Lithocholic acid is an endogenous inhibitor of MDM4 and MDM2

Abstract

The proteins MDM2 and MDM4 are key negative regulators of the tumor suppressor protein p53, which are frequently upregulated in cancer cells. They inhibit the transactivation activity of p53 by binding separately or in concert to its transactivation domain. MDM2 is also a ubiquitin ligase that leads to the degradation of p53. Accordingly, MDM2 and MDM4 are important targets for drugs to inhibit their binding to p53. We found from in silico screening and confirmed by experiment that lithocholic acid (LCA) binds to the p53 binding sites of both MDM2 and MDM4 with a fivefold preference for MDM4. LCA is an endogenous steroidal bile acid, variously reported to have both carcinogenic and apoptotic activities. The comparison of LCA effects on apoptosis in HCT116 p53 þ∕þ vs. p53 −∕− cells shows a predominantly p53-mediated induction of caspase-3/7. The dissociation constants are in the μM region, but only modest inhibition of binding of MDM2 and MDM4 is required to negate their upregulation because they have to compete with transcriptional coactivator p300 for binding to p53. Binding was weakened by structural changes in LCA, and so it may be a natural ligand of MDM2 and MDM4, raising the possibility that MDM proteins may be sensors for specific steroids.HDMX | virtual screening | natural product | cancer pathways | bile acid sensor T he tumor-suppressor protein p53 plays a pivotal role in cancer (1, 2). Often, its function is severely impaired by upregulation of its two key negative regulators, MDM2 and MDM4 (1-3). The N-terminal domains of MDM2 and MDM4 are structurally very similar and both bind to the same sequence in the intrinsically disordered N terminus of p53 (2, 4, 5). The binding cavities within MDM4 and MDM2 are important targets for drug therapy that releases them from p53 (6-10). Nutlins (11), for example, are potent MDM2 inhibitors and are potential therapeutics (12, 13) as well as being invaluable research tools for probing p53 pathways (12,14), as are spiro-oxindoles, which were found by in silico methods (15,16). MDM4 has a different specificity for small molecules, binding nutlins, for example, less tightly. Accordingly, MDM4-selective and dual MDM4/MDM2 inhibitors are also being sought (7,17,18). We searched for MDM4 inhibitors by structure-based in silico screening of binding (6, 19) and identified LCA as an endogenous inhibitor of both MDM4 and MDM2. LCA is a secondary bile acid formed by bacteria in the gut from its precursor chenodeoxycholic acid (CDCA, Fig S1). It has been variously reported to show both carcinogenic and apoptotic activities (20,21). LCA is a rare example of a toxic endobiotic that is efficiently detoxicated by conjugation with taurine or glycine, sulfation at C-3 by the sulfotransferase SULT2A1, or metabolism through cytochrome P450 CYP3A enzymes (20). It induces its own metabolism by activating nuclear receptors like the vitamin D receptor (22) (VDR) and the farnesoid X receptor (23) (FXR). Thereby, it inhibits the synthesis of bile acids and promotes the transcription of gene...

Wild type p53 reactivation: From lab bench to clinic

“…LCA binds to the p53 binding sites of both Mdm2 and MdmX with a fivefold preference for MdmX. These findings raise an interesting possibility that LCA could be a natural ligand for Mdm proteins [47].…”

Bile acid receptors as targets for drug development

“…LCA is a cytotoxic bile acid formed mainly by bacterial deconjugation and 7α-dehydroxylation of conjugated CDCA, and is passively absorbed in the colon where it is considered pro-oncogenic. 91 LCA induces experimental cholestasis that results in extensive liver damage. PXR serves as a xenobiotic sensor, and induces phase I and II metabolism of many endogenous and exogenous compounds, including bile acids.…”

2-Sulfonylpyrimidines: Mild alkylating agents with anticancer activity toward p53-compromised cells

“…Caspase-3/7 activity and cytotoxicity were measured using the ApoTox-Glo Triplex assay (Promega), performed as described (67). Briefly, cells were incubated with compound or DMSO control for 6 h. After 1 h incubation with Caspase-Glo 3/7 reagent, we recorded luminescence using a Centro XS 3 …”