small molecule – Drug Discovery Opinion

The BMK1 Pathway in Oncology – a Road Less Travelled

Of the four mammalian MAP kinase pathways (ERK1/2, JNK, p38 and BMK1), BMK1 is the least studied. BMK1 and ERK1/2 pathways are both activated by mitogens and oncogenic signals and are therefore implicated in tumorigenesis. Indeed, the ERK1/2 pathway has received significant attention for the development of chemotherapeutic drugs. Deregulated BMK1 activity has been associated with a variety of human malignancies including chemoresistance of breast tumours, metastasis of prostate tumour cells and tumour-associated angiogenesis. Conditional knockout of endothelial BMK1 in mice, however, led to lethal vascular instability, discouraging exploration of BMK1 as a therapeutic target.

A new study from scientists at the Scripps Research Institute has revealed more detail on the role of BMK1 in oncogenesis and suggests that BMK1 inhibition could be a viable therapeutic strategy. The study found that BMK1 is associated with the tumour suppressor, PML (promyelocytic leukemia protein), and suppresses its anti-cancer activity. In cellular studies, reduced expression of BMK1 resulted in induced expression of p21, a downstream effector of PML and modulator of cell proliferation.

The team’s serendipitous discovery of a selective inhibitor of BMK1, XMD8-92, permitted further studies in animal models. XMD8-92 significantly inhibited the growth of xenografted human tumours in mice, with no obvious adverse effects. More specifically, in contrast to the BMK1 conditional knockout studies, no vascular instability was observed in response to pharmacological inhibition of BMK1.

The study is published in Cancer Cell.

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.

First Small Molecules Active Against Ricin, Shiga Toxin

Castor Oil Seeds — Seeds of the castor oil plant, *Ricinus communis*

Ricin, one of the world’s deadliest toxins, was infamously used to assassinate the Bulgarian dissident writer Georgi Markov in 1978 near Waterloo Bridge in London. Markov died a few days after a ricin-filled pellet was fired into his leg using a modified umbrella. It has been estimated that as little as 500 micrograms of ricin could be fatal in humans if delivered by injection. Since ricin is easily extracted from the seeds of castor oil plants which are widely grown as an ornamental species and commercial crop, it has obvious potential for bioterrorist attacks. Until now, there has been no antidote to ricin poisoning, but French researchers have now described the first small molecules that are able to protect mice against its lethal effects.

Ricin and bacterial Shiga-like toxins exert their lethal effects by blocking protein synthesis. The proteins comprise two subunits, the A chain and the B-chain, which are linked by a disulphide bond. The B-chain facilitates cell entry and intracellular transport, and is reductively cleaved to free the A-chain which inactivates ribosomes and shuts down protein synthesis. To reach their cytosolic target, ribosomal RNA, the toxins follow the retrograde transport route from the plasma membrane to the endoplasmic reticulum, via endosomes and the Golgi apparatus.

Using a cell-based screen of over 16,000 compounds, the French team found two compounds that were able to block the transport of the toxins within the cell whilst showing very little cytotoxicity. The two compounds, Retro-1 and Retro-2, were shown to selectively block the intracellular transport of the toxins between early endosomes and the Golgi apparatus. Unlike other compounds that are known to block retrograde transport, Retro-1 and Retro-2 do not affect other intracellular trafficking and do not show any toxicity. Retro-2 was found to fully protect mice against a lethal dose of ricin, but only if administered before the ricin. Since the compounds act on host cell pathways rather than on the toxin itself, they should also defend against the Shiga-like toxins produced by pathogens such as E. coli, Shigella and Cholera.

The study is published in the journal Cell.

Closantel May be Useful in Fighting River Blindness

Adult black fly with Onchocerca volvulus emerging from the insect’s antenna — Adult black fly with *Onchocerca volvulus* emerging from the insect’s antenna Photo: United States Department of Agriculture

Onchoceriasis – also known as river blindness – is the world’s second leading infectious cause of blindness. The disease is caused by the nematode, Onchocerca volvulus, and is transmitted to humans through the bite of a blackfly. Once inside the body, the female worm produces thousands of larval worms (microfilariae) which migrate to the skin and eyes. When the microfilariae die, they cause intense itching and a strong immune response that can destroy nearby tissue, leading eventually to blindness and disfiguring skin lesions. Control programmes have involved the use of larvicides to reduce blackfly populations and the use of ivermectin to treat infected people and limit the spread of disease. Ivermectin is most effective against the larval stage of the worm and is believed to kill the parasites by activating glutamate-gated chloride channels which are specific to invertebrates.

A team led by researchers at the Scripps Institute has now focused on a new way to kill the parasite. The protective outer cuticle of the worms is made of chitin and two classes of enzymes – chitin synthases and chitinases – are known to be critical for chitin formation and remodelling. One chitinase, OvCHT1, is expressed only in the infective third-stage larvae and is believed to be involved in development and host transmission. The team screened a small library of compounds for activity against OvCHT1 and found that closantel was able to inhibit the enzyme. When closantel was tested on cultured third-stage larvae, the compound prevented the larvae from moulting and developing into adult worms. Since the mechanism of action of closantel is completely different to that of ivermectin, it – or other chitinase inhibitors – could potentially be used to treat ivermectin-resistant worms. Closantel is a broad-spectrum anti-parasitic agent currently used in some countries in veterinary medicine.

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

Serotonin Synthesis Inhibitor Could Treat Osteoporosis

In 2008, researchers led by a team at Columbia University showed that, by turning on or off production of serotonin in the gut, they could control bone formation. Serotonin signals to cells in the skeleton to slow production of new bone and, by turning off the intestine’s release of serotonin, the team was able to prevent osteoporosis in mice undergoing menopause. The team have now shown that daily oral administration of LP-533401 for 6 weeks is effective both prophylactically and therapeutically against osteoporosis in ovariectomized mice.

LP-533401 inhibits tryptophan hydroxylase-1 (TPH-1), the first enzyme in gut-derived serotonin biosynthesis. TPH-1 is mostly expressed in peripheral tissues such as the gut, whilst TPH-2 is the major isoform in the central nervous system. Although LP-533401 inhibits human TPH-1 and TPH-2 with similar potency (K_i ~ 0.7µM) in vitro, it selectively lowers serotonin levels in the gut whilst leaving levels in the brain unchanged, likely because the compound does not cross the blood-brain barrier. LP-533401 and an ethyl ester pro-drug were originally developed to treat gastrointestinal diseases such as irritable bowel syndrome and to reduce chemotherapy-induced vomiting and nausea.

Although much work will need to be done before trials can be carried out in patients, the present study, which is published in Nature Medicine, demonstrates that pharmacological inhibition of synthesis of gut-derived serotonin could become a new anabolic treatment for osteoporosis. Most osteoporosis drugs only prevent the breakdown of old bone and are not able to stimulate the growth of new bone.

New ‘Broad-Spectrum’ Antiviral

Bowling strike — Image: Flickr – Russ J Taylor

Although a variety of broad-spectrum antibiotics have been developed, broad-spectrum antiviral agents have proved more difficult to identify. Effective treatments have been developed for individual viruses such as HIV, herpes viruses and influenza viruses – and vaccines have also been developed against papilloma viruses and herpes viruses – but there is a need for small molecules that are able to treat a range of viral infections and could also be used against newly emerging viruses.

Researchers led by a team at UCLA have now identified a compound, LJ-001, that can treat a range of enveloped viruses. The team screened a library of around 30,000 compounds against Nipah virus, a pathogen that was first identified in 1998 and causes severe disease in both animals and humans. Further tests showed that LJ-001 was also effective against other enveloped viruses including Ebola virus, HIV, hepatitis C virus, West Nile virus, Rift Valley fever virus, yellow fever virus and influenza A virus, but had no effect against non-enveloped viruses. The compound interacts with the viral lipid envelope and inhibits viral entry at a step after virus binding but before virus–cell fusion.

Although LJ-001 also binds to cellular membranes, the team believe that its low toxicity can be attributed to the fact that metabolically active cells are able to repair their membranes whilst static viruses are not. LJ-001showed no overt toxicity at effective anti-viral concentrations in either in vitro or in vivo studies, and pretreatment of mice with LJ-001 prevented virus-induced mortality from Ebola and Rift Valley fever viruses.

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

Neuroprotective Small Molecule TrkB Agonist

Roles have been suggested for brain-derived neurotrophic factor (BDNF) – which helps to support neurons and also stimulates and controls neurogenesis – in preventing or treating degenerative diseases such amyotrophic lateral sclerosis, Parkinson’s disease, and Alzheimer’s disease. The use of BDNF itself in therapy is limited by a poor pharmokinetic profile including rapid metabolism and poor CNS penetration. BDNF elicits at least some of its effects through binding to the high affinity tyrosine kinase receptor B, TrkB, and investigators at Emory University School of Medicine have now identified a small, high-affinity molecule that can also activate signalling through TrkB.

7,8-dihydroxy flavone structure — 7,8-dihydroxyflavone

7,8-Dihydroxyflavone was shown to protect wild-type, but not TrkB-deficient, neurons from apoptosis. Following intraperitoneal administration, the compound was also found to activate TrkB in the brain and to be protective in animal models of seizure, stroke and Parkinson’s disease. The compound was also found to have low toxicity on chronic dosing. Although favonoids such as 7,8-dihydroxyflavone occur in a wide range of foodstuffs, levels obtained from a normal diet are believed to be insufficient for a sustained effect.

The study is published in the online early edition of PNAS.

Correct Protein Folding

Neurons are particularly sensitive to the toxic effects of misfolded proteins and the accumulation of such species has been associated with neurodegenerative diseases including Parkinson’s disease, amyotropic lateral sclerosis (ALS), Alzheimer’s disease and transmissible spongiform encephalopathies (prion diseases). Hereditary protein conformational disorders such as Huntington’s disease are characterised by trinucleotide repeats that result in the insertion of poly-glutamine (polyQ) stretches which adopt β-sheet structures and make the protein prone to incorrect folding and aggregation. The ability to stabilise native protein conformations would likely prevent the neurotoxicity linked to misfolding and scientists at Duke University Medical Center have now discovered compounds that may be able to achieve this.

Since increasing the levels of protein chaperones has been shown to suppress protein misfolding, the team focussed on identifying small molecule activators of heat shock transcription factor 1 (HSF1), the master regulator of protein chaperone gene transcription. HSF1A was discovered using a humanised yeast-based high throughput screen and shown to activate HSF1 in mammalian and fruit fly cells, to elevate protein chaperone expression, and to reduce protein misfolding. HSF1A was also shown to prevent cell death in polyQ-expressing neuronal precursor cells and to protect against cytotoxicity in a fruit fly model of polyQ-mediated neurodegeneration.

Previous screens that have identified activators of HSF1 have not been able to discriminate against compounds that promote HSF1 activation through the proteotoxic accumulation of unfolded proteins or through the inhibition of Hsp90, a central chaperone involved in cell growth, signalling, and proliferation. HSF1A is structurally distinct from other small molecule activators of HSF1 and, although the precise mechanism by which the compound activates human HSF1 is not yet understood, it could lead to new therapies for neurodegenerative diseases caused by protein misfolding.

The study is published in PLoS Biology.

How Small Molecule Restores Aldehyde Dehydrogenase Function

Human Aldehyde Dehydrogenase, apo form (PDB ID = 1O05)

The lower alcohol tolerance of some Asian groups compared with people of European descent is caused, in part, by a mutant copy of the aldehyde dehydrogenase gene, ALDH2. As well as carrying out the second step in the oxidative metabolism of alcohol, the conversion of acetaldehyde to acetic acid, ALDH2 metabolises toxic species created by lack of oxygen in the wake of a heart attack and is involved in the metabolism of nitroglycerine which is used to treat angina. People with a deficiency in the activity of ALDH2 are at increased risk of cardiovascular damage and scientists at Indiana University and Stanford University reported in 2008 that a small molecule activator of ALDH2, Alda-1, could reduce infarct size in rats if administered before ischaemic damage. In vitro, Alda-1 was found to be a particularly effective activator of the inactive form of the enzyme found in some East Asian populations, suggesting that treatment with Alda-1 could be of benefit to individuals with either wild-type or mutant ALDH2 who are subjected to cardiac ischaemia by a heart attack or by procedures such as coronary bypass surgery.

Writing in Nature Structural & Molecular Biology, the team have now described mechanistic details of the activation of ALDH2 by Alda-1, a discovery which should lead to more potent and selective analogues of Alda-1 with better promise as drug candidates to minimize ischaemic heart damage. The structures of bound Alda-1 reveal how the compound activates the wild-type enzyme and how it restores the activity of the inactive form by acting as a structural chaperone.

Allosteric PDE4 Inhibitors with Reduced Potential for Side Effects

Crystal structure of PDE4D catalytic domain (tetramer) complexed with Rolipram - pdb ID = 1Q9M. Data from Emerald Biostructures are not yet released in the pdb.

The cyclic nucleotide phosphodiesterases (PDEs) are important regulators of signal transduction and selective inhibitors of the different subtypes have great clinical potential. PDE4 inhibitors are expected to be beneficial in the treatment of inflammatory and respiratory diseases such as asthma and COPD as well as CNS disorders including schizophrenia, depression, and Alzheimer’s disease but their potential has so far been limited by the incidence of side effects, particularly emesis. The emetic response is mediated in part by a brainstem noradrenergic pathway and, for non-CNS indications, can be reduced by limiting distribution of inhibitors to the brain. Active site directed PDE4 inhibitors completely inhibit enzyme activity at high concentrations but researchers at Emerald Biostructures (formerly deCODE biostructures) have now identified allosteric small molecule modulators of PDE4 with reduced potential for side effects. The four PDE4 variants (PDE4A, B, C, and D) all contain signature regulatory domains called upstream conserved regions 1 and 2 (UCR1 and UCR2). UCR2 is needed for high-affinity binding of the PDE4 inhibitor rolipram and X-ray crystallographic structures revealed that small molecule inhibitors bind to UCR2, thereby controlling access to the active site. The team used the structural data together with supporting mutational data to design PDE4 allosteric modulators that only partially inhibit cAMP hydrolysis. The modulators were shown to be potent in cellular assays as well as in vivo cognition tests and to have greatly reduced potential for emesis in several species. The authors hope that their work will lead to the identification of PDE4 modulators with reduced potential for emesis that can be used to treat disorders where brain distribution is needed. The study is published in the December 27^th advance online issue of Nature Biotechnology.