New structural and mechanistic understanding for cancer drug design

A University of Iowa-led study has revealed the unexpected structure adopted by the DNA repair protein RAD52 as it binds and protects replicating DNA in dividing cells. This new structural and mechanistic understanding of the RAD52-DNA complex may help researchers develop new anti-cancer drugs. 

"RAD52 is a coveted drug target for treating cancers that have DNA repair deficiencies, including breast and ovarian cancers, and some glioblastomas," explains Maria Spies, PhD, professor of biochemistry and molecular biology in the UI Carver College of Medicine, and senior author of the new study that was published April 2 in Nature.

This protein is an attractive target for new anti-cancer drugs because while it is dispensable in healthy human cells, RAD52 becomes essential for survival of cancer cells, which are deficient in DNA repair function, such as those with defects in BRCA1 and BRCA2 genes."

Maria Spies, Professor, Biochemistry and Molecular Biology, Carver College of Medicine, University of Iowa Health Care

Cancers with DNA repair deficiencies depend on other proteins to provide backup pathways for DNA repair, which allows the cancer cells to proliferate fast and survive despite DNA damage. RAD52 is one of those proteins. This means that molecules that block RAD52 and prevent it from functioning could be useful for treating these types of cancer. 

It has already been shown that RAD52 inhibitors can selectively kill cancerous cells and minimize the toxicity associated with radiation and chemotherapy. This ability is similar to the action of the first drugs approved to target BRCA1/2 deficient cancers, the so-called PARP (poly-ADP-ribose polymerase) inhibitors, which are now in clinical use. While almost 15% of patients treated with the PARP inhibitor olaparib remain disease free for more than five years, many develop resistance within the first year. 

"Targeting RAD52 (independent of or together with PARP inhibition) will increase the repertoire of available therapies," Spies says. "However, to develop drugs that will inhibit RAD52 in cancer cells, we first need to understand how RAD52 functions at the molecular, structural, and cellular level." 

A new shape reveals possible targets for drug therapy 

The fact that RAD52 appears to be dispensable in normal human cells but essential for survival of cancer cells experiencing defective DNA repair creates both an advantage and a challenge. The advantage is that inhibiting RAD52 should kill cancer cells with minimal negative effect on the patient's healthy cells. The challenge is figuring out what functions and features of RAD52 should be targeted. 

In the new study Spies and her UI team, collaborating with Pietro Pichierri, PhD, professor of molecular medicine, at the Istituto Superiore di Sanità, in Rome, Italy, and M. Ashley Spies, PhD, professor of drug discovery and experimental therapeutics in the UI College of Pharmacy, have discovered structural and functional information about RAD52 that may help them develop new, specific ways to inhibit this protein. 

Double ring structure protects DNA 

Spies and Pichierri had previously discovered that RAD52 is important in protecting stalled DNA replication forks. Their work suggested that this new function of RAD52 facilitates the survival of cancer cells. 

In the new study, Spies' team used cryogenic electron microscopy (CryoEM) to show that RAD52 proteins form an unexpected spool-like structure composed of two rings of RAD52, each containing 11 copies of protein, that engages all three arms of the "DNA replication fork," rearranges the fork structure, and protects it from excessive degradation. 

To obtain this image, the team created a DNA substrate, which resembles a stalled DNA replication fork. The substrate fixes the RAD52 complex in place by bringing the two rings together with all three DNA arms. Both single and double stranded DNA features interact with RAD52 and hold the structure in place, allowing the team to obtain a detailed 3D structure of the whole protein-DNA complex. 

Using specialized microscopes built in Spies' lab, the researchers were also able to monitor the RAD52-DNA transactions at the single-molecule level, revealing that the fork protection occurs through dynamic protein-DNA interactions. 

"Although the single ring structure had been observed previously, this is the first structure showing the two rings together on the DNA, doing something unexpected," Spies says. "This new structure provides clues about which important areas of the protein can be targeted for future drug discovery." 

Targeting RAD52 to create new cancer drugs 

Spies' team already has small molecules that bind and inhibit RAD52, but to develop these molecules into testable drugs, they need to be further refined and modified to make them more effective and more specific. 

The results of Spies lab's structural and biophysical work were complemented by computational studies by M. Ashley Spies, and cell-based and super-resolution imaging by the Pichierri group in Rome. In combination, the labs' efforts revealed the importance of the two-ring RAD52 architecture to its ability to act as a DNA replication gatekeeper and to the survival of cancer cells. 

"This work and our structure-activity knowledge gained in this study sets up future work on understanding the RAD52 activities and regulation and offers new targets for its inhibition," Spies says. "Hopefully, this information will help us develop new inhibitors of this protein and tap the potential of RAD52 as an anti-cancer drug target." 

In addition to Maria Spies, Pichierri and Ashley Spies, the study team also included co-lead authors Masayoshi Honda, PhD, Mortezaali (Ali) Razzaghi, PhD, both research scientists in the Spies UI lab. In addition, Spies credits the expertise of Nick Schnicker, PhD, and Lokesh Gakhar, PhD, the current and former directors of the UI Protein and Crystallography Core for their assistance in starting the structural studies of RAD52. 

The study was funded in part by grants from the National Cancer Institute, part of the NIH.