4 minute read
Giuseppe La Regina
TREATMENT OF CHRONIC MYELOID LEUKEMIA BY INHIBITION OF TUBULIN POLYMERIZATION
Giuseppe La Regina
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RESEARCH AREA: Novel therapeutic interventions
Dipartimento di Chimica e Tecnologie del Farmaco Sapienza Università di Roma, Rome, Italy giuseppe.laregina@uniroma1.it
Long-term survivors of glioblastoma multiforme (GBM) are at high risk of developing second primary neoplasms, including leukemia. For these patients, the use of classic tyrosine kinase inhibitors (TKIs), such as imatinib mesylate (IM), is strongly discouraged, since this treatment causes a tremendous increase of tumor and stem cell migration and invasion. We aimed to develop agents useful for the treatment of patients with GBM and chronic myeloid leukemia (CML) using an alternative mechanism of action from the TKIs, specifically based on the inhibition of tubulin polymerization.
We identified two new derivatives, (4-(thiophen-3-yl)-1-(p-tolyl)-1H-pyrrol-3-yl)(3,4,5trimethoxyphenyl)methanone and (1-(4-fluorophenyl)-4-(furan-3-yl)-1H-pyrrol-3-yl)(3,4,5trimethoxyphenyl)methanone, that, as planned, not only inhibited tubulin polymerization, but also inhibited the proliferation of both GMB and CML cells, including those expressing the T315I mutation, at low nanomolar concentrations.
Derivatives (4-(thiophen-3-yl)-1-(p-tolyl)-1H-pyrrol-3-yl)(3,4,5trimethoxyphenyl)methanone and (1-(4-fluorophenyl)-4-(furan-3-yl)-1H-pyrrol-3-yl)(3,4,5trimethoxyphenyl)methanone were compared with the TKIs IM and third-generation ponatinib (PN) in the Bcr-Abl-expressing KU812 cells, in the IM-sensitive KBM5 cells and in the IM-resistant KBM5-T315I cells expressing the threonine for isoleucine mutation at position 315 of the oncoprotein. The IC50 values of both compounds were at least one order of magnitude superior to IM. Derivative (4-(thiophen-3-yl)-1-(p-tolyl)-1H-pyrrol-3yl)(3,4,5-trimethoxyphenyl)methanone showed strong inhibition at single digit nanomolar concentrations of the KBM5-wt (IC50 = 8.2 nM) and KBM5-T315I (IC50 = 8.8 nM) cell lines, and the same compound was 1.2 fold superior to PN against both cell lines (IC50 = 10.2 and 10.6 nM, respectively).
Combination of derivative (4-(thiophen-3-yl)-1-(p-tolyl)-1H-pyrrol-3-yl)(3,4,5trimethoxyphenyl)methanone with PN showed a large increase in the percentage of apoptotic cells, greater than in the combinations of PN with either IM or nilotinib, in the three CML lines. Most importantly, in the KBM5-T315I cells, the PN+(4-(thiophen-3-yl)1-(p-tolyl)-1H-pyrrol-3-yl)(3,4,5-trimethoxyphenyl)methanone combination yielded 70% apoptotic cells, as compared with 35% for PN alone. The same derivative was compared with IM and PN alone and in combination in the KBM5-T315I cells for mitochondrial membrane depolarization. In this assay, derivative (4-(thiophen-3-yl)-1-(p-tolyl)-1Hpyrrol-3-yl)(3,4,5-trimethoxyphenyl)methanone was superior to IM and slightly inferior to PN. Combining PN+(4-(thiophen-3-yl)-1-(p-tolyl)-1H-pyrrol-3-yl)(3,4,5-
trimethoxyphenyl)methanone strongly increased mitochondrial membrane depolarization by augmenting the PN-mediated effects.
The synergy of PN in combination with derivative (4-(thiophen-3-yl)-1-(p-tolyl)-1Hpyrrol-3-yl)(3,4,5-trimethoxyphenyl)methanone was evaluated for effects on the phosphorylation of Bcr-Abl and for the expression of apoptosis/mitochondrial damagerelated proteins. Derivative (4-(thiophen-3-yl)-1-(p-tolyl)-1H-pyrrol-3-yl)(3,4,5trimethoxyphenyl)methanone altered mitochondrial functionality even when used alone, affecting the expression of Mcl-1 and the amount of cytoplasmatic cytochrome C, which are major effectors of the cellular apoptotic machinery. Mcl-1 is an antiapoptotic protein of the Bcl2 family originally isolated from human myeloid leukemia cells, and Mcl-1 plays a key role in regulating the mitochondrial pathway leading to apoptosis in a wide variety of tissues. These proteins manage mitochondrial permeability to cytochrome C and hence activate the initiation of a caspase cascade that leads to programmed cell death. Inhibition of tyrosine phosphorylation of Bcr-Abl was boosted by the combination of derivative (4(thiophen-3-yl)-1-(p-tolyl)-1H-pyrrol-3-yl)(3,4,5-trimethoxyphenyl)methanone with PN, while the total level of the Bcr-Abl oncoprotein was little affected. These results indicate that derivative (4-(thiophen-3-yl)-1-(p-tolyl)-1H-pyrrol-3-yl)(3,4,5trimethoxyphenyl)methanone used as a single agent did not have a significant effect on Bcr-Abl activation, suggesting that an upstream inhibition of a “Bcr-Abl-addicted pathway” is required to prompt the proapoptotic effect.
In experiments in BALB/cnu/nu mice injected subcutaneously with U87MG cells, derivative (4-(thiophen-3-yl)-1-(p-tolyl)-1H-pyrrol-3-yl)(3,4,5trimethoxyphenyl)methanone, when administered intraperitoneally at 25 mg/kg, significantly inhibited GBM cancer cell proliferation, in vivo tumorigenesis, and tumor angiogenesis. The same compound had a calculated improvement of metabolic stability to phase II metabolism and higher lipophilicity compared to the previously reported derivatives.
Publications
Puxeddu M, Shen H, Bai R, Coluccia A, Bufano M, Nalli M, Sebastiani J, Da Pozzo E, Tremolanti C, Martini C, Orlando V, Biagioni S, Sinicropi MS, Ceramella J, Iacopetta D, Coluccia AML, Hamel E, Liu T, Silvestri R, La Regina G. Discovery of pyrrole derivatives for the treatment of glioblastoma and chronic myeloid leukemia. Eur J Med Chem 2021;221:e113532, doi: 10.1016/j.ejmech.2021.113532, IF 6.514.
Passirani C, Vessieres A, La Regina G, Link W, Silvestri R. Modulating undruggable targets to overcome cancer therapy resistance. Drug Resist Update 2022;60:e100788, doi: 10.1016/j.drup.2021.100788, IF 18.500.
Van Dycke J, Puxeddu M, La Regina G, Mastrangelo E, Tarantino D, Rymenants J, Sebastiani J, Nalli M, Matthijnssens J, Neyts J, Silvestri R, Rocha-Pereira J. Discovery
of a novel class of norovirus inhibitors with high barrier of resistance.
Liu T, Wu J, Han C, Gong Z, La Regina G, Chen J, Dou F, Silvestri R, Chen C, Yu Z.
RS-5645 attenuates inflammatory cytokine storm induced by SARS-CoV-2 spike
protein and LPS by modulating pulmonary microbiota. Int J Biol Sci 2021;17:33053319, doi: 10.7150/ijbs.63329, IF 6.582.
Li S, Xu A, Li Y, Tan C, La Regina G, Silvestri R, Wang H, Qi W. RS4651 suppresses lung fibroblast activation via the TGF- 1/SMAD signalling pathway. Eur J Pharmacol 2021;903:e174135, doi: 10.1016/j.ejphar.2021.174135, IF 4.432.
Daniele S, La Pietra V, Piccarducci R, Pietrobono D, Cavallini C, D'Amore VM, Cerofolini L, Giuntini S, Russomanno P, Puxeddu M, Nalli M, Pedrini M, Fragai M, Luchinat C, Novellino E, Taliani S, La Regina G, Silvestri R, Martini C, Marinelli L.
CXCR4 antagonism sensitizes cancer cells to novel indole-based MDM2/4
inhibitors in glioblastoma multiforme. Eur J Pharmacol 2021;897:e173936, doi: 10.1016/j.ejphar.2021.173936, IF 4.432.
Research Group
Giuseppe La Regina, Antonio Coluccia: Researchers. Michela Puxeddu, Jessica Sebastiani, Marianna Bufano: PhD Students.
