Drug Discovery Opinion

Although normally clinically silent, persistent hypertension and atherosclerosis are leading risk factors for strokes, heart attacks, heart failure, aneurysms and chronic renal failure. How blood pressure is regulated is still not fully understood, but a team lead by scientists at the University of Pennsylvania School of Medicine has now suggested a role for prostaglandin F2α (PGF2α) in increasing blood pressure and accelerating atherosclerosis. Prostaglandins are a group of hormone-like substances that mediate many physiological and pathophysiological processes, and the team found that mice lacking the receptor for PGF2α had lower blood pressure and less atherosclerosis than wild-type mice. Knocking out the PGF2α receptor was found to suppress activity of the renin-angiotensin system which plays a key role in regulating blood pressure. When blood pressure is low, the liver secretes a protein, angiotensinogen, which is cleaved by renin to give angiotensin I. Further cleavage by angiotensin converting enzyme (ACE) produces angiotensin II which increases blood pressure by narrowing blood vessels and by stimulating release of aldosterone which leads in turn to retention of sodium and water.

If the results seen in mice translate to humans, blocking the PGF2α receptor may provide a novel strategy for controlling blood pressure and reducing atherosclerosis. The study is published in full in the Proceedings of the National Academy of Sciences.

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The discovery of the cannabis receptors CB₁ and CB₂ and the subsequent discovery of anandamide (N-arachidonoylethanolamine), the first endogenous agonist of both receptors, provided a rationale for the known analgesic properties of Cannabis sativa. Anandamide is rapidly hydrolyzed and inactivated by the serine hydrolase, fatty acid amide hydrolase (FAAH), leading to the hypothesis that inhibition of FAAH would be an effective analgesic strategy. Writing in the journal Chemistry & Biology, scientists at the Scripps Research Institute and Pfizer Inc. have now described the discovery of PF-3845, a highly efficacious and selective inhibitor of FAAH. Mechanistic and structural studies confirmed that PF-3845 acts as a covalent inhibitor and carbamylates the active site serine of FAAH. Initial experiments showed that PF-3845 is 10 to 20 times more potent than other FAAH inhibitors and has superior pharmacokinetic properties. In animal studies, PF-3845 raised brain anandamide levels for up to 24 hours and produced a significant cannabinoid receptor-dependent reduction in inflammatory pain. Even if PF-3845 is not itself progressed to the clinic, the team believe that it will be a valuable pharmacological tool for further in vivo characterization of the endocannabinoid system.

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Heart disease is a leading cause of death and illness in the developed world and, once damaged, the heart has very limited capacity for regeneration. Following a heart attack, if blood flow is not restored to the heart muscle within 20-40 minutes, the muscle cells (cardiomyocytes) will die. The dead cells are replaced by scar tissue which does not contract or pump as well as healthy heart tissue.

Writing in the journal Nature, Jun Takeuchi and Benoit Bruneauat at the Gladstone Institute of Cardiovascular Disease have now identified a cocktail of three proteins that can turn mouse mesoderm into cardiac muscle cells (cardiomyocytes). Mesoderm is one of the three primary germ cell layers in the very early embryo – the others are the ectoderm and the endoderm – that can differentiate to give a number of tissues such as bone, blood, and muscle, including heart muscle. The three key proteins are the cardiac transcription factors, GATA4 and TBX5, which are believed to be involved in heart development and function, and a cardiac-specific subunit of BAF chromatin-remodelling complexes, Baf60c. Defects in the genes for these proteins have been linked to abnormal development and defects in the heart.

A combination of all three proteins was shown to direct differentiation of mouse mesoderm specifically into cardiac muscle cardiomyocytes that beat rhythmically, just like normal heart cells. Although, so far, only cells from very early mouse embryos have been turned into cardiomyocytes, Takeuchi and Bruneauat hope that their work will help to understand how new cardiomyocytes can be produced for use in regenerative medicine to treat heart disease.

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An old adage says ‘we must all eat a peck of dirt before we die’, and there is increasing evidence linking the elimination of ‘dirt’ with a rising incidence of allergies and autoimmune disorders – the so-called ‘hygiene hypothesis’. More recently, a link between parasitic infections and the development of healthy immunoregulatory networks has been proposed.

There is evidence that worm infections in humans cause immunosuppression, and a team of scientists from the University of Nottingham has now shown a similar effect in populations of wild mice. Unlike laboratory animals, these mice were ‘naturally’ exposed to a variety of infections and so may provide a better insight into how the immune system functions in its natural context. The researchers used toll-like receptor (TLR) assays to provide an overall measure of immune function in response to a variety of parasites. After correcting for other variables, infection with both the nematode, H. polygyrus, and the louse, P. serrata, were found to suppress innate immunity, with a stronger effect produced by louse infections. It is perhaps surprising that the louse, just by attaching and feeding on the surface of the body can cause such a stronger immune response and the authors suggest that P. serrata could also secrete substances into the mouse that interfere directly with immune function or that the lice could act as a vector for an unidentified bacterial pathogen. The dampening of immune responsiveness associated with parasitic infections in wild mice supports the view that levels of innate immune activation in modern parasite-free human populations may be much greater than would have been typical during their recent evolutionary history, perhaps leading to an increased incidence of immune diseases.

The study is published in the journal BMC Biology.

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As well as being used as a spice to add flavour and colour to a variety of dishes, turmeric has long been valued in parts of Asia for its medicinal properties. Although turmeric has been used for centuries as an antiseptic and has been claimed to be effective in a wide range of conditions including autoimmune diseases, heart disease, Alzheimer’s disease and cancer, it is only recently that scientists have begun to explore its properties in detail.

Curcumin, a molecule with a broad spectrum of antioxidant and anti-inflammatory properties, has been identified as the main active ingredient of turmeric and scientists at the University of Michigan have now described how curcumin acts in the body. Instead of interacting directly with numerous unrelated membrane proteins, the team found that curcumin regulates the action of membrane proteins indirectly by changing the physical properties of the bilayer.

Writing in the Journal of the American Chemical Society, the researchers describe in detail how curcumin inserts deep into the membrane in a transbilayer orientation, anchored by hydrogen bonds to the phosphate groups of lipids. The insertion into the membrane is similar to that seen with cholesterol and, like cholesterol, curcumin induces segmental ordering in the membrane. Using a combination of solid-state NMR and differential scanning calorimetry experiments, the team showed that curcumin has a strong effect on membrane structure even at low concentrations. The team plan to use similar experiments to explore the action of other drugs, such as capsaicin, which also interact with membranes.

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Although cannabis has a long history of use in rituals, as a medicine and as a psychoactive agent, it wasn’t until the late 1980s that the first receptor for the major psychoactive component, Δ9-tetrahydrocannabinol, was identified. This receptor, which came to be known as the CB₁ receptor, is expressed mainly in the brain and is responsible for the psychotropic effects of cannabis. Shortly afterwards, a second receptor, the CB₂ receptor, was discovered on cells of the immune system. Since then, lipids such as anandamide and 2-arachidonoylglycerol have been shown to be endogenous ligands for cannabis receptors.

More recently, a peptide antagonist of the CB₁ receptor was identified, and now US and Brazilian scientists have discovered peptide agonists in the brains of mice. The antagonist, hemopressin (PVNFKFLSH), is a 9-amino acid residue peptide derived from α-hemoglobin and the newly discovered agonists are N-terminally extended variants of hemopressin, incorporating two or three extra amino acids. The peptide agonists were found to activate a signal transduction pathway distinct from that activated by the endocannabinoid, 2-arachidonoylglycerol, or the classic CB₁ agonist, Hu-210.

The study, which is published in the FASEB Journal, suggests an additional mode of regulation of endogenous cannabinoid receptor activity, and the authors hope that this could lead to the discovery of new drugs for managing pain, stimulating appetite and preventing cannabis abuse.

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Tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is one of several members of the TNF gene superfamily that induce apoptosis through engagement of death receptors. The protein is a potentially attractive treatment for cancer since it induces apoptosis in a variety of cancer cells. However, not all cells of a particular type react uniformly to anti-cancer treatments and, although a promising drug candidate, TRAIL is not 100% successful. Genetic explanations have been put forward to explain the differences in cell responses to treatment but a team of scientists at Harvard University wanted to investigate the mechanisms involved in more detail. They exposed both cancer cells and normal cells to varying concentrations of TRAIL and found that a proportion of them always survived. When the surviving cells were isolated, they and their immediate progeny remained highly resistant to the apoptotic effects of TRAIL for a short time. After reproducing for several days, however, the sensitivity of the cells to TRAIL reverted to that of the original colonies with around 90% of the cells dying and 10% surviving. Using a variety of imaging techniques, the team showed that levels of proteins involved in TRAIL-induced apoptosis were different in the sensitive and resistant cells, despite the fact that the cells were genetically identical. They found that the altered protein levels were initially inherited by progeny cells but that inheritance was transient. The initial differences in protein expression between the cells were completely random – cells do not produce proteins uniformly but rather in bursts, with the timing and level of production varying from cell to cell.

The findings offer an alternative explanation to the cancer stem-cell hypothesis about why some cells are more resistant to chemotherapy or radiation treatment and the team hope that the new insight may contribute to the design of more effective treatments.

The study was published online on April 12^th in Nature.

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Recent research has shown that gene expression can be regulated at the level of mRNA by riboswitches. A riboswitch is an aptamer region on an mRNA molecule that can specifically bind a small effector molecule, causing changes in the structure of the expression platform and so regulating the activity of the mRNA. Riboswitches most usually switch off the ability of mRNA to carry out protein synthesis but can also switch it on. A variety of riboswitch classes have been identified, with most of the examples being discovered in bacteria including E. coli and streptococcus as well as bacteria causing anthrax, gonorrhoea, meningitis and dysentry.

Researchers at the University of Rochester Medical Center have now solved the crystal structure of the smallest known riboswitch, the preQ₁ riboswitch. The preQ₁ riboswitch controls the ability of bacteria to produce queuosine (Q), a molecule which enables accurate gene expression by overcoming an inbuilt defect in the mRNA-ribosome-tRNA system known as tRNA wobble, and which is essential for the survival of many important pathological bacteria. The preQ₁ riboswitch ‘senses’ the level of preQ₁, a precursor to Q. If too much preQ₁ is present, genes responsible for producing preQ₁, or for its transport, are shut down. The preQ₁ precursor, known as preQ₀, has the same effect in reducing production of Q. One gene that is regulated by the preQ₁ riboswitch is that which codes for the enzyme queF, which converts preQ₀ into preQ₁.

The structure of the preQ₁ riboswitch from Thermoanaerobacter tengcongensis complexed with preQ₀ shows preQ₀ bound in a buried pocket. The structure also reveals how the first base of the mRNA ribosome binding site binds to a loop of the riboswitch, and how the loop end of the preQ₁ riboswitch aptamer domain binds to preQ₀. Binding of the preQ₁ aptamer loop to the first base in the ribosome binding site was found to be mediated by a standard G to C base pairing interaction. The preQ₁ aptamer (34 nucleotides) is about 2.5-fold shorter than functionally related riboswitches that recognize similar metabolites.

An understanding of how bacterial species sequester their ribosome binding sites using divergent preQ₁ riboswitch aptamers could lead to the design of a new class of antibiotics. There is evidence that some existing antibiotics act – in part at least – by targeting riboswitches and, since riboswitches have not yet been found in human cells, the hope is that antibiotics acting on riboswitches will have a low propensity for side effects. The study is published in the Journal of Biological Chemistry.

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Research recently published in the journal PNAS describes a new role for the hormone, oxytocin. This nine-residue peptide is best known for its role in female reproduction and in mediating trusting behaviours and emotional experiences, but the new study reports a key role in bone metabolism. Deletion of oxytocin or the oxytocin receptor caused osteoporosis in both male and female mice. The underlying mechanism in osteoporosis is an imbalance between bone formation and bone resorption. Under normal physiological conditions, bone undergoes constant remodelling by osteoclasts which resorb bone, and osteoblasts which deposit new bone. Oxytocin was found to stimulate differentiation of osteoblasts to a mineralising phenotype by up-regulating bone morphogenic protein 2. The effect of oxytocin on osteoclasts was found to be more complex – on the one hand, osteoclast formation was stimulated but, on the other hand, bone resorption by mature osteoclasts was inhibited. The discovery that oxytocin plays a role in regulating bone mass could have implications for the treatment of osteoporosis, a condition that affects one in three women and one in twelve men. Oxytocin itself is not orally bioavailable and is administered either by injection or as a nasal spray but a number of groups are developing orally bioavailable non-peptidic oxytocin agonists.

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A new slow-release anaesthetic drug-delivery system could potentially revolutionize the treatment of pain during and after surgery, and may also have a large impact on chronic pain management. Researchers at Children’s Hospital Boston have developed a liposomal delivery system for the ultra-potent local anaesthetic, saxitoxin. Saxitoxin is one of a family of neurotoxic alkaloids produced by aquatic microorganisms which block voltage-gated sodium channels on nerve cells. In studies in rats, liposomal delivery of saxitoxin blocked sciatic nerve transmission without causing significant nerve or muscle damage. The team found that liposomes containing only saxitoxin produced nerve blocks that lasted for two days whilst liposomes containing saxitoxin together with dexamethasone – a steroid known to enhance the action of encapsulated anaesthetics – caused a nerve block that lasted for seven days without significant damage to surrounding nerves or muscle. Despite the extreme potency of saxitoxin, systemic toxicity occurred only with high loadings of dexamethasone which increased release of the anaesthetic. Previous attempts to develop slow-release anaesthetics have been limited by short duration of action, toxicity to the surrounding tissue and/or systemic toxicity. The team hope that eventually a single injection that could cause a nerve block lasting for weeks – or even months – could be used to manage chronic pain and are optimising a formulation that would be suitable for clinical trials.

The research is published in the April 13th online edition of PNAS.

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