bluuebunny/crossref_metadata_2025 huggingface dataset:
Genomics in target and drug discovery
Authors: van Duin M. , Woolson H. , Mallinson D. , Black D.
Published: June 2004

Genomics-based discovery of novel therapeutic drug targets requires the design of well-controlled biological or pharmacological experiments with experimental questions and hypotheses that relate to the therapeutic area of interest. This will aid the validation level of differentially expressed genes and hence facilitate the de-selection of the genes that are identified in microarray experiments. We here provide an example of how this approach is followed in the manipulation of human macrophage foam cells towards the discovery of novel drug targets for treatment of atherosclerosis.
The role of therapeutic antibodies in drug discovery
Authors: Stockwin L.H. , Holmes S.
Published: June 2004

The last 5 years have seen a major upturn in the fortune of therapeutic monoclonal antibodies (mAbs), with nine mAbs approved for clinical use during this period and more than 70 now in clinical trials beyond phase II. Sales are expected to reach $4 billion per annum worldwide in 2002 and $15 billion by 2010. This success can be related to the engineering of mouse mAbs into mouse/human chimaeric antibodies or humanized antibodies, which have had a major effect on immunogenicity, effector function and half-life. The issue of repeated antibody dosing at high levels with limited toxicity was essential for successful clinical applications. Emerging technologies (phage display, human antibody-engineered mice) have created a vast range of novel, antibody-based therapeutics, which specifically target clinical biomarkers of disease. Modified recombinant antibodies have been designed to be more cytotoxic (toxin delivery), to enhance effector functions (bivalent mAbs) and to be fused with enzymes for prodrug therapy and cancer treatment. Antibody fragments have also been engineered to retain specificity and have increased the penetrability of solid tumours (single-chain variable fragments). Radiolabelling of antibodies has now been shown to be effective for cancer imaging and targeting. This article focuses on developments in the design and clinical use of recombinant antibodies for cancer therapy.
A new paradigm for drug discovery: integrating clinical, genetic, genomic and molecular phenotype data to identify drug targets
Authors: Schadt E.E. , Monks S.A. , Friend S.H.
Published: June 2004

Application of statistical genetics approaches to variations in mRNA transcript abundances in segregating populations can be used to identify genes and pathways associated with common human diseases. The combination of this genetic information with gene expression and clinical trait data can also be used to identify subtypes of a disease and the genetic loci specific to each subtype. Here we highlight results from some of our recent work in this area and further explore the many possibilities that exist in employing a more comprehensive genetics and functional genomics approach to the functional annotation of genomes, and in applying such methods to the validation of targets for complex traits in the drug discovery process.
A study of mitochondrial DNA mutations in peripheral lymphocytes in an aging cohort
Authors: Zhang B. , Ye S. , Sayer A.A. , Hammans S.R. , Adio S. , Hinks L.J. , Smythe P.J. , Groot D. , Cooper C. , Day I.N.M.
Published: June 2004

Somatic mutation in the mitochondrial genome occurs much more rapidly than in the nuclear genome and is a feature, possibly contributory, of the aging of cells and tissues. Identifying mitochondrial sequence changes in blood DNA of elderly subjects may provide a maker for the epigenetic changes of mitochondrial DNA known to occur in tissues with lower cellular turnover, and would also have implications for immunosenescence. No large-scale epidemiological studies have been reported previously. In this study we have established long-PCR banks of the mitochondrial genome from peripheral lymphocytes for an elderly cohort of 716 individuals with a range of measured aging phenotypes, and we have established assays for three widely reported mutations: the 4977 bp and 8048 bp deletions and point mutation A3243G. No individuals were identified with detectable heteroplasmy for these changes. Implications for tissue and population prevalence are discussed. The mitochondrial long-PCR DNA banks established will be useful for a wide range of studies of somatic mutation and of germline haplotypes in relation to aging.
Effects of age on antibody affinity maturation
Authors: Dunn-Walters D.K. , Banerjee M. , Mehr R.
Published: June 2004

The elderly are more susceptible to infectious diseases. Mortality and morbidity from infections increase sharply over the age of 65 years. At the same time, the efficacy of vaccinations in the elderly is decreased. The elderly also have an increased incidence of cancer and inflammatory diseases. All the above indicate an age-related dysregulation of the immune system. Evidence suggests that the change in the humoral immune response with age is a qualitative rather than a quantitative one, i.e. it is the affinity and specificity of the antibody that changes, rather than the quantity of antibody produced. There are a number of possible causes of this failure, one of which is a defect in the mechanism of hypermutation of immunoglobulin genes. We have studied individual clonal responses within germinal centres of spleen and Peyer's patches in young and old patient groups. Our results indicate that there is no difference in the actual mechanism of hypermutation with age. There are, however, differences that are due either to a change in selection processes or to a change in the founder cells available for activation.
Osteoporosis: pathogenesis and clinical intervention
Authors: Mueller G. , Russell R.G.G.
Published: June 2004

Osteoporosis is a very common disorder and much has been learnt in recent years about the many pathogenic processes that contribute to bone loss and fragility. Drug treatments are now available to prevent bone loss and reduce fracture, and there are prospects for modifying some of the pathogenic processes themselves. In common with other structures, the tissues of the musculoskeletal system undergo many changes with aging, and some of the commonest skeletal disorders are seen in the elderly. The changes in bone lead to osteoporosis and fractures, whereas muscle changes (sarcopenia) contribute to frailty, and changes in cartilage lead to osteoarthritis.
An interaction map of proteasome subunits
Authors: Coux O.
Published: June 2004

Despite the central role of the 26 S proteasome in eukaryotic cells, many facets of its structural organization and functioning are still poorly understood. To learn more about the interactions between its different subunits, as well as its possible functional partners in cells, we performed, with Marc Vidal's laboratory (Dana-Farber Cancer Institute, Boston, MA, U.S.A.), a systematic two-hybrid analysis using Caenorhaditis elegans 26 S proteasome subunits as baits [Davy, Bello, Thierry-Mieg, Vaglio, Hitti, Doucette-Stamm, Thierry-Mieg, Reboul, Boulton, Walhout et al. (2001) EMBO Rep. 2, 821–828]. A pair-wise matrix of all subunit combinations allowed us to detect numerous possible intra-complex interactions, among which some had already been reported by others and eight were novel. Interestingly, four new interactions were detected between two ATPases of the 19 S regulatory complex and three α-subunits of the 20 S proteolytic core. Possibly, these interactions participate in the association of these two complexes to form the 26 S proteasome. Proteasome subunit sequences were also used to screen a cDNA library to identify new interactors of the complex. Among the interactors found, most (58) have no clear connection to the proteasome, and could be either substrates or potential cofactors of this complex. Few interactors (7) could be directly or indirectly linked to proteolysis. The others (12) interacted with more than one proteasome subunit, forming ‘interaction clusters’ of potential biological interest.
Protecting p53 from degradation
Authors: Laín S.
Published: June 2004

Inactivation of the p53 function is a common event in cancer. Approx. 50% of human tumours express mutant p53 and there is evidence that in others, including many childhood tumours, p53 function is impaired in other ways. These defects on p53 function may be due to the alteration of cellular factors that modulate p53 or to the expression of viral oncoproteins. Radiotherapy and many of the chemotherapeutic drugs currently used in cancer treatment are potent activators of p53. However, most of these therapies have a serious drawback; that is, the long-term consequences of their DNA-damaging effects. Understanding the mechanisms regulating p53 stability is crucial for the development of new strategies to activate p53 non-genotoxically. Here we describe the effect of a potent activator of the p53 response, the nuclear export inhibitor leptomycin B, on Mdm2 degradation and we provide evidence for the oligomerization of the p14ARF tumour suppressor and Mdm2 inhibitor in response to oxidative stress.
Electron transfer in human cytochrome P450 reductase
Authors: Gutierrez A. , Grunau A. , Paine M. , Munro A.W. , Wolf C.R. , Roberts G.C.K. , Scrutton N.S.
Published: June 2004

Cytochrome P450 reductase (CPR) is a diflavin enzyme responsible for electron donation to mammalian cytochrome P450 enzymes in the endoplasmic reticulum. Dissection of the enzyme into functional domains and studies by site-directed mutagenesis have enabled detailed characterization of the mechanism of electron transfer using stopped-flow and equilibrium-perturbation methods, and redox potentiometry. These studies and the mechanism of electron transfer in CPR are reported herein.
The role of iron and 2-oxoglutarate oxygenases in signalling
Authors: Hewitson K.S. , McNeill L.A. , Elkins J.M. , Schofield C.J.
Published: June 2004

Sensing of ambient dioxygen levels and appropriate feedback mechanisms are essential processes for all multicellular organisms. In animals, moderate hypoxia causes an increase in the transcription levels of specific genes, including those encoding vascular endothelial growth factor and erythropoietin. The hypoxic response is mediated by hypoxia-inducible factor (HIF), an αβ heterodimeric transcription factor in which both the HIF subunits are members of the basic helix–loop–helix PAS (PER-ARNT-SIM) domain family. Under hypoxic conditions, levels of HIFα rise, allowing dimerization with HIFβ and initiating transcriptional activation. Two types of dioxygen-dependent modification to HIFα have been identified, both of which inhibit the transcriptional response. Firstly, HIFα undergoes trans-4-hydroxylation at two conserved proline residues that enable its recognition by the von Hippel-Lindau tumour-suppressor protein. Subsequent ubiquitinylation, mediated by an ubiquitin ligase complex, targets HIFα for degradation. Secondly, hydroxylation of an asparagine residue in the C-terminal transactivation domain of HIFα directly prevents its interaction with the co-activator p300. Hydroxylation of HIFα is catalysed by enzymes of the iron(II)- and 2-oxoglutarate-dependent dioxygenase family. In humans, three prolyl hydroxylase isoenzymes (PHD1–3) and an asparagine hydroxylase [factor inhibiting HIF (FIH)] have been identified. The role of 2-oxoglutarate oxygenases in the hypoxic and other signalling pathways is discussed.
Haem-oxygen reactive intermediates: catalysis by the two-step
Authors: Makris T.M. , Denisov I.G. , Sligar S.G.
Published: June 2004

The catalytic schemes of a variety of haem enzymes, including the P450 mono-oxygenases, consist of a number of common reactive haem-oxygen adducts. The characterization of these intermediates by optical and EPR spectroscopies has reinforced the similarity of these intermediate states in a number of haem enzyme systems. Furthermore, the reactivity of these states in P450 and horseradish peroxidase, in which multiple potent oxidants are formed, provides a paradigm for many other haem enzymes.
Mapping the conformational itinerary of β-glycosidases by X-ray crystallography
Authors: Davies G.J. , Ducros V.M.-A. , Varrot A. , Zechel D.L.
Published: June 2004

The conformational agenda harnessed by different glycosidases along the reaction pathway has been mapped by X-ray crystallography. The transition state(s) formed during the enzymic hydrolysis of glycosides features strong oxocarbenium-ion-like character involving delocalization across the C-1–O-5 bond. This demands planarity of C-5, O-5, C-1 and C-2 at or near the transition state. It is widely, but incorrectly, assumed that the transition state must be 4H3 (half-chair). The transition-state geometry is equally well supported, for pyranosides, by both the 4H3 and 3H4 half-chair and 2,5B and B2,5 boat conformations. A number of retaining β-glycosidases acting on gluco-configured substrates have been trapped in Michaelis and covalent intermediate complexes in 1S3 (skew-boat) and 4C1 (chair) conformations, respectively, pointing to a 4H3-conformed transition state. Such a 4H3 conformation is consistent with the tight binding of 4E- (envelope) and 4H3-conformed transition-state mimics to these enzymes and with the solution structures of compounds bearing an sp2 hybridized anomeric centre. Recent work reveals a 1S5 Michaelis complex for β-mannanases which, together with the 0S2 covalent intermediate, strongly implicates a B2,5 transition state for β-mannanases, again consistent with the solution structures of manno-configured compounds bearing an sp2 anomeric centre. Other enzymes may use different strategies. Xylanases in family GH-11 reveal a covalent intermediate structure in a 2,5B conformation which would also suggest a similarly shaped transition state, while 2S0-conformed substrate mimics spanning the active centre of inverting cellulases from family GH-6 may also be indicative of a 2,5B transition-state conformation. Work in other laboratories on both retaining and inverting α-mannosidases also suggests non-4H3 transition states for these medically important enzymes. Three-dimensional structures of enzyme complexes should now be able to drive the design of transition-state mimics that are specific for given enzymes, as opposed to being generic or merely fortuitous.
Molecular mechanism for inhibition of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase by rosuvastatin
Authors: Holdgate G.A. , Ward W.H.J. , McTaggart F.
Published: June 2004

The statins are inhibitors of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase (HMG-CoAR), and are utilized to decrease levels of atherogenic lipoproteins in patients with, or who are at high risk of, cardiovascular disease. This study describes the inhibition of a recombinant, catalytic fragment of human HMG-CoAR by a new statin, rosuvastatin (CRESTOR®). Binding is reversible and involves an initial complex [inhibition constant involving the enzyme–inhibitor complex (E.I), Ki, ≈1 nM], which undergoes a slow transition (t1/2 to reach steady state is 33–360 s) to give tighter association [steady-state inhibition constant involving E.I and the second E.I complex in a two-step mechanism (E.I*), Ki*, ≈0.1 nM]. At steady state, rosuvastatin is at least as potent as atorvastatin, cerivastatin and simvastatin. It is more potent than fluvastatin and pravastatin. For rosuvastatin, inhibition kinetics are competitive with respect to HMG-CoA and non-competitive when NADPH is varied. At 37°C, binding is linked to a large favourable enthalpy change [ ΔH° =−69.0 kJ/mol (−16.5 kcal/mol)] and a small entropic penalty [T ΔS°=−9.6 kJ/mol (−2.3 kcal/mol)]. These characteristics, and the high affinity relative to that of 3S-HMG-CoA (Kd≈6.6 μM), are discussed in relation to the crystal structures of complexes with HMG-CoAR.
A structural perspective on the enzymes that convert dTDP-d-glucose into dTDP-l-rhamnose
Authors: Dong C. , Beis K. , Giraud M.-F. , Blankenfeldt W. , Allard S. , Major L.L. , Kerr I.D. , Whitfield C. , Naismith J.H.
Published: June 2004

Bacteria have a rich collection of biochemical pathways for the synthesis of complex metabolites. These conversions often involve chemical reactions that are hard to reproduce in the laboratory. An area of considerable interest is in the manipulation and synthesis of carbohydrates. In contrast with amino acids, carbohydrates are densely functionalized (each carbon atom is attached to at least one heteroatom) and this holds out the prospect of discovering novel enzyme mechanisms. The results from the study of the biosynthetic dTDP-l-rhamnose pathway are discussed. dTDP-l-rhamnose is a key intermediate in many pathogenic bacteria, as it is the donor for l-rhamnose, which is found in the cell wall of important human pathogens, such as Mycobacteria tuberculosis and Salmonella typhimurium. All four enzymes have been structurally characterized; in particular, the acquisition of structural data on substrate complexes was extremely useful. The structural data have guided site-directed-mutagenesis studies that have been used to test mechanistic hypotheses. The results shed light on three classes of enzyme mechanism: nucleotide condensation, short-chain dehydrogenase activity and epimerization.
Structure and reactivity in the non-mevalonate pathway of isoprenoid biosynthesis
Authors: Hunter W.N. , Bond C.S. , Gabrielsen M. , Kemp L.E.
Published: June 2004

The function, structure and mechanism of two Escherichia coli enzymes involved in the non-mevalonate route of isoprenoid biosynthesis, 2C-methyl-d-erythritol 4-phosphate cytidylyltransferase and 2C-methyl-d-erythritol 2,4-cyclodiphosphate synthase, are reviewed. Comparisons of each with enzymes from microbial pathogens highlight important conservation of sequence suggestive of similarities in secondary structure, subunit folds, quaternary structure and active sites. Since both enzymes are validated drug targets, the models provide templates for structure-based design of anti-microbial agents targeting a number of serious human diseases.
Twists and turns: a tale of two shikimate-pathway enzymes
Authors: Brown K.A. , Carpenter E.P. , Watson K.A. , Coggins J.R. , Hawkins A.R. , Koch M.H.J. , Svergun D.I.
Published: June 2004

We are studying two enzymes from the shikimate pathway, dehydroquinate synthase (DHQS) and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Both enzymes have been the subject of numerous studies to elucidate their reaction mechanisms. Crystal structures of DHQS and EPSPS in the presence and absence of substrates, cofactors and/or inhibitors are now available. These structures reveal movements of domains, rearrangements of loops and changes in side-chain positions necessary for the formation of a catalytically competent active site. The potential for using complementary small-angle X-ray scattering (SAXS) studies to confirm the presence of these structural differences in solution has also been explored. Comparative analysis of crystal structures, in the presence and absence of ligands, has revealed structural features critical for substrate-binding and catalysis. We have also analysed these structures by generating GRID energy maps to detect favourable binding sites. The combination of X-ray crystallography, SAXS and computational techniques provides an enhanced analysis of structural features important for the function of these complex enzymes.
Experiences with the shikimate-pathway enzymes as targets for rational drug design
Authors: Coggins J.R. , Abell C. , Evans L.B. , Frederickson M. , Robinson D.A. , Roszak A.W. , Lapthorn A.P.
Published: June 2004

The background and current context of work on the shikimate-pathway enzymes as potential targets for anti-bacterial, anti-fungal and anti-parasitic drugs is reviewed. Recent work on the third enzyme of the pathway, dehydroquinase, which occurs in two structurally and mechanistically distinct forms, is used to illustrate the present state of studies into rational drug design.
A two-faced molecule offers NO explanation: the proximal binding of nitric oxide to haem
Authors: Lawson D.M. , Stevenson C.E.M. , Andrew C.R. , George S.J. , Eady R.R.
Published: June 2004

Cytochrome c´ (cyt c´) is found in the periplasmic space of denitrifying bacteria where it is thought to mediate the transfer of NO between the nitrogen-cycle enzymes dissimilatory nitrite reductase and nitric oxide reductase. It contains a 5-coordinate (5c) His-ligated haem that shares spectroscopic and ligand-binding properties with the haem group in the sensory domain of soluble guanylate cyclase (sGC). The latter is an extremely important enzyme involved in the control of vasodilation and blood clotting. Curiously, the enzyme is activated up to 200-fold by the binding of NO to the haem, whereas the binding of CO gives rise to only a mild stimulation of activity. Through X-ray crystallography we have studied NO and CO binding to cyt c´. CO binds to the distal face to give a 6-coordinate (6c) adduct. By contrast, NO binding gives rise to a 5c adduct through the displacement of the proximal His, to give a novel and unexpected proximal binding mode for NO. These results are also supported by a range of spectroscopies. In the absence of a crystal structure for sGC we propose that cyt c´ provides a structural model for the haem domain of this enzyme and thereby helps to explain the differential effects of NO and CO on its activity.
Engineering substrate recognition in catalysis by cytochrome P450cam
Authors: Bell S.G. , Chen X. , Xu F. , Rao Z. , Wong L.-L.
Published: June 2004

We have a continuing interest in applying the current knowledge of cytochrome P450cam substrate recognition to engineer the enzyme for the biotransformation of unnatural substrates with the long-term aim of applications in the synthesis of fine chemicals and bioremediation of environmental contaminants. Comparisons of the structure of target substrates with that of camphor, the natural substrate, led to the design of active-site mutants with greatly enhanced activity for the oxidation of chlorinated benzenes and selectivity of (+)-α-pinene oxidation. The crystal structures of the F87W/Y96F/V247L mutant with 1,3,5-trichlorobenzene or (+)-α-pinene bound have revealed the enzyme–substrate contacts and provided insights into the activity and selectivity patterns. The structures have also provided a novel basis for further engineering of P450cam for increased activity in the oxidation of the highly inert pentachlorobenzene and hexachlorobenzene, and increased selectivity of (+)-α-pinene oxidation.
Aspects of tuberous sclerosis complex (TSC) protein function in the brain
Authors: Ramesh V.
Published: June 2004

Tuberous sclerosis complex (TSC), an autosomal dominant disease caused by mutations in either TSC1 or TSC2, is characterized by the development of hamartomas in a variety of organs. Concordant with the tumour-suppressor model, loss of heterozygosity (LOH) is known to occur in these hamartomas at both TSC1 and TSC2 loci. LOH has been documented in renal angiomyolipomas, but loss of the wild-type allele in cortical tubers appears very uncommon. We analysed 24 hamartomas from 10 patients for second-hit mutations by several methods, and found no evidence for the inactivation of the second allele in many of the central nervous system (CNS) lesions, including tumours that appear to be clonally derived. We believe that somatic mutations in TSC1 and TSC2 resulting in the loss of wild-type alleles may not be necessary in some tumour types, and other mechanisms may contribute to tumorigenesis in this setting. We have shown that hamartin interacts with neurofilament light chain (NF-L) and could integrate the neuronal cytoskeleton through its direct interaction with NF-L and ERM (ezrin/radixin/moeisin) proteins. Our unpublished work further documents the binding of tuberin with Pam, a protein associated with c-Myc, which is enriched in brain. All these observations suggest that the tuberin–hamartin complex is likely to have distinct functions in the CNS.
Regulation of tuberous sclerosis complex (TSC) function by 14-3-3 proteins
Authors: Nellist M. , Goedbloed M.A. , Halley D.J.J.
Published: June 2004

Tuberous sclerosis complex (TSC) is a genetic disorder characterized by seizures, mental disability, renal dysfunction and dermatological abnormalities. The disease is caused by inactivation of either hamartin or tuberin, the products of the TSC1 and TSC2 tumour-suppressor genes. Hamartin and tuberin form a complex and antagonise phosphoinositide 3-kinase/protein kinase B/target of rapamycin signal transduction by inhibiting p70 S6 kinase, an activator of translation, and activating 4E-binding protein 1, an inhibitor of translation initiation. Phosphorylation-dependent binding between tuberin and members of the 14-3-3 protein family indicates how the tuberin–hamartin complex may interact with upstream and downstream effectors, and suggests how phosphorylation-dependent regulation of the complex may be controlled.
TSC1 and TSC2: genes that are mutated in the human genetic disorder tuberous sclerosis
Authors: Sampson J.R.
Published: June 2004

The tuberous sclerosis complex genes TSC1 and TSC2 were first identified by positional cloning strategies in the heritable human disorder tuberous sclerosis. They encode previously unknown proteins, termed hamartin and tuberin respectively, that form a functional complex. The phenotypic manifestations of tuberous sclerosis are extremely diverse and suggest normal roles for TSC1 and TSC2 in regulating the growth, proliferation, migration and differentiation of many cell types. Investigations of TSC1 and TSC2 in a number of model organisms and cell-culture systems have provided new insights into the mechanisms through which these roles are effected. Most promisingly, the hamartin–tuberin complex has been shown to function as a negtive regulator of the insulin receptor/phosphoinositide 3-kinase/S6 kinase pathway. Drugs that act to inhibit this pathway may have therapeutic potential for tuberous sclerosis and the related disorder lymphangioleiomyomatosis.
Targeting metabolic pathways in microbial pathogens: oxidative stress and anti-folate drug resistance in trypanosomatids
Authors: Hunter W.N. , Alphey M.S. , Bond C.S. , Schüttelkopf A.W.
Published: June 2004

The large quantity of genomic, biochemical and metabolic data on microbial pathogens provides information that helps us to select biological problems of interest and to identify targets, metabolic pathways or constituent enzymes, for therapeutic intervention. One area of potential use in developing novel anti-parasitic agents concerns the regulation of oxidative stress, and we have targeted the trypanothione peroxidase pathway in this respect. In order to characterize this pathway, we have determined crystal structures for each of its components, and are now studying enzyme–ligand complexes of the first enzyme, trypanothione reductase. Also with regard to trypanosomatids, a question that arose was: why do anti-folates not provide useful therapies? The enzyme pteridine reductase has been shown to contribute to anti-folate drug resistance, and we have determined the enzyme structure and mechanism to understand this aspect of drug resistance.
In silico approaches to predicting drug metabolism, toxicology and beyond
Authors: Ekins S.
Published: June 2004

The discovery and optimization of new drug candidates is becoming increasingly reliant upon the combination of experimental and computational approaches related to drug metabolism, toxicology and general biopharmaceutical properties. With the considerable output of high-throughput assays for cytochrome-P450-mediated drug–drug interactions, metabolic stability and assays for toxicology, we have orders of magnitude more data that will facilitate model building. A recursive partitioning model for human liver microsomal metabolic stability based on over 800 structurally diverse molecules was used to predict molecules with known log in vitro clearance data (Spearman's rho −0.64, P<0.0001). In addition, with solely published data, a quantitative structure–activity relationship for 66 inhibitors of the potassium channel human ether-a-gogo (hERG) that has been implicated in the failure of a number of recent drugs has been generated. This model has been validated with further published data for 25 molecules (Spearman's rho 0.83, P<0.0001). If continued value is to be realized from these types of computational models, there needs to be some applied research on their validation and optimization with new data. Some relatively simple approaches may have value when it comes to combining data from multiple models in order to improve and focus drug discovery on the molecules most likely to succeed.
Arylamine N-acetyltransferases: a pharmacogenomic approach to drug metabolism and endogenous function
Authors: Sim E. , Pinter K. , Mushtaq A. , Upton A. , Sandy J. , Bhakta S. , Noble M.
Published: June 2004

The arylamine N-acetyltransferases (NATs) are a unique family of enzymes that catalyse the transfer of an acetyl group from acetyl-CoA to the terminal nitrogen of hydrazine and arylamine drugs and carcinogens. The NATs have been shown to be important in drug detoxification and carcinogen activation, with humans possessing two isoenzymes encoded by polymorphic genes. This polymorphism has pharmacogenetic implications, leading to different rates of inactivation of drugs, including the anti-tubercular agent isoniazid and the anti-hypertensive drug hydralazine. Mice provide a good model for human NAT, allowing genetic manipulation of expression to explore possible endogenous roles of these enzymes. The first three-dimensional NAT structure was resolved for NAT from Salmonella typhimurium, and subsequently the structure of NAT from Mycobacterium smegmatis has been elucidated. These identified a ‘Cys-His-Asp’ catalytic triad (conserved in all NATs), which is believed to be responsible for the activation of the active site cysteine residue. As more genomic data become available, NAT homologues continue to be found in prokaryotic species, many of which are pathogenic, including Mycobacterium tuberculosis. The discovery of NAT in M. tuberculosis is particularly significant, since this enzyme participates in inactivation of isoniazid in the bacterium, with implications for isoniazid resistance. Structural studies on NAT proteins and phenotypic analyses of organisms (both mice and prokaryotes) following genetic modifications of the nat genes are leading to an understanding of the potentially diverse roles of NAT in endogenous and xenobiotic metabolism. These studies have indicated that NAT, particularly in Mycobacteria, has the potential to be a drug target. Combinatorial chemical approaches, together with in silico structural studies, will allow for advances in the identification of NAT substrates and inhibitors, both as experimental tools and as potential drugs.
Cytochromes P450: novel drug targets in the war against multidrug-resistant Mycobacterium tuberculosis
Authors: Munro A.W. , McLean K.J. , Marshall K.R. , Warman A.J. , Lewis G. , Roitel O. , Sutcliffe M.J. , Kemp C.A. , Modi S. , Scrutton N.S. , Leys D.
Published: June 2004

Novel drug strategies are desperately needed to combat the global threat posed by multidrug-resistant strains of Mycobacterium tuberculosis (Mtb). The genome sequence of Mtb has revealed an unprecedented number of cytochrome P450 enzymes in a prokaryote, suggesting fundamental physiological roles for many of these enzymes. Several azole drugs (known inhibitors of cytochromes P450) have been shown to have potent anti-mycobacterial activity, and the most effective azoles have extremely tight binding constants for one of the Mtb P450s (CYP121). The structure of CYP121 has been determined at atomic resolution, revealing novel features of P450 structure, including mixed haem conformations and putative proton-relay pathways from protein surface to haem iron. The structure provides both a platform for investigation of structure/mechanism of cytochrome P450, and for design of inhibitor molecules as novel anti-tubercular agents.
Circular dichroism and synchrotron radiation circular dichroism spectroscopy: tools for drug discovery
Authors: Wallace B.A. , Janes Robert W.
Published: June 2004

CD spectroscopy is an established and valuable technique for examining protein structure, dynamics and folding. Because of its ability to sensitively detect conformational changes, it has important potential for drug discovery, enabling screening for ligand and drug binding, and detection of potential candidates for new pharmaceuticals. The binding of the anti-tumour agent Taxol to the anti-apoptosis protein Bcl-2 [Rodi, Janes, Sanganee, Holton, Wallace and Makowski (1999) J. Mol. Biol. 285, 197–204] and the binding of the anti-epileptic drug lamotrigine to voltage-gated sodium channels [Cronin, O'Reilly, Duclohier and Wallace (2003) J. Biol. Chem. 278, 10675–10682] are used as examples to show changes detectable by CD involving secondary structure, and are contrasted with the binding of the agonist carbamylcholine to acetylcholine receptors [Mielke and Wallace (1988) J. Biol. Chem. 263, 8177–8182], an example where binding does not involve a secondary structural change. Synchrotron radiation CD spectroscopy offers significant enhancements with respect to conventional CD spectroscopy, which will enable its usage for high-throughput screening and as a tool in ‘chemical genomics’ or ‘reverse chemical genetics’ strategies for ligand identification. The lower wavelength data available enable more detailed, sensitive and accurate detection, the higher light intensity permits much smaller amounts of both proteins and drug candidates to be used in the screening, and future technological developments in sample handling and detection should enable automated high-throughput screening to be performed.
Nicotinic acetylcholine receptors: α-conotoxins as templates for rational drug design
Authors: Janes Robert W.
Published: June 2004

Nicotinic acetylcholine receptors (nAChRs) mediate the passage of potassium and sodium ions across synaptic membranes. Two classes of receptors exist: the neuromuscular nAChRs, which mediate signals between nerve and muscle cells, and the neuronal nAChRs, which are found throughout the nervous system. For treatment of diseases involving nAChRs, drugs must be designed with a high level of selectivity towards only one of these classes or subclasses (in the case of neuronal receptors). α-Conotoxins, small polypeptides isolated from the venoms of marine snails, represent molecules with just this type of selectivity, with specificity even towards certain subclasses of nAChRs. The availability of high-resolution crystal structures of α-conotoxins provides the opportunity to examine the structural features that orchestrate their preferential blocking action. In the present study of a neuromuscular- and a neuronal-specific α-conotoxin, SI and EpI respectively, important and significant differences can be seen in the shapes of the molecules, which must reflect topological features of the different types of target receptor subunits. These then provide a template for computational docking studies with the homologous acetylcholine-binding protein, whose structure is known, so drug analogues of the naturally occurring toxins can be developed with the desired specificities.
Polymorphisms in Toll-like receptor 4 and the systemic inflammatory response syndrome
Authors: Child N.J.A. , Yang I.A. , Pulletz M.C.K. , de Courcy-Golder K. , Andrews A.-L. , Pappachan V.J. , Holloway J.W.
Published: June 2004

The systemic inflammatory response syndrome (SIRS) is a major cause of morbidity and mortality, and is thought to be due to an over-amplification of an inflammatory response. The Toll-like receptor 4 (TLR4) Asp-299→Gly polymorphism has been shown to reduce lipopolysaccharide responsiveness. We examined whether this TLR4 polymorphism is associated with severity of SIRS. A trend was found between the minor allele and mortality in SIRS (odds ratio of 4.3; P=0.076), suggesting a role for TLR4 signalling in the severity of SIRS.
Toll-like receptor 9: modulation of recognition and cytokine induction by novel synthetic CpG DNAs
Authors: Kandimalla E.R. , Zhu F.-G. , Bhagat L. , Yu D. , Agrawal S.
Published: June 2004

Bacterial and synthetic DNA containing unmethylated 2´-deoxyribo(cytidine-phosphate-guanosine) (CpG) dinucleotides in specific sequence contexts activate the vertebrate innate immune system. A molecular pattern recognition receptor, Toll-like receptor 9 (TLR9), recognizes CpG DNA and initiates the signalling cascade, although a direct interaction between CpG DNA and TLR9 has not been demonstrated yet. TLR9 in different species exhibits sequence specificity. Our extensive structure–immunostimulatory activity relationship studies showed that a number of synthetic pyrimidine (Y) and purine (R) nucleotides are recognized by the receptor as substitutes for the natural nucleotides deoxycytidine and deoxyguanosine in a CpG dinucleotide. These studies permitted development of synthetic YpG, CpR and YpR immunostimulatory motifs, and showed divergent nucleotide motif recognition pattern of the receptor. Surprisingly, we found that synthetic immunostimulatory motifs produce different cytokine induction profiles compared with natural CpG motifs. Importantly, we also found that some of these synthetic immunostimulatory motifs show optimal activity in both mouse and human systems without the need to change sequences, suggesting an overriding of the species-dependent specificity of the receptor by the use of synthetic motifs. In the present paper, we review current understanding of structural recognition and functional modulation of TLR9 receptor by second-generation synthetic CpG DNAs and their potential application as wide-spectrum therapeutic agents.
Evolutionary relationships, but functional differences, between the Drosophila and human Toll-like receptor families
Authors: Gangloff M. , Weber A.N.R. , Gibbard R.J. , Gay N.J.
Published: June 2004

The Toll receptor was first found to function in the dorsoventral patterning pathway of Drosophila embryos. It is activated by a specific protein ligand, Spätzle, generated at ventral positions in the early embryo. Drosophila Toll (dToll) also functions in innate immune responses to Gram-positive bacteria and fungi, and Spätzle is required for this response. We have shown that Spätzle is necessary and sufficient for activation of the dToll pathway, and that it probably acts by cross-linking two molecules of Toll to form homodimers. In the present paper, we contrast this mode of regulation with that proposed for the vertebrate Toll-like receptor family, which mediate analogous responses to pathogen pattern antigens. In contrast with dToll, these receptors appear to be activated by direct exposure to pathogen patterns, such as peptidoglycan and lipopolysaccharide. We discuss the evolutionary basis of this functional divergence of the vertebrate and invertebrate Toll-like receptors.
Toll-like receptor 4 signalling: new perspectives on a complex signal-transduction problem
Authors: Vogel S.N. , Fenton M.
Published: June 2004

We previously reported that Toll-like receptor-2 (TLR2) agonists induce expression of a more limited repertoire of pro-inflammatory genes than TLR4 agonists. Murine macrophages stimulated with the TLR4 agonist, Escherichia coli lipopolysaccharide, induced signal transducer and activator of transcription 1 (‘STAT1’) tyrosine phosphorylation that was secondary to the autocrine/paracrine action of interferon (IFN)-β, an immediate early gene. In contrast, TLR2 agonists failed to activate IFN-β gene expression. TLR4-induced IFN-β mRNA was found to be MyD88- and PKR (double-stranded RNA-dependent protein kinase)-independent, but TIRAP (Toll/interleukin-1 receptor domain-containing adapter protein)/Mal (MyD88-adapter-like)-dependent. In the present paper, we outline the recent controversy over the role of TIRAP/Mal in TLR2 and TLR4 signalling in the context of the current molecular tools used for such studies. Collectively, our findings provide the first mechanistic basis for differential patterns of gene expression activated by TLR4 and TLR2 agonists.
Initiation of archaeal DNA replication
Authors: Jenkinson E.R. , Chong J.P.J.
Published: June 2004

The identification of DNA as the genetic material and the elucidation of its structure by Watson and Crick [Watson and Crick, (1953) Nature (London) 171, 737–738], which has its 50th anniversary this year, first suggested the simple elegance with which the problem of passing on precise genetic information from one generation to the next could be solved. Semi-conservative replication is perhaps one of the simplest biological concepts to explain and understand. However, despite an enormous amount of effort in the intervening years, details of the way in which this process is regulated and performed are still unclear in many organisms. Recent work suggests that, due to their simplicity, the Archaea may make a good model for understanding some of the aspects of eukaryotic replication that still elude us.
Halophilic adaptation of protein–DNA interactions
Authors: Bergqvist S. , Williams M.A. , O'Brien R. , Ladbury J.E.
Published: June 2004

Pyrococcus woesei (Pw) is an archaeal organism adapted to living in conditions of elevated salt and temperature. Thermodynamic data reveal that the interaction between the TATA-box-binding protein (TBP) from this organism and DNA has an entirely different character to the same interaction in mesophilic counterparts. In the case of the PwTBP, the affinity of its interaction with DNA increases with increasing salt concentration. The opposite effect is observed in all known mesophilic protein–DNA interactions. The halophilic behaviour can be attributed to sequestration of cations into the protein–DNA complex. By mutating residues in the PwTBP DNA-binding site, potential sites of cation interaction can be removed. These mutations have a significant effect on the binding characteristics, and the halophilic nature of the PwTBP–DNA interaction can be reversed, and made to resemble that of a mesophile, in just three mutations. The genes of functionally homologous proteins in organisms existing in different environments show that adaptation is most often accompanied by mutation of an existing protein. However, the importance of any individual residue to a phenotypic characteristic is usually difficult to assess amongst the multitude of changes that occur over evolutionary time. Since the halophilic nature of this protein can be attributed to only three mutations, this reveals that the important phenotype of halophilicity could be rapidly acquired in evolutionary time.
The chaperonins: perspectives from the Archaea
Authors: Lund P.A. , Large A.T. , Kapatai G.
Published: June 2004

Heat-shock protein (Hsp) 60 chaperones are almost ubiquitous and almost always essential. They can be divided on the basis of sequence homology into two broad types: group I (found in bacteria, mitochondria and chloroplasts) and group II (found in Archaea and the eukaryotic cytosol). Of the two, the group I chaperones are the better understood. Data on their structure, mechanism of action and cellular role will be briefly presented. The group II chaperones are less well studied. In eukaryotes they form large complexes with 8-fold symmetry containing eight different subunits, all of which are essential. They appear to have a major role in the folding of actin and tubulin, although they may also act on other substrates. No crystal structures are available for these complexes. The situation in the Archaea is simpler, with organisms containing between one and three genes for these chaperones. A 2.6 Å structure exists for one archaeal group II chaperone complex. Some progress has been made in defining the reaction cycle of the archaeal group II chaperones and this has shown that they have some properties distinct from the group I chaperones. To date, the in vivo role and importance of the archaeal group II Hsp60 chaperones has not been determined. We have now shown that in the halophilic archaeon Haloferax volcanii not all the genes for these proteins are essential. Further analysis of these proteins in the Archaea should be very productive in yielding more information about these important chaperones and their cellular functions.
The archaeal twin-arginine translocation pathway
Authors: Hutcheon G.W. , Bolhuis A.
Published: June 2004

The twin-arginine translocation (Tat) pathway is a system with the unique ability to export proteins in a fully folded conformation. Its main components are TatA, TatB and TatC, all of which are required for Tat-dependent export. The Tat pathway is found in several Archaea, and in most of them a moderate number of predicted Tat-dependent substrates are present. Putative substrates include those binding cofactors such as iron–sulphur clusters and molybdopterin. In these Archaea, the role of the Tat pathway seems to be similar to that of bacteria: the export of a small subset of proteins that fold before translocation across the cytoplasmic membrane. The exception to this is the Tat system of the halophilic archaeon Halobacterium sp. NRC-1. In this organism, the majority of extra-cytoplasmic proteins are predicted to use the Tat pathway, which is, most likely, a specific adaptation to its particular lifestyle in highly saline conditions.
Repair of UV damage in Halobacterium salinarum
Authors: McCready S. , Marcello L.
Published: June 2004

Halobacterium is one of the few known Archaea that tolerates high levels of sunlight in its natural environment. Photoreactivation is probably its most important strategy for surviving UV irradiation and we have shown that both of the major UV photoproducts, cyclobutane pyrimidine dimers (CPDs) and (6–4) photoproducts, can be very efficiently repaired by photoreactivation in this organism. There are two putative photolyase gene homologues in the published genome sequence of Halobacterium sp. NRC-1. We have made a mutant deleted in one of these, phr2, and confirmed that this gene codes for a CPD photolyase. (6–4) photoproducts are still photoreactivated in the mutant so we are currently establishing whether the other homologue, phr1, codes for a (6–4) photolyase. We have also demonstrated an excision repair capacity that operates in the absence of visible light but the nature of this pathway is not yet known. There is probably a bacteria-type excision-repair mechanism, since homologues of uvrA, uvrB, uvrC and uvrD have been identified in the Halobacterium genome. However, there are also homologues of eukaryotic nucleotide-excision-repair genes (Saccharomy cescerevisiae RAD3, RAD25 and RAD2) so there may be multiple repair mechanisms for UV damage in Halobacterium.
Uracil recognition by archaeal family B DNA polymerases
Authors: Connolly B.A. , Fogg M.J. , Shuttleworth G. , Wilson B.T.
Published: June 2004

Archaeal family-B DNA polymerases possess a novel uracil-sensing mechanism. A specialized pocket scans the template, ahead of the replication fork, for the presence of uracil; on encountering this base, DNA synthesis is stalled. The structural basis for uracil recognition by polymerases is described and compared with other uracil-recognizing enzymes (uridine-triphosphate pyrophophatases and uracil-DNA glycosylases). Remarkably, protein–protein interactions between all three archaeal uracil sensors are observed; possibly the enzymes co-operate to efficiently eliminate uracil from archaeal genomes.