Research Highlights

Pocket Full of Promise for Drug Target p53

box

Figure 1: Stictic acid (green) bound to the transient pocket L1/S3 in p53 mutant protein.

Cancer remains the second leading cause of death, after heart diseases, in the US, and millions of people die from different types of cancer worldwide each year. The known tumor suppressor protein p53 is an important regulator of cell growth in normal cells and its dysfunction is closely related to tumor development and progression. Frequently tumor cells contain a mutated p53 protein that reduces or abolishes p53 anti-tumor activities. One of the current small molecules called PRIMA-1 (APR-246, aprea.com) has been shown to reactivate p53 activity and induce cell death preferentially in tumor cells. In phase I/II clinical trials, healthy volunteers and affected patients are given the candidate drug and the safety and efficacy of the drug are monitored. PRIMA-1 is reported to have shown promising safety and efficacy profiles. A new phase II study is under preparation for ovarian cancer patients with 60% carrying the p53 mutations. However, the precise mode of action of PRIMA-1 compounds are not fully characterized. 

Prof. Rommie Amaro, Director of NBCR, and the principal investigators of her collaborative project, Prof. Peter Kaiser and Prof. Richard Lathrop, at UC Irvine, have been studying reactivation of p53 mutant proteins to deter the growth of tumor cells and identify novel anti-cancer drug leads. "What's very exciting about the reactivation of the p53 would be that it could ideally lead to therapies that will not kill all cells," she continued. "It would be a drug that would react only when interacting with a cancer cell. This means less aggressive, more effective treatments, with the patient having to bear very little cytotoxic danger." 

Christopher Wassman, a graduate student in computer science at the UC Irvine wanted to understand how p53 would be reactivated by PRIMA-1. When he tried docking it in the human p53 protein but did not find any docking sites. Together the two teams have applied a series of computer aided drug discovery tools and identified new lead compounds for p53 reactivation in a study published in a January 2013 article in Nature Communications. 

Using existing human and mouse crystal structures of wild type p53 from the Protein Data Bank, Dr. Ozlem Demir, a Turkish postdoctoral fellow in the Amaro lab, constructed several well characterized cancer p53 mutants in genetic and nuclear magnetic resonance (NMR) experiments. She then utilized a technique pioneered by Prof. Amaro, the relaxed complex scheme, to identify novel pockets in p53 not found in crystal structures. These binding pockets found only through molecular dynamic simulations could be the “real” target binding sites for small molecules such as PRIMA-1. Indeed, all the known p53 reactivation compounds tested are able to bind to a transiently open pocket called the L1/S3 pocket found in both wild type and mutant p53’s. After screening several thousand compounds using structures with the open pocket conformation, stictic acid was identified as a novel p53 reactivation compound. The stictic acid is able to stably bind to the L1/S3 pocket through an extended MD simulation, suggesting that the interaction is relatively stable.

Dr. Wassman and Dr. Roberta Baronio from UC Irvine provided experimental demonstration of PRIMA-1 reactivation of p53 activity in a cell line (Saos-2) without p53 genes but express p53 mutants using expression vectors. These mutations, e.g., R175H (Argnine 175 to histidine mutation), is found in the same L1/S3 pocket identified in the molecular dynamics (MD) simulation. One cysteine residue located within the pocket, Cys124, is shown to be essential for p53 reactivation. In human cells, reactivation of p53 induces the expression of p21, a cell cycle inhibitor, a known effect of p53 activation. Stictic acid is shown to increase the expression of p21 in the same Saos-2 cell lines using several p53 mutant constructs.

The computer simulations carried out by Dr. Demir changed the team’s structural understanding of p53 and revealed what wasn't found in the experimental studies. "We were able to use the newly-exposed pocket to show where these agents can bind," she said. Stictic acid is a natural product found in some species of lichens. The molecule fit deeper into the L1/S3 pocket, and "substantially expanded the initial network of favorable direct and water-mediated interactions, and shifted the ligand and receptor exposure patterns." Prof. Peter Kaiser notes that, "while stictic acid cannot be developed into a viable drug, the work suggests that a comprehensive screening of small molecules with similar traits may uncover a usable compound that binds to this specific p53 pocket."

NBCR works with collaborators like Profs. Kaiser and Lathrop to continuously improve the relaxed complex scheme and make such tools available to the broader biomedical research community. For more information, please see related references.

References: 

  1. Wassman CD, Baronio R, Demir O, Wallentine BD, Chen CK, Hall LV, Salehi F, Lin DW, Chung BP, Hatfield GW, Richard Chamberlin A, Luecke H, Lathrop RH, Kaiser P and Amaro RE (2013) Computational identification of a transiently open L1/S3 pocket for reactivation of mutant p53. Nat Commun 4:1407. doi: 10.1038/ncomms2361
  2. TACC News
  3. National Biomedical Computation Resource
  4. Amaro Lab
  5. Kaiser Lab
  6. Lathrop Lab

Researchers: NBCR: Rommie Amaro, Ozlem Demir; Collaborators: Peter Kaiser, Richard Lathrop, Christopher Wassman, Roberta Baronio (all from UC Irvine)

Figure 1: Stictic acid (green) bound to the transient pocket L1/S3 in p53 mutant protein.