Drug Discovery Opinion

Seasonal epidemics of the influenza virus continue to kill hundreds of thousands of people annually, and the increasing incidence of resistance to approved drugs means that there is a pressing need for new therapies. The viral polymerase is an attractive target for drug development and a newly reported high-resolution crystal structure of the polymerase PA domain should provide useful insights for inhibitor design.

In eukaryotic cells, mRNA must be capped at the 5’-end for efficient translation. Cap structures, consisting of N⁷-methylated guanine units, also assist in transport of mRNA from nucleus to cytoplasm and protect mRNA from degradation by 5′ exonucleases. The viral polymerase ‘steals’ caps from cellular mRNA in a process known as ‘cap-snatching’ and attaches them to viral mRNAs so that these can be translated into new viral proteins.

The polymerase is a heterotrimer comprising three subunits, PA, PB1 and PB2, and whilst previous studies had shown that PB2 plays a role in cap-binding, PB1 was believed to be responsible for ‘cap-snatching’. The new study, published online on February 4^th in the journal Nature, clearly shows, however, that the PA subunit contains the endonuclease active site and plays a crucial role in cleaving the cap from host mRNA. The active site, which is conserved in all influenza viruses, contains a histidine residue together with a cluster of three acidic residues that bind two manganese ions in a configuration similar to that observed in other two-metal-dependent endonucleases. Inhibition of cap-cleavage by the endonuclease would be an effective anti-viral strategy since it would effectively block synthesis of viral proteins.

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Sphingosine-1-phosphate (S1P), the product of phosphorylation of sphingosine by sphingosine kinase, is an important lipid signalling molecule for which biological functions continue to be defined. A new study by researchers working at the National Institute of Allergy and Infectious Diseases (NIAID) has now demonstrated a key role for S1P in bone remodelling. This process involves two major types of bone cells: osteoclasts, which break down bone and osteoblasts, which make new bone. Bone resorption and bone formation are normally finely balanced but, in conditions such as osteoporosis and rheumatoid arthritis, osteoclast activity exceeds osteoblast activity leading to a net decrease in bone density.

Immature osteoclasts circulate within the blood and migrate to the surface of the bones, where they mature and start to degrade the bone matrix. These immature cells were shown to express functional S1P₁ receptors and migrate along an S1P gradient in vitro. Specialised imaging techniques were used to show that the S1P₁ agonist, SEW2871, also stimulated motility in vivo. In a mouse model of postmenopausal osteoporosis, treatment of ovariectomized mice with the S1P₁ agonist, FTY720, reduced osteoporosis by reducing the number of mature osteoclasts attached to the bone surface. FTY720 also acts as an immunosuppressant by preventing egress of lymphocytes from the lymph nodes, and is undergoing phase III clinical trials for the treatment of relapsing-remitting multiple sclerosis.

Most current treatments for osteoporosis target mature osteoclasts, and regulating migration of immature osteoclasts to the bone surface may offer a new approach to treating the disease.

The study, published in the journal Nature, was available online on February 8^th.

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The malaria parasite, Plasmodium falciparum, has limited capacity for de novo amino acid synthesis and relies on degradation of host haemoglobin for a supply of these essential building blocks. Haemoglobin is first degraded into di- and tri-peptides by the action of a number of cysteine-, aspartyl-, and metallo-proteases. These small peptide fragments are then further hydrolysed to release free amino acids by the action of the metallo-exopeptidases, PfA-M1 (an alanyl aminopeptidase) and PfA-M17 (a leucine aminopeptidase).

A team from the University of Monash has recently described the X-ray crystallographic structure of truncated recombinant PfA-M1 at a resolution of 2.1 Å. Comparison of structures of PfA-M1 bound to the known inhibitors, bestatin (K_i 500nM) and hPheP[CH₂]Phe (K_i 80nM), with the native structure showed that the enzyme did not undergo any global conformational rearrangements on binding either inhibitor. It is proposed that substrate access is achieved by means of the C-terminal domain vortex, and that control of substrate hydrolysis can be achieved, and depends on, the size of this channel. hPheP[CH₂]Phe, which provides effective protection in a murine model of malaria, also inhibits PfA-M17 and the authors suggest that inhibiting both PfA-M1 and PfA-M17 may be less likely to allow the development of drug-resistant malaria. The fact that the site of action of PfA-M1 is outside the digestive vacuole, together with the comparative ease of identifying drug-like inhibitors of metallo-proteases, makes PfA-M1 an attractive target for new anti-malarial therapies.

The study is published in the February 5^th Early Edition of PNAS.

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Tea drinking is a tradition dating back thousands of years and green tea, particularly, is thought to have many health-giving properties. Green tea has also been investigated for possible use in the prevention or treatment of cancer, but a new study by researchers at the University of Southern California has shown that a component of green tea blocks the anti-tumour effect of the proteasome inhibitor, bortezomib (Velcade®), which is used to treat multiple myeloma. Before the study, the researchers believed that epigallocatechin gallate (EGCG), or other components of green tea, would enhance the effectiveness of bortezomib and were surprised to find the opposite effect.

In cell culture experiments, 10µM EGCG provided complete protection from the cytotoxic effects of 10nM bortezomib and 2.5µM EGCG also afforded significant (~80%) protection. In tumours from nude mice implanted subcutaneously with multiple myeloma cells, treatment with bortezomib alone (0.5mg/kg) caused a significant increase in apoptosis. In tumours from mice treated with bortezomib and EGCG (25 or 50mg/kg), however, there was no increase in apoptotic cell death when compared to tumours from untreated control mice.

The authors showed that EGCG was exerting its inhibitory effect on the cytotoxicity of bortezomib by a direct covalent interaction between a 1,2-dihydroxybenzene group of EGCG with the boronic acid group of bortezomib, resulting in the formation of a cyclic boronate ester. EGCG also blocked the cytotoxicity of other boronic acid containing proteasome inhibitors, but had no effect on the cytotoxicity of structurally different proteasome inhibitors.

Because EGCG concentrations of 5-8µM can be achieved by consuming concentrated green tea extracts, the authors strongly advise that cancer patients receiving bortezomib therapy should avoid green tea products, especially the concentrated liquid or capsule forms. Studies show that green tea may improve the effectiveness of other cancer therapies.

The study is published in the February 3^rd First Edition of the journal Blood.

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Loss-of-function mutations of human DJ-1/PARK7 are associated with autosomal recessive, early onset Parkinson’s disease. In contrast, amplification of DJ-1 has been observed in numerous tumours and its expression correlated with poor prognosis. Experimental data suggest that DJ-1 contributes to cell survival by enhancing the phosphorylation of Akt, resulting in inhibition of PTEN function. Further data support a role in protection from oxidative stress – knock-out of DJ-1 enhances hydrogen peroxide mediated cytotoxicity.

Researchers have now identified DJ-1 as an upstream activator of hypoxia inducible factor-1(HIF1) function in cancer cells, conferring resistance to hypoxia-induced apoptosis. HIF1 is a transcription factor that plays a key role in a tumour’s ability to adapt under hypoxic conditions, critical for survival and progression. The authors’ experiments demonstrate that loss of DJ-1 in human cell lines decreases transcription of all HIF1-responsive genes examined and that expression of DJ-1 is critical to the activities of Akt and mTOR that are required to maintain HIF1 stability. In addition, DJ-1 has been shown to regulate the activity of the metabolic sensor, AMPK.

The authors conclude that their results strengthen the case for therapeutic intervention at the level of DJ-1 in cancer cells. It will be necessary, however, to consider the loss-of-function consequences. The full study is reported in the January 27^th issue of PNAS.

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Cardiac hypertrophy, or thickening of the heart muscle, occurs naturally in athletes but may also be a pathological response to high blood pressure or stenosis of the aortic valve. Pathological hypertrophy allows the heart to continue pumping blood against the increased pressure but, over time, can lead to loss of function and heart failure. Researchers at the University of California, Davis, have now identified soluble epoxide hydrolase (sEH) as a key enzyme in the development of angiotensin II (Ang II)-mediated cardiac hypertrophy. sEH hydrolyses the blood pressure-regulating epoxyeicosatrienoic acids (EETs) into dihydroxyeicosatrienoic acids (DHETs), which show reduced biological activity. The study, which is published in the January 13^th issue of the Proceedings of the National Academy of Sciences showed that levels of sEH in the heart were elevated in two rodent models of Ang II-induced hypertrophy. The Ang II receptor blocker, losartan, prevented the increase in sEH and administration of an inhibitor of sEH, 1-(1-acetyl-piperidin-4-yl)-3-(4-trifluoromethoxy-phenyl)-urea (TUPS), prevented the pathogenesis of Ang II-induced hypertrophy.

The authors suggest that inhibition of sEH could be a useful approach to treat Ang II-induced cardiac hypertrophy. Existing treatments for heart failure include β-blockers, angiotensin converting enzyme (ACE) inhibitors or Ang II receptor blockers, diuretics and drugs that improve the heart’s ability to pump.

In another study, published in the journal Clinical Science, administration of a different sEH inhibitor, 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA), was found to reduce kidney damage caused by Ang II-induced hypertension in spontaneously diabetic rats.

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Synta Pharmaceuticals has recently announced enrolment of 630 patients for a pivotal Phase III trial of elesclomol (STA-4783) in patients with stage IV metastatic melanoma. Although melanoma is one of the rarer forms of skin cancer, it causes the majority of skin cancer-related deaths – around 50,000 worldwide each year. Early diagnosis and surgical removal of the lesion whilst it is limited to the uppermost layer of the skin is currently the most effective treatment for melanoma. Prognosis is very poor if the cancer has progressed to the deeper layers of the skin or spread to other tissues: limited treatment options are available and expected survival is only six to nine months.

Elesclomol is a first-in-class oxidative stress inducer that triggers apoptosis (programmed cell death) in cancer cells. Oxidative stress is the result of an accumulation of reactive oxygen species such as superoxide, hydrogen peroxide and hydroxide radicals within the cell. Reactive oxygen species are normal products of cell metabolism and play a role in cell signalling but high levels can lead to oxidative stress, resulting in significant damage to the cell or cell death. Normal cells are able to protect themselves against this damage by using enzymes to remove the reactive species and repair any damage, but many types of cancer cells have a diminished anti-oxidant capacity and typically operate under higher levels of oxidative stress than normal cells. This leaves cancer cells especially vulnerable to increases in oxidative stress. Elesclomol exploits this difference between cancer cells and normal cells and is able to induce apoptosis in cancer cells whilst having little or no effect on cancer cells. Elesclomol showed potent anti-cancer activity against a broad range of cancer cell types and enhanced the efficacy of certain chemotherapy agents with minimal additional toxicity.

In a double-blind, randomised, controlled Phase IIb study in 81 patients with stage IV metastatic melanoma, elesclomol in combination with paclitaxel doubled the median time patients survived without their disease progressing, compared to paclitaxel alone. Elesclomol is being developed and commercialised in partnership with GlaxoSmithKline and is also being investigated as a treatment for other cancers.

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The practice of calorific restriction as a means to health, improved mental faculties and a longer life is controversial and two recent studies have contributed more fuel to the debate. A study carried out by scientists at the University of Southern California, and published online on January 13^th in The Journal of Nutrition, compared metabolic rates in two strains of mice, one genetically engineered to be fat and the other lean. Lifespan of the ‘fat’ strain is increased by calorie restriction whereas that of the ‘lean’ strain is not. When both groups of mice were allowed to eat as much as they wanted, they ate similar amounts and had similar body weights at 4 months old. Later in life, however, the ‘fat’ strain mice were significantly heavier than the ‘lean’ strain mice, a difference linked to lower metabolic rate in the ‘fat’ mice. Only the mice with the lower metabolic rate benefitted from a reduced calorie diet. The study authors conclude that calorie restriction may be pointless – and possibly even dangerous – for non-obese individuals and that, ideally, energy expenditure and energy intake should be in balance.

A second study, published in the Proceedings of the National Academy of Sciences, looked at the effect of calorie restriction and intake of unsaturated fatty acids on cognitive performance in older people. Fifty healthy, normal to overweight subjects, with an average age of sixty, were assigned to one of three groups: 30% calorie reduction, increased intake of unsaturated fatty acids or control. Memory performance was measured before the trial began and after three months. A significant improvement (20%) in verbal memory scores was seen in the group eating fewer calories whereas no significant changes were seen in the other two groups. The improvements in memory correlated with decreases in fasting plasma insulin levels and a marker of inflammation, and were most pronounced in individuals who stuck strictly to the diet. The researchers plan to repeat the study in a larger group of people and also to study the effects of calorie restriction in patients with mild cognitive impairment. It is not clear whether reducing calorie intake would improve memory in lower weight individuals.

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The Kinetic Isotope Effect (KIE) refers to the dependence of rate of reaction on the isotopic identity of an atom participating in the reaction. The largest effect is observed when the isotopic replacement is made to an atom involved in bond breaking or formation in the rate-limiting step – a primary isotope effect. When the isotopic substitution is made to an atom that does not participate in the rate-determining step, the magnitude of the effect is smaller – a secondary isotope effect. This property has long been used in the elucidation of reaction mechanisms.

The KIE is most pronounced when the relative mass change of the substituted isotope is large. For example, a change from hydrogen to deuterium represents a 100% increase in mass, whilst a ¹²C to ¹³C change represents only an 8% increase. Reactions involving C-H bonds are typically 6-10 times faster than the corresponding C-D bonds, whereas a reaction involving ¹²C is only marginally faster than the corresponding ¹³C reaction.

In addition to its utility in elucidation of reaction mechanisms, deuterium has also found application as a non-radioactive isotopic tracer for studying metabolic pathways. Because of the KIE, however, care has to be taken that the substitution does not modify metabolism. Turning this property to advantage, two US companies are targeting deuterated versions of clinically proven drugs to identify analogues with improved properties.

In October 2008, Auspex Pharmaceuticals announced positive results from its Phase 1 clinical trial of SD-254, claiming the first validation in humans of targeted deuterium substitution. SD-254 is a deuterium substituted version of venlafaxine, a selective serotonin-norepinephrine reuptake inhibitor (SNRI). The trial indicated a superior pharmacokinetic profile for SD-254 compared to venlafaxine.

At around the same time, Concert Pharmaceuticals reported preclinical data on CTP-347, a deuterium substituted version of the selective serotonin reuptake inhibitor (SSRI), paroxetine. CTP-347 is being developed to treat vasomotor symptoms (hot flashes) caused by tamoxifen treatment or the menopause. CTP-347 does not cause mechanism-based inactivation of CYP2D6 at clinically relevant concentrations and may be more suitable for co-administration with tamoxifen which is activated by metabolism by CYP2D6. CTP-347 is currently in Phase I clinical testing.

Both companies have numerous other deuterated compounds in their pipelines targeting a wide range of therapeutic indications.

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In a study published in the 21^st January early edition of the journal PNAS, a team led by scientists at the Scripps Research Institute have shown that sphingosine analogues may be able to prevent some of the lung damage caused by the influenza virus. During infection, the influenza virus directly damages lung cells but further tissue damage is also caused by the immune response that is triggered to clear the virus. T-cells and dendritic cells respond to the virus by releasing cytokines and chemokines, which in turn attract polymorphonuclear leukocytes (PMLs) and macrophages to the infected site. The cytokines and chemokines also activate dendritic cells and trigger T-cell expansion, causing further local damage. Some strains of virus, such as H5N1 (bird flu), can provoke an especially strong immune response – a ‘cytokine storm’ – in which the lungs become so clogged with immune cells that they are unable to absorb oxygen efficiently. Permanent lung damage may result even if the individual survives.

In the study, mice were infected with the WSN strain of influenza and the effect of administration of the sphingosine analogue, AAL-R, directly into the respiratory tract was examined. AAL-R reduced the release of cytokines and chemokines, inhibited infiltration of macrophages and PMLs into the lungs and reduced tissue damage, even if administered 4 days after infection. Levels of anti-influenza antibodies and cytotoxic T-cell responses were unaffected, and viral replication was not increased by administration of AAL-R.

In a previous study, published in the journal Molecular Pharmacology, the team showed that immunosuppression caused by local administration of AAL-R results from modulation of receptors other than S1P₁ or S1P₂ on dendritic cells in the lungs, and is independent of naive lymphocyte recirculation.

Sphingosine analogues such as AAL-R may be especially effective if used in combination with specific antiviral drugs such as Tamiflu™ or Relenza™, to both reduce viral load and limit damage to the lung.

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