drug discovery – Page 2 – Drug Discovery Opinion

LRRK2 Inhibitor Protective in Parkinson’s Disease Model

"Moondance" photographed by Tena (Madmoiselle Lavender), a Parkinson's veteran

The main symptoms of Parkinson’s disease are tremor, rigidity and involuntary movement, caused by loss of dopaminergic neurons in the brain. Leucine-rich repeat protein kinase-2 (LRRK2) is mutated in a significant number of Parkinson’s disease cases, both familial and sporadic late-onset. A common mutation in which a glycine residue in the active site is altered to serine enhances catalytic activity of the kinase, suggesting that LRRK2 inhibitors might be useful for the treatment of Parkinson’s disease, although it is not entirely clear why enhanced LRRK2 activity causes loss of dopamine-producing neurons. Scientists led by a team at the Johns Hopkins University School of Medicine have now shown that inhibitors of the G2019S variant of LRRK2 can protect the nerve cells of mice genetically modified to produce the mutated kinase. Three weeks twice daily injections of GW5074 provided almost complete protection against loss of dopaminergic neurons compared with placebo treatment.

Although GW5074 is not an especially potent inhibitor of wild type or G2019S LRRK2 (IC₅₀ 0.2µM – 1.0µM depending on substrate) and is not selective (IC₅₀ vs cRaf ca 10nM), the study, which is published in Nature Medicine, provides encouragement that a more potent and selective inhibitor could lead to a new disease-modifying treatment for Parkinson’s disease. The John Hopkins team are collaborating with researchers at Southern Methodist University to design more selective inhibitors and many other groups in both industry and academia are engaged in the search for potent and selective LRRK2 inhibitors.

Converting Pancreatic α-Cells to β-Like-Cells

alpha beta There are four main cell types in the islets of Langerhans in the pancreas; α-cells which secrete glucagon, β-cells which secrete insulin, δ-cells which secrete somatostatin, and PP cells which secrete pancreatic polypeptide. Type I diabetes is an autoimmune disease in which the insulin-producing β-cells are destroyed and could potentially be treated by the creation of new β-cells, either from stem or stem-like cells or by conversion of another mature cell type. It has recently been shown that the transcription factor, Pax4, induces transdifferentiation of pancreatic α-cells into β-cells in adult mice and a team led by researchers at the Broad Institute of Harvard and MIT has now shown that a similar effect can be achieved with a small molecule.

Using a mouse α-cell line, the team screened over 30,000 compounds and found that one of them, BRD7389, induced insulin expression after 3 days treatment. Induction of insulin gene expression in α-cells peaked at 5 days (ca 50-fold at a BRD7389 concentration of 0.85µM) and the cells adopted a β-cell-like morphology. Insulin protein levels were also increased from a basal α-cell state, although were much lower than the levels produced by mature β-cells. BRD7389 also increased insulin secretion in primary human islet cells although since there are many different cells types in the human tissue samples, it is also not possible to attribute the increase in insulin levels exclusively to conversion of α-cells to β-like-cells.

Follow-up studies suggested that upregulation of insulin expression potentially involved inhibition of multiple members of the RSK family of protein kinases, but more experiments are needed to fully elucidate the mechanism of action of BRD7389. The study demonstrates, however, that a small molecule can induce insulin expression in α-cells and suggests that such a strategy could be used to increase β-cell mass by transdifferentiation in vivo. The team now want to identify other small molecules that could be used to enhance the effects of BRD7389, and boost insulin production in people with type I diabetes.

The study is published in PNAS.

Potential New Drugs for Epilepsy

Lightning — Image: Flickr - Stephan Sachs

Over 30% of the 50 million people who are affected by epilepsy do not have their seizures adequately controlled, even with the best available medicines. The 29 amino acid neuropeptide, galanin, is a potent endogenous anticonvulsant that activates galanin receptors type 1 (GalR1) and type 2 (GalR2) and a number of groups, including researchers at the Scripps Research Institute, have been trying to develop drugs that mimic the effects of galanin as novel anticonvulsants. Galnon is an example of a systemically active nonpeptide galanin receptor ligand with affinity for the three galanin receptors which has been shown to reduce seizures in animal models. The Scripps team have now identified a compound that acts appears to act as a selective positive allosteric modulator of the galanin receptor type 2 (GalR2) which they hope will have a reduced potential for side effects compared with galnon. The compound, CYM2503, potentiated the effects of galanin in cells stably expressing the GalR2 receptor, but had no detectable affinity for the galanin binding site. In rodent models of epilepsy, intraperitoneal administration of CYM2503 increased the time to seizure, reduced the duration of seizures, and increased survival rate at 24 h.

The study is published in the Proceedings of the National Academy of Sciences.

PPARγ– A New Twist in the Tale

Obesity and related disorders such as diabetes have reached epidemic proportions. Although the anti-diabetic thiazolidinediones (glitazones) are effective insulin sensitizers, some members of the class have been withdrawn or had their use restricted because of safety concerns. Increased responsiveness to insulin is believed to be mediated by activation of the nuclear receptor, PPARγ but differences in clinically important side effects suggest subtle differences in pharmacology, even amongst full agonists.

Researchers at the Scripps Research Institute and the Dana-Farber Cancer Institute at Harvard University have now shown that cyclin-dependent kinase 5 (Cdk5) in adipose tissue is activated in obese mice fed a high-fat diet, resulting in phosphorylation of PPARγ. This has no effect on the adipogenic capacity of PPARγ but does alter the expression of a large number of obesity-related genes, including a reduction in expression of the insulin-sensitizing adipokine, adiponectin. Phosphorylation of PPARγ by Cdk5 was blocked both in vitro and in vivo by the full agonist, rosiglitazone, and by the partial agonist, MRL-24, leading to increased adiponectin production. The anti-diabetic effect of rosiglitazone in obese patients was also found to be closely associated with inhibition of PPARγ phosphorylation, suggesting that this may be a mechanism of insulin resistance. The authors of the study, which is published in the journal Nature, suggest that drugs that inhibit PPARγ phosphorylation by Cdk5, without necessarily activating the receptor, may provide an improved generation of anti-diabetic drugs.

Study Finds Compounds that Boost Neurogenesis: Possible moa for Dimebon

Following from positive phase II results, the announcement earlier this year that Dimebon (latrepirdine) failed to show a significant effect in a phase III clinical trial in Alzheimer’s patients was a major blow to patients, families and doctors. A study by researchers at UT Southwestern Medical Center has now shown that Dimebon can increase neurogenesis in adult rodent brains and have identified other, more potent compounds.

An in vivo screen of 1000 small molecules in adult mice identified eight compounds that were able to enhance neuron formation in the subgranular zone of the hippocampal dentate gyrus. One of the compounds, P7C3, was selected for further study on the basis of favourable ADME predictions. Daily administration of P7C3 to aged rats for 7 days was shown to enhance hippocampal neurogenesis relative to control animals and, after 2 months, treated rats performed significantly better in the Morris water maze test which provides a measure of learning and memory.
Dimebon and P7C3 structures
P7C3 exerts its proneurogenic effects by protecting newborn neurons from apoptosis and the team next compared the activity of P7C3 with that of Dimebon, which is also believed to have anti-apoptotic activity. Dimebon was found to be proneurogenic in vivo, albeit at levels 10-30 times higher than P7C3, raising the possibility that the two compounds may share a common mechanistic pathway. Although this idea can only be rigorously tested after identification of the molecular target(s), the study raises the hope that more potent analogues of Dimebon with improved clinical efficacy could be identified and also provides appropriate assays.

The study is published in the journal Cell.

Breathe Easy

Researchers from the Wyss Institute for Biologically Inspired Engineering at Harvard University, Harvard Medical School and Children’s Hospital Boston have created a lung-on-a-chip which may help to speed pharmaceutical development by reducing reliance on animal models. The device consists of a layer of human alveolar cells separated from a layer of human endothelial cells by a porous membrane and mimics the boundary between the lung’s air sacs and capillaries. The cells on the chip can also be made to ‘breathe’ by cyclically applying a vacuum to increase the width of the membrane and stretch the cells before allowing them to contract again.

The, lung-on-a-chip has the potential to model the effects of environmental toxins, the inflammatory response to inhaled pathogens and the effectiveness of new drugs. Because the chip is transparent, responses can be captured in real time using high-resolution fluorescence microscopy. When E.Coli bacteria were introduced into the air on the ‘lung’ side of the chip, white blood cells on the ‘blood’ side of the chip migrated through the porous membrane into the air chamber to destroy the bacteria. ‘Breathing’ was found to enhance absorption of nanoparticles, some of which induced an inflammatory response and overproduction of free radicals by the lung cells.

The team are now exploring whether the system can mimic gas exchange between alveolar cells and the bloodstream and believe that the device provides proof-of-principle for the concept that organs-on-chips could replace many animal studies in the future.

The study is published in the journal Science.

Oestrogen – Better Out than In for Cardiovascular Health

Although oestrogen replacement lowers cardiovascular risk in post-menopausal women, treatment is associated with an increased risk of uterine and breast cancer.

The increased cancer risk is linked to oestrogen’s action at nuclear receptors but researchers at UT Southwestern Medical Center have now found that a subpopulation of oestrogen receptors outside the cell nucleus mediate the beneficial cardiovascular effects. The extra-nuclear receptors in endothelial cells are important for blood vessel maintenance and repair and also regulate production of nitric oxide which has a number of beneficial cardiovascular effects. The team have found that an oestrogen-macromolecule complex which is excluded from the nucleus is highly effective in stimulating the extra-nuclear receptors. Similar dendrimer conjugates have been successfully used as drug delivery device in animal models and the oestrogen complex was shown to provide cardiovascular protection in high cholesterol ovariectomized female mice without stimulating growth of breast or uterine cancer. The team believe that such oestrogen-macromolecule complexes could provide cardiovascular protection for both men and women and are creating molecules that may be suitable for use in humans.

The study is published in the Journal of Clinical Investigation.

For cells, too, 2-D is not the same as 3-D

Cell culture experiments to screen for compounds that can inhibit cell migration – and potentially metastasis of cancer cells – are typically carried out in a 2-D environment, but researchers at Johns Hopkins University and the University of Washington suggest that results from such experiments may be, at best, misleading. The team has shown that the way in which cells move in a 3-D environment, such as the human body, is different both qualitatively and quantitatively from the way they move in a 2-D environment such as a culture dish. When cells are grown in 2-D, they develop broad fan-shaped protrusions called lamella along their leading edge which help them to move forward. Macromolecular assemblies known as focal adhesions which can last for up to several minutes are also formed. These focal adhesions mediate cell signalling, force transduction and adhesion. In 3-D, the cells take on a more spindle-like appearance, with two pointed protrusions at opposite ends and focal adhesions – if they form at all – are so small and short-lived that they cannot be resolved by microscopy. The authors suggest that the shape and movement of cells in 2-D culture experiments are artifacts of the environment and could produce misleading results in studies to test the effects of drugs on cell motility. This may explain why positive results from cell culture experiments do not always translate into efficacy in animal models.

Even in cell culture systems designed to more closely mimic a 3-D environment, the cells may be only partially embedded in a matrix and produce misleading results. Using live-cell microscopy, the team showed that, when cells are fully embedded in a 3-D matrix, focal adhesion proteins do not form aggregates, but are distributed throughout the cytoplasm. The focal adhesion proteins still modulate cell motility, but not in the same way as in a 2-D environment. Because loss of adhesion and increased motility are hallmarks of cancer cells, it is important to understand cell motility under physiological conditions and to use culture techniques that most closely mimic this.

The study is published in Nature Cell Biology.

NSAIDs and Cancer – Another Piece of the Puzzle

Regular use of NSAIDS has been linked to reduced incidence of certain types of cancer but the underlying protective mechanisms are unclear. Some of the anticancer effects are believed to be mediated through inhibition of COX-2, but a study led by investigators at Sanford-Burnham Medical Research Institute has now identified another mechanism by which the sulindac sulfide (the NSAID metabolite of sulindac) inhibits tumour growth. The team found that sulindac sulfide induces apoptosis by binding to retinoid X receptor-α (RXRα), a member of the nuclear hormone receptor family which had been already been identified as a potential target for cancer therapy. In cancer cells, levels of RXRα are often reduced, at least in part because of proteolytic processing to a truncated form, tRXRα. As with other nuclear receptors, RXRα regulates transcription of target genes by binding to DNA response elements but accumulating evidence suggests that RXRα may also have extranuclear activity. Both RXRα and tRXRα can exist in the cytoplasm and the study showed that cytoplasmic tRXRα can activate the PI3K/AKT survival pathway by interaction with the p85a subunit of PI3K, leading to anchorage-independent cell growth in vitro, and tumour growth in animals. Sulindac sulfide was found to inhibit the tRXRα-mediated PI3K/AKT activation, suggesting that the compound could provide a useful lead for anti-cancer drugs targeting this pathway.

The use of NSAIDs to reduce the incidence of cancer has been limited by the risk of major cardiovascular events and the Sanford-Burnham have identified an analogue of sulindac sulfide, K-80003 which has improved affinity for RXRα but lacks significant COX-2 inhibitory activity. K-80003 inhibited the growth of cancer cells in vitro and in animals and would be expected to have reduced COX-2-associated side effects.

The study is published in the journal Cancer Cell.

Targeting Influenza A Nucleoprotein

Although the recent sporadic outbreaks of influenza A virus H5N1 and of a new variant of H1N1 in 2009 were less serious than initially feared, public health responses gave an indication of the potential for pandemic influenza A to wreak havoc amongst human populations. Timely development of vaccines should help to contain future outbreaks, but effective antiviral medicines will also be needed. Circulating strains of influenza A virus with resistance to existing neuraminidase inhibitors have already been discovered, and new molecular targets would provide additional protection in the event of a fresh outbreak.

Researchers led by a team at the University of Hong Kong have now identified a compound, nucleozin, which can aggregate the viral nucleoprotein and prevent its transport into the nucleus. The nucleoprotein plays critical roles in viral RNA replication and genome assembly, and nucleozin was shown to block replication of H1N1, H3N2, and H5N1 viruses in cell culture experiments and also to protect mice from lethal challenge with highly pathogenic avian influenza virus A H5N1.

The study, which is published in Nature Biotechnology, shows that the nucleoprotein is a viable drug target and could lead to the development of new treatments to control the impact of future influenza A outbreaks. Potential binding sites for nucleozin on the influenza nucleoprotein were also predicted using molecular docking models.