Drug Discovery Opinion

Although definitive proof of a causative link between aggregation of amyloid peptides (Aβ) and Alzheimer’s disease (AD) remains elusive, much effort has been devoted to devising means of lowering Aβ levels. The ability to assess the effectiveness of such therapies in the clinic has so far been hampered by the lack of a method to determine drug effects on Aβ production or clearance from the human CNS. Aβ is produced by the action of β- and γ-secretases on amyloid precursor protein (APP) and is degraded by a number of enzymes, including insulin-degrading enzyme (IDE) and neprilysin. Inhibitors of both β- and γ-secretase have been developed as potentially disease-modifying treatments, although it is unclear whether increased production, reduced clearance, or a combination of both is responsible for elevated levels of Aβ ?in AD patients. Writing in the Annals of Neurology, researchers at Washington University School of Medicine have now described the use of a recently developed technique known as stable isotope-linked kinetics (SILK) to determine the effect of a γ-secretase inhibitor, LY450139 (semagacestat), on production of Aβ. In a double-blind study, 20 healthy volunteers were assigned to receive varying doses of LY450139 or placebo (n = 5 per group). The volunteers also received an intravenous infusion of a labelled form of the amino acid leucine which, over the course of a couple of hours, became incorporated into newly synthesised proteins, including Aβ. By periodically sampling cerebrospinal fluid (CSF), the team were able to monitor the proportion of labelled Aβ to give a measure of the rate of production. CSF sampling was continued after the labelled leucine infusion was switched off to provide a measure of the rate of Aβ clearance. The results suggested that LY450139 caused a dose-dependent decrease in Aβ production, with an 84% reduction at the highest dose (280mg). There were no differences in Aβ clearance between the placebo and drug treated groups. The SILK procedure takes 36 hours but offers the potential for clearer interpretation of drug effects since analysis of measurements of unlabelled Aβ in CSF is confounded by natural fluctuations in levels.

Ongoing clinical studies are looking at the effect of LY450139 on cognitive function and biochemical and brain imaging biomarkers in AD patients.

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The ability to selectively switch enzymes on and off in a particular tissue could offer many improved treatment options, and scientists at the University of Florida have now devised a way of achieving this goal. Writing in the journal PNAS, they describe a molecule consisting of a DNA aptamer attached by a polyethylene glycol linker to a complementary strand of DNA with azobenzene molecules attached. The aptamer was chosen to bind selectively to the enzyme thrombin and block its role in blood coagulation. In visible light, the azobenzene double bonds are in the trans-conformation and the aptamer is prevented from binding to thrombin by hybridisation with the complementary strand of DNA. When irradiated with ultraviolet light, however, the azobenzene molecules flip into the cis-conformation causing dissociation of the duplex and unveiling the aptamer which can then bind to thrombin and inhibit blood clotting.

The team hope that the technique could one day be used to cut off the blood supply to solid tumours or to produce controlled release versions of drugs which would be activated only in the target tissue, thus reducing the risk of side effects. Endoscopic lights could be used to activate the drugs or, in some cases, it may be sufficient to irradiate skin close to the target site with near-infrared light which penetrates the skin.

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Animal parasites such as malaria have complex life cycles and, so far, most attempts to control infection have centred on preventing the parasite from entering host cells. Writing in the journal Science, a team led by Dr Doron Greenbaum at the University of Pennsylvania has now focussed on an alternative treatment approach – locking the parasites inside the host cell. The team found that Plasmodium falciparum, the species responsible for the majority of human infections, and also the one that causes the most virulent form of malaria, uses a host protease to escape from cells. The protozoa replicate within a vacuole in infected cells and must escape to begin a new lytic cycle. The team used a variety of techniques to show that P falciparum makes use of host cell calpain proteases to facilitate escape.

The team were also interested to find out whether the distantly related parasite, Toxoplasma gondii adopts a similar strategy. Disease caused by T gondii infection is usually mild and self-limiting, but can be fatal to the unborn child if contracted during pregnancy. They found that in the absence of calpain, the parasites could not escape the infected cell, just as they had observed for malaria parasites.

Greenbaum plans to continue to explore the practicality of calpain as a target for anti-parasitic drugs. P falciparum has become increasingly resistant to anti-malarial drugs and targeting a host protein may afford less scope for the development of resistance. Calpains are a family of calcium-dependent cysteine proteases whose physiological roles are poorly understood.

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P-glycoprotein (Pgp), with its ability to transport a wide range of xenobiotic compounds – including drug molecules – across cell membranes, is the bane of medicinal chemists. Pgp, which probably evolved as a defense mechanism against toxic substances, is an ATP-dependent integral membrane protein that is particularly highly expressed in cells of the gut and kidney, and also in capillary endothelial cells that make up the blood-brain barrier. This means that as well as preventing absorption of drugs from the gut after oral dosing, Pgp can limit entry of drugs into the brain. Expression of Pgp by tumour cells also results in decreased accumulation of anti-cancer drugs in the cells, and contributes to multi-drug resistance in chemotherapy.

A team led by scientists at the Scripps Research Institute have now succeeded in solving the X-ray crystallographic structure of murine Pgp, a result that they hope will help chemists to design more effective drugs. The 3.8Šstructure of the apo protein revealed an internal cavity of ca 6000ų, with a 30Šseparation of the two nucleotide-binding domains. Two additional structures with bound inhibitors showed that hydrophobic and aromatic amino acids form distinct drug-binding sites which are capable of stereo-recognition. The apo and drug-bound Pgp structures are open to the cytoplasm and the inner leaflet of the lipid bilayer for drug entry, representing initial stages of the transport cycle. The overall structure of Pgp is very similar to that of the bacterial protein, MsbA, which transports lipids out of bacteria, suggesting that Pgp may work in a similar way. In the bacterial transporter, binding of ATP changes the accessibility of the carrier from cytoplasmic (inward) facing to extracellular (outward) facing so that substances caught inside the cavity are ejected from the cell. The cavities of both transporters are lined with hydrophobic amino acids, but Pgp contains a larger number of highly varied amino acids which perhaps explains its broader substrate specificity.

The study, which is published in full in the journal Science, should help chemists to better understand, if not tame, the beast.

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High-throughput screening (HTS) to identify small-molecule enzyme inhibitors is standard practice, but the substrate-based assays that are typically used are not available for poorly characterised enzymes. Writing in the journal Nature Biotechnology, scientists at the Scripps Institute have now described a substrate-free method for identifying inhibitors of such enzymes.

The new technique combines Activity-Based Protein Profiling (ABPP) with Fluorescence Polarisation (FP) to provide a system compatible with HTS methodology. ABPP utilises reactive chemical probes to covalently modify the active sites of enzymes, exploiting catalytic or recognition features of the site. By using ABPP in competitive mode, small molecules can be screened to identify those able to block the covalent modification of the enzyme by the probe. Since the probes are generally applicable to mechanistically related target proteins, parallel screening can be carried out to simultaneously identify selective inhibitors. The downside of ABPP is the low throughput nature, since readouts are typically based on one-dimensional SDS-PAGE gels. However, by combining ABPP with fluorescence polarisation technology (fluopol-ABPP), the Scripps team has greatly increased throughput.

The team reports application of fluopol-ABPP to identify the alkaloid emetine as a selective inhibitor of the hydrolytic enzyme retinoblastoma-binding protein-9 (RBBP9) and electrophilic inhibitors of glutathione S-transferase omega 1 (GSTO1).

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The first scientific discovery made independently by a robot has just been reported.

The robot, named ADAM, was developed by scientists at Aberystwyth and Cambridge Universities. Rather than a humanoid robot like Honda’s ASIMO, scientist ADAM is actually a fully automated robotic laboratory that can formulate hypotheses and then design experiments to test them. ADAM was set to work to identify genes responsible for producing orphan enzymes in yeast. Using existing knowledge and algorithms programmed by human scientists, ADAM came up with hypotheses about the genes encoding the enzymes and, without any further intervention, selected yeast mutants to carry out cell growth experiments to test his theories. ADAM was able to confirm 12 out of 20 hypotheses and trace the previously unknown genes responsible for producing the yeast enzymes. One of the orphan enzymes is involved in the synthesis of the amino acid lysine and, although it has been suggested as a target for fungicides, no one had previously identified the gene. Adam was able to suggest three possible candidate genes and all were found to produce an enzyme capable of catalysing the correct reaction.

Although robots like ADAM may never don a lab coat and join their colleagues at the bench, as automated systems develop it is likely that they will play an increasing role in making new scientific discoveries, especially those needing repetitive experiments and the manipulation of large data sets. The next robotic scientist, EVE, is being created specifically to help search for new drugs to treat tropical diseases such as malaria and schistosomiasis.

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Bisphosphonates prevent loss of bone mass and are used to treat postmenopausal osteoporosis and also to reduce bone loss in metastatic disease in cancer patients. Bisphosphonates can be divided into two sub-classes, those that do not contain nitrogen such as etidronic acid, and those that do contain nitrogen such as zoledronic acid.

Bisphosphonates that do not contain nitrogen interfere with ATP-linked energy metabolism in osteoclasts whereas the activity of nitrogenous bisphosphonates is related to their ability to inhibit farnesyl diphosphate synthase (FPPS) a key enzyme in the HMG-CoA reductase (mevalonate) pathway. Farnesyl diphosphate (FPP) is used for the post-translational prenylation of small GTPases such as Ras, and depletion of FPP is thought to be the primary mechanism for inhibition of osteoclast function. The anticancer activity of nitrogenous bisphosphonates has also been attributed to inhibition of FPPS and results from recent clinical trials in oestrogen-receptor positive breast cancer and hormone-refractory prostate cancer have been very encouraging. The transition states involved in FPP biosynthesis by FPPS are expected to be broadly similar to those of other prenyltranferases and inhibition of geranylgeranyl diphosphate synthase (GGPPS) has been suggested to be more important than inhibition of FPPS for the anti-cancer activity of bisphosphonates.

An international team led by Professor Eric Oldfield at the University of Illinois set out to design compounds that would inhibit both FPPS and GGPPS but have lower affinity for bone tissue and so be more effective in reaching other tissues. Writing in the Journal of the American Chemical Society, they describe potent inhibitors of FPPS and GGPPS that are effective in blocking tumour cell growth and invasiveness, both in vitro and in vivo. One of the compounds, BPH-715, is about 200 times more active in killing tumour cells than zoledronic acid and, since it is more lipophilic, has a lower affinity for bone. Studies showed that although BPH-175 binds to both FPPS and GGPPS, it inhibits GGPPS more strongly.

As well as inhibiting FPPS and GGPPS, bisphosphonates such as zoledronic acid and BPH-715 also stimulate γδ T-cells which help to kill tumour cells.

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Topical application of viral entry inhibitors or other microbicides is an attractive strategy to prevent sexual transmission of the human immunodeficiency virus (HIV). Griffithsin, a protein isolated from the red algae Griffithsia sp which grows off the coast of New Zealand, has been shown in vitro to be a potent HIV entry inhibitor, but the cost of production has so far hampered its development.

Writing in the March 30^th Early Edition of PNAS, a multinational team of scientists has now described a breakthrough which should allow the manufacture and isolation of significant amounts of griffithsin as an agricultural crop. Griffithsin was shown to accumulate to a level of more than 1 gram of recombinant protein per kilogram of leaf material of Nicotiana benthamiana when expressed via an infectious tobacco mosaic virus vector. Nicotiana benthamiana, which is native to Australia, is a close relative of the tobacco plant and the authors were able to produce more than 60g of pure griffithsin from a single greenhouse with an area of 5000 square feet. The biophysical characteristics of griffithsin and the nature of the plant host allowed isolation of 99% pure protein after a simple 3-step purification procedure.

The plant-produced protein was found to have broad and potent activity against a panel of primary sexually transmitted HIV-1 isolates representative of viruses prevalent in sub-Saharan Africa, Asia and the West. The recombinant griffithsin was further shown to be non-irritating and non-inflammatory, and to have no mitogenic activity. Since viral entry inhibitors are not commonly used in resource-poor countries, griffithsin produced cost-effectively in Nicotiana benthamiana plants has the potential for prevention and treatment of multi-drug resistant viral infections in developing countries.

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The World Health Organisation estimates that one third of the world’s population is latently infected with Mycobacterium tuberculosis (MTB), and that ten per cent of infected individuals will develop active disease. Current treatments for tuberculosis are effective only during active infection, and the emergence of drug-resistant strains of MTB is compromising the efficacy of existing drugs. Nicotinamide adenine dinucleotide (NAD⁺) synthetase is an attractive target for control of MTB since the enzyme is essential for survival of both active and latent mycobacteria, but drug discovery efforts against this enzyme have so far been hampered by a lack of structural information.

University of Maryland scientists have now characterised the structure and mechanism of the MTB synthetase and hope that their work will facilitate the discovery of new drugs that will be able to combat both latent and active MTB infections. All living cells need the coenzyme, NAD⁺, which regulates many physiological processes including redox reactions. A number of biochemical pathways are able to synthesise NAD⁺ but, since MTB has only two pathways – both involving NAD⁺ synthetase – and humans have pathways that avoid NAD⁺ synthetase, an inhibitor of this enzyme should be effective against MTB but have minimal side effects.

The MTB NAD⁺ synthetase is a multifunctional enzyme which catalyzes the ATP-dependent formation of NAD⁺ at the synthetase domain using ammonia derived from L-glutamine in the glutaminase domain. The study, which is published in full in Nature Structural & Molecular Biology, revealed a homooctameric subunit organization, suggesting a tight dependence of catalysis on the quaternary structure.

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The Janus family of protein tyrosine kinases is comprised of four members: JAK-1, JAK-2, JAK-3 and Tyk-2. These kinases provide membrane proximal signalling through association with type 1 and type 2 cytokine receptors, phosphorylating and activating Signal Transducers and Activators of Transcription proteins (STATs) in response to cytokine binding. Expression of JAK-3 is predominantly restricted to haematopoietic cells, whilst the other family members are ubiquitously expressed. Inhibitors of these kinases have received interest since aberrant JAK activity has been associated with a variety of hematopoietic malignancies, cardiovascular diseases and immune-related disorders. A number of clinical studies are in progress to evaluate JAK inhibitors in transplantation, myelofibrosis, polycythaemia vera and essential thrombocythaemia.

Crystal structures of the kinase domains of JAK-2 and JAK-3 have previously been solved, enabling structure-based design of inhibitors. Now collaborators at Cytopia Research and Monash University have published the high-resolution crystal structure of the JAK-1 kinase domain. Whilst the ATP-binding sites of protein kinases are highly conserved, particularly amongst family members, the researchers have identified subtle differences surrounding the JAK1 and JAK2 ATP-binding sites. There is no doubt that developing JAK-1 or JAK-2 selective inhibitors (at the ATP-site) will be challenging, but the new data at least suggest that it could be possible. Whether it is necessary or desirable to achieve such selectivity is, as yet, unclear.

Full details are published in the Journal of Molecular Biology.

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