The impact of molecular biology on drug discovery.

The discipline of molecular biology has become increasingly important in recent times for the process of drug discovery. We describe the impact of molecular biology across the whole process of drug discovery and development, including (i) the identification and validation of new drug targets, (ii) the development of molecular screens to find new candidate drugs, and (iii) the generation of safety data and competences leading to enhanced clinical efficacy. We also speculate on emerging developments in drug discovery where it seems likely that molecular biology will play an even more vital role in the generation of future therapies.

Plasma and whole blood taurine in normal dogs of varying size fed commercially prepared food.

The objective of the present study was to examine the effect of signalment, body size and diet on plasma taurine and whole blood taurine concentrations. A total of 131 normal dogs consuming commercially prepared dog food had blood drawn 3-5 h post-prandially to be analysed for plasma amino acids and whole blood taurine. Body weight and morphometric measurements of each dog were taken. Plasma and whole blood taurine concentrations were 77 +/- 2.1 nmol/ml (mean +/- SEM) and 266 +/- 5.1 nmol/ml (mean +/- SEM), respectively. No effect of age, sex, body weight, body size, or diet was seen on plasma and whole blood taurine concentrations. Mean whole blood taurine concentrations were lower in dogs fed diets containing whole grain rice, rice bran or barley. The lowest whole blood concentrations were seen in dogs fed lamb or lamb meal and rice diets. Plasma methionine and cysteine concentrations were lower in dogs fed diets with animal meals or turkey, and whole grain rice, rice bran or barley. Fifteen of 131 dogs had plasma taurine concentrations lower than, or equal, to the previously reported lowest mean food-deprived plasma taurine concentration in normal dogs of 49 +/- 5 nmol/ml (mean +/- SEM) (Elliott et al., 2000). These findings support the theory that taurine deficiency in dogs may be related to the consumption of certain dietary ingredients. Scientific and clinical evidence supports the hypothesis that dilated cardiomyopathy is associated with low blood taurine concentration in dogs; therefore, further work is indicated to determine the mechanism by which diet can affect taurine status in dogs.

Dietary crude protein concentration does not affect the leucine requirement of growing dogs.

The objective of the present study was to examine the interaction between graded levels of leucine and dietary crude protein. Dose-response curves were generated using four 3 x 3 Latin squares (two dogs/square). Each square represented one of two concentrations of crude protein (140 or 280 g/kg diet) and one of two combinations of three concentrations of leucine (5.0, 7.0 and 9.0 g/kg diet or 9.0, 11 and 13 g/kg diet). An additional experiment was performed by feeding crude protein at 210 g/kg diet with either 7.0 or 11 g leucine/kg diet. Weight gain, food intake, nitrogen retention, plasma albumin and plasma amino acids were measured. The requirement was determined to be the minimum leucine concentration required to maximize weight gain and nitrogen retention. For 8-14-week-old male Beagle dogs, 140 g crude protein/kg diet in a diet containing 18 kJ metabolizable energy/g does not appear to support maximal growth. The leucine requirement was not affected by doubling the dietary crude protein level from 140 to 280 g/kg diet. From these results, the leucine requirement of 8-14-week-old Beagle dogs appears to be 11 g leucine/kg diet independent of the level of dietary crude protein, whereas dogs over 14 weeks require only 7 g leucine/kg diet for maximal nitrogen retention.

Murine CFTR channel and its role in regulatory volume decrease of small intestine crypts.

Cystic fibrosis (CF) is caused by mutations in the secretory Cl(-) channel CFTR (cystic fibrosis transmembrane conductance regulator). Variation in the severity of disease has been attributed to mutations in the CFTR gene that cause different degrees of dysfunction of the CFTR Cl(-) channel. However, studies of mouse models of CF indicate that the severity of intestinal pathology is not correlated with activity of the CFTR chloride channel. This observation suggests that other 'environmental' factors might be important in determining the severity of disease. In this respect, we have identified and characterised an additional cellular defect in intestinal epithelial cells of CF mice, the inability of these cells to regulate their volume after hypotonic challenge. Here, we review the function of murine CFTR as both a Cl(-) channel and as a regulator of volume-dependent homeostatic cell mechanisms.

G551D cystic fibrosis mice exhibit abnormal regulation of inflammation in lungs and macrophages.

The major cause of death in cystic fibrosis (CF) is chronic lung disease associated with persistent infection by the bacterium, Pseudomonas aeruginosa. S100A8, an S-100 calcium-binding protein with chemotactic activity, is constitutively expressed in the lungs and serum of CF patients. Levels of S100A8 mRNA were found to be three to four times higher in the lungs of mice carrying the G551D mutation in CF transmembrane conductance regulator compared with littermate controls. Intravenous injection of bacterial LPS induced S100A8 mRNA in the lung to a greater extent in G551D mice than in wild-type littermates. Localization of S100A8 mRNA and protein in the lung indicate that it is a marker for neutrophil accumulation. Bone marrow-derived macrophages from G551D mice were shown to also exhibit hypersensitivity to LPS, measured by induction of TNF-alpha. These results provide evidence that the pathology of CF relates to abnormal regulation of the immune system.

The in vivo effects of milrinone on the airways of cystic fibrosis mice and human subjects.

Previous studies have indicated that milrinone, a specific type III phosphodiesterase inhibitor, may be able to induce chloride secretion in cystic fibrosis (CF) tissues. We have now assessed the effect of this agent in vivo on the nasal epithelium of CF mutant mice and also in the nose and lungs of human subjects with CF. Wild-type mice showed a small hyperpolarization of the nasal potential difference (PD) in response to milrinone (100 microM, 1.6 +/- 0.6 mV, n = 8, P < 0.05). In contrast, CF mice carrying either the most common human mutation of the gene for the CF transmembrane regulator (CFTR), DeltaF508 (protein mislocalized), or the G551D mutation (protein normally localized) failed to demonstrate this response. Milrinone perfused alone had no significant effect on the baseline nasal PD of human subjects without CF (14.7 +/- 4.0 mV preperfusion; 15.3 +/- 4.6 mV postperfusion), but significantly (P < 0.05) augmented the hyperpolarization induced by a subsequently perfused low-chloride solution (with milrinone, 36.8 +/- 3.0 mV, n = 6; without milrinone, 18.1 +/- 2.2 mV, n = 19). In contrast, in human subjects with CF (n = 6), milrinone alone significantly (P < 0. 05) altered the nasal baseline PD (52.2 +/- 3.3 mV preperfusion; 57. 4 +/- 4.2 mV, postperfusion) but not the subsequent responses to the low-chloride solution (with milrinone, 1.1 +/- 2.2 mV, n = 4; without milrinone, 0.6 +/- 0.5 mV, n = 28) or to isoproterenol (100 microM). In a separate study in subjects (n = 6) with the DeltaF508 mutation, nasal coadministration of milrinone with isoproterenol produced no effect in the presence of amiloride and a low-chloride solution (-0.8 +/- 0.5 mV). This was also the case in the nasal epithelium of CF subjects (n = 4) carrying at least one G551D allele (-0.3 +/- 0.8 mV). Similarly, milrinone did not hyperpolarize the PD of either the tracheal (n = 6) or segmental (n = 6) airways of CF subjects (DeltaF508) when applied topically in vivo in the presence of amiloride, isoproterenol, or adenosine triphosphate (all 100 microM) in a low-chloride solution. These data do not support the use of milrinone to induce chloride secretion in CF airways in vivo.

Regulation of murine cystic fibrosis transmembrane conductance regulator Cl- channels expressed in Chinese hamster ovary cells.

1. We investigated the effect of protein kinases and phosphatases on murine cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels, expressed in Chinese hamster ovary (CHO) cells, using iodide efflux and the excised inside-out configuration of the patch-clamp technique. 2. The protein kinase C (PKC) activator, phorbol dibutyrate, enhanced cAMP-stimulated iodide efflux. However, PKC did not augment the single-channel activity of either human or murine CFTR Cl- channels that had previously been activated by protein kinase A. 3. Fluoride, a non-specific inhibitor of protein phosphatases, stimulated both human and murine CFTR Cl- channels. However, calyculin A, a potent inhibitor of protein phosphatases 1 and 2A, did not enhance cAMP-stimulated iodide efflux. 4. The alkaline phosphatase inhibitor, (-)-bromotetramisole augmented cAMP-stimulated iodide efflux and, by itself, stimulated a larger efflux than that evoked by cAMP agonists. However, (+)-bromotetramisole, the inactive enantiomer, had the same effect. For murine CFTR, neither enantiomer enhanced single-channel activity. In contrast, both enantiomers increased the open probability (Po) of human CFTR, suggesting that bromotetramisole may promote the opening of human CFTR. 5. As murine CFTR had a low Po and was refractory to stimulation by activators of human CFTR, we investigated whether murine CFTR may open to a subconductance state. When single-channel records were filtered at 50 Hz, a very small subconductance state of murine CFTR was observed that had a Po greater than that of human CFTR. The occupancy of this subconductance state may explain the differences in channel regulation observed between human and murine CFTR.

Comparison of the gating behaviour of human and murine cystic fibrosis transmembrane conductance regulator Cl- channels expressed in mammalian cells.

1. To investigate the function of the murine cystic fibrosis transmembrane conductance regulator (CFTR), a full-length cDNA encoding wild-type murine CFTR was assembled and stably expressed in Chinese hamster ovary (CHO) cells. 2. Like human CFTR, murine CFTR formed Cl- channels that were regulated by cAMP-dependent phosphorylation and intracellular ATP. However, murine CFTR Cl- channels had a reduced single-channel conductance and decreased open probability (Po) compared with those of human CFTR. 3. Analysis of the dwell time distributions of single channels suggested that the reduced Po of murine CFTR was caused by both decreased residence in the open state and transitions to a new closed state, described by an intermediate closed time constant. 4. For both human and murine CFTR, ATP and ADP regulated the rate of exit from the long-lived closed state. 5. 5'-Adenylylimidodiphosphate (AMP-PNP) and pyrophosphate, two compounds that disrupt cycles of ATP hydrolysis, stabilized the open state of human CFTR. However, neither agent locked murine CFTR Cl- channels open, although AMP-PNP increased the Po of murine CFTR. 6. The data indicate that although human and murine CFTR have many properties in common, some important differences in function are observed. These differences could be exploited in future studies to provide new understanding about CFTR.

Effect of IBMX and alkaline phosphatase inhibitors on Cl- secretion in G551D cystic fibrosis mutant mice.

Some cystic fibrosis transmembrane conductance regulator (CFTR) mutations, such as G551D, result in a correctly localized Cl- channel at the cell apical membrane, albeit with markedly reduced function. Patch-clamp studies have indicated that both phosphatase inhibitors and 3-isobutyl-1-methylxanthine (IBMX) can induce Cl- secretion through the G551D mutant protein. We have now assessed whether these agents can induce Cl- secretion in cftrG551D mutant mice. No induction of Cl- secretion was seen with the alkaline phosphatase inhibitors bromotetramisole or levamisole in either the respiratory or intestinal tracts of wild-type or cftrG551D mice. In contrast, in G551D intestinal tissues, IBMX was able to produce a small CFTR-related secretory response [means +/- SE: jejunum, 1.8 +/- 0.9 microA/cm2, n = 7; cecum, 3.7 +/- 0.8 microA/cm2, n = 7; rectum (in vivo), 1.9 +/- 0.9 mV, n = 5]. This was approximately one order of magnitude less than the wild-type response to this agent and, in the cecum, was significantly greater than that seen in null mice (cftrUNC). In the trachea, IBMX produced a transient Cl- secretory response (37.3 +/- 14.7 microA/cm2, n = 6) of a magnitude similar to that seen in wild-type mice (33.7 +/- 4.7 microA/cm2, n = 9). This response was also present in null mice and therefore is likely to be independent of CFTR. No effect of IBMX on Cl- secretion was seen in the nasal epithelium of cftrG551D mice. We conclude that IBMX is able to induce detectable levels of CFTR-related Cl- secretion in the intestinal tract but not the respiratory tract through the G551D mutant protein.

Targeted disruption of the Wnt2 gene results in placentation defects.

Wnt genes have been implicated in a range of developmental processes in the mouse including the patterning of the central nervous system and limbs. Reported here for the first time is the expression of Wnt2 in the early heart field of 7.5-8.5 dpc (days post-coitum) mouse embryos, making Wnt2 a potentially useful gene marker for the early stages of heart development. Expression was also detected in the allantois from 8.0 dpc and at later stages in the placenta and umbilicus. Mice deficient in Wnt2, generated by gene targeting, displayed runting and approximately 50% died perinatally. Histological analysis revealed alterations in the size and structure of placentas from these mice from 14.5 dpc. The placental defects were associated primarily with the labyrinthine zone and included oedema and tissue disruption and accumulation of maternal blood in large pools. There was also an apparent decrease in the number of foetal capillaries and an increase in the amount of fibrinoid material in the Wnt2 mutant placentas. These results suggest that Wnt2 is required for the proper vascularisation of the mouse placenta and the placental defects in Wnt2-deficient mice result in a reduction in birthweight and perinatal lethality.

A demonstration using mouse models that successful gene therapy for cystic fibrosis requires only partial gene correction.

Quantifying the level of transgene expression necessary for phenotypic effect is an important consideration in designing somatic gene therapy protocols. A nonlinear relationship between phenotype and gene activity is predicted by control analysis for any autosomal recessive condition. The unaffected phenotype of heterozygotes for autosomal recessive disorders demonstrates that 50% of the normal level of gene expression is sufficient to prevent disease. By extension, an exaggerated and positive effect on the mutant phenotype is predicted to arise from only a small addition of normal transgene expression delivered by gene therapy. We tested this expectation directly by intercrossing mice carrying different Cftr alleles which modulate Cftr gene expression from 0 to 100%. We demonstrate that 5% of the normal level of Cftr gene expression results in a disproportionately large correction of the chloride ion transport defect (50% of normal) and essentially complete rescue of the intestinal disease (100% survival). It follows that even modest levels of transgene expression and only partial correction of CFTR channel activity may have a significant clinical impact.

Cystic fibrosis mice carrying the missense mutation G551D replicate human genotype-phenotype correlations.

We have generated a mouse carrying the human G551D mutation in the cystic fibrosis transmembrane conductance regulator gene (CFTR) by a one-step gene targeting procedure. These mutant mice show cystic fibrosis pathology but have a reduced risk of fatal intestinal blockage compared with 'null' mutants, in keeping with the reduced incidence of meconium ileus in G551D patients. The G551D mutant mice show greatly reduced CFTR-related chloride transport, displaying activity intermediate between that of cftr(mlUNC) replacement ('null') and cftr(mlHGU) insertional (residual activity) mutants and equivalent to approximately 4% of wild-type CFTR activity. The long-term survival of these animals should provide an excellent model with which to study cystic fibrosis, and they illustrate the value of mouse models carrying relevant mutations for examining genotype-phenotype correlations.

Cystic fibrosis transmembrane conductance regulator splice variants are not conserved and fail to produce chloride channels.

In the human CFTR only the rare exon 4- splice variant is conserved in mice. We have discovered two novel murine variants, exon 5- and exon 11b+. The exon 5- variant represents up to 40% of mRNA in all CFTR-expressing tissues and leaves the reading frame intact. The exon 11b+ variant inserts a novel exon between exons 11 and 12 with expression restricted to the testis. Two variants of 11b have been found and both introduce premature stop codons. When we expressed human CFTR variants lacking either exon 5 or exon 9 in HeLa cells, they failed to generate cAMP-mediated chloride transport, due to defective intracellular processing. The lack of conservation of splice variants between species and the inability of the more abundant splice variants to generate protein that is correctly processed argue against a physiological role and may simply represent aberrant splicing that is tolerated by the cell and organism.

The sluggish-A gene of Drosophila melanogaster is expressed in the nervous system and encodes proline oxidase, a mitochondrial enzyme involved in glutamate biosynthesis.

Certain gene mutations in Drosophila melanogaster cause sluggish motor activity. We have localized the transcription unit of the sluggish-A gene to a 14.7-kb region at the base of the X chromosome and have cloned corresponding cDNAs. The predicted protein product has significant sequence similarity to Saccharomyces cerevisiae proline oxidase (EC 1.5.99.8), a mitochondrial enzyme which catalyzes the first step in the conversion of proline to glutamate. In the mutant fly, mitochondrial proline oxidase activity is reduced and has kinetic properties different from those of the wild type, providing further evidence that the gene encodes proline oxidase. Indeed, the free proline level in mutant flies is elevated. When the mutant is rescued by transformation, the proline oxidase and free proline levels, as well as the motor and phototactic behavior, are restored to normal. During embryonic development the sluggish-A transcript is predominantly expressed in the nervous system. Significantly, it has previously been reported that a mouse mutant, PRO/Re, which has reduced proline oxidase activity and elevated free proline levels, also exhibits sluggish behavior.

Molecular cloning and analysis of small optic lobes, a structural brain gene of Drosophila melanogaster.

Mutations in the small optic lobes (sol) gene of Drosophila melanogaster cause specific cells to degenerate in the developing optic lobes, resulting in the absence of certain classes of columnar neurons. These neuronal defects lead to specific alterations in behavioral characteristics, particularly during flight and walking maneuvers. We have isolated the wild-type sol locus by microcloning and chromosomal walking and have established its genetic and molecular limits. Two major transcripts of 5.8 and 5.2 kilobases are produced from this locus by alternative splicing and are present throughout the entire life cycle. Sequence analyses of cDNAs corresponding to these two classes of transcripts predict two proteins of 1597 and 395 amino acids. The first shows similarity in its carboxyl-terminal region to the catalytic domain of a vertebrate calcium-activated neutral protease (calpain), whereas its amino-terminal region contains several zinc-finger-like repeats of the form WXCX2CX10-11CX2C. The second predicted protein contains only the first two of the zinc-finger-like repeats and is missing the calpain domain. By constructing transgenic flies carrying a single wild-type copy of the sol gene in a homozygous sol mutant background, we have restored the normal neuroanatomical phenotype to individuals that would have developed mutant brains.

Cis-acting sequences sufficient for correct tissue and temporal specificity of larval serum protein 1 genes of Drosophila.

We have constructed hybrid genes in which the coding region of the bacterial gene chloramphenicol acetyl transferase (CAT) has been linked to varying lengths of upstream sequences of Drosophila genes for larval serum sequence 1 (LSP1). These have been inserted into a P-element transformation vector and subsequently transferred into the germ-line of recipient flies. Transformants carrying the CAT gene linked to 1650 bp, 570 bp or 377 bp of upstream LSP1alpha sequences, or 745 bp or 471 bp of upstream beta sequences express CAT with the same developmental and tissue specificity as the endogenous LSP1 genes. Constructs having only 66 bp of upstream LSP1beta sequences, however, show extremely low levels of CAT expression in tissues and at developmental stages in which LSP1 is not expressed. We discuss the significance of short regions of homology between the DNA upstream of the alpha and beta genes, which lie within the regions identified by the transformation experiments as being required for the cis-regulation of LSP1 synthesis.

Sequence conservation around the 5' ends of the larval serum protein 1 genes of Drosophila melanogaster.

We have determined the nucleotide sequence at the 5' ends of the genes for the alpha, beta and gamma polypeptides of larval serum protein 1 (LSP1) of Drosophila melanogaster. In their upstream regions, the three genes share homology around the TATA boxes. There is also a homologous region of about 20 nucleotides at positions 200, 216 and 377 upstream from the alpha, beta and gamma genes, respectively. Another 18-nucleotide homology occurs between a sequence 111 nucleotides upstream from the alpha gene and 130 nucleotides upstream of the beta gene. This contains a seven-nucleotide match with a sequence 180 nucleotides upstream from the gamma gene. The sequences corresponding to the 5' non-translated regions of the RNA show two regions of strong homology: one being within the first 20 nucleotides at the very 5' end of the RNA, and the other being between nucleotides 27 and 52 of the three transcripts. The first AUG codon to precede a long open reading frame is found at nucleotides 89, 86 and 83 downstream from the 5' end of the alpha, beta and gamma RNAs, respectively. An extremely conserved nucleotide sequence with an exact homology of 66 nucleotides between the alpha and beta genes, and sharing 27 nucleotides with the gamma gene, is contained within this long open reading frame in the first exon. Conceptual translation of the long open reading frame shows that the hydrophobic nature of the first 20 amino acids of the three polypeptides has been conserved whereas the exact sequence has not. This suggests that the N termini contain signal sequences required for secretion of the protein into the haemolymph. The three genes have intervening sequences ranging from 65 to 68 nucleotides in length at comparable locations close to the 5' end of the genes.

Expression of the prokaryotic gene for chloramphenicol acetyl transferase in Drosophila under the control of larval serum protein 1 gene promoters.

We have linked the protein coding region of the prokaryotic gene for chloramphenicol acetyl transferase (CAT) to the promoter region of the Drosophila genes for larval serum protein 1 (LSP1). These regions consist of 1.65 X 10(3) and 2.25 X 10(3) base long DNA segments upstream from the LSP1 alpha and beta genes, respectively. The hybrid genes have been inserted into a P-element transformation vector and the constructs introduced into cultured Drosophila Kc cells by calcium phosphate-mediated transfection, or into the germ-lines of flies by P-element-mediated transformation. CAT expression occurs in approximately 1% of the cultured cells following transfection and the accumulation of protein is maximal three to four days after transfection; and it is dependent upon the presence of the LSP1 sequences. We have also obtained several lines of transformed flies that carry the LSP1-CAT genes in their germ-line. The onset of synthesis of functional chloramphenicol acetyl transferase in these organisms occurs in the late third larval instar, rising to a maximum at puparium formation. We continue to detect CAT until the first few hours of adulthood. Assays of CAT activity in the homogenates of dissected tissues indicated that the genes are only expressed in the fat body, as are the endogenous LSP1 genes. We have confirmed this tissue-specific localization of CAT in indirect immunofluorescence using an anti-CAT monoclonal antibody. Northern blots indicate that CAT transcripts are found only in the fat body but their abundance is an order of magnitude lower than endogenous LSP1 transcripts. Primer extension experiments show that transcription of the hybrid genes is initiated at the same nucleotide as the endogenous LSP1 genes. Taken together these data indicate that the 1.65 X 10(3) and 2.25 X 10(3) base segments of DNA upstream from the LSP1 alpha and LSP1 beta genes contain cis-acting regulatory elements necessary for correct tissue and temporal specificity of LSP1 gene expression.