- Email: [email protected]
One in the eye for herpes simplex virus
News & Comment
TRENDS in Pharmacological Sciences Vol.23 No.8 August 2002
355
Journal Club
‘S-SXR-RMs’? Selective SXR response modulators – the future of designer drug interactions? When is an inhibitor not just an inhibitor? When it binds to a nuclear receptor (NR), that’s when! Cytochromes P450, the main phase I metabolic enzymes in drug metabolism, are traditionally thought to be modulated by drugs and other xenobiotics at the level of either enzyme activity or gene expression. This modulation often causes profound complications for the safe use of drugs because the clearance of a drug might increase or decrease in ways that are often difficult to predict following the administration of another drug. At the enzyme level, inhibitors can compete with substrates or irreversibly inactivate the P450. Stimulation of enzyme activity, although less common, also occurs. At the gene-expression level, most interactions involve induction of expression via activation of certain NRs. However, repression of P450 expression is also possible. A new study by Takeshita et al. [1] reveals a novel subtlety in P450 regulation: a classical inhibitor could act as a selective modulator of P450 induction by affecting the interactions of an NR with coregulators. P450 3A4 is both the most important and the most problematic enzyme in human drug metabolism because any one of its
wide range of substrates can potentially inhibit the metabolism of any other substrate. Moreover, P450 3A4 is induced by numerous xenobiotics via the steroid X receptor (SXR; or pregnane X receptor), an NR that responds to many diverse ligands. Like other NRs [e.g. the estrogen receptor (ER)], the SXR is presumed to bind a selection of coregulators – coactivators and corepressors – that help mediate its disparate effects on the expression of target genes. Again by analogy with the ER, it follows that ligands could affect the binding of particular coregulators differentially, depending on the peculiarities of the structure of the ligand-bound receptor. Takeshita et al., in the process of examining the interaction of SXR with the coactivator SRC-1 (steroid receptor coactivator 1) and the corepressor SMRT (silencing mediator for retinoid and thyroid hormone receptor), have found that the classical P450 3A4 inhibitor ketoconazole could act as a selective modulator of the SXR, possibly in much the same way as selective estrogen response modulators (SERMs) act at the ER. In reporter gene assays ketoconazole appeared to have limited ability to enhance transcription by itself through the SXR,
but it blocked the inductive effect of the more powerful inducer corticosterone. Moreover, ketoconazole appeared to interrupt the interaction of the SXR with both the coactivator SRC-1 and the corepressor SMRT. Clearly, much remains to be elucidated regarding the interactions of coregulators with the SXR and how they might be modulated by xenobiotics, including ketoconazole. However, this initial study suggests that we should expect the subtleties of NR modulation that have been revealed in other NRs to apply to the SXR as well. The question will be whether we can make sense of this extra complexity in rationalizing and predicting drug interactions. Every ligand that interacts with the SXR is likely to have a different spectrum of effects and with such a promiscuous receptor there are many possibilities. 1 Takeshita, A. et al. (2002) Putative role of the orphan nuclear receptor SXR in the mechanism of CYP3A4 inhibition by xenobiotics. J. Biol. Chem. 10.1074/jbc.M111245200
Elizabeth M.J. Gillam [email protected]
One in the eye for herpes simplex virus Approximately 90% of the population are infected with herpes simplex virus (HSV) type I. Infections usually occur around the mouth and are generally dormant but become activated in times of stress, producing what most people know as cold sores. Occasionally, infections occur around the eye and can lead to a potentially blinding condition known as stromal keratitis. One of the early major events in stromal keratitis is the formation of new blood vessels; however, the cause of such angiogenesis has puzzled researchers because none of the proteins expressed by HSV possesses angiogenic activity. Zheng et al. [1] now provide evidence that HSV DNA is responsible for this activity, and their work suggests novel therapeutic approaches not only to treat stromal http://tips.trends.com
keratitis, but also to induce angiogenesis in situations in which it might be beneficial. The genome of HSV, like the genome of bacteria but unlike our genome, contains a high number of CpG motifs. Such CpG motifs have been well known to stimulate the immune system and are often used as immunization adjuvants. This observation suggested to Zheng and colleagues that HSV DNA might be the mystery angiogenic agent. To determine whether HSV DNA is responsible for induction of new blood vessels in stromal keratitis, the authors used beads coated with either HSV DNA or synthetic oligodeoxynucleotides (ODNs) containing CpG motifs. These beads induced angiogenesis in the corneas of mice whereas control beads, uncoated or coated with herring sperm DNA, did not induce
angiogenesis. ODNs containing G-tetrad motifs have been shown to antagonize the stimulatory ability of CpG motifs and the authors showed that these neutralizing ODNs were able to antagonize the angiogenic activity of HSV DNA. The authors also provide evidence that angiogenesis induced by HSV DNA is mediated by the production of vascular endothelial growth factor (VEGF). Inflammatory cells near the corneal region treated with HSV DNA or CpG ODN were stained with an anti-VEGF antibody, revealing that VEGF was being produced in this area. Furthermore, these antibodies to VEGF inhibited the angiogenesis, demonstrating that the newly synthesized VEGF is indeed responsible for this new blood vessel formation. Finally, using a macrophage cell line and fluorescent
0165-6147/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved.
356
News & Comment
CpG ODNs, Zheng et al. demonstrate that stimulation of VEGF is direct because it is those cells that take up the CpG ODN that are induced to express VEGF. The authors conclude that a neutralizing ODN could be a potential therapy for
TRENDS in Pharmacological Sciences Vol.23 No.8 August 2002
HSV-induced stromal keratitis, whereas a CpG ODN could be useful to stimulate angiogenesis artificially where it would be beneficial, such as in tissue grafts, in the treatment of vascular disease or, more lucratively, as a treatment for baldness.
1 Zheng, M. et al. (2002) DNA containing CpG motifs induces angiogenesis. Proc. Natl. Acad. Sci. U. S. A. 10.1073/pnas.132605599
Ian Wood [email protected]
In Brief
Novel ligands at IP3 receptors The phosphoinositide pathway is an important source of cytoplasmic Ca2+ signals that uses inositol (1,4,5)trisphosphate [Ins(1,4,5)P3] generated by hydrolysis of plasma membrane phospholipids to activate Ins(1,4,5)P3 receptors in the endoplasmic reticulum (ER) and release stored Ca2+. A recent study intriguingly suggests, however, that Ins(1,4,5)P3 is not a unique activator of the Ins(1,4,5)P3 receptor, and identifies endogenous neuronal Ca2+-binding proteins that bind to these receptors and open their Ca2+ channels in the absence of Ins(1,4,5)P3. These novel ligands have homology with calmodulin, and their binding is sensitive to cytosolic Ca2+. They can therefore mediate amplification of trigger Ca2+ signals arising from other sources. Because these ligands are synaptically located, it is tempting to speculate that they might be involved in the ‘sculpting’ of synaptic connections that underlies memory formation. [Yang, J. et al. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7711–7716] VR
Genentech loses lawsuit A Los Angeles jury has found Genentech guilty of fraud in a lawsuit brought by City of Hope Medical Research Center (Los Angeles, CA, USA). At the heart of the lawsuit was a dispute about royalties on protein products sold by the biotech giant. The superior court ruled that Genentech willfully withheld royalities on drugs developed from City of Hope research. Susan Harriman (an Attorney for Genentech) said that the jury’s finding of fraud or malice represented ‘sufficient punishment’. City of Hope have been awarded more than US$300 million in compensatory damages; punitive damages are still to be determined. Glenn Krinsky http://tips.trends.com
(in-house counsel for City of Hope) made it clear that he hoped for a ‘significant’ award to deter future wrongdoing by other companies. AB
effects of lithium and valproic acid, probably by regulating inositol metabolism. [Williams, R.S. et al. (2002) Nature 417, 292–295] AB
Circadian output molecule Zinc – link to plaque formation identified? in Alzheimer’s disease The circadian clock, Increasing evidence implicating zinc as a culprit in the pathogenesis of Alzheimer’s disease (AD) comes from a recent study reporting a marked decrease in β-amyloid (Aβ) deposition in the brains of genetically engineered mice created by crossing human mutant amyloid precursor protein (APP) transgenics with mice lacking the transporter required for zinc transport into synaptic vesicles. Zinc is known to promote aggregation of synthetic Aβ in vitro, with a histidine at residue 13 of the Aβ crucially involved. In vivo, zinc is released during neurotransmission, principally by glutamatergic corticofugal fibres, and hence the ensuing rise in extracellular zinc concentration to ~300 µM could account for Aβ precipitation in plaques being limited to the neocortex in human AD. That zincinduced aggregation of Aβ in vitro is reversed by chelation raises the possibility that chelation therapy might be an additional strategy in the prevention or treatment of AD. [Lee, J-Y. et al. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7705–7710] VR
Three mood-stabilizers, one target What do lithium, carbamazepine and valproic acid have in common, apart from their efficacy in bipolar affective disorder? All three drugs deplete inositol from sensory neurone growth cones, according to a recent article published in Nature. The drugs reduce the frequency of growth-cone collapse, an effect that is reversed by the addition of inositol to the culture medium. Using a genetically engineered form of the mould Dictyostelium, the authors have shown that loss of the gene encoding prolyl oligopeptidase conferred resistance to the
located in the suprachiasmatic nucleus, regulates various biological rhythms. Although several clock genes are known to display circadian patterns of expression within the suprachiasmatic nucleus, it is not clear how the rhythm is transmitted to other parts of the brain. A recent article published in Nature identifies a secreted protein, prokineticin 2 (PK2), as a potential output molecule. The gene is expressed in a circadian fashion, peaking during the light phase. Experiments using knockout mice have shown that transcription of the gene encoding PK2 is regulated by known clock genes. Intracerebroventricular administration of recombinant PK2 to rats alters their pattern of locomotor activity, indicating that it mediates the circadian behavioural output. [Cheng, M.Y. et al. (2002) Nature 417, 405–410] AB
Prostate cancer to become no. 1 in UK Researchers predict that prostate cancer is to become more common than lung cancer in British males within the next couple of years. This is partly because of the decreased incidence of smoking-related lung cancers, and partly because of increased testing for prostate cancer, which leads to earlier diagnosis. It is estimated that 22 000 new cases of prostate cancer are diagnosed in the UK each year. Scientists at the Institute of Cancer Research in London, UK said that one of the main challenges in prostate cancer research is to determine which of the patients diagnosed with the disease need aggressive treatment. AB
0165-6147/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved.
TRENDS in Pharmacological Sciences Vol.23 No.8 August 2002
355
Journal Club
‘S-SXR-RMs’? Selective SXR response modulators – the future of designer drug interactions? When is an inhibitor not just an inhibitor? When it binds to a nuclear receptor (NR), that’s when! Cytochromes P450, the main phase I metabolic enzymes in drug metabolism, are traditionally thought to be modulated by drugs and other xenobiotics at the level of either enzyme activity or gene expression. This modulation often causes profound complications for the safe use of drugs because the clearance of a drug might increase or decrease in ways that are often difficult to predict following the administration of another drug. At the enzyme level, inhibitors can compete with substrates or irreversibly inactivate the P450. Stimulation of enzyme activity, although less common, also occurs. At the gene-expression level, most interactions involve induction of expression via activation of certain NRs. However, repression of P450 expression is also possible. A new study by Takeshita et al. [1] reveals a novel subtlety in P450 regulation: a classical inhibitor could act as a selective modulator of P450 induction by affecting the interactions of an NR with coregulators. P450 3A4 is both the most important and the most problematic enzyme in human drug metabolism because any one of its
wide range of substrates can potentially inhibit the metabolism of any other substrate. Moreover, P450 3A4 is induced by numerous xenobiotics via the steroid X receptor (SXR; or pregnane X receptor), an NR that responds to many diverse ligands. Like other NRs [e.g. the estrogen receptor (ER)], the SXR is presumed to bind a selection of coregulators – coactivators and corepressors – that help mediate its disparate effects on the expression of target genes. Again by analogy with the ER, it follows that ligands could affect the binding of particular coregulators differentially, depending on the peculiarities of the structure of the ligand-bound receptor. Takeshita et al., in the process of examining the interaction of SXR with the coactivator SRC-1 (steroid receptor coactivator 1) and the corepressor SMRT (silencing mediator for retinoid and thyroid hormone receptor), have found that the classical P450 3A4 inhibitor ketoconazole could act as a selective modulator of the SXR, possibly in much the same way as selective estrogen response modulators (SERMs) act at the ER. In reporter gene assays ketoconazole appeared to have limited ability to enhance transcription by itself through the SXR,
but it blocked the inductive effect of the more powerful inducer corticosterone. Moreover, ketoconazole appeared to interrupt the interaction of the SXR with both the coactivator SRC-1 and the corepressor SMRT. Clearly, much remains to be elucidated regarding the interactions of coregulators with the SXR and how they might be modulated by xenobiotics, including ketoconazole. However, this initial study suggests that we should expect the subtleties of NR modulation that have been revealed in other NRs to apply to the SXR as well. The question will be whether we can make sense of this extra complexity in rationalizing and predicting drug interactions. Every ligand that interacts with the SXR is likely to have a different spectrum of effects and with such a promiscuous receptor there are many possibilities. 1 Takeshita, A. et al. (2002) Putative role of the orphan nuclear receptor SXR in the mechanism of CYP3A4 inhibition by xenobiotics. J. Biol. Chem. 10.1074/jbc.M111245200
Elizabeth M.J. Gillam [email protected]
One in the eye for herpes simplex virus Approximately 90% of the population are infected with herpes simplex virus (HSV) type I. Infections usually occur around the mouth and are generally dormant but become activated in times of stress, producing what most people know as cold sores. Occasionally, infections occur around the eye and can lead to a potentially blinding condition known as stromal keratitis. One of the early major events in stromal keratitis is the formation of new blood vessels; however, the cause of such angiogenesis has puzzled researchers because none of the proteins expressed by HSV possesses angiogenic activity. Zheng et al. [1] now provide evidence that HSV DNA is responsible for this activity, and their work suggests novel therapeutic approaches not only to treat stromal http://tips.trends.com
keratitis, but also to induce angiogenesis in situations in which it might be beneficial. The genome of HSV, like the genome of bacteria but unlike our genome, contains a high number of CpG motifs. Such CpG motifs have been well known to stimulate the immune system and are often used as immunization adjuvants. This observation suggested to Zheng and colleagues that HSV DNA might be the mystery angiogenic agent. To determine whether HSV DNA is responsible for induction of new blood vessels in stromal keratitis, the authors used beads coated with either HSV DNA or synthetic oligodeoxynucleotides (ODNs) containing CpG motifs. These beads induced angiogenesis in the corneas of mice whereas control beads, uncoated or coated with herring sperm DNA, did not induce
angiogenesis. ODNs containing G-tetrad motifs have been shown to antagonize the stimulatory ability of CpG motifs and the authors showed that these neutralizing ODNs were able to antagonize the angiogenic activity of HSV DNA. The authors also provide evidence that angiogenesis induced by HSV DNA is mediated by the production of vascular endothelial growth factor (VEGF). Inflammatory cells near the corneal region treated with HSV DNA or CpG ODN were stained with an anti-VEGF antibody, revealing that VEGF was being produced in this area. Furthermore, these antibodies to VEGF inhibited the angiogenesis, demonstrating that the newly synthesized VEGF is indeed responsible for this new blood vessel formation. Finally, using a macrophage cell line and fluorescent
0165-6147/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved.
356
News & Comment
CpG ODNs, Zheng et al. demonstrate that stimulation of VEGF is direct because it is those cells that take up the CpG ODN that are induced to express VEGF. The authors conclude that a neutralizing ODN could be a potential therapy for
TRENDS in Pharmacological Sciences Vol.23 No.8 August 2002
HSV-induced stromal keratitis, whereas a CpG ODN could be useful to stimulate angiogenesis artificially where it would be beneficial, such as in tissue grafts, in the treatment of vascular disease or, more lucratively, as a treatment for baldness.
1 Zheng, M. et al. (2002) DNA containing CpG motifs induces angiogenesis. Proc. Natl. Acad. Sci. U. S. A. 10.1073/pnas.132605599
Ian Wood [email protected]
In Brief
Novel ligands at IP3 receptors The phosphoinositide pathway is an important source of cytoplasmic Ca2+ signals that uses inositol (1,4,5)trisphosphate [Ins(1,4,5)P3] generated by hydrolysis of plasma membrane phospholipids to activate Ins(1,4,5)P3 receptors in the endoplasmic reticulum (ER) and release stored Ca2+. A recent study intriguingly suggests, however, that Ins(1,4,5)P3 is not a unique activator of the Ins(1,4,5)P3 receptor, and identifies endogenous neuronal Ca2+-binding proteins that bind to these receptors and open their Ca2+ channels in the absence of Ins(1,4,5)P3. These novel ligands have homology with calmodulin, and their binding is sensitive to cytosolic Ca2+. They can therefore mediate amplification of trigger Ca2+ signals arising from other sources. Because these ligands are synaptically located, it is tempting to speculate that they might be involved in the ‘sculpting’ of synaptic connections that underlies memory formation. [Yang, J. et al. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7711–7716] VR
Genentech loses lawsuit A Los Angeles jury has found Genentech guilty of fraud in a lawsuit brought by City of Hope Medical Research Center (Los Angeles, CA, USA). At the heart of the lawsuit was a dispute about royalties on protein products sold by the biotech giant. The superior court ruled that Genentech willfully withheld royalities on drugs developed from City of Hope research. Susan Harriman (an Attorney for Genentech) said that the jury’s finding of fraud or malice represented ‘sufficient punishment’. City of Hope have been awarded more than US$300 million in compensatory damages; punitive damages are still to be determined. Glenn Krinsky http://tips.trends.com
(in-house counsel for City of Hope) made it clear that he hoped for a ‘significant’ award to deter future wrongdoing by other companies. AB
effects of lithium and valproic acid, probably by regulating inositol metabolism. [Williams, R.S. et al. (2002) Nature 417, 292–295] AB
Circadian output molecule Zinc – link to plaque formation identified? in Alzheimer’s disease The circadian clock, Increasing evidence implicating zinc as a culprit in the pathogenesis of Alzheimer’s disease (AD) comes from a recent study reporting a marked decrease in β-amyloid (Aβ) deposition in the brains of genetically engineered mice created by crossing human mutant amyloid precursor protein (APP) transgenics with mice lacking the transporter required for zinc transport into synaptic vesicles. Zinc is known to promote aggregation of synthetic Aβ in vitro, with a histidine at residue 13 of the Aβ crucially involved. In vivo, zinc is released during neurotransmission, principally by glutamatergic corticofugal fibres, and hence the ensuing rise in extracellular zinc concentration to ~300 µM could account for Aβ precipitation in plaques being limited to the neocortex in human AD. That zincinduced aggregation of Aβ in vitro is reversed by chelation raises the possibility that chelation therapy might be an additional strategy in the prevention or treatment of AD. [Lee, J-Y. et al. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7705–7710] VR
Three mood-stabilizers, one target What do lithium, carbamazepine and valproic acid have in common, apart from their efficacy in bipolar affective disorder? All three drugs deplete inositol from sensory neurone growth cones, according to a recent article published in Nature. The drugs reduce the frequency of growth-cone collapse, an effect that is reversed by the addition of inositol to the culture medium. Using a genetically engineered form of the mould Dictyostelium, the authors have shown that loss of the gene encoding prolyl oligopeptidase conferred resistance to the
located in the suprachiasmatic nucleus, regulates various biological rhythms. Although several clock genes are known to display circadian patterns of expression within the suprachiasmatic nucleus, it is not clear how the rhythm is transmitted to other parts of the brain. A recent article published in Nature identifies a secreted protein, prokineticin 2 (PK2), as a potential output molecule. The gene is expressed in a circadian fashion, peaking during the light phase. Experiments using knockout mice have shown that transcription of the gene encoding PK2 is regulated by known clock genes. Intracerebroventricular administration of recombinant PK2 to rats alters their pattern of locomotor activity, indicating that it mediates the circadian behavioural output. [Cheng, M.Y. et al. (2002) Nature 417, 405–410] AB
Prostate cancer to become no. 1 in UK Researchers predict that prostate cancer is to become more common than lung cancer in British males within the next couple of years. This is partly because of the decreased incidence of smoking-related lung cancers, and partly because of increased testing for prostate cancer, which leads to earlier diagnosis. It is estimated that 22 000 new cases of prostate cancer are diagnosed in the UK each year. Scientists at the Institute of Cancer Research in London, UK said that one of the main challenges in prostate cancer research is to determine which of the patients diagnosed with the disease need aggressive treatment. AB
0165-6147/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved.