CLIPS™ technology: “Powerful, elegant, and full of potential”

Mastering peptides can enable life-improving breakthroughs in the life sciences. Constraining peptides in well-defined 3D conformations is a powerful method. Pepscan developed a surprisingly simple, yet elegant principle to do so. Its CLIPS™ technology can create mono-, bi- and, in combination with click chemistry, even up to hexa-cyclic formats. “A super powerful tool. We know this from our own work, and it has been proven as well by Bicycle Therapeutics, to whom we granted a global CLIPS™ Technology license for two of their product candidates last year”, says Pepscan’s CSO Peter Timmerman, discussing the advantages in a dual interview with Michael Goldflam, Director of Peptide Discovery at Pepscan.

Michael Goldflam

Peptides are gaining popularity in biotechnology and biomedicine worldwide. Their uses range from fundamental research to applications in disease diagnostics and treatment. Peptides are usually designed to mimic certain parts of protein surfaces. “In order to work effectively, their 3D conformation is crucial”, explains Peter Timmerman, Chief Scientific Officer at Pepscan. “When they represent the curved part of a protein surface, for instance, they don’t perform well if they have a linear structure. Antibodies won’t recognize them. When we were developing methods for our epitope mapping platform, we realized that we needed to cyclize peptides in order to deliver optimal results.”

Peter Timmerman

To address this need, Pepscan developed an innovative scaffold technology, called CLIPS™ (Chemical LInkage of Peptides onto Scaffolds, see text box below). “When we first developed this solution around 2004, we found that double or triple looped peptides have an enormously enhanced proteolytic stability”, says Timmerman. “And with the scaffold we developed in cooperation with the University of Amsterdam, we’re currently working on making tri- to hexa-cyclic peptides.”

“The potential is vast,” adds Michael Goldflam, Pepscan’s Director of Peptide Discovery. “In many applications, such as phage display, it helps enormously. It significantly boosts many interesting developments.”

What are the scaffolds made of?

Michael: “They are small organic molecules; many of these contain an aromatic core. They carry two or more methyl bromide groups, which react with the thiol groups of the cysteines on the peptide. One limitation is, if you want to get a single product, you need a C2-symmetric CLIPS™. We now have a large collection of C2-symmetric molecules, which also cover a bigger set of distances between the cross-linkers. All in all, this allows you to play with the 3D structure of your peptides, and hence with their characteristics, such as affinity, specificity, stability and solubility.”

Peter: “We tried different types of scaffolds, with benzylic, allylic, and alkylic bromides. We found that both benzylic and allylic bromides react exclusively, and rapidly, with the thiol group of peptides – they don’t get hydrolyzed by water equally fast. Water molecules aren’t strong enough to compete with the thiols on the peptide, even though they are present in more than a thousand-fold excess! When we claimed this almost 20 years ago, nobody believed us. It was just a wild experiment to try. But now this technology allows us to perform our CLIPS™ reactions in water, and in the presence of side-chain-unprotected amino acids at very dilute conditions. That is key. That is the reason why others have also embraced our technology.”


What about the reaction speeds?

Michael: “We achieve some of the highest reaction speeds in chemical biology. If you work at picomolar concentrations, like in phage display libraries, speed is a crucial feature.”

Peter: “Yes, absolutely. Click chemistry uses ‘spring-loaded’ azide and alkyne molecules, and speeds up once you add a copper catalyst. CLIPS™ is even ten to one hundred times ‘more spring-loaded’ than Click chemistry.”

Michael: “Truly one of the most reactive and selective functionalities out there.”

Peter: “The combination of this technology with applications like phage display is extremely powerful. Early work in phage display, with linear peptides, was not very successful. The exploration of cyclization technology in phage display opens up a wide range of possibilities. There is huge diversity and trillions of compounds to screen. CLIPS™ is a super powerful tool for doing this. We know this from our own work, but also from the work of Bicycle Therapeutics, to whom we granted a global CLIPS™ Technology license for two of their product candidates last year. Bicycle Therapeutics has become a front-runner in applying our technology for the benefit of patients – something to be proud of.”

How would you summarize the advantages of CLIPS?

Michael: “What makes this technology unique is the combination of features: it is fast, selective, and stabilizes peptides in a well-defined 3D structure. If you want to develop a peptide drug, and are narrowing down your number of candidates from trillions to a handful, the cost aspect is crucial. Once you’ve chosen your peptide, the cost of production becomes decisive. CLIPS™ allows you to keep the production costs low. There are not so many cross-linking technologies out there that can do all that. Click chemistry can do a lot, but it uses copper; CLIPS™ is a lot cleaner.”

Peter: “And it’s simple. I can do a CLIPS™ reaction in a vial on my table during a lecture. It’s beautiful to see how such a powerful technology can be so simple. And it’s not just useful for phage display. Some companies use this technology as an inspiration for developing new small molecules. Several of our partners say: this is a really big field. Drug design using peptides as an inspiration.”

And tomorrow?

Michael: “Our discovery platform has resulted in several leads, which our customers are bringing to the next phases of development.”

Peter: “Our technology absolutely has the potential to significantly improve the lives of patients. We keep working with our customers to achieve this.”

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How CLIPS™ works

CLIPS™ technology involves the (multiple) cyclization of linear peptides via reaction with a small rigid entity (chemical scaffold) that carries two or more reactive anchor points. The anchors react exclusively with the thiols of the cysteines in the peptide and attach to the peptide via multiple covalent bonds. The peptide folds around the scaffold and loses flexibility while adopting a well-defined 3D structure, with the scaffold entity in the center. The better-defined 3D structure combined with the non-natural covalent thioether bonds provide enhanced stability for degradative enzymes, as well as enhanced affinity, selectivity and proteolytic stability.

Pepscan has developed a toolbox of several different types of fully synthetic, tailor-made CLIPS™ scaffolds, varying mainly in size, polarity, rigidity, solubility, functionality, and ”SS spanning” distance. When positioned appropriately within the peptide sequence, the resulting CLIPS™ peptide resembles the 3D structure of the corresponding region on the intact protein much better than the linear sequence.