Current Drug Targets - Volume 5, Issue 2, 2004

The value of structure analysis of proteins in rational drug design is undisputed. The ability to visualize the three dimensional structure of a receptor or enzyme provides a unique view of the residues that make up the protein's ligand binding or catalytic site. The analysis of complexes with ligands or inhibitors provides many clues on the roles that these residues play in the functioning of the molecule. The information derived from this 'picture' can be used to design the next generation of inhibitors with improved complementarity to the ligand-binding site. Whilst many other considerations such as solubility, lipophilicity, chemical stability and bioavailability also 'shape' the drug design process, crystallography can assist in designing compounds which minimize the problems of resistance that arise, for example, when drugs are used to treat infections by viruses that undergo rapid mutation. In this volume, Elspeth Garman (Oxford) and Graeme Laver (Canberra) describe the impact which structural studies have had on the design of influenza drugs. Despite the fact that influenza is a disease which affects millions of people, sometimes with fatal consequences, there has not, until recently, been any drug effective against all strains and vaccines may be relatively or totally ineffective. Screening of many thousands of compounds by pharmaceutical companies has resulted in only two compounds, amantadine and rimantidine, which target the M2 ion channel on the virus and these drugs have major disadvantages. Knowledge of the crystal structure of influenza virus neuraminidase, on the other hand, has allowed the rational design of four “plug-drugs” which bind to the active site of flu neuraminidase and stop replication of the virus. Two of these compounds, Relenza and Tamiflu, are now being used world wide and, although effective when used properly, suffer from problems of delivery. They need to be given very soon after infection to be effective, they only inhibit the influenza virus and none of the other respiratory agents which cause flu-like symptoms, and they are very expensive. The review by Leo Brady and Gus Cameron (Bristol) details the application of protein crystallography and other structural techniques to identifying novel drug targets from plasmodia, the causative agent of malaria. The current use of structure-based design in developing effective drugs for plasmodial targets is discussed. The review by Steve Wood and Simon Kolstoe (Southampton) summarises the known properties of amyloid fibres and proteins found associated with them. The role of X-ray crystallography in analyzing the amyloid proteins themselves and proteins that protect the fibres is discussed. In addition the analysis of complexes with compounds that inhibit fibre formation and compounds that inhibit the processing enzymes will be covered. In the next article, Chris Dealwis and Jonathan Wall (Knoxville) describe structural approaches to combating the pathogenicity of amyloid disease characterised by the deposition of monoclonal free immunoglobulin light chain proteins (LC) as amyloid fibrils within vital organs. Finally, in the review by Leighton Coates (Southampton) and Dean Myles (Grenoble), the manifold benefits of atomic resolution X-ray diffraction analysis of proteins are described. The ability to locate hydrogen atoms of interest and thus define the protonation states of many side chains including the active site groups is potentially of enormous benefit to the drug design process. In addition, the ability to improve definition of disordered regions of the molecule, to analyse ligands which bind with poor occupancy and to study anisotropic movements within the molecule provides further important data. Recent developments in neutron data collection mean that complementary information on proton positions can be provided for an increasing number of proteins. There are several hundred Xray crystal structures that have been refined to or beyond atomic resolution in the protein databank. A number of these structures are of proteins that are currently targets for drug discovery and these are surveyed to illustrate the benefits of atomic resolution X-ray analysis.

Despite the fact that influenza is a disease which affects millions of people, sometimes with fatal consequences, there has not, until recently, been any drug effective against all strains. Vaccines may be relatively or totally ineffective, so drugs are needed. Random screening of many thousands of compounds by pharmaceutical companies has resulted in only two compounds, amantadine and rimantidine, which target the M2 ion channel on the virus. These drugs have major disadvantages. Knowledge of the crystal structure of influenza virus neuraminidase, on the other hand, has allowed the rational design of four “plug-drugs” which bind to the active site of flu neuraminidase and stop replication of the virus. Two of these compounds, Relenza and Tamiflu, are now being used worldwide and, although effective when used properly, suffer from problems of delivery. They need to be given very soon after infection to be effective, they only inhibit the influenza virus and none of the other respiratory agents which cause flu-like symptoms, and they are very expensive.

Malaria remains a major disease of mankind, and resistance to existing therapeutics is rapidly emerging. Limited financial investment to develop new therapeutics requires the careful selection of well-defined targets from the causative parasite, Plasmodium falciparum. In these circumstances, protein crystallography can provide valuable structural detail to facilitate both the selection of suitable targets and the development of compounds to provide novel drug candidates. This review summarises the current involvement of crystallographic studies in anti-malarial drug development programmes. Protein crystallography is increasingly central to the exploitation of a number of potential Plasmodial targets, including the aspartic acid proteases (plasmepsins) and cysteine proteases (falcipains) involved in haem degradation within the parasite food vacuole. Lead compounds are being identified from collections previously synthesised against homologous human enzymes. Plasmodium have an unusual dependence on the glycolytic pathway relative to their human hosts, and this is reflected in subtle structural differences identified in the crystal structures of a number of parasite glycolytic enzymes including aldolase and lactate dehydrogenase. Other enzymes from a range of biosynthetic pathways have also been targeted in crystallographic studies. These include dihydrofolate reductase, the target of existing anti-folate therapeutics, and enoyl reductase from the fatty acid biosynthesis pathway which is already the target of effective bacteriocides. Crystal structures of these drug-enzyme complexes not only allow visualisation and improvement of inhibitor-protein contacts, but in the former case have also been used to probe the molecular basis of emerging antimalarial drug resistance. Crystallography is similarly proving valuable as a tool to facilitate the development of inhibitors of purine salvage, isoprenoid synthesis and utilisation, and protein processing mechanisms.

Amyloid fibres are stable, persistent and highly ordered aggregates of mis-folded protein that accumulate in tissues and are a prominent feature of the pathology of a wide range of human diseases. The presumed role of amyloid as a causative factor of tissue damage is based largely on 'guilt by association'. However, growing understanding of the nature of amyloid, its formation by a nucleated growth mechanism from destabilised and partially unfolded precursors and its persistence at sites of deposition has provided the foundation for the development of approaches to inhibit amyloid formation and enable its clearance. In spite of intensive study, our understanding of the detailed structure of amyloid itself remains incomplete although 'crossed-β' structure is clearly a common constituent. On the other hand detailed structural understanding of transthyretin, β-secretase and serum amyloid P component is contributing to the design of small molecule compounds to target amyloid. Thyroxin mimetics stabilise the native tetrameric protein structure. β-secretase inhibitors will limit the production of the amyloidogenic Aβ1-42 polypeptide. Compounds that crosslink serum amyloid P component rapidly deplete the plasma and amyloid-βound pool of this protein. The efficacy of these compounds as drugs to prevent formation or enable removal of amyloid will provide a stringent test of the 'amyloid hypothesis' of disease.

Immunoglobulin light chain (LC) proteins exhibit the greatest sequence variability of all proteins associated with amyloid disease. The hallmark event in amyloidogenesis is a change in the secondary and / /or tertiary structure of a normal, soluble protein, that fosters selfaggregation and fibril formation. The structural heterogeneity of light chain proteins has hampered understanding of the precise mechanisms involved in fibril formation. The development of effective therapeutics will be benefited by a fundamental understanding of mechanisms and structural prerequisites which govern amyloidogenesis. This review focuses on light chain (AL) amyloidosis resulting from the aggregation of κ and λ LCs. Specifically the thermodynamic and structural data of several WT and mutant amyloidogenic LCs have been carefully examined. Moreover, we discuss the importance of hydrophobic and ionic interactions on amyloidosis by comparing several available three-dimensional structures of amyloidogenic and highly homologous non-amyloidogenic proteins that can be destabilized to become amyloidogenic by site specific mutations.

The number of protein crystal structures being refined to atomic resolution is increasing each year as well as the size of proteins being studied. There are currently 346 structures in the protein data bank which have been refined to or beyond atomic resolution. The benefits of atomic resolution X-ray data are discussed along with a number of structural examples of biomedically relevant proteins. The complementary role of neutron diffraction will also be discussed.

Cancer is a growing problem for human health world-wide. Dramatic breakthroughs have increased our understanding of the molecular mechanisms involved in the process of tumorigenesis, allowing us to develop more refined anti-cancer treatments, expanding the repertoire of available anti-cancer drugs, and increasing the efficiency of their delivery to malignant cells. Nevertheless, even with improved understanding of the complex origins of cancer cells, there is a dearth of efficient and above all specific anti-cancer treatments. Apoptin (viral protein 3 - VP3), a gene product derived from the Chicken Anaemia Virus (CAV) represents a novel anti-cancer tool. It appears to have innate tumour-specific p53-independent, Bcl-2-enhanced proapoptotic activity, and hence may be of great utility in the endeavour to achieve specific and efficient elimination of cancer cells, particularly in cases of drug resistance through Bcl-2 overexpression / loss of p53 function etc. This review will examine the unique aspects of apoptin's properties, and in particular, its ability to localise specifically in the nucleus of transformed but not normal cells. The latter ability, importantly, appears to be integrally related to its tumour-specific pro-apoptotic action.

DNA polymerase is one of the most important target molecules of antitumor agents, especially for antimetabolite nucleosides that include 1-β-D-arabinofuranosylcytosine (araC) and 2'-deoxy-2',2'-difluorocytidine (gemcitabine). There are several subtypes of mammalian DNA polymerases and their localization and function have been clarified. DNA polymerase α, δ and ε have been implicated to be responsible for DNA replication, whereas DNA polymerase β, δ and e have been suggested to work in DNA repair. DNA polymerase γ is encoded in the nucleus but localizes in the mitochondria, and is responsible for the mitochondrial DNA replication. Recently, several antiviral nucleoside analogs were reported to inhibit DNA polymerase g after intracellular phosphorylation and cause severe chronic toxicity. 1-(2-Deoxy-2-fluoro-4-thio-β-D-arabinofuranosyl)cytosine (4'-thio-FAC), an antimetabolite similar to araC and gemcitabine, is recently shown by us to be a very promising agent because of its potent antitumor activity by oral administration to mice. We tested for the inhibitory activities of the triphosphates of 4'-thio-FAC and gemcitabine against DNA polymerase α, β and γ. The triphosphates of 4'-thio-FAC (4'-thio-FACTP) exhibited the potent inhibitory action against DNA polymerase a, whereas it showed moderate inhibition against DNA polymerase β and little inhibition against DNA polymerase γ. The triphosphate of gemcitabine (dFdCTP) did not show potent inhibition against these three DNA polymerases. Thus, the effect on ribonucleotide reductase was suggested to be more responsible for the antitumor action of gemcitabine. The differences in the mechanisms of action against DNA polymerases between these drugs and other nucleosides were also discussed.

Parkinson's disease (PD) is a serious motor disorder and it is the second most common brain degenerative disease in human. PD is known to be caused by degeneration of dopamine neurons in the substantia nigra but the cause of cell death is largely unknown. Mammalian neurokinins [NKs] are a group of neuropeptides that include substance P (SP; neurokinin-1, NK-1), substance K (SK; NK-2; neurokinin A), and neuromedin K (NK; NK-3; neurokinin B). Their biological effects as neurotransmitters, neuromodulators, or neurotrophic-like factors are mediated by three distinct neurokinin receptors, namely SP receptor (SPR: NK-1 receptor, NK-1R), SKR (NK-2R), and NKR (NK-3R). Several lines of evidence have indicated that neurokinins are implicated in the pathogenesis of PD. First, decreases of SP level and SP-immunoreactivity have been found in nigral and striatal tissues of animals with PD and postmortem PD patients. Second, NKs exert neuroprotective effects on neurons. In addition, NK receptors, namely NK-1 and NK-3 receptors, are abundantly localized in dopaminergic and cholinergic neurons of the basal ganglia, indicating that these neurons are under the physiological regulation of NKs. Moreover, modulation in motor activity occurred in 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP)-treated mice, PD animal model, after systemic administration of NK receptor agonists. NKs and NK receptors, therefore, might be important molecules that are associated with functions and survival of neurons in the basal ganglia, in particular the dopamine neurons. Further studies should be devoted to elucidate the functional roles of NK systems in (a) the neuropathogenesis and neuroprotection during the course of PD, (b) the efficacy of NK receptor drugs towards PD, and (c) potential therapeutic intervention that targets at the prevention or treatment of PD.