The ubiquitin system represents a rich source of molecular targets for cancer, prominent among these is the p53 ubiquitin ligase, Hdm2. Our initial attempt to identify inhibitors of this E3 yielded three related 5-deazaflavins, the HLI98s, which were limited in utility by solubility and overall potency (42
). We have now identified a related compound, referred to as HLI373, as a water soluble and more potent inhibitor of Hdm2. This small molecule preferentially induces apoptosis in transformed cells and a number of tumor cell lines that express wild type p53. Maximal stabilization of p53 is observed in the low micromolar range when endogenous p53 and Hdm2 were assessed (IC50
~ 3 μM). Therefore, HLI373 represents a potential lead for development of anti-cancer therapeutics.
Due to its essential role in controlling cell growth, its frequent inactivation in human tumors and its propensity to induce apoptosis in tumor cells, activation of p53 is well recognized as being a highly desirable goal in the treatment of cancer. For example, p53 delivered by adenovirus effectively prevents the growth of certain tumors in culture and nude mice (42
). More recently, it has been demonstrated that restoration of expression of p53 in animal models using genetic method blocks tumor growth (22
). Therefore, developing small molecules that increase p53 is of great clinical importance. Many studies have shown that the level of p53 in cells is tightly controlled through ubiquitylation and proteasomal degradation (2
As a consequence of its central role in regulation of p53, Hdm2 represents a particularly attractive molecular target for inactivation so as to increase p53 activity in cancers that express active p53. One approach to this is to prevent the binding of p53 to Hdm2. Accordingly, small peptides derived from p53 have been shown to inhibit the interaction of Hdm2 with p53, resulting in accumulation of p53 in cells (43
). At least three non-peptidic small molecules, inhibit the interaction of p53 and Hdm2, resulting in p53-dependent apoptosis (20
). In particularly, Nutlin-3 has shown potential promise in tumors that express wild type p53 (45
). We and others (24
) have taken an alternative approach, by screening for small molecules that inhibits Hdm2's E3 activity so as to prevent the ubiquitylation and subsequent proteasomal degradation of p53. Our data first with the HLI98s and now with HLI373 demonstrate that inhibition of Hdm2-mediated ubiquitylation in cells can result in stabilization of both p53 and Hdm2 and preferential killing of tumor cells expressing wild type p53.
Although the HLI98s can stabilize p53 and Hdm2, they have limited solubility in aqueous solution and relative low potency, which prevent their further application in animal models of human tumors. Recently the 7-nitro group present in HLI98s was found to be dispensable for p53 activation (47
). Similarly, HLI373 lacks the 7-nitro group and unlike previously described members of this family, it has the added benefit of water solubility. This property makes it potentially suitable for administration through a variety of routes in vivo
and makes if more amenable to cellular studies than the HLI98s.
There are a number of issues that remain regarding the HLI compounds. One relates to mechanism of action. The HLI compounds inhibit Hdm2 ubiquitin ligase activity in cells, but whether this is a consequence of blocking E2-Hdm2 interactions or altering the RING finger structure remains to be determined. A second issue relates to specificity, it is unlikely that without substantial medicinal chemistry surrounding these leads that a highly specific compound could be generated. However, as we have previously shown, the HLI98s are relatively specific, and the limited analysis we have undertaken with HLI373 also suggests a degree of specificity for Hdm2/Mdm2. While a high degree of specificity is generally considered desirable, it is evident that drugs possessing a certain level of off-target activity can be acceptable (48
) and, in some cases, might even contribute to therapeutic efficacy. A third important issue relates to potency. In some cells we observe stabilization of p53 at 1 μM of HLI373 and activation of a discernable p53 response at 3 to 5 μM. While it is common wisdom that highly potent reagents with activity in the nanomolar levels are essential in therapeutics, this may not necessarily the case when targeting p53. Animal studies have demonstrated that minimal increases in p53 activity are capable of resulting in a substantial p53 response (22
). When p53 levels are acutely increased massive toxicity, particularly in proliferating tissues, can occur (50
). Thus, it may be that agents such as HLI373 that may have an appropriate level of bioavailability are of sufficient activity to be of clinical utility.
In conclusion, HLI373 is a potentially drug-able compound that may serve as a potential lead for therapeutics targeting the E3 activity of Hdm2.