Drug Discovery Opinion

Whitehead Institute researchers have described a new drug discovery technique which uses yeast cells to both synthesise and screen novel compounds. Writing in the journal Nature Chemical Biology, the team have demonstrated that they can negate the toxic effects of α-synuclein in a yeast model that mimics much of the cellular pathology of Parkinson’s disease. α-Synuclein accumulates in vulnerable brain cells in patients with Parkinson’s disease, and the team had previously shown that yeast cells engineered to express large amounts of α-synuclein do not survive. In the new study, yeast cells were engineered to produce cyclic peptides – which target protein-protein interactions – and the α-synuclein was then switched on. The cells that produced cyclic peptides that protect against α-synuclein toxicity survived: the rest died. Out of a library of millions of cyclic peptides, only two were found to rescue yeast cells from α-synuclein toxicity. Although it is not yet clear how the cyclic peptides protect the cells – they were shown not to affect vesicle trafficking – both peptides share a structural motif with thioredoxins, proteins that act as antioxidants; metal transport proteins and proteins that regulate gene activity. The team are now working to determine the precise mechanism of action and to develop new analogues of the peptides.

In a follow-on study carried out by researchers at the University of Alabama, these two cyclic peptides were also found to protect dopaminergic neurones in a C. elegans model of Parkinson’s disease.

The new technique is rapid and inexpensive compared with other methods of lead discovery, and should be applicable to other diseases where key aspects of the pathology can be modelled in yeast or mammalian cells.

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Researchers at the University of California in San Diego have developed computational tools that will allow scientists to quickly and easily determine whether newly isolated nonribosomal peptides (NRPs) are novel or already known. NRPs are a diverse family of secondary metabolites produced by microorganisms such as bacteria and fungi and have a wide range of biological activities: examples of NRPs in clinical use include the antibiotics, penicillin, cephalosprorin and vancomycin; the immunosuppressant, cyclosporine; and the cytostatic drug, bleomycin. NRPs, which are produced independently of mRNA by nonribosomal peptide synthetases, often have cyclic and/or branched structures and may contain non-proteinogenic amino acids as well as numerous other chemical modifications. The lack of genomic DNA information and complex chemical structures have made it very difficult, time-consuming and costly to determine the structure of NPRs, but the new algorithms should replace manual annotations and allow much more rapid characterization. The algorithms are able to calculate the chemical structure of a newly isolated cyclic NRP from fragments generated by mass spectrometry and can be used alongside ‘dereplication’ tools that can calculate the mass spectrometry signature of known NRPs to determine if the newly isolated NRP has already been described.

The study is published in full in the journal Nature Methods, and the web-based tools for sequencing NRPs are available (at no cost to researchers at non-profit organizations) at: bix.ucsd.edu/nrp.

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The primary function of the thymus is to produce mature T-cells and to implement controls to prevent auto-immunity. Lymphocyte precursors migrate from the bone-marrow to the thymus, where they become thymocytes and subsequently mature into T-cells. Since the T-cell repertoire is generated relatively early in life, the thymus is most active during childhood, begins to atrophy around puberty, and is barely detectable in the elderly.

A team led by researchers at the Children’s Hospital of Pittsburgh have now reported knock-out mice that live 30% longer than their wild-type counterparts and in which the thymus remains intact throughout life. The scientists knocked out the gene encoding pregnancy-associated plasma protein A (PAPPA), a recently identified zinc metalloprotease that degrades insulin-like growth factor binding proteins (IGFBP). The knockout mice exhibit proportional dwarfism, similar to IGF1, IGF2 and IGF-receptor (IGFR) knockouts (although IGF1 and IGFR knockout mice are not viable).

The team suggests that the PAPPA-knockout mice benefit from reduced IGF signalling in tissues as a consequence of increased levels of IGFBPs. Indeed, maintenance of thymic structure with ageing correlated with lower steady-state levels of IGF1 in the thymus. Elderly knockout mice continued to generate new T-cells and maintained a robust immune system. Whilst the subtleties of the model require further elucidation, the work so far suggests that manipulation of PAPPA may be a route to modulation of immune competence and healthy ageing.

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

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US scientists have discovered that rapamycin, first discovered in a soil sample from Easter Island, can significantly extend life expectancy in genetically heterogeneous populations of mice. Rapamycin, which is used primarily to prevent rejection following kidney transplants, is an immunosuppressant that interferes with TOR (target of rapamycin) signalling. Inhibition of TOR signalling was already known to extend lifespan in invertebrates but a similar effect in mammals had not been directly demonstrated, although calorie restriction – which has been shown to enhance life expectancy in mammals – is believed to interfere with TOR signalling. The original aim of the present study was to begin treating the mice at 4 months of age but problems in developing a suitable formulation of rapamycin meant that treatment was delayed until they were 20 months old, the equivalent of 60 years old in human terms. Although the team were doubtful that the study would still provide a clear-cut result they went ahead with dosing anyway and found that, compared with untreated animals, the maximal lifespan (age at which 10% survive) was increased by 14% for female mice and by 9% for male mice. In terms of life expectancy, this is equivalent to a 38% increase for the females and a 28% increase for the males. The disease patterns and causes of death in treated and untreated mice were found to be similar. In a separate study, rapamycin was also found to increase survival in both male and female mice when administered from the age of 9 months.

If similar effects on longevity were seen in humans, inhibition of mTOR signalling, even from age 60, could still significantly enhance lifespan, although an increased susceptibility to infections and possible risk of developing lymphoma or other malignancies as a result of immunosuppression would need to be considered.

The study is published in the journal Nature.

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Crohn’s disease is an inflammatory disease of the gastrointestinal tract that causes abdominal pain, diarrhea and vomiting. First described by Burrill Bernard Crohn and co-workers in 1932, the disease is believed to be an autoimmune disorder but the precise causes are unknown. Treatment options focus on management of acute symptoms and maintenance of remission, since no known cure is available.

Crohn himself was convinced that the disease was caused by Mycobacterium paratuberculosis (MAP), the same pathogen responsible for the related Johne’s disease in cattle. Whilst his research was unable to establish the involvement of MAP, the theory has received more attention in recent years.

New research from McGill University Health Centre (MUHC), Quebec, has established a link between the human NOD2 gene and mycobacteria. Mutations in NOD2 have been observed in approximately 25% of Crohn’s disease patients, but the nature of the effect of these mutations has not been understood. Normally, NOD2 codes for a receptor that recognises invading bacteria, triggering an immune response. The MUHC study demonstrates that the NOD2 receptor preferentially recognises a peptide, N-glycolylated peptidoglycan-derived muramyl dipeptide (MDP), which is only found in mycobacteria. When mycobacteria invade the human body, they cause an immediate and very strong immune response via the NOD2 receptor. This new discovery, published in the Journal of Experimental Medicine, associates the predisposition for Crohn’s disease with both the NOD2 mutation and the presence of mycobacteria, but researchers must still determine the precise combination of these factors to understand how the disease develops.

In a separate study, researchers from Case Western Reserve University School of Medicine have identified a novel link between ITCH, a gene known to regulate inflammation in the body and NOD2. ITCH,which encodes an E3 ubiquitin ligase, can cause a variety of inflammatory diseases when malfunctioning. The team at Case Western found that ITCH also influences NOD2-induced inflammation. These findings, to be published in the August 11^th issue of Current Biology, suggest a common pathophysiology exists between multiple inflammatory diseases. The unexpected finding of the interaction between these genes offers the possibility of new drug targets for intervention in Crohn’s disease.

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Lupus erythematosus is a chronic autoimmune disease that, worldwide, is more common than leukaemia, multiple sclerosis, and muscular dystrophy. There are three recognised forms of lupus: discoid (cutaneous) lupus which affects the skin, systemic lupus (SLE) which attacks multiple organ systems, and drug-induced lupus which generally resolves once the drug is discontinued. The symptoms of lupus, which can flare and subside, vary from patient to patient but include severe fatigue, joint pain, muscle aches, anaemia, and general malaise. Approximately 10% of discoid lupus cases develop into systemic lupus which can result in the destruction of vital organs.

The causes of lupus are poorly understood, but researchers at the Scripps institute have now shown that specific Toll-like receptors (TLRs) play a key role in the development of this disease. One of the principal diagnostic indicators of lupus is a high level of self-recognising antinuclear antibodies. Antinuclear antibodies normally form part of the immune response and are produced when bacteria or viruses are degraded in the endolysosome. TLRs inside this compartment specifically detect viral RNA and viral and bacterial DNA and stimulate immune cells to produce antibodies against these molecules.

Writing in the Proceedings of the National Academy of Sciences, the Scripps team have shown that three TLRs in the endolysosome are necessary for the generation of antinuclear antibodies in mice. Previous studies had pointed to endolysosomal TLRs – TLR3, TLR7, TLR8 (in humans but not mice) and TLR9 – as important for the production of antinuclear antibodies but, in mouse models of lupus, knocking out only TLR 7 or TLR9 had not dramatically reduced symptoms. The Scripps team wanted to eliminate all three TLRs in mice and achieved this by knocking out a transmembrane endoplasmic reticulum protein, UNC-93B, which is necessary for correct functioning of the endolysosomal TLRs. Knocking out UNC-93B in strains of laboratory mice that spontaneously develop many of the same symptoms as human lupus sufferers produced animals with fewer antinuclear antibodies and fewer and less severe symptoms of lupus. Even lipid A stimulation of TLR4 – which is known to promote production of autoantibodies – did not produce symptoms of lupus in the knockout animals. Nucleic acid-sensing TLRs may thus provide a critical pathway in the development of systemic autoimmunity by reducing tolerance to nucleic acid-containing antigens.

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Maybe not quite as numerous as the stars in the sky, but chemists at the University of Berne have created a database of almost 1 billion drug-like molecules with 13 or fewer heavy atoms. Writing in the Journal of the American Chemical Society, Jean-Louis Reymond and Lorenz Blum describe a new searchable database, GDB13, of molecules containing up to 13 atoms of C, N, O, S, and Cl.

The search for novel leads is one of the key challenges in drug discovery and Reymond and Blum have previously developed chemical universe database GDB-11 which describes 26.4 million structures containing 11 or fewer atoms of C, N, O, and F and satisfying simple chemical stability and synthetic feasibility rules. The limiting factor in computing GDB-11 was elimination from the initial list of unstable or chemically impossible molecules, most of which contained multiple heteroatoms. To speed up computation of GDB-13, a very fast ‘element-ratio’ filter was used, with cut-off values of (N + O)/C < 1.0, N/C < 0.571, and O/C < 0.666. Fluorine was eliminated from GDB-13 since it was rarely found and had not proved attractive to the group when following up output from GDB-11.

With these modifications, the algorithm was sufficiently fast to compute the database up to 13 heavy atoms, producing 910 million molecules in 40,000 CPU h. The molecular enumeration was dominated by monocyclic, bicyclic, and tricyclic molecules, most of which were heterocyclic – 54% of GDB-13 molecules have at least one three- or four-membered ring. Essentially all of the molecules are drug-like (Lipinski or Vieth criteria) and many were also lead-like or fragment-like.

A chlorine/sulphur set (67.3 million compounds) that enumerates molecules with chlorine atoms as aromatic substituents and sulphur atoms in aromatic heterocycles, sulphones, sulphonamides, and thioureas was also generated. This set is felt to be of particular interest for virtual screening because of the distinct molecular shapes and functional groups that are possible with these larger atoms.

Despite a large fraction of chemical space being excluded to accelerate computation, the authors believe that, with 977,468,314 entries, GDB-13 is the largest publicly available database of virtual molecules ever reported. The database is available free of charge at http://www.gdb.unibe.ch and should provide a rich source of inspiration for previously un-described bioactive fragments. For those unable to resist, fluorine atoms can be added during optimisation.

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Brain infections such as bacterial meningitis and encephalitis can cause death or serious disability and are difficult to treat with conventional antibiotics because of poor CNS-penetration and bacterial drug-resistance. Small cationic antimicrobial peptides form an important part of the host defence system and have potential as therapeutic agents. Unlike conventional antibiotics, antimicrobial peptides appear to be bacteriocidal rather than bacteriostatic, require a short contact time to induce killing, and do not easily induce resistance. Most small antimicrobial peptides form α-helices or β-sheet-like structures that can insert into, and subsequently disrupt, negatively charged bacterial cell surfaces.

Scientists at Singapore’s Institute of Bioengineering and Nanotechnology have described novel antimicrobial peptide nanoparticles that are able to readily cross the blood brain barrier. The nanoparticles are self-assembled from an amphiphilic oligopeptide containing a motif that promotes cell penetration. The nanoparticles were effective against a range of bacteria, yeasts and fungi in vitro and were also more effective against Staphylococcus aureus infection in mice than their unassembled peptide counterparts. Importantly, the nanoparticles were also able to cross the blood brain barrier and suppress bacterial growth in rabbits with Staphylococcus aureus meningitis. The study showed that antimicrobial nanoparticles are effective against bacterial brain infections in animals and do not damage red blood cells, liver or kidneys at the tested doses. The next challenge will be to develop nanoparticles that can be used in people.

The study is published in the journal Nature Nanotechnology.

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Multiple sclerosis (MS), an autoimmune disease that results in damage to oligodendrocytes which maintain insulating myelin sheaths around nerve fibres in the central nervous system, is the most common disabling neurological disorder affecting young adults. The inflammatory process in MS is initiated by T-cells which recognise myelin as ‘foreign’ and attack it as if it were an invading virus. Disease-modifying agents are now available to treat MS but these are not effective for all patients and advanced forms of MS remain difficult to treat. Analysis of tissue samples from MS patients has revealed that two pathways more usually associated with blood pressure regulation – the renin-angiotensin system and the kallikrein-kinin system – are altered compared with samples from normal subjects.

A team led by researchers in Berlin have now shown that the bradykinin B1 receptor specifically controls infiltration of immune cells into the CNS. In the mouse EAE model of MS, activating the B1 receptor resulted in reduced disease severity whereas blocking the receptor led to more rapid disease onset. The protective effect of the B1 receptor was found to be mediated by its expression on T-cells. In an in vitro model of CNS penetration, a B1 receptor agonist, R838, considerably reduced the number of migrated T-cells and additional application of a B1 antagonist, R715, restored migration. Analysis of immune cells from the CNS of mice with EAE revealed that, in mice lacking the B1 receptor, the proportion of infiltrating CD4⁺ T-cells was increased and, significantly, the proportion of T_H17 cells – believed to be crucial for autoimmune neuroinflammation – was also greater. The study suggests that the B1 receptor acts as an endogenous modulator of recruitment of pathogenic lymphocytes into the CNS and limits harmful immune responses. The authors hope that selective B1 receptor agonists could one day play a role in the management of chronic inflammatory diseases such as MS.

The study is published in the journal Nature Medicine.

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Rheumatoid arthritis (RA) is a chronic, typically progressive, autoimmune disease that primarily affects the joints although it can damage other tissues, including heart, lung, and eyes. There is currently no cure for RA and the goal of treatment is reduce joint pain and inflammation, maximise function, and minimise joint destruction and deformity. Newer biological treatments targeting components of the immune system in combination with disease-modifying antirheumatic drugs (DMARDs) are effective in preventing joint damage in some patients, but not all sufferers respond to these treatments and some may relapse despite treatment. The cause of RA remains unknown but both genetic and environmental factors are suspected to play a role.

Researchers at Imperial College have now identified a new immune trigger that may contribute to the pathology of RA. Tenascin-C is an extracellular matrix glycoprotein specifically expressed at areas of inflammation and tissue damage in inflamed rheumatoid joints. The team found that injection of tenascin-C into the joint cavity in mice caused severe joint inflammation and damage and, in a separate experiment, that mice lacking tenascin-C were protected from erosive arthritis. In cultures of cells from rheumatoid arthritis patients, tenascin-C induced synthesis of pro-inflammatory cytokines via activation of Toll-like receptor 4 (TLR4). TLR4 is one of a family of receptors that play a key role in pathogen recognition and activation of innate immunity. Stimulation of TLR4 is known to activate macrophages leading to release of TNF-α, one of the targets of existing biological agents used to treat RA patients. Previous studies had shown that mice lacking TLR4 do not show chronic joint inflammation, and blocking the interaction between tenascin-C and TLR4 may provide a new way to combat RA.

The study is published in the journal Nature Medicine.

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