Three new articles from the Shin research group are pushing the boundaries of protein design, creating new possibilities for cancer treatment and biosensors.
“Rational design, directed evolution, and computational methods, done in collaboration, are the trifecta that we think will be key to protein design in the 21st century,” said Jumi Shin, an Associate Professor whose research group is located on the Mississauga campus. “We have already shown the merits of using proteins—which nature uses—as tools in the lab for applications like synthetic biology and drugs.”
Rational design is a strategy where chemists design or modify proteins based on detailed knowledge of their structure and function. Using principles from chemistry and biophysics, researchers can develop their know-how to engineer new proteins useful in the drug arena and synthetic biology, to name some applications.
In “HinZip: Combining Hin Recombinase and FosW to Mimic HD-Zip Plant Proteins,” for example, Shin and PhD students Raneem Akel and Rama Edaibis used rational design to create a customized protein that can target a given genetic sequence, a useful trait for turning genes on or off.
“Our work is extremely novel,” Akel said. “With HinZip, we have made a tool for synthetic biology that combines elements from different biological domains and kingdoms. Thus, HinZip is expected to be orthogonal to all other processes in any organism it is introduced to. It has a wide applicability to be used in gene circuits to control the expression of other proteins."
What kind of applicability can these kinds of proteins have? In Designer Frankenproteins That Halt the Proliferation of Myc-Driven Cancer Cells, one of the Shin Group’s custom proteins has been shown to inhibit or block a protein complex called Myc/Max from binding to the E-box DNA target site.
“This is good because Myc, in particular, goes rogue in many cancers,” explains Shin, “And currently there is no small-molecule drug that can tackle the Myc/Max/E-box network.”
What does this mean? “Our protein drugs are potentially the next-generation arsenal against cancers.”
Maryam Ali, a UofT PRiME Fellow, outlined the potential of this work. “Our collaboration with Micheline Piquette-Miller in UofT Pharmacy has given extremely positive results, where we see our MEF protein and variants specifically targeting the Myc/Max/E-box network in multiple triple-negative breast cancer cell lines. We are excited to see the results of the animal studies our collaborators are now obtaining with our proteins.”
This project was funded by the Cancer Research Society and is now being funded by Ontario Institute of Cancer Research Cancer Therapeutic Innovation Pipeline. The CTIP Late Accelerator program provides up to $1M over 2 years to develop and translate new anti-cancer treatments and provides resources that enable assessment of "go/no-go" decisions.
"This project is developing a new class of drugs that can enter cancer cells and disrupt a major cancer network that is active in more than 70 per cent of tumours and that has resisted conventional drug approaches," says the official OICR announcement, made April 10th.
Professor Shin says her team is delighted to partner with OICR CTIP. "This generous funding allows us to enlarge our collaboration and move our proteins forward."
Developing those new proteins can be streamlined by using directed evolution, in this case by using phage that expresses a mutable protein-of-interest and pushing it to express the most efficient version of that protein.
“Phages are highly infectious particles that carry the DNA harboring the gene expressing the protein we are trying to mutate and improve,” said Shin.
The phage DNA is constantly mutated, if the mutated protein shows the desired function—in this case, binding to the specified DNA target with increasing binding strength—then phages carrying that good-performing gene are produced to then infect a new round of bacteria cells. The winners survive into the next generation and produce offspring.
The group’s third paper, Unlocking genetic potential: harnessing phage for targeted mutagenesis in phage-assisted evolution, also developed this breakthrough technique for optimizing protein development. Shin explains "We collected as many mutations as possible just to give our system a good start, generating a lot of variants (offspring). Some will be clear winners—dominating the pool, continuing to mutate and producing even better offspring.
The work with phages referenced in the paper has also lately cracked what seemed to be a previously impossible problem. Shin explained: “We fused phage-assisted evolution with MutaT7-targeted mutagenesis. These two systems were previously incompatible. Phages are highly infectious and a great way to accumulate mutations that promote survival in the next generation, while MutaT7 allows targeted mutagenesis by bringing a mutator enzyme only to the gene-of-interest. In the original system, the problem was that mutators also mutate the host's genome, other DNA, etc, and this is not what you want.”
“People can make libraries, even large libraries, of mutations. However, with our system, not only can you make large libraries of the particular protein you are trying to mutate and improve for future generations, but the system will also "choose" the winners. “
“We don't have to manually look at every single protein variant and make decisions, as this would be extraordinarily time and reagent/cost-consuming. The biological system does the analysis for us. Then we take a winner and then continue to refine it in subsequent generations by mutation and selecting even stronger binders.”
"This is the great advantage of a true directed evolution system where you can continually have mutagenesis and selection occurring,” said Shin. “Most systems are manual—you mutate, then you look at variants—it's lots of work, and this is just round one!”
Asked when she knew the fusion of the previously incompatible systems would work, Ali said, “I think most researchers have that “Eureka” moment when they finally see positive results after years of troubleshooting, and let me say, it is a wonderful moment! When our eMPAE system finally started giving us the mutations we were expecting, I triple checked my data before I led myself to believe it. I then sent more samples for analysis to validate our results to make sure it wasn’t random or due to chance. I remember leaving for a conference later that week, feeling like I finally made it, ready to share my research with anyone who would listen."
The potential for this work is considerable, according to Maryam Ali, who says the Shin Lab is the only lab in Canada that uses phage-assisted evolution for protein modification. “Recently with our new eMPAE system, we have seen extremely positive results for both library generation and directed evolution, outperforming previous systems with high mutation rates and targeted mutagenesis, which hadn’t been done before."
Asked how their educational journeys had taken them into Shin’s world of frankenproteins, both students said they wanted to move forward with biological chemistry research with therapeutic applications, and, as they were researching potential research supervisors, were intrigued by Shin's term “frankenproteins” and further pursuing the research possibilities of using the protein scaffold to provide useful, interesting molecules.
Akel and Ali also shared their sense of privilege to be working on multiple projects involving these proteins, with their mixture of protein engineering, synthetic biology and cancer therapeutics.
The future looks bright for the Shin Lab’s proteins. Akel talked about the potential to use HinZip and its variants as a biosensor: “We are looking into incorporating it into complex synthetic biology systems. More specifically, we are collaborating with Dr. Radhakrishnan Mahadevan from UofT Chemical Engineering and Applied Chemistry to use HinZip as a part of a biological sensor. In short, HinZip would participate in a cascade where the presence of a particular chemical would induce a visible signal.”
Maryam Ali, meanwhile, expressed high hopes for the MEF variants: “We are expecting our proteins to be used as cancer drugs, as the pathway they inhibit is overexpressed in over 70% cancers. Currently, the only other drug that inhibits this pathway suffers from a weaker binding affinity and suboptimal pharmacokinetic properties. Already our MEF proteins have shown higher binding affinity, and structural stability, and through our collaboration with the Piquette-Miller group, we have been testing our proteins in mice. So far, the mice studies have been promising, with preliminary results showing our proteins are non-toxic to mice.”
The MEF variants have an advantage over some current medical strategies, because they have such a strong and specific binding affinity to their cellular target in comparison with most drugs. "These characteristics, together with data clearly showing MEF and its variants are not toxic to mice, make MEF a desirable cancer drug worth pursuing in further studies,” said Ali.
Protein action lies at the heart of many biological processes, and for some time researchers have known synthesized proteins can play a vast role in medical, environmental, and industrial applications. As the Shin research group pushes this work forward, they are pioneering innovative approaches to protein engineering that promise to unlock new therapeutic strategies, enhance sustainability efforts, and revolutionize manufacturing processes.
Our protein drugs are potentially the next-generation arsenal against cancers. -Jumi Shin