HLM006474

HLM006474 is a small molecular inhibitor of E2F with an IC50 value of 30μM [1].

HLM006474 has been reported to inhibit all DNA-binding of all E2F complexes including trimeric E2F, free forms of E2F, E2F4 and non-specific complex in EMSA assay. In addition, HLM006474 has been exhibited to induce apoptosis in multiple drug resistant cell lines with IC50 values of 36±6μM，31±4μM and 46±6μM for 8226-s, 8226-dox40, and 8226-MR20 cell lines, respectively, by a MTS viability assay. Moreover, HLM006474 has been revealed to powerfully inhibit growth in a number of cell lines, especially leukemia cell lines [1].

References:
[1] Cress D W, Ma Y. Small molecule E2F inhibitor: U.S. Patent 8,202,886[P]. 2012-6-19.

Improved synthesis, resolution, absolute configuration determination and biological evaluation of HLM006474 enantiomers.[Pubmed:30578036]

Bioorg Med Chem Lett. 2019 Feb 1;29(3):380-382.

An improved green synthesis of the E2F inhibitor HLM0066474 is described, using solvent-free and microwave irradiation conditions. The two enantiomers are separated using semi-preparative separation on Chiralpak ID and their absolute configuration is determined by vibrational circular dichroism (VCD) analysis. Biological evaluation of both enantiomers on E2F1 transcriptional activity reveals that the (+)-R, but not the (-)-S enantiomer is biologically active in repressing E2F1 transcriptional activity.

Inhibition of Skp2 sensitizes lung cancer cells to paclitaxel.[Pubmed:28176922]

Onco Targets Ther. 2017 Jan 18;10:439-446.

S-phase kinase-associated protein 2 (Skp2) is an E3 ubiquitin ligase and plays an important role in the control of cell cycle progression. Skp2 is upregulated in several cancers, including lung cancers, but the role of Skp2 in the tumorigenesis and anticancer drug resistance in human lung cancer remains to be determined. We report here that Skp2 positively regulated mitotic arrest deficient 2 (MAD2) expression and that inhibition of Skp2 sensitizes human lung cancer cells to paclitaxel. Knockdown of Skp2 by small interfering RNA (siRNA) decreased Mad2 messenger RNA (mRNA) and protein levels in A549 and NCI-H1975 cells, accompanied with upregulation of p27 but decrease of the phosphorylation of retinoblastoma (Rb). In contrast, ectopic overexpression of Skp2 increased Mad2 mRNA and protein levels and phosphorylation of Rb, while it decreased p27. Pharmacological inhibition of CDK1/2 by flavopiridol or E2F1 with HLM006474 led to downregulation of Mad2 expression and prevented the increase of Mad2 expression by Skp2. Most importantly, pharmacological inhibition of Skp2 sensitized A549 and NCI-H1299 cells to paclitaxel. Our results demonstrated that SKP2 positively regulates the gene expression of MAD2 through p27-CDKs-E2F1 signaling pathway and that inhibition of Skp2 sensitizes A549 and NCI-H1299 cells to paclitaxel, suggesting that small molecule inhibitors of Skp2 are potential agents for the treatment of lung cancer with upregulation of Skp2.

E2F inhibition synergizes with paclitaxel in lung cancer cell lines.[Pubmed:24831239]

PLoS One. 2014 May 15;9(5):e96357.

The CDK/Rb/E2F pathway is commonly disrupted in lung cancer, and thus, it is predicted that blocking the E2F pathway would have therapeutic potential. To test this hypothesis, we have examined the activity of HLM006474 (a small molecule pan-E2F inhibitor) in lung cancer cell lines as a single agent and in combination with other compounds. HLM006474 reduces the viability of both SCLC and NSCLC lines with a biological IC50 that varies between 15 and 75 microM, but with no significant difference between the groups. Combination of HLM006474 with cisplatin and gemcitabine demonstrate little synergy; however, HLM006474 synergizes with paclitaxel. Surprisingly, we discovered that brief treatment of cells with HLM006474 led to an increase of E2F3 protein levels (due to de-repression of these promoter sites). Since paclitaxel sensitivity has been shown to correlate with E2F3 levels, we hypothesized that HLM006474 synergy with paclitaxel may be mediated by transient induction of E2F3. To test this, H1299 cells were depleted of E2F3a and E2F3b with siRNA and treated with paclitaxel. Assays of proliferation showed that both siRNAs significantly reduced paclitaxel sensitivity, as expected. Taken together, these results suggest that HLM006474 may have efficacy in lung cancer and may be useful in combination with taxanes.

Established and new mouse models reveal E2f1 and Cdk2 dependency of retinoblastoma, and expose effective strategies to block tumor initiation.[Pubmed:22286767]

Oncogene. 2012 Nov 29;31(48):5019-28.

RB(+/-) individuals develop retinoblastoma and, subsequently, many other tumors. The Rb relatives p107 and p130 protect the tumor-resistant Rb(-/-) mouse retina. Determining the mechanism underlying this tumor suppressor function may expose novel strategies to block Rb pathway cancers. p107/p130 are best known as E2f inhibitors, but here we implicate E2f-independent Cdk2 inhibition as the critical p107 tumor suppressor function in vivo. Like p107 loss, deleting p27 or inactivating its Cdk inhibitor (CKI) function (p27(CK-)) cooperated with Rb loss to induce retinoblastoma. Genetically, p107 behaved like a CKI because inactivating Rb and one allele each of p27 and p107 was tumorigenic. Although Rb loss induced canonical E2f targets, unexpectedly p107 loss did not further induce these genes, but instead caused post-transcriptional Skp2 induction and Cdk2 activation. Strikingly, Cdk2 activity correlated with tumor penetrance across all the retinoblastoma models. Therefore, Rb restrains E2f, but p107 inhibits cross talk to Cdk. While removing either E2f2 or E2f3 genes had little effect, removing only one E2f1 allele blocked tumorigenesis. More importantly, exposing retinoblastoma-prone fetuses to small molecule inhibitors of E2f (HLM006474) or Cdk (R547) for merely 1 week dramatically inhibited subsequent tumorigenesis in adult mice. Protection was achieved without disrupting normal proliferation. Thus, exquisite sensitivity of the cell-of-origin to E2f and Cdk activity can be exploited to prevent Rb pathway-induced cancer in vivo without perturbing normal cell division. These data suggest that E2f inhibitors, never before tested in vivo, or CKIs, largely disappointing as therapeutics, may be effective preventive agents.

A small-molecule E2F inhibitor blocks growth in a melanoma culture model.[Pubmed:18676853]

Cancer Res. 2008 Aug 1;68(15):6292-9.

HLM006474 was identified using a computer-based virtual screen and the known crystal structure of the DNA-bound E2F4/DP2 heterodimer. Treatment of multiple cell lines with HLM006474 resulted in the loss of intracellular E2F4 DNA-binding activity as measured by electrophoretic mobility shift assay within hours. Overnight exposure to HLM006474 resulted in down-regulation of total E2F4 protein as well as known E2F targets. The effects of HLM006474 treatment on different cell lines varied but included a reduction in cell proliferation and an increase in apoptosis. HLM006474 induced apoptosis in a manner distinct from cisplatin and doxorubicin. E2F4-null mouse embryonic fibroblasts were less sensitive than wild-type counterparts to the apoptosis-inducing activity of the compound, revealing its biological specificity. A375 cells were extremely sensitive to the apoptosis-inducing activity of the compound in two-dimensional culture, and HLM006474 was a potent inhibitor of melanocytes proliferation and subsequent invasion in a three-dimensional tissue culture model system. Together, these results suggest that interference with E2F activity using small molecules may have clinical application in cancer therapy.

The RB family is required for the self-renewal and survival of human embryonic stem cells.[Pubmed:23212373]

Nat Commun. 2012;3:1244.

The mechanisms ensuring the long-term self-renewal of human embryonic stem cells are still only partly understood, limiting their use in cellular therapies. Here we found that increased activity of the RB cell cycle inhibitor in human embryonic stem cells induces cell cycle arrest, differentiation and cell death. Conversely, inactivation of the entire RB family (RB, p107 and p130) in human embryonic stem cells triggers G2/M arrest and cell death through functional activation of the p53 pathway and the cell cycle inhibitor p21. Differences in E2F target gene activation upon loss of RB family function between human embryonic stem cells, mouse embryonic stem cells and human fibroblasts underscore key differences in the cell cycle regulatory networks of human embryonic stem cells. Finally, loss of RB family function promotes genomic instability in both human and mouse embryonic stem cells, uncoupling cell cycle defects from chromosomal instability. These experiments indicate that a homeostatic level of RB activity is essential for the self-renewal and the survival of human embryonic stem cells.