Potent Inhibitor of Insulin Degrading Enzyme Reported

Ii1 structure
A new study from a Mayo Clinic-led research team has identified novel, potent inhibitors of insulin degrading enzyme (IDE). Despite an interest in IDE for over 50 years, because of its involvement in insulin catabolism, these are the first potent and selective inhibitors of the enzyme to be described. Given their peptidic nature the current compounds are unlikely to be drugs themselves, but the team hope that their findings will enable further exploration of IDE inhibition as a therapy for diabetes.

IDE is a ubiquitously expressed, secreted enzyme belonging to a small superfamily of zinc-metalloproteases that evolved independently of conventional zinc-metalloproteases. This difference is emphasised by the team’s finding that potent, non-selective hydroxamate inhibitors of zinc metalloproteases did not inhibit IDE.

Crystal structure of the Ii1-IDE complex (PDB ID 3E4A)
Crystal structure of the Ii1-IDE complex (PDB ID 3E4A)
A high-throughput screening campaign failed to identify useful hits, so the researchers turned to a substrate-based approach leading to identification of Ii1 (Inhibitor of IDE 1), with a Ki of 1.7nM. Additional biostructural work identified the distinctive mechanism of IDE inhibition.

In vitro studies with the inhibitors, which included equipotent retro-inverso peptide analogues, demonstrated potent inhibition of extracellular insulin catabolism. In addition, and somewhat unexpectedly, IDE inhibition also enhanced insulin signalling, suggesting IDE involvement in intracellular degradation of insulin.

As well as cleaving insulin, IDE degrades a number of other substrates including atrial natriuretic peptide, glucagon and amyloid-β protein (Aβ). Indeed there has been considerable interest in up-regulating IDE activity as a potential therapy for Alzheimer’s disease (AD). The authors of the current study, published in PLoS ONE, suggest that any concern regarding negative impacts of IDE inhibition on AD could be addressed by developing inhibitors that do not cross the blood-brain barrier. Further, in light of the recent finding that intranasal insulin improves cognition in early AD patients, and given insulin’s beneficial effects on learning and memory, it may be overly simplistic to assume that IDE’s role in AD pathogenesis is limited to its predicted effects on Aβ alone.

Another Activity for Vinpocetine

Vinpocetine is the semi-synthetic derivative (ethyl ester) of the alkaloid, apovincamine, found in the Lesser Periwinle (Vinca minor). It has previously been reported to have neuroprotective properties and to increase cerebral blood flow. Although it is used in Eastern Europe to treat cerebrovascular disorders and age-related memory loss, clinical studies have not conclusively demonstrated efficacy. Nevertheless, the compound is also widely used as a dietary supplement.

Vinpocetine is known to inhibit sodium-gated ion channels and has also been identified as a weak inhibitor (IC50 ~ 10µM) of phosphodiesterase-1 (PDE-1). These activities have been used to explain the neuroprotective and vasorelaxant properties of the molecule.

vinpocetine structure
Researchers at University of Rochester Medical Center have now reported potent activity of vinpocetine in a mouse model of lung inflammation, showing promise for the treatment of chronic inflammatory diseases such as COPD, rheumatoid arthritis and psoriasis. The scientists have demonstrated that the mechanism of action is via inhibition of IκB kinase (IKK) resulting in suppression of the proinflammatory transcription factor, NFκB.

Since the compound has a long history of human use, the researchers hope that development of vinpocetine as an antiinflammatory therapy will be easier than for a novel molecule. The university has applied for a patent for vinpocetine for use as an IKK-inhibitor for the treatment of COPD and Yan and Berk, lead scientists of the study, have formed a start-up company, Rock Pharmaceuticals, with the hope of licensing the intellectual property rights.

Full details of the study are currently in press at PNAS, entitled “Vinpocetine inhibits inflammation via an IKK-dependent but PDE-independent mechanism”.

HCV NS5A Inhibitor Shows Early Promise in Clinic

Crystal structure of the N-terminal zinc-binding domain of HCV NS5A (PDB ID=1ZH1), the proposed target of BMS-790052 (inset)
Hepatitis C virus (HCV) is a leading cause of liver disease and the current ‘standard of care’ treatment, a combination of pegylated interferon-α and ribavirin, is expensive, can cause serious side effects and is only effective for some patients. There are at least 6 known HCV genotypes, with several subtypes and quasispecies within each genotype and these all respond differently to treatment – the combination of pegylated interferon-α and ribavirin is able to clear the virus from about half of patients infected with genotype 1 HCV. In the search for more effective treatments, a number of groups are developing compounds that specifically target HCV. Inhibitors of the viral NS3 serine protease and the NS5B4 RNA-dependent RNA polymerase have already entered the clinic and researchers at Bristol-Myers Squibb have now reported that a compound with a new mechanism of action, BMS-790052, led to dramatic reductions in viral load and produced few side effects in a phase I clinical study. BMS-790052 targets NS5A, an HCV non-structural protein that possesses no enzymatic activity and is thought to regulate viral replication and host cell interactions. The compound, which was identified using a chemical genetics strategy, is active at picomolar concentrations in vitro towards replicons expressing a broad range of HCV genotypes and acts additively to synergistically with interferon and other small molecule antiviral compounds.

The study, which is published in the journal Nature, provides the first clinical evidence that targeting NS5A may be an effective strategy for treating patients who are chronically infected with HCV. Future treatments for HCV infection may involve ‘cocktails’ of drugs that target specific stages in the viral lifecycle, just as HIV infection is managed now, although whether it will be possible to drop interferon from HCV treatment combinations remains to be seen.

Stopping Cancer Spread

Fascin crystal structure
Human fascin complexed with macroketone, PDB ID=3LNA
The deadliest feature of cancer is its ability to spread, or metastasise. Migrastatin, a compound isolated from Streptomyces, was found to weakly inhibit tumour cell migration and, in 2005, researchers from Weill Medical College of Cornell University and the Sloan-Kettering Institute for Cancer Research described simplified analogues of migrastatin, including a compound they called macroketone, that inhibit mammary tumour metastasis in mice. Although the compounds were effective in preventing the spread of cancer cells, it wasn’t known how they achieved this. In a new study, published in the journal Nature, the team have revealed that macroketone exerts its anti-metastatic effect by targeting the actin-bundling protein, fascin. Cancer cells use invasive finger-like protrusions called invadopodia to spread into and degrade extracellular matrix and recent studies have shown that fascin is important for their assembly and stability.

Macroketone structure
Mice implanted with cancer cells and treated with macroketone lived out a full lifespan without any spread of the cancer whilst untreated animals all died from metastases. When treatment was delayed for one week after introduction of the cancer cells, metastasis was still blocked by more than 80%. Macroketone did not prevent implanted cancer cells from forming tumours or growing, suggesting that such compounds would need to be used in combination with chemotherapy drugs acting on the primary tumour. Because fascin is overexpressed in metastatic tumour cells but is only expressed at very low levels in normal epithelial cells, treatments that target fascin should have comparatively little effect on normal cells and may have fewer side effects than other treatments.

X-ray studies showed that macroketone binds to one of the actin-binding sites on fascin which prevents the actin fibres from bundling together and could form the basis for further drug design.

Open Access Drug Target Database Launched

Image: Flickr – psyberartist

SciClips has launched an online database of drug targets, based on information collected from US and International patent applications. Currently the database contains around 4000 targets linked to their respective diseases and the data are updated on a weekly basis. As well as the association of the biological targets with diseases, the database is also organised according to type of drug (such as antibodies, proteins, siRNA, miRNA or small molecules).

All targets are linked to PubMed, Google Scholar, GeneBank, UniProt, the USPTO database, WO(PCT) database and Google Patents, allowing the user to access additional information, and searches can be conducted on the biological target, drug type or disease.

Whilst the web interface seems a little idiosyncratic, feedback from users will allow SciClips to refine it.

According to the SciClips website:

“SciClips is a web-based scientific platform for open innovation as well as for sharing information and ideas related to various scientific areas. Currently we are offering services and inviting ideas in several areas involving stem cells, proteomics, biomarkers, metabolomics and drug discovery tools. …. We believe that by offering scientific services to cutting edge research areas, we will be able to attract scientists (from) all over the world to think collectively, share ideas and achieve scientific breakthroughs. We will add services in other scientific areas in (the) coming months.”

Vive la Différence

Image: Flickr - Andresmoschini
A year ago, when researchers at Purdue University argued that environmental standardisation in laboratory experiments involving mice was likely to lead to more, rather than less, variation between different laboratories, they met with some resistance since it was not clear what factors should be varied to improve reproducibility. Following an analysis of data from behavioural tests commonly used in drug discovery studies, they have now shown that introducing only two controlled environmental variables to preclinical studies using mice can greatly reduce false positives and the number of animals needed for testing. The tests, which compared behaviours between two inbred strains of mice, were repeated in four different model laboratories that varied in details such as background noise, the age of the mice, environmental enrichment, familiarity with handler, lighting levels and cage size. In each laboratory, one group of mice (standardised) were treated identically whilst the other group (heterogenised) were tested under four different sets of conditions produced by varying two environmental factors in a controlled manner. Mice of the same strain would have been expected to show the same behaviours in each laboratory but, in 33 out of 36 behavioural characteristics such as fear and curiosity, the standardised group showed as much as five times more variation between laboratories compared with the heterogenised group.

When conditions are highly standardised, the variation in data produced within a particular laboratory will be very low, but variations between laboratories will be large and unpredictable. The researchers believe that tests in mice using a heterogeneous test design more closely resemble human clinical trials and should reduce both the number of animals needed for preclinical studies and the number of false positives. A reduction in false positives could have important implications for reducing the number of compounds that fail in expensive downstream clinical trials.

The study is published in Nature Methods.

Upside Down and Back to Front

upside down and back to front
Image: Flickr - Irargerich
In back-to-back papers published in the Proceedings of the National Academy of Sciences, researchers describe retro-inverted peptide mimetics targeting the vascular endothelial growth factor receptor (VEGFR) and epidermal growth factor receptor (EGFR) pathways. The team begin by screening a phage display library to identify peptides that bind to the target receptor and refining the hits by structural and functional analysis. Since natural peptides are often unstable to proteolysis, the amino acid retro-inversion method (ie substitution of D-amino acids for L-amino acids in conjunction with reversal of chain direction) was used to produce molecules with greater resistance to proteolysis. The side chain topology of the resulting peptide mimetics is similar to that of the original peptide – albeit with the amide bonds and terminal charges reversed.

The formation of new blood vessels has been associated with a number of pathologies including cancer and diseases of the eye such as diabetic retinopathy and age-related macular degeneration. VEGF comprises a family of five growth factors that promote angiogenesis by binding to, and selectively activating, several membrane-bound tyrosine kinase receptors (VEGFR-1, -2, and -3) and neurophilins (NRP-1 and -2). The minimal structural requirement for binding of a small peptide fragment to VEGFR-1 and NRP-1 was determined to be the tripeptide, RPL. The retro-inverted tripeptidomimetic, D-LPR was found to be resistant to proteolysis and to bind effectively to VEGFR-1 and NRP-1. In competition binding experiments, the IC50 values of RPL for VEGFR-1 and NRP-1 were 30 nM and 4 pM and the IC50 values of D-LPR for VEGFR-1 and NRP-1 were 2 pM and 2 pM. D-LPR was shown to be effective in three animal models of angiogenesis, including a mouse model of retinopathy. When administered topically, D-LPR led to a significant (ca 50%) reduction in angiogenesis, suggesting that the peptide mimetic can penetrate the vitreous humour and may provide a lead for the development of soluble and permeable small drug molecules that can be administered in eye drops.

EGFR, a tyrosine kinase receptor, is abnormally activated in many types of epithelial tumours, including including lung, colon, and head and neck cancers. Although, theoretically, the EGFR pathway could be blocked by three drug classes – antibodies, kinase inhibitors or soluble ligand receptor traps/decoys – so far no EGFR decoy has been developed. In the second paper, the team describe small molecules that mimick EGFR and behave as soluble decoys for EGFR ligands, EGF and TGFα. The cysteine-bridged pentapeptide, CVRAC, was found to bind specifically to EGFR ligands and both CVRAC and the retro-inverted derivative, D-CARVC, markedly reduced proliferation of tumour cell lines. In immunocompetent female mice bearing mammary tumours, mice treated with D-CARVC had significantly smaller tumour volumes than control mice.

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.

closantel structure
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

Image: Flickr - Mrs. Bones
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 structure
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 (Ki ~ 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.

LJ-001 structure
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.