experimental Results: Tumor volume Dramatically diminished. Compared with bortezomib, the antitumor activity is enhanced, manifested by smaller tumor size.

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Animals and the murine xenograft model [1]
The study method was described previously.29 Briefly, 0.5 × 107 RPMI8226 cells were inoculated subcutaneously into SCID mice previously administered with rabbit antiasialo-GM1 intraperitoneally 1 day before tumor inoculation. At 10 days after tumor inoculation, the tumor-bearing mice were divided into four groups of five mice each, such that the mean tumor volumes were approximately equal in the four groups. Tumor volume was calculated by the following formula: tumor volume (mm3)=0.5 × (major diameter) × (minor diameter)2. Mice were treated by intraperitoneal injection of 0.5 mg/kg Toyocamycin twice weekly, 1.0 mg/kg toyocamycin once weekly, or 1.0 mg/kg BTZ twice weekly for 2 weeks. Volumes of tumors in Toyocamycin-treated mice were compared with untreated or BTZ-treated animals during the treatment period.

11824 Mouse LD50 Oral 8 mg/kg Index of Actinomycete Antibiotics, edited by Hiroshi Umezawa et al., University of Tokyo Press. Tokyo Press, 1967, -(805), 1967
11824 Mouse Intraperitoneal LD50 20 mg/kg CRC Handbook of Antibiotic Compounds, Volume 1-, Berdy, J., Boca Raton, Florida, CRC Press, 1980, 5(315), 1981
11824 Mouse Subcutaneous LDLo 10 mg/kg Antibiotics Journal, Series A, 9(60), 1956
11824 Mouse Intravenous LD50 1500 μg/kg CRC Handbook of Antibiotic Compounds, Volume 1-, Berdy, J., Boca Raton, Florida, CRC Press, 1980, 5(318), 1981

[1]. Identification of Toyocamycin, an agent cytotoxic for multiple myeloma cells, as a potent inhibitor of ER stress-induced XBP1 mRNA splicing. Blood Cancer J. 2012 Jul;2(7):e79.

[2]. Selective CDK9 Inhibition by Natural Compound Toyocamycin in Cancer Cells. Cancers (Basel). 2022 Jul 8;14(14):3340.

[3]. Toyocamycin induces apoptosis via the crosstalk between reactive oxygen species and p38/ERK MAPKs signaling pathway in human prostate cancer PC-3 cells. Pharmacol Rep. 2017 Feb;69(1):90-96.

Toyocamycin is an N-glycosylpyrrolopyrimidine compound, a derivative of tuberculin, in which the hydrogen atom at the 5-position of the pyrrolopyrimidine ring is replaced by a cyano group. It possesses antimetabolite, antitumor, antibacterial, and apoptosis-inducing activities. It belongs to the nitrile, ribonucleoside, and antibiotic-antifungal class of compounds.
4-Amino-5-cyano-7-(D-furanoribosyl)-7H-pyrrolo(2,3-d)pyrimidine. An antibiotic antimetabolite isolated from Streptomyces toicae cultures. It is an adenosine analogue that blocks RNA synthesis and ribosome function, primarily used as a biochemical tool.
Toyocamycin has been reported in Streptomyces toyocaensis, Streptomyces diastatochromogenes, and other organisms with relevant data.
4-Amino-5-cyano-7-(D-rifuranosyl)-7H-pyrrolo(2,3-d)pyrimidine. An antibiotic antimetabolite isolated from Streptomyces toyocaensis cultures. It is an adenosine analogue that blocks RNA synthesis and ribosome function, primarily used as a biochemical tool.

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An earlier phase I study of toyocamycin monotherapy also reported its potential antitumor activity in patients with advanced solid tumors. ⁵¹However, no further clinical evaluation was planned because no significant clinical response was observed in that study. In this study, tocamycin did not show systemic side effects such as organ dysfunction and cytopenia; only local necrosis at the infusion site was reported when the drug was infused into soft tissue. This suggests that adverse reactions to tocamycin may be manageable if administered via a central venous catheter. In addition, since this study was a phase I clinical trial, the assessment of the disease stabilization phase was lacking (while the disease stabilization phase is usually used in recent clinical trials of molecularly targeted therapy), so this study did not rule out the potential clinical efficacy of tocamycin in solid tumors. In summary, we demonstrated that the adenosine analog tocamycin has a potent inhibitory effect on IRE1-XBP1 in endoplasmic reticulum stress tumors and multiple myeloma cells and can induce dose-dependent apoptosis in these cells. These results provide preclinical evidence for clinical trials of tocamycin and other adenosine analogs as monotherapy or in combination with bortezomib (BTZ) for the treatment of multiple myeloma. [1] In the mid-1960s, based on its good anti-cancer effect on cancer cell lines, but whose mechanism of action was not fully understood, toyocamycin was tested in a phase I clinical trial (NSC-63701). 23 cancer patients received toyocamycin for 5 days at a dose of 10-200 µg/kg (intravenous infusion, 1-2 hours). No systemic toxicity was observed, but local toxicity (severe phlebitis) at the injection site was observed in patients receiving the highest dose. Our study shows that Toyocamycin is a potent and selective CDK9 inhibitor at low doses. This natural product can be used as a small molecule tool to modulate CDK9 activity in vitro, and its specific binding may inspire interest in designing novel CDK9 inhibitors. [2] Toyocamycin is an adenosine analog derived from Streptomyces and belongs to the class of antibiotics. Toocamycin is a potent inhibitor of RNA self-cleavage and phosphatidylinositol kinase in mammalian cells. In addition, it has been reported to inhibit the activity of kinases such as protein kinase C (PKC), cdc2, or phosphatidylinositol 3-kinase (PI3K). It also inhibits endoplasmic reticulum stress-mediated X-box binding protein 1 (XBP-1) splicing in multiple myeloma (MM) cells. Many adenosine analogs, such as toocamycin, sanguivaricin, and MCS-C2, have been investigated as anticancer drugs for a variety of cancer cells. However, to our knowledge, the link between toyocamycin activation of the ROS-mediated MAPK signaling pathway and apoptosis has not been reported. In this study, our results show for the first time that toyocamycin induces apoptosis in human prostate cancer cells by increasing ROS production, activating MAPKs and subsequently disrupting mitochondrial function, activating caspase-3, and cleaving PARP. [3]

C12H13N5O4

291.26272

291.097

C, 49.48; H, 4.50; N, 24.04; O, 21.97

606-58-6

11824

White to off-white solid powder

1.91g/cm3

721.1ºC at 760 mmHg

389.9ºC

1.849

-1.6

4

8

2

21

443

4

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

XOKJUSAYZUAMGJ-WOUKDFQISA-N

InChI=1S/C12H13N5O4/c13-1-5-2-17(11-7(5)10(14)15-4-16-11)12-9(20)8(19)6(3-18)21-12/h2,4,6,8-9,12,18-20H,3H2,(H2,14,15,16)/t6-,8-,9-,12-/m1/s1

4-amino-7-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]pyrrolo[2,3-d]pyrimidine-5-carbonitrile

TOYOCAMYCIN; Vengicide; Antibiotic 1037; Uramycin B; Siromycin; Cyanotubericidin; Ahygroscopin-B; 7-Deaza-7-cyanoadenosine;

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)

DMSO : ~100 mg/mL (~343.34 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.58 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (8.58 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (8.58 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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