New pathway for molecular cancer drug therapies

 A fundamental challenge in drug development is the balance between optimizing a drug's lock-and-key fit with its target and the drug's ability to make its way across the cellular membrane and access that target. The search for cell-permeable drugs has conventionally focused on low-molecular weight molecules with rigid, nonpolar chemical structures. However, emerging therapeutic strategies break traditional drug design rules by employing larger, flexibly linked chemical entities.

In a study publishing December 8, 2022 in Science, UCSF researchers Kevin Lou, an MD-PhD student, Luke Gilbert, PhD, and Kevan Shokat, PhD, reveal the discovery of a cellular uptake pathway important for larger molecules. These large and complex molecules bind in unconventional ways to their targets, are efficiently taken up by target cells, and can be harnessed to create new drugs for the treatment of cancer and other diseases.

Through a combination of functional genomics and chemical methods, the scientists uncovered an endogenous pathway involving interferon-induced transmembrane (IFITM) proteins that promote the cellular uptake of diverse linked chemotypes. These proteins are found in plasma membranes and often provide cellular resistance to viruses.

Most traditional pharmaceuticals are small molecules that follow simple molecular rules including limits on the molecular size and number of sticky chemical groups on the molecule's surface. Many key drug targets, such as kinase enzymes often involved in cancer, are difficult to selectively target with traditional drugs.

"There are over 500 human kinase enzymes that are very similar in the pocket where the drug binds, making it a challenge to selectively target a single member of this family and leading to undesirable medication side effects," explains the study's first author Kevin Lou. "Increasingly, it has been found that certain linked molecules outside this traditional framework can maintain drug-like properties and gain new mechanisms of action."

There are many important intracellular drug targets that researchers have been unable to be target with small, compact, and rigid molecules. To address this challenge, scientists have taken to linking multiple ligands into a single chemical entity (a linked chemotype). These linked chemotypes can have enhanced potency, greater selectivity, and the capacity to induce the association of more than one target.

"Given this discrepancy between the favorable biological activity of many large, bivalent molecules and traditional concepts of passive permeability, we inferred that linked chemotypes might hijack cellular processes to assist with passage through the cell membrane," wrote Lou. We selected as an example a bitopic inhibitor of mTOR, RapaLink-1, whose molecular weight falls well beyond common guidelines."

The team designed two new linked drugs that they hypothesized might take advantage of this cellular entry pathway. They generated DasatiLink-1 through a linker-joined combination of two known inhibitors of the leukemia protein BCL-ABL1, known as dasatinib and asciminib. Since each drug binds a distinct pocket on the target protein, the researchers reasoned that the linked version could affix itself to two points of contact like a two-pronged key inserting into two locks, enhancing its specificity and effectiveness.?

They also designed BisRoc-1 by linking two molecules of the chemotherapy drug rocaglamide together in a way that would allow it to bridge two copies of the drug's protein target. Despite the fact that both of these drugs violate traditional drug design principles, the team showed that both drugs enter cells, bind tightly to their intended targets, and work just as well as the unlinked versions. The linked versions were uniquely dependent on IFITM protein expression in the target cells, supporting a general role for the IFITM pathway across many types of linked molecules. The researchers showed that DasatiLink-1 is specific for only the BCL-ABL1 kinase, unlike the more relaxed specificity of its two constituent drugs when unlinked.

"Linked inhibitors that require a multi-pronged binding mechanism are much more selective," Lou explains. "They offer substantial advantages as long as they can enter cells efficiently."

"We discovered that IFITM proteins enable bitopic inhibitors to enter cells and this will likely allow us to target previously untargetable proteins in disease," said Luke Gilbert, PhD, co-corresponding author and the Goldberg-Benioff Endowed Professorship in Prostate Cancer Translational Biology at UCSF. "Hopefully, our study will generate new clues for how IFITM proteins function mechanistically that can be pursued by drug design scientists and virologists."

The scientists are working on chemically optimizing the properties of the linked BCR-ABL inhibitors to increase their potency and position them as next-generation therapeutics for BCR-ABL mutant cancers. "We are also excited to expand the scope of intracellular targets amenable to bitopic inhibition," said Gilbert.

Authors: In addition to first author Kevin Lou and co-corresponding authors Luke Gilbert and Kevan Shokat, the other authors of the paper are Douglas Wassarman, Ziyang Zhang, and Megan Moore of the University of California, San Francisco and the Howard Hughes Medical Institute; Tangpo Yang, Thomas O'Loughlin, and Jack Taunton of the University of California, San Francisco; YiTing Paung and Markus Seeliger of Stony Brook University; Regina Egan and Patricia Greninger of the Massachusetts General Hospital; and Cyril Benes of the Massachusetts General Hospital and Harvard Medical School.

Funding: This research was supported by funding from the National Institutes of Health, the Damon Runyon Cancer Foundation, the Pew-Stewart Scholars program, the Goldberg-Benioff Endowed Professorship, the Howard Hughes Medical Institute, the Samuel Waxman Cancer Research Foundation, Wellcome Trust, the Ono Pharma Foundation, Pfizer, and Arc Institute.

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Source: science daily

Novel spatial-omics technology enables investigation of diseases at their early stages

 How can you trace a single diseased cell in an intact brain or a human heart? The search resembles looking for a needle in a haystack. The teams of Ali Ertürk at Helmholtz Munich and LMU Munich and Matthias Mann at the Max Planck Institute of Biochemistry in Martinsried near Munich have now developed a new technology named DISCO-MS that solves the problem. DISCO-MS uses robotics technology to obtain proteomics data from 'sick' cells precisely identified early in the disease.

Most diseases are asympotomatic initially and the affected persons usually still feel fine -- symptoms are not yet present, or still too mild to realize. However, a change has already happened within the body: a virus may have started replicating, or a rogue cell might have divided more often than it should have. But how can these changes be perceived?

Researchers face a similar dilemma when they are studying the early development of diseases. Even when working with animal models, scientists can rarely pinpoint the small sites of disease initiation or characterize the exact molecular changes that drive disease progression.

With the development of DISCO-MS by the research teams of Ertürk and Mann in Germany, this task has become much easier. DISCO-MS combines methods to turn mouse and human tissues transparent with the latest robotics and proteomics technologies to determine their molecular makeup.

DISCO-MS: Transparency to detect early molecular changes

DISCO-MS starts with the so-called DISCO tissue clearing, which renders the mouse body or human organs transparent -- making them accessible to imaging. Thereby, fluorescently labeled cells can be readily identified in intact tissues of specific sites using high-resolution three-dimensional microscopy.

Once the regions of interest have been identified, they are isolated using a new robotics technology called DISCO-bot, developed by mechanical engineer Furkan Öztürk, a Ph.D. student in Ertürk's lab. The robot-assisted extracted tissues are processed for their proteome analysis using advanced mass spectrometry (MS) methods developed by Andreas-David Brunner, a former Ph.D. student in Mann's lab. This high-tech approach allows complete molecular characterization of any desired tissue region identified in 3D in whole mouse bodies or human organs.

Early detection catches the diseases

To showcase the method's power, first author Harsharan Singh Bhatia and colleagues applied DISCO-MS to Alzheimer's disease (AD) mouse model and to atherosclerotic plaques (pathological hardening and narrowing of blood vessels) in the human heart. In the tissue samples of the AD model, the team applied artificial intelligence (AI) to identify the typical AD plaques at the early stages of the disease, which had been difficult to detect by any other method. Subsequent proteomics analyses of the plaques provided an unbiased and large scale study of proteins affected in AD, revealing new molecular players that could be biomarkers for Alzheimer's disease.

In the human heart, the researchers were interested in the composition of the tissues around atherosclerotic plaques, which were quickly visible after tissue clearing. AI detection and robotics extraction of the tissues again allowed the identification of dysregulated molecular pathways in human heart cells related to aortic plaques. These results are key findings, as they form the basis for potential therapeutic targets.

DISCO-MS is the first spatial-omics technology in intact 3D volumes and accelerates studying complicated diseases ranging from cancer to metabolic disorders. As DISCO-MS works with pre-clinical and clinical tissues, it enables the study of diseases at their earliest stages and, subsequently, the development of potential new therapeutics.

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Source: science daily

Eye scratching mechanisms in rats

 Anyone suffering dry skin, eczema, or insect bites will know the unpleasant effects of itchy skin. While the physiological mechanisms behind itching sensations on the skin are well understood, corresponding signals for the eyes are obscure. A research group led by Associate Professor SAKAMOTO Hirotaka from Okayama University (Ushimado Marine Institute; UMI) and Associate Professor TAKANAMI Keiko from Nara Women's University has now investigated the neural mechanisms behind itchy eyes in rodents. What's more, the group also provides insights on the limb tactics rodents adopt to mitigate this eye trouble.

Histamine, a chemical released under allergic and inflammatory conditions, is the trigger that sets into motion itching sensations. Skin-related itch signals are transmitted in neurons via a biochemical pathway known as the spinal Gastrin-releasing Peptide-Gastrin-releasing Peptide receptor (GRP-GRPR) pathway. Now, the research team has already revealed the presence of the GRP-GRPR pathway in nerves transmitting signals from the eyes to the brain, known as trigeminal ganglion (TG) nerves. Thus, this system was the main target of their study.

The researchers first studied scratching behavior in rats when their eyes were provoked with histamine drops. They found that when histamine was instilled in either the left or the right eye, the rats used the same-sided (as the troubled eye) hindfoot for scratching and soothing that eye. However, when histamine was used to trigger itchiness in both eyes together, the rats relied primarily on the right hindfoot for rubbing. The rats seemed to show a right-sided preference when it came to eye scratching.

Next, the focus moved on to biochemical pathways and the GRP-GRPR system. It was found that histamine intervention in either eye resulted in an increase in activity of GRPR-containing neurons in that side of the brainstem. It seemed likely, then, that the GRPR-containing neurons played a role in relaying itch signals from the eyes. To confirm this further, the team used a toxin to lesion these GRPR-containing neurons in the right side of the brainstems of rats. When these rats were then given histamine drops in the eyes, the incidence and duration of scratching was indeed less. A lack of active GRPR neurons resulted in diminished itch signals from their eyes.

"These findings could open up a new field of research on the mechanisms of the laterality in vertebrates and also offer new potential therapeutic approaches…," suggest the researchers. Besides revealing the right-sided preference animals have when using their hindlimbs, the eye scratching test used here could also be used for determining the footedness of individual rodents in future experiments. In addition, the role of the GRP-GRPR system in TG nerves revealed could be the basis for treating eye conditions that cause itchiness.

Background

Gastrin-releasing Peptide-Gastrin-releasing Peptide receptor (GRP-GRPR) pathway: GRP is a neural chemical that plays a role in transmitting signals across the nervous system. Based on the location of the nerves carrying GRP, the signals vary. Nerves carrying GRP are connected to another set of nerves which contain GRPR -- a partner for the GRP molecules being transmitted. When the two connect, biochemical signals are triggered in the brain.

Trigeminal ganglion (TG) neurons from the eyes carry GRP molecules into the bottom part of the brain. A region known as the spinal trigeminal nucleus caudalis (Sp5C) in this part of the brain is found to be rich in incoming GRP-containing nerve endings. Moreover, the Sp5C is abundant in GRPR. Thus, in this study the researchers targeted GRPR neurons in the Sp5C using a toxin to observe the role of this pathway in eye itchiness.

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Source: science daily

Understanding the 'eating just one potato chip is impossible' gene

 High-calorie foods -- high in fat, oil, and sugar -- can taste good but often cause overeating, leading to obesity and major health problems. But what stimulates the brain to cause overeating?

Recently, it has become clear that a gene called CREB-Regulated Transcription Coactivator 1 (CRTC1) is associated with obesity in humans. When CRTC1 is deleted in mice, they become obese, indicating that functioning CRTC1 suppresses obesity. However, since CRTC1 is expressed in all neurons in the brain, the specific neurons responsible for suppressing obesity and the mechanism present in those neurons remained unknown.

To elucidate the mechanism by which CRTC1 suppresses obesity, a research group led by Associate Professor Shigenobu Matsumura from the Graduate School of Human Life and Ecology at Osaka Metropolitan University focused on neurons expressing the melanocortin-4 receptor (MC4R). They hypothesized that CRTC1 expression in MC4R-expressing neurons suppressed obesity because mutations in the MC4R gene are known to cause obesity. Consequently, they created a strain of mice that expresses CRTC1 normally except in MC4R-expressing neurons where it is blocked to examine the effect that losing CRTC1 in those neurons had on obesity and diabetes.

When fed a standard diet, the mice without CRTC1 in MC4R-expressing neurons showed no changes in body weight compared to control mice. However, when the CRTC1-deficient mice were raised on a high-fat diet, they overate, then became significantly more obese than the control mice and developed diabetes.

"This study has revealed the role that the CRTC1 gene plays in the brain, and part of the mechanism that stops us from overeating high-calorie, fatty, and sugary foods," said Professor Matsumura. "We hope this will lead to a better understanding of what causes people to overeat."

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 Source: science daily

Scientists use machine learning to get an unprecedented view of small molecules

 A new machine learning model will help scientists identify small molecules, with applications in medicine, drug discovery and environmental chemistry. Developed by researchers at Aalto University and the University of Luxembourg, the model was trained with data from dozens of laboratories to become one of the most accurate tools for identifying small molecules.

Thousands of different small molecules, known as metabolites, transport energy and transmit cellular information throughout the human body. Because they are so small, metabolites are difficult to distinguish from each other in a blood sample analysis -- but identifying these molecules is important to understand how exercise, nutrition, alcohol use and metabolic disorders affect wellbeing.

Metabolites are normally identified by analysing their mass and retention time with a separation technique called liquid chromatography followed by mass spectrometry. This technique first separates metabolites by running the sample through a column, which results in different flow rates -- or retention times -- through the measurement device. Mass spectrometry is then used to fine-tune the identification process by sorting metabolites according to their mass. Researchers can also break metabolites into smaller pieces to analyse their composition using a technique called tandem mass spectrometry.

'Even the best methods can't identify more than 40% of the molecules in samples without making some additional assumptions about the candidate molecules,' says Professor Juho Rousu of Aalto University.

Now, Rousu's group has developed a novel machine learning model to identify small molecules. It was recently published in Nature Machine Intelligence.

'This new open-source model offers the whole research community an enriched view of small molecules. It will help research into methods to identify metabolic disorders, such as diabetes, or even cancer,' says Rousu.

The new approach elegantly sidesteps one of the challenges facing conventional methods. Because the retention times of molecules vary from lab to lab, data cannot be compared between labs. Eric Bach, a doctoral student at Aalto, came up with an alternative during his PhD research that solved the problem.

'Our research shows that while absolute retention times may vary, the retention order is stable across measurements by different labs,' Bach explains. 'This allowed us to merge all publicly available data on metabolites for the first time ever and feed it into our machine learning model.'

With the incorporation of data from dozens of laboratories around the globe, the machine learning model is accurate enough to distinguish between mirror image molecules, known as stereochemical variants. So far, identification tools have not been able to tell stereochemical variants apart, and the new capability is expected to open up new avenues in drug design and other fields.

'The fact that using stereochemistry improved the identification performance is a revelation for all developers of metabolite identification methods' says Emma Schymanski, associate professor at the Luxembourg Centre for Systems Biomedicine (LCSB) of the University of Luxembourg. 'This method could also be used to help identify and trace micropollutants in the environment or characterise new metabolites in plant cells.'

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Source:science daily

Ethereal color variant of mysterious plant is actually a new species

 Green leaves and photosynthesis were once considered essential characteristics of plants. However, some plants have stopped performing photosynthesis and take the nutrients they need from other organisms instead. One such mycoheterotrophic plant is ghostly-looking Monotropastrum humile that is widely found across East and Southeast Asia. It often grows in woodlands where there is little sunlight, obtaining the nutrients it needs by feeding off the hyphae of fungi. Despite its wide distribution, it was previously believed that only one species of this plant existed in the world. However, Professor SUETSUGU Kenji and colleagues have discovered that a variant found in Japan is actually a new species, shaking up our understanding of this unusual-looking genus of plants.

It has rosy pink petals and stems resembling milk glass, giving it a beautiful, otherworldly appearance. As it was first found around Kirishima in Kagoshima Prefecture, Japan, the new species has been named Monotropastrum kirishimense.

Originally, this new species was tentatively treated as a color variant of M. humile, known as M. humile f. roseum. Thus began an extensive and multifaceted 20-year study to determine how exactly these plants differed. Specimens were collected from throughout Japan and Taiwan, as well as Vietnam.

Results of various analyses revealed morphological differences, including the following; M. kirishimense flowers and ovaries are more rounded than those of M. humile, and its rootball is more obscured by the surrounding soil (in contrast to M. humile's protruding root tips). M. kirishimense individuals are shorter above ground (under 5cm) and longer below ground (over 10cm). The flowering season is different too; M. humile flowers bloom approximately 40 days earlier than M. kirishimense. As the two plant species have the same primary pollinator (the bumblebee Bombus diversus), this difference in flowering times can reduce heterospecific pollen deposition, helping to ensure conspecific mating, and thereby preventing them from producing hybrids.

There are several other possible reasons why M. kirishimense and M. humile may have evolved into separate species. One possibility is that they have become specialized in feeding on different fungi, which has led to reproductive isolation, or the inability to produce offspring together. This process is known as resource partitioning and is one of the major ways that species can evolve from a common ancestor. Genetic analysis of mycobionts revealed that M. kirishimense has a consistent, specialized association with a particular lineage of fungi, whereas M. humile is associated with different lineages. Therefore, this study suggests that M. kirishimense may have evolved into a new species by relying on a specific type of fungus. In fact, the phylogenetic tree (a 'family tree' of the evolutionary history of a group of organisms) of the plants themselves shows that the genetic characteristics of M. kirishimense and M. humile can be separated into two clades. Based on the researchers' analysis of various characteristics, it has been revealed that M. kirishimense is distinct from M. humile in terms of its appearance, flowering patterns, evolutionary history, and ecological relationships. Therefore, the researchers concluded that it should be recognized as an independent species.

Overall, the research group not only revealed that M. kirishimense is a distinct species but also deepened understanding of plants in the Monotropastrum genus. Mycohetrotrophic plants are very vulnerable to extinction as they rely on specific ecosystems to survive and are usually found in old-growth forests. The newly recognized species, M. kirishimense, is rare and presumably endangered. Now that it has been identified as a new species, conservation efforts can be made to protect it. This study emphasizes the importance of combining various analysis methods, called integrative taxonomy, to fully understand and protect biodiversity. This work was conducted by a multi-institutional research group, including Professor Suetsugu (Graduate School of Science, Kobe University), Professor SUYAMA Yoshihisa (Graduate School of Agricultural Science, Tohoku University), and Dr. Tian-Chuan Hsu (Taiwan Forestry Research Institute). The paper was published online in Journal of Plant Research on November 30, 2022.

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Source: science daily

Alveolar macrophages help CD8+ T cells go (anti-)viral

 The human immune system is a highly complex network of cells, signals, and responses that is tightly regulated to ensure that the body can fight off infection without damaging its own tissues. Now, researchers from Japan report a new way in which the immune system protects lung tissue from viral infections.

In a study published in Cell Reports, researchers from Nara Institute of Science and Technology (NAIST) have revealed that antigen-specific killer T cells (CD8+ T cells) rapidly expand in the lungs when they encounter antigen-presenting alveolar macrophages (AMs) to protect against viral infection.

CD8+ T cells confer protective immunity against infection with respiratory viruses, such as influenza A virus (IAV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), by killing infected cells. In order to target the correct cells for killing, naive CD8+ T cells must be primed by contact with antigen-presenting cells (APCs), which mediate the uptake of virus-infected cells and present their antigens, in a process known as cross-presentation. The primed CD8+ T cells then clonally expand and differentiate into effector or long-lived antigen-specific memory T cells.

"Multiple cell types can present antigen to CD8+ T cells in the lungs, although the role of tissue-resident macrophages in this process is unclear," explains Takumi Kawasaki, lead author of the study. "AMs are the first cells in the lungs that encounter infectious materials, environmental particles, surfactants, and dying cells, and they are important for the host defense against bacterial and fungal infection, so we suspected that they were also important in protecting against respiratory virus infection."

To test this, the researchers explored the mechanisms by which APCs instruct antigen-specific CD8+ T cells in the lungs. First, mice were primed by vaccination with a specific antigen or infection with IAV, and then they were subjected to secondary immunization or re-infection.

"We determined that antigen-presenting AMs present inhaled antigen to memory CD8+ T cells," says senior author of the study, Taro Kawai, "and that this resulted in a rapid expansion of antigen-specific CD8+ T cells in the lungs."

Furthermore, the researchers found that AMs help to develop resident memory-type cell population by producing interleukin 18. Importantly, administration of antigen-loaded AMs to mice induced the proliferation of resident memory-type CD8+ T cells.

"This strategy may improve the efficacy of CD8+ T cell-dependent cellular immunity," says Kawai.

Given that the lung is a major tissue for IAV and SARS-CoV-2 infection, the findings from this study regarding the mechanism of lung-resident memory CD8+ cell expansion are expected to lead to the development of new vaccines that induce cellular immunity. Virus-specific antigen-presenting AMs could be delivered as a type of "cell transplant vaccine" in the future.

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Source: science daily