WO2001016603A2 - Analysis and treatment of body weight and eating disorders - Google Patents
Analysis and treatment of body weight and eating disorders Download PDFInfo
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- WO2001016603A2 WO2001016603A2 PCT/GB2000/003277 GB0003277W WO0116603A2 WO 2001016603 A2 WO2001016603 A2 WO 2001016603A2 GB 0003277 W GB0003277 W GB 0003277W WO 0116603 A2 WO0116603 A2 WO 0116603A2
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- A61P3/00—Drugs for disorders of the metabolism
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- A61P3/00—Drugs for disorders of the metabolism
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- G01N2800/00—Detection or diagnosis of diseases
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- G01N2800/044—Hyperlipemia or hypolipemia, e.g. dyslipidaemia, obesity
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- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/30—Psychoses; Psychiatry
- G01N2800/303—Eating disorders, e.g. anorexia, bulimia
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
Definitions
- the present invention relates to methods and compositions relating to body weight disorders and eating disorders in conditions such as obesity. Specifically, the present invention identifies and describes proteins that are differentially expressed in body weight and/or eating disordered states relative to their expression in normal or non-body weight disordered states and in particular identifies and describes proteins associated with the regulation of appetite and body weight. Further, the present invention identifies and describes proteins via their ability to interact with gene products involved in the regulation of body weight and appetite. Still further, the present invention provides methods, particularly experimental paradigms for the identification of differentially expressed proteins that are potential molecular targets for compounds to treat body weight and/or eating disorders including, but not limited to, obesity. Still further, the present invention provides methods for the identification and therapeutic use of compounds for the treatment of body weight and/or eating disorders including, but not limited to, obesity.
- Body weight disorders including eating disorders, are one of the major public health problems in all industrialised countries and is a growing problem in countries undergoing rapid acculturalisation. Obesity, the most prevalent of eating disorders, is the most common nutritional disorder in the Western world and can have a prevalence of up to 50% in middle-aged and elderly populations. However, it is also an increasing problem in children. Other weight disorders, such as anorexia nervosa and bulimia nervosa are also serious health risks and affect approximately 0.2% of the population, particularly female, in Western countries. Anorexia and cachexia are major features of other diseases such as cancer, AIDS and trauma.
- Obesity defined as an excess body fat relative to lean body mass, also contributes to many other diseases. For example, obesity is responsible for the increased incidence of diseases such as coronary artery disease, stroke and non-insulin dependent diabetes. Obesity is not merely a behavioural problem, i.e. the result of voluntary hyperphagia . Rather, the differential body composition observed between obese and non-obese subjects results from differences in both metabolism and neurological/metabolic interactions. These differences are to some extent genetically inherited but the nature of the gene products that control body weight and body composition are unknown. Attempts to identify protein molecules involved in the control of body weight have been largely empiric and the nature of the mechanisms by which body composition and/or substrate flux are monitored have not yet been identified.
- PWS Prader-Willi syndrome
- mice which contain the autosomal recessive mutations ob/ob (obese) or db/db (diabetes) . These mutations are on chromosome 6 and four respectively, but lead to clinically similar pictures provided the genes are expressed on the same background strain.
- the ob gene product has been identified as 16kDa polypeptide produced primarily by adipose tissue that provides a signal to the brain on the adipose tissue fat stores.
- mice with a mutation, resulting in no circulating protein are hyperphagic, obese, have poor thermo-regulation and non- shivering thermogenesis and are insulin resistant with impaired glucose tolerance. Treatment of these mice with recombinant leptin reduces food intake and stimulates energy expenditure so that the mice become less obese .
- the db/db mice have a mutation in the receptor for leptin so that normal signal transduction via the JAK/STAT pathway does not occur. This mutation, when on the
- C57BI/6 background is phenotypically identical to the ob mutation, but causes additionally frank diabetes when on the C57BI/Ks background.
- Other single gene mutations in mice associated with obesity include the yellow mutation at the agouti locus, mutations at the fat and tubby loci and an autosomal dominant mutation at the adipose locus on chromosome 7.
- mutant animal models include fa/fa (fatty) rats and ZDF fatty rats, which bear strong respective similarities with the ob/ob and db/db mice.
- fa/fa rat is hyperphagic, obese, insulin resistant, very hyperinsulinaemic and glucose intolerant
- ZDF rat is hyperphagic, obese, insulin resistant and hyperinsulinaemic, but develops frank diabetes after approximately 6 weeks of age .
- Inbred mouse strains such as the NZO mouse, the Japanese KK mouse are models of obesity. Further, desert rodents, such as spiny mice and sand rats are neither insulin resistant nor diabetic in their natural habitats, but do become insulin resistant and glucose intolerant when fed on a standard laboratory diet.
- Obesity is a common feature of elderly rodents and the development of obesity can be accelerated by feeding diets with a high fat content, whether these diets are synthetic homogenous diets or are the result of supplementation of replacement of the normal rat chow by human food with a high fat content (cafeteria diet) .
- Farm animal studies have clearly demonstrated that selective breeding can be undertaken for common traits such as food efficiency, growth rate, leanness.
- studies in mice have been undertaken to selectively breed for obesity and leanness so that over several generations of brother/sister matings colonies of mice are produced from a common parent stock that are significantly different in the level of fatness.
- the present invention relates to methods and compositions for the treatment of body weight and eating disorders, including but not limited to, obesity. More specifically, the present invention identifies and describes proteins that are differentially expressed in body weight and/or eating disordered states relative to their expression in normal, or non-body weight disordered states and also identifies proteins that are differentially expressed in response to manipulations relevant to body weight regulation and/or control of food intake. Such differentially expressed proteins may represent ⁇ target proteins' and/or fingerprint proteins. Further, the present invention identifies and describes proteins termed pathway proteins via their ability to interact with proteins involved in the regulation of body weight and/or food intake. Pathway proteins may also exhibit target protein and/or fingerprint protein characteristics.
- the present invention provides a method of screening an agent to determine its usefulness in treating a condition characterised by body weight disorders and/or eating disorders, the method comprising:
- an agent is selected if it changes the expression of a differentially expressed protein towards that of a normal weight subject.
- the present invention provides a method for the identification of an agent or agents for use in the treatment of body weight and/or eating disorders comprising the steps of :
- the present invention provides the use of an agent identified by the above method for the preparation of a medicament for the treatment of a condition characterised by body weight or eating disorders.
- a condition characterised by body weight or eating disorders include obesity, non-insulin dependent diabetes (type 2 diabetes) , Cushing syndrome, anorexia nervosa and bulimia.
- the present invention provides a method of treating a condition characterised by body weight or eating dysfunction in a patient, the method comprising administering a therapeutically or prophylactically effective amount of such an agent identified by the above method.
- the present invention provides a method of determining the nature or degree of body weight and/or eating dysfunction in a human or animal subject, the method comprising: (a) establishing a paradigm in which at least one protein is differentially expressed in relevant tissue from, or representative of, subjects having a body weight and/or eating disorder;
- the patient sample used in method can be a tissue sample or body fluid sample or urine.
- This method allows the causes of body weight or eating disorder dysfunction of a patient to be correlated to different types to prophylactic or therapeutic treatment available in the art, thereby enhancing the likelihood of obtaining a beneficial response in the patient to the therapy.
- the present invention provides a method of treatment by the use of an agent that will restore the expression of one or more differentially expressed proteins in the body weight or eating disorders dysfunction state to that found in the normal state in order to maintain a reduced body weight (prevent weight gain) or to prevent the re-emergence of an eating disorder .
- the present invention provides a method whereby the pattern or differentially expressed proteins in a tissue sample or body fluid sample or urine of an individual with body weight and/or eating disorder dysfunction is used to predict the most appropriate and effective therapy to alleviate the dysfunction state and to monitor the success of that treatment .
- the present invention provides a protein which is differentially expressed in relevant tissue from, or representative of subjects having differential levels of obesity or leanness or nutrient intake states or macronutrient selection and which is as obtainable by the method of two-dimensional gel electrophoresis carried out on said tissue or a protein- containing extract thereof, the method comprising:
- differentially expressed proteins are described in the examples below.
- fingerprint proteins may be used in methods for identifying compounds useful for the treatment of body weight and/or eating disordered states.
- 'Target protein' refers to a differentially expressed protein involved in body weight regulation and/or the control feeding such that modulation of the expression of that protein may act to prevent or ameliorate the body weight and/or eating disordered state including, but not limited to, obesity.
- This invention is based, in part, on systematic search strategies involving modulations of body weight and/or eating behaviours and obesity experimental paradigms, coupled with sensitive detection of proteins by 2D- electrophoresis .
- the invention further provides methods for the identification of compounds that modulate the expression of proteins involved in body weight and feeding processes relevant to the regulation of energy balance. Still further, the present invention describes methods for the prevention and/or treatment of obesity, which may involve the administration of such compounds to individuals predisposed to or exhibiting obesity.
- the present invention describes methods for the prognostic and diagnostic evaluation of subjects with body weight and eating disordered states in order to make a prognosis of the most effective therapy for each subject .
- Differential expression refers to at least one recognisable difference in tissue protein expression. It may be a quantitatively measurable, semi- quantitatively estimatable or qualitatively detectable difference in tissue protein expression.
- a differentially expressed protein herein DEP
- DEP differentially expressed protein
- tissue in the normal state and less strongly expressed or not expressed at all in tissue in the body weight or eating disordered state.
- it may be strongly expressed in tissue in the disorder state but less strongly expressed or not expressed at all in the normal state.
- the differential expression can be either way around in the comparison between untreated and treated tissue. Further, expression may be regarded as differential if the protein undergoes any recognisable change between the two states under comparison.
- the present invention is based on methods which directly determine differentially expressed proteins present in tissue samples, by employing techniques such as 2D gel electrophoresis .
- allem means a prototype example, test model or standard.
- Body weight and/or eating disordered state includes conditions in which the body mass is either below normal or above normal and conditions in which eating behaviour is abnormal .
- Conditions characterised by body weight and/or eating disordered state include obesity, anorexia, bulimia and cathexic states associated with cancer, ' AIDS and trauma.
- Nutrient intake status includes any paradigm providing a difference in nutrient intake such as differences between fed and fasting, over-feeding v normal feeding, meal eating v ad-libitum eating, sated v unsated.
- Micronutrient selection includes paradigms in which individuals or animals select or are provided different macronutrients such as high fat v low fat, fat preferring v carbohydratre preferring.
- Relevant tissue means any tissue which undergoes a biological change in the body weight or eating disordered state or the experimental paradigm.
- tissue ... representative of ... subjects means any tissue in which the above-mentioned biological change can be simulated for laboratory purposes and includes, for example, a primary cell culture or cell line derived ultimately from relevant tissue.
- subjects includes human and animal subjects.
- the treatments referred to above can comprise the administration of one or more drugs or foodstuffs, and/or other factors such as diet or exercise.
- the differentially expressed proteins include "fingerprint proteins", “target proteins” or “pathway proteins” .
- fingerprint protein means a DEP, the expression of which can be used, alone or together with other DEPs, to monitor or assess the condition of a patient suspected of suffering from a bodyweight or eating disordered state. Since these proteins will normally be used in combination, especially a combination of four or more, they are conveniently termed “fingerprint proteins", without prejudice to the possibility that on occasions they may be used singly or along with only one or two other proteins for this purpose. Such a fingerprint protein or proteins can be used, for example, to diagnose a particular type of body weight or eating disorder and thence to suggest a specific treatment for it.
- diagnosis includes the provision of any information concerning the existence, non-existence or probability of the disorder in a patient. It further includes the provision of information concerning the type or classification of the disorder or of symptoms, which are or may be experienced in connection with it. It encompasses prognosis of the medical course of the disorder.
- target protein means a DEP, the level or activity of which can be modulated by treatment to alleviate a body weight or eating disordered state. Modulation of the level or activity of the target protein in a patient may be achieved, for example, by administering the target protein, another protein or gene which interacts with it, or an agent which counteracts or reduces it, for example an antibody to the protein, competitive inhibitor of the protein or an agent which acts in the process of transcription or translation of the corresponding gene.
- the DEPs can interact with at least one other protein or which a gene involved in the regulation of body weight or eating.
- Such other proteins are termed herein “pathway proteins” (PPs) .
- the term is applied to the protein with which it, the DEP, interacts, not the DEP itself, although a pathway protein can be another DEP .
- Figures 1A-E show computer images of stained 2-DGE gels from the liver tissue of lean mice identifying spots thereon, including DEPs.
- Figures 2-8 show DEPs which are underexpressed in ob/ob mouse liver tissue relative to expression in lean mouse lean mouse liver tissue.
- Figures 9-11 show DEPs which are underexpressed in ob/ob mouse skeletal muscle relative to expression in lean mouse skeletal muscle, and DEPs which are overexpressed in ob/ob skeletal muscle relative to lean mouse skeletal muscle .
- Figures 12-23 show DEPs which are underexpressed in ob/ob mouse adipose tissue relative to lean mouse adipose tissue, and DEPs which are overexpressed in obese mouse adipose tissue relative to adipose tissue of lean mice.
- Figures 24-29 show DEPs which are underexpressed in ob/ob mouse brown adipose tissue relative to lean mouse brown adipose tissue, and DEPs which are overexpressed in obese mouse brown adipose tissue relative to brown adipose tissue of lean mice.
- Proteins termed 'target proteins' and/or fingerprint proteins are described which are differentially expressed in body weight and/or eating disordered states relative to their expression in normal states and/or which are differentially expressed in response to manipulations relevant to the regulation of body weight and/or eating. Additionally, proteins termed 'pathway proteins' are described which interact with proteins involved in regulation of body weight and/or eating. Methods for the identification of such fingerprint target and pathway proteins are also described.
- Described below are methods for the identification of compounds, which modulate the expression of proteins, involved in the regulation of body weight and/or eating. Additionally described below are methods for the treatment of body weight and/or eating disordered states including, but not limited to, obesity.
- the present invention concerns methods for the identification of proteins which are involved in body weight and/or eating disorders and/or which are involved in obesity.
- proteins may represent proteins, which are differentially expressed in body weight and/or eating disordered states relative to their expression in normal states. Further, such proteins may represent proteins that are differentially expressed or regulated in response to manipulation relevant to modulating body weight and/or food intake.
- differentially expressed proteins may represent 'target' or 'fingerprint' proteins. Methods for the identification of such proteins are described in section 1.1. Methods for the further characterisation of such differentially expressed proteins and for their identification as target and/or fingerprint proteins are presented below in section 1.3.
- pathway proteins involved in body weight and/or eating disordered states and/or obesity.
- Pathway proteins refer to a protein, which exhibits the ability to interact with other proteins relevant to body weight and/or eating disordered states.
- a pathway protein may be differentially expressed and therefore may have the characteristics of a target or fingerprint protein.
- 'Differential expression' refers to both qualitative as well as quantitative differences in protein expression.
- a differentially expressed protein may qualitatively have its expression activated or completely inactivated in normal versus body weight and/or eating disordered state or under control versus experimental conditions.
- Such a qualitatively regulated protein will exhibit an expression pattern within a given tissue or cell type, which is detectable in either control or body weight and/or eating disordered subject, but not detectable in both.
- Alternatively, such a qualitatively regulated protein will exhibit an expression pattern within one or more cell types, which is detectable in either control or experimental subjects but not detectable in both.
- 'Detectable' refers to a protein expression pattern, which is detectable by the technique of differential display 2D electrophoresis, which are well known to those of skill in the art .
- a differentially expressed protein may have its expression modulated, i.e. quantitatively increased or decreased, in normal versus body weight or eating disorder states or under control versus experimental conditions.
- the degree to which expression differs in normal versus body weight and/or eating disordered states or control versus experimental states need only be large enough to be visualised via standard characterisation techniques, such as silver staining of 2D-electrophoretic gels.
- standard characterisation techniques such as silver staining of 2D-electrophoretic gels.
- Other such standard characterisation techniques by which expression differences may be visualised are well known to those skilled in the art. These include successive chromatographic separations of fractions and comparisons of the peaks, capillary electrophoresis and separations using micro-channel networks, including on a microchip.
- Chromatographic separations can be carried out by high performance liquid chromatography as described in
- Capillary electrophoresis is a technique described in many publications, for example in the literature "Total CE Solutions” supplied by Beckman with their P/ACE 5000 system. The technique depends on applying an electric potential across the sample contained in a small capillary tube.
- the tube has a charged surface, such as negatively charged silicate glass.
- Oppositely charged ions in this instance, positive ions
- the appropriate electrode in this instance, the cathode
- EEF electro-osmotic flow
- Micro-channel networks function somewhat like capillaries and can be formed by photoablation of a polymeric material.
- a UV laser is used to generate high energy light pulses that are fired in bursts onto polymers having suitable UV absorption characteristics, for example polyethylene terephthalate or polycarbonate.
- the incident photons break chemical bonds with a confined space, leading to a rise in internal pressure, mini-explosions and ejection of the ablated material, leaving behind voids which form micro- channels.
- the micro-channel material achieves a separation based on EOF, as for capillary electrophoresis. It is adaptable to micro-chip form, each chip having its own sample injector, separation column and electrochemical detector: see J.S. Rossier et al , 1999, Electrophoresis 20 : pp. 727-731.
- Differentially expressed proteins may be further described as target proteins and/or fingerprint proteins.
- 'Fingerprint proteins' refer to a differentially expressed protein whose expression pattern may be utilised as part of a prognostic or diagnostic body weight and/or eating disorder evaluation or which, alternatively, may be used in methods for identifying compounds useful for the treatment of body weight and/or eating disordered states.
- a fingerprint protein may also have characteristics of a target protein or a pathway protein.
- 'Target protein' refers to a differentially expressed protein involved in body weight and/or eating disordered states and/or obesity such that modulation of the level or activity of the protein may act to prevent the development of the disordered states including, but not limited to, obesity.
- a target protein may also have the characteristics of a fingerprint protein or a pathway protein.
- Section 1.1.1 A variety of methods may be used for the identification of proteins, which are involved in body weight and/or eating disordered states and/or which may be involved in obesity. Described in Section 1.1.1 are several experimental paradigms, which may be utilised for the generation of subjects, and samples, which may be used for the identification of such proteins. Material from the paradigm control and experimental subjects may be characterised for the presence of differentially expressed protein sequences as discussed below in Section 1.1.2.
- tissue from normal and body weight and/or eating disorder subjects would be compared.
- Such subjects could include, but would not be limited to, subjects with obesity. It could also involve a comparison of normal subjects and subjects who have resisted the development of obesity despite overfeeding.
- Appropriate tissues would include, but not be limited to, blood and adipose tissue. It could also include postmortem samples from subjects. Particularly useful tissues would include brain particularly the hypothalamus, skeletal muscle, liver and adipose tissue.
- Among additional paradigms would include a comparison of obese subjects and obese subjects whose body weight had been reduced by, but not limited to, dietary restriction or dietary modification, anorexic drugs such as sibutramine, Y5 receptor antagonists, leptin and leptin mimetics, MC4 agonists, exercise and thermogenic drugs such as b 3 -adrenoceptor agonists.
- anorexic drugs such as sibutramine, Y5 receptor antagonists, leptin and leptin mimetics, MC4 agonists, exercise and thermogenic drugs such as b 3 -adrenoceptor agonists.
- adipose tissue from various fat depots within the same individuals but typically to be a comparison of subcutaneous and omental adipose tissue since omental adipose tissue is associated with the metabolic and cardiovascular complications of obesity, whereas excess subcutaneous adipose tissue does not carry a similar high risk.
- a further paradigm which may be utilised for the identification of differentially expressed proteins involved in body weight and/or eating disorders are paradigms designed to analyse those proteins which may be involved in genetic models of obesity. Accordingly, such paradigms are referred to as 'genetic obesity paradigms' .
- such paradigms may identify the proteins regulated either directly or indirectly by the ob/ob, db/db, tub or fat gene products.
- rats such a paradigm may identify proteins regulated either directly or indirectly by the fa gene product.
- test subjects may include ob/ob, db/db, tub/tub or fat/fat experimental mice and lean littermate controls.
- Test subjects could also include fa/fa and male and female ZDF rats. Samples of tissues such as skeletal muscle, whole brain, hypothalamus, adipose tissue and liver would be obtained. The examples below demonstrate the use of such genetic paradigms in identifying proteins which are differentially expressed in obese animals versus normal animals .
- ob/ob, db/db, tub/tub and/or fat/fat mice and/or fa/fa and ZDF rats and lean control animals and inbred or outbred strains of rodents may be treated with drugs that reduce body weight, particularly body fat mass.
- Such drugs include, but are not limited to, appetite suppressants such as sibutramine, fenfluramine, NPY antagonists, melanocortin-4 receptor agonists, orexin antagonists, MCH antagonists, thermogenic drugs such as b 3 -adrenoceptor agonists and anti-obesity agents such as leptin, leptin mimetics and other cytokines such as axokine and brain derived neurotrophic factor.
- appetite suppressants such as sibutramine, fenfluramine, NPY antagonists, melanocortin-4 receptor agonists, orexin antagonists, MCH antagonists, thermogenic drugs such as b 3 -adrenoceptor agonists and anti-obesity agents such as leptin, leptin mimetics and other cytokines such as axokine and brain derived neurotrophic factor.
- ob/ob, db/db, tub/tub and/or fat/fat mice and/or fa/fa and/or ZDF rats and lean controls or inbred or outbred strains of rodents may be offered dietary treatments to either worsen the obese state or reduce the level of obesity or to identify animals that resist dietary obesity.
- lean or obese animals could be provided with a high fat diet to exacerbate the obese state.
- Sprague Dawley rats would be fed on a high fat diet or a cafeteria diet consisting of human snack foods. Some of the rats become obese whilst others resist obesity.
- this paradigm can be used to select proteins that are associated with a predisposition to the development of obesity and to proteins associated with an ability to resist dietary obesity.
- This paradigm can be further refined by incorporating drug treatment or exercise paradigms.
- obese rodents that had been weight reduced by dietary restriction would be allowed access to ad-libitum food whilst a control group of obese rodents would remain dietary restricted. Such a paradigm would allow the identification of proteins associated with the regain of obesity following withdrawal of dietary restriction.
- Some native animal strains do not exhibit obesity in the wild but do when fed a laboratory chow or other laboratory diets. These include the desert rodents, the spiny mouse and the sand rat. Comparison of animals fed on the natural diet and those fed on a laboratory diet allows identification of proteins associated with body weight disorders and obesity.
- Selective breeding can also be used to obtain genetically closely related animals that exhibit different degrees of adiposity and/or metabolic efficiency. Comparisons between these closely related animals allows the identification of proteins associated with body weight disorders and obesity.
- hypothalamus A particularly important tissue with respect to the control of feeding behaviour and the regulation of energy balance is the hypothalamus. It is clear that many substances act in this area of the brain to regulate feeding and/or energy balance. These include catecholamines, serotinergic agents, leptin, orexins, melanocyte concentrating hormone, melanocortin receptor agonists and antagonists. It is also clear that there are complex interactions between these various substances . However, no systematic evaluation of the changes in protein composition of the hypothalamus and its constituent nuclei, which underlay the changes in feeding behaviour, has yet been undertaken.
- hypothalamic nuclei from fed v fasted rodents; sated v non-sated rodents; obese v lean rodents; fat preferring v non-fat preferring rodents may be used to identify the hypothalamic protein patterns associated with particular feeding behaviours.
- paradigms include systems in which isolated cells such as adipocytes, are incubated in vitro with agents such as leptin.
- tissue from subjects utilised in paradigms such as those described above in 1.1.1 are obtained.
- blood and body fluids may be analysed since the differentially expressed proteins might be released into the circulations.
- Whole tissue or isolated cells may be used.
- Sub-cellular fractions of cells might also be used.
- Particularly useful sub-cellular fractions include the nuclear protein fraction.
- pathway protein refers to a protein which exhibits the ability to interact with differentially expressed proteins involved in body weight and/or eating disorders and/or to interact with differentially expressed proteins which are relevant to obesity.
- a pathway protein may be differentially expressed and, therefore, may have the characteristics of a target and/or fingerprint protein.
- Any method suitable for detecting protein-protein interactions may be employed for identifying pathway proteins by identifying interactions between proteins and proteins known to be differentially expressed in body weight and/or eating disordered states and/or obesity regulation.
- Such differentially expressed proteins may be cellular or extracellular proteins. Those proteins, which interact with such differentially expressed proteins, represent pathway gene products.
- a pathway protein may be used, in conjunction with standard techniques, to identify its corresponding pathway gene. For example, at least a portion of the amino acid sequence of the pathway gene product may be ascertained using techniques well known to those of skill in the art, such as via the Edman degradation technique (see, e.g. Creighton (1983) 'Proteins: Structures and Molecular Principles', W.H. Freeman & Co., N.Y., pp. 34-49).
- the amino acid sequence obtained may be used as a guide for the generation of oligonucleotide mixtures that can be used to screen for pathway gene sequences. Screening may be accomplished, for example, by standard hybridisation or PCR techniques. Techniques for the generation of oligonucleotide mixtures and the screening are well-known (see, e.g. Ausubel, supra, and PCR Protocols: A Guide to Methods and Applications (1990) Innis, M. et al . , eds. Academic Press Inc . , New York) .
- yeast two-hybrid system detects protein interactions in vivo, the yeast two-hybrid system.
- yeast two-hybrid system One method, which detects protein interactions in vivo, the yeast two-hybrid system, is described in detail for illustration only and not by way of limitation.
- One version of this system has been described (Chien et al (1991) Proc. Natl. Acad. Sci. USA, 88, 9578-9582) and is commercially available from Clontech (Palo Alto, Calif.).
- plasmids are constructed that encode two hybrid proteins : one consists of the DNA-binding domain of a transcription activator protein fused to a known protein, in this case, the differentially expressed protein known to be involved in body weight and/or eating disordered states and/or obesity regulation, and the other consists of the transcription activator protein's activation domain fused to an unknown protein that is encoded by a cDNA which has been recombined into this plasmid as part of a cDNA library.
- the plasmids are transformed into a strain of the yeast Saccharomyces cerevisiae that contains a reporter gene (e.g. lacZ) whose regulatory region contains the transcription activator's binding sites.
- the yeast two-hybrid system or related methodology may be used to screen activation domain libraries for proteins that interact with a known differentially expressed 'bait' protein.
- Total genomic or cDNA sequences are fused to the DNA encoding an activation domain.
- This library and a plasmid encoding a hybrid of the bait protein product fused to the DNA-binding domain are co- transformed into a yeast reporter strain, and the resulting transformants are screened for those that express the reporter gene.
- the bait gene can be cloned into a vector such that it is translationally fused to the DNA encoding the DNA-binding domain of the GA 4 protein. These colonies are purified and the library plasmids responsible for reporter gene expression are isolated. DNA sequencing is then used to identify the proteins encoded by the library plasmids.
- a cDNA library of the cell line from which proteins that interact with bait proteins are to be detected can be made using methods routinely practised in the art.
- the cDNA fragments can be inserted into a vector such that they are translationally fused to the activation domain of GAL4.
- This library can be co- transformed along with the bait-gene GAL4 fusion plasmid into a yeast strain, which contains a lacZ gene driven by a promoter which contains GAL4 activation sequence.
- a cDNA encoded protein, fused to GAL4 activation domain, that interacts with bait gene product will reconstitute an active GAL4 protein and thereby drive expression of the lacZ gene.
- Colonies which express lacZ can be detected by their blue color in the presence of X-gal .
- the cDNA can then be purified from these strains, and used to produce and isolate the bait gene-interacting protein using techniques routinely practised in the art.
- pathway protein Once a pathway protein has been identified and isolated, it may be further characterised as, for example, discussed below, in Section 1.3.
- Differentially expressed proteins such as those identified via the methods discussed, above, in Section 1.1, and pathway genes, such as those identified via the methods discussed, above, in Section 1.2, above, as well as genes identified by alternative means, may be further characterised by utilising, for example, methods such as those discussed herein. Such proteins will be referred to herein as 'identified proteins'.
- Analyses such as those described herein yield information regarding the biological function of the identified proteins.
- An assessment of the biological function of the differentially expressed proteins in addition, will allow for their designation as target and/or fingerprint proteins.
- any of the differentially expressed proteins whose further characterisation indicates that a modulation of the proteins expressed or a modulation of the proteins activity may ameliorate any of the body weight and/or eating disorders will be designated 'target proteins', as defined above, in Section 1.
- target proteins along with those discussed below, will constitute the focus of the compound discovery strategies discussed below, in Section 3.
- target proteins and/or modulating compounds can be used as part of the treatment and/or prevention of body weight and/or eating disorders and/or obesity.
- any of the differentially expressed proteins whose further characterisation indicates that such modulations may not positively affect body weight and/or eating disorders, but whose expression pattern contributes to a protein 'fingerprint' pattern correlative of, for example, a body weight and/or eating disordered state, will be designated a 'fingerprint protein'.
- 'Fingerprint patterns' will be more fully discussed below, in Section 7.1. It should be noted that each of the target proteins may also function as fingerprint proteins, as well as may all or a portion of the pathway proteins.
- pathway proteins may also be characterised according to techniques such as those described herein. Those pathway proteins which yield information indicating that they are differentially expressed and that modulation of the proteins expression or a modulation of the proteins expression or a modulation of the proteins activity may ameliorate any of the body weight and/or eating disorders of interest will also be designated 'target proteins' . Such target proteins, along with those discussed above, will constitute the focus of the compound discovery strategies discussed below, in Section 3 and can be used as part of the treatment methods described in Section 4, below.
- pathway proteins characterisation indicates that modulation of gene expression or gene product activity may not positively affect body weight and/or eating disorders of interest, but whose expression is differentially expressed and contributes to a gene expression fingerprint pattern correlative of, for example, a body weight and/or eating disordered state
- pathway genes may additionally be designated as fingerprint genes.
- the corresponding nucleotide sequence of the identified protein may be obtained by utilising standard techniques well known to those of skill in the art, may, for example, be used to reveal homologies to one or more known sequence motifs which may yield information regarding the biological function of the identified protein.
- In vivo systems may include, but are not limited to, animal systems, which naturally exhibit body weight and/or eating disorder-like symptoms, or ones which have been engineered to exhibit such symptoms. Further, such systems may include systems for the further characterisation of body weight and/or eating disorders, and/or obesity, and may include, but are not limited to, naturally occurring and transgenic animal systems such as those described above, in Section 1.1.1, and Section
- In vitro systems may include, but are not limited to, cell-based systems comprising cell types known to be associated with energy storage and metabolism. Such cells may be wild type cells, or may be non-wild type cells containing modifications known to, or suspected of, contributing to a body weight a /or eating disorder of interest. Such systems are discussed in detail below, in Section 2.2.2.
- the expression of these proteins may be modulated within the in vivo and/or in vitro systems, i.e. either overexpressed or underexpressed in, for example, transgenic animals and/or cell lines, and its subsequent effect on the system then assayed.
- the activity of the identified protein may be ⁇ modulated by either increasing or decreasing the level of activity in the in vivo and/or in vitro system of interest, and its subsequent effect then assayed.
- treatment may include a modulation of protein expression and/or protein activity. Characterisation procedures such as those described herein may indicate where such modulation should involve an increase or a decrease in the expression or activity of the protein of interest. Such methods of treatment are discussed below, in Section 4.
- Identified proteins which include, but are not limited to, differentially expressed proteins such as those identified in Section 1.1 above, and pathway proteins, such as those identified in Section 1.2 above, are described herein. Specifically, the amino acid sequences of such identified proteins are described. Further, antibodies directed against the identified protein, and cell- and animal-based models by which the identified proteins may be further characterised and utilised are also discussed in this Section.
- antibodies capable of specifically recognising one or more differentially expressed or pathway protein epitopes.
- Such antibodies may include, but are not limited to, polyclonal antibodies, monoclonal antibodies (mAbs) , humanised or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') 2 fragments, fragments produced by a FAb expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.
- mAbs monoclonal antibodies
- Such antibodies may be utilised as part of body weight and/or eating disorder treatment methods, and/or may be used as part of diagnostic techniques whereby patients may be tested for abnormal levels of fingerprint, target, or pathway gene proteins, or for the presence of abnormal forms of such proteins.
- various host animals may be immunised by injection with a differentially expressed or pathway protein, or a portion thereof.
- Such host animals may include, but are not limited to, rabbits, mice and rats, to name but a few.
- Various adjuvants may be used to increase the immunological response, depending on the host species, including active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyamin, dinitrophenol , and potentially useful human adjuvant such as BCG bacille Calmette-Fuerin) and Corynebacterium parvum.
- Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunised with an antigen, such as target proteins, or an antigenic functional derivative thereof.
- an antigen such as target proteins, or an antigenic functional derivative thereof.
- host animals such as those described above, may be immunised by injection with differentially expressed or pathway protein supplemented with adjuvants as also described above.
- Monoclonal antibodies which are homogeneous populations of antibodies to a particular antigen, may be obtained by any technique, which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique of Kohler and Milstein (1975, Nature 256; 495- 497; and US Pat. No. 4,376,110), the human b-cell hybridoma technique (Kosbor, et al . , 1983, Immunology
- Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof.
- the hybridoma producing the mAb of this invention may be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo makes this the presently preferred method of production.
- a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region.
- Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide.
- Antibody fragments which recognise specific epitopes, may be generated by known techniques.
- such fragments include, but are not limited to, the F(ab') 2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab') 2 fragments.
- Fab expression libraries may be constructed (Huse, et al . , 1989, Science 246: 1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
- Cell- and animal-based model systems Described herein are cell- and animal-based systems, which act as models for body weight and or eating disorders. These systems may be used in a variety of applications.
- the animal-based model systems can be utilised to identify differentially expressed proteins via one of the paradigms described above, in Section 1.1.1.
- Cell- and animal-based model systems may be used to further characterise differentially expressed and pathway proteins, as described above in Section 1.3. Such further characterisation may, for example, indicate that a differentially expressed protein is a target protein.
- Such assays may be utilised as part of screening strategies designed to identify compounds which are capable of ameliorating body weight and/or eating disorder symptoms, as described below.
- the animal - and cell-based models may be used to identify drugs, pharmaceuticals, therapies and interventions which may be effective in treating such body weight and/or eating disorders.
- such animal models may be used to determine the LD 50 and the ED 50 in animal subjects, and such data can be used to determine the in vivo efficacy of potential body weight and/or eating disorder treatments, including treatments for obesity.
- Animal-based model systems of body weight and/or eating disorders may include, but are not limited to, non- recombinant and engineered transgenic animals.
- Non-recombinant animal models for body weight and/or eating disorders may include, for example, genetic models.
- Such genetic body weight and/or eating disorder models may include, for example, mouse models of obesity such as mice homozygous for the autosomal recessive ob, db, fat or tub alleles. It could also include rat models, for example fa/fa rats.
- Non-recombinant, non-genetic animal models of body weight and/or eating disorder may include, for example, rats or mice fed on a diet containing a large amount of fat.
- Such diets could be synthetic diets in which the fat content (by calorific value) is more than 50%.
- animal models exhibiting body weight and/or eating disorder-like symptoms may be engineered by utilising, for example, the gene sequences of target proteins such as those described above, in Section 2, in conjunction with techniques for producing transgenic animals that are well known to those of skill in the art.
- gene sequences of target proteins may be introduced into, and overexpressed in, the genome of the animal of interest, or, if endogenous gene sequences of target proteins are present, they may either be overexpressed or, alternatively, may be disrupted in order to underexpress or inactivate gene expression of target proteins.
- the coding portion of the target gene sequence may be ligated to a regulatory sequence, which is capable of driving gene expression in the animal and cell type of interest.
- a regulatory sequence which is capable of driving gene expression in the animal and cell type of interest.
- Such regulatory regions will be well known to those of skill in the art, and may be - utilised in the absence of undue experimentation.
- an endogenous gene sequence of a target protein such a sequence may be isolated and engineered such that when reintroduced into the genome of the animal of interest, the endogenous gene alleles of the target protein will be inactivated.
- the engineered gene sequence of the target protein is introduced via gene targeting such that the endogenous sequence is disrupted upon integration of the engineered target gene sequence into the animal ' s genome .
- Animals of any species including, but not limited to, mice, rats, rabbits, guinea pigs, pigs, mini -pigs, goats and non-human primates, e.g. baboons, squirrel, monkeys, rhesus monkeys and chimpanzees may be used to generate body weight and/or eating disorder animal models.
- Any technique known in the art may be used to introduce a target gene transgene of a target protein into animals to produce the founder lines of transgenic animals.
- Such techniques include, but are not limited to, pronuclear microinjection (Hoppe, P.C. and Wagner, T.E., 1989, US Pat. No. 4,873,191); retrovirus mediated gene transfer into germ lines (Van der Putten et al . , 1985, Proc. Natl. Acad. Sci., USA 82: 6148-6152); gene targeting in embryonic stem cells (Thompson et al . , 1989, Cell 56: 313-321); electroporation of embryos (Lo, 1983, Mol. Cell Biol.
- the present invention provides for transgenic animals that carry the transgene in all their cells, as well as animals which carry the transgene in some, but not all their cells, i.e. mosaic animals (see, for example, techniques described by Jakobovits, 1994, Curr. Biol. 4: 761-763) .
- the transgene may be integrated as a single transgene or in concatamers, e.g. head-to-head tandems or head-to-tail tandems.
- the transgene may also be selectively introduced into and activated in a particular cell type by following, for example, the teaching of
- the target gene transgene be integrated into the chromosomal site of the endogenous target gene
- gene targeting is preferred.
- vectors containing some nucleotide sequences homologous to the gene of the endogenous target protein of interest are designed for the purpose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of, the nucleotide sequence of the endogenous target gene.
- the transgene may also be selectively introduced into a particular cell type, thus inactivating the endogenous gene of interest in only that cell type, by following, for example, the teaching of Gu et al . (Gu, H. et al., 1994, Science 265: 103-106).
- the regulatory sequences required for such a cell-type specific inactivation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art .
- the expression of the recombinant target gene and protein may be assayed utilising standard techniques. Initial screening may be accomplished by Southern blot analysis or PCR techniques to analyse animal tissues to assay whether integration of the transgene has taken place. The level of mRNA expression of the transgene in the tissues of the transgenic animals may also be assessed using techniques which include, but are not limited to, Northern blot analysis of tissue samples obtained from the animal, in situ hybridisation analysis, and RT-PCR. Samples of target protein-expressing tissue may also be evaluated immunocytochemically using antibodies specific for the transgene protein of interest .
- target protein transgenic animals that express target gene mRNA or target protein transgene peptide (detected immunocytochemically, using antibodies directed against target protein epitopes) at easily detectable levels should then be further evaluated to identify those animals which display characteristic body weight and/or eating disorder-like symptoms.
- Such symptoms may include, for example, obesity, hyperphagia, hypophagia, leanness, glucose intolerance, hyperinsulinaemia, and/or non-insulin dependent diabetes.
- specific cell types within the transgenic animals may be analysed and assayed for cellular phenotypes characteristic of body weight and/or eating disorders.
- Such cellular phenotypes may include an assessment of a particular cell types fingerprint pattern of expression and its comparison to known fingerprint expression profiles of the particular cell type in animals exhibiting body weight and/or eating disorders.
- Such transgenic animals serve as suitable model systems for body weight and/or eating disorders.
- target protein transgenic founder animals i.e. those animals which express target proteins in cells or tissues of interest and which, preferably, exhibit symptoms of body weight and/or eating disorders
- they may be bred, inbred, outbred or crossbred to produce colonies of the particular animal.
- breeding strategies include, but are not limited to, outbreeding of founder animals with more than one integration site in order to establish separate lines; inbreeding of separate lines in order to produce compound target protein transgenics that transgenically express the target protein of interest at higher levels because of the effects of additive expression of each target gene transgene; crossing of heterozygous transgenic animals to produce animals homozygous for a given integration site in order to both augment expression and eliminate the possible need for screening of animals by DNA analysis; crossing of separate homozygous lines to produce compound heterozygous or homozygous lines; breeding animals to different inbred genetic backgrounds so as to examine effects of modifying alleles on expression of the target protein and the development of body weight and/or eating disorder-like symptoms.
- One such approach is to cross the target protein transgenic founder animals with a wild type strain to produce an FI generation that exhibits body weight and/or eating disorder-like symptoms, such as hyperphagia, hypophagia, obesity and leanness.
- the FI generation may then be inbred in order to develop a homozygous line, if it is found that homozygous target protein transgenic animals are viable.
- Cells that contain and express target gene sequences which encode target proteins and, further, exhibit cellular phenotypes associated with an obesity disorder may be utilised to identify compounds that exhibit an ability to ameliorate body weight and/or eating disorder symptoms.
- Cellular phenotypes, which may indicate an ability to ameliorate body weight and/or eating disorders may include, for example, resistance to insulin .
- cell lines which may be used for such assays may also include recombinant, transgenic cell lines.
- the body weight and/or eating disorder animal models of the invention discussed above, in Section 2.2.1 may be used to generate cell lines, containing one or more cell types involved in body weight and/or eating disorders, that can be used as cell culture models for this disorder. While primary cultures derived from the body weight and/or eating disorder transgenic animals of the invention may be utilised, the generation of continuous cell lines is preferred.
- techniques which may be used to derive a continuous cell line from the transgenic animals see Small, et al . , 1985, Mol. Cell Biol. 5: 642-648.
- cells of a cell type known to be involved in body weight and/or eating disorders may be transfected with sequences capable of increasing or decreasing the amount of target protein within the cell.
- gene sequences of target proteins may be introduced into, and overexpressed in, the genome of the cell of interest, or, if endogenous gene sequences of the target protein are present, they may either be overexpressed or, alternatively, be disrupted in order to underexpress or inactivate target protein expression.
- the coding portion of the target gene sequence may be ligated to a regulatory sequence, which is capable of driving gene expression in the cell type of interest.
- a regulatory sequence which is capable of driving gene expression in the cell type of interest.
- the gene sequence may be isolated and engineered such that when reintroduced into the genome of the cell type of interest, the endogenous target gene alleles will be inactivated.
- the engineered target gene sequence is introduced via gene targeting such that the endogenous target sequence is disrupted upon integration of the engineered target gene sequence into the cell's genome. Gene targeting is discussed above, in Section 2.2.1.
- Transfection of target protein gene sequence nucleic acid may be accomplished by utilising standard techniques. See, for example, Ausubel, 1989, supra. Transfected cells should be evaluated for the presence of the recombinant target gene sequences, for expression and accumulation of target gene mRNA, and for the presence of recombinant target protein production. In instances wherein a decrease in target protein expression is desired, standard techniques may be used to demonstrate whether a decrease in endogenous target gene expression and/or in target protein production is achieved.
- the following assays are designed to identify compounds that bind to target proteins, bind to other cellular proteins that interact with a target proteins, and to compounds that interfere with the interaction of the target proteins with other cellular proteins.
- Such compounds may include, but are not limited to, other cellular proteins. Methods for the identification of such cellular proteins are described below, in Section 3.2
- Compounds may include, but are not limited to, peptides such as, for example, soluble peptides, including, but not limited to, Ig-tailed fusion peptides, comprising extracellular portions of target protein transmembrane receptors, and members of random peptide libraries (see, e.g. Lam, K.S. et al . , 1991, Nature 354: 82-84; Houghten, R. et al . , 1991, Nature 354: 84-86) made of D- and/or L-configuration amino acids, phosphopeptides (including, but not limited to, member of random or partially degenerate, directed phosphopeptide libraries: se, e.g., Songyang, Z.
- peptides such as, for example, soluble peptides, including, but not limited to, Ig-tailed fusion peptides, comprising extracellular portions of target protein transmembrane receptors, and members of random peptide libraries (see,
- antibodies including, but not limited to, polyclonal, monoclonal, humanised, anti-idiotypic, chimeric or single chain antibodies, and FAb, F(ab') 2 an d FAb expression library fragments, and epitope-binding fragments thereof) and small organic or inorganic molecules.
- Compounds identified via assays such as those described herein may be useful, for example, in elaborating the biological function of the target protein, and for ameliorating body weight and/or eating disorders.
- compounds that interact with the target protein may include ones which accentuate or amplify the activity of the bound target protein. Such compounds would bring about an effective increase in the level of target protein activity, thus ameliorating symptoms.
- compounds that bind target protein may be identified that inhibit the activity of the bound target protein.
- Assays for testing the effectiveness of compounds identified by, for example, techniques such as those described in Section 3.1 to 3.3, are discussed below, in Section 3.4.
- In vitro screening assays for compounds that bind to the target proteins may be designed to identify compounds capable of binding the target proteins of the invention.
- Compounds identified may be useful, for example, in modulating the activity of wild type and/or mutant target proteins, may be useful in elaborating the biological function of the target protein, may be utilised in screens for identifying compounds that disrupt normal target protein interactions, or may in themselves disrupt such interactions.
- the principle of the assays used to identify compounds that bind to the target protein involves preparing a reaction mixture of the target protein and the test compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex which can be removed and/or detected in the reaction mixture.
- These assays can be conducted in a variety of ways. For example, one method to conduct such an assay would involve anchoring target protein or the test substance onto a solid phase and detecting target protein/test compounds complexes anchored on the solid phase at the end of the reaction.
- the target protein may be anchored onto a solid surface, and the test compound, which is not anchored, may be labelled, either directly or indirectly.
- microtiter plates may conveniently be utilised as the solid phase.
- the anchored component may be immobilised by non-covalent or covalent attachments. Non-covalent attachment may be accomplished by simply coating the solid surface with a solution of the protein and drying.
- an immobilised antibody preferably a monoclonal antibody, specific for the protein to be immobilised may be used to anchor the protein to the solid surface.
- the surfaces may be prepared in advance and stored.
- the non-immobilised component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g. by washing) under conditions such that any complexes formed will remain immobilised on the solid surface.
- the detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously non-immobilised component is pre-labelled, the detection of label immobilised on the surface indicates that complexes were formed. Where the previously non- immobilised component is not pre-labelled, an indirect label can be used to detect complexes anchored on the surface, e.g. using a labelled antibody specific for the previously non-immobilised component (the antibody, in turn, may be directly labelled or indirectly labelled with a labelled anti-Ig antibody) .
- a reaction can be conducted in a liquid phase, the reaction products separated from unreacted components, and complexes detected, e.g. using an immobilised antibody specific for target protein or the test compound to anchor any complexes formed in solution, and a labelled antibody specific for the other component of the possible complex to detect anchored complexes.
- Any method suitable for detecting protein-protein interactions may be employed for identifying novel target protein-cellular or extracellular protein interactions. These methods are outlined in Section 1.2, above, for the identification of pathway proteins, and may be utilised herein with respect to the identification of proteins which interact with identified target proteins.
- the target proteins of the invention may, in vivo, interact with one or more cellular or extracellular macromolecules, such as proteins.
- macromolecules may include, but are not limited to, nucleic acid molecules and those proteins identified via methods such as those described above, in Section 3.2.
- cellular and extracellular macromolecules are referred to herein as 'binding partners' .
- Compounds that disrupt such interactions may be useful in regulating the activity of the target protein, especially mutant target proteins.
- Such compounds may include, but are not limited to, molecules such as antibodies, peptides, and the like, as described, for example, in Section 3.1 above.
- the basic principle of the assay systems used to identify compounds that interfere with the interaction between the target protein and its cellular or extracellular binding partner or partners involves preparing a reaction mixture containing the target protein, and the binding partner under conditions and for a time sufficient to allow the two to interact and bind, thus forming a complex.
- the reaction mixture is prepared in the presence and absence of the test compound.
- the test compound may be initially included in the reaction mixture, or may be added at a time subsequent to the addition of target protein and its cellular or extracellular binding partner. Control reaction mixtures are incubated without the test compound or with a placebo. The formation of any complexes between the target protein and the cellular or extracellular binding partner is then detected.
- the formation of a complex in the control reaction, but not in the reaction mixture containing the test compound indicates that the compound interferes with the interaction of the target protein and the interactive binding partner. Additionally, complex formation within reaction mixtures contains the test compound and a mutant target protein. This comparison may be important in those cases wherein it is desirable to identify compounds that disrupt interactions of mutant but not normal target proteins .
- the assay for compounds that interfere with the interaction of the target and binding partners can be conducted in a heterogeneous or homogeneous format . Heterogeneous assays involve anchoring either the target protein or the binding partner onto a solid phase and detecting complexes anchored on the solid phase at the end of the reaction. In homogeneous assays, the entire reaction is carried out in a liquid phase.
- test compounds that interfere with the interaction between the target protein and the binding partners can be identified by conducting the reaction in the presence of the test substance, i.e. by adding the test substance to the reaction mixture prior to or simultaneously with the target protein and interactive cellular or extracellular binding partner.
- test compounds that disrupt pre-formed complexes e.g. compounds with higher binding constants that displace one of the components from the complex, can be tested by adding the test compound to the reaction mixture after complexes have been formed.
- the various formats are described briefly below.
- either the target protein or the interactive cellular or extracellular binding partner is anchored onto a solid surface, while the non-anchored species is labelled, either directly or indirectly.
- the anchored species may be immobilised by non-covalent or covalent attachments. Non-covalent attachment may be accomplished simply by coating the solid surface with a solution of the target gene product or binding partner and drying. Alternatively, an immobilised antibody specific for the species to be anchored may be used to anchor the species to the solid surface. The surfaces may be prepared in advance and stored.
- the partner of the immobilised species is exposed to the coated surface with or without the test compound. After the reaction is complete, unreacted components are removed (e.g. by washing) and any complexes formed will remain immobilised on the solid surface.
- the detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the non-immobilised species is pre-labelled, the detection of label immobilised on the surface indicates that complexes were formed. Where the non-immobilised species is not pre-labelled, an indirect label can be used to detect complexes anchored on the surface, e.g.
- the antibody in turn, may be directly labelled or indirectly labelled with a labelled anti-Ig antibody.
- test compounds which inhibit complex formation or which disrupt pre-formed complexes can be detected.
- the reaction can be conducted in a liquid phase in the presence or absence of the test compound, the reaction products separated from unreacted components, and complexes detected, e.g. using an immobilised antibody specific for one of the binding components to anchor any complexes formed in solution, and a labelled antibody specific for the other partner to detect anchored complexes.
- test compounds which inhibit complex or which disrupt pre- formed complexes can be identified.
- a homogeneous assay can be used.
- a preformed complex of the target protein and the interactive cellular or extracellular binding partner is prepared in which either the target protein or its binding partners is labelled, but the signal generated by the label is quenched due to complex formation (see, e.g. US Pat. No. 4,109,496 by Rubenstein which utilises this approach for immunoassays) .
- the addition of a test substance that competes with and displaces one of the species from the pre-formed complex will result in the generation of a signal above background. In this way, test substances, which disrupt target protein/cellular or extracellular binding partner interaction, can be identified.
- the target protein can be prepared for immobilisation using recombinant DNA techniques described in Section 2.1 above.
- the target protein gene coding region can be fused to a glutathione-S-transferase (GST) gene using a fusion vector, such as pGEX-5X-l, in such a manner that its binding activity is maintained in the resulting fusion protein.
- GST glutathione-S-transferase
- the interactive cellular or extracellular binding partner can be purified and used to raise a monoclonal antibody, using methods routinely practised in the art and described above, in Section 2.1.
- This antibody can be labelled with the radioactive isotope 125 , for example, by methods routinely practised in the art.
- a heterogeneous assay e.g.
- the GST-target protein gene fusion protein can be anchored to glutathione-agarose beads.
- the interactive cellular or extracellular binding partner can then be added in the presence or absence of the test compound in a manner that allows interaction and binding to occur.
- unbound material can be washed away, and the labelled monoclonal antibody can be added to the system and allowed to bind to the complexed components.
- the interaction between the target protein and the interactive cellular or extracellular binding partner can be detected by measuring the amount of radioactivity that remains associated with the glutathione-agarose beads. A successful inhibition of the interaction by the test compound will result in a decrease in measured radioactivity.
- the GST-target protein gene fusion protein and the interactive cellular or extracellular binding partner can be mixed together in liquid in the absence of the solid glutathione-agarose beads.
- the test compound can be added either during or after the species are allowed to interact. This mixture can then be added to the glutathione-agarose beads and unbound material is washed away. Again the extent of inhibition of the target protein/binding partner interaction can be detected by adding the labelled antibody and measuring the radioactivity associated with the beads.
- these same techniques can be employed using peptide fragments that correspond to the binding domains of the target protein and/or the interactive cellular or extracellular binding partner (in cases where the binding partner is a protein) , in place of one or both of the full length proteins.
- Any number of methods routinely practised in the art can be used to identify and isolate the binding sites. These methods include, but are not limited to, mutagenesis of the gene encoding one of the proteins and screening for disruption of binding in a co- immunoprecipitation assay. Compensating mutations in the gene encoding the second species in the complex can then be selected. Sequence analysis of the genes encoding the respective proteins will reveal the mutations that correspond to the region of the protein involved in interactive binding.
- one protein can be anchored to a solid surface using methods described in this Section above, and allowed to interact with and bind to its labelled binding partner, which has been treated with a proteolytic enzyme, such as trypsin. After washing, a short, labelled peptide comprising the binding domain may remain associated with the solid material, which can be isolated and identified by amino acid sequencing. Also, once the gene coding for the cellular or extracellular binding partner is obtained, short gene segments can be engineered to express peptide fragments of the protein, which can then be tested for binding activity and purified or synthesised.
- a proteolytic enzyme such as trypsin
- a target protein can be anchored to a solid material as described above, in this Section by making a GST-target protein gene fusion protein and allowing it to bind to glutathione agarose beads.
- the interactive cellular or extracellular binding partner can be labelled with a radioactive isotope, such as 35 S, and cleaved with a proteolytic enzyme such as trypsin. Cleavage products can then be added to the anchored GST-target protein gene fusion protein and allowed to bind. After washing away unbound peptides, labelled bound material, representing the cellular or extracellular binding partner binding domain, can be eluted, purified and analysed for amino acid sequence by well-known methods. Peptides so identified can be produced synthetically or fused to appropriate facilitative proteins using recombinant DNA technology.
- binding compounds including but not limited to, compounds such as those identified in the foregoing assay systems, may be tested for the ability to prevent or ameliorate body weight and/or eating disorder symptoms, which may include, for example, obesity.
- body weight and/or eating disorder symptoms which may include, for example, obesity.
- Cell- based and animal model-based assays for the identification of compounds exhibiting such an ability to ameliorate body weight and/or eating disorder symptoms are described below.
- cell-based systems such as those described above, in Section 2.2.2, may be used to identify compounds, which may act to prevent or ameliorate body weight and/or eating disorder symptoms.
- such cell systems may be exposed to a compound, suspected of exhibiting an ability to ameliorate body weight and/or eating disorder symptoms, at a sufficient concentration and for a time sufficient to elicit a response in the exposed cells.
- the cells are examined to determine whether one or more of the body weight and/or eating disorder-like cellular phenotypes has been altered to resemble a more normal or more wild type phenotype, or a phenotype more likely to produce a lower incidence or severity of disorder symptoms.
- animals-based body weight and/or eating disorder systems such as those described above, in Section 2.2.1, may be used to identify compounds capable of ameliorating body weight and/or eating disorder-like symptoms.
- animal models may be used as test substrates for the identification of drugs, pharmaceuticals, therapies and interventions which may be effective in treating such disorders.
- animal models may be exposed to a compound, suspected of exhibiting an ability to prevent or ameliorate body weight and/or eating disorder symptoms, at a sufficient concentration and for a time sufficient to elicit such a prevention or amelioration of the body weight and/or eating disorder symptoms in the exposed animals.
- the response of the animals to the exposure may be monitored by assessing the reversal of disorders associated with body weight and/or eating disorders such as obesity.
- any treatments that reverse any aspect of body weight and/or eating disorder-like symptoms should be considered as candidates for human or companion animal body weight and/or eating disorder therapeutic intervention including the treatment of obesity.
- Dosages of test agents may be determined by deriving dose-response curves, as discussed in Section 6.1 below.
- any treatments that can prevent the development or redevelopment of body weight and/or eating disorders should be considered as candidates for the prevention of human or companion animal body weight and/or eating disorder therapeutic intervention.
- Such disorders include, but are not limited to, obesity.
- Protein expression patterns may be utilised in conjunction with either cell -based or animal-based systems to assess the ability of a compound to ameliorate body weight and/or eating disorder-like symptoms.
- the expression pattern of one or more fingerprint proteins may form part of a fingerprint profile, which may then be used in such as assessment.
- Fingerprint profiles are described below, in Section 7.1. Fingerprint profiles may be characterised for known states, either body weight and/or eating disorder or normal states, within the cell- and/or animal-based model systems. Subsequently, these known fingerprint profiles may be compared to ascertain the effect a test compound has to modify such fingerprint profiles, and to cause the profile to more closely resemble that of a more desirable fingerprint.
- administration of a compound may cause the fingerprint profile of a body weight and/or eating disorder model system to more closely resemble the control system.
- Administration of a compound may, alternatively, cause the fingerprint profile of a control system to begin to mimic a body weight and/or eating disorder state, which may, for example, be used in further characterising the compound of interest, or may be used in the generation of additional animal models.
- body weight and/or eating disorder symptoms may be ameliorated. It is possible that body weight and/or eating disorders may be brought about, at least in part, by an abnormal level of target protein, or by the presence of a target protein exhibiting an abnormal activity. As such, the reduction in the level and/or activity of such target protein would bring about the amelioration of body weight and/or eating disorder-like symptoms. Techniques for the reduction of target protein gene expression levels or target protein activity levels are discussed in Section 4.1 below.
- body weight and/or eating disorders may be brought about, at least in part, by the absence or reduction of the level of target protein expression, or a reduction in the level of a target proteins activity.
- an increase in the level of target protein gene expression and/or the activity of such proteins would bring about the amelioration of body weight and/or eating disorder-like symptoms.
- target proteins involved in body weight and/or eating disorders may cause such disorders via an increased level of target protein activity.
- a variety of techniques may be utilised to inhibit the expression, synthesis, or activity of such target genes and/or proteins.
- such molecules may include, but are not limited to, peptides (such as, for example, peptides representing soluble extracellular portions of target protein transmembrane receptors), phosphopeptides, small organic or inorganic molecules, or antibodies (including, for example, polyclonal, monoclonal, humanised, anti- idiotypic, chimeric or single chain antibodies, and FAb, F(ab') 2 and FAb expression library fragments, and epitope- binding fragments thereof) .
- peptides such as, for example, peptides representing soluble extracellular portions of target protein transmembrane receptors
- phosphopeptides small organic or inorganic molecules
- antibodies including, for example, polyclonal, monoclonal, humanised, anti- idiotypic, chimeric or single chain antibodies, and FAb, F(ab') 2 and FAb expression library fragments, and epitope- binding fragments thereof.
- antisense and ribosome molecules which inhibit expression of the target protein gene, may also be used in accordance with the invention to inhibit the aberrant target protein gene activity. Such techniques are described below, in Section 4.1.1; triple helix molecules may be utilised in inhibiting the aberrant target protein gene activity.
- antisense ribosome and triple helix molecules.
- Such molecules may be designed to reduce or inhibit either wild type, or if appropriate, mutant target protein gene activity. Techniques for the production and use of such molecules are well known to those of skill in the art.
- Antisense RNA and DNA molecules act to directly block the translation of mRNA by hybridising to targeted mRNA and preventing protein translation.
- antisense DNA oligodeoxy-ribonucleotides derived from the translation initiation site, e.g. between the -10 and +10 regions of the target gene nucleotide sequence of interest, are preferred.
- Ribosomes are enzymatic RNA molecules capable of catalysing the specific cleavage of RNA.
- the mechanism of ribosome action involves sequence specific hybridisation of the ribosome molecule to complementary target RNA, followed by an endonucleolytic cleavage.
- the composition of ribosome molecules must include one or more sequences complementary to the target protein mRNA, and must include the well known catalytic sequence responsible for mRNA cleavage. For this sequence, see US Pat. No. 5,093,246, which is incorporated by reference herein in its entirety.
- engineered hammerhead motif ribosome molecules that specifically and efficiently catalyse endonucleolytic cleavage of RNA sequences encoding target proteins.
- Specific ribosome cleavage sites within any potential RNA target are initially identified by scanning the molecule of interest for ribosome cleavage sites which include the following sequences, GUA, GUU and GUC. Once identified, short TNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target protein gene, containing the cleavage site may be evaluated for predicted structural features, such as secondary structure, that may render the oligonucleotide sequence unsuitable. The suitability of candidate sequences may also be evaluated by testing their accessibility to hybridise with complementary oligonucleotides, using ribonuclease protection assays.
- Nucleic acid molecules to be used in triplex helix formation for the inhibition of transcription should be single stranded and composed of deoxynucleotides .
- the base composition of these oligonucleotides must be designed to promote triple helix formation via Hoogsteen base pairing rules, which generally require sizeable stretches of either purines or pyrimidines to be present on one strand of a duplex.
- Nucleotide sequences may be pyrimidine-based, which will result in TAT and CGC + triplets across the three associated strands of the resulting triple helix.
- the pyrimidine-rich molecules provide base complementary to a purine-rich region of a single strand of the duplex in a parallel orientation to that strand.
- nucleic acid molecules may be chosen that are purine-rich, for example, containing a stretch of G residues. These molecules will form a triple helix with a DNA duplex that is rich in GC pairs, in which the majority of the purine residues are located on a single strand of the targeted duplex, resulting in GGC triplets across the three strands in the triplex.
- the potential sequences that can be targeted for triple helix formation may be increased by creating a so-called "switchback" nucleic acid molecule.
- Switchback molecules are synthesised in an alternating 5' -3 ' , 3 '-5' manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex.
- Anti-sense RNA and DNA, ribosome and triple helix molecules of the invention may be prepared by any method known in the art for the synthesis of DNA and RNA molecules. They include techniques for chemically synthesising oligodeoxyribonucleotides and oligo- ribonucleotides well known in the art such as, for example, solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences may be incorporated into a wide variety of vectors, which incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Alternatively, antisense cDNA constructs that synthesise antisense RNA constitutively inducibly, depending on the promoter used, can be introduced stably into cell lines.
- DNA molecules may be introduced as a means of increasing intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences or ribo- or deoxy-nucleotides to the 5 ' and/ or 3 ' ends of the molecule or the use of phosphorothioate or 2 ' O-methyl rather than phosphodiesterase linkages within the oligodeoxy-ribonucleotide backbone.
- Antibodies that are both specific for target protein and interfere with its activity may be used to inhibit target protein function. Where desirable, antibodies specific for mutant target protein, which interferes with the activity of such mutant target product, may also be used. Such antibodies may be generated using standard techniques described in Section 2.3, supra, against the proteins themselves or against peptides corresponding to portions of the proteins.
- the antibodies include, but are not limited to, polyclonal, monoclonal, Fab fragments, single chain antibodies, chimeric antibodies, etc.
- the target gene protein is intracellular and whole antibodies are used, internalising antibodies may be preferred.
- lipofectin or liposomes may be used to deliver the antibody or a fragment of the Fab region, which binds to the target protein epitope into cells. Where fragments of the antibody are used, the smallest inhibitory fragment, which binds to the target protein's binding domain, is preferred.
- peptides having an amino acid sequence corresponding to the domain of the variable region of the antibody that binds to the target protein may be used. Such peptides may be synthesised chemically or produced via recombinant DNA technology using methods well known in the art (e.g. see Creighton, 1983, supra; and Sambrook et al , 1989, supra).
- single chain neutralising antibodies which bind to intracellular target protein epitopes, may also be administered.
- Such single chain antibodies may be administered, for example, by expressing nucleotide sequences encoding single-chain antibodies within the ⁇ 5 target cell populating by utilising, for example, techniques such as those described in Marasco et al (Marasco, W. et al , 1993, Proc. Natl. Acad. Sci. USA, 90: 7889-7893) .
- target protein is extracellular, or is a transmembrane protein
- any of the administration techniques described below, in Section 6, which are appropriate for peptide administration may be utilised to effectively administer inhibitory target protein
- Target proteins that cause body weight and/or eating disorders may be underexpressed within body weight and/or
- target protein may be increased to levels
- the level of target protein activity may be increased, for example, by either increasing the level of target protein present or by increasing the level of active target protein, which is
- a target protein at a level sufficient to ameliorate body weight and/or eating disorder symptoms may be administered to a patient exhibiting such symptoms. Any of the techniques discussed below, in Section 6, may be utilised for such administration.
- One of skill in the art will readily know how to determine the concentration of effective, non-toxic doses of the normal target protein, utilising techniques such as those described below, in Section 4.6.1.
- patients may be treated by gene replacement therapy.
- One or more copies of a normal target protein gene or a portion of the gene that directs the production of a normal target protein with target protein gene function may be inserted into cells, using vectors which include, but are not limited to, adenovirus, adeno- associated virus, and retrovirus vectors, in addition to other particles that introduce DNA into cells, such as liposomes. Additionally, techniques such as those described above may be utilised for the introduction of normal target protein gene sequences into human cells.
- Cells preferably autologous cells, containing normal target protein gene sequences may then be introduced or reintroduced into the patient at positions which allow for the prevention or amelioration of body weight and/or eating disorder symptoms.
- Such cell replacement techniques may be preferred, for example, when the target protein is a secreted, extracellular protein.
- antibodies may be administered which specifically bind to a target protein and by binding, serve to, either directly or indirectly, activate the target protein function.
- Such antibodies can include, but are not limited to, polyclonal, monoclonal, FAb fragments, single chain antibodies, chimeric antibodies and the like.
- the antibodies may be generated using standard techniques such as those described above, in Section 2.3, and may be generated against the protein themselves or against proteins corresponding to portions of the proteins.
- the antibodies may be administered, for example, according to the techniques described above, in Section 4.1.2.
- the identified compounds, nucleic acid molecules and cells that affect target protein expression, synthesis and/or activity can be administered to a patient at therapeutically effective doses to prevent or to treat or to ameliorate body weight and/or eating disorders.
- a therapeutically effective dose refers to that amount of the compound sufficient to result in amelioration of symptoms of body weight and/or eating disorder, including obesity, or alternatively, to that amount of a nucleic acid molecule sufficient to express a concentration of protein which results in the amelioration of such symptoms .
- Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g. for determining by ED S0 (the dose therapeutically effective in 50% of the population) and by determining the ED 50 of any side-effects (toxicity - TD50) .
- the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio TD 50 /ED 50 .
- Compounds, which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimise potential damage to uninfected cells and, thereby, reduce side effects.
- the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
- the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
- the dosage may vary within this range depending upon the dosage form employed and the route of administration utilised.
- compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers or excipients.
- the compounds and their physiologically acceptable salts and solvates may be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or oral, buccal , parenteral and rectal administration.
- the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g. pre- gelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methyl-cellulose); fillers (e.g. lactose, microcrystaklline cellulose or calcium hydrogen phosphate) ; lubricants (e.g. magnesium, stearate, talc or silica); disintegrants (e.g. potato starch or sodium starch glycollate); or wetting agents (e.g. sodium lauryl sulphate) .
- binding agents e.g. pre- gelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methyl-cellulose
- fillers e.g. lactose, microcrystaklline cellulose or calcium hydrogen phosphate
- lubricants e.g. magnesium, stearate, talc or silica
- Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use.
- Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g. sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g. lecithin or acacia); and preservatives (e.g. methyl or propyl-p-hydroxybenzoates or sorbic acid) .
- the preparations may also contain buffer salts, flavouring, colouring and sweetening agents as appropriate.
- Preparations for oral administration may be suitably formulated to give controlled release of the active compound.
- compositions may take the form of tablets or lozenges formulated in conventional manner .
- the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurised packs or a nebuliser, with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane, carbon dioxide or other suitable gas.
- a suitable propellant e.g. dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane, carbon dioxide or other suitable gas.
- the dosage unit may be determined by providing a valve to deliver a metered amount.
- Capsules and cartridges of, e.g. gelatin, for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
- the compounds may be formulated for parenteral administration by injection, e.g. by bolus injection or continuous infusion.
- Formulations for injection may be presented in unit dosage form, e.g. in ampoules or in multi-dose containers, with an added preservative.
- the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilising and/or dispersing agents.
- the active ingredient may be in powder form for constitution with a suitable vehicle, e.g. sterile pyrogen-free water, before use.
- the compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g. containing conventional suppository bases such as cocoa butter or other glycerides .
- the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation, for example, subcutaneously or intramuscularly) or by intramuscular injection.
- the compounds may be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
- compositions may, if desired, be presented in a pack or dispenser device, which may contain one or more unit dosage forms containing the active ingredient.
- the pack may, for example, comprise metal or plastic foil, such as blister pack.
- the pack or dispenser device may be accompanied by instructions for administration.
- a variety of methods may be employed for the diagnosis of the nature of the body weight and/or eating disorders, the predisposition to body weight and/or eating disorders, and for monitoring the efficacy of any body weight and/or eating disorder compounds during, for example, clinical trials and for monitoring patients undergoing clinical evaluation for the treatment of such disorders .
- the methods include conventional methods such as waist/hip ratios (BMI) , weighing and c.t. or dexascans .
- BMI waist/hip ratios
- Obesity may be detected and the efficacy of treatment monitored by methods and parameters identified by such bodies as the World Health Organization and the International Obesity Task Force.
- Methods may also, for example, utilise reagents such as the fingerprint protein described in Section 4.1, and antibodies directed against differentially expressed and pathway proteins, as described above, in Sections 1.3 (peptides) and 2.3 (antibodies).
- reagents such as the fingerprint protein described in Section 4.1, and antibodies directed against differentially expressed and pathway proteins, as described above, in Sections 1.3 (peptides) and 2.3 (antibodies).
- reagents may be used, for example, for: (1) the detection of the presence of target protein mutations, or (2) the detection of either an over- or an under- abundance of target protein relative to the normal state.
- the methods described herein may be performed, for example, by utilising pre-packaged diagnostic kits comprising at least one specific finger print protein or anti-fingerprint protein antibody reagent described herein, which may be conveniently used, e.g. in clinical settings, to diagnose patients exhibiting body weight and/or eating abnormalities.
- any cell type or tissue in which the fingerprint protein is expressed may be utilised in the diagnostics described below.
- suitable samples types include cell samples, tissue samples, and fluid samples such as blood, urine or plasma.
- Such methods are methods for monitoring the efficacy of compounds in clinical trials for the treatment of body weight and/or eating disorders.
- Such compounds can, for example, be compounds such as those described above, in Section 4.
- Such a method comprises detecting, in a patient sample, a protein, which is differentially expressed in the body weight and/or eating disorder state relative to its expression in a normal state.
- the expression of a single fingerprint protein, or alternatively, a fingerprint pattern of a cell involved in a body weight and/or eating disorder can be determined in the presence or absence of the compound being tested.
- the efficacy of the compound can be followed by comparing the expression data obtained to the corresponding known expression patterns in a normal state.
- Compounds exhibiting efficacy are those which alter the single fingerprint protein expression and/or the fingerprint pattern to more closely resemble that of the normal state.
- the detection of the protein differentially expressed in a body weight and/or eating disorder state relative to their expression in a normal state can also be used for monitoring the efficacy of potential body weight and/or eating disorder compounds and compounds for the treatment of obesity during clinical trials.
- the level and/or activity of the differentially expressed protein can be determined in relevant cells and/or tissues in the presence or absence of the compound being tested.
- the efficacy of the compound can be followed by comparing the protein level and/or activity data obtained to the corresponding known levels/activities for the cells and/or tissues in a normal state.
- Compounds exhibiting efficacy are those which alter the pattern of the cell and/or tissue involved in the body weight and/or eating disorder to more closely resemble that of the normal state.
- Non-fasted lean and obese, 8 week old, female C57 B1/6J ob/ob mice were anaesthetised with 50% "Hyponovel” and 50% “Hyponorm” and then killed humanely with carbon dioxide gas. Liver was removed and snap frozen between tongs in liquid nitrogen. The tissues were lyophilised for 48h, crushed in a mortar with liquid nitrogen and the resultant dried powder stored at -80°C
- a non-linear immobilised pH gradient of IGP strips (3.5- 10.0 NL IPG 18cm) was used as the first dimension. It offered high resolution, great reproducibility and allowed high protein loads. Based on specifications of the Geneva University Hospital, the non-linear pH gradient strips were prepared by Amersham-Pharmacia Biotechnology AB and are commercially available. The strips were 3mm wide and 180mm long.
- the strips were transferred to the Pharmacia strip tray. After placing IPG strips, humid electrode wicks, electrodes and sample cups in position, the strips and cups were covered with low viscosity paraffin oil. Samples were applied in the cups at the cathodic end of the IPG strips in a slow and continuous manner, without touching the gel .
- the voltage was linearly increased from 300 to 3500 V during 3 hours, followed by 3 additional hours at 3500 V, whereupon the voltage was increased to 5000 V.
- a total volt hour product of lOOkvh was used in an overnight run.
- the IPG strips were equilibrated in order to resolubilise the proteins and to reduce -S-S- bonds.
- the strips were thus equilibrated within the strip tray with 100ml of a solution containing Tris-HCI (50mM) , pH 6.8, urea (6M) , glycerol (30% v/v), SDS (2% w/v) and DTE (2% w/v) for 12 min.
- the SH groups were subsequently blocked with 100 ml of a solution containing Tris-HCI (50mM) pH 6.8, urea (6M) , glycerol (30% v/v), SDS (2% w/v), iodoacetamide (2.5% w/v) and a trace of bromophenol blue for 5 min.
- PDA Piperazine-diacrylyl
- the gel composition and dimensions were as follows:
- Trailing buffer Tris-glycine-SDS (25mM- 198mM-0.1% w/v) pH 8.3
- the gels were poured until 0.7cm from the top of the plates and over-layered with sec-butanol for about two hours. After the removal of the overlay and its replacement with water the gels were left overnight.
- the IPG gel strips were cut to size. Six mm were removed from the anodic end and 14mm from the cathodic end. The second dimension gels were over-layered with a solution containing agarose (0.5% w/v) and Tris-glycine-SDS (25mM-198mM-0.1% w/v) pH 8.3 heated at about 70 °C and the IPG gel strips were immediately loaded through it .
- the gel was run at 8-12°C for 5 hours at a constant current of 40mA/gel.
- the voltage is non-limiting, but usually requires 100 to 400 V.
- Step 1 At the end of the second dimension run, the gels were removed from the glass plates and washed in deionised water for 5 min.
- Step 2 The gels were soaked in ethanol : acetic acid:water (40:10:50 volume ratio) for 1 hour.
- Step 3 The gels were soaked in ethanol : acetic acid:water (5:5:90 volume ratio) for 2 hours or overnight .
- Step 4 They were washed in deionised water for 5 min.
- Step 5 They were soaked in a solution containing glutaraldehyde (1% w/v and sodium acetate (0.5M) for 30 min.
- Step 6 They were washed 3 times in deionised water for 10 min.
- Step 7 In order to obtain homogenous dark brown staining of proteins, gels were soaked twice in a 2 , 7- naphthalenedisulphonic acid solution (0.05% w/v) for 30 min .
- Step 8 The gels were then rinsed 4 times in deionised water for 15 min.
- Step 9 The gels were stained in a freshly made ammoniacal silver nitrate solution for 30 minutes. To prepare 750ml of this solution, 6g of silver nitrate were dissolved in 30ml of deionised water, which was slowly mixed into a solution containing 160ml of water, 10ml of concentrated ammonia (25%) and 1.5ml o sodium hydroxide (ION) . A transient brown precipitate might form. After it cleared, water was added to give the final volume.
- ION o sodium hydroxide
- Step 10 After staining, the gels were washed 4 times in deionised water for 4 min.
- Step 11 The images were developed in a solution of citric acid (0.01% w/v) and formaldehyde (0.1% v/v) for 5 to 10 min.
- Step 12 When a slight background stain appeared, development was stopped with a solution of Tris (5% w/v) and acetic acid (2% v/v) .
- the Laser Densitometer (4000 x 5000 pixels; 12 bits/pixel) from Molecular Dynamics and the GS-700 from
- Bio-Rad have been used as scanning devices. These scanners were linked to "Sparc" workstations and
- Two-dimensional polyacrylamide gels may be digitised and analysed by computer to allow quantitative image analysis and automatic gel comparison. Since the 2 -D-PAGE technique was first developed in 1975 several computer systems have been manufactured, mainly by academic 2-D- PAGE related laboratories. In the present work, “Melanie II”, developed at the University Hospital of Geneva was used. It is available for “Unix” workstations, as well as for "Power Macintosh” and IBM-compatible computers.
- mice spot detection, quantitation and matching, gel image extraction, zooming, warping and printing as well as gel stacking and flipping were carried out with the "MelView" program.
- Figure IE shows the entire map from the 2-D-PAGE of liver tissue of lean mice. (It so happens that all the differentially expressed proteins identified are over- expressed in the lean control mice, relative to the ob/ob control mice.)
- the map is divided into four quadrants in the manner :
- the quadrant of Figure IA shows the spots corresponding to proteins of the lowest pi and highest relative molecular mass and the quadrant of Figure ID those of the highest pi and lowest relative molecular mass.
- the DEPs are marked as LOM16, LOM17, LOM18, LOM19, LOM20, LOM 21, LOM22, LOM23, LOM24, LOM25, LOM26, LOM27, LOM28, and LOM29.
- the other spots identified are for reference purposes and have numbers of five digits preceded by "P" or "Q" , corresponding to the SWISS-PROT accession number, thus linking the spots to their genes.
- Tables 1 and 2 list the characteristics of the DEP spots in terms of relative volumes, areas, and optical densities and apparent pi and relative molecular mass.
- FIGS. 2-8 show images of lean controls (left) and ob/ob (“obese") controls (right) relating to one mouse. It should be appreciated that these images cannot fully represent to the eye the differences in expression measurable by computer. Thus, underneath, a bar chart is provided in which the volume of the spot as a percentage of the total volume of all spots is shown on the y-axis. All bar charts relate to 4 mice and resulted from a student T test (P ⁇ 0.01) . The following proteins have been identified as liver markers .
- PVDF polyvinylidene difluoride
- the procedure was as follows. After second-dimensional electrophoresis, the gels were soaked in deionized water for 3 min. Then they were equilibrated in a solution containing lOmM CAPS pH 11 for 30 min. At the same time, PVDF membranes were wetted in methanol for 1 min and equilibrated in a solution containing lOmM CAPS pH 11 and methanol (10% v/v) also for 30 min. Electroblotting was carried out in a semi- dry apparatus with a solution containing lOmM CAPS pH 11 and methanol (20% v/v anodic side; 5% v/v cathodic side) at 1 mA/cm 2 constant current for 3 hours at 15°C.
- the PVDF membranes were stained in a solution containing Coomassie Brilliant Blue R-250 (0.1% w/v) and methanol (50% v/v) for 15 min. Destaining was done in a solution containing methanol (40% v/v) and acetic acid (10% v/v) . The same method was used for preparative gels that did not need electrotransfer for further post-separation analysis, such as peptide mass fingerprinting .
- the PVDF stained membranes were either air-dried or dried on a 3mm thick plate onto a heating plate at 37°C for 10 min.
- amino acid derivatives are sequentially cleaved one at a time from the protein. Proteins with a chemically inaccessible alpha-amino group cannot be sequenced directly by this procedure and are termed N-terminally blocked. The best way to overcome the blocked proteins is to generate individual fragments by chemical or proteolytic cleavage. Routinely, ten to twelve Edman degradation cycles were performed for each spot. A search in the SWISS-PROT database was made to detect identity to known protein sequences .
- the Amido Black stained proteins were excised with a razor blade and N-terminal sequencing was performed using an ABI model 473A or 477A microsequencer from Applied Biosystems equipped with "'Problott" cartridges.
- the spots of interest were excised and soaked for two hours in a solution containing acetic acid (lOOmM), methanol (10% v/v) and PVP-40 (1% v/v) at 37°C. After three washes in deionized water, the PVDF spots were cut into small pieces (about 1mm 2 ) and incubated in 25 microlitres of a solution containing sodium phosphate (lOOmM) pH 8.0 and lysyl endopeptidase (1 microgram) . Following overnight digestion at room temperature, guanidine-HCl (28 mg) and DTT (100 micrograms) were added.
- acetic acid lOOmM
- methanol % v/v
- PVP-40 1% v/v
- PVDF membranes were first stained to visualise proteins, following which the immunodetection was undertaken. This allowed matching of proteins detected with ECL against those detected with the non-specific protein stain through computer comparison of both images. The mechanical strength of PVDF was also exploited as the same 2-D gel can be used many times for different antibodies.
- the membranes were incubated in 10 ml of a solution containing PBS-"Tween" 20 (0.5% v/v), non-fat dry milk (5% w/v) and the secondary peroxidase-conjugated antibody (1.1000; for example, if the primary antibody was sheep anti-mouse, then goat anti-sheep IgG was used as the secondary antibody) for 1 hour.
- the membranes were transferred to a clean glass plate and covered with 10 ml of developing solution (for example ECL from Amersham International or
- the 2-DGE method was repeated, but using a Coomassie blue stain.
- the 2-DGE spots were destained with 100 microlitres of 30% acetonitrile in 50mM ammonium bicarbonate at 37°C for 45 min. The supernatant was discarded and the gel spots dried in a "SpeedVac” for 30 min.
- the gel spots were rehydrated with 25 microlitres of a solution containing 0.2 micrograms of porcine trypsin and 50mM ammonium bicarbonate for 2 hours at 35°C. Then the gel spots and supernatant were dried in a "SpeedVac" for 30 min, rehydrated with 20 microlitres of H 2 0 for 30 min at 35°C and dried again for 30 min.
- the peptide mixtures were analysed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (Perseptive Biosystems Voyager Elite MALDI -TOF-MS) with a nitrogen laser (337nm) and operated in reflectron delayed extraction mode.
- Protein identification has been carried out using "Peptldent” (htt ; //www,expasv. ch/tools/peptid ⁇ nt.html) . It is a tool that allows the identification of proteins using pi, relative molecular mass and peptide mass fingerprinting data.
- the mouse SWISS-2-DPAGE database SWISS-2-DPAGE is an annotated 2-D-PAGE database in which all the data are easily retrieved by computer programs and stored in a format similar to that of the SWISS-PROT Protein Sequence Database, one of the most updated and annotated protein sequence databases presently available.
- the SWISS-2- DPAGE database assembles data on proteins identified on various 2-D-PAGE maps. Each SWISS-2 -DPAGE entry contains data on one protein, including mapping procedures, physiological and pathological data and Bibliographical references, as well as several 2-D-PAGE images showing the protein location. Cross-references are provided to SWISS- PROT and, through the latter, to other databases (EMBL, Genbank, PROSITE, OMIM, etc) .
- Example 1 The method of Example 1 was repeated, substituting for the liver tissue 200 micrograms (analytical scale) or 4 mg (preparative scale) of dried gastrocnemius muscle tissue and using the same respective volumes of the protein-solubilising solution as in Example 1. A 2-DGE map was thus obtained, from which DEPs were identified and isolated.
- the muscle DEPs were: MOM 31
- MOM 36 which were underexpressed in ob/ob mouse skeletal muscle relative to expression in lean mouse skeletal muscle and
- MOM 35 which were overexpressed in ob/ob skeletal muscle relative to lean. These are shown in Figures 9-11 inclusive.
- the location of the DEPs has been determined by running the liver extract from lean mice in the same gel as the muscle extract, so that liver protein reference spots appear in the muscle extract gel to serve as reference spots.
- Table 3 gives the characteristics of the DEP spots in terms of relative volumes, areas, optical densities and apparent pi and relative molecular mass.
- MOM31 0.259 0 .075 0.919 1 225 1.515 6.21 118405
- Example 1 The method of Example 1 was repeated, substituting for the liver tissue 16mg (analytical scale) or 160mg (preparative scale) of white adipose tissue and using the same respective volumes of the protein-solubilising solution as in Example 1.
- a 2-DGE map was thus obtained, from which DEPs were identified and isolated.
- the location of the proteins has been determined by running the liver extract from lean mice in the same gel as the white adipose extract, so that the liver protein reference spots appear in the white adipose extract get to serve as reference spots for the latter also.
- WOMT50 9. .187 1, .529 13.934 1, .094 3, .069 6.97 32129
- Example 1 The method of Example 1 was repeated, substituting for the liver tissue 400 micrograms (analytical scale) or 4mg (preparative scale) of brown adipose tissue and using the same respective volumes of the protein-solubilising solution as in Example 1.
- the weight of sample loaded was 150 micrograms (analytical) or 1.5mg (preparative) .
- a 2-DGE map was thus obtained, from which DEPs were identified and isolated.
- the location of the proteins has been determined by running the liver extract from lean mice in the same gel as the brown adipose extract, so that the liver protein reference spots appear in the brown adipose extract gel to serve as reference spots for the latter also.
- the brown adipose tissue DEPs which are overexpressed in ob/ob mouse brown adipose tissue relative to lean mouse brown adipose tissue are: BOM 66 BOM 67 BOM 68 BOM 75 BOM 76 BOM 77
- brown adipose tissue DEPs that are underexpressed in ob/ob mouse brown adipose tissue relative to lean mouse adipose tissue are: BOM 69
- mice and ob/ob mice either fed ad-lib or fasted overnight were anaesthetised with 50% hypnovel and 50% hyponorm and then killed humanely with carbon dioxide gas. The hypothalamus was removed and frozen in liquid nitrogen. The method of example 1 was then followed but using 200mg of hypothalamic tissue rather than 200mg liver.
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Priority Applications (4)
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EP00954779A EP1210605A2 (en) | 1999-09-02 | 2000-08-24 | Analysis and treatment of body weight and eating disorders |
CA002383381A CA2383381A1 (en) | 1999-09-02 | 2000-08-24 | Methods and compositions relating to body weight and eating disorders |
AU67134/00A AU782425B2 (en) | 1999-09-02 | 2000-08-24 | Methods and compositions relating to body weight and eating disorders |
JP2001520107A JP2003508754A (en) | 1999-09-02 | 2000-08-24 | Methods and compositions relating to weight and eating disorders |
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GBGB9920745.8A GB9920745D0 (en) | 1999-09-02 | 1999-09-02 | Methods and compositions relating to body weight and eating disorders |
GB9920745.8 | 1999-09-02 | ||
GB0002975.1 | 2000-02-09 | ||
GB0002975A GB0002975D0 (en) | 2000-02-09 | 2000-02-09 | Methods and compositions relating to body weight and eating disorders |
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JP (1) | JP2003508754A (en) |
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2004029624A1 (en) | 2002-09-24 | 2004-04-08 | Masaomi Iyo | Diagnostic and examination method for eating disorder |
CN112199370A (en) * | 2020-09-02 | 2021-01-08 | 安徽深迪科技有限公司 | BOM (bill of material) accelerated settlement worker method capable of effectively improving settlement efficiency |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5861485A (en) * | 1994-08-23 | 1999-01-19 | Millennium Pharmaceuticals, Inc. | Polypeptides involved in body weight disorders, including obesity |
WO1999041612A1 (en) * | 1998-02-13 | 1999-08-19 | Oxford Glycosciences (Uk) Ltd. | Methods and compositions for diagnosis of hepatoma |
-
2000
- 2000-08-24 WO PCT/GB2000/003277 patent/WO2001016603A2/en not_active Application Discontinuation
- 2000-08-24 EP EP00954779A patent/EP1210605A2/en not_active Withdrawn
- 2000-08-24 JP JP2001520107A patent/JP2003508754A/en not_active Withdrawn
- 2000-08-24 AU AU67134/00A patent/AU782425B2/en not_active Ceased
- 2000-08-24 CA CA002383381A patent/CA2383381A1/en not_active Abandoned
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
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US5861485A (en) * | 1994-08-23 | 1999-01-19 | Millennium Pharmaceuticals, Inc. | Polypeptides involved in body weight disorders, including obesity |
WO1999041612A1 (en) * | 1998-02-13 | 1999-08-19 | Oxford Glycosciences (Uk) Ltd. | Methods and compositions for diagnosis of hepatoma |
Non-Patent Citations (5)
Title |
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EDVARDSSON U ET AL: "A PROTEOMA ANALYSIS OF LIVERS FROM OBESE(OB/OB) MICE TREATED WITH THE PEROXISOME PROLIFERATOR WY14,643" ELECTROPHORESIS,WEINHEIM,DE, vol. 20, no. 4-5, April 1999 (1999-04), pages 935-942, XP000964564 ISSN: 0173-0835 * |
JITRAPAKDEE SARAWUT ET AL: "Regulation of rat pyruvate carboxylase gene expression by alternative promoters during development, in genetically obese rats and in insulin-secreting cells: Multiple transcripts with 5'-end heterogeneity modulate translation." JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 273, no. 51, 18 December 1998 (1998-12-18), pages 34422-34428, XP002157931 ISSN: 0021-9258 * |
LEE RIVERA IRENE ET AL: "Cloning and characterization of a 35-kDa mouse mitochondrial outer membrane protein MOM35 with high homology to Tom40." JOURNAL OF BIOENERGETICS AND BIOMEMBRANES, vol. 32, no. 1, February 2000 (2000-02), pages 111-121, XP000980184 ISSN: 0145-479X * |
LYNCH CHRISTOPHER J ET AL: "Carbonic anhydrase III in obese Zucker rats." AMERICAN JOURNAL OF PHYSIOLOGY, vol. 264, no. 4 PART 1, 1993, pages E621-630, XP000980239 ISSN: 0002-9513 * |
STANTON L W ET AL: "EXPRESSION OF CA III IN RODENT MODELS OF OBESITY" MOLECULAR ENDOCRINOLOGY, vol. 5, no. 6, 1991, pages 860-866, XP000980348 ISSN: 0888-8809 * |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2004029624A1 (en) | 2002-09-24 | 2004-04-08 | Masaomi Iyo | Diagnostic and examination method for eating disorder |
EP1548435A1 (en) * | 2002-09-24 | 2005-06-29 | Masaomi Iyo | Diagnostic and examination method for eating disorder |
EP1548435B1 (en) * | 2002-09-24 | 2012-05-23 | Masaomi Iyo | Diagnostic method for eating disorders |
CN112199370A (en) * | 2020-09-02 | 2021-01-08 | 安徽深迪科技有限公司 | BOM (bill of material) accelerated settlement worker method capable of effectively improving settlement efficiency |
CN112199370B (en) * | 2020-09-02 | 2024-01-26 | 安徽深迪科技有限公司 | BOM accelerating settlement work method capable of effectively improving settlement efficiency |
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JP2003508754A (en) | 2003-03-04 |
WO2001016603A3 (en) | 2001-09-20 |
AU6713400A (en) | 2001-03-26 |
EP1210605A2 (en) | 2002-06-05 |
CA2383381A1 (en) | 2001-03-08 |
AU782425B2 (en) | 2005-07-28 |
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