WO2017149307A1 - Screening assay using drosophila lines - Google Patents

Screening assay using drosophila lines Download PDF

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WO2017149307A1
WO2017149307A1 PCT/GB2017/050554 GB2017050554W WO2017149307A1 WO 2017149307 A1 WO2017149307 A1 WO 2017149307A1 GB 2017050554 W GB2017050554 W GB 2017050554W WO 2017149307 A1 WO2017149307 A1 WO 2017149307A1
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drosophila
gene
candidate agents
parkinson
genetically modified
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PCT/GB2017/050554
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French (fr)
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Lysimachos ZOGRAFOS
Katarzyna STYCZYNSKA-SOCZKA
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Parkure Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5085—Supracellular entities, e.g. tissue, organisms of invertebrates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
    • A01K67/0333—Genetically modified invertebrates, e.g. transgenic, polyploid
    • A01K67/0337—Genetically modified Arthropods
    • A01K67/0339—Genetically modified insects, e.g. Drosophila melanogaster, medfly
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/11—Protein-serine/threonine kinases (2.7.11)
    • C12Y207/11001—Non-specific serine/threonine protein kinase (2.7.11.1), i.e. casein kinase or checkpoint kinase
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A01K2217/052—Animals comprising random inserted nucleic acids (transgenic) inducing gain of function
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/20—Animal model comprising regulated expression system
    • A01K2217/203—Animal model comprising inducible/conditional expression system, e.g. hormones, tet
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/20—Animal model comprising regulated expression system
    • A01K2217/206—Animal model comprising tissue-specific expression system, e.g. tissue specific expression of transgene, of Cre recombinase
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/70—Invertebrates
    • A01K2227/706—Insects, e.g. Drosophila melanogaster, medfly
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0306—Animal model for genetic diseases
    • A01K2267/0318—Animal model for neurodegenerative disease, e.g. non- Alzheimer's

Definitions

  • the present invention is concerned with methods of using genetically modified Drosophila to screen chemicals for potential use in treating neurodegenerative conditions, such as Parkinson's disease.
  • Parkinson's disease is a common neurodegenerative syndrome characterized by formation of filamentous intraneuronal inclusions (Lewy bodies) comprised of the protein a- synuclein (asyn), dopaminergic neuron death and a movement disorder accompanied by other debilitating symptoms.
  • PD affects 1 in 100 people over 60 years old and currently there is no cure for it, only symptomatic treatment.
  • Drosophila melanogaster commonly fruit fly
  • PD Parkinson's disease
  • a number of further documents (14 - 20) describe the use of mutant drosophila in drug screening assays. These may be in terms of behavioural assays based on climbing assay. However, there is no realisation or suggestion of how to address the issue of throughput by carrying out such assays and/or how one may combine a behavioural response with a different type of response.
  • Drosophila offers some distinct advantages in terms of being an in vivo model and being able to screen many drug candidates at once, the ability to be a truly high- throughput model is quite limited. Moreover, a large library of test candidates may nevertheless generate a large number of potential hits and choosing which of these hits to carry forward can be difficult. Thus there is a need to be able to screen drugs more efficiently in order that initial hits are able to be taken forward more effectively or quickly to a clinical setting.
  • the present invention is based on the use of a plurality of genetically modified Drosophila lines in the development of an improved screening method for identifying lead candidates for further lead optimisation and/or more rapid progression towards the clinic.
  • the present invention is based on a multi-layer screening process using Drosophila models of neurodegenerative disease, such as Parkinson's disease.
  • the starting point in this process is a library of candidate drugs and the end point is a reduced selection of drugs from the library which are active based on showing evidence of a specific behavioural change displayed by the Drosophila, which may be indicative of a drug showing neuroprotection or repair.
  • the reduced selection of drugs may be taken forward for further rounds of screening, administration to other disease models, such as animal models, and/or administered to test subjects.
  • the present invention is ideally directed to the use of a first screen which employs a first library of candidate drugs and then second or further screens which employ a reduced number of candidate drugs from the first library of candidate drugs.
  • a method for screening candidate agents for potential use and/or further development as a drug for treating or preventing a neurodegenerative disorder such as Parkinson's diseases
  • the method comprising: a) administering candidate agents to Drosophila from a first Drosophila line which has been genetically modified to express a wild-type mammalian gene or mutant mammalian gene associated with the development and/or progression of a neurodegenerative disorder in a mammal and observing an effect the candidate agents have on a behaviour of the Drosophila; selecting candidate agents which display a positive effect on the behaviour of the Drosophila; and b) administering the selected candidate agents to Drosophila from a further Drosophila line which has been genetically modified to express a different wild-type mammalian gene or mutant mammalian gene associated with the development and/or progression of the neurodegenerative disorder and observing an effect the selected candidate agents have on the behaviour of the Drosophila; and further selecting candidate agents which display a positive effect on the behaviour
  • the second or further Drosophila lines may display more severe behavioural abnormalities as compared to the first Drosophila line. As it may be more difficult to conduct assays using drosophila which display more severe behavioural abnormalities, there may be an advantage of using such drosophila to screen fewer compounds.
  • the method may optionally include one or more further rounds of screening using further different genetically modified Drosophila lines and/or observing brain tissue samples in order to view any effect the selected candidate agents have on brain morphology, such as neurons in the brain.
  • the present invention is particularly suited for the identification of drug candidates for treating Parkinson's disease or related conditions.
  • Such related conditions include a number of other conditions with Parkinson-like symptoms known as Parkinsonism. These conditions include multiple system atrophy, progressive supranuclear palsy, corticobasal degeneration, Lewy body dementia, stroke, encephalitis (inflammation of the brain), and head trauma.
  • Other similar conditions include essential tremor, dystonic tremor, vascular Parkinsonism, and drug-induced Parkinsonism.
  • the present invention is based upon the development of a method which employs a climbing assay known the art.
  • the present invention may also be used in the identification of drug candidates for diseases or conditions where a climbing assay may be used for observing a behavioural change.
  • the present invention may find application in developing drugs for use in treating any of the above conditions.
  • the present invention permits the potential identification of drug candidates without have to screen full libraries of compounds multiple times. It may also be possible to identify drug candidates which may be active against related, or even unrelated neurological conditions. Certain conditions may in fact currently be thought of as being unrelated, but it may be the case that such an assumption is incorrect in a physiological and/or biochemical sense and it may be possible to identify drugs which act positively in such "unrelated" conditions. Additionally, it will be appreciated that drug screening using such fly models is quite time and resource consuming.
  • the Drosophila lines for use in the present invention may be generated by introducing into a Drosophila genome a wild-type or mutant gene which has been identified as being associated with the development or progression of the particular neurodegenerative disorder.
  • Techniques for genetically modifying Drosophila are known to the skilled addressee and described generally in Bischof (2007) for example. A specific non-limiting example of genetically modifying Drosophila is described hereinafter.
  • the first Drosophila line is genetically modified to express a first gene known to be associated with the development and/or progression of Parkinson's disease and the second Drosophila line is genetically modified to express a different gene or form of the first gene known to be associated with the development and/or progression of Parkinson's.
  • the first Drosophila line may be modified to express a wild-type version of a first gene associated with Parkinson's disease and the second line may be modified to express a mutant version of the first gene associated with Parkinson's disease.
  • the first gene may be a-synuclein.
  • mutant forms of a-synuclein which may be used in the present invention include A30P, A53T, E46K, H50Q and G51 D (numbering according to the sequence identified as P37840 in the UniProtKB database
  • LRKK2 including mutants G2019S and R1441 G
  • VPS35 including mutant D620N
  • EIF4G1 including mutant R1205H
  • sequence identified Q04637 in the UniProtKB database PRKN, PINK1 and DJ-1 among others and any of these genes alone or in addition to the use of ⁇ -synuclein may be used in accordance with the present invention.
  • Third or further genetically modified Drosophila lines may be generated to express further mutant forms of the aforementioned genes, in order that such third or further genetically modified Drosophila lines may be tested in accordance with the present invention.
  • it may be expected that a reduced number of candidate drugs is carried forward for further screening, leading to an improved or more rapid throughput for screening.
  • Double mutants may also be provided and tested.
  • a Drosophila line expressing two mutant genes such as VPS35 (D620N) and asyn.
  • Two separate genetic constructs including each mutant can be generated and then each construct integrated into the Drosophila genome at to two different genome insertion sites, one on the 2 nd (AttP40) and one on the 3 rd (AttP2) chromosome, allowing modular recombination of any two genes or mutants.
  • the candidate agents to be tested will typically be small chemical molecules, which may have a molecular weight of less than 500 Daltons.
  • the assay can also be used to test RNAi and biologic agents (such as peptides, antibodies, antibody fragments and the like).
  • the candidate agents may conveniently added to the food given to the Drosophila, so that the candidate agents are ingested by the Drosophila through feeding.
  • Expression of the genes and/or mutants described herein may typically result in an observable phenotypic response, such as reduction of movement, such as may be observed by way of climbing ability compared to an appropriate control (e.g. a Drosophila line which does not express a human gene).
  • an appropriate control e.g. a Drosophila line which does not express a human gene.
  • a positive effect on Drosophila behaviour in the context of the present invention may be seen as an improvement or reduction in the degree or severity of the reduction of movement, such as climbing ability.
  • Climbing ability and hence an improvement or reducing in the degree or severity may be tested using a known climbing assay. See for example Friggi-Grelin, et al (2003), or modified versions thereof.
  • An improvement in climbing ability may be determined in comparison to a control.
  • the control may be the same test carried out in the absence of the candidate drug, or a test carried out in the presence of a known Parkinson's disease drug, in order to identify candidate drugs which are better
  • Candidate drugs which are identified as providing a positive effect, such as an improvement in climbing ability, as compared to a control, are carried forward to a further test. Typically there may be a significant reduction from the original library of compounds tested, which are carried forward to the further test. The further test as carried out using a different Drosophila line, will enable a further significant reduction in the number of positive compounds being identified. Any further rounds are expected to reduce the number of positive compounds even further.lt is possible to determine whether candidate compounds are neuroprotective, or simply relieve symptoms of Parkinson's disease, such as the observed climbing ability.
  • Neuroprotective compounds will prevent neurons from being destroyed, or reducing other morphological changes, such as a reduction in formation of Lewy-body like aggregations of synuclein.; lower levels of cell death (as measured by molecular markers) and/or increased survival of dopaminergic neurons which may be observed in the brain of the Drosophila.
  • Such an effect may be observed by studying brain samples from Drosophila observed as responding favourably to test candidate agents, that is Drosophila which display a positive behavioural response, such as better climbing ability, to a candidate compound. It is possible through dissection of the Drosophila brain to visualize whether or not a compound prevents or reduces neuron destruction or other brain perturbations from occurring.
  • Drugs which are identified in accordance with the present invention as providing a positive effect, in terms of relieving symptoms and optionally being neuroprotective, may be carried forward to further testing in animal models of the neurodegenerative disorder, such as Parkinson's disease, and/or given to man in order to test their effectiveness.
  • lead candidates may be subjected to chemical modification and such chemically modified candidates tested in accordance with the present invention in order to ascertain if better candidates can be identified.
  • Figure 1 shows in schematic form an embodiment of the present invention
  • Figure 2 shows an example of results which may be obtained by testing various Drosophila lines in accordance with the present invention using a variety of different drugs, at different concentrations and time points;
  • Figure 3 shows a rationalisation of the results obtained in relation to Figure 2 into a more simple positive/negative effect
  • Figure 4 shows similar results to Figure 3, but further including further results observed following testing of a double mutant Drosophila, histological staining of Drosophila brain tissue, or mouse histology/behavioural data where applicable.
  • transgenic Drosophila lines were generated using a phiC31 based system [8]. This allows the insertion of different transgenic constructs into the same insertion site of the Drosophila genome across different transgenic lines, making the results comparable.
  • a number of appropriate upstream activation sequence (UAS) activated genetic constructs were synthesised, each encoding a specific version of a-synuclein (asyn), including the standard 'wild type' and 2 mutant sequences (A30P E46K, numbering as previously defined above).
  • the constructs were inserted in flies using a standard protocol (Genetic Services Inc.). This process generated 3 different models of PD (asyn wt, asyn A30P, asyn E46K).
  • a candidate drug is introduced at a suitable concentration into the fruit fly media.
  • Control groups are treated with the vehicle only.
  • a treatment group (TG) is defined as a specific line of flies expressing a form of asyn and treated with a certain candidate drug at a certain concentration.
  • the relevant control group (CG) is a group a group of flies treated with vehicle only.
  • the climbing assay is performed as described in [12] and also used in [1 ].
  • replicate groups of adult flies are anaesthetised and placed in a vertical plastic column (25 cm long, 1 .5 cm diameter) with a conic bottom end.
  • flies are suddenly tapped to the bottom of the column to startle them and force them to pack in a narrow space.
  • Overexcited flies remained whirling at the bottom and started climbing after a longer delay.
  • flies having reached the top of the column and flies remaining at the bottom were separately counted. Multiple trials are performed, one for each replicate set.
  • Samples were washed 3 ⁇ for 20 min each with PAT and then incubated overnight with goat secondary antibodies from Molecular Probes/I nvitrogen (1 :1000 dilution). Samples were washed again 3 ⁇ for 20 min each with PAT, rinsed with 1 ⁇ PBS and mounted in VectaShield.
  • the anti-TH antibody stains dopaminergic neurons by attaching on Tyrosine Hydroxilase (TH) and then sequestered and stained by the secondary antibody.
  • TH Tyrosine Hydroxilase
  • the brain preparations can be scanned using a confocal microscope. The images of brains acquired are used to quantify the number of dopaminergic cells active (stained) in each treatment and control group.
  • active drug we mean a small molecule that appears to be a good candidate for further development according to the evidence obtained from experiments described here.
  • Figure 1 shows in schematic fashion a representation of how the present invention may be carried out.
  • a library of candidate drugs is provided as represented by the multi-well assay plates shown in the Figure.
  • Each of the drugs within the library is tested in transgenic flies expressing the wild type asyn gene by feeding the each drug to a subset of flies from the line line expressing the wild type asyn gene and thereafter subjecting the flies to the described climbing assay.
  • By comparing the results with a vehicle control it is possible to identify an initial selection of drug candidates from the library, which can be carried forward to a second screen.
  • positive active drugs identified from the first screen are feed to a different line of transgenic Drosophila, such as A30P or E46K mutants and again it is determined whether or not the active drugs from the first screen still remain positive following the second screen.
  • Brain sections from Drosophila which have been identified as being rescued following the second screen may be observed in order to view dopaminergic neuron survival (as shown).
  • Drugs identified as being positive from the second round of screening may be carried forward for further screens or further developed.
  • Further screens may include further testing using further Drosophila lines which express other wild-type or mutant genes known to be associated with neurodegeneration and/or used in animal models, such as mice, rats etc of Parkinson's disease, before being tested in man. Histology testing may optionally be carried out in order to see what effect if any the active/positive drugs have on brain morphology, in particular neuron survival. This can add a further layer of evidence in terms of whether or not a particular drug can be considered as neuroprotective or drugs which are considered to relieve the symptoms of the neurodegenerative disorder.
  • identified active drugs may be used to develop a number of related chemical entities, through appropriate chemical modification of the active drug, in order to provide a number of derivatives and the derivatives tested using the screens of the present invention.
  • Figure 2 shows results obtained following the testing of a number of drugs (6 different drugs) at a variety of concentrations and time points. The drugs where administered to 3 different Drosophila lines. Underlined positive values represent a statistically significant positive or improved response as observed in the climbing test, as compared to a vehicle only control. All other values represent a statistically insignificant improvement, or a poorer response as observed using the climbing assay as compared to the vehicle only control.
  • Figure 3 simplifies the results shown in Figure 2, in order that where any particular subset (drug concentrations tested and the various time points) displayed one or more statistically significant responses, this is identified as a positive result (Y) and the drug can be carried forward to a further test using a different Drosophila line. It will be appreciated that the stringency of such determination can be increased or decreased as deemed appropriate by the investigator. Drugs which are identified as providing a positive effect following the first test may be further tested in a further Drosophila line, optionally brain histology investigated and/or in an animal model, for example. Exemplary results in this regard are shown in Figure 4.
  • the many faces of a-synuclein from structure and toxicity to therapeutic target. (2013).
  • the many faces of ⁇ -synuclein from structure and toxicity to therapeutic target., 74(1 ), 38-48.

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Abstract

The present invention is concerned with methods of using genetically modified Drosophila to screen chemicals for potential use in treating neurodegenerative conditions, such as Parkinson's disease.

Description

SCREENING ASSAY USING DROSOPHILA LINES
Field of the invention
The present invention is concerned with methods of using genetically modified Drosophila to screen chemicals for potential use in treating neurodegenerative conditions, such as Parkinson's disease.
Background to the invention
Parkinson's disease (PD) is a common neurodegenerative syndrome characterized by formation of filamentous intraneuronal inclusions (Lewy bodies) comprised of the protein a- synuclein (asyn), dopaminergic neuron death and a movement disorder accompanied by other debilitating symptoms. PD affects 1 in 100 people over 60 years old and currently there is no cure for it, only symptomatic treatment.
The use of Drosophila melanogaster (common fruit fly) as a model for Parkinson's disease (PD) has been established for more than a decade with the seminal work of Feany & Bender [1 ]. In their paper the authors describe how expression of the key effector protein in PD, asyn (reviewed in [2]) - in wild type and mutated forms (A30P, A53T) is associated with severe forms of PD [3], causes a series of phenotypes. These include reduced motor ability, Lewy body-like asyn inclusions in the brain, retinal degeneration and reduced survival of dopaminergic neurons. As a natural continuation of this work many research groups have created genetically engineered fruit flies expressing other mutant forms of asyn and highlighted their potential use in drug discovery. These models, along with validation data and their advantages as in-vivo drug discovery models - have been covered and reviewed in the literature numerous times, e.g. [4, 5, 6]. As described in extent in the aforementioned reviews, the use of fruit flies for in-vivo drug discovery has the obvious advantages of using a living organism to test drugs in combination with the throughput potential.
A number of further documents (14 - 20) describe the use of mutant drosophila in drug screening assays. These may be in terms of behavioural assays based on climbing assay. However, there is no realisation or suggestion of how to address the issue of throughput by carrying out such assays and/or how one may combine a behavioural response with a different type of response. Although the use of Drosophila offers some distinct advantages in terms of being an in vivo model and being able to screen many drug candidates at once, the ability to be a truly high- throughput model is quite limited. Moreover, a large library of test candidates may nevertheless generate a large number of potential hits and choosing which of these hits to carry forward can be difficult. Thus there is a need to be able to screen drugs more efficiently in order that initial hits are able to be taken forward more effectively or quickly to a clinical setting.
It is amongst the objects of the present invention to obviate and/or mitigate one or more of the aforementioned disadvantages. Summary of the invention
The present invention is based on the use of a plurality of genetically modified Drosophila lines in the development of an improved screening method for identifying lead candidates for further lead optimisation and/or more rapid progression towards the clinic.
The present invention is based on a multi-layer screening process using Drosophila models of neurodegenerative disease, such as Parkinson's disease. The starting point in this process is a library of candidate drugs and the end point is a reduced selection of drugs from the library which are active based on showing evidence of a specific behavioural change displayed by the Drosophila, which may be indicative of a drug showing neuroprotection or repair. The reduced selection of drugs may be taken forward for further rounds of screening, administration to other disease models, such as animal models, and/or administered to test subjects. The present invention is ideally directed to the use of a first screen which employs a first library of candidate drugs and then second or further screens which employ a reduced number of candidate drugs from the first library of candidate drugs.
In a first aspect there is provided a method for screening candidate agents for potential use and/or further development as a drug for treating or preventing a neurodegenerative disorder, such as Parkinson's diseases, the method comprising: a) administering candidate agents to Drosophila from a first Drosophila line which has been genetically modified to express a wild-type mammalian gene or mutant mammalian gene associated with the development and/or progression of a neurodegenerative disorder in a mammal and observing an effect the candidate agents have on a behaviour of the Drosophila; selecting candidate agents which display a positive effect on the behaviour of the Drosophila; and b) administering the selected candidate agents to Drosophila from a further Drosophila line which has been genetically modified to express a different wild-type mammalian gene or mutant mammalian gene associated with the development and/or progression of the neurodegenerative disorder and observing an effect the selected candidate agents have on the behaviour of the Drosophila; and further selecting candidate agents which display a positive effect on the behaviour of the Drosophila.
In one embodiment the second or further Drosophila lines may display more severe behavioural abnormalities as compared to the first Drosophila line. As it may be more difficult to conduct assays using drosophila which display more severe behavioural abnormalities, there may be an advantage of using such drosophila to screen fewer compounds.
The method may optionally include one or more further rounds of screening using further different genetically modified Drosophila lines and/or observing brain tissue samples in order to view any effect the selected candidate agents have on brain morphology, such as neurons in the brain.
The present invention is particularly suited for the identification of drug candidates for treating Parkinson's disease or related conditions. Such related conditions include a number of other conditions with Parkinson-like symptoms known as Parkinsonism. These conditions include multiple system atrophy, progressive supranuclear palsy, corticobasal degeneration, Lewy body dementia, stroke, encephalitis (inflammation of the brain), and head trauma. Other similar conditions include essential tremor, dystonic tremor, vascular Parkinsonism, and drug-induced Parkinsonism. In one embodiment the present invention is based upon the development of a method which employs a climbing assay known the art. Thus, the present invention may also be used in the identification of drug candidates for diseases or conditions where a climbing assay may be used for observing a behavioural change. As well as the above identified diseases/conditions, this may also be used in looking for drugs for treating Alzheimer's and Huntington's diseases. Thus, the present invention may find application in developing drugs for use in treating any of the above conditions. By employing a layered screening approach, the present invention permits the potential identification of drug candidates without have to screen full libraries of compounds multiple times. It may also be possible to identify drug candidates which may be active against related, or even unrelated neurological conditions. Certain conditions may in fact currently be thought of as being unrelated, but it may be the case that such an assumption is incorrect in a physiological and/or biochemical sense and it may be possible to identify drugs which act positively in such "unrelated" conditions. Additionally, it will be appreciated that drug screening using such fly models is quite time and resource consuming. Thus, by reducing the amount of drug candidates going forward to a secondary screen, saves considerable time and resources. It can also permit a higher number of replicates to be carried out, leading to a better statistical significance; this would be impractical or extremely costly when using a large library of compounds.
The Drosophila lines for use in the present invention may be generated by introducing into a Drosophila genome a wild-type or mutant gene which has been identified as being associated with the development or progression of the particular neurodegenerative disorder. Techniques for genetically modifying Drosophila are known to the skilled addressee and described generally in Bischof (2007) for example. A specific non-limiting example of genetically modifying Drosophila is described hereinafter.
In accordance with the invention the first Drosophila line is genetically modified to express a first gene known to be associated with the development and/or progression of Parkinson's disease and the second Drosophila line is genetically modified to express a different gene or form of the first gene known to be associated with the development and/or progression of Parkinson's. For example, the first Drosophila line may be modified to express a wild-type version of a first gene associated with Parkinson's disease and the second line may be modified to express a mutant version of the first gene associated with Parkinson's disease. The first gene may be a-synuclein. Examples of mutant forms of a-synuclein which may be used in the present invention include A30P, A53T, E46K, H50Q and G51 D (numbering according to the sequence identified as P37840 in the UniProtKB database In addition to a- synuclein there are other established genetic causes of Parkinson's disease such as LRKK2 (including mutants G2019S and R1441 G) (numbering according to the sequence identified as Q5S007 in the UniProtKB database), VPS35 (including mutant D620N)(numbering according to the sequence identified Q96QK1 in the UniProtKB database) and EIF4G1 (including mutant R1205H) (numbering according to the sequence identified Q04637 in the UniProtKB database, PRKN, PINK1 and DJ-1 among others and any of these genes alone or in addition to the use of α-synuclein may be used in accordance with the present invention. Third or further genetically modified Drosophila lines may be generated to express further mutant forms of the aforementioned genes, in order that such third or further genetically modified Drosophila lines may be tested in accordance with the present invention. In each further round of screening, it may be expected that a reduced number of candidate drugs is carried forward for further screening, leading to an improved or more rapid throughput for screening. Double mutants may also be provided and tested. For example it is possible to make a Drosophila line expressing two mutant genes, such as VPS35 (D620N) and asyn. Two separate genetic constructs including each mutant can be generated and then each construct integrated into the Drosophila genome at to two different genome insertion sites, one on the 2nd (AttP40) and one on the 3rd (AttP2) chromosome, allowing modular recombination of any two genes or mutants.
The candidate agents to be tested will typically be small chemical molecules, which may have a molecular weight of less than 500 Daltons. The assay can also be used to test RNAi and biologic agents (such as peptides, antibodies, antibody fragments and the like). The candidate agents may conveniently added to the food given to the Drosophila, so that the candidate agents are ingested by the Drosophila through feeding.
Expression of the genes and/or mutants described herein may typically result in an observable phenotypic response, such as reduction of movement, such as may be observed by way of climbing ability compared to an appropriate control (e.g. a Drosophila line which does not express a human gene). Thus, a positive effect on Drosophila behaviour in the context of the present invention may be seen as an improvement or reduction in the degree or severity of the reduction of movement, such as climbing ability. Climbing ability and hence an improvement or reducing in the degree or severity may be tested using a known climbing assay. See for example Friggi-Grelin, et al (2003), or modified versions thereof. An improvement in climbing ability may be determined in comparison to a control. The control may be the same test carried out in the absence of the candidate drug, or a test carried out in the presence of a known Parkinson's disease drug, in order to identify candidate drugs which are better than existing ones.
Candidate drugs which are identified as providing a positive effect, such as an improvement in climbing ability, as compared to a control, are carried forward to a further test. Typically there may be a significant reduction from the original library of compounds tested, which are carried forward to the further test. The further test as carried out using a different Drosophila line, will enable a further significant reduction in the number of positive compounds being identified. Any further rounds are expected to reduce the number of positive compounds even further.lt is possible to determine whether candidate compounds are neuroprotective, or simply relieve symptoms of Parkinson's disease, such as the observed climbing ability. Neuroprotective compounds will prevent neurons from being destroyed, or reducing other morphological changes, such as a reduction in formation of Lewy-body like aggregations of synuclein.; lower levels of cell death (as measured by molecular markers) and/or increased survival of dopaminergic neurons which may be observed in the brain of the Drosophila. Such an effect may be observed by studying brain samples from Drosophila observed as responding favourably to test candidate agents, that is Drosophila which display a positive behavioural response, such as better climbing ability, to a candidate compound. It is possible through dissection of the Drosophila brain to visualize whether or not a compound prevents or reduces neuron destruction or other brain perturbations from occurring.
Drugs which are identified in accordance with the present invention as providing a positive effect, in terms of relieving symptoms and optionally being neuroprotective, may be carried forward to further testing in animal models of the neurodegenerative disorder, such as Parkinson's disease, and/or given to man in order to test their effectiveness. Alternatively or optionally, lead candidates may be subjected to chemical modification and such chemically modified candidates tested in accordance with the present invention in order to ascertain if better candidates can be identified.
The present invention will now be further described with reference to the figures and following non-limiting examples. Figure 1 shows in schematic form an embodiment of the present invention;
Figure 2 shows an example of results which may be obtained by testing various Drosophila lines in accordance with the present invention using a variety of different drugs, at different concentrations and time points;
Figure 3 shows a rationalisation of the results obtained in relation to Figure 2 into a more simple positive/negative effect; and
Figure 4 shows similar results to Figure 3, but further including further results observed following testing of a double mutant Drosophila, histological staining of Drosophila brain tissue, or mouse histology/behavioural data where applicable.
Materials and Methods
Transgenic Drosophila melanogaster (fruit fly) models of PD:
All transgenic Drosophila lines were generated using a phiC31 based system [8]. This allows the insertion of different transgenic constructs into the same insertion site of the Drosophila genome across different transgenic lines, making the results comparable. A number of appropriate upstream activation sequence (UAS) activated genetic constructs were synthesised, each encoding a specific version of a-synuclein (asyn), including the standard 'wild type' and 2 mutant sequences (A30P E46K, numbering as previously defined above). The constructs were inserted in flies using a standard protocol (Genetic Services Inc.). This process generated 3 different models of PD (asyn wt, asyn A30P, asyn E46K). In order to use these models for testing drugs 3-4 males are crossed with 10 virgin female elav- Gal4 flies. In the first progeny (F1 generation) of this cross the interaction between the UAS and Gal4 under the expression control of the pan-neuronal elav promoter means that the form of synuclein in each model is expressed only in the central nervous system (CNS) - a standard application of the Gal4/UAS system [1 1 ].
Treating fruit flies with candidate drugs:
A candidate drug is introduced at a suitable concentration into the fruit fly media. Control groups are treated with the vehicle only. A treatment group (TG) is defined as a specific line of flies expressing a form of asyn and treated with a certain candidate drug at a certain concentration. The relevant control group (CG) is a group a group of flies treated with vehicle only.
Climbing assay:
The climbing assay is performed as described in [12] and also used in [1 ]. In summary: At the time point of interest (e.g x, y and z days post eclosion) and for the treatment group of interest, replicate groups of adult flies are anaesthetised and placed in a vertical plastic column (25 cm long, 1 .5 cm diameter) with a conic bottom end. After 30 min recovery from C02 exposure, flies are suddenly tapped to the bottom of the column to startle them and force them to pack in a narrow space. Overexcited flies remained whirling at the bottom and started climbing after a longer delay. After 30s, flies having reached the top of the column and flies remaining at the bottom were separately counted. Multiple trials are performed, one for each replicate set.
The score (climbing score or CS) is a mean / S.E.M. of values obtained in the these trials, expressed in arbitrary units from 0 to 4 and representing as percentages of the total number of flies. If the CS of a treatment group is higher than that of the control group and this difference is significant (ANOVA p-value < 0.05) then this is defined for the purposes of the present invetion as a climbing phenotype reversal or rescue. This can be further quantified by an "effect size" metric where effect size = (Average CS of drug treated group - Average CS of vehicle treated group)/Standard deviation of pooled drug and vehicle treated group CS measurements). Measuring the survival of dopaminergic neurons in fruit fly brains:
Fruit fly brains are dissected and fixed according to [13]. In summary adult brains are dissected in 1 x PBS with Inox 5 forceps on Sylgard dishes loosely following established protocols and fixed with 2% paraformaldehyde (Sigma) overnight (4 °C). Samples were then washed 3χ for 20 min each with PAT (1 x PBS, 1 % BSA (Sigma) and 0.5% Triton X-100 (Sigma)) and then blocked with 3% normal goat serum (NGS; BioSource) in PAT for 1 h at room temperature. Primary antibody (anti-TH) incubations were performed overnight at 4 °C, nutating in 500 μΙ PAT plus NGS. Samples were washed 3χ for 20 min each with PAT and then incubated overnight with goat secondary antibodies from Molecular Probes/I nvitrogen (1 :1000 dilution). Samples were washed again 3χ for 20 min each with PAT, rinsed with 1 χ PBS and mounted in VectaShield.
Measuring the survival of dopaminergic neurons in fruit fly brains:
The anti-TH antibody stains dopaminergic neurons by attaching on Tyrosine Hydroxilase (TH) and then sequestered and stained by the secondary antibody. The brain preparations can be scanned using a confocal microscope. The images of brains acquired are used to quantify the number of dopaminergic cells active (stained) in each treatment and control group.
If the average of a treatment group value is higher than the one of the control group and this difference is significant (ANOVA p-value < 0.05) then we define that as an improvement in the survival of dopaminergic neurons which is also evidence of neuroprotection of a particular drug.
Selection of an active drug
By active drug we mean a small molecule that appears to be a good candidate for further development according to the evidence obtained from experiments described here. An active drug, after treatment of a specific treatment group gives satisfies:
• A climbing phenotype rescue at any of the time points of interest (x, y or z days post eclosion) and optionally
• An improvement in the survival level of dopaminergic neurons at the final (z days post eclosion) time point of interest (neuroprotective effect).
Figure 1 shows in schematic fashion a representation of how the present invention may be carried out. A library of candidate drugs is provided as represented by the multi-well assay plates shown in the Figure. Each of the drugs within the library is tested in transgenic flies expressing the wild type asyn gene by feeding the each drug to a subset of flies from the line line expressing the wild type asyn gene and thereafter subjecting the flies to the described climbing assay. By comparing the results with a vehicle control, it is possible to identify an initial selection of drug candidates from the library, which can be carried forward to a second screen. In the second screen, positive active drugs identified from the first screen are feed to a different line of transgenic Drosophila, such as A30P or E46K mutants and again it is determined whether or not the active drugs from the first screen still remain positive following the second screen. Brain sections from Drosophila which have been identified as being rescued following the second screen may be observed in order to view dopaminergic neuron survival (as shown). Drugs identified as being positive from the second round of screening may be carried forward for further screens or further developed.
Further screens may include further testing using further Drosophila lines which express other wild-type or mutant genes known to be associated with neurodegeneration and/or used in animal models, such as mice, rats etc of Parkinson's disease, before being tested in man. Histology testing may optionally be carried out in order to see what effect if any the active/positive drugs have on brain morphology, in particular neuron survival. This can add a further layer of evidence in terms of whether or not a particular drug can be considered as neuroprotective or drugs which are considered to relieve the symptoms of the neurodegenerative disorder. Alternatively identified active drugs may be used to develop a number of related chemical entities, through appropriate chemical modification of the active drug, in order to provide a number of derivatives and the derivatives tested using the screens of the present invention. This would allow the possible development of better potential drug candidates from those tested using the original chemical library. It is envisaged that the multi-layered approach of the present invention will be more informative than simple in vitro screening for candidate drugs and also be quicker and less costly than using animal disease models. Although the use of animal disease models may be required, it is envisaged that they may be employed later on in any screening process, such that less drugs and/or animals may be required. In accordance with the present invention Figure 2 shows results obtained following the testing of a number of drugs (6 different drugs) at a variety of concentrations and time points. The drugs where administered to 3 different Drosophila lines. Underlined positive values represent a statistically significant positive or improved response as observed in the climbing test, as compared to a vehicle only control. All other values represent a statistically insignificant improvement, or a poorer response as observed using the climbing assay as compared to the vehicle only control.
Figure 3 simplifies the results shown in Figure 2, in order that where any particular subset (drug concentrations tested and the various time points) displayed one or more statistically significant responses, this is identified as a positive result (Y) and the drug can be carried forward to a further test using a different Drosophila line. It will be appreciated that the stringency of such determination can be increased or decreased as deemed appropriate by the investigator. Drugs which are identified as providing a positive effect following the first test may be further tested in a further Drosophila line, optionally brain histology investigated and/or in an animal model, for example. Exemplary results in this regard are shown in Figure 4.
Feany, M. B., & Bender, W. W. (2000). A Drosophila model of Parkinson's disease. Nature, 404(6776), 394-398
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Irwin DJ, Lee V, Trojanowski JQ. Parkinson's disease dementia: convergence of [alpha]-synuclein, tau and amyloid-[beta] pathologies. Nat Rev Neurosci. 2013 E Rincon-Limas D, Jensen K, Fernandez-Funez P. Drosophila models of proteinopathies: the little fly that could. Curr Pharm Des. 2012;18(8) :1 108-22.
Mizuno, H., Fujikake, N., Wada, K., & Nagai, Y. (2010). a-Synuclein Transgenic Drosophila As a Model of Parkinson's Disease and Related Synucleinopathies. Parkinson's Disease, 201 1 , 212706.
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Bischof, J., Maeda, R. K., Hediger, M., Karch, F., & Basler, K. (2007). An optimized transgenesis system for Drosophila using germ-line-specific phiC31 integrases. Proceedings of the National Academy of Sciences of the United States of America, 104(9), 3312-3317. http://doi.org/10.1073/pnas.061 151 1 104
Markstein, M., Pitsouli, C, Villalta, C, Celniker, S. E., & Perrimon, N. (2008). Exploiting position effects and the gypsy retrovirus insulator to engineer precisely expressed transgenes. Nature Genetics, 40(4), 476-483.
Kruger, R., Kuhn, W., Muller, T., Woitalla, D., Graeber, M., Kosel, S., et al. (1998). Ala30Pro mutation in the gene encoding alpha-synuclein in Parkinson's disease. Nature Genetics, 18(2), 106-108.
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Duffy, J. B. (2002). GAL4 system in Drosophila: a fly geneticist's Swiss army knife. Genesis (New York, NY : 2000), 34(1 -2), 1-15. http://doi.org/10.1002/gene.10150 Friggi-Grelin, F., Coulom, H., Meller, M., Gomez, D., Hirsh, J., & Birman, S. (2003). Targeted gene expression in Drosophila dopaminergic cells using regulatory sequences from tyrosine hydroxylase. Journal of Neurobiology, 54(4), 618-627. Hampel, S., Chung, P., McKellar, C. E., Hall, D., Looger, L. L, & Simpson, J. H. (201 1 ). Drosophila Brainbow: a recombinase-based fluorescence labeling technique to subdivide neural expression patterns. Nature Methods, 8(3), 253-259.
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WO2016/051 157.

Claims

Claims
1 . A method for screening candidate agents for potential use and/or further development as a drug for treating or preventing a neurodegenerative disorder, such as Parkinson's diseases, the method comprising: a) administering candidate agents to Drosophila from a first Drosophila line which has been genetically modified to express a wild-type mammalian gene or mutant mammalian gene associated with the development and/or progression of a neurodegenerative disorder in a mammal and observing an effect the candidate agents have on a behaviour of the Drosophila; selecting candidate agents which display a positive effect on the behaviour of the Drosophila; and b) administering the selected candidate agents to Drosophila from a further Drosophila line which has been genetically modified to express a different wild-type mammalian gene or mutant mammalian gene associated with the development and/or progression of the neurodegenerative disorder and observing an effect the selected candidate agents have on the behaviour of the Drosophila; and further selecting candidate agents which display a positive effect on the behaviour of the Drosophila.
2. The method according to claim 1 wherein the observed behaviour is an alteration in movement, such as the ability of Drosophila to climb.
3. The method according to claims 1 or 2 wherein the second or further Drosophila lines may display more severe behavioural abnormalities as compared to the first Drosophila line.
4. The method according to any preceding claim further including one or more further rounds of screening using further different genetically modified Drosophila lines and/or observing brain tissue samples in order to view any effect the selected candidate agents have on brain morphology, such as neurons in the brain.
5. The method according to any preceding claim for use in identifying drug candidates for treating Parkinson's disease or related conditions including multiple system atrophy, progressive supranuclear palsy, corticobasal degeneration, Lewy body dementia, stroke, encephalitis (inflammation of the brain), and head trauma, essential tremor, dystonic tremor, vascular Parkinsonism, and drug-induced Parkinsonism.
6. The method according to any of claims 1 - 4 for use in identifying drug candidates for treating Alzheimer's and Huntington's diseases.
7. The method according to any preceding claim wherein the Drosophila lines are generated by introducing into a Drosophila genome a wild-type or mutant gene which has been identified as being associated with the development or progression of the particular neurodegenerative disorder.
8. The method according to claim 7 wherein the first Drosophila line is genetically modified to express a first gene known to be associated with the development and/or progression of Parkinson's disease and the second Drosophila line is genetically modified to express a different gene or form of the first gene known to be associated with the development and/or progression of Parkinson's.
9. The method according to claim 8 wherein the first Drosophila line is genetically modified to express a wild-type gene associated with Parkinson's disease and the second line is genetically modified to express a mutant version of the first gene associated with Parkinson's disease.
10. The method according to claims 8 or 9 wherein the gene and/or mutant gene is a- synuclein.
1 1 . The method according to claim 10, wherein the mutant version of a-synuclein comprises the mutation A30P, A53T, E46K, H50Q and/or G51 D.
12. The method according to claims 7 -9 wherein the gene and/or mutant gene is LRKK2, VPS35, EIF4G1 , PRKN, PINK1 or DJ-1 .
13. The method according to any preceding claim comprising administering the selected candidate agents to Drosophila from a further Drosophila line which has been genetically modified to express a different wild-type mammalian gene or mutant mammalian gene associated with the development and/or progression of the neurodegenerative disorder and observing an effect the selected candidate agents have on the behaviour of the Drosophila; and further selecting candidate agents which display a positive effect on the behaviour of the Drosophila.
14. The method according to any preceding claims further comprising studying brain samples from the Drosophila used in the method in order to detect whether or not the selected drug candidates cause an observable change in the brain of the Drosophila.
15. The method according to any preceding claim further comprising testing selected drug candidates in an animal model of the disease.
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