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In silico discovery of novel retinoic acid receptor agonist structures.

Schapira M, Raaka BM, Samuels HH, Abagyan R - BMC Struct. Biol. (2001)

Bottom Line: Thirty ligand candidates were tested in vitro.Two novel agonists resulting from the predicted receptor model were active at 50 nM.One of them displays novel structural features which may translate into the development of new ligands for cancer therapy.

View Article: PubMed Central - HTML - PubMed

Affiliation: Structural Biology, Skirball Institute of Biomolecular Medicine, New York, USA. schapira@saturn.med.nyu.edu

ABSTRACT

Background: Several Retinoic Acid Receptors (RAR) agonists have therapeutic activity against a variety of cancer types; however, unacceptable toxicity profiles have hindered the development of drugs. RAR agonists presenting novel structural and chemical features could therefore open new avenues for the discovery of leads against breast, lung and prostate cancer or leukemia.

Results: We have analysed the induced fit of the active site residues upon binding of a known ligand. The derived binding site models were used to dock over 150,000 molecules in silico (or virtually) to the structure of the receptor with the Internal Coordinates Mechanics (ICM) program. Thirty ligand candidates were tested in vitro.

Conclusions: Two novel agonists resulting from the predicted receptor model were active at 50 nM. One of them displays novel structural features which may translate into the development of new ligands for cancer therapy.

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Related in: MedlinePlus

In vitro activity of the novel RAR agonists. HeLa cells were separately transfected with a vector expressing each isoform of the full-length receptor and a ΔMTV-IR-CAT reporter gene. The CAT activity induced by each ligand at concentrations from 50 nM to 20 μM was measured. The activity induced by the natural hormone all-trans RA at 1 μM was used as the reference for maximal induction for RARs, and 1 μM 9-cis RA was used for RXR-β. The activity plotted is the observed percentage of this maximal induction.
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Figure 4: In vitro activity of the novel RAR agonists. HeLa cells were separately transfected with a vector expressing each isoform of the full-length receptor and a ΔMTV-IR-CAT reporter gene. The CAT activity induced by each ligand at concentrations from 50 nM to 20 μM was measured. The activity induced by the natural hormone all-trans RA at 1 μM was used as the reference for maximal induction for RARs, and 1 μM 9-cis RA was used for RXR-β. The activity plotted is the observed percentage of this maximal induction.

Mentions: HeLa cells were separately transfected with either of the three wild type hRAR isoforms, and a ΔMTV-IR-CAT reporter gene [22, 23]. The cells were incubated with each ligand at concentrations from 50 nM to 20 μM to stimulate CAT activity. Possible toxicity of the compounds was deduced from the amount of cellular protein extract after 2 days of incubation. The percentage of conversion induced by 1 μM all-trans RA (RARs) or 1 μM 9-cis RA (RXR-β) was used as a positive control to determine the maximum induction. Fig. 3 shows all-trans RA induced conversion of RAR-α as a function of acid concentration. RAR-β could induce 20% of the maximum CAT activity when activated by 50 nM agonist 1 (chemical structure shown Fig. 1c), while RAR-γ was only 12% active and RAR-α not active at all under the same conditions (Fig. 4). At 200 nM of agonist 1, RAR-β was 50% active, RAR-γ 25% and RAR-α 5% active. Similarly, RAR-β could induce 22% of the maximum CAT activity when activated by 50 nM agonist 2 (chemical structure shown Fig. 1d). RAR-α and RAR-γ were 10% and 14% active at the same concentration of agonist, respectively. At 200 nM of agonist 2, RAR-β displayed 48% of its maximal activity, RAR-α 40% and RAR-γ 17%. At 20 μM, agonist 1 induced full activation of RAR-β and RAR-γ, and 80% activation of RAR-α, showing that this compound is a full agonist. Agonist 2 was toxic for the cells at 20 μM, as shown by protein content of cell culture dishes, which is why little transcriptional activity was observed at this concentration. However, little or no toxicity was observed at 8 μM, a concentration at which agonist 2 induced about 50% maximal activity of RAR-α and RAR-β, and 35% maximal activity of RAR-γ. Agonist 1 was not toxic at 20 μM. Finally, RXR-β was activated only weakly by both agonists 1 and 2 at 2 μM, but was significantly activated by agonist 1 at 20 μM (Fig. 4). Comparison of Figs. 3 and 4 also shows that at 200 nM of agonists 1 and 2, RAR-β exhibits the same activity as the positive control RAR-α induced by all-trans RA at the same concentration.


In silico discovery of novel retinoic acid receptor agonist structures.

Schapira M, Raaka BM, Samuels HH, Abagyan R - BMC Struct. Biol. (2001)

In vitro activity of the novel RAR agonists. HeLa cells were separately transfected with a vector expressing each isoform of the full-length receptor and a ΔMTV-IR-CAT reporter gene. The CAT activity induced by each ligand at concentrations from 50 nM to 20 μM was measured. The activity induced by the natural hormone all-trans RA at 1 μM was used as the reference for maximal induction for RARs, and 1 μM 9-cis RA was used for RXR-β. The activity plotted is the observed percentage of this maximal induction.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC32304&req=5

Figure 4: In vitro activity of the novel RAR agonists. HeLa cells were separately transfected with a vector expressing each isoform of the full-length receptor and a ΔMTV-IR-CAT reporter gene. The CAT activity induced by each ligand at concentrations from 50 nM to 20 μM was measured. The activity induced by the natural hormone all-trans RA at 1 μM was used as the reference for maximal induction for RARs, and 1 μM 9-cis RA was used for RXR-β. The activity plotted is the observed percentage of this maximal induction.
Mentions: HeLa cells were separately transfected with either of the three wild type hRAR isoforms, and a ΔMTV-IR-CAT reporter gene [22, 23]. The cells were incubated with each ligand at concentrations from 50 nM to 20 μM to stimulate CAT activity. Possible toxicity of the compounds was deduced from the amount of cellular protein extract after 2 days of incubation. The percentage of conversion induced by 1 μM all-trans RA (RARs) or 1 μM 9-cis RA (RXR-β) was used as a positive control to determine the maximum induction. Fig. 3 shows all-trans RA induced conversion of RAR-α as a function of acid concentration. RAR-β could induce 20% of the maximum CAT activity when activated by 50 nM agonist 1 (chemical structure shown Fig. 1c), while RAR-γ was only 12% active and RAR-α not active at all under the same conditions (Fig. 4). At 200 nM of agonist 1, RAR-β was 50% active, RAR-γ 25% and RAR-α 5% active. Similarly, RAR-β could induce 22% of the maximum CAT activity when activated by 50 nM agonist 2 (chemical structure shown Fig. 1d). RAR-α and RAR-γ were 10% and 14% active at the same concentration of agonist, respectively. At 200 nM of agonist 2, RAR-β displayed 48% of its maximal activity, RAR-α 40% and RAR-γ 17%. At 20 μM, agonist 1 induced full activation of RAR-β and RAR-γ, and 80% activation of RAR-α, showing that this compound is a full agonist. Agonist 2 was toxic for the cells at 20 μM, as shown by protein content of cell culture dishes, which is why little transcriptional activity was observed at this concentration. However, little or no toxicity was observed at 8 μM, a concentration at which agonist 2 induced about 50% maximal activity of RAR-α and RAR-β, and 35% maximal activity of RAR-γ. Agonist 1 was not toxic at 20 μM. Finally, RXR-β was activated only weakly by both agonists 1 and 2 at 2 μM, but was significantly activated by agonist 1 at 20 μM (Fig. 4). Comparison of Figs. 3 and 4 also shows that at 200 nM of agonists 1 and 2, RAR-β exhibits the same activity as the positive control RAR-α induced by all-trans RA at the same concentration.

Bottom Line: Thirty ligand candidates were tested in vitro.Two novel agonists resulting from the predicted receptor model were active at 50 nM.One of them displays novel structural features which may translate into the development of new ligands for cancer therapy.

View Article: PubMed Central - HTML - PubMed

Affiliation: Structural Biology, Skirball Institute of Biomolecular Medicine, New York, USA. schapira@saturn.med.nyu.edu

ABSTRACT

Background: Several Retinoic Acid Receptors (RAR) agonists have therapeutic activity against a variety of cancer types; however, unacceptable toxicity profiles have hindered the development of drugs. RAR agonists presenting novel structural and chemical features could therefore open new avenues for the discovery of leads against breast, lung and prostate cancer or leukemia.

Results: We have analysed the induced fit of the active site residues upon binding of a known ligand. The derived binding site models were used to dock over 150,000 molecules in silico (or virtually) to the structure of the receptor with the Internal Coordinates Mechanics (ICM) program. Thirty ligand candidates were tested in vitro.

Conclusions: Two novel agonists resulting from the predicted receptor model were active at 50 nM. One of them displays novel structural features which may translate into the development of new ligands for cancer therapy.

Show MeSH
Related in: MedlinePlus