Drug Discovery Opinion

The human body is host to a plethora of microorganisms and, for the most part, their presence has no ill effects. Some, particularly intestinal bacteria, even provide benefit. From a microbial perspective, harming the host does not have any obvious survival benefit (unless it enables infection to spread, such as the sneezing induced by the cold virus). So why is it that inoffensive organisms occasionally turn nasty, evolving properties that are damaging or even deadly to us? A study funded by the US Public Health Service and the Wellcome Trust provides one answer to the question.

Since many pathogens interact with their host at mucosal surfaces and have to compete with other microflora, scientists at the University of Pennsylvania School of Medicine and Oxford University used a mouse model of nasal infection to investigate whether competition between microbes promoted virulence. They found that Haemophilus influenzae was able to out-compete Streptococcus pneumoniae by recruiting the host’s immune system. S. pneumoniae is normally harmless and ignored by the immune system, but the immune response stimulated by H. influenzae has unintended consequences. S. pneumoniae strains with polysaccharide capsules that confer resistance to the immune attack are able to survive at the expense of non-resistant strains, resulting in a S. pneumoniae population dominated by the resistant phenotype. Unfortunately, the resistant strains are also more dangerous – if they are able to enter the bloodstream they can multiply unchecked and go on to cause pneumonia, septicaemia and meningitis. So in this battle between S. pneumoniae and H. influenzae, with weapons provided by the host, S. pneumoniae prevails at the expense of the host.

The study is published in Current Biology.

The process of cell competition is believed to provide a mechanism to optimise tissue ‘fitness’ during development by eliminating weaker cells from the overall cell population. First described in Drosophila, a number of genes have been linked to cell competition but the precise details of the process are poorly understood. A new study conducted by scientists at the Spanish National Cancer Centre (CNIO), however, has furthered our understanding.

Using a combination of genomic analysis and functional assays, the team investigated how cells of Drosophila wing imaginal discs distinguished ‘winner’ and ‘loser’ cells. They found that six genes were upregulated early in loser cells and five of these encoded cell membrane proteins, suggesting that cell-cell communication is critical in the initial stages of cell competition. One of these membrane proteins, Flower (Fwe), was examined in detail.

Fwe is conserved in multicellular organisms and in the Drosophila study was found to be required and sufficient to label cells as winners or losers. The win/lose decision is mediated by three differentially expressed forms of fwe (fwe^ubi, fwe^LoseA and fwe^LoseB) and cells are identified as losers when relative differences in fwe^ubi and fwe^Lose levels are detected – stress conditions that uniformly affect the entire population result in cell survival. Although further work is necessary to elucidate the detail, the team proposes that, in outcompeted cells, the fwe transcript is alternatively spliced and fwe^Lose isoforms are induced at the expense of fwe^ubi. It is likely that downregulation of fwe^ubi and upregulation of fwe^Lose both contribute to establish the lose/win decision.

The cellular tagging by Flower isoforms may have biomedical implications beyond cell competition since imbalances in cell fitness also occur during ageing, cancer formation and metastasis.

The study is published in Developmental Cell.

Iminosugars, where the endocyclic oxygen of sugars is replaced by a basic nitrogen, constitute a class of carbohydrate analogues that has received considerable attention in recent years.

The discovery that the natural product, 1-deoxy nojirimycin, had potent inhibitory properties toward alpha-glucosidases sparked exploration of synthetic approaches to, and biological properties of, the class. This has led to the approval of miglitol (Glyset®) for treatment of type II diabetes and miglustat (Zavesca®) for treatment of type I Gaucher’s disease in patients for whom enzyme replacement therapy is not suitable. More recently, Zavesca® is the first drug to be approved for treatment of progressive neurological manifestations in adult or paediatric patients with Niemann-Pick type C disease.

Subsequent research has identified iminosugars as inhibitors of a broader range of enzymes and the class is providing leads for a variety of diseases including viral infections and tumour metastasis as well as diabetes and lysosomal storage disorders.

A forthcoming meeting, organised by Summit Corporation plc, will cover both historical and future perspectives of iminosugars. Entitled “Iminosugars: Past, Present and Future – Medicines for tomorrow”, the meeting will be held at St John’s College, Oxford (UK) on 28^th June. The full programme and registration details can be found on the Summit website.

MicroRNAs (miRNAs) are small (21-23 nucleotides), single-stranded RNA molecules that function as regulators of gene expression. The human genome encodes several hundred miRNAs and abnormal expression of these has been associated with cancer progression. We have previously reported on miRNA involvement in cholesterol regulation, amyotrophic lateral sclerosis and liver cancer. Now a collaboration between Rosetta Genomics, NYU Langone Medical Center and Vanderbilt School of Medicine has identified the potential utility of miR-33 for development of therapies targeting malignant mesothelioma (MM).

MM is a rare cancer that has been associated with exposure to asbestos dust (although a small proportion of patients have never been exposed). Taking anywhere between 20 and 50 years for symptoms to develop, the cancer affects the mesothelium, a thin layer of tissue surrounding the internal organs. The most common form of MM is pleural, involving the lining of the lungs, but it may also affect other tissues such as the peritoneum. Current treatment options, including surgery and chemotherapy, are limited and often confounded by late diagnosis because of the absence of symptoms.

This latest study found that MM cell lines derived from patients with aggressive disease failed to express miR-31, a microRNA that has also been linked to suppression of breast cancer metastasis. Functional studies, where miR-31 was reintroduced to the cells, showed that the microRNA could inhibit proliferation, migration, invasion, and clonogenicity of mesothelioma cells. miR-31 suppressed expression of a number of factors associated with maintenance of DNA replication and cell cycle progression, including the pro-survival phosphatase PPP6C. The mRNA for PPP6C, which contains three miR-31-binding sites in its 3′-UTR, was down-regulated when miR-31 was present and up-regulated in clinical MM specimens compared to matched normal tissues.

Whilst the study, published in the Journal of Biological Chemistry, reveals a key role for miR-31 in MM, considerable challenges remain to exploit this finding for therapy of the disease.

The eukaryotic translation initiation factor eIF5A, which exists in two isoforms, was originally thought to be involved in formation of the first peptide bond during mRNA translation, but more recent work has implicated it as a translation elongation factor. In mammalian cells it has variously been associated with mediation of proliferation, apoptosis and inflammatory responses, although its mechanisms of action have remained vague. It has also been identified as a cofactor of the Rev trans-activator protein of HIV-1. eIF5A is unique in that it is the only known protein to contain the amino acid hypusine, formed posttranslationally via the sequential action of deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH) acting at a specific lysine residue.

Based on the role of eIF5A in inflammation, a multi-institutional research team led by scientists at Indiana University School of Medicine has explored involvement of the protein in pancreatic islet dysfunction during the development of diabetes. In a low-dose streptozotocin mouse model of diabetes the team found that depletion of eIF5A (using siRNA) protected the mice from pancreatic β-cell loss and hyperglycemia. The depletion of eIF5A resulted in impaired translation of inducible nitric oxide synthase (iNOS)-encoding mRNA within islet cells. Further studies using an inhibitor of DHS, N¹-guanyl-1,7-diaminoheptane (GC7), demonstrated a requirement for hypusination in the action of eIF5A.

The study, published in the Journal of Clinical Investigation, demonstrates a role for eIF5A in inflammation-induced damage to islet cells. Since the negative effects of eIF5A depend on hypusination, DHS may represent a viable therapeutic target for diabetes. Further work will be necessary to establish the role of this pathway in development and progression of the human disease.

Plasmodium parasites, responsible for malaria in humans, have a complex lifecycle that is dependent on mosquito and human hosts. In human blood, the merozoite stage of the parasite invades red blood cells (erythrocytes), growing and multiplying before rupturing the cell and escaping to infect other erythrocytes. It is this profound effect on erythrocytes that is responsible for the symptoms of malaria – fevers, chills and anaemia. Untreated, the disease can be fatal and drug resistance is an increasing problem. With up to half a billion people infected each year and nearly a million deaths, mostly in sub-Saharan Africa, there is an urgent need for new treatments.

Researchers at Harvard School of Public Health (HSPH) were attempting to identify the mechanism by which Plasmodium falciparum merozoites enter erthyrocytes, but instead found a parasite protein that is essential for escape from the cells. When the protein, P. falciparum calcium-dependent protein kinase (PfCDPK5), was suppressed the parasites were trapped in the host cell and unable to infect new cells. In further experiments the team showed that these merozoites were still able to invade erythrocytes if released from their host cell by other means, indicating separate mechanisms for invasion and egress from erythrocytes.

The findings reveal an essential step in the biology of P. falciparum and suggest a new, parasite-specific, drug target for fighting one of the world’s most common and dangerous infections. Whilst many scientists are looking for inhibitors of parasite egress and invasion of red blood cells, no anti-malarial drugs yet target these stages of the parasite lifecycle.

The study is published in Science.

Cholesterol is an essential component of all cellular membranes and is also required for synthesis of vitamin D and steroid hormones. Since it is poorly soluble in water, it is mainly transported through the bloodstream within lipoproteins – complex spherical particles composed of amphiphilic proteins and lipids whose outward-facing surfaces are water-soluble and inward-facing surfaces are lipid-soluble. Triglycerides and cholesterol esters are carried internally whilst phospholipids and cholesterol are transported in the surface monolayer of the lipoprotein particle. Several types of lipoproteins are found in blood, comprised of different apolipoproteins (which target specific tissues via receptor recognition) and with different capacities for cholesterol. These are usually referred to by their densities – the higher the ratio of cholesterol to lipoprotein, the lower the density. This gives rise to the so-called “bad cholesterol” (low density lipoprotein, LDL-cholesterol) and “good cholesterol” (HDL-cholesterol).

Problems arise when levels of the various lipoproteins are out of balance. Increased circulating levels of LDL-cholesterol are associated with the formation of foam cells, which can become trapped in the walls of blood vessels and contribute to artherosclerotic plaque formation leading to heart attacks and strokes. Conversely, HDL transports lipids to the liver for disposal and removes cholesterol from peripheral tissues, including the foam cells that form atherosclerotic plaques.

A new study by researchers at Massachusetts General Hospital (MGH) has now identified micro RNAs (miRNAs) that appear to play an important role in regulation of cholesterol/lipid levels. The team found that two members of the miR-33 family (miR-33a and miR-33b) target the ATP-binding cassette transporter A1 (ABCA1), an important regulator of HDL synthesis and reverse cholesterol transport, for posttranscriptional repression. Using antisense inhibition of miR-33 in mouse and human cell lines the researchers demonstrated up-regulation of ABCA1 expression and increased cholesterol efflux. Further, treatment of mice on a western-type diet with the antisense inhibitor resulted in elevated plasma HDL without affecting levels of LDL. The findings suggest that miR-33 may represent a therapeutic target for cardiovascular diseases.

The study is published in Science.

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.

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 K_i 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.

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

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”.

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