Discovery
Fast folding for synthetic peptides and microproteins
Certain types of peptides and microproteins for drug discovery research can be made more efficiently and quickly using a reaction solvent that helps mimic nature’s way.
Chemists can now produce an important class of small proteins called cysteine-rich peptides in their naturally folded 3D structure more reliably and much faster, thanks to methods that mimic what happens inside cells. The advance, achieved by researchers at Xi’an Jiaotong-Liverpool University (XJTLU) in China and Nanyang Technological University (NTU) in Singapore, is published in the journal Angewandte Chemie.
Cysteine is one of the many different amino acid molecules that can become linked together to form protein chains. Peptides are chains that are shorter than many natural proteins. Cysteine molecules each contain a sulfur atom that can become bonded to the sulfur of another cysteine elsewhere in a protein, holding different parts of the chain together.
“Re-creating the 3D shapes of cysteine-rich peptides has always been a big problem in their manufacturing,” says Dr Shining Loo of the XJTLU team. Many bioactive proteins and peptides have multiple disulfide bonds between cysteine amino acids, which are crucial for maintaining their precise 3D folded structure. Drugs like linaclotide for constipation and ziconotide for chronic pain are examples of cysteine-rich peptide drugs on the market.
“Our procedure should unlock new opportunities for drug discovery and cost-effective manufacturing of cysteine-rich microproteins and peptides as therapeutic agents,” adds researcher Dr Antony Kam of the XJTLU team.
Nature’s influence
Inspired by how nature quickly folds proteins inside cells, the researchers tried a different approach for the ‘oxidative’ folding reactions that form the disulfide bonds. Instead of using water-based (aqueous) solutions they used a mixture of organic solvents. This method imitates the natural enzyme that mediates the disulfide bond formation, by creating a highly reactive environment to greatly speed up the formation and rearrangement of these bonds.
By learning from nature in this way, the team was able to make 15 different peptides and microproteins, between 14 to 58 amino acids long with two to five disulfide bonds, at rates more than 100,000 times faster than could be achieved in aqueous solvents.
The image shows two round molecules connected with a line to represent a disulfide bond. Disulfide bond common in protein structures. Credit: A7davis (Public domain)
A schematic summary of the peptide/microprotein folding creating the final structure held in place by disulfide bonds. Credit: Dr Kam and Dr Loo (XJTLU)
“The folding was efficiently completed within one second,” Dr Loo remarks, “And the range of microproteins we produced demonstrates that our method should be effective with a much larger range of peptides and microproteins in future investigations.”
This discovery is the latest advance from the XPad (XJTLU Peptide and Drug) research group, jointly established by Dr Loo and Dr Kam. This group is committed to using tools from chemical biology, synthetic biology, and molecular pharmacology to advance the application of peptides for developing therapeutic agents.
“The future of peptide research holds great promise, and we are committed to delivering even more valuable advancements in this field,” Dr Kam concludes.
External Link: https://www.xjtlu.edu.cn/en/news/2024/03/fast-folding-for-synthetic-peptides-and-microproteins
DOI: https://onlinelibrary.wiley.com/doi/10.1002/anie.202317789
Reference: Dr. Antony Kam, Dr. Shining Loo, Dr. Yibo Qiu, Prof. Chuan-Fa Liu, Prof. James P. Tam, Ultrafast Biomimetic Oxidative Folding of Cysteine-rich Peptides and Microproteins in Organic Solvents. Angewandte Chemie, 2024; 63 (14)
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Discovery
Scientists tame chaotic protein fueling 75% of cancers
Discovery opens window to more effective treatment
Meet MYC, the shapeless protein responsible for making the majority of human cancer cases worse. UC Riverside researchers have found a way to rein it in, offering hope for a new era of treatments.
In healthy cells, MYC helps guide the process of transcription, in which genetic information is converted from DNA into RNA and, eventually, into proteins. “Normally, MYC’s activity is strictly controlled. In cancer cells, it becomes hyper active, and is not regulated properly,” said UCR associate professor of chemistry Min Xue.
“MYC is less like food for cancer cells and more like a steroid that promotes cancer’s rapid growth,” Xue said. “That is why MYC is a culprit in 75% of all human cancer cases.”
At the outset of this project the UCR research team believed that if they could dampen MYC’s hyperactivity, they could open a window in which the cancer could be controlled.
The MYC proteins (grey ribbons) bind to DNA and promote cancer progression. UCR researchers developed a molecule (orange pretzel-like shape) that binds to MYC, inhibiting its cancer-promoting function. (Min Xue/UCR)
However, finding a way to control MYC was challenging because unlike most other proteins, MYC has no structure. “It’s basically a glob of randomness,” Xue said. “Conventional drug discovery pipelines rely on well-defined structures, and this does not exist for MYC.”
A new paper in the Journal of the American Chemical Society, on which Xue is the senior author, describes a peptide compound that binds to MYC and suppresses its activity.
In 2018, the researchers noticed that changing the rigidity and shape of a peptide improves its ability to interact with structureless protein targets such as MYC.
“Peptides can assume a variety of forms, shapes, and positions,” Xue said. “Once you bend and connect them to form rings, they cannot adopt other possible forms, so they then have a low level of randomness. This helps with the binding.”
In the paper, the team describes a new peptide that binds directly to MYC with what is called sub-micro-molar affinity, which is getting closer to the strength of an antibody. In other words, it is a very strong and specific interaction.
“We improved the binding performance of this peptide over previous versions by two orders of magnitude,” Xue said. “This makes it closer to our drug development goals.”
Currently, the researchers are using lipid nanoparticles to deliver the peptide into cells. These are small spheres made of fatty molecules, and they are not ideal for use as a drug. Going forward, the researchers are developing chemistry that improves the lead peptide’s ability to get inside cells.
Once the peptide is in the cell, it will bind to MYC, changing MYC’s physical properties and preventing it from performing transcription activities.
This work is possible in part with funding from the U.S. Department of Defense and congressionally directed medical research and from the National Institutes of Health.
Xue’s laboratory at UC Riverside develops molecular tools to better understand biology and uses that knowledge to perform drug discovery. He has long been interested in the chemistry of chaotic processes, which attracted him to the challenge of taming MYC.
The MYC proteins (grey ribbons) bind to DNA and promote cancer progression. UCR researchers developed a molecule (orange pretzel-like shape) that binds to MYC, inhibiting its cancer-promoting function. (Min Xue/UCR)
“MYC represents chaos, basically, because it lacks structure. That, and its direct impact on so many types of cancer make it one of the holy grails of cancer drug development,” Xue said. “We are very excited that it is now within our grasp.”
External Link: https://news.ucr.edu/articles/2024/01/04/scientists-tame-chaotic-protein-fueling-75-cancers
DOI: https://pubs.acs.org/doi/10.1021/jacs.3c09615
Reference: Zhonghan Li, et al. MYC-Targeting Inhibitors Generated from a Stereodiversified Bicyclic Peptide Library. Journal of the American Chemical Society 2024; 146 (2): 1356-1363
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DEVELOPMENT
A new path to drug diversity
Research on protein evolution reveals new starting points for the rapid and targeted development of future drugs
Many important medicines, such as antibiotics and anticancer drugs, are derived from natural products made by Bacteria. The enzyme complexes that produce these active ingredients have a modular design that makes them ideal tools for synthetic biology. By exploring protein evolution, a team led by Prof. Dr. Helge Bode has found new “fusion sites” that enable faster and more targeted drug development.
Industry often follows the assembly line principle: components are systematically assembled into complex products, with different production lines yielding different products. However, not humans are the actual inventors of this principle, but bacteria. Non-ribosomal peptide synthetases (NRPS) are bacterial enzymes that, like production lines, produce an immense variety of natural products. They enable bacteria to survive in a wide variety of natural habitats. Humans have benefited significantly from these enzyme complexes, as they are the origin of many important drugs like antibiotics.
Multitude of enzyme variants generates diversity of natural substances
The research group of Prof. Dr. Helge Bode at the Max Planck Institute for Terrestrial Microbiology in Marburg is investigating the use of these enzyme systems for the targeted production of drugs in the laboratory. The researchers modify parts of the enzymes and thus the functional properties of the entire enzyme complexes (NRPS engineering) in order to produce products with new properties. However, although this concept has been pursued for several years, it has not yet worked as hoped. “We realized that there is a great opportunity in taking nature as a model. If we understand the natural processes, we will know which areas of the enzyme are best suited for NRPS engineering,” explains Dr. Kenan Bozhüyük, one of the lead authors of the study, which was now published in the journal “Science”.
Recombination following the natural model
To find out which subunits of the enzyme work particularly well together, the team focused on the question: What are the positions that evolution itself applies to establish or change the new “assembly lines” to create the required active compounds? Together with the group of Dr. Georg Hochberg (also MPI) and Prof. Dr. Michael Groll (TU Munich), the team screened for “hotspots” of natural recombination. “We analyzed several tens of thousands of enzymes bioinformatically and then combined the analysis with laboratory experiments to verify the predicted target sites,” explain the first authors Leonard Präve and Dr. Carsten Kegler.
In fact, the team found a new “fusion point” for the targeted production of functional NRPS hybrids. They were even able to combine NRPS sequences from completely different organisms, such as bacteria and fungi.
The researchers then tested their new knowledge in a medical context: They constructed a new, pharmacologically active peptide. The comprehensive study demonstrates the great potential of bacterial natural products as the basis for new drugs.
The aim is to create customised medicines
“Research in both, synthetic biology and evolutionary biochemistry, has made enormous progress in recent years,” said Prof. Helge Bode, Director at the Max Planck Institute in Marburg. “The key advantage of our approach is that we are using evolutionary processes that have proven themselves over millions of years. Our evolution-inspired fusion sites are more versatile and have higher success rates.”
The team’s concept combines synthetic biology with the high-throughput methods needed to discover biologically active compounds faster and more cost-effectively. In this way, the researchers hope to develop customized biological drugs with improved therapeutic properties – something that is becoming increasingly important in view of the rise in drug resistance and drug intolerance.
External Link: https://www.mpi-marburg.mpg.de/1367868/2024-03-b?c=11478
DOI: https://www.science.org/doi/10.1126/science.adg4320
Reference: Kenan A. J. Bozhüyük et al., Evolution-inspired engineering of nonribosomal peptide synthetases. Science 2024; 383 (6689)
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DELIVERY
Oral peptides: A new era in drug development
Scientists at EPFL have developed stable orally available cyclic peptides with a much higher bioavailability than current ones, virtually creating a generation of drugs that can target previously untreatable diseases like certain types of cancer.
For decades, a substantial number of proteins, vital for treating various diseases, have remained elusive to oral drug therapy. Traditional small molecules often struggle to bind to proteins with flat surfaces or require specificity for particular protein homologs. Typically, larger biologics that can target these proteins demand injection, limiting patient convenience and accessibility.
“We have now succeeded in generating cyclic peptides that bind to a disease target of our choice and can also be administered orally.” – Professor Christian Heinis, EPFL
In a new study published in Nature Chemical Biology, scientists from the laboratory of Professor Christian Heinis at EPFL have achieved a significant milestone in drug development. Their research opens the door to a new class of orally available drugs, addressing a long-standing challenge in the pharmaceutical industry.
“There are many diseases for which the targets were identified but drugs binding and reaching them could not be developed,” says Heinis. “Most of them are types of cancer, and many targets in these cancers are protein-protein interactions that are important for the tumor growth but cannot be inhibited.”
The study focused on cyclic peptides, which are versatile molecules known for their high affinity and specificity in binding challenging disease targets. At the same time, developing cyclic peptides as oral drugs has proven difficult because they are rapidly digested or poorly absorbed by the gastrointestinal tract.
“Cyclic peptides are of great interest for drug development as these molecules can bind to difficult targets for which it has been challenging to generate drugs using established methods,” says Heinis. “But the cyclic peptides cannot usually be administered orally – as a pill – which limits their application enormously.”
Cyclizing Breakthrough
The research team targeted the enzyme thrombin, which is a critical disease target because of its central role in blood coagulation; regulating thrombin is key to preventing and treating thrombotic disorders like strokes and heart attacks.
To generate cyclic peptides that can target thrombin and are sufficiently stable, the scientists developed a two-step combinatorial synthesis strategy to synthesize a vast library of cyclical peptides with thioether bonds, which enhance their metabolic stability when taken orally.
“We have now succeeded in generating cyclic peptides that bind to a disease target of our choice and can also be administered orally,” says Heinis. “To this end, we have developed a new method in which thousands of small cyclic peptides with random sequences are chemically synthesized on a nanoscale and examined in a high-throughput process.”
Two steps, one pot
The new method process involves two steps, and takes place in the same reactive container, a feature that chemists refer to as “one pot”.
The first step is to synthesize linear peptides, which then undergo a chemical process of forming a ring-like structure – in technical terms, being “cyclized”. This is done with using “bis-electrophilic linkers” – chemical compounds used to connect two molecular groups together – to form stable thioether bonds.
In the second phase, the cyclized peptides undergo acylation, a process that attaches carboxylic acids to them, further diversifying their molecular structure.
The technique eliminates the need for intermediate purification steps, allowing for high-throughput screening directly in the synthesis plates, combining the synthesis and screening of thousands of peptides to identify candidates with high affinity for specific disease targets – in this case, thrombin.
Using the method, the PhD student leading the project, Manuel Merz, was able to generate a comprehensive library of 8,448 cyclic peptides with an average molecular mass of about 650 Daltons (Da), only slightly above the maximum limit of 500 Da recommended for orally available small molecules. The cyclic peptides also showed a high affinity for thrombin.
When tested on rats, the peptides showed oral bioavailability up to 18%, which means that when the cyclic peptide drug is taken orally, 18% of it successfully enters the bloodstream and to have a therapeutic effect. Considering that orally administered cyclic peptides generally show a bioavailability below 2%, increasing that number to 18% is a substantial advance for drugs in the biologics category – which includes peptides.
Setting targets
By enabling the oral availability of cyclic peptides, the team has opened up possibilities for treating a range of diseases that have been challenging to address with conventional oral drugs. The method’s versatility means it can be adapted to target a wide array of proteins, potentially leading to breakthroughs in areas where medical needs are currently unmet.
“To apply the method to more challenging disease targets, such as protein-protein interactions, larger libraries will likely need to be synthesized and studied,” says Manuel Merz. “By automating further steps of the methods, libraries with more than one million molecules seem to be within reach.”
In the next step of this project, the researchers will target several intracellular protein-protein interaction targets for which it has been difficult to develop inhibitors based on classical small molecules. They are confident that orally applicable cyclic peptides can be developed for at least some of them.
Other contributors: EPFL Center of Phenogenomics
External link: https://actu.epfl.ch/news/oral-peptides-a-new-era-in-drug-development/
DOI: https://doi.org/10.1038/s41589-023-01496-y
Reference: Merz, M.L., Habeshian, S., Li, B. et al. De novo development of small cyclic peptides that are orally bioavailable. Nat Chem Biol 2024; 20: 624–633
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DELIVERY
New chemical method advances toward targeted RNA medicine
In close collaboration with Novo Nordisk, a novel method has been developed to enhance the synthesis of therapeutic oligonucleotides for targeted drugs. This method holds importance in the creation of customised medicine aimed at precisely targeting diseased cells, representing a pivotal stride towards more efficient and gentle treatments to optimise patients’ quality of life.
Targeted drugs aim to pinpoint the exact location in the body where diseased tissue is located and where the medicine is required. The manifold benefits of administering a targeted drug include heightened efficacy, as the drug is meticulously designed for specificity, thereby reducing side effects and minimising damage to healthy tissue. Consequently, this approach enhances the patient’s quality of life during treatment.
Oligonucleotides (ONs), specifically designed short chains of DNA or RNA, have emerged as a crucial tool with immense potential in personalised medicine. These therapeutic ONs are already in use for conditions, such as certain types of muscular dystrophy and liver disease, which conventional drugs cannot address.
Depending on the type, ONs can function by, preventing or changing the production of a protein in the cell, particularly beneficial in diseases caused by the overproduction of a specific protein.
Peptide conjugates as a precise drug delivery solution
However, a persistent challenge lies in precisely delivering therapeutic ONs to a broader range of tissues where treatment of diseased cells is needed. Researchers have attempted to overcome this hurdle by attaching small, specific protein markers to these ONs, known as peptide conjugates, recognizable by the diseased cells.
One such peptide is glucagon-like peptide-1 (GLP1), which has been used to specifically target therapeutic ONs to the pancreas. This peptide is the natural analog of the diabetes and obesity drug Semaglutide sold as Ozempic, Rybelsus and Wegovy.
The positioning of chemical modifications on the therapeutic ON is crucial for the clinical success of this class of drugs. Likewise, the placement of GLP1 in peptide-ON conjugates could be of absolute importance. However, placing the ligand inside the ON sequence has, until now, required specialized and costly ON building blocks.
Development of peptide conjugates without expensive building blocks
In close collaboration with Novo Nordisk, Professor Kurt Gothelf and his research group have now devised a method to simplify the construction of an entire library of therapeutic ON – peptide conjugates. As a central part of the collaboration, iNANO PhD student Jakob M. Smidt visited Specialist Lennart Lykke at Novo Nordisk in Måløv. The expertise led to a highly successful collaboration with novel discoveries that are beneficial for researchers in the pharmaceutical industry and at universities. In this collaboration, the researchers have discovered a synthesis method for ON conjugates that incorporates built-in handles and a special linker, enabling easy linkage of ONs to a peptide marker by adjusting the pH.
The noteworthy aspect of this method is the elimination of the need for specialized and expensive ON building blocks to integrate peptides into the oligonucleotide sequence.
Potential for effective medicine with multiple functions
The significance of this method lies in its streamlining of the process, making the production of these conjugates more accessible and cost-effective. This breakthrough holds the potential to produce therapeutic ONs with multiple functions, paving the way for more effective drugs.
By joining the expertise in ON chemistry, including novel functionalization technology, of the Gothelf lab together with the peptide science legacy of Novo Nordisk, new and robust conjugation chemistry has emerged. The method has enabled insights into the structure-activity relationship of ON conjugates and is thus a very important scientific contribution to understanding this promising class of therapeutic molecules.
This work is the product of a fruitful public-private collaboration where knowledge and new ideas have openly been shared and discussed with the common ambition to make a scientific impact, and potentially make a difference to people living with disease.
Gothelf envisions a future where this method directs ON-based drugs to specific tissues in the body. The development of this method marks a significant stride towards more effective and targeted drugs, indicating the potential for customised therapeutic ONs to deliver drugs precisely to the intended location in the body.
External link: https://inano.au.dk/about/news-events/news/show/artikel/new-chemical-method-advances-toward-targeted-rna-medicine
DOI: https://doi.org/10.1093/nar/gkad1015
Reference: Jakob Melgaard Smidt, Lennart Lykke, Carsten Enggaard Stidsen, Nuša Pristovšek, Kurt V Gothelf, Synthesis of peptide–siRNA conjugates via internal sulfonylphosphoramidate modifications and evaluation of their in vitro activity. Nucleic Acids Research 2024; 52, (1): 49–58
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Oligonucleotides Section
Next-generation treatments hitch a ride into cancer cells
Antisense oligonucleotides (ASOs) are next-generation drugs that can treat disease by blocking the transfer of harmful messages from our genes. In people with cancer, ASOs have the potential to block messages that encourage the growth and spread of the tumor. However, ASOs aren’t used for treating cancer yet. They must first get delivered inside cancer cells, but the cancer cells won’t let them in.
Finding an effective ASO delivery system is a major challenge. Cancer cells have gatekeeper molecules that stop unwanted substances from entering. Although investigators have tried many ways of getting ASOs past the gatekeepers, success has been limited.
Now, in a study recently published in the journal Nucleic Acids Research, researchers from Osaka University have discovered a way to deliver ASOs to their targets inside cancer cells. The team synthesized a new compound, named L687, which opens specific calcium permeable channels on the surface of cancer cells. When the calcium flows into cells through the open channels it tells the cells to let in the ASOs.
“We discovered that we could selectively activate the TRPC3/C6 calcium permeable channels 1) with the activator L687,” explains lead author Hiroto Kohashi. “We then found that combination treatment with L687 and ASO promoted efficient uptake of ASO into cancer cells during laboratory tests and tumor cells inside the mouse. As a result, target gene activity was suppressed and ASO efficacy was enhanced.”
Until now, ASOs have mainly been used to treat incurable diseases and had to be delivered into the liver or spinal fluid. According to the Osaka team’s research, L687 is an effective drug delivery system that may extend the benefits of ASO treatment to other parts of the body.
“We hope that the results of our research will lead to significant progress in the development and delivery of ASOs and similar gene-targeting drugs for treating cancer,” says senior author Masahito Shimojo.
The team believes that L687 could be a particularly effective way of delivering ASO therapy to lung or prostate cancers. These cancers have many TRPC3/C6 calcium permeable channels 1) that can be opened by L687, potentially revealing new targets for these next-generation therapies.
1) TRPC3/C6 channels belong to a Transient Receptor Potential Canonical (TRPC) Channel subfamily of a TRP channel superfamily.
External link: https://resou.osaka-u.ac.jp/en/research/2024/20240416_2
DOI: https://doi.org/10.1093/nar/gkae245
Reference: Hiroto Kohashi, et al. A novel transient receptor potential C3/C6 selective activator induces the cellular uptake of antisense oligonucleotides. Nucleic Acids Research 2024; 1-15
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RNA
Antisense oligonucleotides targeting lnc-HLX-2-7 as a therapy to G3 Medulloblastoma
Johns Hopkins Kimmel Cancer Center investigators have identified a non-coding RNA, called lnc-HLX-2-7, as a potential therapeutic target in group 3 (G3) medulloblastoma.
Their experiment was conducted on mice, using antisense oligonucleotides targeting lnc-HLX-2-7 and the result was a tumor growth inhibition.
Their discovery is based on the fact that lnc-HLX-2-7 RNA specifically accumulates in the promoter region of HLX, a sense-overlapping gene of lnc-HLX-2-7, which activates HLX expression.
By doing so, several other genes (including TBX2, LIN9, HOXM1, and MYC) known to be involved in cancer development are activated.
When this therapy was combined with a standard chemotherapy drug called cisplatin, tumor growth was further inhibited, and the survival of the mice was significantly extended compared to using the therapy alone. These findings suggest that targeting the lnc-HLX-2-7-HLX-MYC pathway could be an effective strategy for treating G3 MBs.
DOI: https://doi.org/10.1016/j.celrep.2024.113938
Reference: Keisuke Katsushima et al., A therapeutically targetable positive feedback loop between lnc-HLX-2-7, HLX, and MYC that promotes group 3 medulloblastoma. Cell reports 2024; 43 (3)
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RNA
RNA – Optimizing Boosters: How COVID mRNA Vaccines Reshape Immune Memory After Each Dose
Researchers show that T cells can reshape their memory and maintain diversity against different COVID variants in response to successive mRNA vaccinations.
mRNA vaccines developed against the spike glycoprotein of severe acute respiratory syndrome type 2 coronavirus (SARS-CoV-2), displayed remarkable efficiency in combating coronavirus 19 (COVID-19). These vaccines work by triggering both cellular and humoral immune responses against the spike protein of the virus. Cellular
