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Tag: Enzyme

  • Understanding obesity’s effects on liver metabolism

    Understanding obesity’s effects on liver metabolism

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    Your liver plays a vital role in your metabolism, the biological process which converts food into energy. We know that being overweight can negatively affect metabolic activity, but not exactly how. To better understand this, researchers compared the livers of mice which were a typical weight with mice which were obese. They were surprised to find that biological regulation of metabolic activity, after a period of feasting and fasting, was reversed between them. In typical mice, allosteric regulation (the process which controls metabolism) was inhibited during feeding and activated when fasting. However, in obese mice, allosteric regulation increased during feeding and decreased when fasting. Investigating the reasons behind this reversed biological behavior could help health professionals understand how obesity affects the body and the development of disease.

    The World Obesity Federation (WOF) estimates that by 2035, over 4 billion people will be overweight or living with obesity. This may lead to a rise in obesity-related health conditions, such as heart disease, nonalcoholic fatty liver disease and Type 2 diabetes. Identifying the causes and effects of obesity, which is now understood to be a complex disease, is key for physicians looking to provide support and help people stay healthy.

    One known way that obesity can affect health is by impacting metabolism, the process by which our bodies take in, store and use energy from our food. Certain organs play key roles in this process, notably the liver. Not only is food processed there to provide energy, but it is one of the places where useful products at the end of the metabolic process are stored until we need them. To better understand the effects of obesity on the liver, researchers compared the livers of typical mice and obese mice after periods of feeding and fasting.

    The team carried out trans-omics analysis, an approach where they gathered data on five sets of biological processes (multi-omics). They then combined these layers of data with information from biological databases to create a trans-omic network. This gave them an overview of how the different layers interacted. “

    We constructed a trans-omic network of metabolic reactions in the livers of mice that could feed freely. We then compared this with data we had previously gathered from mice that had fasted for 16 hours. While enzyme and allosteric regulation which controls metabolism was suppressed in typical mice during feeding, we were surprised to find that the reverse occurred in obese mice and that this activity increased.”

    Professor Shinya Kuroda, Graduate School of Science, University of Tokyo

    When we eat, our liver builds up stores of energy which is then released as needed, a system known as metabolic homeostasis. However, the researchers saw that in obese mice this equilibrium became dysregulated, i.e., normal function was disrupted, indicating a potential breakdown of the system. This could lead to metabolic disorders such as tiredness, lack of energy and decreased appetite. By contrast, they saw that transcriptional regulation, a process which regulates metabolism and controls cell activity at a genetic level, did not change much between feeding and fasting. This means that, compared to allosteric regulation, it is more stable and less affected by what we eat.

    The team noted that what they observed may not only be evidence of disruption within the liver alone, but a change to broader metabolic cycles throughout the body. “Obesity is a metabolic disease, so to understand it, it is important to construct a trans-omic network with metabolome (the complete set of small-molecule chemicals) at its center,” said Kuroda. “We are interested not only in the liver, but also how the products of metabolic reactions circulate between liver and muscle through the blood in obese mice, which is what we will be working on now.”

    Source:

    Journal reference:

    Bai, Y., et al. (2024) Trans-omic analysis reveals opposite metabolic dysregulation between feeding and fasting in liver associated with obesity. iScience. doi.org/10.1016/j.isci.2024.109121.

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  • SARS-CoV-2-infection and vaccine-induced antibodies wane initially but stabilize for lasting protection

    SARS-CoV-2-infection and vaccine-induced antibodies wane initially but stabilize for lasting protection

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    In a recent observational study published in the journal Immunity, researchers from the United States of America investigated the longevity of antibody responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and vaccination. They found that the humoral responses to SARS-CoV-2 infection and vaccination were long-lasting and biphasic, with an initial decline followed by stabilization after seven to nine months.

    Study: SARS-CoV-2-infection- and vaccine-induced antibody responses are long lasting with an initial waning phase followed by a stabilization phase. Kateryna Kon / ShutterstockStudy: SARS-CoV-2-infection- and vaccine-induced antibody responses are long lasting with an initial waning phase followed by a stabilization phase. Kateryna Kon / Shutterstock

    Background

    The COVID-19 pandemic, which began four years ago, prompted the rapid development of messenger RNA (mRNA) vaccines, including the BNT162b2 and mRNA-1273, helping save millions of lives. However, emerging variants of SARS-CoV-2 and the waning immunity against them pose challenges. Although mRNA-based vaccine-induced immunity is perceived to decline rapidly, this perception is based on limited data, primarily from short-term studies.

    Amidst the exponential rise of SARS-CoV-2 cases in March 2020, the New York metropolitan area faced a crisis, with essential healthcare workers at a high infection risk. In response, a specific and sensitive SARS-CoV-2 enzyme-linked immunosorbent assay (ELISA) was developed, and the Protection Associated with Rapid Immunity to SARS-CoV-2 (PARIS) study was launched. This initiative tracked antibody responses, reinfection rates, and immunity factors in healthcare workers, offering vital insights into pandemic dynamics. Researchers in the present study utilized data from the PARIS study, one of the most comprehensive investigations on SARS-CoV-2 immunity longevity, and analyzed the humoral responses to SARS-CoV-2 infection and vaccination.

    About the study

    The PARIS study was an observational, longitudinal study conducted from April 2020 to March 2023 and enrolled 501 healthcare workers. Their mean age was 41 years, and 67% of them were female. Weekly saliva samples and bi-weekly blood samples were collected for the first two months. Nasopharyngeal/ante-near swabs were taken for respiratory symptoms or after vaccination. About 38% of participants showed baseline SARS-CoV-2-spike-binding immunoglobulin G (IgG) antibodies. A total of 93% of participants were vaccinated– 0.2% received four mRNA boosters, 2.6% had three boosters, 16.6% had two boosters, and 53.7% had one booster. Approximately 21.3% of the participants chose not to receive boosters.

    The study utilized REDCap for monthly surveys on general health and SARS-CoV-2 risk, focusing on side effects after mRNA vaccinations and booster doses. Data from 228 participants were analyzed, and severity scoring was conducted, revealing reported incidence and severity trends across doses and subgroups.

    Antibody titers in serum were assessed using enzyme-linked immunosorbent assay (ELISA) and optical density at 490 nm (OD490). Statistical and quantitative analysis involved the use of the Wilcoxon test, Mann-Whitney U test, log-rank test, unweighted pair group method with arithmetic mean (UPGMA) clustering, antibody kinetic modeling including nonlinear mixed-effects (NLME) models, and demographic factor assessment in post-vaccine and post-boost models.

    Results and discussion

    While 38% of the participants had detectable spike-binding IgG antibodies at baseline, 62% were seronegative at the first visit. Vaccination-naïve individuals exhibited low antibody titers after the first mRNA vaccine dose, with a substantial increase after the second dose. However, individuals with pre-existing immunity reached higher and faster peak titers, maintaining over threefold higher responses after primary immunization.

    Seven to nine months post-primary vaccination, antibody titers were found to achieve a steady state. Individuals with hybrid immunity maintained higher and more stable titers compared to naïve recipients, indicating the induction of long-lasting serum antibodies. Furthermore, vaccine type and age were found to affect the antibody titers in participants without hybrid immunity modestly. As per the study, the administration of booster doses elevated the threshold at which long-term serum antibody responses reached a stable state.

    A total of 225 SARS-CoV-2 infections were observed in the study period, predominantly occurring after immunization, with breakthrough infections more prevalent during the Omicron wave. In individuals with vaccine-only immunity, breakthrough infections acted as equivalent boosts to antibody responses, while in those with hybrid immunity, vaccination had a more robust boosting effect compared to a second infection.

    Participants with pre-existing immunity experienced more side effects after the first vaccine dose, with overall reactogenicity decreasing after subsequent doses. Booster doses induced fewer systemic side effects than the second dose in naïve participants, while those with hybrid immunity had a different pattern, showing slightly increased side effects with booster doses.

    However, the study is limited by the inability to analyze mucosal immune responses, the lack of measuring neutralizing antibodies or antibodies to specific epitopes, and the lack of assessment of later variant spikes or nucleoprotein.

    Conclusion

    In conclusion, the present study provides evidence that antibody responses to SARS-CoV-2 mRNA vaccination exhibit a classical biphasic decay, transitioning from rapid waning to stabilization. The findings emphasize the prolonged protection provided by hybrid immunity against several variants and the potential booster-like effect of breakthrough infections in enhancing immunity.

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  • Novel treatment strategy shows promise against common liver cancer in children

    Novel treatment strategy shows promise against common liver cancer in children

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    Hepatoblastoma (HB) is the most common liver cancer in children. Researchers and physicians in the field are concerned because in the last decade HB has been rising rapidly worldwide and has seen the most rapid increase among all pediatric solid tumors.

    A team led by researchers at Baylor College of Medicine has been working on improving therapies for this devastating disease. They recently reported in the Journal of Hepatology a novel treatment strategy that produced encouraging results in animal models.

    “High-risk disease leads to high rates of relapse and mortality,” said first author Dr. Andrés F. Espinoza, general surgery resident in Baylor’s Michael E. DeBakey Department of Surgery.

    “In this study, we investigated the potential benefits of a new combination therapy that included an inhibitor of the enzyme histone deacetylase (HDAC) present at elevated levels in HB cells,” Espinoza said. “Previous studies have suggested that HDAC inhibitors may provide treatment options for HB, but there is no published preclinical data on such therapies.”

    To assess the effects of the novel combination therapy, the researchers developed a preclinical testing pipeline with clinically relevant HB models.

    Identifying an effective new combination therapy

    First, the researchers tested several HDAC inhibitors on their ability to kill patient-derived HB cell lines grown in the lab. They found that HDAC inhibitor panobinostat was the most effective at eliminating cancer cells.

    The team then tested whether adding panobinostat to current chemotherapy regimens would improve tumor response. They screened these treatments in patient-derived spheroids, spherical cellular aggregates derived from human tumor samples. Their results show that adding panobinostat to the combination treatment including vincristine and irinotecan eliminated more tumor cells than any of the other combinations.

    Finally, they tested the new combination therapy of vincristine, irinotecan and panobinostat (VIP) in four aggressive animal models of HB the researchers had derived from high-risk, relapsed and treatment refractory HBs. The molecular characterization of these tumors in the animal models showed that they conserved all mutations found in the human tumors of origin as well as other molecular markers present in patient tumor samples. This indicated that the tumors in the animal models closely matched those in the patients. Importantly, in contrast to other studies that tested treatments on small-sized tumors, this study tested VIP in large tumors.

    After just one week of therapy, the tumors that were treated with VIP had a significant decrease in volume and alpha fetoprotein levels, the tumor marker. These findings are very encouraging as they suggest that VIP therapy may be a promising and effective option for patients with currently untreatable high-risk, relapsed or refractory HB.”


    Dr. Sanjeev A. Vasudevan, corresponding author, associate professor of surgery at Baylor

    Other contributors to this work include: Roma H. Patel, Kalyani R. Patel, Andrew A. Badachhape, Richard Whitlock, Rohit K. Srivastava, Saiabhiroop R. Govindu, Ashley Duong, Abhishek Kona, Pavan Kureti, Bryan Armbruster, Dina Kats, Ramakrishnan R. Srinivasan, Lacey E. Dobrolecki, Xinjian Yu, Mohammad J. Najaf Panah, Barry Zorman, Stephen F. Sarabia, Martin Urbicain, Angela Major, Karl-Dimiter Bissig, Charles Keller, Michael T. Lewis, Andras Heczey, Pavel Sumazin, Dolores H. López-Terrada and Sarah E. Woodfield.

    The authors are affiliated with Baylor College of Medicine, Texas Children’s Hospital, Children’s Cancer Therapy Development Institute at Beaverton or Duke University.

    This work was supported by a U.S. Department of Defense Career Development Award (W81XWH-21-1-0396 /CD201061), a Baylor College of Medicine Michael E. DeBakey Department of Surgery Faculty Research Award, a Macy Easom Cancer Research Foundation Grant, a Cancer Prevention and Research Institute of Texas (CPRIT) Multi-Investigator Research Award (RP180674), a P30 Cancer Center Support Grant NCI-CA125123, a CPRIT Core Facilities Support Grant RP220646 and an NIH Ruth L. Kirschstein National Research Service Award Individual Postdoctoral Fellowship (1F32CA278313-01).

    Source:

    Baylor College of Medicine

    Journal reference:

    Espinoza, A. F., et al. (2024). A Novel Treatment Strategy Utilizing Panobinostat for High-Risk and Treatment-Refractory Hepatoblastoma. Journal of Hepatology. doi.org/10.1016/j.jhep.2024.01.003.

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  • Discovery of novel enzyme family holds potential for antibiotic development

    Discovery of novel enzyme family holds potential for antibiotic development

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    Researchers at Umeå University in Sweden, led by Professor Felipe Cava, have identified a new family of enzymes that creates a unique type of cross-linking between the building blocks of bacterial cell walls. This discovery could help develop new antibiotics against infectious diseases.

    Bacterial cell walls form mesh-like structures, shielding cells from rupturing under high internal pressure and safeguarding against external threats. The cell wall is comprised of sugar and amino acid molecules interconnected by various types of cross-links. These cross-links play a crucial role in providing strength and stability to the cell wall, while also enabling bacteria to adapt to diverse environments and stressors.

    In a groundbreaking study recently published in the esteemed journal Nature Communications, researchers from Umeå University and international institutions have unveiled a novel family of enzymes responsible for generating a unique cross-linkage between L-alanine and meso-diaminopimelic acid. These amino acids are integral components of the peptide chains constituting the cell wall of numerous bacterial species. Termed LD1,3-transpeptidase, this enzyme has been identified across various groups of alpha and beta proteobacteria, including opportunistic pathogens such as Burkholderia and Achromobacter.

    The researchers utilized Gluconobacter oxydans, a model organism employed in vinegar production, to identify the novel LD1,3-transpeptidase enzyme and elucidate its three-dimensional structure. They have demonstrated that this enzyme possesses unique characteristics distinguishing it from other known enzymes involved in cell wall cross-linkage. These distinctive properties enable the enzyme to utilize various substrates and execute diverse reactions, critical for maintaining the cell wall’s integrity. Specifically, their findings indicate that cells lacking these cross-links exhibit heightened sensitivity to β-lactam antibiotics, underscoring the potential of LD1,3-transpeptidases as promising targets for therapeutic interventions, particularly those aimed at enhancing antibiotic effectiveness.

    The principal investigator of the study is Felipe Cava, Professor of Infection Biology at Umeå University and Director of the Umeå Hypoxic Research Facility. With extensive expertise in bacterial cell wall research and its implications in bacterial survival and disease progression, Professor Cava has spearheaded investigations into this field for a significant duration.

    The bacterial cell wall stands as one of the most remarkable structures, yet much remains to be uncovered about its diversity and dynamics. Through the identification and characterization of novel enzyme families like LD1,3-transpeptidase, we not only expand our understanding of bacterial biology but also discover fresh targets for developing antibiotics to combat infectious diseases.”


    Felipe Cava

    The study was funded by, among others, the Swedish Research Council, the Knut and Alice Wallenberg Foundation, the Kempe Foundations.

    Source:

    Journal reference:

    Espaillat, A., et al. (2024). A distinctive family of L,D-transpeptidases catalyzing L-Ala-mDAP crosslinks in Alpha- and Betaproteobacteria. Nature Communications. doi.org/10.1038/s41467-024-45620-5.

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  • Enzyme released by immune cells may play role in depression

    Enzyme released by immune cells may play role in depression

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    Mount Sinai researchers have shown for the first time that immune cells called monocytes, derived in the bone marrow and released into the bloodstream, can be drawn during stress into sites in the brain that control emotional behaviors. There, they release an enzyme called matrix metalloproteinase 8 (MMP8) that breaks down proteins and restructures the brain to alter the function of neurons and, ultimately, impair social behavior and reward.

    These data establish a novel mechanism by which the immune system can affect central nervous system function and behavior in the context of stress, potentially opening the door to novel therapeutic targets for stress-related disorders. The study appears in the February 7 issue of Nature.

    Psychosocial stress is a major factor for developing major depressive disorder and post-traumatic stress disorder (PTSD) and has been shown to have profound effects on the body, including the immune system and the brain. These data are the first to show that immune cells derived in the bone marrow-;and not the brain-;can be recruited during stressful circumstances to the brain, setting off a cascade of other mechanisms that alter brain function and behavior.”


    Flurin Cathomas, MD, lead author, Instructor of Neuroscience and member of the Brain-Body Research Center at Mount Sinai

    To explore these mechanisms, the research team performed comparative cross-species analyses in mice and humans and found that MMP8 is elevated in the serum of study subjects with major depressive disorder, as well as in stress-susceptible mice following chronic social defeat stress, a model of social trauma. Studies in mice confirmed that peripheral MMP8 enters the brain through damaged blood vessels to restructure the brain’s extracellular tissue matrix, which leads to altered function of neurons that ultimately impairs social behavior and reward.

    Prior to this work, most hypotheses about the role of the immune system in stress disorders such as depression have centered on mechanisms related to the brain’s resident immune cells, called microglia, and their ability to release pro-inflammatory molecules such as interleukins to control neural function and behavior.

    Using single-cell RNA sequencing to look at gene expression profiles in circulating monocytes as compared to microglia, the team found that, contrary to popular belief, the microglia did not exhibit a pro-inflammatory gene signature. The team found no evidence that they upregulate genes that code for interleukins. This is in stark contrast to circulating monocytes found within the blood vessel lining of brain regions that control mood and emotion.

    “There are no existing medications to target MMP8, and while it’s not yet clear if such treatments will ultimately be effective in treating depression, my hope is that this study will lead to renewed effort in developing such drugs,” said Scott Russo, PhD, Mount Sinai Professor in Affective Neuroscience, Leon Levy Director of the Brain-Body Research Center, and Center for Affective Neuroscience at Mount Sinai. “It’s also possible that non-pharmacological ‘lifestyle’ strategies to promote positive immune health might be helpful in treating these stress-related disorders.”

    The disturbances in the immune system identified in this study were only found in a subset of patients, which highlights the heterogeneous nature of such illnesses in terms of etiology. Additionally, the studies performed in human subjects were purely correlative, so the team does not yet know if treatments targeting monocytes or MMP8 directly will be effective for human stress disorders. Importantly, there are several additional MMPs that can be derived directly in the brain and it remains unclear whether they play complementary or opposing roles.

    “The brain and the body are unequivocally connected and we are really at the precipice of a markedly deeper understanding of how the connections between the brain and peripheral organ systems like the immune system, cardiovascular system, and others can affect a person’s health,” said Dr. Russo. “Our work suggests that strategies to promote immune health can benefit one’s emotional well-being and possibly prevent stress-related illnesses like depression and PTSD. Additional research for continued understanding and potential treatment development is warranted.”

    The Mount Sinai research team is currently testing therapeutic strategies to inhibit MMP8 as novel antidepressants. They are also investigating MMP8 as a novel immune biomarker for depression patients.

    Source:

    Mount Sinai Health System

    Journal reference:

    Cathomas, F., et al. (2024). Circulating myeloid-derived MMP8 in stress susceptibility and depression. Nature. doi.org/10.1038/s41586-023-07015-2.

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  • New treatment target found for CDKL5 deficiency disorder

    New treatment target found for CDKL5 deficiency disorder

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    Scientists at the Francis Crick Institute have found a new treatment target for CDKL5 deficiency disorder (CDD), one of the most common types of genetic epilepsy. 

    CDD causes seizures and impaired development in children, and medications are limited to managing symptoms rather than tackling the root cause of the disease. The disorder involves losing the function of a gene producing the CDKL5 enzyme, which phosphorylates proteins, meaning it adds an extra phosphate molecule to alter their function.

    Following recent research from the same lab showing that a calcium channel could be a target for therapy for CDD, the team has now identified a new way to potentially treat CDD by boosting another enzyme’s activity to compensate for the loss of CDKL5. 

    In research published today in Molecular Psychiatry, the scientists studied mice that don’t make the CDKL5 enzyme. These mice show similar symptoms to people with CDD like impaired learning or social interaction. 

    The researchers first identified that CDKL5 is active in nerve cells in mice but not in another type of brain cell called an astrocyte. In the nerve cells, they measured the level of phosphorylation of EB2, a molecule known to be targeted by CDKL5, to understand what happens when CDKL5 isn’t produced. 

    Interestingly, even in mice that don’t produce CDKL5, there was still some EB2 phosphorylation taking place, which suggested that another similar enzyme must also be able to phosphorylate it.

    By looking at enzymes similar to CDKL5, the researchers identified that one called CDKL2 also targets EB2 and is present in human neurons. In mice without both CDKL5 and CDKL2, the remaining EB2 phosphorylation almost fully dropped off.

    The researchers concluded that, although most activity comes from CDKL5, about 15% is from CDKL2, and the remaining <5% from another enzyme yet to be identified. 

    Their research suggests that increasing the level of CDKL2 in people who are deficient in CDKL5 could potentially treat some of the effects on the brain in early development. 

    CDD is a devastating condition that impacts young children from birth, and we don’t know a huge amount about why losing this one enzyme is so disastrous for the developing brain. Through this research, we’ve identified a potential way to compensate for the loss of CDKL5. If we can increase levels of CDKL2, we might one day be able to stop symptoms from developing or getting worse.”


    Sila Ultanir, Group Leader of the Kinases and Brain Development Laboratory, The Francis Crick Institute

    The researchers are now investigating if mice without CDKL5 can be treated by stimulating their brain cells to produce more CDKL2. The lab is also working with biotechnology companies to identify molecules that increase CDKL2 for potential new medicines for CDD. 

    Margaux Silvestre, former PhD student at the Crick and now postdoctoral researcher at the Max Planck Institute for Brain Research in Frankfurt, said: “Our discoveries offer fresh insights into the expression and regulation of CDKL5 in the brain. Moreover, the identification of CDKL2 as a potential compensatory enzyme provides hope for uncovering better treatments that could truly make a difference in the lives of the children with this devastating condition. This research owes its success to all the authors involved in the publication but also the unwavering support we received from the technical teams at the Crick – a big shoutout to them!”

    The research was funded by the Loulou Foundation, a private foundation dedicated to the development of therapeutics and eventual cures for CDD.

    Source:

    The Francis Crick Institute

    Journal reference:

    Silvestre, M., et al. (2024). Cell type-specific expression, regulation and compensation of CDKL5 activity in mouse brain. Molecular Psychiatry. doi.org/10.1038/s41380-024-02434-7.

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  • Paxlovid enhances treatment options for COVID-19 patients

    Paxlovid enhances treatment options for COVID-19 patients

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    In a recent review published in the Pharmaceutics, a group of authors explored the design, synthesis, and mechanism of action of Paxlovid, a Protease inhibitor (PI) drug combination for treating coronavirus disease 2019 (COVID-19).

    Study: The Design, Synthesis and Mechanism of Action of Paxlovid, a Protease Inhibitor Drug Combination for the Treatment of COVID-19. Image Credit: Tobias Arhelger/Shutterstock.com

    Background 

    The COVID-19 pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, significantly challenged global healthcare systems and medical science.

    In response, researchers worldwide developed vaccines with innovative mechanisms and small-molecule antivirals targeting crucial viral proteins.

    Among these, PaxlovidTM, a blend of nirmatrelvir and ritonavir PIs, stands out for its effectiveness in treating COVID-19.

    Nirmatrelvir inhibits SARS-CoV-2’s main protease, vital for viral replication, while ritonavir boosts nirmatrelvir’s effectiveness by inhibiting Cytochrome P450 3A4 (CYP3A4), an enzyme that would otherwise degrade nirmatrelvir quickly.

    Further research is needed to develop alternative main protease (MPro) inhibitors despite the success of the nirmatrelvir-ritonavir combination, ensuring continued effectiveness against COVID-19.

    PIs as antivirals for Hepatitis C virus (HCV) and Human immunodeficiency virus (HIV) 

    PI Drugs for HCV and HIV Infections

    PIs are key in treating HCV and HIV infections. HCV, a small ribonucleic acid (RNA) virus causing hepatic diseases, is targeted by PIs like asunaporevir, telaprevir, and boceprevir, focusing on the nonstructural (NS)3/4A serine protease.

    These inhibitors are peptidomimetics, containing peptide bonds and a ‘warhead’ group that binds covalently but reversibly to the enzyme’s active site.

    HIV PIs target the virus’s aspartic acid protease, which is crucial for viral replication. They are used in antiretroviral therapy, transforming HIV from fatal to chronic.

    Development and mechanism of Nirmatrelvir

    Nirmatrelvir, developed from Pfizer’s earlier SARS-CoV-1 PI .. PF-00835231, faced challenges in oral absorption.

    Modifications like altering the warhead and substituting various molecular components enhanced its binding affinity and antiviral activity, eventually leading to nirmatrelvir with a nitrile warhead, improving solubility and synthesis.

    Despite different warheads, its structural similarity to boceprevir, and its role as a covalent inhibitor of SARS-CoV-2 Mpro makes it significant in COVID-19 treatment.

    Synthesis of nirmatrelvir

    Nirmatrelvir’s synthesis involves coupling the P1 building block and the P2-P3 dipeptide, with the final step being the formation of the nitrile warhead.

    The process starts with protected amino acid derivatives, proceeding through stages like Boc-deprotection, ester cleavage, and dipeptide formation.

    The synthesis yields nirmatrelvir with high efficiency and introduces a new approach involving a Ugi-type three-component reaction for higher diastereoselectivity.

    Synthesis and structure-activity relationship (SAR) study of nirmatrelvir analogs

    Research by Chia and co-workers led to the synthesizing nirmatrelvir analogs with different P1′ moieties, examining the role of the warhead in antiviral activity.

    These studies revealed varying levels of effectiveness in protease inhibition and antiviral activity, with some derivatives showing similar or superior effects to nirmatrelvir. However, challenges in cell penetration and specificity to SARS-CoV-2 limited the broader application of these analogs.

    Novel covalent and non-covalent inhibitors of SARS-CoV-2 Mpro

    Recent developments in SARS-CoV-2 Mpro inhibitors have introduced both peptidomimetic and non-peptidic inhibitors.

    These include warheads, such as epoxide rings and fluoromethyl groups, offering alternative mechanisms of covalent binding to the enzyme.

    Non-covalent inhibitors, like ensitrelvir, show lower reactivity but better selectivity due to their secondary interaction nature. These developments represent crucial steps in diversifying therapeutic options against COVID-19 and its evolving strains.

    Ritonavir as a pharmacokinetic enhancer

    Structure, activity, and interactions of ritonavir

    Originally an HIV protease inhibitor, Ritonavir is known for its efficacy at low doses (~100 mg) in inhibiting the CYP3A4 enzyme, a crucial element in drug metabolism.

    While high doses of Ritonavir are poorly tolerated, its low-dose effectiveness is leveraged in combination therapies with other HIV protease inhibitors, enhancing their half-lives and thus reducing required dosages.

    This unique use of Ritonavir has been explored even in early COVID-19 treatments. However, its use poses risks of significant drug–drug interactions, especially with medications metabolized by CYP3A4, potentially elevating their levels to toxic concentrations.

    Additionally, Ritonavir’s effect on other enzymes and transport proteins is noted, albeit of lesser importance in Paxlovid treatment.

    Synthesis of ritonavir

    developed at Abbott Laboratories, Ritonavir’s synthesis involves complex chemical processes, combining chiral amine and carboxylic acid building blocks.

    The synthesis starts with a cyclocondensation reaction involving thioformamide and ethyl 2-chloroacetate, followed by a series of steps leading to the formation of ritonavir.

    This intricate process involves various intermediate compounds and chemical reactions, including triethylamine and 4-dimethylaminopyridine, highlighting the sophistication required in pharmaceutical synthesis.

    The production of Ritonavir demonstrates the intricate chemical engineering necessary to develop effective pharmaceutical agents.

    Paxlovid—application and activity against mutant variants

    Paxlovid, combining nirmatrelvir and ritonavir, has shown significant efficacy in reducing COVID-19-related hospitalizations and mortality.

    While it has gained emergency use authorization in various regions, its effectiveness against emerging strains and mutant variants is under continuous scrutiny.

    The evolving landscape of SARS-CoV-2 mutations necessitates ongoing monitoring to ensure the sustained efficacy of treatments like Paxlovid.

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  • Caltech Scientists Engineer Enzyme To Degrade Silicon-Carbon Bonds in Silicones

    Caltech Scientists Engineer Enzyme To Degrade Silicon-Carbon Bonds in Silicones

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    Artificially Evolved Enzyme Breaking a Silicon Carbon Bond

    Scientists have engineered an enzyme to break down silicon-carbon bonds in siloxanes, marking a significant step towards making these widespread and environmentally persistent chemicals biodegradable. This innovation, achieved through directed evolution, paves the way for more effective management of silicone waste. Above is an artist’s depiction of an artificially evolved enzyme breaking a silicon-carbon bond. Credit: Caltech/Dow

    Scientists have successfully engineered an enzyme capable of breaking down the stubborn man-made bonds between silicon and carbon that exist in widely used chemicals known as siloxanes, or silicones. This breakthrough marks an initial step towards making these persistent environmental chemicals biodegradable.

    “Nature is an amazing chemist, and her repertoire now includes breaking bonds in siloxanes previously thought to evade attack by living organisms,” says Frances Arnold, the Linus Pauling Professor of Chemical Engineering, Bioengineering and Biochemistry at Caltech and winner of the 2018 Nobel Prize in Chemistry for her pioneering work in directed evolution, a method for engineering enzymes and other proteins using the principles of artificial selection. Arnold and her colleagues, including Dimitris (Dimi) Katsoulis of Michigan-based Dow Inc. used directed evolution to create the new silicon–carbon bond-cleaving enzyme. The results are published in the January 26 issue of the journal Science.

    The researchers say that while practical uses for their engineered enzyme could still be a decade away or more, its development opens the possibility that siloxanes could one day be degraded biologically. “For example, natural organisms could evolve in siloxane-rich environments to catalyze a similar reaction, or further improved versions of laboratory-evolved enzymes such as this one could possibly be used to treat siloxane contaminants in wastewater,” Arnold says.

    Katsoulis explains that nature doesn’t use silicon–carbon bonds, “but we do and have been for about 80 years. The volatile nature of some of these compounds warrants health and environmental research to properly understand the degradation mechanisms of these materials in the environment.”

    Silicones in Everyday Products

    Siloxane chemicals can be found in countless products, including those used in household cleaning, personal care, and the automotive, construction, electronics, and aerospace industries. The compounds’ chemical backbone is made of silicon–oxygen bonds, while carbon-containing groups, often methyl, are attached to the silicon atoms. “The silicon–oxygen backbone gives the polymer an inorganic-like character while the silicon–methyl groups give the polymer organic-like characteristics. Thus, these polymers have unique material properties, such as high thermal and oxidative stability, low surface tension, and high backbone flexibility among others,” Katsoulis says.

    Siloxanes are believed to persist in the environment for days to months, and, therefore, ongoing research aims to provide greater scientific understanding of the health and environmental safety of silicone materials. The chemicals naturally start to fragment into smaller pieces, especially in soil or aquatic environments, and those fragments become volatile or escape into the air, where they undergo degradation by reacting with free radicals in the atmosphere. Of all the bonds in siloxanes, the silicon–carbon bonds are the slowest to break down.

    Katsoulis approached Arnold to collaborate on efforts to speed up siloxane degradation after he read about her lab’s work in coaxing nature to produce silicon–carbon bonds. In 2016, Arnold and her colleagues used directed evolution to engineer a bacterial protein called cytochrome c to form silicon–carbon bonds, a process that does not occur in nature. “We decided to get nature to do what only chemists could do—only better,” Arnold said in a Caltech news release. The research demonstrated that biology could make these bonds in ways that are more environmentally friendly than those traditionally used by chemists.

    In the new study, the researchers wanted to find ways to break the bonds rather than create them. The scientists used directed evolution to evolve a bacterial enzyme called cytochrome P450. Directed evolution is similar to breeding dogs or horses in that the process is designed to bring out desired traits. The researchers first identified a variant of cytochrome P450 in their collection of enzymes that had a very weak ability to break silicon–carbon bonds in so-called linear and cyclic volatile methylsiloxanes, a common subgroup of the siloxane family.

    Overcoming Obstacles in Enzyme Evolution

    They mutated the DNA of the cytochrome P450 and tested the new variant enzymes. The best performers were then mutated again, and the testing was repeated until the enzyme was active enough to enable the researchers to identify the products of the reaction and study the mechanism by which the enzyme works.

    “Evolving enzymes to break these bonds in siloxanes presented unique hurdles. With directed evolution, we must evaluate hundreds of new enzymes in parallel to identify a few enzyme variants with improved activity,” says Tyler Fulton (PhD ’22), co-lead author of the study and a postdoctoral scholar at Caltech in Arnold’s lab. One challenge involved the siloxane molecules leaching plastic components from the 96-well plates used to screen the variants. To solve the problem, the team created new plates made from common lab supplies.

    “Another challenge was finding the starting enzyme for the directed evolution process, one with even just a tiny amount of the desired activity,” Arnold says. “We found it in our unique collection of cytochrome P450s evolved in the laboratory for other types of new-to-nature silicon chemistry.”

    The final improved enzyme does not directly cleave the silicon–carbon bond but rather oxidizes a methyl group in the siloxanes in two sequential steps. Basically, this means that two carbon–hydrogen bonds are replaced with carbon–oxygen bonds, and this change allows the silicon–carbon bond to break more readily.

    The research draws parallels to studies involving a plastic-eating enzyme, explains Fulton, referring to a polyethylene terephthalate (PET)-degrading enzyme discovered in the bacteria Ideonella sakaiensis in 2016 by a different group of researchers. “While the PET-degrading enzyme was discovered by nature rather than by engineers, that enzyme inspired other innovations that are finally coming to fruition for plastic degradation. We hope this demonstration will similarly inspire further work to help break down siloxane compounds,” he says.

    Reference: “Directed evolution of enzymatic silicon-carbon bond cleavage in siloxanes” by Nicholas S. Sarai, Tyler J. Fulton, Ryen L. O’Meara, Kadina E. Johnston, Sabine Brinkmann-Chen, Ryan R. Maar, Ron E. Tecklenburg, John M. Roberts, Jordan C. T. Reddel, Dimitris E. Katsoulis and Frances H. Arnold, 25 January 2024, Science.
    DOI: 10.1126/science.adi5554

    The research was funded by Dow’s University Partnership Initiative and the National Science Foundation. Other Caltech authors include co-lead author Nicholas Sarai (PhD ’23), as well as graduate student Ryen L. O’Meara, Kadina E. Johnston (PhD ’23), and Arnold lab manager Sabine Brinkmann-Chen. Other Dow authors include Ryan R. Maar, Ron E. Tecklenburg, John M. Roberts, and Jordan C. T. Reddel.



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