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Baloxavir Antiviral to Shorten and Prevent Influenza

Baloxavir is an antiviral drug that was FDA approved in 2018 for the treatment of influenza. It works by inhibiting an enzyme that influenza needs to replicate its DNA. When taken early, it can shorten the course of influenza by a day.

But can it also prevent influenza?

Researchers working for Shionogi, the manufacturer of baloxavir, set up a clinical trial in Japan to test this question. During the 2018-2019 flu season, they identified patients with influenza when they went to see their primary care doctor.

The researchers offered the household contacts of these patients either baloxavir or a placebo. Ten days later, the researchers tested the household contacts for influenza RNA (clinical influenza) using RT-PCR.

1.9% of household contacts who took the active treatment had clinical influenza whereas 13.6% of household contacts who took the placebo had clinical influenza. This is a successful demonstration that Baloxavir is effective at preventing influenza and as such was recently FDA approved for this use.

The authors of this study were not able to rule out whether this chemical can contribute to the rise of drug-resistant influenza. Further research is needed on this topic.

 

Baloxavir

Baloxavir Marboxil

 

Ikematsu H, Hayden FG, Kawaguchi K, et al. Baloxavir marboxil for prophylaxis against influenza in household contacts. N Engl J Med. 2020 Jul 23; 383(4):309-320. doi 10.1056/NEJMoa1915341. PMID: 32640124.

 

Rosiglitazone for Neuroinflammatory Abnormalities

Gulf War Illness is a chronic disorder brought on by exposure to sand storms, prophylaxis against chemical warfare, and chronic unpredictable stress. This illness is characterized by neuropsychological and cognitive problems affecting the central nervous system as well as specific organs.

Animal studies of this illness have shown neuroinflammation in several regions of the brain. Treatments used to reduce the neuroinflammation have led to improvements of the neurofunctional abnormalities.

Rosiglitazone and pioglitazone are compounds from the thiazolidinedione family that both have demonstrated neuroprotection in animal models. A new study recently completed at the University of Maryland looked at Gulf War Illness in a rat model and the impact of treatment with rosiglitazone.

Simulation

In order to simulate a Gulf War type of exposure, the rats were subjected to a variety of intense daily stressors for 33 days. Stressors included subjection to restraint, cold, wet, hunger, thirst, and various other types of unpredictably timed environmental stresses. During the 33 days the experimental treatment groups were also administered an oral solution of rosiglitazone each day.

Live Testing

Beginning on day 35, a battery of neurofunctional evaluations was begun. Evaluations included open-field exploration, maze exploration, new object recognition, sucrose preferences, tail suspension response, coat hygiene, splash testing, and forced swimming. These test methods were used as a gauge for cognition, anxiety-like behavior, and depression-like behavior.

Neurofunctional evaluation of the groups showed the development of significant abnormalities in rats exposed to the stressors compared to rats not subjected to such stress. The results of rats exposed to daily stressors and treatment with rosiglitazone were less conclusive. Rosiglitazone treatment seemed to improve measures related to cognition and anxiety, while not having much impact on weight gain or depression.

Tissue Testing

Following the neurofunctional evaluations, the rats were sacrificed so that the brain tissues could be analyzed. Examination of astrocytes, translocator protein, and microglia showed results consistent with a reduced inflammatory response in the samples from the rosiglitazone-treated group.

This modelling of Gulf War Illness successfully induced critical neurofunctional abnormalities, while the concurrent treatment with rosiglitazone reduced the development or abnormalities and neuroinflammation. The model will be useful for further study of the impact and potential treatments for Gulf War Illness.

 

Learn more about Gulf War Illness on Wikipedia

 

Thiazolidinediones Available:

Ciglitazone

Pioglitazone

Rosiglitazone

Rosiglitazone Maleate

Troglitazone

 

Keledjian K, Tsymbalyuk O, Semick S, et al. The peroxisome proliferator-activated receptor gamma (PPARγ) agonist, rosiglitazone, ameliorates neurofunctional and neuroinflammatory abnormalities in a rat model of Gulf War Illness. PLoS One. 2020 Nov 13;15(11):e0242427. PMID: 33186383

Lovastatin Impact on Brain Cholesterol

Lovastatin’s impact on brain cholesterol levels is found to produce neuroprotective effects.

Statins are known to reduce cholesterol synthesis in the liver, easily cross the blood-brain barrier, and have various additional effects on brain cells.

Lovastatin, aka Mevinolin, is commonly used as a medication to slow the production of cholesterol in the body, leading to reduced concentration of cholesterol in the blood, and thereby preventing buildup on artery walls.

Interestingly, in the average healthy adult, 25% of total cholesterol is in the brain.

This cholesterol in the brain is necessary to maintain the nicotinic acetylcholine receptors (nAChRs) at the cell membranes and for moving ions across the membranes.

There are different kinds of nAChRs present in the central nervous system, with both homomeric and dimeric species. The most abundant of the homomeric species in the central nervous system is α7 nAChR, while the most abundant dimeric species is the combination of α4 and β2 subunits.

These nAChR species play roles in a variety of cognitive functions, including learning, memory, and involvement in the development of Alzheimer’s disease. At the same time, the nAChRs all seem to be quite sensitive to membrane cholesterol levels. As has been demonstrated, the levels of nAChRs may be altered by manipulating the cholesterol level.

Cholesterol is known to have a big impact on muscle-type nAChR, however, its impact on neuronal-type nAChR is less studied.

Consequently, a study was formed to evaluate the impact of ongoing lovastatin treatment on cholesterol levels and the α7 and α4-containing nAChRs.

When cultured hippocampal neurons were incubated with varying levels of lovastatin for up to 14 days the total and surface cholesterol levels were significantly reduced, in a dose-dependent manner. No signs of neuronal damage were observed at the concentrations used, 0, 10, 50, 100, and 1000 nM.

The levels of α7 nAChR found in the cultured hippocampal nerves were monitored and found to be increased with the lovastatin treatments.

Previous studies have suggested that individuals with long-term statin treatment are less susceptible to developing Alzheimer’s disease. The nAChR species in the central nervous system are potentially a key to this phenomenon.

The lingering question remains: is it the impact on cholesterol biosynthesis, which occurs in the liver, and the resulting decreased level of cholesterol interacting with the nAChRs in the brain, or is it the statin itself entering the brain, that is responsible for the neuroprotective effect? It is quite possibly a combination of both factors and more research may be able to tease this information apart.

 

Additional Statins Available…

 

Borroni V, Kamerbeek C, Pediconi M, et al. Lovastatin differentially regulates α7 and α4 neuronal nicotinic acetylcholine receptor levels in rat hippocampal neurons. Molecules. 2020 Oct 20;25(20):4838. doi: 10.3390/molecules25204838. PMID: 33092257.

https://www.scientificamerican.com/article/this-is-your-brain-on-cholesterol/

 

Vinca Alkaloids and Their Biosynthesis Enzymes

Vinca alkaloids such as vincristine and vinblastine are crucial for the treatment of several types of cancers. However, these drugs are also in short supply, because they can only be found in small amounts in one plant species, the Madagascar periwinkle.

Unfortunately, the biosynthetic pathway that the Madagascar periwinkle uses to produce vincristine and vinblastine isn’t yet fully understood. However, if we find a way to manipulate that pathway, then we may be able to upregulate production of these molecules or transfer the pathway into another plant species that is easier to grow. Recently, a team of researchers identified the last two enzymes in the vinblastine biosynthesis pathway.

To begin, this team searched periwinkle RNA expression data for genes that are expressed at the same time as known vinblastine synthesis enzymes. Consequently, they found two candidate genes, which they named taberosine synthase (TS) and catharanthine synthase (CS). They first tried knocking out the genes’ expression in periwinkle plants. As a result, plants without TS and CS expression made much less precursors to vinblastine.

Next, they expressed TS and CS in the leaves of tobacco plants. Under these circumstances, the leaves formed detectable levels of vinblastine precursors.

Conceivably, these enzymes could be used to produce vinblastine and other medically important vinca alkaloids in other plant species.

 

 

Caputi L, Franke J, Farrow SC, et al. Missing enzymes in the biosynthesis of the anticancer drug vinblastine in Madagascar periwinkle. Science. 2018 Jun 15;360(6394):1235-1239. PMID: 29724909

Lopinavir Antiretroviral Delivery Using NLC

Lopinavir is an antiretroviral drug that is important for the treatment of HIV. Unfortunately, it has a low bioavailability because it dissolves poorly in water.

The drug might be absorbed better if it was encased in a nanostructured lipid carrier (NLC). An NLC is a particle containing a mixture of solid and liquid lipids- which should deliver drugs better than solid-only lipid particles. Recently, researchers in Malaysia sought to do a proof-of-concept of an NLC to deliver lopinavir.

To determine optimal conditions for creating NLCs, they varied homogenization time and amounts of solid lipid, liquid lipid, and surfactant. They picked the most promising sets of conditions for further study.

The researchers tested the NLCs for drug release rate in simulated gastric fluid and in simulated intestinal fluid. The NLCs released drug better than straight lopinavir, which would not dissolve in either fluid. Next, they applied the NLCs to a cell line that is popular for studying intestinal epithelium. NLC lopinavir entered the cells faster than straight lopinavir.

Finally, the researchers fed an NLC lopinavir suspension to rats. The rats reached higher blood lopinavir concentrations than rats fed straight lopinavir.

Lopinavir is an antiretroviral drug with poor bioavailability. Nanostructured lipid carriers show potential for improved drug delivery of lopinavir.

Khan AA, Mudassir J, Akhtar S, et al. Freeze-dried lopinavir-loaded nanostructured lipid carriers for enhanced cellular uptake and bioavailability: statistical optimization, in vitro and in vivo evaluations. Pharmaceutics. 2019; 11(2):97. PMID: 30823545

Baricitinib: Inhibitor of JAK-STAT Pathway

Baricitinib inhibits Janus kinases 1 and 2. These kinases are the first enzymes of the JAK-STAT signaling pathway. This pathway is important for many cell functions. Disorders of JAK-STAT can play a role in cancer and immune disease. For example, baricitinib, a JAK-STAT signaling disrupter, is approved in Europe to treat rheumatoid arthritis.

Progeria and JAK-STAT

Recently, researchers at the Technical University of Munich using a text mining approach had suspicions that a rare premature aging disorder, Hutchinson-Gilford progeria syndrome, is associated with the JAK-STAT pathway. It is known that this syndrome has four main symptoms that mimic normal aging: vascular disease, arthritis, lipodistrophy, and alopecia. In most cases of this disease, a single de novo mutation is responsible for causing the production of progerin, leading to a shortened life span. Unfortunately, sufferers rarely live past the age of thirteen.

The research team decided to run a literature meta-analysis on genes associated with each symptom of progeria. In all, seventeen genes implicated in all four symptoms were found, and of those seventeen, fourteen were part of the JAK-STAT pathway.

Baricitinib Inhibits Janus Kinases

Henceforth, to test the role of the JAK-STAT pathway in progeria experimentally, the team grew cells in culture from progeria patients and also from unaffected controls. The progeric cells expressed abnormal levels of the fourteen identified genes. When the researchers applied baricitinib to the cell culture, the gene expression levels normalized. After one month of baricitinib treatment, progeric cells were less likely to become senescent.

Furthermore, etoposide was also applied to the cell culture, which is known to worsen cell senescence. As anticipated, etoposide treatment disrupted expression of the fourteen genes.

In conclusion, this research shows that the JAK-STAT pathway is likely part of the Hutchinson-Gilford progeria disease mechanism, and baricitinib may have potential as a therapy.

 

Liu C, Arnold R, Henriques G, et al. Inhibition of JAK-STAT signaling with baricitinib reduces inflammation and improves cellular homeostasis in progeria cells. Cells. 2019 Oct 18;8(10):1276. doi: 10.3390/cells810276. PMID: 31635416.

Impact of 2-DG on Triple-Negative Breast Cancers

Triple-negative breast cancers present a difficult problem. Most existing therapies for breast cancer target hormone receptors on the cell surface to inhibit cell division. As such, breast cancers that lack these receptors, known as triple-negative cancers, are harder to treat. Physicians need more pharmaceutical options for treating these cancers.

A Possibility?

One promising molecule is 2-Deoxy-D-Glucose, which takes advantage of cancer cells’ altered metabolism. Some types of cancer cells depend on glycolysis as their source of energy, rather than oxidative phosphorylation, and so require more glucose than normal cells, which depend on oxidative phosphorylation for energy. Interestingly, 2-Deoxy-D-glucose is a toxic glucose analog that enters cells through normal glucose transporters. The toxicity should be more impactful to cancer cells than to normal cells because of the increased rate of uptake.

One Team’s Investigation

Recently, a team of researchers in Dublin tested 2-Deoxy-D-glucose’s effectiveness at treating breast cancer. They hypothesized that if 2-Deoxy-D-glucose worked as expected, then it should be more toxic to more aggressive, metabolically active cancers than less aggressive ones.

They tested two breast cancer cell lines, Hs578T, and its more aggressive variant, Hs578Ts(i)8. 2-Deoxy-D-glucose reduced the ability of the breast cancer cells to spread and migrate in vitro. The more aggressive cell line was more affected.

Normally when epithelial cells such as breast cancer travel through the blood or lymph system, they undergo apoptosis. Hence, cancer cells must have resistance to apoptosis in order to metastasize. The researchers tested the lines’ resistance to apoptosis. 2-Deoxy-D-glucose reduced the ability of the aggressive cell line to metastasize, but not the less aggressive one. Further evidence also suggests that the cancer stem cells present were directly affected by 2-Deoxy-D-Glucose treatment, in both cell lines.

Therefore, these results suggest that 2-Deoxy-D-glucose has the potential to treat triple-negative breast cancer, particularly the more aggressive variant in this study. Further studies may have a positive impact on developing treatments for the most aggressive and difficult to treat triple-negative breast cancers.

O’Neill S, Porter RK, McNamee N, et al. 2-Deoxy-D-Glucose inhibits aggressive triple-negative breast cancer cells by targeting glycolysis and the cancer stem cell phenotype. Sci Rep. 2019. 9:3788. doi: 10.1038/s41598-019-39789-9. PMID: 30846710.

 

 

Promising Mycobacteria Treatment: Bedaquiline

Bedaquiline, a potential treatment for several kinds of mycobacteria, was the first new drug to be approved for the treatment of tuberculosis in forty years back in 2012. This drug is especially important for the treatment of multidrug-resistant tuberculosis when first line treatments fail. It works by preventing the mycobacterium from making ATP, which is a different mechanism from older treatments.

Bedaquiline has attracted interest from public health researchers for the treatment of non-tuberculous mycobacteria. These bacteria infect lungs and wounds, especially in immunocompromised patients. In rich countries, non-tuberculous mycobacterial infections cause a greater health burden than tuberculosis itself does. These infections are difficult to treat because, as with tuberculosis, no effective drugs have been developed recently.

Researchers at the University of Texas Health Science Center have begun investigating the potential for developing a new treatment for this type of infection using bedaquiline. To begin, they first collected isolates of non-tuberculous mycobacteria from lungs and wounds of infected patients and grew them in nutrient broth. The isolates were then tested using broth microdilution antimicrobial susceptibility testing (AST). They calculated the minimum amount of bedaquiline they needed to add to the broth to inhibit bacterial growth, the minimum inhibitory concentration. They found that the required concentrations should be achievable in patients’ blood, so bedaquiline has potential to treat non-tuberculous mycobacterial infections.

In considering bedaquiline for the treatment of mycobacteria, researchers caution that bedaquiline should not be used as a monotherapy, because of the potential for bacteria to develop resistance.

 

Bedaquiline Fumarate

 

Brown-Elliot BA, Wallace RJ. 2018. In vitro susceptibility testing of bedaquiline against mycobacterium absceccus complex. Antimicrobial Agents and Chemotherapy. 29:63(2). doi: 10.1128/AAC.01919-18. PMID: 30509936

 

Now that you’ve read about it, find out how to pronounce it too!

 

Okadaic Acid for Alzheimer’s Research

New discoveries are one step closer with a new application using okadaic acid in Alzheimer’s research. Alzheimer’s disease is difficult to model in non-human animals. Recently, a collaboration between Indian and American researchers proposed a treatment combination that could produce rats with more biomarkers of Alzheimer’s disease than previous models.

Hypoxia increases the amount of misfolded amyloid beta aggregates in the brain, a well-known biomarker of Alzheimer’s disease. However, hypoxia does not cause tau tangles, which is another biomarker of Alzheimer’s disease. Okadaic acid is a toxin that is known for causing diarrhetic shellfish poisoning. It selectively inhibits phosphatases, so it is often used as a research chemical to study phosphorylation. As it turns out, in the brain it hyperphosphorylates tau, which causes it to tangle.

A combination of hypoxia and okadaic acid treatment therefore should produce rats with more biomarkers of Alzheimer’s disease than either treatment alone.

This collaborative research team microinjected okadaic acid into live rat brains, then placed the rats in a hypoxic chamber with 10% oxygen (atmosphere contains about 21%) for three days. The treated rats fared worse at an electrical shock avoidance test and a water maze memory test.

The team then sacrificed the rats and analyzed their brain biochemistry. As predicted, hypoxia increased the amount of amyloid beta aggregates in the rat brains, and okadaic acid increased the number of hyperphosphorylated tau tangles. Rats that received the combination treatment also had decreased acetylcholine esterase activity and increased oxidative damage, which are also biochemical hallmarks of Alzheimer’s disease.

This combination treatment of hypoxia plus okadaic acid in rats should be useful for further advancing the research into Alzheimer’s disease.

 

Okadaic Acid

Okadaic Acid Ammonium

Okadaic Acid Sodium

 

Kaushal A, Wani WY, Bal A, Gill KD, Kaur J. Okadaic acid and hypoxia induced dementia model of Alzheimer’s type in rats. 2019. Neurotoxicity Research.  PMID: 30729451

 

 

The Metabolic Impact of Kynurenic Acid

Background

Kynurenic acid is a metabolite of tryptophan. This chemical is found throughout the body, and appears to act on glutamate receptors in the brain. One effect of Kynurenic acid on Gpr35 signaling that has been discovered is enhanced cellular respiration.

Interestingly, high levels of kynurenic acid have been found in the brains of patients with schizophrenia. Further research is ongoing to understand its role in that disease. Less is known about its role in the rest of the body.

Kynurenic Acid Combined with Consistent Exercise and Feeding

One relationship that has been discovered is that exercise causes the levels of kynurenic acid to increase in skeletal muscle. Recently, Agudelo et al. sought to tease the effects of kynurenic acid on metabolism apart from the effects of exercise alone.

To accomplish this, mice were dosed with kynurenic acid without changing their exercise habits. Their metabolisms then sped up, but their level of exercise and feeding stayed the same. As a result, the mice lost some weight after two weeks, which came from a reduction in their white fat stores.

Kynurenic Acid Combined with Altered Diets and Genetics

Previous research has shown that kynurenic acid activates the receptor protein Gpr35. Agudelo et al. wanted to test whether Gpr35 was involved in the relationship between kynurenic acid and metabolism.

To test the effect of Kynurenic acid on Gpr35 signaling, they put mice on a high-fat diet that causes them to gain weight. When they dosed the mice on the high-fat diet with kynurenic acid, they prevented the mice from gaining weight.

When they dosed mice that lack Gpr35 with kynurenic acid, the mice gained weight as usual. These results suggest that Gpr35 signaling is important for kynurenic acid’s role in raising metabolism.

 

In conclusion, this study increases the understanding of both kynurenic acid and Gpr35. A therapeutic application for kynurenic acid is a long way off, but worth studying.

 

K977545  Kynurenic Acid

 

Agudelo LZ, Ferreia DMS, Cervenka I, et al. Kynurenic acid and Gpr35 regulate adipose tissue energy homeostasis and inflammation. 2018. Cell Metabolism. 27(2):378-392. PMID: 29414686.

Chemicals for Pharmaceutical Research for the Novel Coronavirus, COVID-19

The coronavirus known as COVID-19 first appeared in December, 2019 and spread around the world within a matter of days. As of this writing, it has infected nearly 400,000 people in 169 countries.

Scientists around the world have stopped their normal research to focus on creating a vaccine and treatment for this illness which has killed more than 17,000 people worldwide. Fortunately, more than 100,000 have recovered.

On March 22, 2020, an international team of scientists published a research paper preprint on biorXiv analyzing the interaction between the novel coronavirus and human proteins.* Their analysis predicts 332 protein-protein interactions and 69 existing pharmaceuticals that might perturb these interactions. The researchers propose that these drugs may be candidates for treating COVID-19 infections.

 LKT Laboratories has a number of these proposed chemicals for COVID-19 research in stock that can be shipped within 24 hours:

We also have various other antiviral compounds available:

See complete list of available antiviral compounds here

*A SARS-CoV-2-Human Protein-Protein Interaction Map Reveals Drug Targets and Potential Drug-Repurposing, David E. Gordon et al., bioRxiv 2020.03.22.002386; doi: https://doi.org/10.1101/2020.03.22.002386.

LKT Laboratories is a biochemical supply company focused on the synthesis, purification, and isolation of small molecules for research applications. All laboratory activities, including synthesis, natural product isolation, and full analytical characterization of our products are performed at our headquarters in St. Paul, MN.  Call or email us if you have questions about the chemicals above or have a need for other chemicals not listed.

Recombinant AMPs May Be More Effective Antibacterials

Background:

Antimicrobial peptides (AMPs) are short strings of amino acids, about 12-50 residues long, that show potent antibacterial, antiviral, and antifungal activity. They are found throughout the tree of life as part of the innate immune system. Many AMPs are in clinical use, such as vancomycin.

A class of AMPs called the cecropins were first isolated from the moth Hyalophora cecropia. Magainins were first isolated from the frog Xenopus laevis. An analog of magainin went through clinical trials as a treatment for infected diabetic foot ulcers, but failed.

Research Inspiration:

Researchers from the Jilin Agricultural University in China are interested in developing AMPs for livestock feed as an alternative to antibiotics. Previous research suggested that the fusion of peptides from two AMPs may enhance their antibacterial activity. Zhang et al. decided to test the properties of a magainin II – cecropin B fusion.

The group was also interested in testing whether the fungus Cordyceps militaris would make a suitable expression vehicle for fusion proteins. They constructed a plasmid that coded for the magainin II – cecropin B fusion and transformed cordyceps to express it. Then they extracted protein from the transformed fungus.

New Testing:

They tested the extract using ELISA, PAGE, and Western blotting and found that the cordyceps did express the fusion protein. Then, they swabbed the protein onto plates containing various species of bacteria to test whether it inhibited bacterial growth. Magainin II – cecropin B inhibited bacterial growth better than cecropin B alone.

The researchers also administered magainin II – cecropin B to mice infected with E. coli. The fusion inhibited E. coli growth in the mouse gut, and the mice suffered less intestinal damage. The same effect was observed when they fed the transformed cordyceps straight to the mice.

Zhang et al. conclude that magainin II – cecropin B is an effective antimicrobial. Cordyceps militaris that expresses magainin II – cecropin B could potentially be added to livestock feed.

 

C1609 Cecropin B
M0124 Magainin 1
M0126 Magainin 2

 

Zhang M, Shan Y, Gau H, Wang B, Liu X, Dong Y, Liu X, Yao N, Zhou Y, Li X, Li H. Expression of a recombinant hybrid antimicrobial peptide magainin II-cecropin B in the mycelium of the medicinal fungus Cordyceps militaris and its validation in mice. 2018. Microb Cell Fact. 17:18. PMID: 29402269.

Uncovering Markers for Parkinson’s Disease with 6-Hydroxydopamine

6-Hydroxydopamine is a dopamine analogue that selectively destroys dopaminergic neurons. It is used in biomedical research to destroy the substantia nigra in lab animals and induce Parkinson’s-like symptoms.

Choi et al. were interested in studying the early stages of Parkinson’s disease. Before the onset of motor symptoms, patients suffer from poor gastric emptying and constipation. Previous research suggests that Parkinson’s disease is also associated with changes in gut microbiome. Choi et al. sought to test whether Parkinson’s-like brain lesions directly caused changes in gut microbiome.

To test this, they injected 6-hydroxydopamine into mice. Two weeks later, they collected fecal samples from the mice, then extracted and sequenced 16S rRNA to identify microbial species.

Injected mice and control mice had about the same microbial species richness in their feces, but species composition changed. Injected mice contained fewer bacteria from the genus Lactobacillus and more from the genus Bacteroides. The researchers suggest that the Parkinson’s-lesioned brain might affect gut microbia through changes in the firing of the vagus nerve.

H966144 6-Hydroxydopamine Hydrobromide

 

Choi JG, Huh E, Kim N, Kim D-H, Oh MS (2019). High-throughput 16S rRNA gene sequencing reveals that 6-hydroxydopamine affects gut microbial environment. PLoS ONE 14(8): e0217194. https://doi.org/10.1371/journal.pone.0217194

HDAC Inhibitor LMK-235 Promotes Odontoblast Differentiation

Dental pulp, the living tissue in the core of teeth, contains stem cells. Dental pulp stem cells are of medical interest because they can differentiate into odontoblasts, osteoblasts, adipocytes, and chondrocytes, and because they can be obtained using minimally invasive methods.

The mechanism of dental pulp stem cell differentiation is not currently understood. Liu et al. suspect that epigenetic modifications controlled by histone deacetylaces (HDACs) play a role. Previous studies have shown that HDAC inhibition promotes stem cell differentiation into odontoblasts.

The molecule LMK-235 is an inhibitor of HDAC4 and HDAC5. Recently, Liu et al. investigated whether LMK-235 can promote dental pulp cell differentiation into odontoblasts. They cultured dental pulp cells from waste wisdom teeth and applied a series of doses of LMK to the culture fluid.

At high doses, LMK-235 inhibits cell proliferation. Liu et al. used a low dose, 100nM, for the rest of the experiment. At 100nM, LMK-235 increased the mRNA expression of genes associated with differentiation such as DSPP, ALP, and Runx2. A Western blot showed that LMK-235 increased the protein expression of DSPP and Runx2. The treated cells also showed more calcified nodules in culture.

Liu et al. acknowledge that their evidence in this study is indirect, because the cultured cells did not fully differentiate into odontoblasts. However, the results show that dental pulp cells hold promise for the field of regenerative medicine.

L493400 LMK-235

 

 

Liu Z, Chen T, Han Q, Chen M, You J, Fang F, Peng L, Wu B. HDAC inhibitor LMK-235 promotes the odontoblast differentiation of dental pulp cells. 2018. Molecular medicine reports. 17:1445-1452.

Dietary Phytochemicals Exhibit Multiple Mechanisms of Action Against Pathogenic Bacteria

Isothiocyanates are a family of biologically active phytochemicals found naturally in cruciferous plants. They have been investigated for their antioxidative, anti-inflammatory, and anticancer effects. They have also shown activity against neurodegenerative diseases. Recently, their properties as antibacterial agents were investigated by Nowicki, et al.

Mechanisms for two isothiocyanates, sulforaphane and phenethyl isothiocyanate, were studied in multiple strains of bacteria.  These strains included clinical isolates of E. coli, K. pneumoniae, S. aureus, S. epidermidis, and E. faecalis. In strains where growth inhibition by isothiocyanates is independent of (p)ppGpp production, the inhibition of nucleic acid synthesis and cell viability may be due to a drop in GTP levels. In strains which do not produce (p)ppGpp upon treatment such as B. subtilis, the isothiocyanates compromise the integrity of the cytoplasmic membrane. They concluded that isothiocyanates likely have more than one target in bacterial cells, and that the mechanisms of action may be species-specific. Although the mechanisms change the resulting inhibition of bacterial growth is the same across multiple strains.

LKT Labs offers a wide variety of isothiocyanate molecules for research use.

 

S8044 R,S-Sulforaphane

E6880 Erucin

I0416 Iberin

I74571-Isothiocyanato-6-(methylsulfinyl)-hexane

Full List of ITCs

 

References:

Nowicki D., Maciag-Dorszynska M. et al. Various modes of action of dietary phytochemicals, sulforaphane and phenethyl isothiocyanate, on pathogenic bacteria. Sci Rep. 9(1):13677 (2019). doi: 10.1038/s41598-019-50216-x.

LY-294002 Shows Promise in Cancer Therapy as an Inhibitor of PKM2 and the Warburg Effect

Most cancer cells get their energy through glycolysis instead of oxidative phosphorylation, even in the presence of oxygen. This process is named the Warburg effect after its discoverer, and it allows cells to proliferate even with damaged mitochondria. In 2008, researchers at Harvard Medical School discovered that one form of pyruvate kinase, PKM2, is required for the Warburg effect. The other form of pyruvate kinase is not involved. These findings make PKM2 an interesting target for developing cancer therapies.

The P13K/Akt/mTOR regulatory pathway is upstream of PKM2. The small molecule LY294002 targets P13K, so recently Lu et al. tested its effect on cancer cell proliferation.

Lu et al. grew several lines of gastric cancer cells in culture. A Western blot analysis found that PKM2 expression was increased in cancer cell lines compared to the control. They chose one cell line to study in detail. LY294002 decreased cell proliferation and increased apoptosis in this line. LY294002 also decreased the activity of lactate dehydrogenase, a biomarker of the Warburg effect.

They wanted to test whether PKM2 was part of the mechanism by which LY294002 decreased cell proliferation, so they used siRNA to knock down PKM2 on its own. siRNA-treated cells behaved similarly to LY294002-treated cells: they had lowered proliferation, increased apoptosis, and lowered lactate dehydrogenase activity. Lu et al. concluded that PKM2 is important to the mechanism of LY294002’s action.

LY294002 shows promise as an inhibitor of PKM2 and the Warburg effect.

L4796 LY-294002

L960002 LY-294002 Hydrochloride

 

 

 

Lu J, Chen M, Gao S, Yuan J, Zhu Z, Zou X. LY294002 inhibits the Warburg effect in gastric cancer cells by downregulating pyruvate kinase M2. 2018. Oncology letters. 15:4358-4364.

Discovering Differences in Human and Mouse STING using DMXAA

The xanthones are a diverse class of natural products that can be extracted from plants, fungi, lichen, and invertebrates. They have a wide range of biological activities, including antitumor and antimicrobial activity.

DMXAA, a synthetic derivative of xanthone, attracted particular interest in the 2000s because it disrupts tumor blood supply in mice. In 2008 it entered Phase III clinical trials for non-small cell lung cancer. It failed to show benefit in humans.

Recently, Shih et al. investigated the failure of DMXAA to translate across species. In mice, DMXAA activates the STING (stimulator-of-interferon-genes) protein, which leads to an immune response against tumors. DMXAA does not activate human STING.

The binding pockets of human and mouse STING are identical. So what causes the difference in DMXAA effectiveness? Shih et al. used molecular dynamics, a computational method that simulates the movements of molecules. When they simulated the binding of DMXAA to human STING and mouse STING, they found that the lid of mouse STING remains closed, preventing bound DMXAA from leaving. In human STING, the DMXAA leaves. A peptide residue in the STING lid region is responsible for the difference.

This work may aid in the rational design of a STING activator, but the researchers conclude that the design of such a drug would be challenging.

D509921 DMXAA

STING Activators

Shih AY, Damm-Ganamet KL, Mirzadegan T. Dynamic Structural Differences between Human and Mouse STING Lead to Differing Sensitivity to DMXAA. 2018. Biophysical Journal. DOI: 114:32-39.

MELK Inhibitor OTSSP167 Reduces Multiple Myeloma Bone Disease

MELK (maternal embryonic leucine zipper kinase) signals cell division and may play a role in several kinds of cancer. In 2012, researchers screened for inhibitors of MELK and found a substance that they named OTSSP167. OTSSP167 shows promise as a chemotherapy drug.

Bone disease is a top cause of morbidity for multiple myeloma patients. Bone lesions cause bone pain, bone breakdown, and encourage further tumor growth. Even when chemotheraphy causes tumor remission, the bone lesions don’t heal. Bisphosphonates, the current standard of care, come with serious side effects.

Researchers Muller and Bolomsky investigated OTSSP167’s potential to treat multiple myeloma. In 2018, Bolomsky et al. found that OTSSP167 reduced tumor load in a multiple myeloma mouse model. As predicted, it worked by inhibiting MELK. The researchers also found that OTSSP167 reduced multiple myeloma bone disease, even at doses that were too low to inhibit tumor growth. They followed up on their findings in another 2018 paper.

In the follow-up, Muller et al. dosed bone cell cultures and multiple myeloma mice with OTSSP167. They found that OTSSP167 inhibits the growth and function of osteoclasts, cells that reabsorb bone, and stimulates osteoblasts, cells that produce bone. The mice dosed with OTSSP167 had fewer bone lesions and better bone volume than controls.

Muller et al. conclude that OTSSP167 has the potential to treat multiple myeloma tumors and to reduce bone lesions.

O783743 OTSSP167 Hydrochloride

Chung S, Suzuki H, Miyamoto T, et al. Development of an orally-administrative MELK-targeting inhibitor that suppresses the growth of various types of human cancer. Oncotarget. 2012 3(12):1629–1640. doi:10.18632/oncotarget.790

Muller J, Bolomsky A, Dubois S, Duray E, Stangelberger K, Plougonven E, Lejeune M, Léonard A, Marty C, Hempel U, Baron F, Beguin Y, Cohen-Solal M, Ludwig H, Heusschen R, Caers J. Maternal embryonic leucine zipper kinase inhibitor OTSSP167 has preclinical activity in multiple myeloma bone disease. Haematologica. 2018 Vol 103(8):1359-1368.

Bolomsky A, Heusschen R, Schlangen K, et al. Maternal embryonic leucine zipper kinase is a novel target for proliferation- associated high-risk myeloma. Haematologica. 2018 103(2):325-335.

Dynasore Shows Potential as a Treatment for Ocular Surface Disease

Rose bengal and fluorescein can stain damaged conjunctival and corneal tissue in the eye. They play an important role in ophthalmology to diagnose dry eye and eye surface damage. The mechanism these stains use to stain damaged cells and not healthy ones is not understood.

Dynasore is a small molecule that inhibits dynamin. Dynamin is essential for endocytosis in eukaryotic cells, so dynasore inhibits endocytosis. Dynasore is used as a cell biology research tool.

Recently, Webster et al. proposed a mechanism for how rose bengal and fluorescein work. Sublethal cell damage causes the cell to repair itself by remodeling its plasma membrane. While remodeling the plasma membrane, the cell might take up stain via endocytosis. They tested their hypothesis by applying oxidative stress to monolayer cultures of human corneal epithelial cells. The oxidative stress caused an increase in both stain uptake and endocytosis. Then they applied the endocytosis inhibitors chlorpromazine hydrochloride, genistein, Dynasore hydrate, and Dyngo-4a (a Dynasore derivative). Stain uptake was blocked.

However, when they repeated the experiment in whole eye tissue harvested from mice, oxidative stress increased stain uptake, but did not increase endocytosis. Most of the endocytosis inhibitors did not block stain uptake. Dynasore and Dyngo-4a did.

If eye cell damage increased stain uptake but not endocytosis, endocytosis must not be the mechanism for cell uptake. Then why did two of the endocytosis inhibitors, Dynasore and its derivative, inhibit stain uptake if endocytosis was not involved?

Webster et al. performed cytotoxicity and Western blotting assays and concluded that Dynasore and Dyngo-4a prevented oxidative damage to the cells. The results have implications for the field of ophthalmology. Dynasore and Dyngo-4a may have therapeutic potential.

 

Webster A, Chintala SK, Kim J, Ngan M, Itakura T, Panjwani N, Argueso P, Barr JT, Jeong S, Fini ME. Dynasore prtects the ocular surface against damaging oxidative stress. PLoS ONE. 2018. 13(10):e0204288. https://doi.org/10.1371/journal.pone.0204288.

Differentiating Embryonic Stem Cells with Dorsomorphin

In 2008, Yu et al. identified a compound that dorsalizes structures in growing zebrafish embryos, which they named dorsomorphin. This compound inhibits the bone morphogenetic protein (BMP) signaling pathway, which plays an important role in development and cell differentiation.

Recently, Valizadeh-Arshad et al. hypothesized that small molecules like dorsomorphin could be used to promote human embryonic stem cells to differentiate into motor neurons. One of the goals of regenerative medicine is to produce tissues such as neurons from stem cells, to heal nerve damage that will not heal on its own. Currently, coaxing embryonic stem cells to differentiate into motor neurons is expensive and inefficient.

The researchers modified an existing motor neuron differentiation protocol to include a cocktail of small molecules that have shown promise in previous studies: all-trans retinoic acid, dorsomorphin, A8301, and XAV939. They cultured the embryonic stem cells on plates, and exposed the cells to the small molecules as well as established differentiation factors.

They then used flow cytometry and immunofluorescence staining on the cells to test for expression of genes associated with neurons, such as NESTIN, PAX6, and MAP2. The cells expressed these marker genes with efficiencies greater than 50%. The cells showed voltage-gated currents in a patch-clamp experiment, suggesting the presence of both K+ and Ca++/Na+ channels. However, the cells were unable to generate more than single action potentials. More research needs to be done before this cell differentiation method can produce mature motor neurons.

 

D582703 Dorsomorphin

D582705 Dorsomorphin Dihydrochloride

R1877 all-trans-retinol, ≥98%

 

 

Valizadeh-Arshad Z, Shahbazi E, Hashemizadeh S, Moradmand A, Jangkhah M, Kiani S. In Vitro Differentiation of Neural-Like Cells from Human Embryonic Stem Cells by A Combination of Dorsomorphin, XAV939, and A8301. Cell J. 2018 19:4. doi: 10.1038/nbt1201-1129.

Yu PB, Hong CC, Sachidanandan C, et al. Dorsomorphin inhibits BMP signals required for embryogenesis and iron metabolism. Nat Chem Biol. 2007;4(1):33–41. doi:10.1038/nchembio.2007.54.