Accelerating Opportunities for New COVID-19 Therapeutics

Today, there are only a few therapies approved to treat COVID-19, but while novel therapies can take decades and billions of dollars to develop, are there opportunities to repurpose existing drugs for new therapies? Our latest CAS Insights Report showcases how CAS Knowledge Graphs reveal new connections and insights that identify drugs to potentially repurpose.

Drug repurposing is critical for faster development of therapies. However, assembling all the critical information and connections around new proteins, viruses, targets, pathways, and clinical information can be challenging. This demonstrates how CAS Knowledge Graphs can identify top clinical candidates to repurpose for COVID-19 therapies.

What is a knowledge graph? 

A knowledge graph combines data from disparate sources to model a particular area. It describes data in nodes and edges. Nodes represent each point of data and edges represent the relationship between them. The image below provides a simplified example of a knowledge graph that predicts which drugs might inhibit vascular inflammation. 

Figure 1. Example of a knowledge graph showing the connections between data using nodes and edges

Traditional databases may only show direct connections (direct inhibitors of transcription factor STAT3), but a knowledge graph can show deeper data connections. In this example, the knowledge graph presents inhibitors that act further down the pathway.

Delving into COVID-19: small molecule drug discovery

The CAS Biomedical Knowledge Graph combines human-curated data from the CAS Content Collection^TM with publicly available biomedical data. 

It contains high-quality data from over 6 million small molecules, 24,000 diseases, and 26,000 human and viral genes. A knowledge graph reveals insights that would not be possible using traditional research methods. 

Our approach included two core components to uncover potential drug candidates for COVID-19:

• CAS scientists identified 20 biological processes linked to COVID-19. These processes included blood coagulation, viral entry, and endocytosis. One disease node represented ‘cytokine storm,’ an important aspect of severe COVID-19 pathology.
• Changes in gene expression as seen in the literature, specifically, genes significantly upregulated by SARS-CoV-2 infection. These were used to identify relevant biological processes and the biological processes associated with ≥4 of these genes. These processes included inflammatory response, angiogenesis, and negative regulation of RNA transcription.

Figure 2. Diagram outlining the two-component approach to identify potential small molecule drug candidates for COVID-19 therapeutics

Using the knowledge graph, we identified:

• Any small molecules with inhibiting or activating relationships to these biological processes
• Any small molecules that inhibited upregulated genes

The analysis identified 1,350 small molecules that could offer potential for repurposing as therapeutics for COVID-19.

Evaluating new potential therapeutics in COVID-19

Once we identified potential molecules, we assessed the power of their connections and boosted scores accordingly. To do this, we used a novel algorithmic method to rank each molecule. The equation evaluated the relationships between the small molecules and the interactions with the genes and biological processes identified in our two-component approach. 

For example, a cytokine storm was considered an important connection. We then evaluated the relationships between the small molecules and the interactions with the genes and biological processes identified in our two-component approach. Score boosts were given to important connections, such as to cytokine storm and to small molecules that have an activating relationship with genes, given the rarity of these occurrences.

Thus, we were able to develop a ranking table of all the small molecules and we present the top 50 in our whitepaper. In Figure 2 below, you can see the top 10 scoring drug candidates from the results. The size of the node corresponds to the number of connections to other nodes.

Figure 3. A network diagram showing the connection of the top 10 scoring drug candidates from the results with the size of the nodes corresponds to the number of connections to other nodes

Out of the top 50 drugs identified in our ranking table, 11 are currently in clinical trials for treating COVID-19. This provides validation of our results. 

Our biomedical knowledge graph uncovers four drug classes that have been linked previously to SARS-CoV-2 or general viral infection mechanisms.  The four drug classes include:

Kinase inhibitors

These were the single largest class of drugs found in our results. Kinases are involved in almost all biological processes and their activities are dysregulated in many diseases. Receptor tyrosine kinases (RTKs) are involved in the cell entry of many viruses. The kinase inhibitors identified included those affecting RTKs such as EGF, FGF, PDGF, and ALK receptors, as well as non-receptor tyrosine kinases such as Bruton tyrosine kinase. Serine-threonine kinase inhibitors targeting receptors B-RAF, PKC, PIM, and GSK-2beta were also identified by our knowledge graph. 

Histone deacetylase inhibitors (HDIs )

HDIs regulate gene expression by reducing histone deacetylation. HDIs reduce the expression of both angiotensin-converting enzyme 2 (ACE2), the main cell surface receptor of SARS-CoV-2, and the ABO glycosyltransferase, an enzyme that helps regulate blood type, which is a known COVID-19 risk factor. HDIs also regulates several of the chemokines and cytokines involved in the immune response in COVID-19.  As such, their inclusion in the results is logical.

Microtubule-regulating agents

Microtubules are filaments composed of tubulin subunits. Studies have shown that SARS-CoV-2 proteins interact with microtubules or microtubules-associated proteins. Our results uncovered that microtubule-regulating agents, such as docetaxel, colchicine, and mebendazole, may be of use in disrupting SARS-CoV-2 infection. Colchicine is already in clinical trials for the treatment of COVID-19 patients.

Protease inhibitors

Of the protease inhibitors identified, most were proteasome inhibitors. Studies have shown that the ubiquitin-proteasome system is involved in viral replication and the cytokine storm, including in diseases associated with coronavirus. Protease inhibitors are a logical choice for exploring in relation to COVID-19. Indeed, several such inhibitors are already being investigated as COVID-19 therapeutics. Some were found in our results, such as bortezomib, carfilzomib, and saxagliptin.

The power of connections

The methodology behind our knowledge graph enhances potential drug identification for COVID-19 treatment and will be of great value for drug discovery in other diseases beyond COVID-19, such as Alzheimer’s disease, Parkinson’s disease, autoimmune diseases, cancer, and even rare diseases.  Our knowledge graphs are both scalable and modular and offer great value to all areas of science, including chemistry, nutrition, and renewable energies. The opportunities are vast.

Lipid Nanoparticles: Versatile, Sophisticated Drug Delivery Systems  

Since the discovery of the first-generation liposomes in the 1960s, lipid nanoparticles (LNPs) have evolved tremendously. The key application of LNPs as therapeutic vehicles is in the pharmaceutical industry, although they serve purpose in other fields such as medical imaging, cosmetics, nutrition, and agriculture, albeit on a smaller scale. 

Lipid nanoparticles have been widely used in the pharmaceutical industry for decades. Compared with other gene and vaccine delivery systems, they are easier to manufacture, less immunogenic, and can carry larger payloads – making them successful and efficient carriers for various kinds of therapeutics, including small molecules, proteins, and nucleic acids. 

Recently, lipid nanoparticles were propelled into the global spotlight for their role in two approved COVID mRNA vaccines, effectively aiding accurate mRNA delivery, which sees them as a cutting-edge technology in vaccine platforms. Apart from mRNA therapeutics, lipid nanoparticles can play a key role in other disease areas. In fact, a number of lipid nanoparticles are already approved for delivering treatments for a variety of diseases (Figure 1). Here, we briefly explore the use of lipid nanoparticles in antitumor therapeutics. 

Applications of Lipid Nanoparticles in Cancer Therapy 

The CAS Content Collection™ has allowed us to review the distribution of treatment areas that use  LNP formulations (Figure 2). We have seen that antitumor therapeutic effects comprise the biggest portion of LNP drug use (46%), indicating their prominent role in this area. The largest single use of antitumor LNP formulations is seen in breast cancer (>25%), followed by ovarian cancer and lung cancer (both 10%).  

Lipid nanoparticles are associated with multiple therapeutic benefits that make them suitable for drug delivery in cancer treatment:  

LNPs have also been shown to improve the efficacy of cancer therapies via what is known as an enhanced permeability and retention (EPR) effect. LNPs can readily pass through tumor blood vessels, owing to their increased permeability resulting from rapid, yet defective, angiogenesis. This allows selective accumulation of LNPs in tumors when they are administered intravenously via direct injection, though data varies for different administration routes. Furthermore, dysfunctional lymphatic drainage in tumors improves LNP retention; the accumulation of LNPs allows the selective release of antitumor agents inside tumor cells.   

To better understand the applicability of LNPs across different therapies, the CAS Content Collection™ was employed to correlate different LNP formulation processes with the therapies that they may be applied to: immunoliposomes and stealth liposomes were found to be the most ubiquitous LNP types for antitumor therapy.  

One example of a highly effective stealth liposome cancer therapy is DOXIL^® (doxorubicin HCl liposome injection), the earliest approved liposomal drug developed for the management of advanced ovarian cancer, multiple myeloma, and HIV-associated Kaposi’s sarcoma. The LNPs used in DOXIL^® employ the EPR effect to overcome the cardiotoxic properties of the potent anticancer agent, doxorubicin, while sterically stabilized nanoparticles extend circulation time in human plasma. 

With regards to current and future research, a number of Phase I/II clinical trials are currently investigating LNP formulations as cancer immunotherapy targets in an array of solid tumors, including melanoma, adult glioblastoma, gastrointestinal cancer, and genitourinary cancer, to name a few – underlining the broad clinical use of these therapies.  

Stimuli-responsive liposomes are another approach being investigated to further enhance drug delivery in tumors, where they are designed to be released under certain physicochemical or biochemical stimuli. Examples include doxorubicin (stimuli: temperature/pH), 5-fluorouracil (stimulus: magnetic field), and AMD3100 (stimulus: laser irradiation). 

The Future of Lipid Nanoparticles in the Emerging Field of Nanomedicine 

The field of nanomedicine has shown remarkable progress as a modern drug therapy with broad clinical applications beyond cancer. Nanomedicine has helped to improve the effectiveness, selectivity, and biodistribution of conventional drug carrier systems while reducing their limitations.  

The use of lipid nanoparticles in medicine is likely to expand, and holds great promise in genetic medicine where gene editing, vaccine development, and immuno-oncology rely on the ability to efficiently deliver nucleic acids into cells. More sophisticated and multifunctional nanocarrier designs are being developed to address the needs of personalized medicines, meaning successful drug delivery, regardless of a patient’s biological barriers related to age, disease status, and comorbidities.  

With continued development, lipid nanoparticles can be recognized as one of the most advantageous and promising areas in modern nanotechnology.

Tenchov R, et al. Lipid nanoparticles - From liposomes to mRNA vaccine delivery, a landscape of research diversity and advancement. ACS Nano. 2021 Jun 28. doi: 10.1021/acsnano.1c04996. Online ahead of print. 

Recent clinical trials show that recreational psychedelics like LSD (Lysergic acid diethylamide), MDMA (3,4-Methylenedioxymethamphetamine), sometimes known as ‘Molly’), and Psilocybin (sometimes known as ‘Shrooms’) when properly controlled for dosing and administration are having positive outcomes. With an increasingly global problem and no new classes of antidepressants in the last 30 years, could psychedelics be another tool in the fight against depression and PTSD?  

Why does mental health matter? A growing problem and opportunity

A healthy population is the cornerstone of a thriving and prosperous economy. But according to the World Health Organization (WHO), over 280 million people suffer from depression, and an estimated 284 million people have suffered from post-traumatic stress disorder (PTSD) across the world. We know that mental illness negatively impacts people’s ability to work, and can significantly limit their participation in the labor market.  In fact, depression is the leading cause of disability worldwide and with remission rates for PTSD ranging from 20-30%, there is a growing population that is seeking care.   

Subsequently, there is a case to be made for the urgency of tackling a growing mental health crisis. The rates of mental illness among adolescents and young adults have increased significantly over the last decade, and the COVID pandemic has accelerated this impact. Despite this, there has not been a significant breakthrough in research or a revolution in the corresponding treatments to alleviate the issue.  In fact, many big pharmaceutical companies backed away from research and development in mental illness drugs and narrowed their investment in neuroscience research programs due to high risks and high failure rates in clinical trials.

The status quo: Treatment resistance with current anti-depressants 

It has been over 30 years without a novel class of anti-depression medications. Selective serotonin reuptake inhibitors (SSRIs), such as Prozac, have been the major class of anti-depression medication for several decades. Serotonin is a key hormone that activates the serotonin receptors in certain regions of the brain to stabilize our mood, feelings, and happiness (Figure 1). 

The SSRI drugs work by inhibiting the reuptake process of serotonin (5-HT) at the neuronal synapses in a way to increase the serotonin level at the synapses (Figure 1). However, many patients have developed treatment resistance to these drugs after many years of prescription, which is resulting in a significant demand to find new ways of treatment. Could psychedelics be one of those ways? 

Promising results in early clinical trials

Since 1990, there has been increased research interest in psychedelic drugs due to the technological advancement in neuroimaging, allowing researchers to link these drugs with an actual materialized outcome in an experimental setting. These drugs are a class of hallucinogenic substances, that exert their neurological effect by binding to some of the neurotransmitter receptors (receptors sensing chemical signals between neurons). After ingestion, they produce changes in people’s perception, mood, and cognitive process and may take one to a mental trip away from reality, called a “psychedelic trip.” These drugs are organic chemical compounds, either synthesized or extracted from natural sources.

Given the increasing awareness of mental health problems and the fact that the current situation of inadequate available treatments, psychedelic drugs are finding their renewed value as a tool for treating a variety of mental illnesses. Here, we take a look at three psychedelic drugs which currently in the late stage of clinical trials:  

• Psilocybin (shrooms) for the treatment of drug-resistant depression in phase 2 trials
• Lysergic acid diethylamide (LSD) for the treatment of major depressive disorder in phase 2 trials
• 3,4-Methylenedioxymethamphetamine (MDMA) a key ingredient in the drug commonly known as ecstasy or molly, for the treatment of PTSD patients in phase 3 trials.

Mushrooming potential for psilocybin

Once ingested, psilocybin is turned into the active drug form of psilocin (Figure 2) and its chemical structure is very similar to serotonin, allowing it to function as a serotonin receptor agonist. The only differences in the structures are the positions of hydroxyl and methyl groups (Figure 3). Over a few hours after ingestion, psilocybin produces profound changes in consciousness with visual and auditory hallucinations. 

From a randomized clinical trial, psilocybin-assisted therapy was found to be efficacious in producing large, rapid, and sustained antidepressant effects in patients with major depressive disorder. Currently, psilocybin is in phase 2 clinical trial for major depressive disorder. In addition, several pilot studies of psilocybin-assisted psychotherapy also have shown positive benefits in treating both alcohol and nicotine addiction. 

Early outcomes showed of LSD

Like psilocybin, LSD can also be found and extracted from mushrooms. However, it was first chemically synthesized by Switzerland scientist Dr. Albert Hofmann in 1938. The psychological effect of LSD was heavily investigated during the years from 1950-1970. Many publications during that period showed positive behavioral and personality changes in patients with various psychiatric disorders. It was also observed that LSD together with suitable accompaniment during its administration, could reduce pain, anxiety, and depression in patients with advanced cancer.

Like psilocybin, LSD mainly works as a serotonin receptor agonist due to its structural similarity with serotonin (Figure 4). However, it is still poorly understood in the mechanisms of interactions between the receptor activation and the resulting impairment in cognition and induction of hallucinations. Nevertheless, the benefit of LSD in treating various mental illnesses is currently being investigated in several pilot clinical studies. More promisingly, LSD with different doses is being tested in a phase 2 trial for major depressive disorder. 

MDMA moves beyond raves

MDMA is a synthetic psychedelic drug (Figure 5). It has been popularly used at nightclubs as a party drug. MDMA primarily acts as an indirect serotonergic agonist to increase the amount of serotonin released into the synapse. It also acts on serotonin storage vesicles and serotonin transporters to increase the amount of serotonin ready to be released and to promote its release. This process can lead to significant increases in serotonin available in the synapse. MDMA has been shown to enhance fear memory extinction, modulate fear memory reconsolidation, and bolster social behavior in animal models. 

More interestingly, recent work done by a research team at Johns Hopkins revealed its therapeutic value and potential mechanism in treating PTSD patients. The team discovered that MDMA was shown to reopen the otherwise closed critical periods for neuronal circuits formation in disease states, allowing the reformation of the neuronal circuits when environmental stress is no longer present. MDMA is currently in phase 3 trial and its phase 2 trials have shown promising safety and efficacy findings in treating PTSD patients. 

Progress, but more work required

While progress has been made, there are still some roadblocks to using psychedelics in the treatment of mental health disorders. First, outside of Oregon, many of these substances are schedule 1 controlled substances and are illegal. Secondly, the potential for abuse, neglect, and misuse of highly controlled substances is high with both patients and providers. Finally, there are physical risks as well. A few patients occasionally may experience a “bad trip,” described as an acute state of anxiety and confusion, or they experience a moderate increase in blood pressure and heart rate.

Although psychedelic drugs do not cause dependency or withdrawal issues as opioids or cannabis substances do, long-term use or frequent use may lead to tolerance. It is advised that psychedelic drugs should be administrated to the patients in a controlled and supervised environment. 

Mental health is not a simple black and white status; it is complex—spectral and continuous. At the positive end, mentally thriving and fulfilling; in the middle, coping and surviving; and at the negative end, daily functions disrupted by illness. Treatment options should also be addressed on a continuum in a highly collaborative environment with the patient and their healthcare providers. This may range from cognitive behavioral therapy to known drugs or procedures, to more experimental approaches depending on the doctor’s diagnosis and assessment. 

As innovation continues to accelerate in this exciting therapeutic area, CAS recently partnered with April 19 Discovery, an AI-driven drug discovery company specializing in psychedelics. This machine learning collaboration has accelerated lead compound development for April 19 Discovery through the CAS Content Collection^TM and customized services. Learn more in the press release.

Gut microbiome as an extra organ in human body

The human body harbors a large collection of microorganisms—predominantly bacteria, but also viruses, protozoa, fungi, and archaea. They are collectively known as the microbiome. Gut microbiota, gut flora, or microbiome are the microorganisms that live in the digestive tracts of humans and other animals. While some bacteria are associated with disease, others are particularly important for many aspects of health. In fact, there are more bacterial cells in the human body than human cells–roughly 40 trillion bacterial cells vs. only 30 trillion human cells. These microbes may weigh roughly as much as the brain. Together, they function as an extra organ in the human body and play a huge role in human health. The collective genome of the gut microbiome exceeds over 100 times the amount of human DNA in the body. Considering this enormous genetic potential of the microbiota, it is anticipated that it plays a role in virtually all physiological processes in the human body. Gut bacteria have been linked to several mental illnesses, and patients with various psychiatric disorders such as depression, bipolar disorder, schizophrenia, and autism have been found to have significant alterations in the composition of their gut microorganisms.

The interest in gut microbiome as related to human health, and specifically to mental health, is exponentially increasing in the years after 2000, as demonstrated by a search in CAS Content Collection^TM. Currently, there are over 7,000 publications on gut microbiome as related to mental health (Figure 1).

Babies acquire their first dose of microbes at birth. Development of the human gut microbiome

It is generally believed that the uterus is a sterile environment, and that bacterial colonization starts during birth. The microbiome of a newborn varies according to mode of delivery: the microbiome of vaginally delivered infants is like the maternal vaginal microbiome and that of infants delivered by cesarean section resembles the maternal skin microbiome. Various other factors affect the developing neonatal microbiome such as premature birth and mode of feeding. The major determinant of gut microbiome composition throughout adulthood seems to be diet. Fast changes in microbiome composition happen in response to changes in dietary intake. Characteristic patterns are noticeable in plant-based versus animal-based diets.   The development and alteration of the gut microbiome are affected by multiple other factors as well. Exposure to stress ranks as the second most important factor (after diet) affecting the gut microbiome composition, according to a search in the CAS Content Collection. Other factors include: mode of delivery and infant feeding method, environmental conditions, medications, stage and mode of lifecycle, comorbid diseases, and medical procedures (Figure 2). A disruption to the microbiota homeostasis caused by an imbalance in their functional composition and metabolic activities, or a shift in their local distribution is termed dysbiosis, indicating microbial imbalance or maladaptation.

Considering the now recognized significant role of diet on gut microbiome composition, and the vital impact of the gut microbiome on health, the million-dollar question remains: –which diet is beneficial and thus recommendable to keep our gut bacteria happy? Although there is not a definitive unambiguous answer pointing out certain food as a specific illness remedy, some major guidelines have been figured out. A high-fiber diet specifically affects the gut microbiota. Dietary fiber can only be digested and fermented by enzymes from microbiota living in the colon. Short chain fatty acids are released because of fermentation, which lowers the pH of the colon. The highly acidic environment determines the type of microbiota that would survive. The lower pH limits the growth of certain harmful bacteria such as Clostridium difficile. High-fiber foods such as inulin, starches, gums, pectins, and fructooligosaccharides have become known as prebiotics because they feed our beneficial microbiota. In general, high amounts of such prebiotic fibers are found in fruits, vegetables, beans, and whole grains like wheat, oats, and barley. Another highly beneficial class of foods contains probiotics, live bacteria that are good for the digestive system and may further amend our gut microbiome. These include fermented foods such as kefir, yogurt with live active cultures, pickled vegetables, kombucha tea, kimchi, miso, and sauerkraut.

Gut microbiota participants

The human gut microbiota is divided into many groups called phyla. The gut microbiota primarily comprises four main phyla including Firmicutes, Bacteriodetes, Actinobacteria, and Proteobacteria, with the Firmicutes and Bacteroidetes representing 90% of gut microbiota. The majority of bacteria reside within the gastrointestinal tract, with most predominantly anaerobic bacteria housed in the large intestine (Figure 3).  

The gut-brain axis – gut microbiome as the “second brain”

It is now well established that gut and brain are in constant bidirectional communication, of which the microbiota and its metabolic production are a major component. Michael Gershon called the digestive system “the second brain” in his 1999 book , at the time when scientists were beginning to realize that the gut and the brain in humans were engaged in constant dialogue and the gut microbes significantly modulate brain function. 

It is now a common belief that gut microbiota communicates with the central nervous system through neural, endocrine, and immune routes, and thereby controls brain function. Studies have demonstrated a substantial role for the gut microbiota in the regulation of anxiety, mood, cognition, and pain. Thus, the emerging concept of a microbiota–gut–brain axis suggests that modulation of the gut microbiota may be an effective strategy for developing novel therapeutics for central nervous system disorders.

Gut microbiota and COVID-19

Recently, correlation has been reported between gut microbiota composition and levels of cytokines and inflammatory markers in patients with COVID-19.  It is suggested that the gut microbiome is involved in the magnitude of COVID-19 severity via modulating host immune responses. Moreover, the gut microbiota dysbiosis could contribute to persistent symptoms even after disease resolution, emphasizing a need to understand how gut microorganisms are involved in inflammation and COVID-19.

Gut microbial neuroactive metabolites

Abnormalities in the gut microbiota-brain axis have come out as a key factor in the pathophysiology of neural disease, therefore increasing amount of research is devoted to understanding the neuroactive potential of the products of gut microbial metabolism. Thus, major neuroactive gut microbial metabolites have appeared as follows.

Neurotransmitters

Gut microbiome produces neurotransmitters, which regulate brain activity. The majority of central nervous system neurotransmitters are also present in the gastrointestinal tract, where they exercise local effects such as modulating gut motility, secretion, and cell signaling. Members of the gut microbiota can synthesize neurotransmitters, e.g., Lactobacilli and Bifidobacteria produce GABA; Escherichia coli produce serotonin and dopamine; Lactobacilli produce acetylcholine.  (Figure 4) They signal the brain via the vagus nerve.

Short-chain fatty acids

Short-chain fatty acids are small organic compounds produced in the cecum and colon by anaerobic fermentation of dietary carbohydrates that feed other bacteria and are readily absorbed in the large bowel.  Short-chain fatty acids are involved in digestive, immune and central nervous system function, though different viewpoints regarding their impact on behavior exist.  The three most abundant short-chain fatty acids produced by gut microbiome are acetate, butyrate, and propionate (Figure 5).  Their administration was demonstrated to alleviate symptoms of depression in mice.  Gram-positive, anaerobic bacteria which ferment dietary fibers to produce short-chain fatty acids are Faecalibacterium and Coprococcus bacteria.  Faecalibacteria are abundant gut microbes, with significant immunological roles and clinical relevance for a variety of diseases, including depression. 

Tryptophan metabolites 

Tryptophan is an essential amino acid participating in protein synthesis. Its metabolic breakdown by bacterial enzymes (tryptophanase) give rise to neuroactive molecules with established mood-modulating properties, including serotonin, kynurenine, and indole (Figure 6). It has been found that dietary intake of tryptophan can modulate central nervous system concentrations of serotonin in humans, and that tryptophan depletion aggravates depression.

Lactic acid

Lactic acid (Figure 6) is an organic acid developing mainly from the fermentation of dietary fibers by lactic acid bacteria (e.g., L. lactis, L. gasseri, and L. reuteri), Bifidobacteria and Proteobacteria. Lactates can be converted by several bacterial species to short-chain fatty acids contributing to the total short-chain fatty acid pool. Lactic acid is absorbed into the bloodstream and can cross the blood-brain barrier. Lactic acid has a well-recognized role in central nervous system signaling in the brain. Due to its ability to be metabolized into glutamate, it is used as an energy substrate by neurons. It also contributes to synaptic plasticity and triggers memory development.

Vitamins 

Most bacteria in the gut, such as Lactobacillus and Bifidobacterium, synthesize vitamins (particularly from the group of B-vitamins and vitamin K) as part of their metabolism in the large intestine. Humans rely on the gut microbiota for vitamin production. Vitamins are key micronutrients with ubiquitous roles in a multitude of physiological processes in the human body, including the brain. Active transporters bring them across the blood-brain barrier. In the central nervous system, their role spreads from energy homeostasis to neurotransmitter production. Vitamin deficiencies can have a significant negative effect on neurological function. Folic acid (vitamin B9) is a vitamin of microbial origin that has been extensively implicated in the pathology of depression. 

Perspective 

A recent innovative investigational treatment, fecal microbiota transplantation, has been tested in clinical trials and found extremely therapeutically promising. In the last five years, ~1,000 documents related to fecal transplants have been included each year in the CAS Content Collection. For example, it has been reported that fecal microbiota transplantation is able to resolve 80-90% of infections caused by recurrent Clostridioides difficile that does not respond to antibiotics. The unique implications for clinical trials using fecal microbiota transplants, which are increasingly investigated as potential treatments for a range of diseases, need to be promptly explored. 

At present, research into the modulation of the gut-brain axis via the gastrointestinal microbiota is an emerging innovative, frontline science. A large portion of the data available is based on either basic science or animal models that may not be adaptable to effective human interventions. Therefore, individualized prescriptions of specific prebiotic compounds and probiotic strains that would represent the ideal of personalization for nutrition and lifestyle medicine remain hopeful. Ongoing efforts to further characterize the functions of the microbiome and the mechanisms underlying host-microbe interactions will provide a better understanding of the role of the microbiome in health and disease.

For more on how emerging trends and new approaches are helping the millions of people who suffer from depression, anxiety, and PTSD see our blog on psychedelics and their progress as a therapeutic approach.

Since the start of the pandemic, more than 11.4 million children have tested positive for COVID-19 in the U.S., with children under 4 accounting for more than 1.6 million of those cases and 3.2% of total hospitalizations due to COVID-19. As the Food and Drug Administration (FDA) and Centers for Disease Control (CDC) have begun reviewing the safety data for Pfizer and BioNTech’s COMIRNATY® COVID vaccines for Emergency Use Authorization (EUA) with children under the age of 5, several key questions have emerged as parents learn more and decide whether to vaccinate their children under the age of 5. While dosage amounts are different for children under the age of 5, understanding the types of ingredients that are in COVID vaccines will enable parents to make more informed decisions.  

For children under 5, are the vaccine ingredients the same?  

Not exactly; while the active ingredients of the Pfizer and BioNTech COVID-19 vaccines are identical to the current adult vaccines, the only difference is in the buffer called tromethamine (Tris) used that allows for the children’s vaccine to remain refrigerated longer. While tromethamine may not sound like a common ingredient, from a scientific perspective, it was introduced into the world’s published literature in 1944 and is often used in cosmetics, serums, and vaccines since 1978.  

The other critical difference is in the dosage of 3 µg that will be administered in 3 doses for children under the age of 5 vs. the 10 µg dose that is given twice to children ages 5 and older and the 30 µg dose given 3 times to those ages 12 and above.   

How common are the ingredients of the COVID-19 vaccines?

To discover how common an ingredient is, CAS offers a unique perspective. For over 100 years, whenever new chemistry-related scientific research has been published, CAS has captured that information in the CAS Content Collection™, allowing us to see when a chemical compound first shows up in research and every time thereafter. As a result, the CAS Content Collection shows us how often a compound is studied or used in research since each compound is given its own Registry Number. By connecting each COVID-19 vaccine ingredient to its prevalence in scientific literature, we can gain insight as to how scientifically common it is.

In fact, ingredients that are most scientifically common also appear in our homes, primarily as ingredients in foods and sometimes skincare products. On the other hand, the vaccine ingredients which appear less in the CAS Content Collection (like lipids) are newer and have fewer and more specific applications. Nonetheless, our content helps us better understand these unique ingredients.

Everyday common household ingredients

The most common ingredients are easily found within our own pantries. Some, in singular form like salt or sugar, or within popular food and drink items like Gatorade or Jell-O. To see how prevalent an ingredient is in the CAS Content Collection, we consider the number of times its Registry number has been referenced in the world’s publications and will categorize them as:

• High >50,000
• Medium: 10,000-50,000
• Low: 0-10,000

* Includes occurrence of ingredient crystallized with one or two water molecules and occurrence without water.

Scientifically common Ingredients

In this category, we find those ingredients that are a little more specialized but still are used in a variety of applications. They are far more common in medicine and/or research than in our cupboards and have been so for several decades at least. The most common ingredient is 2-hydroxypropyl beta-cyclodextrin(HPBCD), a fascinating ring-shaped compound, derived from beta-cyclodextrin (BCD) that forms naturally from starch. Not only has BCD been studied over 50,000 times, but there are over 26,000 compounds based off it. Newer on the scene is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), a phosphatidylcholine that can occur naturally—in a mixture of phosphatidylcholines and other lecithins—in foods like soybeans. Its pure form, whether isolated or synthetic, has been studied in vaccines or lipid nanoparticles for over two decades. Tromethamine and tromethamine HCl, stabilizers in the Moderna vaccine, also make the list as established vaccine ingredients as well as cosmetics ingredients.

Unique Ingredients

Ingredients that are less common are the specialized lipids for the Moderna and Pfizer mRNA vaccines. These lipids make up the lipid nanoparticles (LPNs) that protect the spike protein mRNA and help carry it safely into our cells. LPN technology has been around for nearly 30 years, with cancer research playing a critical role in its innovation. To make mRNA vaccines a reality, the right lipids were needed to be discovered and developed. It is important to note that, while still new, these lipid ingredients still pre-date the COVID-19 pandemic.

The virus-related particles are the only truly new ingredients of vaccines, having been developed after the beginning of the pandemic. For Pfizer and Moderna, this consists of an mRNA strand encoding the viral spike protein of COVID-19. The mRNA used is based off the original variant of SARS-CoV-2; if newer vaccines are released that target later variants of the coronavirus, such as Omicron, this can be done by using a newer sequence of mRNA. The mRNA vaccines do not cause any genetic changes to our cells, because mRNA only stays in the cytosol of the cells and does not interfere with DNA in the cell nucleus. Like the mRNA vaccines, the Johnson & Johnson vaccine provides a genetic template to cells for producing the spike protein of the coronavirus, using a modified adenovirus-26 (ad26) vector virus that carries a piece of DNA. Because the mRNA and DNA are specific to COVID-19, these ingredients have been developed since the start of the pandemic. Similar mRNA vaccines using the same LPN technology have been studied since 2016, and an Ebola viral vector vaccine using ad26 was in development also as early as 2016.

Owing to the intense research surrounding the COVID-19 pandemic, the prevalence of these unique ingredients in the CAS Content Collection is growing all the time. No doubt, as time goes on, more uses for the LPNs and ad26 viral vectors will be developed.

Summary

The formulations and ingredients of the COVID-19 vaccines have been under a lot of scrutiny but as the EUA is potentially pending for Pfizer and BioNTech’s COMIRNATY^® children under the age of 5, understanding how common some of these ingredients may prove helpful in allowing parents to make educated decisions. For those who are seeking greater detail on all ingredients, download this table of all the ingredients by vaccine.

For even more on COVID-19, visit the CAS Covid-19 Resources collection for the latest data sets, bioindicator explorer, and peer-reviewed articles.  

As of December 2021, over 8 billion vaccine doses for coronavirus disease 2019 (COVID-19) have been administered, including around 217 million ‘booster’ shots. The main target for these vaccines has been the so-called “spike,” or “S,” protein, an essential viral protein that plays a key role in allowing the virus to invade host cells.

While vaccines are critical, the development of COVID-19 therapeutics has revealed that intrinsically disordered proteins may play a critical pathological role. Historically, biologists believed the amino acid sequence of each protein determines its three-dimensional structure, which, in turn, determines its function. However, there is a large group of proteins and regions that lack a fixed or ordered 3D structure, yet they still exhibit essential biological activities—so-called intrinsically disordered proteins and regions (Figure 1).

This protein disorder is encoded into amino acid sequences and is abundant in all living organisms and viruses. A deeper understanding of these noteworthy regions within the SARS-CoV-2 protein characteristics could enable faster progress for therapeutic development in COVID-19.
 

Examples of intrinsically disordered proteins

The natural variability found in the “intrinsically disordered proteins” (IDPs) or the “intrinsically disordered regions” (IDPRs) within proteins can be noticed in all three kingdoms of life. They are linked to important processes such as enzyme catalysis, allosteric regulation, cell signaling, transcription, and others. 

However, they also play a role in disease, including neurodegeneration, diabetes, cardiovascular disease, amyloidosis, genetic diseases, and cancer. Furthermore, viral proteins often contain such regions, which have been correlated with virulence because they endow the viral proteins’ ability to easily and promiscuously bind to host proteins. 

Interest in IDPs/IDPRs in protein science has been rapidly increasing since the year 2000, as demonstrated by a search in the CAS Content Collection^TM (Figure 2), and their roles in drug design, including in COVID-19, are beginning to be explored. 

Intrinsically disordered proteins in SARS-CoV-2

SARS-CoV-2 forms a virion including its genomic RNA bundled in a particle comprising: the S protein, important for entry into host cells; the membrane (M) protein facilitating viral assembly; the ion channel small envelope (E) protein; and the nucleocapsid (N) protein, which assembles with viral RNA to form the nucleocapsid (Figure 3). 

IDPs/IDPRs are not common in the SARS-CoV-2 proteome. As a matter of fact, SARS-CoV-2 proteome exhibits significant levels of structural order — except for the nucleocapsid (N) protein, SARS-CoV-2 proteins are highly ordered proteins containing a few intrinsically disordered protein regions. Noteworthy, however, the existing disordered regions contribute significantly to the functioning and virulence of the virus and are thus promising drug targets for antiviral drug discovery; such an approach has already proven to be valuable in identifying new drug candidates.

The nucleocapsid (N) protein

The RNA-binding N protein stabilizes the genomic RNA inside the virus particle and regulates the viral genome transcription, replication, and packaging. The N protein is highly disordered—its average percentage of predicted intrinsic disorder is around 65%. These disordered regions seem to be important in maintaining the nucleocapsid and so could serve as targets for drug design. Disordered regions within the N protein also appear to be important in enabling the protein to aggregate via a process termed ‘liquid-liquid phase separation’, potentially as a way of disrupting the natural formation of stress granules, important in host cell immunity. Thus, disruption of the N protein liquid-liquid phase separation process holds promise for antiviral intervention and offers new targets and strategies for the development of drugs to combat COVID-19.

The spike (S) protein

The S protein ornaments the viral surface like a crown. It is critical for viral entry into the host (Figure 4) and as such, has been a commonly-utilized drug target in the development of COVID-19 vaccinations. The receptor binding and membrane fusion, the initial steps in infection, are both related to regions of substantial intrinsic disorder.
 
Analysis of the S protein indicates that both S subunit cleavage sites associated with S maturation, and the S fusion peptide, are associated with IDPRs. Considering that proteolytic digestion is considerably faster in unstructured relative to structured protein regions, this structural specificity of the SARS-CoV-2 S protein might be of high functional importance.  

During SARS-CoV-2 virus infection, IDPRs can be detected at the interface of the spike protein and ACE2 receptor, the receptor found in human tissues to which the virus binds. The key residues of the spike protein have a strong binding affinity to ACE2, one likely reason for the high transmissibility of SARS-CoV-2.

Thus, receptor binding and membrane fusion, the initial and important steps in the coronavirus infection, are both related to regions of substantial intrinsic disorder in the S protein. They are primary targets for inhibiting SARS-CoV-2 infection.  

The membrane (M) protein

The M protein is a major transmembrane protein that is found in large numbers in the virion. SARS-CoV-2 has one of the hardest protective outer shells among coronaviruses–this is potentially related to the low intrinsic disorder of the M protein (6%) and may be responsible for the high resilience and transmissibility of the virus. Indeed, a correlation has been shown between the virulence of various viruses and the percentage of intrinsic disorder of their M proteins, with less disordered M proteins being associated with more contagious viruses. 

Future outlook: the frontiers of drug design

The appearance of novel viruses and associated epidemics around the globe are currently a major concern. Knowledge of the structures and functions of viral proteins is thus of high significance for identification of novel therapeutic targets for prevention and treatment of disease.  

In our peer-reviewed publication in ACS Infectious Diseases, we summarize the information available on the SARS-CoV-2 proteome with regards to the occurrence of intrinsic disorder in its proteins. In fact, it has been recognized that the SARS-CoV-2 proteome exhibits substantial levels of structural order–only the N protein is highly disordered. Although other SARS-CoV-2 proteins are characterized by lower degrees of disorder, their existing IDPRs contribute significantly to the functioning and virulence of the virus and are promising drug targets for antiviral drug design.

IDPs are widespread and have numerous crucial biological functions that complement the functionality of ordered proteins. However, when misfunction occurs (e.g., misexpression, misprocessing, or misregulation), IDPs/IDPRs tend to engage in undesirable interactions and become involved in the development of various pathological states. In fact, many proteins associated with neurodegeneration, diabetes, cardiovascular disease, amyloidosis, and genetic diseases, as well as most of the human cancer-related proteins, are either IDPs or contain long IDPRs. 

Although structural biology techniques can be utilized in drug development, the practice of rational drug design has traditionally underrepresented the presence of intrinsic disorder in target proteins. Understanding the structure of these regions in the SARS-CoV-2 and other pathogenic proteomes would clearly be of great benefit for drug development in COVID-19 and beyond, continuing to push the boundaries of drug design.  

"We are in the midst of a therapeutic revolution," according to the authors of a recent review article in Frontiers in Bioengineering and Biotechnology. They were commenting on the rapid expansion of RNA therapeutics in modern research and clinical development, driven in part by interest in RNA COVID-19 vaccines in the ongoing SARS-CoV-2 pandemic.  

Traditionally, pharmaceutical drug development has been dominated by so-called small molecule drugs (defined as any organic compound with low molecular weight), which still have numerous applications across medicine. However, advances in biotechnology and molecular biology have since enabled researchers to design macromolecular agents ranging from monoclonal antibodies and recombinant proteins to oligonucleotides and genes/gene fragments as drug candidates. As a result, ‘biologics’ have been established as key players in the therapeutic toolbox available today. As of early 2020, biologics comprised seven of the top 10 best-selling drugs globally. 

In addition, nucleic-acid-based drug design has emerged – and is currently enjoying rapid expansion. While clinical development of RNA therapeutics  has traditionally been hindered by challenges such as efficiency and immunogenicity, the recent success of the mRNA COVID-19 vaccines and the approval of several RNA-based drugs has lent substantial momentum to the field. Using the CAS Content Collection™ – the largest human-curated collection of published scientific knowledge – we examine the application of RNA in modern medicine. 

Read the related CAS Insights Report: RNA-Derived Medicines: A review of the research trends and developments" 

Advantages and challenges of RNA therapeutics

Targeting the "undruggable"

One great advantage of RNA therapy is that RNA drugs can be used to target ‘undruggable’ molecules, which are otherwise hard or impossible to target using small molecule-based drugs. Only around one-fifth of proteins can be targeted by commonly used drugs, including small molecules and antibodies, and it is not possible to target noncoding RNAs using traditional small molecules or monoclonal antibodies (which bind to active-site pockets of protein receptors or enzymes, thus requiring translation to have occurred).  

Ease of synthesis  

RNA products can have major manufacturing advantages over proteins in terms of the simplicity, cost-effectiveness, and speed of the manufacturing processes made particularly relevant in recent RNA vaccine development. Nucleic-acid-based strategies can also circumvent the requirement for complex synthesis processes such as post-translational modifications by utilizing the cellular machinery of the mammalian cell. 

In addition, the sequence of the RNA can be rapidly adjusted, delivering custom molecules for different targets. This greatly expedites the development process, as has clearly been the case with the rapid development of the COVID-19 RNA vaccines.  

Safety and side effects 

Because they enter the nucleus, DNA drugs raise safety concerns due to potential integration into the host genome. Aside from CRISPR-Cas system RNAs that edit the genome, RNAs do not alter the genomic material and carry no risk of genomic integration.  

However, RNA therapeutics can have specificity issues that risk side effects, and their susceptibility to degradation can result in poor pharmacodynamics, complicating their use. Some of these issues can be mitigated by chemically modifying the RNA, a topic on which research has focused. 

Delivery 

RNA therapeutics tend to have a large size compared with small molecule therapeutics and have a high electric charge, making their intracellular delivery in their native forms challenging.  

Research trends in RNA therapeutics 

Since 1995, there has been a steady increase in the number of journals and patents containing information on RNA therapeutics – with a peak in patients around 2001 (possibly related to the first human clinical trial using dendritic cells transfected with mRNA encoding tumor antigens) and a spike in journal numbers in 2020 (most likely resulting from interest in the COVID-19 mRNA vaccines) (Figure 1).

RNA research has gradually become more diversified as new types of RNA are being discovered, particularly in the areas of siRNA, miRNA, lncRNA, and CRISPR (Figure 2). The rates of increase of publication volumes of circRNA, exosome RNA, lncRNA, and CRISPR are significantly faster than the rest. Notably, CRISPR technology accounts for 20% of the overall RNA-related patent applications in 2020. This coincides with an accelerating number of approvals for CRISPR-based therapies to enter clinical trials. 

Interfering with the functionality of newly discovered RNAs is considered a promising therapeutic tool to overcome the weaknesses of traditional therapeutic approaches (Table 1).

Which therapy areas are being targeted by RNA therapeutics?

Infectious diseases and cancer show the largest growth and the greatest number of therapeutics in research phases (Figures 3 and 4). The COVID-19 pandemic has escalated RNA medicines for infectious disease in both research phases and the number of approved therapeutics (Figure 4), bringing the first approved mRNA therapeutics to market. 

Solving challenges in RNA therapeutics

RNA chemical modification can be used to protect RNA from degradation and improve target specificity, lowering the risk of side effects due to off-target effects. In addition to chemical modification, delivery vehicles comprising nanomaterials can be used to protect the RNA from degradation and aid the transport of the therapeutic to its desired target.

According to the data in the CAS Content Collection, the use of RNA modification began to take off in 1995 and is associated with smaller sequence lengths (Figure 5). The predominance of modified 18-27-nucleotide RNAs reflects the use of this sequence length in particular forms of RNA (siRNAs and ASOs). Examination of modifications in FDA-approved RNA medicines confirms the correlation between the type of RNA and its modifications.

RNA base modifications 

Non-canonical nucleotides with modifications that interfere with the formation of hydrogen bonds can thermally destabilize the formation of a duplex with the target and thus improve target specificity by limiting off-target binding. In addition, modification can improve the performance of therapeutic RNA. The use of the modified base N1-methylpseudouridine in therapeutic mRNAs, such as in COVID-19 mRNA vaccines, improves translation and lowers cytotoxic side effects and immune response to the mRNA. Both Pfizer’s Comirnaty and Moderna’s Spikevax mRNA vaccines also use 7-methylguanosine caps linked by a 5’ triphosphate to the 5’ end of the mRNA, duplicating the mRNA caps found in nature that prevent degradation of the 5’ end of the mRNA

Modifications on ribose

Modifications at the 2’ position of the ribose in RNA can increase stability and reduce off-target effects. The most common modifications at the 2’ position include 2’-O-methyl, 2’-fluoro, 2’-O-methoxyethyl (MOE), and 2’-amine.

Backbone modifications

Modifications at the phosphate group in the sugar-phosphate backbone can improve RNA delivery by neutralizing the negative charge that can interfere with transport across membranes and confer increased resistance to nucleases, thereby extending their tissue elimination half-lives. One of the most widely used backbone modifications is phosphorothioate.

RNA nanocarrier-related research

While biological barriers such as immunogenicity and nuclease stability are usually addressed by modifying the chemical structure of the RNA, additional delivery systems are necessary to overcome other barriers in the body. Encapsulation of RNA into nanoparticles is a successful way to protect and deliver RNA. Currently, there are nearly 7,000 scientific publications in the CAS Content Collection related to the RNA delivery systems. RNA carrier-related studies are dominated by the lipid nanoparticles, closely followed by the polymeric nanocarriers (Figure 6).

Conclusions

RNA therapeutics represent a rapidly-expanding category of drugs that are expected to change the standard of care for many diseases. They have several advantages compared with traditional medications based on small molecules and biological molecules, being cost-effective, relatively simple to manufacture, and capable of targeting previously ‘undruggable’ sites. The classical challenges experienced with their stability, delivery, and off-site effects can be eliminated or reduced via chemical modifications and RNA nanocarriers. A search in the CAS Content Collection has highlighted infectious diseases such as COVID-19 and cancer as key therapeutic areas for RNAs and shown that rates of increase of publication volumes of circRNA, exosome RNA, lncRNA, and CRISPR are particularly high – with a notable current explosion in CRISPR-related research. 

Hear from the experts

To gain additional insights, view the recent ACS webinar to see what today’s leaders in RNA therapeutics are most excited by. This panel discussion features experts from diverse research backgrounds, including:

• Dr. John P. Cooke, Medical Director of the Center for RNA Therapeutics
• Dr. Robert DeLong, Associate Professor of the Nanotechnology Innovation Center at Kansas State University
• Dr. Barb Ambrose, Senior Information Scientist at CAS
• Dr. Ramana Doppalapudi, Vice President of Chemistry at Avidity Biosciences
• Moderated by Dr. Gilles Georges, Vice President and Chief Scientific Officer at CAS

With more and more molecular glues, induced proximity, and targeted protein degradation approaches crowding the clinic, a landscape view of this emerging field has critical implications in therapeutic areas like cancer, autoimmune, and neurodegenerative diseases. Learn more in our latest white paper with unique CAS insights, and a view to future opportunities.

With heightened viral awareness due to the COVID-19 pandemic, the news of monkeypox outbreaks around the world is raising many red flags. Monkeypox is a virus that is regularly found and confined to Central and West Africa. It has spread in unusual ways with this outbreak and amongst populations that have not been vulnerable in the past. Currently there are over 300 confirmed or suspected cases in at least 19 countries outside of Africa with many more under investigation. The CAS Content Collection™ enables unique insights on monkeypox, the research landscape, therapeutic options, and scientific profiles with similar viruses.   

What is Monkeypox? 

Monkeypox virus is classified in the family Poxviridae with the genus Orthopoxvirus. The CAS Content Collection shows the phylogeny of the monkeypox virus. It is within the same family as a common childhood skin disease Molluscum contagiosum and within the same genus as viruses such as the vaccinia virus (cowpox virus) and the variola virus (smallpox virus). It is not however related to the commonly known chicken pox virus (Varicella). It was first discovered in 1958 in colonies of research monkeys that developed a pox-like disease, with the first human case reported in the Democratic Republic of the Congo (DRC) in 1970. All subsequent cases outside of endemic countries have been limited to travel from these countries or infected imported animals. Monkeypox is classified as a zoonotic disease with transmission being primarily from animal to human or vice versa. This current outbreak however has shifted to human-to-human transmission in non-endemic countries, baffling many and bringing this rare disease to the headlines.

Landscape view of the published science on monkeypox 

Analyzing the CAS Content Collection, research for Orthopoxvirus started to increase during the late 1980s with 30,000 journal articles and patents present under this category.  As expected, the publication volume for monkeypox is much smaller with around 1200 journal articles and patents, with research increasing slightly in the early 2000s, and remaining relatively stable from 2003 through 2021. 

Table 1. Top 10 Companies and Research Institutes researching the monkeypox virus

How is monkeypox transmitted?   

Monkeypox virus is a double‐stranded DNA (dsDNA) virus with a genome size of around 190 kb. In contrast to SARS-CoV-2 which is a single stranded RNA virus with a genome size of ≈30 kb. As we know all too well, SARS-CoV-2 is so small that it can be aerosolized and travel over 6 feet in the air. Monkeypox virus in contrast is much larger in size, it does not aerosolize, and travels only a few feet before dropping in the air. The Monkeypox virus also does not linger in the air as the SARS-CoV-2 virus does. For human-to-human transmission through air, it takes prolonged face to face contact with an infected individual.  It can also be spread through direct contact with body fluids or lesions or indirect exposure to lesion material such as through clothes or bedding. Animal-to-human transmission can occur by bite or scratch, bush meat preparation, and direct or indirect contact with body fluids or lesions. The virus enters the body through broken skin, the respiratory tract, or mucous membranes.  Another positive contrast from the SARS-CoV-2 virus is that the Monkeypox virus mutates at a much slower rate, given that it is a DNA virus with a much larger size.  Which makes historical and current vaccines highly effective. 

Genetic profiles of current monkeypox 

DNA viruses are usually stable and mutate extremely slowly compared to RNA viruses. Researchers from Portugal shared the first draft genome on May 19, 2022 and released an additional nine genome sequences of the Monkeypox virus that is causing the current multi-country outbreak on May 23.  The current preliminary draft of genomic sequencing shows the current outbreak belongs to the standard West African strain and is closely related to the monkeypox strain that was associated with animal exportation from Nigeria to several countries in 2018 and 2019. Researchers observed that the current outbreak most likely came from a single origin, but also diverged from the 2018/2019 sequence with 50 small nucleic acid polymorphisms (SNP).  They also discovered the first signs of microevolution within this outbreak cluster, with the emergence of 7 SNPs leading to the creation of 3 decedent branches which included a further subcluster of 2 sequences.  This two sequence subcluster was determined to have a 913bp frameshift deletion which appears to correlate to human-to-human transmission. This microevolution may allow this genome sequence to have enough resolution to track the virus dissemination with this outbreak, which is often not possible with other dsDNA viruses.   

Vaccinations and Possible Monkeypox treatments 

While vaccines are not immediately available to the general public, the US government is currently releasing JYNNEOS vaccines from the nation’s strategic national stockpile for some high-risk contacts of early patients. Current vaccinations and possible treatments are summarized and shown below in Table 2.    

Table 2. Monkeypox vaccine and possible treatments.  

Outlook 

With the World Health Organization announcing the eradication of smallpox in 1980, the smallpox eradication program ended worldwide. As with any vaccination, immunity wanes over time and anyone born after 1980 did not receive the smallpox vaccination and therefore is not protected from the monkeypox virus. Research shows that 30 years after the smallpox vaccination campaigns ended in the DRC, there was a major increase in the incidence of human monkeypox infections. This along with less research on monkeypox may seem troubling to some but there has been extensive research on other viruses within the same family. Vaccination, treatment options, along with less transmissibility all point to a controllable situation for this current outbreak that should minimize global impact. All cases of the outbreak have not been identified yet but with the viral awareness and public health prevention measures, the monkeypox virus should stop spreading amongst non-endemic countries.   

What are molecular glues?

Molecular glues are small chemical entities that use a ground-breaking strategy known as proximity-induced target protein degradation (TPD) in which pathogenic proteins of interest (POIs) are linked to natural digestive enzymes and are destroyed (Figure 1). This approach effectively ‘sticks’ or links POIs to E3 ubiquitin ligases of the ubiquitin-proteasome system, which normally functions as a cellular trash processing system. This approach has created new classes of therapeutic agents with entirely novel modes with exciting potential activity against various serious diseases such as cancer, inflammatory and immune diseases, and infections, many of which are driven by the aberrant expression of a pathogenic protein. There are more than 600 E3 ubiquitin ligases encoded in the human genome, and so far, only a few have been exploited using the TPD approach, so there is considerable potential to develop many more. 

Ask the experts from Dana-Farber, the CEO of Neomorph, and CAS as they explore emerging trends in molecular glues, targeted protein degradation, and induced proximity in a live ACS webinar on Oct. 5, 2022 at 2 pm EDT.  Register here. 

Protein degraders are advantageous because they act via transient binding rather than competitive occupancy and dissociate after promoting polyubiquitination of the POI. A single degrader can therefore destroy many copies of a pathogenic protein providing high efficiency against previously ‘undruggable’ proteins at low doses. Protein inhibitor drugs block the active site of a pathogenic protein, whereas degraders remove all of its functions, providing higher sensitivity to POIs, and a better chance to affect the non-enzymatic protein interactions.

The development of TPD compounds that use ubiquitin-driven natural protein degradation for therapeutic purposes has advanced considerably over the past two decades. The first patent of a therapeutic chimeric degrader to link a POI to E3 ligase was filed by Proteinix in1999. This was followed by a proof-of-concept study in which a cancer-associated protein was successfully degraded by a proteolysis-targeting chimera (PROTAC). The early PROTACs were large-molecular structures; the first report of a small molecule androgen receptor (AR) degrader using nutlin-3 for recruiting of MDM2 was published in 2008. The subsequent discovery of small-molecule mimetics of the HIF1α peptide expedited the rational design of small molecular PROTACs. So far, few proximity-induced TPDs have reached clinical testing, but two PROTACs (ARV-110 and ARV-471, which target the androgen and estrogen receptor, have progressed to Phase II trials, and many others are now in development.

Molecular glues and PROTACs have different properties. Molecular glues are small molecules that interact with a variety of target proteins that are difficult to predict, exhibiting distinct biological activities by inducing and enhancing the interaction of two proteins that otherwise do not show an intrinsic affinity for each other. PROTACs are bivalent molecules consisting of two moieties, one binding to the POI and the other to E3 ligase, joined by a linker. Molecular glues are smaller and are expected to have better pharmacological properties, higher membrane permeability, better cellular uptake, and better penetration of the blood-brain barrier than PROTACs. Molecular glue identification has relied primarily on serendipity, but rational design approaches to targeting undruggable proteins are emerging. Following the initial discovery, the pathway proceeds to scaffold definition, optimization, and validation (Table 1).

Table 1. Pathway of molecular glue degrader discovery and structure-guided drug design.
 

Molecular glues of varying types have been discovered; the best known is probably thalidomide and its analogs, and lenalidomide and pomalidomide, which target the E3 ligase cereblon. Molecular glues have been identified with various other mechanisms of action, including autophagy-mediated protein degradation, MEK sub-complexes stabilization, KRAS mutant inhibition, α-tubulin polymerization stabilization, and FK506-binding protein 12 (FKBP12) degradation.  

The landscape of molecular glue research - as found in CAS Content Collection™ 

The CAS Content Collection™ is the largest human-curated collection of published scientific knowledge, suitable for quantitative analysis of global scientific publications against variables such as time, research area, formulation, application, and chemical composition. To assess recent advances in molecular glue research, particularly in medicinal chemistry and drug discovery, a new CAS Insights Report has examined the relevant publication data from 2012 to 2021 from the CAS Content Collection. During this period, there was explosive growth in published articles and patents related to protein degraders (Figure 2). 

Journal publications on protein degraders were mostly from the United States, China, United Kingdom, Japan, Germany, and others (Table 2A). The Dana-Farber Cancer Institute and the University of Dundee published the largest number of TPD-related journal articles (Table 2B). The largest numbers of protein degrader-related patent filings were from China and United States (Table 2C). The journals that frequently publish TPD-related articles are presented in Table 2D, highlighting the importance of TPD in medical research; the Journal of Medicinal Chemistry and the European Journal of Medicinal Chemistry have the greatest number of TPD-related articles.

Table 2. Top countries (A), organizations (B), and scientific journals (D) publishing TPD-related journal articles, and top countries filing TPD-related patents (C).

The most frequent type of TPD in publications were small molecules (85.8%), followed by biosequences (7.7%), including peptides, proteins, nucleic acids, and salts (Figure 3 left panel). This indicates a change from the early protein-targeting chimeric molecules, which were peptide-based. Protein degraders are synthesized via multistep chemical reactions, which explains the dominance of the synthesis-related roles of SPN (synthetic preparation) and RCT (reactant) in the literature analysis results (Figure 3, right panel). Significantly greater numbers of compounds indexed in the CAS Content Collection originated from patents.

In the literature analysis, CRBN, VHL, and MDM2 were found to be the most popular types of E3 ligases being used to recruit TPDs to induce ubiquitination and subsequent proteasomal degradation of target proteins in cancer, inflammation, neurodegenerative, autoimmune, and infectious diseases (Table 3).

Table 3. Correlation of the number of protein degraders-related publications in the CAS Content Collection for the three most widely used E3 ligases with the targeted diseases. Percentages are from the total number of publications related to protein degraders.

The largest number of publications during 2017-2021 were on PROTACs and E3 ubiquitin ligases, followed by cereblon, proteasome, and ubiquitination (Figure 4A). Documents on molecular glues and protein degraders were less numerous but showed explosive growth and interest from 2019 onwards. Other concepts, including ubiquitination, E3 ligase, cereblon, PROTAC, and proteasome, also showed continuing strong growth throughout 2017-2021 (Figure 4B).

An analysis of the diseases targeted by protein degraders found in the CAS Content Collection showed the largest portion (44%) of the publications were associated with varied cancers (e.g., breast and prostate cancer, multiple myeloma, and leukemia). In addition, infectious (11%), neurodegenerative (10%), inflammatory (10%), autoimmune (8%), metabolic (6%), and cardiovascular (5%) diseases were also highly represented (Figure 5).

Molecular glues - discovered and in development

The number of known molecular glue types and structures is already considerable and is growing. The most extensively investigated molecular glues are small molecules that bind the E3 ligase CRBN and the aryl sulfonamides that engage DCAF15. Other molecular glues that induce protein degradation through various non-E3 ligase mechanisms of action include autophagy-mediated protein degradation, protein-protein interaction stabilization, KRAS mutant inhibition, microtubule polymerization stabilization, and inhibition of the mammalian target of rapamycin (mTOR). In addition, there are natural compounds such as cyclosporin A and sanglifehrin A, which have been found to function as molecular glues. These developments indicate an important broadening of molecular glue approaches and a potentially more diverse range of action.

While many potential molecular glues have been identified, so far, very few have been assessed for therapeutic efficacy in the clinic, and even fewer have received regulatory approval. Various companies worldwide have a series of molecular glue products in pre-clinical development for use in a variety of cancers and neurodegenerative and inflammatory diseases. A snapshot of these companies includes:

• Ranok (Hangzhou, China) - Drug candidate RNK05047 entering clinical trials first half of 2022 for the treatment of solid tumors and lymphomas
• Monte Rosa Therapeutics (Boston, MA, USA) - Initiated IND-enabling activities for its lead program targeting GSPT1 for oncology treatment and beyond. IND application to be submitted to the FDA mid-2022. Drug discovery phase for other molecular glues targeting solid/liquid tumors, autoimmune diseases, and blood diseases
• Plexium/Partnered with Amgen (San Diego, CA, USA) - Lead optimization phase for a cereblon molecular glue targeting IKZF2 for the treatment of immune disease and cancer
• Frontier Medicines/Partnered with AbbVie (San Francisco, CA, USA) - Drug discovery phase to develop small molecule covalent drugs against intractable immunology and oncology targets
• f5 Therapeutics (San Diego, CA, USA) - molecular candidates for multiple different cancers, multiple sclerosis, rheumatoid arthritis, nonalcoholic steatohepatitis, and liver fibrosis
• Ambagon Therapeutics/Partnered with BMS and Merck (San Carlos, CA, USA) - Drug discovery phase with five early discovery oncology treatment compounds
• Amphista Therapeutics (London, UK) – Cancer treatments with non-E3 ligase mode of action
• Proxygen/Partnered with Boehringer Ingelheim (Vienna, Austria) - Drug discovery phase treating lung and gastrointestinal cancers
• Neomorph/Partnered with Dana Farber Cancer Institute (San Diego, CA, USA) - Drug discovery phase to advance their molecular glue development pipeline against undruggable targets
• Seed Therapeutics/Partnered with Lilly (New York, NY, USA) - Drug discovery phase with molecular glue pipeline candidates treating cancers, neurodegenerative diseases, and infectious diseases. Their lead compound targets the KRAS oncogene
• Pin Therapeutics (Seoul, South Korea) – Drug discovery phase
• Venquis Therapeutics (San Diego, CA, USA) - Drug discovery phase for cancer and degenerative diseases
• IRB Barcelona/Partnered with Almirall (Barcelona, Spain) - Drug discovery phase for skin disease treatment
• Shanghai Dage Biomedical Technology Co., Ltd. (Shanghai, China) - Pipeline of molecular glues addressing targets for cancers, inflammatory disease, and metabolic disease. Lead optimization phase for oncology molecular glue candidates 
• Triana Biomedicines (Waltham, MA, USA) - Launched recently in April 2022 to establish a rationally designed molecular glue pipeline to treat inadequately addressed diseases
• Evotec/Partnered with BMS (Hamburg, Germany) - Drug discovery phase to develop a pipeline of molecular glue degraders

Several other companies/institutions have a promising range of molecular glue compounds in various stages of clinical development that aim to treat many different solid and liquid tumors along with inflammatory conditions and autoimmune diseases such as systemic lupus erythematosus (Figure 6).

Prospects for molecular glues as therapeutic agents – can they address unmet medical needs?

Inducing the proximity of target proteins to degradative enzymes that would not otherwise come into close contact using molecular glues is an exciting approach with the potential to create large numbers of highly novel therapeutic agents against a wide range of serious diseases. The challenge is to now develop the TPD approach by identifying candidate compounds with useful activities and progressing them to clinically useful products.   

The mechanism of action of molecular glues and their design principles are insufficiently understood and further research is needed to create new compounds and exploit them fully. The methods of novel molecular glue discovery have largely relied on intensive high-throughput screening, followed by systematic validation. There is a need for efficient and rational design strategies to enhance the development of new and more efficient compounds and to assess their activity in varying indications. Computational tools to model and predict molecular glue binding and advances in crystallization to improve understanding of protein docking are needed to transition from serendipity to rational design. 

The CAS Content Collection searches show that inducing protein degradation using molecular glues is attracting widespread interest and explosive growth both in terms of research articles and patents. While E3 ligase-targeting compounds continue to predominate, there is also strong interest in non-E3 ligase compounds and some natural molecular glue degraders, which indicates a large diversity of potential agents. The outlook for molecular glues, therefore, appears to be promising with the likely appearance of many more therapeutic agents using this approach in the near future, which have the intriguing potential to address a diverse range of medically unmet needs. 

Download our CAS Insights Report to discover the explosive growth of molecular glue degraders, their therapeutic applications, and how the research interest has evolved over the last decade.